Aggrecanase Structure
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 60/711,457,
filed August 25,2005, and U.S. Provisional Application No. 60/711,458, also filed
August 25,2005. The contents of both provisional applications are incorporated herein by
reference in their entirety.
TECHNICAL FIELD
This invention relates to aggrecanase polypeptides, aggrecanase polypeptide/ligand
complexes, crystals of aggrecanase polypeptides, crystals of aggrecanase
polypeptide/ligand complexes, and related methods and software systems.
BACKGROUND
Aggrecanases are enzymes that can cleave cartilage aggrecan, a component of the
extracellular matrix. Cartilage aggrecan generally includes a core protein with multiple
functional domains that allow the cartilage to resist compressive forces. When the
degradation of extracellular matrix components exceeds the synthesis of extracellular
matrix components, there is a loss of aggrecan and a subsequent disruption of cartilage,
resulting in a disruption of the structure and function of certain tissue types. The
degradation of aggrecan is believed to be pathophysiological event that is seen in the earlier
stages of joint diseases such as osteoarthritis (OA) and rheumatoid arthritis.
SUMMARY
In one aspect, the invention features a crystallized aggrecanase polypeptide.
In another aspect, the invention features a crystallized polypeptide-ligand complex
that includes an aggrecanase polypeptide and a ligand.
In another aspect, the invention features a crystallized polypeptide-ligand complex
that includes an aggrecanase-1 polypeptide and a ligand.
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In yet another aspect, the invention features a crystallized polypeptide-ligand
complex that includes an aggrecanase polypeptide and a polycyclic ligand having a Zinc-
chelating moiety.
In another aspect, the invention features a composition that includes a crystal. The
crystal includes an aggrecanase polypeptide and a ligand.
In another aspect, the invention features a method that includes using a three-
dimensional model of a complex that includes an aggrecanase polypeptide bound to a
ligand. The three-dimensional model is used to design an agent that binds the aggrecanase
polypeptide.
In a further aspect, the invention features a method that includes using a three-
dimensional model of an aggrecanase polypeptide to design an agent that binds the
aggrecanase polypeptide.
In another aspect, the invention features a method that includes selecting an agent
by performing rational drug design with a three-dimensional structure of a crystalline
complex. The agent is contacted with an aggrecanase polypeptide, and an ability of the
agent to bind the aggrecanase polypeptide is detected. The crystalline complex includes an
aggrecanase polypeptide.
In yet another aspect, the invention features a method that includes contacting an
aggrecanase polypeptide with a ligand to form a composition and crystallizing the
composition to form a crystalline complex where the ligand is bound to the aggrecanase
polypeptide. The crystalline complex can diffract X-rays to a resolution of at least
about 3.5 A.
In another aspect, the invention features a software system that includes instructions
for causing a computer system to accept information relating to the structure of an
aggrecanase polypeptide bound to a ligand, accept information relating to a candidate
agent, and determine binding characteristics of the candidate agent to the aggrecanase
polypeptide. Determination of the binding characteristics is based on the information
relating to the structure of the aggrecanase polypeptide bound to the ligand and the
information relating to the candidate agent.
In another aspect, the invention features a computer program on a computer
readable medium on which is stored a plurality of instructions. When the instructions are
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executed by one or more processors, the processors accept information relating to the
structure of a complex that includes an aggrecanase polypeptide bound to a ligand. The
processors further accept information relating to a candidate agent and determine binding
characteristics of the candidate agent to the aggrecanase polypeptide. Determination of the
binding characteristics is based on the information related to the structure of the
aggrecanase polypeptide and the information related to the candidate agent.
In another aspect, the invention features a method that includes accepting
information relating to the structure of a complex including an aggrecanase polypeptide
bound to a ligand and modeling the binding characteristics of the aggrecanase polypeptide
with a candidate agent. Such a method is implemented by a software system.
In another aspect, the invention features a computer program on a computer
readable medium on which is stored a plurality of instructions. When the instructions are
executed by one or more processors, the processors accept information relating to a
structure of a complex that includes an aggrecanase polypeptide bound to a ligand. The
processors further model the binding characteristics of the aggrecanase polypeptide with a
candidate agent.
In another aspect, the invention features a software system that includes instructions
for causing a computer system to accept information relating to a structure of a complex
that includes an aggrecanase polypeptide bound to a ligand. The instructions also cause a
computer system to model the binding characteristics of the aggrecanase polypeptide with a
candidate agent.
In another aspect, the invention features a method of modulating aggrecanase
activity in a subject. The method includes using rational drug design to select an agent that
is capable of modulating aggrecanase activity, and administering a therapeutically effective
amount of the agent to the subject.
In another aspect, the invention features a method of treating a subject having a
condition associated with aggrecanase activity. The method includes using rational drug
design to select an agent that is capable of affecting aggrecanase activity and administering
a therapeutically effective amount of the agent to a subject in need such an agent.
In another aspect, the invention features a method of prophylactically treating a
subject susceptible to a condition associated with aggrecanase activity. The method
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includes determining that the subject is susceptible to the condition associated with the
activity, using rational drug design to select an agent that is capable of effecting
aggrecanase activity, and administering a therapeutically effective amount of the agent to
the subject.
In another aspect, the invention features a crystallized polypeptide-ligand complex
that includes an aggrecanase-2 polypeptide and a ligand.
In another aspect the invention features a crystallized polypeptide-ligand complex
that includes an aggrecanase-2 polypeptide and a peptidomimetic ligand having a metal
chelating moiety.
In yet another aspect, the invention features a composition that includes a crystal,
which includes an aggrecanase-2 polypeptide and a ligand.
In another aspect, the invention features a method that includes using a three-
dimensional model of a complex to design an agent that binds the aggrecanase-2
polypeptide. The complex includes an aggrecanase-2 polypeptide bound to a ligand.
In a further aspect, the invention features a method to design an agent that binds the
aggrecanase-2 polypeptide. The method includes using a three-dimensional model of an
aggrecanase-2 polypeptide
In another aspect, the invention features a method of selecting an agent by
performing rational drug design with a three-dimensional structure of a crystalline complex
that includes an aggrecanase-2 polypeptide. The agent is contacted with an aggrecanase-2
polypeptide, and an ability of the agent to bind the aggrecanase-2 polypeptide is detected.
In another aspect, the invention features a method that includes contacting an
aggrecanase-2 polypeptide with a ligand to form a composition and crystalhzing the
composition to form a crystalline complex in which the ligand is bound to the aggrecanase-
2 polypeptide. The crystalline complex can diffract X-rays to a resolution of at least about
3.5 A.
In another aspect, the invention features a software system that includes instructions
for causing a computer system to accept information relating to the structure of an
aggrecanase-2 polypeptide bound to a ligand. The instructions further cause a computer
system to accept information relating to a candidate agent, and determine binding
characteristics of the candidate agent to the aggrecanase-2 polypeptide. The determination
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is based on the information relating to the structure of the aggrecanase-2 polypeptide bound
to the ligand and to the information relating to the candidate agent.
In a further aspect, the invention features a computer program residing on a
computer readable medium on which is stored a plurality of instructions. When the
instructions are executed by one or more processors, the processors accept information
relating to the structure of a complex that includes an aggrecanase-2 polypeptide bound to a
ligand. The processors further accept information relating to a candidate agent and
determine binding characteristics of the candidate agent to the aggrecanase-2 polypeptide.
Such determination is based on the information relating to the structure of the
aggrecanase-2 polypeptide and to the information relating to the candidate agent
In another aspect, the invention features a method that includes accepting
information relating to the structure of a complex including an aggrecanase-2 polypeptide
bound to a ligand. The method further includes modeling the binding characteristics of the
aggrecanase-2 polypeptide with a candidate agent. Such a method is implemented by a
software system.
In another aspect, the invention features a computer program residing on a computer
readable medium on which is stored a plurality of instructions. When the instructions are
executed by one or more processors, the processors accept information relating to a
structure of a complex that includes an aggrecanase-2 polypeptide bound to a ligand and
model the binding characteristics of the aggrecanase-2 polypeptide with a candidate agent.
In another aspect, the invention features a software system that includes instructions
for causing a computer system to accept information relating to a structure of a complex
including an aggrecanase-2 polypeptide bound to a ligand. The instructions further cause
the computer system to model the binding characteristics of the aggrecanase-2 polypeptide
with a candidate agent.
In another aspect, the invention features a method of modulating aggrecanase-2
activity in a subject. The method includes using rational drug design to select an agent
capable of modulating aggrecanase-2 activity and administering a therapeutically effective
amount of the agent to the subject.
In another aspect, the invention features a method of treating a subject having a
condition associated with aggrecanase-2 activity. The method includes using rational drug
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design to select an agent that is capable of affecting aggrecanase-2 activity and
administering a therapeutically effective amount of the agent to a subject in need of such an
agent.
In yet another aspect, the invention features a method of prophylactically treating a
subject susceptible to a condition associated with aggrecanase-2 activity. The method
includes determining that the subject is susceptible to the condition, using rational drug
design to select an agent that is capable of effecting aggrecanase-2 activity, and
administering a therapeutically effective amount of the agent to the subject.
Other features and advantages of the invention will be apparent from the
description, drawings and claims.
DESCRIPTION OF DRAWINGS
FIG. 1A is the amino acid sequence (SEQ ID NO:l) of a fragment of a human
Agg-1 polypeptide (Agg-l-AlC2) that includes the catalytic domain (amino acids 214-428)
and the disintegrin-like domain (amino acids 437-509) and a mutation at amino acid 362
(Glu362Gln) that makes the polypeptide more amenable to crystallization. The glutamine
at position 362 is indicated in bold and underlined. A FLAG-Tag (indicated in bold) fused
to the C-terminus of the polypeptide facilitated purification.
FIG IB is the wildtype amino acid sequence (SEQ ID NO:2) of a fragment of a
human Agg-1 polypeptide corresponding to the mutant FLAG-tagged fragment described
in FIG. 1A. The wildtype sequence includes the catalytic domain (amino acids 214-428)
and the disintegrin-like domain (amino acids 437-509). The wildtype glutamate at position
362 is indicated in bold and underlined.
FIG. 2 is a ribbon diagram illustrating the structure of the Agg-1 -A1C2 polypeptide.
Calcium atoms and zinc atoms are also indicated.
FIG 3 is the amino acid sequence (SEQ ID NO:3) of a fragment of a human Agg-2
polypeptide including the catalytic domain (amino acids 265-476), disintegrin-like domain
(amino acids 486-556), and thrombospondin-like domain (amino acids 557-628). A strep-
tag is fused to the C-terminus of the polypeptide and is indicated in bold.
FIG 4 is the chemical structure of 2-[4,-(4-Isobutyryl-phenoxymethyl)-biphenyl-4-
sulfonylamino]-3-methyl-butyric acid (Compound 1).
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FIG. 5 is a ribbon diagram illustrating the structure of the human Agg-l-AlC2
polypeptide bound to the inhibitor Compound 1. Structural helices are identified by "aA"
through "aH." Structural sheets are indicated by "PA" through "pK." Calcium atoms and
zinc atoms are also indicated.
FIG. 6 is a ribbon diagram illustrating the structure of the catalytic domain of an
Agg-l-AlC2/Compound 1 complex. The disulfide bonds in the Agg-l-AlC2 polypeptide
are shown as sticks.
FIG 7 is the chemical structure of batimastat.
FIG. 8 is a ribbon diagram illustrating the structure of a human Agg-2 polypeptide
(SEQ ID NO:3) bound to the inhibitor batimastat. Structural helices are identified by
"aA," through "aH." Structural sheets are indicated by "pA" through "pK." Calcium
atoms and zinc atoms are also indicated.
FIG. 9 is a ribbon diagram illustrating the structure of the disintegrin-like domain of
anAgg-l-AlC2/Compound 1 complex. The disulfide bonds in the Agg-l-AlC2
polypeptide are shown as sticks.
FIG. 10 is an electron density map of the active site of unliganded Agg-l-AlC2.
FIG. 11 is a superposition of active site structures of unliganded Agg-l-AlC2 and
the Agg-l-AlC2/Compound 1 complex.
FIG. 12 is the structure of the inhibitor Compound 1. Interactions between
Compound 1 and the Agg-l-AlC2 polypeptide, and the active zinc atom are indicated. SI
and SI' represent successive substrate binding pockets.
FIG. 13 is the structure of batimastat. Interactions between batimastat and amino
acid residues of the human Agg-2 polypeptide (SEQ ID NO:3) and the active site zinc atom
are indicated. SI, S2', S3' and SI' represent successive substrate binding pockets.
Like reference symbols in the various drawings indicate like elements.
DETAILED DESCRIPTION
In general, this invention relates to aggrecanase polypeptides, aggrecanase
polypeptide/ligand complexes, crystals of aggrecanase polypeptides, crystals of
aggrecanase polypeptide/ligand complexes, and related methods and software systems.
Without wishing to be bound by theory, it is believed that crystal structures of aggrecanase
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polypeptides and/or aggrecanase polypeptide/ligand complexes can be useful for designing .
or identifying other ligands that can interact with aggrecanase polypeptides.
As an example, Agg-1 and Agg-2 aggrecanases can cleave between Glu373 and
Ala374 of aggrecan, and aggrecan fragments resulting from such cleavage have been
predominantly found in synovial fluids of patients with osteoarthritis and joint injury.
Therefore, it is believed that identification of aggrecanase inhibitors may be useful for
treatment of these disorders.
An exemplary aggrecanase polypeptide is a human Agg-1 polypeptide. FIG. 1A is
the amino acid sequence (SEQ ID NO:l) of a fragment of a human Agg-1 polypeptide
(Agg-l-AlC2) that includes the catalytic domain (amino acids 214-428) and the
disintegrin-like domain (amino acids 437-509) and a mutation at amino acid 362
(Glu362Gln) that makes the polypeptide more amenable to crystallization. The glutamine
at position 362 is indicated in bold and underlined. A FLAG-Tag (indicated in bold) fused
to the C-terminus of the polypeptide facilitated purification. FIG. IB is the wildtype amino
acid sequence (SEQ ID NO:2) of a fragment of a human Agg-1 polypeptide corresponding
to the mutant FLAG-tagged fragment described in FIG. 1 A. The wildtype sequence
includes the catalytic domain (amino acids 214-428) and the disintegrin-like domain
(amino acids 437-509). The wildtype glutamate at position 362 is indicated in bold and
underlined. FIG. 2 is a ribbon diagram illustrating the structure of the Agg-l-AlC2
polypeptide (Calcium atoms and zinc atoms are also indicated). The coordinates of the
crystal structure of the Agg-1 -A1C2 polypeptide are provided below at Table 4.
Another exemplary aggrecanase polypeptide is a human Agg-2 polypeptide. FIG. 3
is the amino acid sequence (SEQ ID NO:3) of a fragment of a human Agg-2 polypeptide
including the catalytic domain (amino acids 265-476), disintegrin-like domain (amino acids
486-556), and thrombospondin-like domain (amino acids 557-628).
An exemplary aggrecanase polypeptide/ligand complex is a human Agg-1
polypeptide bound to the aggrecanase inhibitor (2-[4'-(4-Isobutyryl-phenoxymethyl)-
biphenyl-4-sulfonylarnino]-3-methyl-butyric acid) ("Compound 1"). FIG. 4 shows the
structure of Compound 1, and FIG. 5 is a ribbon diagram illustrating the structure of the
human Agg-l-AlC2 polypeptide bound to the inhibitor Compound 1. Structural helices
are identified by "aA" through "aH", and structural sheets are indicated by "PA" through
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"J3K." Calcium atoms and zinc atoms are also indicated. FIG. 6 is a ribbon diagram
illustrating the structure of the catalytic domain of an Agg-l-AlC2/Compound 1 complex.
The disulfide bonds in the Agg-l-AlC2 polypeptide are shown as sticks. The coordinates
of the crystal structure of the human Agg-1 polypeptide/Compound 1 complex are provided
below at Table 5.
Another exemplary aggrecanase polypeptide/ligand complex is a human Agg 2
polypeptide bound to the metalloproteinase inhibitor, batimastat. FIG. 7 shows the
structure of batimastat, and FIG. 8 is a ribbon diagram illustrating the structure of a human
Agg-2 polypeptide (SEQ ID NO:3) bound to the inhibitor batimastat. Structural helices are
identified by "aA," through "aH." Structural sheets are indicated by "p\A" through "pK."
Calcium atoms and zinc atoms are also indicated. FIG. 9 is a ribbon diagram illustrating
the structure of the disintegrin-like domain of an Agg-1-AlC2/Compound 1 complex. The
disulfide bonds in the Agg-l-AlC2 polypeptide are shown as sticks. The coordinates of
the crystal structure of the human Agg-2 polypeptideatimastat complex are provided
below at Table 6.
To determine the structure of an aggrecanase, such as Agg-1 or Agg-2, a human
Aggl-polypeptide or a human Agg-2 polypeptide can be prepared and crystallized as
described below. In general, the human Agg-1 polypeptide or the human Agg-2
polypeptide can be prepared as desired. For example, in some embodiments, the human
Agg-1 polypeptide is expressed from a DNAplasmid. The expression can be driven by a
promoter, such as an inducible promoter. The human Agg-1 polypeptide can be expressed
as a fusion protein with a suitable tag, such as a glutathione-S-transferase (GST), myc, HA,
hexahistidine, Strep, or FLAG tag. The tag can facilitate isolation of the human Agg-1
polypeptide from cells, such as from bacterial cells or from a mammalian cell line. For
example, the human Agg-1 polypeptide can be expressed in and isolated from Chinese
Hamster Ovary (CHO) cells. A fusion protein can be cleaved at a protease site engineered
into the fusion protein, such as at or near the site of fusion between the polypeptide and the
tag. When it is desirable to form a complex between the human Agg-1 polypeptide and a
ligand, such as Compound 1, the human Agg-1 polypeptide can be contacted with the
ligand following cleavage and purification. For example, the human Agg-1 polypeptide
can be mixed with Compound 1 prior to purification (e.g., prior to cleavage of a
9

polypeptide tag), or the human Agg-1 polypeptide can be mixed with Compound 1 after
purification. In some embodiments, Compound 1 can be mixed with the human Agg-1
polypeptide prior to purification and again following purification.
The described methods can also be used for the expression and purification of the
human Agg-2 polypeptide. A ligand such as batimastat can be mixed with the human
Agg-2 polypeptide prior to purification, after purification, or both prior to and following
purification.
The human Agg-1 polypeptide or the human Agg-2 polypeptide can be placed in
solution for collecting spectral data, NMR data, or for growing a crystal. For example, the
human Agg-1 polypeptide or the human Agg-2 polypeptide can be crystallized in the
presence of a salt {e.g., a sodium salt), a polymer (e.g., polyethylene glycol (PEG)), and/or
an organic solvent. Crystals can be grown by various methods, such as, for example,
sitting or hanging drop vapor diffusion. In general, crystallization can be performed at a
temperature of from about 4°C to about 60°C (e.g., from about 4°C to about 45°C, such as
at about 4°C, about 15°C, about 18°C, about 20°C, about 25°C, about 30°C, about 32°C,
about 35°C, about 37°C).
In certain embodiments, the human Agg-1 polypeptide and Compound 1, or the
human Agg-2 polypeptide and batimastat, can be combined in a solution for collecting
spectral data for the human Agg-1 polypeptide/Compound 1 complex or the human Agg-2
polypeptide/batimastat complex, for collecting NMR data for either of these two
complexes, or for growing a crystal of either of these two complexes as described above.
In general, a crystal of the human Agg-1 polypeptide or the human Agg-2
polypeptide can diffract X-rays to a resolution of about 3.5 A or less (e.g., about 3.2 A or
less, about 3.0 A or less, about 2.5 A or less, about 2.4 A or less, about 2.3 A or less, about
2.2 A or less, about 2.1 A or less, about 2.0 A or less, about 1.9 A or less, about 1.8 A or
less, about 1.7 A or less, about 1.6 A or less, about 1.5 A or less, or about 1.4 A or less), m
some embodiments, a crystal of the human Agg-1 polypeptide or the human Agg-2
polypeptide can diffract X-rays to a resolution of from about 1.7 A to about 3.0 A (e.g., the
crystal of the human Agg-1 polypeptide can diffract X-rays to about 2.0 to about 2.8 A).
In general, a crystal of the human Agg-1 polypeptide bound to Compound 1 or the
the human Agg-2 polypeptide bound to batimastat can diffract X-rays to a resolution of
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about 3.5 A or less (e.g., about 3.2 A or less, about 3.0 A or less, about 2.5 A or less, about
2.4 A or less, about 2.3 A or less, about 2.2 A or less, about 2.1 A or less, about 2.0 A or
less, about 1.9 A or less, about 1.8 A or less, about 1.7 A or less, about 1.6 A or less, about
1.4 A or less, or about 1.4 A or less). In some embodiments, a crystal of the human Agg-1
polypeptide bound to Compound 1 or the human Agg-2 polypeptide bound to batimastat
can diffract X-rays to a resolution of from about 1.7 A to about 3.0 A (e.g., the crystal of
the human Agg-1 polypeptide bound to Compound 1 can diffract X-rays to about 2.8 A,
and the crystal of the human Agg-2 polypeptide bound to batimastat can diffract X-rays to
about 2.9 A).
In certain embodiments, a crystal of the human Agg-1 polypeptide belongs to space
group P2i with unit cell parameters a=128.28 A, b=83.63 A, c=150.16 A, p=l 12.409°. In
other embodiments, a crystal of the human Agg-1 polypeptide bound to Compound 1
belongs to space group P2i with unit cell parameters a=82.07A, b=83.96A, c=98.95A,
P=89.9°. In other embodiments, a crystal of the human Agg-2 polypeptide bound to
batimastat belongs to space group P3i with unit cell parameters a=93.64A, b=93.64A,
c=92.59A, Y=120°. The space group refers to the overall symmetry of the crystal, and
includes point symmetry and space symmetry. In certain embodiments, a crystal of the
human Agg-1 polypeptide can contain eight molecules of the human Agg-1 polypeptide in
the asymmetric unit, a crystal of the human Agg-1 polypeptide bound to Compound 1 can
contain four molecules of the complex in the asymmetric unit, or a crystal of the human
Agg-2 polypeptide bound to batimastat can contain two molecules of the complex in the
asymmetric unit. The asymmetric unit is the smallest unit from which the crystal structure
can be generated by making use of the symmetry operations of the space group. A crystal
is generally made up of the motif defined by the space-group symmetry operations on the
asymmetric units, and a translation of that motif through the crystal lattice.
Structural data describing a crystal can be obtained, for example, by X-ray
diffraction. X-ray diffraction data can be collected by a variety of sources, X-ray
wavelengths and detectors. In some embodiments, rotating anodes and synchrotron sources
(e.g., Advanced Light Source (ALS), Berkeley, California; or Advanced Photon Source
(APS), Argonne, Illinois) can be used as the source(s) of X-rays. In certain embodiments,
X-rays for generating diffraction data can have a wavelength of from about 0.5 A to about
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1.6 A (e.g., about 0.7 A, about 0.9 A, about 1.0 A, about 1.1 A, about 1.3 A, about 1.4 A,
about 1.5 A, or about 1.6 A). In some embodiments, area detectors and/or charge-couple
devices (CCDs) can be used as the detector(s).
X-ray diffraction data of a crystal of the human Agg-1 polypeptide or the human
Agg-2 polypeptide, or a complex of the human Agg-1 polypeptide bound to Compound 1
or the human Agg-2 polypeptide bound to batimastat can be used to obtain the structural
coordinates of the atoms in the complex. The structural coordinates are Cartesian
coordinates that describe the location of atoms in three-dimensional space in relation to
other atoms in the complex. For example, the structural coordinates listed in Table 4 are
the structural coordinates of a crystalline human Agg-1 polypeptide. The structural
coordinates listed in Tables 5 and 6 are the structural coordinates of a crystalline complex
of the human Agg-1 polypeptide bound to Compound 1 and the human Agg-2 polypeptide
bound to batimastat, respectively. The structural coordinates of Table 4 describe the
location of atoms of the human Agg-1 polypeptide in relation to each other and the
structural coordinates of Table 5 describe the location of atoms of the human Agg-1
polypeptide in relation to each other when the human Agg-1 polypeptide is bound to
Compound 1. The structural coordinates of Table 5 also describe the location of atoms in
the human Agg-1 polypeptide in relation to the atoms in Compound 1, and the location of
atoms in Compound 1 in relation to each other. The structural coordinates of Table 6
describe the location of atoms of the human Agg-2 polypeptide in relation to each other
when the human Agg-2 polypeptide is bound to batimastat, the location of atoms in the
human Agg-2 polypeptide in relation to the atoms in batimastat, and the location of atoms
in batimastat in relation to each other. The structural coordinates can be modified by
mathematical manipulation, such as by inversion or integer additions or subtractions. As
such, structural coordinates are relative coordinates. For example, structural coordinates
describing the location of atoms in the human Agg-1 polypeptide, or the human Agg-1
polypeptide bound to Compound 1, or the human Agg-2 polypeptide bound to batimastat
are not specifically limited by the actual x, y, and z coordinates of Tables 4, 5, and 6,
respectively.
The structural coordinates of the human Agg-1 polypeptide can be used to derive a
representation of the polypeptide or a fragment of the polypeptide. In addition, the
12

structural coordinates of a complex of the human Agg-1 polypeptide bound to Compound 1
or the human Agg-2 polypeptide bound to batimastat can be used to derive a representation
(e.g., a two dimensional representation or three dimensional representation) of the complex,
a fragment of the complex, the human Agg-1 polypeptide or the human Agg-2 polypeptide,
or a fragment of the human Agg-1 polypeptide or the human Agg-2 polypeptide. Such
representations can be useful for a number of applications, including, for example, the
visualization, identification and characterization of an active site of the polypeptide. In
certain embodiments, a three-dimensional representation can include the structural
coordinates of the human Agg-1 polypeptide according to Tables 4 or 5, ± a root mean
square (rms) deviation from the alpha carbon atoms of amino acids of not more than about
1.5 A {e.g., not more than about 1.0 A, not more than about 0.5 A). In certain other
embodiments, a three-dimensional representation can include the structural coordinates of
the human Agg-2 polypeptide according to Table 6.
RMS deviation is the square root of the arithmetic mean of the squares of the
deviations from the mean, and is a way of expressing deviation or variation from structural
coordinates. Conservative substitutions (see discussion below) of amino acids can result in
a molecular representation having structural coordinates within the stated rms deviation.
For example, two molecular models of polypeptides that differ from one another by
conservative amino acid substitutions can have coordinates of backbone atoms within a
stated rms deviation, such as less than about 1.5 A (e.g., less than about 1.0 A, less than
about 0.5 A). Backbone atoms of a polypeptide include the alpha carbon (Ca or CA)
atoms, carbonyl carbon (C) atoms, and amide nitrogen (N) atoms.
Various software programs allow for the graphical representation of a set of
structural coordinates to obtain a representation of the human Agg-1 polypeptide, a
complex of the human Agg-1 polypeptide bound to Compound 1 or the human Agg-2
polypeptide bound to batimastat, or a fragment of one of these complexes. In general, such
a representation should accurately reflect (relatively and/or absolutely) structural
coordinates, or information derived from structural coordinates, such as distances or angles
between features. In some embodiments, the representation is a two-dimensional figure,
such as a stereoscopic two-dimensional figure. In certain embodiments, the representation
is an interactive two-dimensional display, such as an interactive stereoscopic two-
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dimensional display. An interactive two-dimensional display can be, for example, a
computer display that can be rotated to show different faces of a polypeptide, a fragment of
a polypeptide, a complex and/or a fragment of a complex. In some embodiments, the
representation is a three-dimensional representation. As an example, a three-dimensional
model can be a physical model of a molecular structure (e.g., a ball-and-stick model). As
another example, a three dimensional representation can be a graphical representation of a
molecular structure (e.g., a drawing or a figure presented on a computer display). A two-
dimensional graphical representation (e.g., a drawing) can correspond to a three-
dimensional representation when the two-dimensional representation reflects three-
dimensional information, for example, through the use of perspective, shading, or the
obstruction of features more distant from the viewer by features closer to the viewer. In
some embodiments, a representation, can be modeled at more man one level. As an
example, when the three-dimensional representation includes a polypeptide, such as a
human Agg-1 polypeptide or a human Agg-2 polypeptide, or a complex, such as a complex
of the human Agg-1 polypeptide bound to Compound 1 or the human Agg-2 polypeptide
bound to batimastat, the polypeptide can be represented at one or more different levels of
structure, such as primary (amino acid sequence), secondary (e.g., a-helices and p-sheets),
tertiary (overall fold), and quaternary (oligomerization state) structure. A representation
can include different levels of detail. For example, the representation can include the
relative locations of secondary structural features of a protein without specifying the
positions of atoms. A more detailed representation could, for example, include the
positions of atoms.
In some embodiments, a representation can include information in addition to the
structural coordinates of the atoms in the human Agg-1 polypeptide, a complex of the
human Agg-1 polypeptide bound to Compound 1 or the human Agg-2 polypeptide bound
to batimastat. For example, a representation can provide information regarding the shape
of a solvent accessible surface, the van der Waals radii of the atoms of the model, and the
van der Waals radius of a solvent (e.g., water). Other features that can be derived from a
representation include, for example, electrostatic potential, the location of voids or pockets
within a macromolecular structure, and the location of hydrogen bonds and salt bridges.
14

An agent that interacts with (e.g., binds) the human Agg-1 polypeptide or the
human Agg-2 polypeptide can be identified or designed by a method that includes using a
representation of either polypeptide or a fragment of either polypeptide, or a complex of the
human Agg-1 polypeptide bound to Compound 1 or the human Agg-2 polypeptide bound
to batimastat, or a fragment of either of these complexes. Exemplary types of
representations include the representations discussed above. In some embodiments, the
representation can be of an analog polypeptide, polypeptide fragment, complex or fragment
of a complex. A candidate agent that interacts with the representation can be designed or
identified by performing computer fitting analysis of the candidate agent with the
representation. In general, an agent is a molecule. Examples of agents include
polypeptides, nucleic acids (including DNA or RNA), steroids and non-steroidal organic
compounds. An agent that interacts with a polypeptide (e.g., a human Agg-1 polypeptide
or a human Agg-2 polypeptide) can interact transiently or stably with the polypeptide. The
interaction can be mediated by any of the forces noted herein, including, for example,
hydrogen bonding, electrostatic forces, hydrophobic interactions, and van der Waals
interactions.
As noted above, X-ray crystallography can be used to obtain structural coordinates
of a complex of the human Agg-1 polypeptide bound to Compound 1 or the human Agg-2
polypeptide bound to batismatat. However, such structural coordinates can be obtained
using other techniques including NMR techniques. Additional structural information can
be obtained from spectral techniques (e.g., optical rotary dispersion (ORD), circular
dichroism (CD)), homology modeling, and computational methods {e.g., computational
methods that can include data from molecular mechanics, computational methods that
include data from dynamics assays).
In some embodiments, the X-ray diffraction data can be used to construct an
electron density map of the human Agg-1 polypeptide, a complex of the human Agg-1
polypeptide bound to Compound 1 or the human Agg-2 polypeptide bound to batimastat, or
a fragment of the polypeptide or a fragment of the complex, and the electron density map
can be used to derive a representation {e.g., a two dimensional representation, a three
dimensional representation) of the human Agg-1 polypeptide, the human Agg-1
polypeptide bound to Compound 1, or the human Agg-2 polypeptide bound to batimastat,
15

or a fragment of the polypeptide or of either complex. Creation of an electron density map
typically involves using information regarding the phase of the X-ray scatter. Phase
information can be extracted, for example, either from the diffraction data or from
supplementing diffraction experiments to complete the construction of the electron density
map. Methods for calculating phase from X-ray diffraction data include, for example,
multiwavelength anomalous dispersion (MAD), multiple isomorphous replacement (MIR),
multiple isomorphous replacement with anomalous scattering (MIRAS), single
isomorphous replacement with anomalous scattering (SIRAS), reciprocal space solvent
flattening, molecular replacement, or any combination thereof. These methods generate
phase information by making isomorphous structural modifications to the native protein,
such as by including a heavy atom or changing the scattering strength of a heavy atom
already present, and then measuring the diffraction amplitudes for the native protein and
each of the modified cases. If the position of the additional heavy atom or the change in its
scattering strength is known, then the phase of each diffracted X-ray can be determined by
solving a set of simultaneous phase equations. The location of heavy atom sites can be
identified using a computer program, such as SHELXS (Sheldrick, Institut Anorg. Chemie,
Gottingen, Germany), and diffraction data can be processed using computer programs such
as MOSFLM, SCALA, SOLOMON, and SHARP ("The CCP4 Suite: Programs for Protein
Crystallography," Acta Crystallogr. Sect. D, 54:905-921,1997; deLa Fortelle and
Brigogne, Meth. Enzym. 276:472-494,1997). The phase of X-ray scatter for a crystalline
human Agg-1 polypeptide bound to Compound 1, for example, can be determined by MAD
using crystals of a selenomethionine substituted protein. To create a selenomethionine
substituted protein, mammalian cells expressing the human Agg-1 nucleic acid can be
cultured in the presence of selenomethionine. The selenomethionine-substituted protein is
purified, contacted with Compound 1, and the complex crystallized by a standard method,
such as by the hanging drop technique. Phases obtained by MAD from crystals of the
native and selenomethionine substituted protein each complexed with Compound 1 can
then be used to create an electron density map of the complex.
The electron density map can be used to derive a representation of a polypeptide, a
complex, or a fragment of a polypeptide or complex by aligning a three-dimensional model
of a polypeptide or complex (e.g., a complex containing a polypeptide bound to a ligand)
16

with the electron density map. For example, the electron density map corresponding to the
human Agg-1 polypeptide can be aligned with the electron density map corresponding to
the human Agg-1 polypeptide/Compound 1 complex derived by an isomorphous
replacement method. The human Agg-2 polypeptide/batimastat complex can be aligned
with the electron density map corresponding to the human Agg-1 polypeptide complexed to
Compound 1.
The alignment process results in a comparative model that shows the degree to
which the calculated electron density map varies from the model of the previously known
polypeptide or the previously known complex. The comparative model is then refined over
one or more cycles (e.g., two cycles, three cycles, four cycles, five cycles, six cycles, seven
cycles, eight cycles, nine cycles, ten cycles) to generate a better fit with the electron density
map. A software program such as CNS (Brunger et at, Acta Crystallogr. D54:905-921,
1998) can be used to refine the model. The quality of fit in the comparative model can be
measured by, for example, an RWOrk or Re value. A smaller value of Rwork or Rfree
generally indicates a better fit. Misalignments in the comparative model can be adjusted to
provide a modified comparative model and a lower Rwork or Rg-ee value. The adjustments
can be based on information (e.g., sequence information) relating to the human Agg-1
polypeptide, the human Agg-2 polypeptide, Compound 1, batimastat, the human Agg-1
polypeptide/Compound 1 complex or the human Agg-2 polypeptide/batimastat complex, as
appropriate. As an example, in embodiments in which a model of a previously known
complex of a polypeptide bound to a ligand is used, such as the human Agg-1 polypeptide
bound to Compound 1, an adjustment can include replacing the Compound 1 of the
complex with a different ligand, such as batimastat. As another example, in certain
embodiments, an adjustment can include replacing an amino acid in the previously known
polypeptide (e.g., the human Agg-1 polypeptide) with the amino acid in the corresponding
site of a different aggrecanase, such as the human Agg-2 polypeptide. When adjustments
to the modified comparative model satisfy a best fit to the electron density map, the
resulting model is that which is determined to describe the polypeptide or complex from
which the X-ray data was derived. Methods of such processes are disclosed, for example,
in Carter and Sweet, eds., "Macromolecular Crystallography" in Methods in Enzvmology,
Vol. 277, Part B, New York: Academic Press, 1997, and articles therein, e.g., Jones and
17

Kjeldgaard, "Electron-Density Map Interpretation," p. 173, and Kleywegt and Jones,
"Model Building and Refinement Practice," p. 208.
Discussed above is a method of deriving a representation of a complex by aligning
a three-dimensional model of a previously known polypeptide or a previously known
complex with a newly calculated electron density map corresponding to a crystal of the
polypeptide or the complex. One adjustment that can be used in this modeling process can
include replacing the compound in the representation of the previously known complex
with Compound 1 orbatimastat.
A machine, such as a computer, can be programmed in memory with the structural
coordinates of the human Agg-1 polypeptide, or a complex of the human Agg-1
polypeptide bound to Compound 1 or the human Agg-2 polypeptide bound to batimastat,
together with a program capable of generating a graphical representation of the structural
coordinates on a display connected to the machine. Alternatively or additionally, a
software system can be designed and/or utilized to accept and store the structural
coordinates. The software system can be capable of generating a graphical representation
of the structural coordinates. The software system can also be capable of accessing
external databases to identify compounds with similar structural features as Compound 1 or
batimastat, and/or to identify one or more candidate agents with characteristics that may
render the candidate agent(s) likely to interact with the human Agg-1 polypeptide or the
human Agg-2 polypeptide.
A machine having a memory containing structure data or a software system
containing such data can aid in the rational design or selection of a human Agg-1
polypeptide agonist, a human Agg-1 polypeptide antagonist, a human Agg-2 polypeptide
agonist, or a human Agg-2 polypeptide antagonist. For example, such a machine or
software system can aid in the evaluation of the ability of an agent to associate with a
complex of the human Agg-1 polypeptide bound to Compound 1 or the human Agg-2
polypeptide bound to batimastat, or can aid in the modeling of compounds or proteins
related by structural or sequence homology to the human Agg-1 polypeptide or the human
Agg-2 polypeptide. As used herein, an agonist refers to a compound that enhances at least
one activity of the human Agg-1 polypeptide or the human Agg-2 polypeptide. An
antagonist refers to a compound that inhibits or counteracts at least one activity of the
18

human Agg-1 polypeptide or the human Agg-2 polypeptide. For example, a compound,
such as Compound 1 or batimastat may function as an antagonist of the human Agg-1
polypeptide or the human Agg-2 polypeptide by, for example, decreasing the rate of
aggrecan cleavage by the human Agg-1 polypeptide or the human Agg-2 polypeptide, or by
inhibiting interaction of the human Agg-1 polypeptide or the human Agg-2 polypeptide
with aggrecan, thereby inhibiting aggrecan cleavage.
The machine can produce a representation (eg., a two dimensional representation, a
three dimensional representation) of a complex of the human Agg-1 polypeptide bound to
Compound 1 or the human Agg-2 polypeptide bound to batimastat or a fragment of either
complex. A software system, for example, can cause the machine to produce such
information. The machine can include a machine-readable data storage medium including
a data storage material encoded with machine-readable data. The machine-readable data
can include structural coordinates of atoms of a complex of the human Agg-1 polypeptide
bound to Compound 1 or the human Agg-2 polypeptide bound to batimastat or a fragment
of either complex. Machine-readable storage media (e.g., data storage material) include,
for example, conventional computer hard drives, floppy disks, DAT tape, CD-ROM, DVD,
and other magnetic, magneto-optical, optical, and other media which may be adapted for
use with a machine (e.g., a computer). The machine can also have a working memory for
storing instructions for processing the machine-readable data, as well as a central
processing unit (CPU) coupled to the working memory and to the machine-readable data
storage medium for the purpose of processing the machine-readable data into the desired
three-dimensional representation. A display can be connected to the CPU so that the three-
dimensional representation can be visualized by the user. Accordingly, when used with a
machine programmed with instructions for using the data (e.g., a computer loaded with one
or more programs of the sort described herein) the machine is capable of displaying a
graphical representation (e.g., a two dimensional graphical representation, a three-
dimensional graphical representation) of any of the polypeptides, polypeptide fragments,
complexes, or complex fragments described herein.
A display (e.g., a computer display) can show a representation of the human Agg-1
polypeptide or the human Agg-2 polypeptide, or a complex of the human Agg-1
polypeptide bound to Compound 1 or a complex of the human Agg-2 polypeptide bound to
19

batimastat, or a fragment the human Agg-1 polypeptide or the human Agg-2 polypeptide or
a fragment of either complex. The user can inspect the representation and, using
information gained from the representation, generate a model of the human Agg-1
polypeptide or polypeptide fragment bound to a ligand, or a complex or fragment thereof
that includes an agent other than Compound 1 or batimastat The model can be generated,
for example, by altering a previously existing representation of the human Agg-1
polypeptide, the human Agg-1 polypeptide/Compound 1 complex or the human Agg-2
polypeptide/batirnastat complex. Optionally, the user can superimpose a three-dimensional
model of an agent on the representation of the human Agg-1 polypeptide, or the human
Agg-1 polypeptide bound to Compound 1 or the human Agg-2 polypeptide bound to
batimastat. The agent can be an agonist (e.g., a candidate agonist) of the human Agg-1
polypeptide or the human Agg-2 polypeptide, or an antagonist (e.g., a candidate antagonist)
of the human Agg-1 polypeptide or the human Agg-2 polypeptide. In some embodiments,
the agent can be a known compound or a fragment of a known compound, m certain
embodiments, the agent can be a previously unknown compound, or a fragment of a
previously unknown compound.
It can be desirable for the agent to have a shape that complements the shape of the
active site. There can be a preferred distance, or range of distances, between atoms of the
agent and atoms of the human Agg-1 polypeptide or the human Agg-2 polypeptide.
Distances longer than a preferred distance may be associated with a weak interaction
between the agent and active site (e.g., the active site of the human Agg-1 polypeptide or
the human Agg-2 polypeptide). Distances shorter than a preferred distance may be
associated with repulsive forces that can weaken the interaction between the agent and the
polypeptide. A steric clash can occur when distances between atoms are too short. A steric
clash occurs when the locations of two atoms are unreasonably close together, for example,
when two atoms are separated by a distance less than the sum of their van der Waals radii.
If a steric clash exists, the user can adjust the position of the agent relative to the human
Agg-1 polypeptide or the human Agg-2 polypeptide (e.g., a rigid body translation or
rotation of the agent) until the steric clash is relieved. The user can adjust the conformation
of the agent or of the human Agg-1 polypeptide or the human Agg-2 polypeptide in the
vicinity of the agent in order to relieve a steric clash. Steric clashes can also be removed by
20

altering the structure of the agent, for example, by changing a "bulky group," such as an
aromatic ring, to a smaller group, such as to a methyl or hydroxyl group, or by changing a
rigid group to a flexible group that can accommodate a conformation that does not produce
a steric clash. Electrostatic forces can also influence an interaction between an agent and a
ligand-binding domain. For example, electrostatic properties can be associated with
repulsive forces that can weaken the interaction between the agent and the human Agg-1
polypeptide or the human Agg-2 polypeptide. Electrostatic repulsion can be relieved by
altering the charge of the agent, e.g., by replacing a positively charged group with a neutral
group.
Forces that influence binding strength between Compound 1 or batimastat and the
human Agg-1 polypeptide or the human Agg-2 polypeptide, respectively, can be evaluated
in the polypeptide/agent model. These can include, for example, hydrogen bonding,
electrostatic forces, hydrophobic interactions, van der Waals interactions, dipole-dipole
interactions, 7i-stacking forces, and cation-7i interactions. The user can evaluate these
forces visually, for example by noting a hydrogen bond donor/acceptor pair arranged with a
distance and angle suitable for a hydrogen bond. Based on the evaluation, the user can
alter the model to find a more favorable interaction between the human Agg-1 polypeptide
or the human Agg-2 polypeptide and the agent. Altering the model can include changing
the three-dimensional structure of the polypeptide without altering its chemical structure,
for example by altering the conformation of amino acid side chains or backbone dihedral
angles. Altering the model can include altering the position or conformation of the agent,
as described above. Altering the model can also include altering the chemical structure of
the agent, for example by substituting, adding, or removing groups. For example, if a
hydrogen bond donor on the human Agg-1 polypeptide or the human Agg-2 polypeptide is
located near a hydrogen bond donor on the agent, the user can replace the hydrogen bond
donor on the agent with a hydrogen bond acceptor.
The relative locations of an agent and the human Agg-1 polypeptide or the human
Agg-2 polypeptide, or their conformations, can be adjusted to find an optimized binding
geometry for a particular agent to the human Agg-1 polypeptide or the human Agg-2
polypeptide. An optimized binding geometry is characterized by, for example, favorable
hydrogen bond distances and angles, maximal electrostatic attractions, rrrinimal
21

electrostatic repulsions, the sequestration of hydrophobic moieties away from an aqueous
environment, and the absence of steric clashes. The optimized geometry can have the
lowest calculated energy of a family of possible geometries for the human Agg-1
polypeptide/agent complex or the human Agg-2 polypeptide/agent complex. An optimized
geometry can be determined, for example, through molecular mechanics or molecular
dynamics calculations.
A series of representations of the human Agg-1 polypeptide, or complexes of the
human Agg-1 polypeptide bound to Compound 1, or complexes of the human Agg-2
polypeptide bound to batimastat, having different bound agents can be generated. A score
can be calculated for each representation. The score can describe, for example, an expected
strength of interaction between the human Agg-1 polypeptide or the human Agg-2
polypeptide and the agent. The score can reflect one of the factors described above that
influence binding strength. The score can be an aggregate score that reflects more than one
of the factors. The different agents can be ranked according to their scores.
Steps in the design of the agent can be carried out in an automated fashion by a
machine. For example, a representation of the human Agg-1 polypeptide or the human
Agg-2 polypeptide can be programmed in the machine, along with representations of
candidate agents. The machine can find an optimized binding geometry for each of the
candidate agents to the active site, and calculate a score to determine which of the agents in
the series is likely to interact most strongly with the human Agg-1 polypeptide or the
human Agg-2 polypeptide.
A software system can be designed and/or implemented to facilitate these steps.
Software systems (e.g., computer programs) used to generate representations or perform
the fitting analyses include, for example: MCSS, Ludi, QUANTA, Insight II, Cerius2,
CHARMm, and Modeler from Accelrys, Inc. (San Diego, CA); SYBYL, Unity, FleXX,
and LEAPFROG from TRIPOS, Inc. (St. Louis, MO); AUTODOCK (Scripps Research
Institute, La Jolla, CA); GRID (Oxford University, Oxford, UK); DOCK (University of
California, San Francisco, CA); and Flo+ and Flo99 (Thistlesoft, Morris Township, NJ).
Other useful programs include ROCS, ZAP, FRED, Vida, and Szybki from Openeye
Scientific Software (Santa Fe, NM); Maestro, Macromodel, and Glide from Schrodinger,
22

LLC (Portland, OR); MOE (Chemical Computing Group, Montreal, Quebec), Allegrow
(Boston De Novo, Boston, MA), CNS (Brunger, et at, Acta Crystal!. Sect. D 54:905-921.
1997) and GOLD (Jones et al, J. Mol. Biol. 245:43-53,1995). The structural coordinates
can also be used to visualize the three-dimensional structure of the human Agg-1
polypeptide, or a complex of the human Agg-1 polypeptide bound to Compound 1 or the
human Agg-2 polypeptide bound to batimastat using MOLSCRJJPT, RASTER3D, or
PYMOL (Kraulis, J. Appl. Crystallogr. 24: 946-950,1991; Bacon and Anderson, J. Mol.
Graph. 6: 219-220,1998; DeLano, The PYMOL Molecular Graphics System (2002)
DeLano Scientific, San Carlos, CA).
The agent can, for example, be selected by screening an appropriate database, can
be designed de novo by analyzing the steric configurations and charge potentials of a
human Agg-1 polypeptide or a human Agg-2 polypeptide in conjunction with the
appropriate software systems, and/or can be designed using characteristics of known
ligands of other aggrecanase enzymes or other metalloproteinases. The method can be
used to design or select agonists or antagonists of the human Agg-1 polypeptide or the
human Agg-2 polypeptide. A software system can be designed and/or implemented to
facilitate database searching, and/or agent selection and design.
Once an agent has been designed or identified, it can be obtained or synthesized and
further evaluated for its effect on the human Agg-1 polypeptide or the human Agg-2
polypeptide activity. For example, the agent can be evaluated by contacting it with the
human Agg-1 polypeptide or the human Agg-2 polypeptide and measuring the effect of the
agent on polypeptide activity. A method for evaluating the agent can include an activity
assay performed in vitro or in vivo. For example, an activity assay performed in vitro can
be a fluorescence-based assay. Agents can be assessed by their ability to inhibit cleavage
of a fluorescent peptide substrate, such as Abz-TEGARGSVI-Dap(Dnp) (Abz:o-
aminobenzoyl; Dnp: 2,4 dinitrophenyl) (Anaspec, Inc., San Jose, California). The peptide
sequence TEGARGSVI is based on the amino acid sequence of the Glu373-Ala374
cleavage site of aggrecan in osteoarthritis. Candidate compounds can be pre-incubated
with a purified human Agg-1 polypeptide for 10 min. and then the peptide substrate can be
added to the combination at temperatures ranging from 25°C to 37°C, typically at 30° C.
23

Cleavage of the Glu-Ala bond releases the fluorophore from internal quenching. This
results in an increase in fluorescence monitored at A.ex 340 ran and XeX 420 ran over a period
of 40 min. The initial rate (v) at each concentration of the substrate is fit to the following
equation:
V=Vrnax-S,,/(S0)/,+S")
where h is the Hill constant and So.s is the substrate concentration at half the Vax.
The percentage activity remaining in the presence of inhibitor is plotted as a function of
inhibitor concentration, and the IC50 value is determined by fitting the data to the following
equation:
% activity = 100IC50/ & + IC50)
where I0 is initial concentration of inhibitor.
An activity assay can be an in vivo assay, such as a cell-based assay. A cell based
assay can include monitoring the effect of a candidate agent on aggrecan cleavage. Such
assays for the inhibitors may involve contacting the inhibitor with cells expressing the
human Agg-1 polypeptide and aggrecan, and then measuring aggrecan cleavage, such as by
detecting and measuring aggrecan fragments produced by cleavage at the aggrecanase
susceptible site. Aggrecan fragments can be detected by standard protein detection
techniques, such as immunohistochemical analysis methods.
Depending upon the action of the agent on the human Agg-1 polypeptide or the
human Agg-2 polypeptide, the agent can act either as an agonist or antagonist of the human
Agg-1 polypeptide activity or the human Agg-2 polypeptide activity. An agonist, for
example, may increase the rate of aggrecan cleavage or increase the binding affinity of the
human Agg-1 polypeptide or the human Agg-2 polypeptide to aggrecan. Conversely, an
antagonist may decrease the rate of aggrecan cleavage or decrease the binding affinity of
the human Agg-1 polypeptide or the human Agg-2 polypeptide to aggrecan. The agent can
be contacted with the human Agg-1 polypeptide or the human Agg-2 polypeptide in the
presence of an aggrecan substrate in order to determine whether or not the agent inhibits
binding of the human Agg-1 polypeptide or the human Agg-2 polypeptide to the aggrecan
substrate. A crystal containing the human Agg-1 polypeptide or the human Agg-2
24

polypeptide bound to the identified agent can be grown and the structure determined by X-
ray crystallography. A second agent can be designed or identified based on the interaction
of the first agent with the human Agg-1 polypeptide or the human Agg-2 polypeptide.
Various molecular analysis and rational drug design techniques are further
i
disclosed in, for example, U.S. Patent Nos. 5,834,228,5,939,528 and 5,856,116, as well as
in PCT Application No. PCT/US98/16879, published as WO 99/09148.
While certain embodiments have been described, other embodiments are also
contemplated.
As an example, while embodiments involving the human Agg-1 polypeptide, the
human Agg-1 polypeptide bound to Compound 1, and the human Agg-2 polypeptide bound
to batimastat have been described, the description herein is more generally directed to any
aggrecanase polypeptide and any ligand.
An aggrecanase polypeptide can be a full-length, mature polypeptide, including the
full-length amino acid sequence of any isoform of an aggrecanase polypeptide. An isoform
is any of several multiple forms of a protein that differ in their primary structure.
An aggrecanase polypeptide can be a fragment of a human Agg-1 polypeptide or a
fragment of a human Agg-2 polypeptide, such as a propeptide domain, a catalytic domain,
a disintegrin-like domain, a trombospondin type-1 domain, a cysteine-rich domain, a spacer
domain, or a combination thereof.
An aggrecanase polypeptide can have an active site. For example, the catalytic
domain is an active site of an aggrecanase. In general, an active site can include a site of
ligand binding, or a site of phosphorylation, glycosylation, alkylation, acylation, or other
covalent modification. A site of ligand binding can be a site of aggrecan binding or a site
of binding of an agonist or antagonist. An active site can include an attachment site for a
sulfated glycosaminoglycan, such as a chondroitin sulfate and keratin sulfate, or a site of
protease cleavage such as a furin cleavage site. The active site can interact with a
component of the extracellular matrix, such as a heparin or an integrin. A ligand binding
site can include accessory binding sites adjacent to or proximal to the actual site of binding
that may affect activity upon interaction with the ligand. An active site of the human
Agg-1 polypeptide can include amino acids of SEQ ID NO: 1 or SEQ ID NO:2 (FIG. 1A or
25

FIG. IB, respectively). For example, an active site of the human Agg-1 polypeptide can
include one or more of amino acids Leu330, Gly33], Ala333, His361, Phe357, and Ala248
as defined by the amino acid positions of SEQ ID NO:l and SEQ E>NO:2. An active site
of the human Agg-2 polypeptide can include amino acids of SEQ ID NO:3 (FIG. 3). For
example, an active site of the human Agg-2 polypeptide can include one or more of amino
acids Glu411, Asp377, Leu379, Ser441, andLeu443 as defined by the amino acid positions
ofSEQIDNO:3(FIG.3).
The numbering of the amino acids of the human Agg-1 polypeptide or the human
Agg-2 polypeptide maybe different than that set forth herein, and the sequence of the
human Agg-1 polypeptide or the human Agg-2 polypeptide may contain certain
conservative amino acid substitutions that yield the same three-dimensional structure. For
example, the numbering of the human Agg-1 polypeptide maybe different than that set
forth in FIG. 1A or FIG. IB, and the sequence of the human Agg-1 polypeptide may
contain conservative amino acid substitutions but yield the same structure as that defined
by the coordinates of Tables 4 and 5 and illustrated in FIGs. 2,5,6,10, and 11. The
numbering of the human Agg-2 polypeptide may be different than that set forth in FIG. 3,
and the sequence of the human Agg-2 polypeptide may contain conservative amino acid
substitutions but yield the same structure as that defined by the coordinates of Table 6 and
illustrated in FIG. 8. Corresponding amino acids and conservative substitutions in other
isoforms or analogs are easily identified by visual inspection of the relevant amino acid
sequences or by using commercially available homology software programs (e.g.,
MODELLAR, MSI, San Diego, CA).
An analog is a polypeptide having conservative amino acid substitutions. A
conservative substitution can include switching one amino acid for another with similar
polarity, steric arrangement, or of the same class (e.g., hydrophobic, acidic or basic), and
includes substitutions having an inconsequential effect on the three-dimensional structure
of the human Agg-1 polypeptide or the human Agg-2 polypeptide with respect to
identification and design of agents that interact with the polypeptide, as well as for
molecular replacement analyses and/or for homology modeling.
An aggrecanase polypeptide, such as an Agg-1 polypeptide and an Agg-2
polypeptide, can originate from a nonmammalian or mammalian species. A mammalian
26

aggrecanase polypeptide can originate from a human, for example. Exemplary nonhuman
mammals include a nonhuman primate (such as a monkey or ape), a mouse, rat, goat, cow,
bull, pig, horse, sheep, wild boar, sea otter, cat, and dog. Exemplary nonmammalian
species include chicken, turkey, shrimp, alligator, and fish.
An agent can be, for example, a chemical compound {e.g., a polypeptide, nucleic
acid, peptidomimetic). A peptidomimetic is a chemical compound that can mimic the
ability of a peptide to recognize certain physiological molecules, such as proteins and
nucleic acids. In some instances, the peptidomimetic includes non-peptidic structural
elements that are capable of mimicking or antagonizing the biological action(s) of a natural
parent peptide. For example, scissile peptide bonds can be replaced with one or more non-
scissile dipeptide isosteres.
In general, agents that interact with an Agg-1 polypeptide may also interact with an
Agg-2 polypeptide, and agents that interact with an Agg-2 polypeptide may also interact
with an Agg-1 polypeptide. For example, the compositions and methods described herein
would be appropriate for use when Compound 1 is bound to an Agg-2 polypeptide, and
when batimastat is bound to an Agg-1 polypeptide.
While embodiments have been described in which Compound 1 or batimastat is a
ligand, more generally other compounds may also be used as ligands.
As an example, based on a representation of the human Agg-1 polypeptide bound to
Compound 1, derived from the structure of the crystalline complexes, and without wishing
to be bound by theory, it is believed that a Zn atom in the active site chelates with one of
the carboxylate oxygen atoms of Compound 1 at a distance of about 2.1 A (see FIG. 12),
and that the other carboxylate oxygen participates in a water-mediated hydrogen bond with
the backbone atoms of Ala333. It is also believed, however, that this water-mediated
interaction is present only in the mutant form of the protein. It is further believed that
carboxylate MMP inhibitors generally bind more favorably when protonated because they
can form a direct hydrogen bond with the carboxylate of the active site Glu (Glu362),
which was replaced with Gin in the mutant crystallized protein (compare FIGs. 1A and
IB). In the mutant crystallized protein, a primary amide replaced the carboxylate (via the
Glu->Gln mutation), and it is therefore believed that the water-mediated hydrogen bond
could not be made. Thus, the second oxygen of Compound 1 is believed to have been free
27

to interact with other portions of the protein. It is believed that a second area of interaction
between Compound 1 and the human Agg-1 polypeptide is at the site of a hydrogen bond
acceptor near the zinc atom within the human Agg-1 polypeptide. It is also believed that
one of the oxygen atoms from the sulfonamide of Compound 1 occupies this area through
interactions with the backbone NHs of both Leu330 and Gly331, at distances of 2.7 A and
3.1 A, respectively. It is further believed that he SI' pocket of the active site, which spans
about 15 A, is filled by the substituted bi-phenyl portion of Compound 1. In addition, it is
believed that within the S1' pocket are favorable K stacking interactions between the i
biphenyl moiety and His361 of the Agg-1 active site (having about 3.7 A separation). It is
believed that an additional favorable % stacking interaction occurs between the phenyl
moiety substituted on the biphenyl and Phe357. It is also believed that the carbonyl
moiety, which is a substituent on the phenyl moiety, forms a water mediated (2.5A)
hydrogen bond with the backbone atoms of ctB.
Based on this information, and without wishing to be bound by theory, it is believed
that other compounds capable of having one or more similar interactions with a human
Agg-1 polypeptide may also be capable of acting as ligands for the human Agg-1
polypeptide. Such compounds may have the structure:

where each A and B represent a ring (e.g., a cyclyl ring, a heterocyclyl ring, an aryl ring, or
a heteroaryl ring), each L, M, and Y are linker moieties, each R1, R2, and R3 are
substituents, X is a hydrogen bond acceptor, and Z is a metal chelating moiety.
In general, each A and B is independently formed of at least five atoms (e.g., five
atoms, six atoms, seven atoms, eight atoms, nine atoms, 10 atoms, 11 atoms, 12 atoms, 13
atoms, 14 atoms). One or more atoms (e.g., one atom, two atoms, three atoms, four atoms)
can independently be heteroatoms {e.g., N, S, O). For example, in some embodiments,
each A and B is independently aryl or heteroaryl moieties. Examples of such aryl and
heteroaryl moieties include phenyl, pyridyl, pyrimidyl, pyridazyl, thiophenyl, furanyl, and
pyrrolyl.
28

In some embodiments, each L and M can be a bond, for example, providing a direct
attachment of A with B. In certain embodiments, each L and M can independently provide
a spacer, for example a one or two atom spacer, between the two moieties linked together.
Examples of such linkers include methylene, ethylene, oxygen, sulfur, amino,
methyleneoxy, memyleneamino, methylenethioyl, sulfoxide, or sulfone.
Y is generally a moiety linking the hydrogen bond acceptor, X, to the metal
chelator, Z. In some embodiments, Y is a linker. Examples of linkers include alkyl linkers,
such as alkyl linkers having a branched side chain (e.g., an isopropyl side chain).
Additional examples of linkers include alkylene linkers (e.g., methylene, ethylene,
propylene, isopropylene, butylene, or isobutylene), oxygen, sulfur, amino linkers,
methyleneoxy, methyleneamino, methylenethioyl, sulfoxide and sulfone. In some
embodiments, Y is a bond.
R1 is generally a moiety on the A ring that can extend into the SI' pocket of the
human Agg-1 polypeptide (see FIG. 12, for example). In some embodiments, R1 is H. In
certain embodiments, Rl is a larger moiety that extends more deeply into the SI' pocket.
As an example, in some embodiments, R1 is a Q-C6 alkyl (eg., C\ alkyl, C2 alkyl, C3 alkyl,
C4 alkyl, C5 alkyl, C6 alkyl), C2-C6 alkenyl (e.g., C\ alkenyl, C2 alkenyl, C3 alkenyl, C4
alkenyl, C5 alkenyl, C6 alkenyl) or C2-C6 alkynyl (e.g., Q alkynyl, C2 alkynyl, C3 alkynyl,
C4 alkynyl, C5 alkynyl, C6 alkynyl). As another example, in certain embodiments R1 is a
ring moiety, such as a cyclyl ring, a heterocyclyl ring, an aryl ring, or a heteroaryl ring. In
some embodiments, R1 is a fused ring system, for example, a fused cylcyl, aryl,
heterocyclyl or heteroaryl ring system. In some embodiments, one or more heteroatoms in
the heterocyclyl or heteroaryl ring system participates in a hydrogen bond (e.g., a water
mediated hydrogen bond) with the peptide backbone of Ala248. In some embodiments, R
is substituted. In certain embodiments, one or more of the substituents can participate as a
hydrogen bond acceptor with the carbonyl backbone of Ala248 (e.g., via a water molecule).
For example, the substituents can be nitro, cyano, alkylcarbonyl, sulfoxide, sulfone,
sulfonamide, carbonyl, carboxamide, carbamate, or carbonate.
In general, each R2 and R3 is independently, a neutral substituent including less
than about eight non-hydrogen atoms. A neutral substituent has no net positive or negative
charge. Examples of such substituents include hydrogen, halogen (e.g., F, CI, Br),
29

OC(halogen)3, C(halogen)3, Cj-Cg alkoxy {e.g., Q alkoxy, C2 alkoxy, C3 alkoxy, C4
alkoxy, C5 alkoxy, C6 alkoxy), Q-C6 alkyl {e.g., C\ alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5
alkyl, Q alkyl), Q-Q, alkylthioyl (e.g., d alkylthioyl, C2 alkylthioyl, C3 alkylthioyl, C4
alkylthioyl, C5 alkylthioyl, C6 alkylthioyl), or CrC6 alkylamino {e.g., Ci alkylamino, C2
alkylamino, C3 alkylamino, C4 alkylamino, Cs alkylamino, C$ alkylamino). In some
embodiments, R2 and R3, taken together with the ring atom to which they are attached,
form a ring {e.g., providing a rased three ring system with A and B). For example R2 and
R3, taken together with the atoms of attachment from A and B can form a cyclyl ring, a
heterocyclyl ring, an aryl ring, or heteroaryl ring. In some embodiments, the neutral
substituent is hydrophobic.
X is generally a hydrogen bond acceptor. Examples of hydrogen bond acceptors
include sulfur, sulfoxide, sulfone, sulfonamide, carbonyl, carboxamide, urea, carbamate
and carbonate.
Z is generally a metal chelating moiety. For example, Z can be a bidentate metal
chelator that can chelate with a metal such as Fe, Mg, Mn, or Zn. Examples of metal
chelating moities include carboxylic acid, carboxylic amide, hydroxamic acid (for example
a reverse hydroxamic acid), hydroxyurea, hydrazide, sulfonic acid, sulfonamide,
hydroxysulfonamide, sulfodiimide, phosphoric acid, phosphonic acid, thiol, thiol carbonyl,
thiirane, dithiol, sulfonylhydrazide, a heterocyclic moiety {e.g., sulfo&imine, thiazoladme
dione, pyrirnidine trionethiadiazine, barbiturate, thiadiazole {e.g., a peptidic thiadiazole or
thiadiazolethione), thiadiazine, imidazolidinedione, pryidhrione, aminomethyl
bermmidazole) napthylhydroxamate, or a heterocyclic moiety bound to an amide or
carbonyl moiety {e.g., pyridinylamide, pyridinylone, or pyrrolylone).
As another example, based on a representation of the human Agg-2 polypeptide
bound to batimastat, derived from the structure of the crystalline complexes, and without
wishing to be bound by theory, it is believed that the hydroxamate moiety of batimastat
interacts with both the active site metal (having, for example, O-Zn distances of 2.1 A and
2.6 A) and the carboxylate sidechain of the catalytic glutamic acid (Glu411 of Agg-2) via
hydrogen bonding (0-0 distance of 2.4 A). It is also believed that the peptidomimetic
inhibitor batimastat interacts with the human Agg-2 polypeptide in an extended, beta-sheet-
like conformation. It is further believed that the three sidechains of batimastat (thiophene,
30

isobutyl, and benzyl) interact with successive substrate binding pockets, while the two
backbone amide groups make four beta-sheet-like hydrogen-bonds with the protein. In
addition, it is believed that the thiophene, isobutyl, and benzyl sidechains occupy the SI,
SI', and S2' sites respectively, while the intervening amide moieties form hydrogen bonds
to the backbone atoms of Asp377, Leu379, Ser441, and Leu443. It is believed that the
heavy atom distances of these hydrogen bonds are 2.8 A, 3.1 A, 2.7 A, and 2.7 A,
respectively.
Based on this information, and without wishing to be bound by theory, it is believed
that other compounds capable of having one or more similar interactions with a human
Agg-2 polypeptide may also be capable of acting as ligands for the human Agg-2
polypeptide. Such compounds may have the structure:

where each of D and E represent an amide bond or other bond, each of R11, R12, R13, and
R14 represent side chain moieties, for example side chains in the naturally occurring amino
acids or side chains found in unnaturally occurring or artificial amino acids; and G is a
metal chelating moiety.
In some embodiments, each D and E is independently amide, sulfonamide,
arninomethylenehydroxyl, carbamate, carbonate, vinyl, or urea.
In general, each of Ru, R12, R13, and R14 is sized and shaped to fill pockets S3', S2SI', and SI of the human Agg-2 polypeptide, respectively (see FIG. 13, for example). For
example, the S3' pocket is relatively small, and therefore, in some embodiments, R11 is a
lower alkyl, such as, for example, a hydrogen, or preferably a methyl, ethyl, or propyl.
Each of pockets S2', SI' and SI are slightly larger than S3' and therefore can
accommodate larger side chain moieties. Accordingly, in some embodiments, each of R ,
R13, and R14 is independently a neutral moiety, such as, for example a ring moiety, a chain
moiety, or a combination of a ring and chain moiety. For example, each of R12, R13, and
R14 can be independently C1-C6 alkyl {e.g., C\ alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl,
31

C6 alkyi), C2-C6 alkenyl (e.g., Ci alkenyl, C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, C6
alkenyl) or C2-C6 alkynyl (e.g., C\ alkynyl, C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl,
Q alkynyl), cyclyl, heterocycly, aryl, heteroaryl cyclyloxy, heterocyclyoxy, aryloxy,
heteroaryloxy, cyclylthio, heterocyclythio, arylthio, heteroarylthio, cyclylalkyl,
heterocyclylalkyl, arylalkyl, or heteroarylalkyl. In some embodiments, R12 is an aryl
moiety (e.g., a phenyl moiety). In some embodiments, R13 is an alkyl moiety (e.g., an
isopropyl moiety). In some embodiments, R14 is a heteroarylthio moiety (e.g., a
thiophenylthio moiety). In some embodiments, the neutral moiety is a hydrophobic moiety.
As described above, generally, G is a metal chelating moiety. For example, G can
be a bidentate metal chelator that can chelate with a metal such as Fe, Mg, Mn, or Zn. In
some instances, G can also participate in hydrogen bonding with the hydrogen bond
acceptor near the catalytic metal (e.g., catalytic Zn) of the human Agg-2 polypeptide. In
some embodiments, this hydrogen bond acceptor can stabilize the substrate through an
amide carbonyl in the peptide backbone. Examples of metal chelating moieties include
carboxylic acid, carboxylic amide, hydroxamic acid (e.g., a reverse hydroxamic acid),
hydroxyurea, hydrazide, sulfonic acid, sulfonamide, hydroxysulfonamide, sulfodiimide,
phosphoric acid, phosphonic acid, thiol, thiol carbonyl, thiirane, dithiol, sulfonylhydrazide,
a heterocyclic moiety (e.g., sulfodiimine, thiazoladine dione, pyrimidine trionethiadiazine,
barbiturate, thiadiazole (e.g., a peptidic thiadiazole or a thiadiazolethione), thiadiazine,
imidazolidinedione, pyridinione, aminomethyl benzimidazole), napthylhydroxamate, or a
heterocyclic moiety bound to an amide or carbonyl moiety (e.g., pyridinylamide,
pyridinylone, or pyrrolylone).
It is believed that a ligand having the structures described above can have a
physiological effect similar to Compound 1 or batimastat. For example, it is believed that
the ligand can inhibit cleavage of aggrecan.
The following examples are illustrative and not intended as limiting.
EXAMPLES
Example 1. Agg-l-AlC2 and Agg-l-AlC2 bound to Compound 1 were crystallized
and their structures determined.
32

A mutant form of a recombinant human Agg-1 polypeptide was cloned into a vector
for expression in Chinese Hamster Ovary (CHO) cells. The construct encoded the A1C2
mutant Agg-1 (hereafter, Agg-1-A1C2), which carried a glutarnine at amino acid
position 362 instead of a glutamate (FIG 1 A), was stable against proteolysis, appeared
more amenable to crystallization than the wildtype counterpart, and had specificity and
inhibitor sensitivity similar to those of the full-length wildtype protein.
To express selenomethionine labeled Agg-1-A1C2, CHO cells were grown in
175 cm2 flasks containing 75 ml of the maintenance medium (Rl medium buffered with
10 mM Hepes, pH 7.3, 1.25 mg/L Fungizone, 10% dialyzed and heat-inactivated Fetal
bovine serum, 1 % Penicillin/Streptomycin, 2 mM Glutarnine, 0.5 g/L G418,
50 nM Methotrexate) in a humidified incubator with 5% C02 at 37°C. Then 2.5 x 107 cells
were transferred to a 1700 cm2 roller bottle containing 400 ml of the maintenance medium.
Cells were grown at 37°C with slow rolling in a Bellco machine (Bellco Glass, Inc.,
Vineland, NJ).
When the cells reached >90% confluence, the medium was discarded and the roller
bottle was washed twice with phosphate-buffered saline. The cells were labeled with
selenomethionine at 37°C with slow rolling in 300 ml of the labeling medium (Methionine-
free DME medium with 1.25 mg/L Fungizone, 1% Penicillin/Streptomycin, 2 mM
Glutarnine, 30 mg/L selenomethionine, 50 mg/L Heparin, 0.5g/L G418, 50nM
Methotrexate) for 4 days. After labeling, the medium was harvested, filtrated, and stored
at -80°C. The cells remaining in the roller bottle were further cultured in 300 ml of fresh
labeling medium for 3 days at 37°C. The medium was then harvested, filtrated and stored
at -80°C. A total of 10 liters of conditioned media containing the secreted
selenomethionine-labeled human Agg-1 polypeptide were prepared. The expression level
was estimated to be 1 mg/L. Mass spectrometry indicated >90% selenium incorporation in
the labeled proteins.
Conditioned CHO media expressing the Agg-l-AlC2 construct was diluted into
25 mM Hepes pH 6.8, 5 mM CaCl2,10 uM ZnCl2, bound to a Poros® HQ column (Applied
Biosystems, Foster City, CA) and eluted with linear gradient 50 mM-lM NaCl. Agg-1-
AlC2-containing fractions were loaded onto a polypropyl aspartamide hydrophobic
33

interaction column (Nest Group, Soufhborough, MA) in 1.2 M (NHSCv Agg-l-AlC2
was eluted by decreasing (NILSC concentration. Subsequent purification steps included
gel filtration (G3000SW) and anti-Flag M2 affinity chromatography. The unbound
material from the Flag affinity column was bound to a Mono Q column (Pharmacia) using
starting buffer 25 mM MMT pH 6.8, 50 mM NaCl and elution with a linear gradient up to
1M NaCl. Protein was dialyzed into final buffer consisting of 25 mM Hepes pH 6.8,
5 mM CaCl2,10 uM ZnCl2,300 mM NaCl. All purification steps were performed at 4°C.
The Agg-l-AlC2 protein was concentrated to 8 mg/mL in 25 mM HEPES pH 6.8,
300 mM NaCl, 10 uM ZnCl2, 5 mM CaCl2. The Agg-l-AlC2/Compound 1 complex was
obtained by incubating the protein with 1.2 molar excess of the inhibitor. Crystals of
unliganded Agg-l-AlC2 and the Agg-l-AlC2/Compound 1 complex were grown by
hanging drop technique at 18°C using 10% PEG 4K, 0.1 M MES pH 6.0 as a precipitating
solution. Optimized crystals were obtained by streak seeding and macro seeding with an
addition of 8-15 mM L-cysteine. Crystals grew to a maximum size of 0.4 x 0.4 x 0.2 mm3
in about 2-3 weeks. Crystals from the selenomethione substituted Agg-l-AlC2 protein
were grown using the same technique, as described above. Crystals of inhibitor-bound
protein belong to the monoclinic space group P2i and have unit cell parameters
a = 82.566 A, b =82.618 A, c = 99.326 A, p = 90.626°, with 4 molecules per
crystallographic asymmetric unit. Unliganded protein crystallized in the space group P2i
with unit cell parameters a = 128.28 A, b = 83.63 A, c = 150,16 A, p = 112.409°, and with
8 molecules per asymmetric unit. For data collection crystals were transferred to the
solution containing the crystallization reagent plus 25% glycerol, and then flash-frozen in
the liquid nitrogen at 100K.
The structure of inhibitor-bound Agg-l-AlC2 was determined with phases obtained
by multiwavelength anomalous diffraction (MAD) from crystals of
selenomethionine-substituted protein. MAD data were collected at three wavelengths on
beam line 5.0.2 at the Advanced Light Source, Berkeley, CA, using a Quantum-4 CCD
detector (Area Detector Systems). The data were integrated with MOSFLM and then scaled
with SCALA ("The CCP4 Suite: Programs for Protein Crystallography," Acta Crystallogr.
Sect. D 50:760-763,1994). Selenium sites were located using SHELXS. Refinement of
anomalous scatterer parameters, phase calculation and density modification by
34

SOLOMON, and all were performed with SHARP (de La Fortelle and Brigogne, Methods
Enzym. 276:472-494,1997). Experimental maps were used to build an initial model
(QUANTA), with subsequent rounds of rebuilding and refinement in CNS (Brunger et ah,
Acta Crystall. Sect D 54:905-921,1997) against native data. The 2.8 A native data set was
collected at the Advanced Light Source, processed with MOSFLM and scaled with SCALA
("The CCP4 Suite: Programs for Protein Crystallography," Acta Crystallogr. Sect. D
50:760-763,1994). Statistics for data collection, phasing and refinement for the Agg-1-
AlC2/Compound 1 complex are summarized in Table 1.
35

Table 1. Statistics of X-Rav Diffraction Data Collection for Agg-l-AlC2/Compound 1


Data

Multiwavelength Anomalous Diffraction

The structural coordinates of the refined model of the Agg-l-AlC2/Compound 1
complex are presented below in Table 5. In Table 5, the "#" column assigns an index to
each atom for which coordinates are given. The "name" column indicates what type of
36

atom, and the "res" column indicates what type of residue the atom belongs to. The
"chain" indicates which polypeptide the atom belongs to. "Res #" gives the residue
number for the atom. For example, atom number 1 (the first row in Table 5) is the beta
carbon (CB) of Ala214. Its x, y, and z structural coordinates are given in the X, Y, and Z
columns, respectively. The column headed "occ" describes the occupancy assigned to the
atom (1.00 = full occupancy), and the "B" column provides B factors (or temperature
factors) in units of A2. Coordinates of bound Compound 1 are denoted with the entry
"WAY" in the res column, water is denoted by "HOH," and zinc and calcium atoms are
denoted by "ZN" and "CA," respectively.
Subsequently, the crystal structure of the unliganded form of the Agg-l-AlC2 was
solved by molecular replacement method with AMORE ("The CCP4 Suite: Programs for
Protein Crystallography," Acta Crystallogr. Sect. D 50:760-763,1994) and CNS (Brunger
et al, Acta Crystallogr. Sect. D 54:905-921,1997), using the refined structure of the
inhibitor-bound form. Diffraction data from crystals of unliganded enzyme were collected
to 3 A resolution at the Advanced Light Source, and processed and reduced with
MOSFLM and SCALA ("The CCP4 Suite: Programs for Protein Crystallography," Acta
Crystallogr. Sect. D 50:760-763,1994). Statistics for data collection and refinement are
shown in Table 2.
The structural coordinates of the refined model of the Agg-l-AlC2 polypeptide are
presented below in Table 4. The columns and designations of Table 4 are as described for
Table 5.
37


Table 2. Statistics of X-Rav Diffraction Data Collection for Agg-l-AlC2
The structure of the Agg-l-AlC2/Compound 1 complex is shown in FIG. 5.. The N-
terminal residues 214-428 represent the catalytic domain of the enzyme, and the C-terminal
residues 438-509 represent the disintegrin-like domain. The two domains are connected by
a 9-residue crossover linker (residues 429-438) that extends across the surface of the
catalytic domain on the side opposite to the zinc-binding region.
38

The catalytic domain of Agg-l-AlC2 reveals a characteristic polypeptide fold that
shares structural features with the zinc-peptidase superfamily. It has an a/p structure
consisting of six a-helices (aA- aF) surrounding a core of five P-strands (PA- PE) and
topologically is more similar to snake venom metalloproteinases (SVMP) than to MMPs.
The catalytic zinc environment involves the characteristic zinc-chelating motif
361HExxHxxxxxH371 with three histidines (His361, His365 and His371) coordinating the
zinc atom and the Met-turn motif xMx with the invariant methionine (Met391)
essential for the structural integrity of the zinc-binding site. Compared with SVMPs and
MMPs that share a conserved glycine residue in the zinc-binding region (HExxHxxGxxH),
the topologically equivalent asparagine (Asn368) in Agg-l-AlC2 is arranged in a similar
conformation to allow for a sharp turn in the polypeptide chain. Accommodation of
glutamine residue (Gln362) in place of the catalytic glutamic acid (Glu362) has no effect
on the architecture of the active site.
As shown in more detail in FIG 6, in contrast to SVMPs that have a range of one-
to three-disulfide bonds, this structural arrangement is supported by four disulfide bridges
formed between cysteines that are conserved in the ADAMTS family: Cys293-Cys345,
Cys322-Cys327, Cys339-Cys423 and Cys377-Cys407. Among these, the Cys339-Cys423
disulfide connection has a structural equivalent in the SVMP structures, but the others seem
to be unique to aggrecanases. The Cys293-Cys345 bridge anchors the long aC helix
(Cys293) to the P-sheet (Cys345), the Cys322-Cys327 disulfide keeps together sequentially
distant parts of the S-shapedloop (S-loop, MMP terminology) and the Cys377-Cys407
connection locks the small aE helix (Cys377) against the C-terminal helix aF (Cys407). In
addition, there are three calcium ions, identified by large peaks in the electron density. One
Ca2+ ion is found near the Cys322-Cys327 disulfide bridge, forming contacts to three
carbonyl oxygens (Leu321, Cys327, Thr329) and three carboxylate oxygens (Asp320,
Glu349). Another site, harboring two calcium ions, is in the vicinity of the Cys339-Cys423
bridge, in a location similar to the calcium-binding site in SVMPs. However, in Agg-1-
A1C2 this region reveals a highly charged environment that allows for two calcium ions as
opposed to the one in SVMPs structures, such as the structures described for atrolysin C
and adamalysin II. The two Ca2+ ions are separated by 4.3 A and collectively are
coordinated with three aspartates (Asp304, Asp311, Asp426), one glutamate (Glu221) and
39

two carbonyl groups (Asp304, Cys423). Comparative amino acid sequence analysis with
the Agg-l-Al C2 structure indicates that the residues coordinating the calcium ions at both
sites have a high level of conservation across the aggrecanase family.
The disintegrin-like domain of Agg-l-AlC2 is made up of two small a-helices
followed by two highly twisted antiparallel P-sheets. Each P-sheet has three short p-strands
interrupted by irregular connections and long loops. This arrangement is held in place by
four disulfide bridges between eight conserved cysteines, as shown in FIG. 8: Cys449-
Cys472, Cys460-Cys482, Cys467-Cys501, Cys495-Cys506. The orientation of this
domain is maintained by a number of electrostatic and hydrophobic interactions within the
catalytic domain.
The active site of Agg-1 is very similar to that of the MMPs and SVMPs (Rush and
Powers, Current Topics in Med. Chem. 4:1311-1327,2004). In general, the active site is
broadly defined by a narrow concave groove on the surface of the catalytic domain that
runs parallel to PD. At the center of this groove is the catalytic Zn, which is key to protease
activity as it activates the water molecule responsible for hydrolysis of the substrate's
peptide bond. As indicated above, themtrogen atoms of Histidines 361, 365 and 371
coordinate the catalytic Zn of Agg-1. Also common to other protease active sites are the
presence of several inward-facing pockets and solvent facing grooves adjacent to the major
active site groove. These features of the protein accommodate the sidechains of the
substrate and are thus useful to discriminate against sidechains for selectivity. Finally, a
hydrogen bond acceptor "hot spot" near the catalytic Zn may stabilize the substrate through
a hydrogen bond with one of the protein's amide carbonyls. In Agg-1, this "hot spot" is
located at a tight turn in the backbone preceding pD and is formed by the two inward facing
backbone NHs of Leu330 and Gly331.
Most known inhibitors of MMPs and SVMPs share several features. The first is a
Zn-chelating group that occupies the fourth coordination site of the active site Zn atom.
This interaction contributes a significant amount of energy to the free energy of binding.
The same interaction is observed for the inhibitor described here within, which chelates the
Zn via one of the carboxylate oxygen atoms at a distance of 2.1 A. The other carboxylate
oxygen is participating in a water-mediated hydrogen bond with the backbone atoms of
Ala333. Carboxylate MMP inhibitors are known to bind more favorably when protonated,
40

because they can form a direct hydrogen bond with the carboxylate of the active site Glu
(Glu362 in Agg-1). Therefore, we predict that the water-mediated interaction with Ala333
is only present in the mutant form of the protein. Since in Agg-1-A1C2 the carboxylate is
substituted by a primary amide (via the Glu362Gln mutation), the same hydrogen bond
cannot be made, and thus the second oxygen of the inhibitor is free to make interactions
elsewhere.
Another common feature of MMP inhibitors is the placement of a hydrogen bond
acceptor at the active site "hot spot" described above. In this case, the hot spot is occupied
by one of the oxygen atoms of the sulfonamide group of Compound 1. The O-N distances
to the backbone NHs of Leu330 and Gly331 are 2.7 A and 3.1 A, respectively.
Typically the side chains of each amino acid of a polypeptide substrate are involved
in the specificity of a substrate/protease interaction. The side chain of each substrate
residue is recognized by regions of the enzyme which are collectively called sub-sites. The
generally accepted nomenclature for the protease sub-sites and their corresponding
substrate residues follows, where the double slash represents the position of bond cleavage.
Protease sub-sites: S4, S3, S2, SI, SI', S2', S3', S4'; substrate residues: P4, P3, P2, PI, //
PI', P2', P3', P4'. Another common feature of known MMP inhibitors is the presence of a
PI' group, an inhibitor group that fills the SI' pocket of the active site. This is likely due to
the fact that the SI' pocket is typically a very large, hydrophobic pocket, and thus inhibitors
that utilize this space can gain free energy by the hydrophobic effect In this Agg-1
structure, the SI' pocket is in fact a channel that spans approximately 15 A, and is
completely filled by the inhibitor.
Several interactions between Compound 1 and Agg-l-AlC2 are less common
among known MMP inhibitors. For example, there is a favorable % stacking interaction
between the biphenyl n system and His361 of the active site (~ 3.7 A separation). There is
also a second n stacking interaction between the PI' phenyl ring and Phe357 (~ 3.7 A
separation). Finally, there is a water mediated (2.5 A) hydrogen bond between the carbonyl
oxygen of the PI' group and the backbone atoms of (xB. The inhibitor-protein interactions
are illustrated in FIG. 12.
The overall structure'of the Agg-l-AlC2 (see FIG. 2) polypeptide is similar to the
Agg-1-A1C2 polypeptide bound to Compound 1. When superimposed, the two structures
41

show an r.m.s. deviation of 1.4 A for the 280 equivalent Ca-pairs. However, the
architecture of the active site in the unbound form shows significant conformational
changes compared to the inhibitor-bound form. These changes include reconfiguration of
the S-loop and positional rearrangement of residues therein. Electron density maps
(FIG 10) revealed that in the unliganded structure the entire region from Leu321 up to
Leu330 is looped toward the active site, completely blocking the entrance to the S' pocket.
Although local, the rearrangement is quite significant with displacements of ~2.7 A and
-6.6 A for the Ca-atoms of Cys322 and Cys327, respectively. In this position, the Cys322-
Cys327 disulfide bridge is maintained, and this bridge stabilizes the displacement of the
loop and the "inhibitory" conformation. An unanticipated feature of this arrangement is
that residues following Cys327, Asp328 and Thr329, insert their side chains into a pocket
near the catalytic Zn2+ ion, where the carboxylate group of Asp328 chelates to the metal
atom (see FIG 11). Hence, the S-loop appears to be an "autoinhibitory" element in two
aspects. First, it precludes enzyme-substrate recognition by physically occupying the space
where peptide substrates would bind, and second, Asp328 prevents the Zn atom from
becoming enzymatically active until Asp328 is removed. These data suggest that the
binding pocket opens upon interaction with ligands.
Example 2. Aggrecanase-2/Batimastat complex was crystallized and its structure
determined.
A recombinant human Agg-2 polypeptide was expressed from CHO cells. The
expressed Agg-2 polypeptide included the enzyme's catalytic domain, disintegrin-like
domain, and thrombospondin-like domain, and a Strep-tag® (IB A, St. Louis, MO) fused to
amino acid Phe628 of the protein, truncating the polypeptide at the C-terminus (FIG 3). A
three amino acid linker was included immediately following the spatially conserved
phenylalanine preceding the Strep-tag®. CHO cell lines expressing Agg-2 were established
by transfecting the Agg-2_Phe628_Strep construct into CHO/ DUKX cells using the
manufacturers recommended protocol for lipofection (InVitrogen, Carlsbad, CA). Clones
were selected in 0.02, 0.05 and 0.1 uM methotrexate. Cell lines expressing the highest
level of the recombinant protein were selected by monitoring the recombinant protein in
CHO conditioned media by Western blotting using an anti-streptavidin antibody conjugated
42

to horseradish peroxidase (HRP) (Southern Biotech, Birmingham, AL) followed by ECL
chemiluminescence (Amersham Biosciences, Piscataway, NJ) and autoradiography.
Concentrated condition media expressing Agg-2_Phe628_Strep were diluted three
fold with buffer A (20 mM Tris-Cl, pH 8.0,5 mM CaCl2,10 uM ZnCl2,50 mM NaCl) and
loaded onto a Poros® HS (Applied Biosystems, Foster City, CA) anion exchange column
pre-equilibrated with buffer A. The column was washed and developed by a NaCl gradient
up to 1.0 M in the same buffer. Agg-2-containing fractions were pooled and subjected to
Strep-Tactin (IBA GmbH, Gottingen, Germany) affinity chromatography in buffer B
(20 mM Tris.Cl, pH 8.0,5 mM CaCl2,10 uM ZnCl2,150 mM NaCl). The column was
washed and Agg-2 protein was eluted with 2.5 mM Desthiobiotin in buffer B. A Superdex-
200 gel filtration column was used to further purify the protein using buffer C (20 mM Tris
pH 8.5, 5 mM CaCl2,10 uM ZnCl2, 50 mM NaCl) as the mobile phase. The resulting
Agg-2 containing fractions were pooled and concentrated to 5 mg/mL for crystallography
studies.
Inhibitor-bound Agg-2 was obtained by incubating the concentrated protein with
1.2 molar excess of the inhibitor, batimastat. Crystals were grown by hanging drop
technique at 18°C using 10% PEG 8K; 0.2 M NaCl; 0.1 M CHES pH 9.5 as a precipitating
solution. Crystals belonged to the space group P3i and had unit cell parameters a = 93.64
A, b = 93.64 A, c = 92.59 A, and y = 120°, with 2 molecules per crystallographic
asymmetric unit. For data collection, the crystal was transferred to the solution containing
the crystallization reagent (10% PEG 8K, 0.2 M NaCl; 0.1 M CHES, pH 9.5) plus
25% glycerol, and then flash-frozen in the liquid nitrogen at 100K.
The structure of the Agg-2/Batimastat complex was determined by molecular
replacement method using AMORE (Navaza, Acta Ciystallogr. A50:157-163,1994) and
the structure of Agg-l-AlC2 bound to Compound 1 as a search model. Diffraction data
were collected to 2.9 A resolution at the Advanced Light Source (Berkeley, California),
processed and reduced with MOSFLM and SCALA ("The CCP4 Suite: Programs for
Protein Crystallography," Acta Crystallogr. Sect. D 50:760-763,1994). Analysis of
probability distribution for intensities showed that the crystal is merohedrally twinned, with
a twinning fraction of 0.42. Rebuilding in QUANTA and refinement in CNS (Brunger et
43

al, Acta Crystaltogr. Sect. D 54:905-921,1997) were performed taking twinning into
account. Statistics for data collection and refinement are shown in Table 3.
Table 3. Statistics of X-Ray Diffraction Data Collection Agg-2/Batimastat complex

The structural coordinates of the refined model of the Agg-2/batimastat polypeptide
are presented below in Table 6. The columns and designations of Table 6 are as described
for Table 5, except the residue designation "WAY" identifies batimastat atoms.
44

A ribbon diagram of the structure of the Agg-2/Batimastat structure is shown in
FIG 8. The N-terminal residues 265-476 form the catalytic domain of the enzyme and the
C-terminal residues 486-556 form the disintegrin-like domain. No electron density was
observed for the thrombospondin-like domain, residues 557-628, suggesting that this
region is disordered in the crystal structure. The catalytic domain and disintegrin-like
domain are connected by a 9-residue crossover linker (residues 477-486) that extends
across the surface of the catalytic domain on the side opposite to the zinc-binding region.
The catalytic domain of Agg-2 reveals a characteristic polypeptide fold that shares
structural features with the zinc-peptidase superfamily. It has an a/p structure consisting of
six a-helices (aA- aF) surrounding a core of five |3-strands (PA-|3E) and topologically is
more similar to snake venom metalloproteinases (SVMP) than to MMPs. The catalytic zinc
environment involves the characteristic zinc-chelating motif 410HexxHxxGxxH420 with
three histidines (His410, His414 and His420) coordinating the zinc atom and the Met-turn
motif 438xMx440 with the invariant methionine (Met439) essential for the structural integrity
of the zinc-binding site.
In contrast to SVMPs that range from one- to three-disulfide proteinases, the
structural arrangement of Agg-2 in the Agg-2/Batimastat complex is supported by four
disulfide bridges formed between cysteines that are conserved in the ADAMTS family:
Cys342-Cys394, Cys371-Cys376, Cys388-Cys471 and Cys426-Cys455. Among these, the
Cys388-Cys471 disulfide connection has a structural equivalent in all of the SVMP
structures, but the others seem to be unique to aggrecanases. The Cys342-Cys394 bridge
anchors the long aC helix (Cys342) to the P-sheet (Cys394), the Cys371-Cys376 disulfide
tethers sequentially distant parts of the S-shaped loop ("S-loop") and the Cys426-Cys455
connection anchors the small aE helix (Cys426) against the C-terminal helix aF (Cys455).
In addition, there are three calcium ions, identified from the large peaks in the electron
density. One Ca2+ ion is found near the Cys371-Cys376 disulfide bridge, forming contacts
to three carbonyl oxygens (Leu370, Cys371, Thr378) and three carboxylate oxygens
(Asp369, Glu398). Another site, harboring two calcium ions, is in the vicinity of the
Cys388-Cys471 bridge, in a location similar to the calcium-binding site in SVMPs.
However, in Agg-2 this region is highly charged, allowing for two calcium ions instead of
the one seen in the SVMPs structures of atrolysin C or adamalysin II. The two Ca2+ ions
45

are separated by 4.8 A and coordinate with three aspartates (Asp353, Asp360, Asp474), one
glutamate (Glu270) and two carbonyl groups (Asp353, Cys471). Comparative amino acid
sequence analysis aligned with the Agg-2 structure indicates that residues coordinating the
calcium ions at both sites have a high level of conservation across the aggrecanase family.
The disintegrin-like domain of Agg-2 reveals a unique structure made up of two
small a-helices followed by two highly twisted antiparallel p-sheets. Each P-sheet has three
short |3-strands interrupted by irregular connections and long loops. This arrangement is
held in place by four disulfide bridges between eight conserved cysteines: Cys497-Cys519,
Cys508-Cys529, Cys514-Cys548, and Cys542-Cys553. The orientation of this domain is
maintained by a number of electrostatic and hydrophobic interactions with the catalytic
domain..
The active site of Agg-2 is very similar to that of the MMPs and SVMPs (Rush and
Powers, Current Topics in Med. Chem. 4:1311-1327,2004; Skiles etal, Current Med.
Chem. 8:425-474,2001). In general, the active site is broadly defined by a narrow concave
groove on the surface of the catalytic domain that runs parallel to PD. At the center of this
groove is the catalytic Zn, which is key to protease activity as it activates the water
molecule responsible for the hydrolysis of the substrate's peptide bond. As indicated
above, the Nitrogen atoms of Histidines 410,414 and 420 coordinate the catalytic Zn of
Agg-2. Also common to other protease active sites are the presence of several inward-
facing pockets and solvent-facing grooves adjacent to the major active site groove. These
features allow the protein to accommodate side chains of the substrate and are therefore •
useful for distinguishing side chains for selectivity. Finally, a hydrogen bond acceptor "hot
spot" near the catalytic Zn may stabilize the substrate via a hydrogen bond to one of the
protein's amide carbonyls. In Agg-2, this "hot spot" is located at a tight turn in the
backbone preceding pD and is formed by the two inward facing backbone NHs of Leu379
andGly380.
The strongest enthalpic interactions between the batimastat and Agg-2 are likely to
be the interaction of the hydroxamic moiety with various components of the active site. In
this and previously reported batimastat/MMP structures, the hydroxamate interacts with
both the active site Zn (O-Zn distances of 2.1 A and 2.6 A) and the carboxylate sidechain
of the catalytic glutamic acid (Glu411 in Agg-2) via Hydrogen bonding (0-0 distance of
46

2.4 A). These interactions are likely to contribute significantly to the enthalpy of the
protein-ligand interaction.
Batimastat is essentially a peptidomimetic inhibitor, and as such, interacts with the
protein in an extended, beta-sheet-like conformation. Its three "sidechains" (the thiophene,
isobutyl and benzyl substituents) interact with successive substrate binding pockets, while
the two backbone amide groups make four beta-sheet-like H-bonds with the protein, hi
Agg-2, the thiophene, isobutyl and benzyl sidechains occupy the SI, SI' and S2' sites
respectively, while the intervening amide groups hydrogen bond to the backbone atoms of
Asp377, Leu379, Ser441, and Leu443 (SI, SI' and S2' represent sub-sites in the Agg-2
binding site) (FIG. 13). The heavy atom distances of these Hydrogen bonds are observed to
be 2.8 A, 3.1 A, 2.7 A and 2.7 A, respectively.
47


48


49


50


51


52


53


54


55


56


57


58


59

WHAT IS CLAIMED IS:
1. A crystallized aggrecanase polypeptide.
2. The crystallized aggrecanase polypeptide of claim 1, wherein the aggrecanase
polypeptide is an aggrecanase-1 polypeptide.
3. The crystallized aggrecanase polypeptide of claim 1, wherein the aggrecanase
polypeptide comprises a catalytic domain.
4. The crystallized aggrecanase polypeptide of claim 1, wherein the aggrecanase
polypeptide consists essentially of a catalytic domain and a disintegrin-lilce domain.
5. The crystallized aggrecanase polypeptide of claim 1, wherein the aggrecanase
polypeptide comprises the amino acid sequence of SEQ ID NO:l.
6. The ciystallized aggrecanase polypeptide of claim 1, wherein the crystallized
aggrecanase polypeptide has space group V2\.
7. The crystallized aggrecanase polypeptide of claim 6, wherein the crystallized
aggrecanase polypeptide has unit cell dimensions a=128.28 A, b=83.63 A, c=150.16 A,
and p=l 12.409°.
8. The crystallized aggrecanase polypeptide of claim 1, wherein the aggrecanase
polypeptide is from a mammalian species.
9. The crystallized aggrecanase polypeptide of claim 1, wherein the aggrecanase
polypeptide is from a nonmammalian species.
596

10. The crystallized aggrecanase polypeptide of claim 1, wherein the aggrecaaase
polypeptide is from a human.
11. The crystallized aggrecanase polypeptide of claim 1, wherein the crystallized
polypeptide is capable of diffracting X-rays to a resolution of at least about 3.5 A.
4
12. The crystallized aggrecanase polypeptide of claim 1, wherein the crystallized
polypeptide comprises the structural coordinates of Table 4, +/- a root mean square
deviation for alpha carbon atoms of not more than 1.5A.
13. A crystallized polypeptide-ligand complex, comprising:
an aggrecanase polypeptide; and
a ligand.
14. The crystallized polypeptide-ligand complex of claim 13, wherein the ligand
is an inhibitor of aggrecanase activity.
15. The crystallized polypeptide-ligand complex of claim 13, wherein the ligand
has the structure:

wherein A and B represent ring systems; L, M, and Y are linker moieties; R1, R2, and R3
are substituents; X is a hydrogen bond acceptor; and Z is a metal chelating moiety.
16. The crystallized polypeptide-ligand complex of claim 15, wherein A is an aryl
moiety having five or more atoms or a heteroaryl moiety having five or more atoms.
17. The crystallized polypeptide-ligand complex of claim 15, wherein B is an aryl
moiety having five or more atoms or a heteroaryl moiety having five or more atoms.
597

AMENDED CLAIMS received by the International Bureau on 10 August 2007
(10.08.2007)
18. The crystallized polypeptide-ligand complex of claim 15, wherein L is a
bond, methylene* ethylene, oxygen, sulfur, amino, methyleneoxy, methyleneamino,
methylenethioyl, sulfoxide, or sulfone.
19. The crystallized polypeptide-ligand complex of claim 15, wherein M is a
methylene, ethylene, oxygen, sulfur, amino, methyleneoxy, methyleneamino,
methylenethioyl, sulfoxide, or sulfone,
20. The crystallized polypeptide-ligand complex of claim 15, wherein Y is a
methylene, ethylene, propylene, isopropylene, butylene, isobutylene, oxygen, sulfur,
amino, methyleneoxy, meihyleneamino, methyrenethioyl, sulfoxide or sulfone.
21. The crystallized polypeptide-ligand complex of claim 15, wherein R1 is a
hydrogen, Ci-Cg alkyL, C2-C$ alkenyh Qj-Cg alkynyl, cyclyl, heterocyclyl, aryl, or
heteroaryl, wherein said Ci-C$ alkyl, C2-C6 alkenyl, QrCs alkyayl, cyclyh heterocyclyL
aryl, and heteroaryl axe each optionally substituted with nitro, cyano, alkylcarbonyL
sulfoxide, sulfone, sulfonamide, carbonyl, carboxamide, carbamate, or carbonate,
22. The crystallized polypeptide-ligand complex of claim 15, wherein R2 is a
hydrogen, fluorine, chlorine, bromine, OC(halogen)3, C(halogen)3, Ci-C6 alkoxy, Ci-C$
alkyl, Ci-C6 alkylthioyi, or CrCg alkylamino.
23. The crystallized polypeptide-ligand complex of claim 15, wherein R3 is a
hydrogen, fluorine, chlorine, bromine, OC(halogen)3, C(halogen)33 Ci-Cg alkoxy, CrCs
alkyl, Cj-Cg alkylthioyi, or Ci-Cs alkylamino.
24. The crystallized polypeptide-Hgand complex of claim 15, wherein X is a
sulfur, sulfoxide, sulfone, sulfonamide, carbonyl, carboxamide, urea, carbamate, or
carbonate.

25. The crystallized polypeptide-ligand complex of claim 15, wherein Z is a
carboxylic acid, hydxoxamic acid, hydroxyurea, hydrazide, sulfonic acid, sulfonamide,

hydroxysulfonarnide, sulfodiimide, phosphoric acid, phosphonic acid, thiol, thiol
carbonyl, thiirane, dithiol, sulfonylhydrazide, sulfodiimine, thiazoladine dione,
pyrimidme trionethiadiazine, barbiturate, thiadiazole, thiadiazine, imidazolidinedione,
pyridinione, amiriornethyl benzimidazole, napthylhydroxamate, pyridinylamide,
pyridinylone or pyrrolylone.
26. The crystallized polypeptide-ligand complex of claim 13, wherein the ligand
is a peptMomimetic compound.
27. The crystallized polypeptide-ligand complex of claim 13, wherein the ligand
is a matrix metalloproteinase inhibitor.
28. The crystallized polypeptide-ligand complex of claim 13, wherein the ligand
is Compound 1.
29. The crystallized polypeptide-ligand complex of claim 13, wherein the
crystallized polypeptide-ligand complex has space group P2i.

30. The crystallized polypeptide-ligand complex of claim 13, wherein the
crystallized polypeptide-ligand complex has unit cell dimensions a=82.07 A, b=83.96 A,
c=98.95 A, and P=89.9°.
31. The crystallized polypeptide-ligand complex of claim 13, wherein the
aggrecanase polypeptide comprises a catalytic domain.
32. The crystallized polypeptide-ligand complex of claim 31, wherein the
aggrecanase polypeptide further comprises a disintegrin-like domain.
3 3. The crystallized polypeptide-ligand complex of claim 31, wherein the
ligand is bound to the catalytic domain.

34, The crystallized polypeptide-ligand complex of claim 13, wherein the
aggrecanase polypeptide is from a mammalian species.
3 5. The crystallized polypeptide-ligand complex of claim 13, wherein the
aggrecanase polypeptide is from a nonmammalian species.
36. The crystallized polypeptide-ligand complex of claim 13, wherein the
aggrecanase polypeptide is from a human.
37. The crystallized polypeptide-hgand complex of claim 13, wherein the
aggrecanase polypeptide comprises the amino sequence defined by SEQ ID NO:l.
3 8. The crystallized polypeptide-ligand complex of claim 13, wherein the
complex is capable of diffracting X-rays to a resolution of at least about 3.5 A.
3 9. The crystallized polypeptide-ligand complex of claim 13, wherein the
complex comprises the structural coordinates of Table 4, ± a root mean square deviation
for alpha carbon atoms of not more than 1.5 A.
40. The crystallized polypeptide-ligand complex of claim 13, wherein the ligand
binds amino acid 362 of the aggrecanase-1 polypeptide, and wherein amino acid 362 is
glutamate or glutarnine.
41. The crystallized polypeptide-ligand complex of claim 13,
wherein the ligand binds a zinc atom located in the active site of the aggrecanase
polypeptide.
42. The crystallized polypeptide-ligand complex of claim 13, wherein the
aggrecanase polypeptide is an aggrecanase-1 polypeptide.
43. The crystallized polypeptide-ligand complex of claim 42,

wherein the ligand binds one or more of Leu330, Gly331, Ala333, His361,
Phe357 and Ala248 of the aggrecanase-1 polypeptide.
44. The crystallized polypeptide-ligand complex of claim 42,
wherein the aggrecanase-1 polypeptide comprises the amino acids of SEQ ID
NO:l, and the crystallized polypeptide-ligand complex diffracts X-rays to a resolution of
at least about 3.5 A.
45. The crystallized polypeptide-ligand complex of claim 42, wherein the
ligand is bound to the aggrecanase polypeptide.
46. A crystallized polypeptide-ligand complex, comprising:
an aggrecanase polypeptide; and
a polycyclic ligand having a Zn chelating moiety.
47. The composition of claim 46, wherein the aggrecanase polypeptide is an
aggrecanase-1 polypeptide.
48. A composition, comprising:
a crystal, comprising:
an aggrecanase polypeptide; and
a ligand.
49. The composition of claim 48, wherein the ligand is bound to the
aggrecanase polypeptide.
50. The composition of claim 48, wherein the aggrecanase polypeptide is an
aggrecanase-1 polypeptide.
51. The composition of claim 48, wherein the ligand is a peptidomimetic.

52. The composition of claim 48, wherein the ligand is a matrix
metalloproteinase inhibitor.
53. The composition of claim 48, wherein the ligand is Compound 1.
54. A method, comprising:
using a three-dimensional model of a complex comprising an aggrecanase
polypeptide bound to a ligand to design an agent that binds the aggrecanase polypeptide.
55. The method of claim 54, wherein the three-dimensional model comprises
a catalytic domain.of the aggrecanase polypeptide.
56. The method of claim 55, wherein the three-dimensional model further
comprises a disintegrin-like domain of the aggrecanase polypeptide.
57. The method of claim 54, wherein the three-dimensional model comprises
structural coordinates of atoms of the aggrecanase polypeptide.
58. The method of claim 57, wherein the structural coordinates are
experimentally determined coordinates.
59. The method of claim 54, wherein the three-dimensional model comprises
structural coordinates of the ligand.
60. The method of claim 54, wherein the ligand is a peptidomimetic.
61. The method of claim 54, wherein the ligand is Compound 1.
62. The method of claim 59, further comprising altering the ligand of the
model.

63. The method of claim 62, wherein altering the ligand comprises changing
the structural coordinates of the ligand.
64. The method of claim 62, wherein altering the ligand comprises changing
the chemical structure of the ligand.
65. The method of claim 54, wherein the aggrecanase polypeptide is an
aggrecanase-1 polypeptide.
66. The method of claim 65, wherein the three-dimensional model comprises
structural coordinates of an atom selected from the group consisting of atoms of amino
acids Leu330, Gly331, Ala333, His361, Phe357, and Ala248 of the aggrecanase-1
polypeptide.
67. The method of claim 65, wherein the three-dimensional model comprises
structural coordinates of an atom of amino acid 362 of the aggrecanase-1 polypeptide,
and wherein amino acid 362 is a glutamate or a glutamine.
68. The method of claim 54, further comprising calculating a distance
between an atom of the aggrecanase polypeptide and an atom of the agent
69. The method of claim 54, further comprising comparing a predicted
interaction between the agent and the aggrecanase polypeptide with the interaction
between the ligand and the aggrecanase polypeptide.
70. The method of claim 54, further comprising providing a composition
comprising the aggrecanase polypeptide.
71. The method of claim 70, wherein the composition includes the agent
designed to bind the aggrecanase polypeptide.

72. The method of claim 54, further comprising experimentally detenriining
the interaction of the agent with the aggrecanase polypeptide.
73. The method of claim 72, further comprising comparing the interaction of
the agent with the aggrecanase polypeptide to an interaction of a second agent with the
aggrecanase polypeptide.
74. A method comprising:
using a three-dimensional model of an aggrecanase polypeptide to design an agent
that binds the aggrecanase polypeptide.
75. The method of claim 74, wherein the aggrecanase polypeptide is an
aggrecanase-1 polypeptide.
76. The method of claim 74, wherein the three-dimensional model includes a
ligand bound to the aggrecanase polypeptide.
77. The method of claim 76, wherein the ligand is a peptidomimetic.
7 8. The method of claim 76, wherein the ligand is a matrix metalloproteinase
inhibitor.
79. The method of claim 76, wherein the ligand is Compound 1.
80. The method of claim 76, wherein the three-dimensional model comprises
structural coordinates of atoms of the ligand.
81. The method of claim 74, wherein the three-dimensional model comprises
a catalytic domain of the aggrecanase polypeptide.
82. The method of claim 74, wherein the agent inhibits aggrecanase activity.

83. The method of claim 74, wherein the three-dimensional model comprises
structural coordinates of atoms of the aggrecanase polypeptide.
84. The method of claim 83, wherein the structural coordinates are
experimentally determined coordinates.
85. The method of claim 84, wherein the structural coordinates are according
to Table 5, +/- a root mean square deviation for alpha carbon atoms of not more than
1.5 A.
86. The method of claim 75, wherein the three-dimensional model comprises
structural coordinates of an atom selected from the group consisting of atoms of amino
acids Leu330, Gly331, Ala333, ffis361, Phe357 andAla248 of the aggrecanase-1
polypeptide.
87. The method of claim 75, wherein the three-dimensional model comprises
structural coordinates of an atom of amino acid 362 of the aggrecanase-1 polypeptide,
and wherein amino acid 362 is a glutamate or ghrtamine.
88. A method, comprising:
selecting an agent by performing rational drug design with a three-dimensional
structure of a crystalline complex, wherein the complex comprises an aggrecanase
polypeptide;
contacting the agent with an aggrecanase polypeptide; and
detecting an ability of the agent to bind the aggrecanase polypeptide.
89. The method of claim 88, wherein the aggrecanase polypeptide is an
aggrecanase-1 polypeptide.

90. The method of claim 88, wherein the agent is selected via computer
modeling.
91. The method of claim 88, further comprising synthesizing the agent.
92. The method of claim 91, further comprising detecting an ability of the
agent to inhibit aggrecanase activity.
93. The method of claim 91, further comprising detecting an ability of the
agent to inhibit or reduce cartilage degradation in vitro or in vivo.
94. The method of claim 88, further comprising:
obtaining a supplemental crystalline complex comprising the aggrecanase
polypeptide and the agent;
determining the three-dimensional structure of the supplemental crystalline
complex;
selecting a second agent by performing rational drug design with the three-
dimensional structure of the supplemental crystalline complex;
contacting the second agent with the aggrecanase polypeptide; and
detecting the ability of the second agent to bind the aggrecanase polypeptide.
95. The method of claim 94, wherein the second agent is selected via
computer modeling.
96. The method of claim 94, further comprising synthesizing the second agent.
97. The method of claim 96, further comprising detecting an ability of the
second agent to inhibit aggrecanase activity.
98. The method of claim 96, further comprising detecting an ability of the
second agent to inhibit or reduce cartilage degradation in vitro or in vivo.

99. A method, comprising:
contacting an aggrecanase polypeptide with a ligand to form a composition; and
crystallizing the composition to form a crystalline complex in which the ligand is
bound to the aggrecanase polypeptide,
< wherein, the crystalline complex can diffract X-rays to a resolution of at least
about 3.5 A.
100. The method of claim 99, wherein the aggrecanase polypeptide is an
aggrecanase-1 polypeptide.
101. The method of claim 99, wherein the method includes using hanging drop
vapor diffusion.
102. The method of claim 99, wherein the ligand is capable of inhibiting
aggrecanase activity.
103. The method of claim 99, wherein the ligand is capable of inhibiting or
reducing cartilage degradation in vitro or invivo.
104. The method of claim 99, wherein the ligand is a peptidomimetic.
105. The method of claim 99, wherein the ligand is a matrix metalloproteinase
inhibitor.
106. The method of claim 99, wherein the ligand is Compound 1.
107. A software system, comprising instructions for causing a computer system
to:
accept information relating to the structure of an aggrecanase polypeptide bound
to a ligand;

accept information relating to a candidate agent; and
determine binding characteristics of the candidate agent to the aggrecanase
polypeptide,
wherein the determination is based on the information relating to the structure of
the aggrecanase polypeptide bound to the ligand, and the information relating to the
candidate agent.
108. The software system of claim 107, wherein the aggrecanase polypeptide is
an aggrecanase-1 polypeptide.
109. A computer program residing on a computer readable medium having a
plurality of instructions stored thereon, which, when executed by one or more processors,
cause the one or more processors to:
accept information relating to the structure of a complex comprising an
aggrecanase polypeptide bound to a ligand;
accept information relating to a candidate agent, and
determine binding characteristics of the candidate agent to the aggrecanase
polypeptide,
wh erein the determination is based on the information relating to the structure of
the aggrecanase polypeptide and the information relating to the candidate agent.
110. The computer program of claim 109, wherein the aggrecanase polypeptide
is an aggrecanase-1 polypeptide.
111. A method, comprising:
accepting information relating to the structure of a complex comprising an
aggrecanase polypeptide bound to a ligand; and
modeling the binding characteristics of the aggrecanase polypeptide with a
candidate agent,
wherein the method is implemented by a software system.

112. The method of claim 111, wherein the aggrecanase polypeptide is an
aggrecanase-1 polypeptide.
113. A computer program residing on a computer readable medium having a
plurality of instructions stored thereon, which, when executed by one or more processors,
cause the one or more processors to:
accept information relating to a structure of a complex comprising an aggrecanase
polypeptide bound to a ligand; and
model the binding characteristics of the aggrecanase polypeptide with a candidate
agent.
114. The computer program of claim 113, wherein the aggrecanase polypeptide
is an aggrecanase-1 polypeptide.
115. A software system, comprising instructions for causing a computer system
to:
accept information relating to a structure of a complex comprising an aggrecanase
polypeptide bound to a ligand; and
model the binding characteristics of the aggrecanase polypeptide with a candidate
agent.
116. The software system of claim 115, wherein the aggrecanase polypeptide is
an aggrecanase-1 polypeptide.
117. Amethod of modulating aggrecanase activity in a subject, comprising:
using rational drug design to select an agent that is capable of modulating
aggrecanase activity; and
adbmnistering a therapeutically effective amount of the agent to the subject.
118. The method of claim 117, wherein the aggrecanase activity is an
aggrecanase-1 activity.

119. The method of claim 117, wherein the agent is capable of inhibiting or
reducing cartilage degradation in vivo.
120. The method of claim 117, wherein the rationaldrug design includes using
a three-dimensional structure of a crystalline complex that comprises an aggrecanase
polypeptide.
121. The method of claim 120, wherein the crystalline complex further
comprises a ligand.
122. The method of claim 120, wherein the ligand is a peptidomimetic.
123. The method of claim 120, wherein the ligand is a matrix metalloproteinase
inhibitor.
124. The method of claim 120, wherein the ligand is Compound 1.
125. A method of treating a subject having a condition associated with
aggrecanase activity, comprising:
using rational drug design to select an agent that is capable of affecting
aggrecanase activity; and
administering a therapeutically effective amount of the agent to a subject in need
thereof.
126. The method of claim 125, wherein the aggrecanase activity is aggrecanase-1
activity.
127. The method of claim 125, wherein the agent is capable of inhibiting
aggrecanase activity.

128. The method of claim 125, wherein the agent is capable of inhibiting
cartilage degradation in vivo.
129. The method of claim 125, wherein the condition is a joint disorder.
130. The method of claim 129, wherein the joint disorder is an arthritic disorder
*
or a joint injury.
131. The method of claim 130 wherein the arthritic disorder is osteoarthritis or
rheumatoid arthritis.
132. Amethod of prophylactically treating a subject susceptible to a condition
associated with aggrecanase activity, comprising:
determining that the subject is susceptible to the condition associated with
aggrecanase activity;
using rational drug design to select an agent that is capable of effecting
aggrecanase activity; and
administering a therapeutically effective amount of the agent to the subject.
133. The method of claim 132, wherein the aggrecanase activity is aggrecanase-1
activity.
134. The method of claim 132, wherein the agent is capable of inhibiting
cartilage degradation.
135. The method of claim 132, wherein the condition is a joint disorder.
136. The method of claim 135, wherein the joint disorder is an arthritic disorder
or a joint injury.

137, The method of claim 136, wherein the arthritic disorder is rheumatoid
arthritis or osteoarthritis.
138, A crystallized polypeptide-ligand complex, comprising:
an aggrecanase-2 polypeptide, and
a ligand.
*
139. The crystallized polypeptide-ligand complex of claim 138, wherein the
ligand is an inhibitor of aggrecanase activity.
140. The crystallized polypeptide-ligand complex of claim 138, wherein the
ligand has the structure:

wherein D and E represent an amide or a rnimie thereof; Ru, R12, R13, and R14 represent
side chain moieties; and G is a metal chelating moiety.
141. The crystallized polypeptide-ligand complex of claim I40j vvherein Rn is
methyl, ethyl or propyl.
142. The crystallized polypeptide-ligand complex of claim 140, wherein one or
more of R12 R13, and RH is selected from the group consisting of a Cj-C6 alkyl, C2-C6
alkenyl, C2~CG alkynyl, cycyl, heterocyclyl, aryl, heteroaryl cyclyloxy, heterocyclyloxy,
aryloxy, heteroaryloxy, cyclyithio, heterocyclylthio, arylthio, heteroarylthio, cyclylalkyl,
heterocyclylalkyl, arylalkyl, and heteroarylalkyl.
143. The crystallized polypeptide-ligand complex of claim 140, wherein R12 is a
phenyl moiety,

144. The crystallized polypeptide-ligand complex of claim 140, wherein R13 is an
isopropyl moiety.
145. The crystallized polypeptide-ligand complex of claim 140, wherein R14 is a
heteroarylthio moiety.
146. The crystallized polypeptide-ligand complex of claim 140, wherein G is a
carboxylic acid, carboxylic amide, or hydroxarnic acid.
147. The crystallized polypeptide-ligand complex of claim 138, wherein the
ligand is a peptidomimetic compound.
148. The crystallized polypeptide-ligand complex of claim 138, wherein the
ligand is a matrix metalloprotemase inhibitor.
149. The crystallized polypeptide-ligand complex of claim 138, wherein the
ligand is batimastat

150. The crystallized polypeptide-ligand complex of claim 138, wherein the
crystallized polypeptide-ligand complex has space group P3i.
151. The crystallized polypeptide-ligand complex of claim 138, wherein the
crystallized polypeptide-ligand complex has unit cell dimensions a=93.64 A, b=93.64 A,
c=92.59A,andy=120°.
152. The crystallized polypeptide-ligand complex of claim 138, wherein the
aggrecanase-2 polypeptide comprises a catalytic domain.
153. The crystallized polypeptide-ligand complex of claim 152, wherein the
aggrecanase-2 polypeptide further comprises a disintegrin-like domain.

154. The crystallized polypeptide-ligand complex of claim 138, wherein the
ligand is bound to the catalytic domain.
155. The crystallized polypeptide-ligand complex of claim 138, wherein the
aggrecanase-2 polypeptide is from a mammalian species.
156. The crystallized polypeptide-ligand complex of claim 138, wherein the
aggrecanase-2 polypeptide is from a nonmammalian species.
157. The crystallized polypeptide-ligand complex of claim 138, wherein the
aggrecanase-2 polypeptide is from a human.
158. The crystallized polypeptide-ligand complex of claim 138, wherein the
aggrecanase-2 polypeptide comprises the amino sequence of SEQ ID NO:3.
159. The crystallized polypeptide-ligand complex of claim 138, wherein the
complex is capable of diffracting X-rays to a resolution of at least about 3.5 A.
160. The crystallized polypeptide-ligand complex of claim 138, wherein the
complex comprises the structural coordinates of Table 6, ± a root mean square deviation
for alpha carbon atoms of not more than 1.5 A.
161. The crystallized polypeptide-ligand complex of claim 138,
wherein the ligand binds a zinc atom located in the active site of the aggrecanase-
2 polypeptide.
162. The crystallized polypeptide-ligand complex of claim 138,
wherein the ligand binds one or more of Glu411, Asp377, Leu379, Ser411, and
Leu443 of the aggrecanase-2 polypeptide.

163. The. crystallized polypeptide-ligand complex of claim 138,
wherein the aggrecanase-2 polypeptide comprises the amino acids of SEQ ID
NO:3, and the crystallized polypeptide-ligand complex diffracts X-rays to a resolution of
at least about 3.5 A.
164. The crystallized polypeptide-ligand complex of claim 138, wherein the
ligand is bound to the aggrecanase-2 polypeptide.
165. A crystallized polypeptide-ligand complex, comprising:
an aggrecanase-2 polypeptide; and
a peptidomimetic ligand having a metal chelating moiety.
166. A composition, comprising:
a crystal, comprising:
an aggrecanase-2 polypeptide; and
a ligand.
167. The composition of claim 166, wherein the ligand is bound to the
aggrecanase-2 polypeptide.
168. The composition of claim 166, wherein the ligand is a peptidomimetic.
169. The composition of claim 166, wherein the ligand is a matrix
metalloproteinase inhibitor.
170. The composition of claim 166, wherein the ligand is batimastat.
171. A method, comprising:
using a three-dimensional model of a complex comprising an aggrecanase-2
polypeptide bound to a ligand to design an agent that binds the aggrecanase-2
polypeptide.

172. The method of claim 171, wherein the three-dimensional model comprises
*
a catalytic domain of the aggrecanase-2 polypeptide.
173. The method of claim 172, wherein the three-dimensional model further
comprises a disintegrin-like domain of the aggrecanase-2 polypeptide.
174. The method of claim 171, wherein the three-dimensional model comprises
structural coordinates of atoms of the aggrecanase-2 polypeptide.
175. The method of claim 174, wherein the structural coordinates are
experimentally determined coordinates.
176. The method of claim 171, wherein the three-dimensional model comprises
structural coordinates of the ligand.
177. The method of claim 171, wherein the ligand is a peptidomimetic.
178. The method of claim 171, wheremmeHgandisbatimastat..
179. The method of claim 176, further comprising altering the ligand of the
model.
180. The method of claim 179, wherein altering the ligand comprises changing
the structural coordinates of the ligand.
181. The method of claim 179, wherein altering the ligand comprises changing
the chemical structure of the ligand.

182. The method of claim 171, wherein the three-dimensional model comprises
structural coordinates of an atom selected from the group consisting of atoms of amino
acids Glu411, Asp377, Leu379, Ser441, and Leu443 of the aggrecanase-2 polypeptide.
183. The method of claim 171, further comprising calculating a distance
between an atom of the aggrecanase-2 polypeptide and an atom of the agent.
184. The method of claim 171, further comprising comparing a predicted
interaction between the agent and the aggrecanase-2 polypeptide with the interaction
between the ligand and the aggrecanase-2 polypeptide.
185. The method of claim 171, further comprising providing a composition
comprising the aggrecanase-2 polypeptide.
186. The method of claim 185, wherein the composition includes the agent
designed to bind the aggrecanase-2 polypeptide.
187. The method of claim 171, further comprising experimentally determining
the interaction of the agent with the aggrecanase-2 polypeptide.
188. The method of claim 187, further comprising comparing the interaction of
the agent with the aggrecanase-2 polypeptide to an interaction of a second agent with the
aggrecanase-2 polypeptide.
189. A method comprising:
using a three-dimensional model of an aggrecanase-2 polypeptide to design an
agent that binds the aggrecanase-2 polypeptide.
190. The method of claim 189, wherein the three-dimensional model includes a
ligand bound to the aggrecanase-2 polypeptide.

191. The method of claim 190, wherein the ligand is a peptidomimetic.
192. The method of claim 190, wherein the ligand is a matrix metalloproteinase
inhibitor.
193. The method of claim 190, wherein the ligand is batimastat.
<
194. The method of claim 190, wherein the three-dimensional model comprises
structural coordinates of atoms of the ligand.
195. The method of claim 189, wherein the three-dimensional model comprises
a catalytic domain of the aggrecanase-2 polypeptide.
196. The method of claim 189, wherein the agent inhibits aggrecanase activity.
197. The method of claim 189, wherein the three-dimensional model comprises
structural coordinates of atoms of the aggrecanase-2 polypeptide.
198. The method of claim 197, wherein the structural coordinates are
experimentally determined coordinates.
199. The method of claim 198, wherein the structural coordinates are according
to Table 6, +/- a root mean square deviation for alpha carbon atoms of not more than
1.5 A.
200. The method of claim 189, wherein the three-dimensional model comprises
structural coordinates of an atom selected from the group consisting of atoms of amino
acids Glu411, Asp377, Leu379, Ser441, andLeu443 of the aggrecanase-2 polypeptide.
201. A method, comprising:

selecting an agent by performing rational drug design with a three-dimensional
structure of a crystalline complex, wherein the complex comprises an aggrecanase-2
polypeptide;
contacting the agent with an aggrecanase-2 polypeptide; and
detecting an ability of the agent to bind the aggrecanase-2 polypeptide.
202. The method of claim 201, wherein the agent is selected via computer
modeling.
203. The method of claim 201, further comprising synthesizing the agent.
204. The method of claim 203, further comprising detecting an ability of the
agent to inhibit aggrecanase-2 activity.
205. The method of claim 203, further comprising detecting an ability of the
agent to inhibit or reduce cartilage degradation in vitro or in vivo.
206. The method of claim 201, further comprising:
obtaining a supplemental crystalline complex comprising the aggrecanase-2
polypeptide and the agent;
deterrrnning the three-dimensional structure of the supplemental crystalline
complex;
selecting a second agent by performing rational drug design with the three-
dimensional structure of the supplemental crystalline complex;
contacting the second agent with the aggrecanase-2 polypeptide; and
detecting the ability of the second agent to bind the aggrecanase-2 polypeptide.
207. The method of claim 206, wherein the second agent is selected via
computer modeling.

208. The method of claim 206, further comprising synthesizing the second
agent.
209. The method of claim 208, further comprising- detecting an ability of the
second agent to inhibit aggrecanase-2 activity.
<
210. The method of claim 208, further comprising detecting an ability of the
second agent to inhibit or reduce cartilage degradation in vitro or in vivo.
211. A method, comprising:
contacting an aggrecanase-2 polypeptide with a ligand to form-a composition; and
crystallizing the composition to form a crystalline complex in which the ligand is
bound to the aggrecanase-2 polypeptide,
wherein the crystalline complex can diffract X-rays to a resolution of at least
about 3.5 A.
212. The method of claim 211, wherein the method includes using hanging
drop vapor diffusion.
213. The method of claim 211, wherein the ligand is capable of inhibiting
aggrecanase-2 activity.
214. The method of claim 211, wherein the ligand is capable of inhibiting or
reducing cartilage degradation in vitro or in vivo.
215. The method of claim 211, wherein the ligand is a peptidomimetic.
216. The method of claim 211, wherein the ligand is a matrix metalloproteinase
inhibitor.
217. The method of claim 211, wherein the ligand is batimastat.

218. A software system, comprising instructions for causing a computer system
to:
accept information relating to the structure of an aggrecanase-2 polypeptide
bound to a ligand;
accept information relating to a candidate agent; and
determine binding characteristics of the candidate agent to the aggrecanase-2
polypeptide,
wherein the determination is based on the information relating to the structure of
the aggrecanase-2 polypeptide bound to the ligand, and the information relating to the
candidate agent.
219. A computer program residing on a computer readable medium having a
plurality of instructions stored thereon, which, when executed by one or more processors,
cause the one or more processors to:
accept information relating to the structure of a complex comprising an
aggrecanase-2 polypeptide bound to a ligand;
accept information relating to a candidate agent; and
determine binding characteristics of the candidate agent to the aggrecanase-2
polypeptide,
wherein the determination is based on the information relating to the structure of
the aggrecanase-2 polypeptide and the information relating to the candidate agent.
220. A method, comprising:
accepting information relating to the structure of a complex comprising an
aggrecanase-2 polypeptide bound to a ligand; and
modeling the binding characteristics of the aggrecanase-2 polypeptide with a
candidate agent,
wherein the method is implemented by a software system.

221. A computer program residing on a computer readable medium having a
plurality of instructions stored thereon, which, when executed by one or more processors,
cause the one or more processors to:
accept information relating to a structure of a complex comprising an
aggrecanase-2 polypeptide bound to a ligand; and
model the binding characteristics of the aggrecanase-2 polypeptide with a
candidate agent.
222. A software system, comprising instructions for causing a computer system
to:
accept information relating to a structure of a complex comprising an
aggrecanase-2 polypeptide bound to a ligand; and
model the binding characteristics of the aggrecanase-2 polypeptide with a
candidate agent
223. A method of modulating aggrecanase-2 activity in a subject, comprising:
using rational drug design to select an agent that is capable of modulating
aggrecanase-2 activity; and
administering a therapeutically effective amount of the agent to the subject.
224. The method of claim 223, wherein the agent is capable of inhibiting or
reducing cartilage degradation in vivo.
225. The method of claim 223, wherein the rational drug design includes using
a three-dimensional structure of a crystalline complex that comprises an aggrecanase-2
polypeptide.
226. The method of claim 225, wherein the crystalline complex further
comprises a ligand.
227. The method of claim 225, wherein the ligand is a peptidomimetic.

228. The method of claim 225, wherein the ligand is a matrix metalloproteinase
inhibitor.
229. The method of claim 225, wherein the ligand is batimastat.
230. A method of treating a subj ect having a condition associated with
aggrecanase-2 activity, comprising:
using rational drug design to select an agent that is capable of affecting
aggrecanase-2 activity; and
administering a therapeutically effective amount of the agent to a subject in need
thereof.
231. The method of claim 230, wherein the agent is capable of inhibiting
aggrecanase-2 activity.
232. The method of claim 230, wherein the agent is capable of inhibiting
cartilage degradation in vivo.
233. The method of claim 230, wherein the condition is a joint disorder..
234. The method of claim 233, wherein the joint disorder is an arthritic disorder
or a joint injury.
235. The method of claim 234, wherein the arthritic disorder is osteoarthritis or
rheumatoid arthritis.
236. A method of prophylactically treating a subject susceptible to a condition
associated with aggrecanase-2 activity, comprising:
determining that the subject is susceptible to the condition associated with
aggrecanase-2 activity,

using rational drug design to select an agent that is capable of effecting aggrecanase-2
activity; and
administering a therapeutically effective amount of fee agent to the subject.
237. The method of claim 236, wherein the agent is capable of inhibiting cartilage
degradation in vivo,
23 8. The method of claim 236, wherein the condition is a joint disorder.
239. The method of claim 238, wherein fee joint disorder is an arthritic disorder or a
joint injury.
240. The method of claim 239, wherein the arthritic disorder is rheumatoid arthritis or
osteoarthritis.

241. (New) The crystallized polypeptide-ligand complex of claim 15, wherein A is an
axyl moiety having five or more atoms, B is an atyl moiety having five or more atoms, L is a
bond, M is methyleneoxy, Y is isopropylene, R1 is aryl optionally substituted with alkylcarbonyl,
Rz is hydrogen, R3 is hydrogen; X is sulfonamide; and Z is carboxylic acid.
242. (New) The crystallized polypeptide-ligand complex of claim 241, wherein A is a
phenyl moiety and B is a phenyl moiety.
243. (New) The crystallized polypeptide-ligand complex of claim 21, wherein R1 is aryl
optionally substituted with alkylcarbonyl.
244. (New) The crystallized polypeptide-ligand complex of claim 13, wherein the ligand
is2-[4'-(4-isobutyryl-phenoxyme

245. (New) The crystallized polypeptide-ligand complex el" claim 140, wherein D is an
amide bond, E is an amides R11 is methyl, R12 is phenyl, R13 is isopropyl, R1"* is heteroarylthio,
and G is hydroxamic acid.

This invention relates to aggrccanasc polypeptides and aggrecanase polypeptide/ligand complexes, crystals of aggrecanase
and aggrecanase polypeptide/ligand complexes, and related methods and software systems.