This invention relates to progesterone receptor/ligand complexes, and related methods
and software systems.
BACKGROUND
Progesterone is a steroidal hormone that regulates many biological processes. Many
of the physiological effects of progesterone are mediated by progesterone receptors (PRs).
Interaction of progesterone with progesterone receptor can cause activation of the receptor.
This can cause the progesterone receptor to be transported from the cytoplasm into the
nucleus. In the nucleus, the progesterone receptor can function as a transcriptional activator,
which can cause increased expression of specific gene targets.
SUMMARY
In genera], the invention relates to crystalline protein/ligand complexes that include a
PR polypeptide bound to a non-steroidal ligand. The PR polypeptide can include the ligand
binding domain of the PR, and the non-steroidal ligand can be an agonist or antagonist of the
receptor. The invention also relates to methods of using a three-dimensional model of a PR
polypeptide/non-sleroidal ligand complex to design an agent, such as an agonist or
antagonist, that can interact with a PR polypeptide. The invention also features related
software methods.
In one aspect, the invention features a crystallized protein-Iigand complex that
includes a progesterone receptor polypeptide and a non-steroidal ligand. The non-steroidal
ligand is an agonist or antagonist of the progesterone receptor polypeptide.
In another aspect, the invention features a crystallized protein-ligand complex that
includes a progesterone receptor polypeptide, which includes the atnino acid sequence of
SEQ ID N0:2, and a non-steroidal ligand. The crystallized protein-ligand complex diffracts
X-rays to a resolution of at least about 3.5 A. The non-steroidal ligand is an agonist or
antagonist of the progesterone receptor polypeptide.
In another aspect, the invention features a composition that includes a crystal, which
includes a progesterone receptor polypeptide and a non-steroidal ligand. The non-steroidal
ligand is an agonist or antagonist of the progesterone receptor polypeptide.
In yet another aspect, the invention features a method that includes using a threedimensional
model to design an agent that interacts with a progesterone receptor polypeptide.
The three-dimensional model includes the progesterone receptor polypeptide bound to a nonsteroidal
ligand that is an agonist or antagonist of the receptor polypeptide.
Another aspect of 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 complex includes a progesterone receptor polypeptide bound to a non-steroidal ligand
that is an agonist or antagonist of the receptor polypeptide, and the method includes
contacting the agent with a receptor polypeptide and detecting the ability of the agent to bind
the polypeptide.
In another aspect, the invention features a method of growing a crystal that includes a
progesterone receptor polypeptide and a non-steroidal ligand that is an agonist or antagonist
of the polypeptide. The method includes contacting the receptor polypeptide with the nonsteroidal
ligand, and the resulting crystal can diffract X-rays to a resolution of at least about
3.5 A.
Another aspect of the invention features a software system for determining binding
characteristics of a candidate agent to a progesterone receptor polypeptide. The software
system includes instructions for causing a computer system to accept information relating to
the structure of a progesterone receptor polypeptide bound to a non-steroidal ligand that is an
agonist or antagonist of the progesterone receptor polypeptide, and to accept information
relating to a candidate agent. The determination of binding characteristics is based on the
information relating to the structure of the receptor polypeptide and the information relating
to the candidate agent.
Another aspect of the invention features a computer program for determining the
binding characteristics of a candidate agent to a progesterone receptor polypcptide. The
computer program resides on a computer readable medium that includes a plurality of
instructions. When executed by one or more processors, the plurality of instructions causes
the one or more processors to accept information relating to the structure of a progesterone
receptor polypeptide bound to a non-steroidal ligand that is an agonist or antagonist of the
progesterone receptor polypeptide, and to accept information relating to a candidate agent.
The determination of binding characteristics is based on the information relating to the
structure of the receptor polypeptide and the information relating to the candidate agent.
In another aspect, the invention features a method for modeling the binding
characteristics of a progesterone receptor polypeptide with a candidate agent. The method
includes a software system that models the binding characteristics by accepting information
relating to the structure of a progesterone receptor polypeptide bound to a non-steroidal
ligand that is an agonist or antagonist of the progesterone receptor polypeptide.
In yet another aspect, the invention features a computer program for modeling the
binding characteristics of a progesterone receptor polypeptide with a candidate agent. The
computer program resides 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 progesterone receptor polypeptide bound to a
non-steroidal ligand that is an agonist or antagonist of the progesterone receptor polypcptide
and the processors model the binding characteristics of the receptor polypeptide with the
candidate agent.
In another aspect, the invention features a software system for modeling the binding
characteristics of a progesterone receptor polypeptide with a candidate agent. The software
system includes instructions for causing a computer system to accept information relating to
the structure of a progesterone receptor polypeptide bound to a non-steroidal ligand that is an
agonist or antagonist of the progesterone receptor polypeptide and to model the binding
characteristics of the receptor polypeptide with a candidate agent.
Structural information of a polypeptide can lead to a greater understanding of how the
polypeptide functions in vivo. For example, knowledge of the structure of a protein can
reveal properties that facilitate the interaction of the protein with its ligands, including other
proteins, effector molecules (e.g., hormones), and nucleic acids. In the case of a
progesterone receptor, an understanding of such interactions can facilitate the design or
selection of ligands (e.g., drugs) that can be useful for modulating the activity of the
progesterone receptor in vivo, and can therefore be useful for treating a human. Structure
based modeling can be used to identify ligands capable of interacting with a PR polypeptide,
thus eliminating the need for screening assays, which can be expensive and time-consuming.
Structural information can also be used to direct the modification of a ligand known to
interact with a PR polypeptide to generate an alternative ligand with more desirable
properties, such as tighter binding or greater specificity.
Unless otherwise defined, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which this invention
pertains. Although methods and materials similar or equivalent to those described herein can
be used in the practice or testing of the present invention, useful methods and materials are
described below. The materials, methods, and examples are illustrative only and not
intended to be limiting. Other features and advantages of the invention will be apparent from
the accompanying drawings and description, and from the claims. The contents of all
references, pending patent applications and published patents, cited throughout this
application arc hereby expressly incorporated by reference. In case of conflict, the present
specification, including definitions, will control.
DESCRIPTION OF DRAWINGS
FIG 1A is an electron density map showing tanaproget bound to the human PR-LBO.
The A, B, and C rings of tanaproget are as designated. The benzoxazine moiety of tanaprogei
is formed of rings B and C.
FIG IB is an electron density map showing tanaproget bound to the human PR-LBD.
FIG 2 A shows the amino acid sequence of the human progesterone receptor ligand
binding domain (human PR-LBD) (sequence underlined) fused to glutathionc-S-transferasc
(GST) (sequence not underlined) (SEQ ID NO:1). A thrombin cleavage site (LVPRG (SEQ
ID NO:3), marked in bold) occurs at the junction between the GST and PR-LBD sequences.
FIG 2B shows the amino acid sequence (SEQ ID NO:2) of the human PR-LBD.
Amino acids are numbered according to the amino acid sequence of the full-length human
progesterone receptor (see FIG 2C).
FIG 2C is the full-length sequence of human progesterone receptor (GenBank
Accession Number NMJXX)926; SEQ ID NO:4). Amino acids of the human PR-LBD are
underlined.
DETAILED DESCRIPTION
The structure of the human progesterone receptor ligand binding domain (PR-LBD)
bound to the non-steroidal hormone tanaproget (1UPAC name: 5-(4,4-dimethyl-2-thioxo-1,4-
dihydro-2H-3,l-benzoxazin-6-yl)-l-mcthyl-lH-pyrrole-2-carbonitrile; tanaproget is the
United States Adopted Name (USAN)) was determined by X-ray crystallography and is
described herein, FIGs. 1A and IB are electron density maps illustrating the structure of the
PR-LBD/tanaprogct complex. The electron density maps provide evidence indicating that
the tertiary structure of the human PR-LBD bound to tanaproget is very similar to that of the
human PR-LBD bound to its natural ligand, progesterone. It is therefore believed that the
crystal structure of the human PR-LBD/tanaproget complex (see Table 2 below) can be
useful for designing or identifying other ligands, such as, for example, non-steroidal 11gauds,
that can also interact with a PR-LBD.
FIGs, 2A and 2B provide information regarding the amino acid sequence of the
human PR-LBD, and FIG. 2C provides information regarding the full length sequence of
human PR.
The chemical structure of tanaproget is shown below:
In general, a complex of the human PR-LBD bound to tanaproget can be prepared as
desired. In some embodiments, such a complex can be prepared as follows. The human PRLBD
is expressed from a DNA plasmid. The expression can be driven by a promoter, such
as an inducible promoter. The human PR-LBD can be expressed as a fusion protein with a
suitable tag, such as a glutathione-S-transferase (GST), myc, HA, hexahistidine, or FLAG
tag. The tag can facilitate isolation of the human PR-LBD from 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. Following cleavage and purification, the human
PR-LBD can be contacted with tanaproget. For example, the human PR-LBD can be mixed
with tanaproget prior to purification (e.g., prior to cleavage of a polypeptide tag), or the
human PR-LBD can be mixed with tanaproget after purification. In some embodiments,
tanaproget can be mixed with the human PR-LBD prior to purification and again following
purification.
The human PR-LBD and tanaproget can be combined in a solution for collecting
spectral data for the human PR-LBD/tanaproget complex, NMR data for the human PRLBD
Aanaproget complex, or for growing a crystal of the human PR-LBD/tanaproget
complex. For example, the human PR-LBD/tanaproget complex 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 4C 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, about20"C, about 25C, about 30C, about 32"C, about 35°C, about
37°C).
In general, a crystal of the human PR-LBD bound to tanaproget can diffract X-rays lo
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). In some embodiments, a crystal of the human
PR-LBD bound to tanaproget can diffract X-rays to a resolution of from about 1.6 A to about
2.5 A (e.g., from about 1.8 A to about 2.2 A).
In some embodiments, a crystal of the human PR-LBD bound to tanaproget belongs
to space group P2| with unit cell parameters a » 57.52 A, b = 64.50 A, c = 70.41 A, and
b 95.76°. In certain embodiments, a crystal of the human PR-LBD bound to tanaproget can
further contain two molecules of the human PR-LBD in the asymmetric unit.
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 1,6 A (e.g.,
about 0.7 A, about 0.9 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 a complex of the human PR-LBD bound to
tanaproget 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 threedimensional
space in relation to other atoms in the complex. For example, the structural
coordinates listed in Table 2 are the structural coordinates of a crystalline complex of the
human PR-LBD bound to tanaproget. These structural coordinates describe the location of
atoms of the human PR-LBD in relation to each other, the location of atoms in the human
PR-LBD in relation to the atoms in tanaproget, and the location of atoms in tanaproget in
relation to each other. The structural coordinates of the complex 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 a PR-LBD bound to tanaproget are not specifically
limited by the actual x, y, and z coordinates of Table 2.
The structural coordinates of a complex of the human PR-LBD bound to tanaproget
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 PR-LBD or a
fragment of the PR-LBD. Such a representation 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-dimensionaLrepresentation can include
the structural coordinates of the human PR-LBD according to Table 2 ± a root mean square
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). Root mean square 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 root mean square 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 (C0or 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 a complex of the human PR-LBD bound to
tanaproget or a fragment thereof. 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-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-andstick
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.t a drawing) can correspond to a
three-dimensional representation when the two-dimensional representation reflects threedimensional
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 than one level. As an example,
when the three-dimensional representation includes a polypeptide, such as a complex of the
human PR-LBD bound to tanaproget, the polypeptide can be represented at one or more
different levels of structure, such as primary (arnino acid sequence), secondary (e.g., uhcliccs
and 0-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 a complex of the human PR-LBD bound to tanaproget.
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
macromolccular structure, and the location of hydrogen bonds and salt bridges.
An agent that interacts with a human PR-LBD can be identified or designed by a
method that includes using a representation of the human PR-LBD or a fragment thereof, or a
complex of human PR-LBD bound to tanaproget or a fragment thereof. 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 can be a ligand, and can act as an agonist or antagonist An agent that interacts with a
polypeptide (e.g., a PR 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 human PR-LBD bound to tanaproget. 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 (eg.,
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 a complex of human PR-LBD bound to tanaproget or a fragment thereof, and
the electron density map can be used to derive a representation (e.g., a two dimensional
representation, a three dimensional representation) of human PR-LBD bound to tanaprogct or
a fragment thereof. 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, multiwavdength 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 a 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. I f 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 Crystalhgr. Sect.
11
A24:905-921,1997;dcLaFortelleandBrigogne,Aferfc£»rym.276:472-494,1997). Upon
determination of (he phase, an electron density map of the polypeptide or the complex can be
constructed.
The electron density map can be used to derive a representation of a polypeptide or a
complex, or a fragment of a polypeptide or complex, by aligning a three-dimensional model
of a previously known polypeptide or a previously known complex (e.g., a complex
containing a polypeptide bound to a ligand) with the electron density map. This 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, 10
cycles) to generate a better fit with the electron density map. A software program such as
CNS (Brunger a ai. Ada 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 R*^ or R,rw
value. A smaller value of R^Kk or Rftec generally indicates a better fit. Misalignments in the
comparative model can be adjusted to provide a modified comparative model and a lower
Kmor Rfrce value. The adjustments can be based on information (e.g., sequence
information) relating to human PR-LBD, tanaproget, the previously known polypeptide
and/or the previously known complex. As an example, in embodiments in which a model of
a previously known complex of a polypeptide bound to a ligand is used, an adjustmenl can
include replacing the ligand in the previously known complex with tanaproget. As another
example, in certain embodiments, an adjustment can include replacing an amino acid in the
previously known polypeptide with the amino acid in the corresponding site of human PRLBD.
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 (e.g,, the PR-LBD/tanaproget complex).
Methods of such processes are disclosed, for example, in Carter and Sweet, eds.,
"Macromolecular Crystallography" in Methods in Enzvmologv. Vol. 277, Pan B, New
York: Academic Press, 1997, and articles therein, e.g., Jones and Kjeldgaard, "Electron-
Density Map Interpretation," p. 173, and Kleywegt and Jones, "Model Building and
Refinement Practice," p. 208,
12
In some embodiments, a representation of human PR-LBD bound to tanaproget can
be derived by aligning a previously determined structural model of progesterone bound to
human PR-LBD (e.g., Protein Databank Identification No. Ia2&) with the electron density
map of human PR-LBD bound to tanaproget derived from X-ray diffraction data. One
adjustment that can be used in the modeling process can include replacing progesterone with
tanaproget.
A machine, such as a computer, can be programmed in memory with the structural
coordinates of a complex of the human PR-LBD bound to tanaproget, 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
(e.g., polypcplides) with similar structural features as human PR-LBD, and/or to identify one
or more candidate agents with characteristics that may render the candidate agcnl(s) likely to
interact with human PR-LBD.
A machine having a memory containing structure data or a software system
containing such data can aid in the rational design or selection of PR agonists and/or PR
antagonists. 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 PR-LBD bound to tanaproget, or
can aid in the modeling of compounds or proteins related by structural or sequence homology
to a PR-LBD.
The machine can produce a representation (e.g., a two dimensional representation, a
three dimensional representation) of a complex of the human PR-LBD bound to tanaproget
or a fragment thereof. 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 machinereadable
data can include structural coordinates of atoms of a complex of the human PRLBD
bound to tanaproget or a fragment thereof. 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
13
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 die 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 threedimensional
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 son described herein) the machine is capable of displaying a
graphical representation (e.g., a two dimensional graphical representation, a threedimensional
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 a complex of human
PR-LBD bound to tanaproget or a fragment thereof. The user can inspect the representation
and, using information gained from the representation, generate a model of a complex or
fragment thereof that includes an agent other than tanaproget. The model can be generated,
for example, by altering a previously existing representation of a human PR-LBD/tanaprogel
complex. Optionally, the user can superimpose a three-dimensional model of an agent on the
representation of human PR-LBD bound to tanaproget. The agent can be an agonist (e.g., a
candidate agonist) of human PR-LBD or an antagonist (e.g., a candidate antagonist) of
human PR-LBD. In some embodiments, the agent can be a known compound or fragment of
a compound. In 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 PR polypeptide. Distances longer than a preferred distance may be
associated with a weak interaction between the agent and active site (e.g., human PR-LBD).
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 PR polypeptidc (eg., 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 PR polypeptide in the vicinity of the agent in order to relieve a steric clash.
Steric clashes can also be removed by 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. E!°strostatic forces can also influence an
interaction between an agent and a Itgand-binding domain. For example, electrostatic
properties can be associated with repulsive forces that can weaken the interaction between
the agent and the PR polypeptide. Electrostatic repulsion can be relieved by altering the
charge of the agent, eg., by replacing a positively charged group with a neutral group.
Forces that influence binding strength between tanaproget and human PR-LBD can
be evaluated in the polypeptidc/agent model These can include, for example, hydrogen
bonding, electrostatic forces, hydrophobic interactions, van der Waals interactions, dipoledipole
interactions, n-stacking forces, and cation-re 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 PR polypeptide and the agent.
Altering the model can include changing the three-dimensional structure of the polypeptidc
without altering its chemical structure, for example by altering the conformation of ami no
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 PR 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 PR polypeptide, or their conformations, can
be adjusted to find an optimized binding geometry for a particular agent to the PR
polypeptide. An optimized binding geometry is characterized by, for example, favorable
hydrogen bond distances and angles, maximal electrostatic attractions, minimal 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 a PR polypeptide/agenl complex. An
optimized geometry can be determined, for example, through molecular mechanics or
molecular dynamics calculations.
A series of representations of complexes of human PR-LBD bound to tanaproget,
where tanaproget is replaced or overlaid with different agents, can be generated. A score can
be calculated for each representation. The score can describe, for example, an expected
strength of interaction between human PR-LBD 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 PR-LBD 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 human PRLBD.
A software system can be designed and/or implemented to facilitate these steps.
Software systems (e.g., computer programs) used to generate such three-dimensional models
or perform the necessary fitting analyses include, but are not limited to: MCSS, Ludi,
QUANTA, Insight 11, Ccrius2, CHARMm, and Modeler from Accclrys. 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 FIo99 (Thistlcsoft,
Morris Township, NJ). Other useful programs include ROCS, ZAP, FRED, Vida, and Szybki
from Opencye Scientific Software (Santa Fe, NM); Maestro, Macromodel, and Glide from
Schrodinger, LLC (Portland, OR); MOE (Chemical Computing Group, Montreal, Quebec),
AJlegrow (Boston De Novo, Boston, MA), CNS (Brunger, et ai, Acta Crystall. Sect. D
54:905-921,1997) and GOLD (Jones el al.t J. Mol. Biol. 245_:43-53,1995), The structural
coordinates can also be used to visualize the three-dimensional structure of PK.C0 using
MOLSCRIPT, RASTER3D, or PYMOL (Kraulis, / Appl Crystalhgr, 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, whether an agonist or antagonist, can, for example, be selected by
screening an appropriate database, can be designed de novo by analyzing the steric
configurations and charge potentials of unbound human PR-LBD in conjunction with the
appropriate software systems, and/or can be designed using characteristics of known agonists
or antagonists of progesterone receptors or other hormone receptors. The method can be
used to design or select agonists or antagonists of human PR-LBD. 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 human PR-LBD activity. For example, the agent can be
evaluated by contacting it with human PR-LBD 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. An activity assay can be a cell-based assay, for example.
Depending upon the action of the agent on human PR-LBD, the agent can act either as an
agonist or antagonist of human PR-LBD activity. The agent also can be contacted with the
polypeptide in the presence of progesterone in order to determine whether or not the agent
inhibits binding of progesterone to the polypeptide. A crystal containing human PR-LBD
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 human PR-LBD.
Various molecular analysis and rational drug design techniques are further 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 PR-LBD and tanaproget
have been described, the description herein is more generally directed to any PR polypeptide
and any non-steroidal ligand.
A PR polypeptide can be a fall-length, mature polypeptide, including the full-length
amino acid sequence of any isoform of a PR polypeptide. An isoform is any of several
multiple forms of a protein that differ in their primary structure. For example, human
progesterone receptor exists in at least two isoforms, Pr-B (full-length PR) and Pr-A (Nterminally
truncated PR). The two isoforms are transcribed from a single gene, but have
different translation Stan sites. Thus the isoforms are identical except that Pr-B contains an
additional 164 N-terminal amino acids. The isoforms have an identical centrally located
DNA binding domain, which is flanked at the N-terminus by a transcriptional activation
function-1 (AIM) domain, and at the C-terminus by a hinge region containing nuclear
localization signals and a iigand binding domain (PR-LBD). A second transcriptional
activation function domain (AF-2) is located in the PR-LBD.
A PR polypeptide can be a fragment of a PR, such as a Iigand binding domain, a
DNA-binding domain, a protein-interaction domain (e.g., an activation domain), or a
combination thereof.
A PR polypeptide can have an active site. In general, an active site can include a site
of Iigand binding, or a site of phosphorylation, glycosylation, alkylation, acylalion, or other
covalent modification. A Iigand binding site can include accessory binding sites adjacent or
proximal to the actual site of binding that may affect activity upon interaction with the
Iigand. An active site of a PR polypeptide can include amino acids of SEQ ID NO:2. For
example, an active site of a PR-polypeplide can include one or more of amino acids Ile699,
A!a701, Leu714, Leu715, Leu718, Asn719, Leu721, Gln725, Trp755, Met756, Met759,
Val760, Leu763, Arg766, Ser767, Tyr777, Phe778, Ala779, Leu782, Phe794, Leu797,
Lys798, MerSOl, IIe804, Leu887, His888, Tyr890, Cys89l, Asn893, Thr894, Phe895,
Ser898, Leu901, Val903, Phe905, Met909, Ile913, and Leu9l7 as defined by SEQ ID NO:2.
The numbering of the amino acids of a PR polypeptide may be different than that set
forth herein, and the sequence of the PR polypeptide may contain certain conservative amino
acid substitutions that yield the same three-dimensional structure. For example, the
numbering of a PR-LBD may be different than that set forth in FIG. 2B, and the sequence of
the PR-LBD may contain conservative amino acid substitutions but yield the same structure
as that defined by the coordinates of Table 2 and illustrated in FIGs. 1A and IB.
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 liomology 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., hydrophobia, acidic or basic), and
includes substitutions ha* ing an inconsequential effect on the three-dimensional structure of
the PR polypeptide with respect to identification and design of agents that interact with the
polypeptide (e.g., a PR-LBD), as well as for molecular replacement analyses and/or for
hotnology modeling.
A PR polypeptide can originate from a nonmammalian or mammalian species. A
mammalian PR polypeptide can originate from a human, for example. Exemplary nonhumun
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.
As another example, while embodiments have been described in which tanaproget is
a ligand, more generally other non-steroidal compounds may also be used as ligands. For
example, based on a representation of the human PR-LBD bound to tanaproget derived from
the structure of the crystalline complex, without wishing to be bound by theory it is believed
that: the carbonitrilo nitrogen of tanaproget forms hydrogen bonds with the side chains of
Gln725 and Arg766 of the human PR-LBD; the benzoxazine nitrogen of tanaproget forms
hydrogen bonds with the side chain oxygen of Asn719 of the human PR-LBD; hydrophobic
interactions occur between Leu797 of the human PR-LBD and the hydrophobic region of the
benzoxazine moiety (which includes the pair of methyl substituents in the 4 position) of
tanaproget; and an electrostatic interaction occurs between Thr894 of the human PR-LBD
and the sulfur of tanaproget.
Based on this information, without wishing to be bound by theory, it is believed that
other non-steroidal compounds capable of having one or more similar interactions with the
human PR-LBD may also be capable of acting as ligands (e.g., agonists, antagonists) for the
human PR-LBD. Such non-sicroidal compounds may have the structure:
where A, B and C represent ring systems, B and C are fused rings, and L is a linker moiety.
In general, rings A, B and C are each independently formed of at least four atoms
(e.g., five atoms, six atoms, seven atoms, eight atoms, nine atoms, 10 atoms. 11 atoms, 12
atoms, 13 atoms, 14 a.oms). One or more atoms (e.g., one atom, two atoms, three atoms,
four atoms) in rings A, B and/or C can independently be heteroatoms (e.g., N, S, O). In some
embodiments, rings B and C form an indolc, an oxindole, a thioindole, a benzothiophene, a
benzofuran, a benzothiazole, a benzinudazole, a benzoxazine or a benzthiazine. In one
embodiment rings B and C can form a benzoxazine ring that can have at least one
hydrophobic substituent at the 4 position. In another embodiment the benzoxazine ring can
have at least one hydrophobic substituent near the 4 position e.g. at the 3 position.
Benzoxazine derivatives are described, for example, in U.S. Patent No. 6,562,857, which is
hereby incorporated by reference. Ring A can be, for example, a pyrrole, a furan, a
thiophene, an imidazole, an oxazole, or a pbenyl. In some embodiments, rings A, B and/or C
can each independently include one or more (c,g., one, two, three, four) substitucnts (e.g.,
one or more substitucnts that provide favorable interaction with the human PR-LBD, such as,
for example, through hydrogen bonding, hydrophobic interaction and/or electrostatic
interaction). Examples of substituents include bydroxy substituents, amino substituents,
cyano substituents, nitro substituents, oxime substituents, thiol substituents, amido
substituents, oxo substituents, alky] substituents e.g. having 1-6 carbon atoms, alkcnyl
substituents e.g. having 2-6 carbon atoms, alkynyl substituents e.g. having 2-6 carbon atoms,
aryl substituents e.g. an aromatic carbocyclic mono- or polycyclic ring system having from
6-20 ring carbon atoms, cydyl substituents e.g. a saturated or partially saturated mono- or
polycyclic carbocyclic ring system having from 3-14 ring atoms, heteroaryl substituents e.g.
an aromatic heterocyclic mono- or polycyclic ring system having from 5-14 ring atoms
wherein 1, 2, 3 or 4 of the ring atoms are selected from N, O or S, heterocyclyl substituents
e.g. a saturated or partially saturated mono- or polycyclic heterocyclic ring system having
from 3-14 ring atoms wherein 1,2,3 or 4 of the ring atoms are selected from N, O or S, and
halogens (e.g., fluorine, chlorine, bromine, iodine). While in some embodiments, a
substituent itself may be a hydrogen bond donor or acceptor with the human PR-LBD, in
other embodiments, the substituent may form a hydrogen bond with a portion of the human
PR-LBD through one or more solvent molecules such as water.
In general, L can be a direct chemical bond, or L can be formed of a chemical moiety,
such as, for example, an atkyl moiety e.g. having 1-6 carbon atoms, an alkenyl moiety e.g.
having 2-6 carbon atoms, an alkynyl moiety e.g. having 2-6 carbon atoms, an ether moiety, a
thioether moiety, an amido moiety, a carbonyl moiety or a sulfonyl moiety. In some
embodiments, L can be formed of multiple moieties (e.g., a sulfonyl moiety bonded to an
alkyl moiety).
Although embodiments have been described in which the non-steroidal compound
includes rings B and C fused together, in some embodiments, rings B and C can be joined by
a chemical linker. Examples of linkers are noted above. In certain embodiments, ring B is
not present. For example, ring B can be replaced with a linker moiety that connects ring C
with ring A. The linker moiety can, for example, be of sufficient length to allow favorable
interactions between ring A and one or both of Glu72S or Arg766 of the human PR-LBD,
and between ring C and one or more of Asn7I9, Thr894, or Cys891 of the human PR-LBD.
For example, ring B can be replaced with an alkenyl linker such as a branched alkene, which
can provide sufficient length, structural rigidity, and, where desired, bulk (e.g., the linker
moiety can include a branched alkenyl moiety).
The following example is illustrative and not intended as limiting.
EXAMPLE
Human progesterone ligand binding domain (PR-LBD) was expressed as an ammo
terminal glutathionc-S-transferase (GST) fusion protein from Escherichia coli BL2I
(Stratagene). The PR-LBD domain coding sequence was cloned into the pGEX plasmid
(Amersham Pharmacia Biotech) under tac transcriplional control. A thrombin cleavage site
(LVPRG, SEQ IP #3) was present at the boundary of the GST and PR-LBD regions
(thrombin cleaves between the R and the G). The sequence of this fusion protein is shown in
FIG. 2A, The sequence of the PR-LBD polypeptide following thrombin cleavage is shown in
FIG. 2B.
Bacterial growth and protein expression were performed in a Biostat 10 liter
fermenter (B. Braun Biotech). A 100 mL preculture was used to inoculate 10 liters of media
(fermenter salts, glucose, ampicillin, trace metals and yeast extract media) and expanded
overnight at 25°C. Fifteen minutes prior to induction, the vessel temperature was lowered to
15°C and 5 mis of 66 mM progesterone (Sigma-Aldrich, St. Louis, MO) in 100% ethanol
was added. The culture was induced at a density of 5.4 OD600 absorbance units with
addition of isopropyl-beta-D-thiogalactopyranoside (IPTG, Fisher) to a final concentration of
1.0 mM. Progesterone was again added in 5ml aliquots of 66mM at the time of induction
and every fifteen minutes thereafter, for the duration of expression (4 hours total, 660 /xM
final concentration of progesterone). After 4 hours of induction, the culture was harvested
yielding 158,76g of wet cell weight The protein of interest represented 5-7% of total cell
protein, as estimated by SDS-PAGE. Successful isolation of PR-LBD depended strongly on
inclusion of a PR ligand during expression (possibly to ensure proper protein folding) and
purification. See, for example, Williams and Sigler, Nature 222:392-396,1998; Tanenbaum
& al.. Proc. Natl. Acad. Sci. USA 95:5998-6003,1998; and Matias et ai, J. Biol. Chem. 275:
26164-26171,2000. Tanaproget was exchanged for progesterone during purification.
20 g of frozen cells were suspended in 300 mL of 50 mM HEPES pH 7.3,
150 mM NaCl, 5 mM EDTA, 10% glycerol, 5 mM dithiothreitol (DTT) with 0.33 mM of the
protease inhibitor aminoethylbenzenesulfonyl fluoride (AEBSF, Sigma-Aldrich), 0-3 mL of
protease inhibitor cocktail (Sigma-Aldrich catalog number 8849) and 5 uM progesterone
(Progesterone was stored as a 50 mM stock solution in dimethylsulfoxide). Cells were
broken by passage through a microfluidizer (Midrofluidics, Newton, MA). Cell debris and
aggregated GST/PR-LBD were removed by centrifugation for 2.5 hrs at approximately
40,000 g. CHAPS (Sigma) was added to 1.5% and the solution was passed through a
0.45 micron cellulose nitrate filter and stored overnight.
An initial purification of GST/PR-LBD fusion protein was carried out by affinity
chromatography. The filtered solution was passed over two 5 mL columns (in tandem) of
GSTrap FF glutathione-Sepharose chromatography media (Amersham Bioscience) at flow
rate of about 1 mL/min. Resin was washed with 50 mL of 50 mM HEPES pH 7.3,
150 mM NaCl, 5 mM EDTA, 10% glycerol, then with SO mL of same solution containing
50 uM tanaproget. GST/PR-LBD was eluted with 12 mM reduced glutathione (Sigma) in
SO mM HEPES pH 7.3,100 mM NaCI, 10% glycerol, 0.1% octyl-p-glucoside, and
50 nM tanaproget. Fractions of 5 raL were collected. Those fractions containing GST/PRLBD
were identified by SDS-PAGE and pooled. Typically the pooled fractions had a total
volume of 30-40 mL. Thrombin was added to 25,000 NIH units/mL The solution was
incubated overnight for specific proteolysis.
The solution was diluted with 4 volumes oflO mM HEPES pH 7.3,10% glycerol,
5 mM DTT, 0.1 % octyl-p-glucoside, and 50 uM tanaproget. The solution was passed over a
1 mL column of HiTrap SP FF sulfopropyl-Sepharose (Amersham Bioscience) at a flow rate
of 1 mL/min. The column was washed with 5 mL of 10 mM HEPES pH 7.3, 20 mM NaCI,
10% glycerol, 0.1% octyl-p-glucosidc, and 1 fiM tanaproget (tanaproget was stored as a SO
mM stock solution in dimethylsulfoxide). PR-LBD was eluted from the column with a IS
mL gradient of sodium chloride, running from 20 mM to 220 mM (other components as
above). Fractions of 1 mL were collected, PR-LBD was located by SDS-PAGE and those
fractions containing PR/LBD at a concentration of 1 to 2 mg/mL were used directly for
crystallization.
Prior to crystallization, the PR-LBD/tanaproget complex was determined to be
homogeneous by SDS-PAGE.. The protein had the expected mass as determined by M ALDI
mass spectrometry (-29,800 Da). The protein behaved as a single species during sizeexclusion
chromatograpby. The retention volume, as compared with reference proteins, was
consistent with a dimer of PR-LBD. Only those preparations of the PR-LBD/tanaproget
complex without any detectable progesterone were used for crystallization. Bound ligand
was analyzed by reverse-phase chromatography following protein denaturation in the
presence of guanidine-HCl.
Crystals were grown by hanging drop vapor diffusion at 18°C. The drops contained
2.0 ML protein stock solution (5 mg/mL protein, 10 mM HEPES pH 7.3,10% glycerol,
5 mM DTT, -100 mM NaCI, 0.1% octyl-p-glucoside, 1 fiM tanaproget) mixed with
1.0 uL well solution (8% PEG 3350 (Hampton Research), 300 mM MgSO4,50 mM PIPES
pH 6.5, 10% glycerol) and 0.5 uL 1,3-propanediol (40% v/v, Hampton Research) and
equilibrated against a I mL well solution. Diamond shaped crystals grew in 2-6 weeks,
measuring -50 (jm across.
Showers of small crystals grew in the conditions described above with a variety of
sulfate salts. The number of nucleation events was reduced by the addition of 1,3-
propanediol (40% y/v) to drops, enabling the growth of fewer, larger, single crystals. The
crystals belonged to space group P2| with unit cell parameters a = 57.52 A, b - 64.50 A, c «=
70.41 A,and p = 95.76°, and contained two molecules of PR-LBD in the asymmetric unit,
implying a solvent content of 44%. Crystals were drawn through a cryoprotectant solution of
20% ethylene glycol and 80% well solution, and cooled rapidly in liquid nitrogen.
Diffraction data were recorded on an R-axis 4 detector. Intensities were integrated and
scaled using the programs Denzo and Scalepack (Otwinowski and Minor, Methods Enzymol.
276:307-326,1997),
The structure was solved by molecular replacement using the protein model of the
PR-LBD/progesterone structure (Williams and Sigler, Nature 393:392-396,1998) as the
search model. After several iterative cycles of refinement using CNS (Brunger et al., Acta
Crystallogr. D54:905-921,1998) and model improvement, tanaproget was placed and
refined. The final values of Rva* and /?/*» were 19.62% and 23.74%, respectively. Table 1
summarizes the data collection parameters and results.
(Table Removed) The tertiary structure of PR-LBD/tanaproget was very similar to that of PRLBD/
progcsteronc. Tanaproget occupied the same binding pocket as progesterone. See
FIGs. 1A and IB, which present views of a 3F0to-2FC(ic experimental map of tanaproget
bound to PR-LBD unbiased by ligand phases and contoured at 1.2 a. Superposition of the
structures of the PR-LBD/tanaproget complex nd the PR-LBD/progesteror,i complex
revealed that the 1 -methyl-1 H-pyrrole-2-carbonitrile ring lay roughly between the A and B
rings of progesterone, and the l,4-dihydro-3,l-benzoxazine-2-thione moiety ("benzoxazine")
lay just above the C and D rings of progesterone in the direction of the protruding methyl
groups of progesterone.
Tanaproget mimicked the interaction of progesterone with the ligand binding pocket
of PR-LBD in many ways. The nitrile group, positioned approximately 0.6 A from the 3-
keto substituent of the progesterone A ring, formed hydrogen bonds with the side chains of
Gln725 and Arg766. Similarly, the progesterone 3-keto group interacted with the side chains
of Gln725 and Arg766, and maintained the hydrogen bonding network that provides
specificity for steroids with an oxygen atom at the 3-position of the A ring. The benzoxazine
moiety occupied approximately the same space as rings C and D of progesterone with similar
hydrophobic interactions. The sulfur of the benzoxazine appeared to extend beyond the
methyl-ketone substituent at C17 of progesterone. Tanaproget had an additional favorable
interaction with the protein in the hydrogen bond formed between the benzoxazine nitrogen
and the side chain oxygen of Asn719. The loop between helix 6 and helix 7, including amino
acids 788-794, was rearranged with respect to the PR-LBD/progesterone structure. This
rearrangement did not appear to be directly due to the different ligand, however, as the
shortest distance between tanaproget and PR-LBD in this region is 5 A, from one tanaproget
methyl substituent of the benzoxazine to the side chain of Phe794. The structure indicated
that the sulfur atom of tanaproget may form a weak interaction with Thr894 of the PR-LBD,
and a hydrophobic region on the benzoxazine molecule forms a favorable hydrophobic
interaction with Leu797 of the receptor LBD.
Several side chains were well defined by the experimental electron density in one
molecule of the asymmetric unit (molecule "A"), but not in the other (molecule "B"). These
side chains include: Met908, Phe794, Trp755, and Leu714. Met908 is somewhat disordered
in molecule "A", with mow than one rotamer represented in the electron density.
OTHER EMBODIMENTS
A number of embodiments of the invention have been described. Nevertheless, it will
be understood that various modifications may be made. For example, a structure of
progesterone receptor can be determined where progesterone receptor is bound to a nonsteroidal
agent other than Unaproget. Accordingly, other embodimenls are within the scope
of the following claims.
WE CLAIM:
1. A crystallized protein-ligand complex, comprising:
a progesterone receptor polypeptide; and
a non-steroidal ligand that is an agonist of the progesterone receptor polypeptide or
an antagonist of the progesterone receptor polypeptide.
2. The crystallized protein-ligand complex of claim 1, wherein the non-sieroidal
ligand Is an agonist of the progesterone receptor polypeptide.
3. The crystallized protein-ligand complex of claim 1, wherein the non-steroidal
ligand is an antagonist of the progesterone receptor polypeptide.
4. The crystallized protein-ligand complex of any one of claims 1 to 3, wherein
the non-steroidal ligand comprises at least two rings.
5. The crystallized protein-ligand complex of claim 4, wherein the at least two
rings are fused.
6. The crystallized protein-ligand complex of claim 5, wherein the fused rings
comprise at least one ring heteroatom.
7. The crystallized protein-ligand complex of claim 6, wherein the at least one
ring heteroatom is selected from the group consisting of N, O, and S.
8. The crystallized protein-ligand complex of any one of claims 1 to 7, wherein
the non-steroidal ligand has the structure:
wherein A, B and C represent ring systems, B and C are fused rings, and L is a linker
moiety.
9. The crystallized protein-ligand complex of any one of claims 1 to 8, wherein
the non-steroidal iigand comprises a benzoxazine derivative.
10. The crystallized protein-ligand complex of claim 9, wherein the benzoxazine
derivative comprises an oxo or thio substituent at the 2 position.
11. The crystallized protein-ligand complex of claim 9 or 10, wherein the
benzoxazine derivative has at least one hydrophobia substituent at or near the 4 position of
the benzoxazine ring.
12. The crystallized protein-ligand complex of claim 1, wherein the non-stcroida ]
ligand is tanaproget.
13. The crystallized protein-ligand complex of any one of claims 1 to 12, wherein
the crystallized protein-ligand complex has space group P2(,
14. The crystallized protein-ligand complex of any one of claims 1 to 13, wherein
the crystallized protein-ligand complex has unit cell dimensions a=57.S A, b=64.5 A, c=70.4
A,and/3=95.8°.
15. The crystallized protein-ligand complex of any one of claims I to 14, wherein
the non-steroi Jal ligand is bound to the progesterone receptor polypeptide.
16. The crystallized protein-ligand complex of any one of claims 1 to 15, wherein
the progesterone receptor polypeptide comprises a ligand binding domain.
17. The crystallized protein-ligand complex of claim 16, wherein ihe nonsteroidal
ligand is bound to the ligand binding domain.
18. The crystallized protein-ligand complex of any one of claims 1 to 17, wherein
the progesterone receptor polypeptide is from a mammalian species.
19. The crystallized protein-ligand complex of any one of claims 1 to 17, wherein
the progesterone receptor polypeptide is from a non-mammalian species.
20. The crystallized protein-ligand complex of claim 18, wherein the
progesterone receptor polypeptide is from a human.
21. The crystallized protein-ligand complex of claim 20, wherein the
progesterone polypeptide comprises the amino acid sequence of SEQ ID NO:2.
22. The crystallized protein-ligand complex of any one of claims 1 to 21, wherein
the complex is capable of diffracting X-rays to a resolution of at least about 3.5 A.
23. The crystallized protein-ligand complex of claim 1, wherein the complex
comprises the structural coordinates ofTable 2 ± a root mean square deviation for alpha
carbon atoms of not more than 1.5 A.
24. The crystallized protein-ligand complex of any one of claims 1 to 22, wherein
the non-steroidal ligand interacts with one or more of Gln725, Leu797, Asn719 and Arg766
of the progesterone receptor polypeptide.
25. A composition, comprising a complex according to any one of claims 1 to 24.
26. A method, comprising:
using a three-dimensional model of a progesterone receptor polypeptide bound to a
non-steroidal ligand to design an agent that interacts with the progesterone receptor
polypeptide,
wherein the non-steroidal ligand is an agonist of the progesterone receptor
polypeptide or an antagonist of the progesterone receptor polypeptide.
27. The method of claim 26, wherein the three-dimensional model comprises a
ligand binding domain of the progesterone receptor polypeptide.
28. The method of claim 26 or 27, wherein the three-dimensional model
comprises structural coordinates of atoms of the progesterone receptor polypeptide.
29. The method of claim 28, wherein the structural coordinates are experimentally
determined coordinates.
30. The method of claim 28 or 29, wherein the atoms include atoms of an active
site of the progesterone receptor polypeptide.
31. The method of claim 30, wherein the active site is a ligand binding domain.
32. The method of claim 28, wherein the structural coordinates are according to
Table 2, ± a root mean square deviation for alpha carbon atoms of not more than 1.5 A.
33. The method of any one of claims 28 to 32, wherein the three-dimensional
model comprises structural coordinates of the non-steroidal ligand.
34. The method of claim 33, further comprising altering the non-stcroidal ligand
of the model.
35. The method of claim 34,-wherein altering the non-steroidal ligand comprises
changing the structural coordinates of the non-steroidal ligand.
36. The method of claim 34, wherein altering the non-steroidal ligand comprises
changing the chemical structure of the non-steroidal ligand.
37. The method of any one Of claims 28 to 36, wherein the three-dimensional
model comprises structural coordinates of an atom selected fron" the group consisting of
atoms of amino acids Gln725 and Arg766 of the progesterone receptor polypeptide as
defined by the amino acids of SEQ ID N0:2.
38. The method of any one of claims 28 to 37, wherein the three-dimensional
model comprises structural coordinates of an atom selected from the group consisting of
atoms of amino acids Ile699, AU701, Leu714, Leu715, Leu718, Asn719, Leu721, Gln725,
Trp755, Met756, Met759, Val760, Lcu763, Arg766, Ser767, Tyr777, Phe778, Ala779,
Le«782, Phe794, Leu797, Lys798, MetSOI, Ile804, Leu887, His888, Tyr890, Cys89I.
Asn893, Thr894, Phe895, Ser898, Leu9j01, Val903, Phe905, Met909, Ile913, and Lcu917 as
defined by the amino acids of SEQ ID NO:2.
39. The method of any one of claims 26 to 38, further comprising calculating a
distance between an atom of the progesterone receptor polypeptide and an atom of the agent.
40. The method of any one of claims 26 to 39, further comprising comparing a
predicted interaction between the agent and the progesterone receptor polypeptide with the
interaction between the non-steroidal ligand and the progesterone receptor polypeptide.
41. The method of any one of claims 26 to 40, further comprising providing a
composition including the progesterone receptor polypeptide.
42. The method of claim 41, wherein the progesterone receptor polypeptide is
crystalline.
43. The method of claim 42, wherein the composition includes the agent that
interacts with the progesterone receptor polypeptide.
44. The method of claim 43, further comprising experimentally determining the
interaction of the agent with the progesterone receptor polypeptide.
45. The method of claim 44, further comprising comparing the interaction of the
agent with the progesterone receptor polypeptide to an interaction of a second agent with the
progesterone receptor polypeptide.
46. A method, comprising:
selecting an agent by performing rational drug design with a three-dimensional
structure of a complex according to any one of claims 1 to 24
contacting the agent with a progesterone receptor polypeptide; and
detecting the ability of the agent to bind the progesterone receptor polypeptide.
47. The method of claim 46, wherein the agent is selected via computer modeling.
48. The method of claim 46 or 47, further comprising synthesizing the agent.
49. The method of any one of claims 46 to 48, fiirther comprising:
obtaining a supplemental crystalline complex comprising the progesterone
polypeptide receptor and the agent;
determining the three-dimensional structure of the supplemental crystalline complex;
selecting a second agent by performing rational drug design with the threedimensional
structure of the supplemental crystalline complex;
contacting the second agent with the progesterone polypeptide receptor, and
detecting the ability of the second agent to bind the progesterone receptor
polypeptide.
50. The method of claim 49, wherein the second agent is selected via computer
modeling.
51. The method of claim 49 or 50, further comprising synthesizing the second
agent.
52. A method, comprising:
contacting a polypeptide with a non-stcroidal ligand to grow a crystal of a complex
according to any one of claims 1 to 24.
53. The method of claim 52, wherein the method includes using hanging drop
vapor diffusion.
54. A software system, comprising instructions for causing a computer system to:
accept information relating to the structure of a progesterone receptor polypeptide
bound to a non-sleroidal ligand that is an agonist of the progesterone receptor polypeptide or
an antagonist of the progesterone receptor polypeptide;
accept information relating to a candidate agent; and
determine binding characteristics of the candidate agent to the progesterone receptor
polypeptide,
wherein the determination is based on the information relating to the structure of the
progesterone receptor polypeptide and the information relating to the candidate agent.
55. The software system of claim 54, wherein the structure of the progesterone
receptor polypeptide bound to the non-steroidal ligand is a crystal structure.
56. The software system of claim 55, wherein the crystal structure comprises the
structural coordinates of Table 2, ± a root mean square deviation for alpha carbon atoms of
not more than 1.5 A.
57. The software system of any one of claims 54 to 56, further comprising
instructions for causing the computer system to:
apply information from a database, the information relating to candidate agents; and
identify a candidate agent in the database that can bind the progesterone receptor
polypeptide,
wherein the identification is based on the infarmation relating to the structure of ihe
progesterone receptor polypeptide and information relating to the candidate agent.
58. The software system of claim 57, further comprising instructions for causing
the computer system to model the binding characteristics of the candidate agent with the
progesterone receptor polypeptide.
59. 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 progesterone receptor polypeptide
bound to a non-steroidal ligand that is an agonist of the progesterone receptor polypeptide or
an antagonist of the progesterone receptor polypeptide;
accept information relating to a candidate agent; and
determine binding characteristics of the candidate agent to the progesterone receptor
polypeptide,
wherein the determination is based on the information relating to the structure of the
progesterone receptor polypeptide and the information relating to the candidate agent.
60. A method, comprising:
accepting information relating to the structure of a progesterone receptor polypepiide
bound to a non-steroidal ligand that is an agonist of the progesterone receptor polypeptide or
an antagonist of the progesterone receptor polypeptide; and
modeling the binding characteristics of the progesterone receptor polypeptide with a
candidate agent,
wherein the method is implemented by a software system.
61. The method of claim 60, farther comprising applying information from a
database of candidate agents to identify a candidate agent that can bind the progesterone
receptor polypeptide,
wherein the identification is based on the information relating to the structure of (he
progesterone receptor polypeptide and information relating to the candidate agent
62. 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 progesterone receptor polypeptide
bound to a twn-steroidal ligand that is an agonist of the progesterone receptor polypeptidc or
an antagonist of the progesterone receptor polypeptide; and
model the binding characteristics of the progesterone receptor polypeptide with a
candidate agent.
63. The computer program of claim 62, further comprising instructions which
cause the one or more processors to:
apply information from a database, the information relating to candidate agents; and
identify a candidate agent in the database that can bind the progesterone receptor
polypeptide,
wherein the identification is based on the information relating to the structure of the
progesterone receptor polypeptide.
64. The computer program of claim 63, further comprising instructions which
cause the one or more processors to model the binding characteristics of the candidate agent
with die progesterone receptor polypeptide.
65. A software system, comprising instructions for causing a computer system to:
accept information relating to the structure of a progesterone receptor polypeptide
bound to a non-steroidal ligand that is an agonist of the progesterone receptor polypeptide or
an antagonist of the progesterone receptor polypeptide; and
model the binding characteristics of the progesterone receptor polypeptide with a
candidate agent.
66. The invention substantially such as herein described.