DESC:Field of the invention

The present invention relates to a method for identifying the selective inhibitors of proteins. More particularly, the method for identifying the selective inhibitors of the human DNA methyltransferase1 (hDNMT1).

Background of the invention:
Epigenetic mechanisms such as chromatin modifications and a DNA methylation provide a heritable and reversible way to modulate gene expression. In mammalian cells, S-adenosyl methionine (SAM) based methylation of the CpG dinucleotide at the 5’ position of the cytosine is performed by DNMT1 and DNMT3 enzymes. The DNMT1 enzyme prefers to methylate hemi-methylated (methylation on one strand) DNA and is hence thought to play a major role in maintenance of methylation, after every cellular DNA replication cycle. Aberrant DNA methylation has been associated with several cancers such as colon, lung, breast, thyroid, hematological malignancies and other syndromes involving chromosomal instabilities and mental retardation. The DNA methylation is reversible and hence inhibition of the human DNMT1 (hDNMT1) is thought to be a good strategy for pharmacological intervention in several diseases related to irregular methylation.

Two DNA methylation inhibitors 5-azacytidine and 5-aza-2'-deoxycytidine have been approved by the U.S. F.D.A. for the treatment of higher-risk myelodysplastic syndrome. These compounds are non-selective nucleoside derivatives that get incorporated into DNA and covalently trap hDNMT1 reducing its activity. Due to the non-selective nature, these compounds have considerable cytotoxic side effects. Several dietary bioactive compounds and other natural compounds such as curcumin and EGCG have been reported as hDNMT1 inhibitors. However, these compounds must be treated with caution since several of them have been identified as pan-assay-interfering and hence are not suitable to develop further as drug candidates. A few approved drugs for other indications such as hypertension, anesthesia and antiarrhythmic have been fortuitously discovered to be hDNMT1 inhibitors. Additionally, competitive inhibitors of the cofactor SAM or cytosine have been reported. However, these inhibitors are likely to suffer from selectivity issue since the SAM binding and cytosine binding pocket are conserved across various proteins. Hence, there is an urgent requirement of a method for identifying inhibitors for an ideal site (location) on the hDNMT1 that would afford selectivity to the protein without causing side effects.

Objects of the invention

An object of the present invention is to provide a method for identification of selective inhibitors of a human DNA methyltransferase1 (hDNMT1).

Another object of the present invention is to provide a method for identifying the selective inhibitors, which acts on a newly identified druggable site of the human DNA methyltransferase1 (hDNMT1) .

Yet another object of the present invention is to provide a method for identifying the selective inhibitors, which act on a newly identified druggable site and causes less side effects compared to existing inhibitors.

Still another object of the present invention is to provide a method for identifying selective inhibitors of human DNA methyltransferase1 (hDNMT1), which is simple and economical in operation.

Summary of the invention:

According to present invention, there is a method for identifying selective inhibitors of a human DNA methyltransferase1 (hDNMT1) that can be further developed as drugs (medical). The method comprises steps of constructing a homology model of an active form of a human DNA methyltransferase1 (hDNMT1) in a homology model constructing tool. In the present embodiment, the homology model constructing tool is a computer software. The homology model is constructed using a protein sequence of hDNMT1 and a structure of mouse DNMT1 (DNA methyltransferase 1). A druggable binding site is identified in the constructed homology model. In the present embodiment, the druggable binding site is a new druggable site, which is not identified in the prior art. The new druggable site is made of residues Trp1498, Cys1499, Leu1500, His 1502, Thr1503, His 1507, Trp1510, Leu1513, Met 1533, Gly 1534, and Lys 1535. The new druggable site is identified based on its functional relevance and structural uniqueness.

Further, the identified druggable site is subjected to a virtual screening process. The components having a set of favourable characteristics are selected from the results of the virtual screening process. The selected components from the virtual screening are subjected to an in vitro and to an in vivo hDNMT1 inhibition assays. The selective inhibitors are identified from the results of the in vitro and the in vivo hDNMT1 inhibition assays.

Brief Description of drawings:

The advantages and features of the present invention will be better understood with reference to the following detailed description and claims taken in conjunction with the accompanying drawings, wherein like elements are identified with like symbols, and in which:

Figure 1 shows a flow chart of a method for identification of the selective inhibitors of a human DNA methyltransferase1 (hDNMT1) in accordance with the present invention;

Figure 2 shows an image of a homology model of an active form of hDNMT1 used in the method shown in figure 1;
he proteins are rendered in
the cartoon representation, the 5-mC binding site is rendered
as surface and colored by atom, and the zinc atom is rendered
as vDW and colored green while the SAM is rendered as lines
and colored by atom.
he proteins are rendered in
the cartoon representation, the 5-mC binding site is rendered
as surface and colored by atom, and the zinc atom is rendered
as vDW and colored green while the SAM is rendered as lines
and colored by atom.
Figure 3a shows a graph of results of an in vitro hDNMT1 inhibition assay carried out in the method of figure 1;

Figure 3b shows a graph of the results for a SAM competitive assay carried out in the method of figure 1;

Figure 4 shows a graph of results of an in vivo human DNMT1 inhibition assay carried out in the method of figure 1;

Figure 5 shows a graph of results from an in vivo MTT assay carried out on MRC-5 for assessment of cell viability after exposure to the selective inhibitor identified from the method of figure 1 and 5-azacytidine;

As
assessed by ELISA, at the concentration of 250 lM in
comparison to control cells (CC), compound A showed
signi?cant (p < 0.05 one tailed t test) reduction in total
genomic methylation
Figure 6 shows an image of an identified selective inhibitor identified from the method of figure 1 and the hDNMT1.

Detailed description of the invention

An embodiment of this invention, illustrating its features, will now be described in detail. The words "comprising," "having," "containing," and "including," and other forms thereof, are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items.

The terms “first,” “second,” and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another, and the terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item.

The present invention provides a method for identifying the selective inhibitors of a human DNA methyltransferase1 (hDNMT1), which binds to a newly identified druggable site. The selective inhibitors identified from the method causes less side effects compared to existing inhibitors. The method is simple and economical in operation.

Referring now to figure 1a, a flow chart of a method 100 for identifying the selective inhibitors of a human DNA methyltransferase1 (hDNMT1) in accordance with the present invention is illustrated.

The method starts at step 10.

At step 20, a homology model of an active form of a human DNA methyltransferase1 (hDNMT1) is constructed in a homology model constructing tool. In the present embodiment, the model constructing tool is a computer software. Usage of the computer software is obvious knowledge to a person skilled in the art for constructing the homology model of the human DNA methyltransferase1 (hDNMT1). In the present embodiment, the trade name of the computer software is “Modeller”. In an alternative embodiment, any other obvious computer software can be used for the constructing the homology model. The homology model of the active form of hDNMT1 is constructed using a protein sequence of hDNMT1 and a structure of mouse DNMT1. The protein sequence of hDNMT1 and the structure of mouse DNMT1 are retrieved from a public protein database.

At step 30, a druggable site is recognized (identified) in the constructed homology model. In the present embodiment, the druggable site is a new druggable site, which is not identified in prior art. The new druggable site is made of residues Trp1498, Cys1499, Leu1500, His 1502, Thr1503, His 1507, Trp1510, Leu1513, Met 1533, Gly 1534, and Lys 1535. The site is identified based on its functional relevance and structural uniqueness. The functional relevance is identified based on the prior mutation experiments. Mutations of Trp 1500, Leu 1502, Trp 1512, Leu 1515, and Met 1535 to Ala led to a reduction in DNMT1 activity varying from 50% to 100%. The uniqueness is identified based on structural comparison with other proteins in the public protein database using a structure comparison software.

At step 40, the identified new druggable site (from here in after referred as the druggable site) is subjected to a virtual screening process. In the present embodiment, the virtual screening process is carried out in a simulation software. Usage of the simulation software is obvious knowledge to a person skilled in the art for the virtual screening process. The components from the results of the virtual screening process are assigned a score by the procedure based on fitting of the inhibitor to the new druggable site. For example: Components with a score higher than a threshold are classified as a “good” and are retained while those with a bad score are discarded.

At step 50, the components having a set of favourable characteristics are selected from the results of the virtual screening process. In the present embodiment, the favourable characteristics are ability of the components (inhibitors) to fit to the druggable site with a score.

At step 60, the selected components are subjected to an in vitro hDNMT1 inhibition assay. The in vitro hDNMT1 inhibition assay is a set of tests, which are performed on the hDNMT1. The results from the in vitro DNMT inhibitors assay provides experimental details such as IC50 for the inhibitors, and identifies those which can act on the hDNMT1 effectively. Further, the ability of an inhibitor to inhibit hDNMT1 in the in vivo assay is assessed by checking the reduction in genomic methylation.

At step 70, the suitable inhibitors are identified from the results of the in vitro and in vivo assays. Further, the identified inhibitors can be used for the treatment of diseases such as cancer, genetic diseases and the like.

The method 100 ends at step 80.
A sample selective inhibitor of human DNA methyltransferase1 (hDNMT1) for further development as drug are identified using the method 100 by following steps 20, 30, 40, 50, 60 and 70.

Referring now to figure 2, an image of a sample homology model 110 (Refer figure 2) of an active form of a human DNA methyltransferase (hDNMT1) constructed using the method 100 in accordance with the present invention is illustrated. The protein is rendered in the cartoon representation. The identified druggable site is rendered as surface and coloured by atom, and the zinc atom is coloured green while the S-adenosyl methionine (SAM) is rendered as lines and coloured by atom.

A new druggable site is recognized in the sample constructed homology model 110 by using step 30. The druggable site is subjected to the virtual screening process. The components having a set of favourable characteristics are selected. The selected components from here in after referred as compound A. The selected components (compound A) are subjected to the in vitro and to the in vivo hDNMT1 inhibition assays.

Figure 3a and figure 3b show the graphs of the experimental validations for biological activity of the compound A from the in vitro and the in vivo hDNMT1 inhibition assays. All the assays were carried out in triplicates and values are represented as mean plus SEM (standard error of the mean). The IC50 of compound A is calculated to be 15 ±1.5 µM. On an average, the inhibition by compound A remains unchanged at increasing concentrations of SAM. These results effectively show that compound is not targeting the SAM binding site.

Referring now figure 4, a graph of results of an invivo human DNMT1 inhibition assay carried out in the method of figure 1 is illustrated. The assay was performed using MRC5 cells. CC stands for control cells. As assessed by ELISA, at the concentration of 250 µM in comparison to control cells (CC), compound A showed significant (p < 0.05 one tailed t test) reduction in total genomic methylation. From results of the in vitro hDNMT1 inhibition assay, a suitable inhibitor is identified. The identified selective suitable inhibitor from the method 100 are 5-[(3,4-dimethoxyphenyl)methyl]-N-[2-(4-methylbenzenesulfonyl) acetyl]-1,3,thiadiazol-2-aminide (The International Union of Pure and Applied Chemistry (IUPAC) names).

Figure 5 shows a graph of in vivo MTT assay carried out on MRC-5 for assessment of cell viability after exposure to Compound A and 5-azacytidine. 5-azacytidine demonstrates toxicity even at a low concentration while Compound A showed toxicity only at very high concentrations. 5-azacytidine demonstrates toxicity even at a low concentration while Compound A showed toxicity only at very high concentrations.

Figure 6 shows an image showing interaction of compound A with hDNMT1. The hDNMT1 protein is rendered in surface representation. The 5-mC binding site is coloured by atom while the remaining protein is coloured red. The selective inhibitors are rendered in the line representation and coloured by atom. The zinc atom is coloured green.

The method 100 has an advantage of identification of the selective inhibitors of the human DNA methyltransferase1 (hDNMT1) 110, which acts on the newly identified druggable site. The results from the experimental tests showed that the inhibitors identified from the method 100 causes less side effects compared to an existing drug 5-azacytidine. The method 100 does not use any costly equipment or materials for identifying the suitable inhibitors. Hence, the method 100 is simple and economical in operation.

The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, to thereby enable others skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omission and substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but such are intended to cover the application or implementation without departing from the spirit or scope of the claims of the present invention.
,CLAIMS:We Claim:

1. A method for identification of the selective inhibitors of human DNA methyltransferase1 (hDNMT1) for developing the drugs, the method comprises steps of:
Constructing a homology model of an active form of the human DNA methyltransferase1 in a homology model constructing tool;
recognising a druggable site in the constructed homology model;
subjecting the recognised druggable site to a virtual screening process;
selecting the components from the virtual screening process having a set of favourable characteristics;
subjecting the selected components to an in vitro hDNMT1 inhibition assay and to an in vivo hDNMT1 inhibition assay subsequently; and
identifying the selective inhibitors from the results of the in vitro and the in vivo hDNMT1 inhibition assays.

2. The method as claimed in claim 1, wherein the model constructing tool is a computer software.

3. The method as claimed in claim 1, wherein the homology model is constructed using a protein sequence of the human DNMT1 and a structure of mouse DNMT1.

4. The method as claimed in claim 1, wherein the new druggable site is made of residues Trp1498, Cys1499, Leu1500, His 1502, Thr1503, His 1507, Trp1510, Leu1513, Met 1533, Gly 1534, and Lys 1535.

5. The method as claimed in claim 1, wherein the site is identified based on its functional relevance and structural uniqueness.