Int J Pharm Pharm Sci, Vol 6, Issue 11, 208-212Original Article

DESIGNING OF ANTI-CANCEROUS HISTONE DEACETYLASE INHIBITORS THROUGH MIMICKING OF PROTEIN-PROTEIN INTERFACES

K. RAJAGANAPATHY1, R. SATHIYASUNDAR1, G. RAMESH KUMAR2, V. K. KALAICHELVAN1*

1Department of Pharmacy, Annamalai University, Annamalai Nagar 608002, 2Department of Bioinformatics, AU-KBC Research Center, MIT, Anna University, Chromepet, Chennai 600044.
Email: vkkalaichelvan@yahoo.co.in

Received: 21 Aug 2014 Revised and Accepted: 20 Sep 2014

ABSTRACT

Objective: The objective of the study was to come up with of the small molecular modulators that inhibit protein – protein interfaces or interaction site in HDAC complexes. The main focus is on the mimicking or forming of tiny molecule wherever by inhibiting the protein-protein interactions in specifically HDAC protein complexes. 

Methods: By mimicking of the interface of the protein interaction site like SIN3A-SMRT complex as well as SIN3A-NcoR complexes.

Results: Totally 10 molecular structures were designed through molecular docking with HDAC2 PDB Id 3MAX and were downloaded from protein data bank.

Conclusion: The results clearly indicate that before synthesis and biochemical testing of new lead and its analogs; one can use molecular modeling based methods for qualitative assessment.

Keywords: HDAC, SIN3-NcoR, Docking and protein-protein interfaces.

INTRODUCTION

Nucleosomes are the elemental units of chromatin and area unit comprised of a histone protein octamer around that 146 base pairs of deoxyribonucleic acid are wound. The octamer contains two copies of the four core histones: H2A, H2B, H3 and H4. Linker histones clamp entry and exit sites of deoxyribonucleic acid round the core histone octamer and, with different nuclear proteins, contribute to the formation of a condensed higher order chromatin structure (1). Core and linker histones are the foremost regulators of chromatin condensation, so their modifications regulate the structural stability of the Chromatin fiber. The orderly packing of eukaryotic deoxyribonucleic acid depends on enzymatic modification of histones by the covalent addition of methyl, acetyl group or phosphate teams to extend or decrease transcription. These epigenetic changes (mitotically hereditary alterations in gene expression that don't seem to be caused by changes within the deoxyribonucleic acid sequence) observed because the “histone code” influence chromatin structure and so regulate the accessibility of transcription factors to deoxyribonucleic acid. Histone acetylation results in chromatin de-condensation and increased transcription of the gene. This is accomplished by 2 enzymes known as HAT and HDAC enzymes alter the four core Histone proteins via acetylation and deacetylation at specific lysine (K) residues in the N-terminal tail of the histones in the nucleus [2,3]. HAT and HDACs are present together at transcriptionally active genes. While the HDACs are involved in the transcriptional repression and removal of acetyl groups from histones to cause condensation of chromatin, the HATs are involved in acetylation and de-condensation of the chromatin resulting in increased gene transcription. Thus, HDACs possess negative regulatory mechanisms to that of the HATs. Protein-protein interactions are exclusively changed or deregulated in cancer cells.

There are four HDAC classes with several different HDACs in each class. Class I HDACs are homologous to yeast Rpd3 and include HDACs 1, 2, 3 and 8. Class I HDACs are expressed ubiquitously in human cell lines and tissues and are found primarily in the nucleus. Class II HDACs are homologous to yeast Hda1 and can be subdivided into two subclasses: ClassIIa (HDAC 4, 7, and9) and ClassIIb (HDAC 6 and 10). Class II HDACs exhibit tissue-specific expressions and can shuttle between the nucleus and cytoplasm. Class I and II HDACs share homology in their catalytic sites. Class HDACs or sirtuins (SIRT1-7) include a group of proteins that are homologous with the yeast Sir2 family of proteins. Class IV HDACs contain HDAC11 and are homologous with both classes I and II. The role of HDACs is not restricted to their contribution to histone deacetylation; they also play a role in the de acetylation of non-histone proteins. HDACs are key elements in the regulation of gene expression, differentiation and development, and the maintenance of cellular homeostasis [4].

HDAC2 is gift within the nucleus of human cells. it's concerned in transcriptional repression regulated by the retinoblastoma protein Rb and is recruited to deoxyribonucleic acid through interaction with transcription factors and nuclear receptors. HDAC2 is additionally found in giant multi-protein complexes known as co-repressor complexes that are recruited to promoters of genes by specific transcription factors to silence gene activity. HDAC2 is one among the histone-modifying enzymes that regulates gene expression by transforming chromatin structure and is gift within the Sin3A and nucleosome remodeling and de acetylase (NuRD) co-repressor complexes (5, 6).

A very important feature of HDAC is that it needs many elements of different multi-protein complexes to perform its cellular activities. This includes transcriptional co-repressors like mSin3A, N-CoR/ SMRT–SIN3–HDACadvanced, PML and others in AML (7, 8). The SIN3–HDAC could be a ubiquitous, abundant and enormous macromolecule advanced with extremely preserved useful domains.

The advanced contains HDAC-1, HDAC-2 and also the system macromolecule SIN3, and is concerned to influence many key restrictive signals. The N-CoR / area unit bridging proteins discharged by RAR together with SIN3-HDAC advanced. The N-CoR/SMRTs act as a linker for the interaction of RAR-RXR with SIN3-HDAC advanced is a. The RAR- a part of the nuclear hormone receptor family that functions as a transcription issue by binding to deoxyribonucleic acid and regulation transcription of its target genes. Where as the carboxyl terminal of the N-CoR /SMRT is to blame for non-redundant external interactions, the amino terminal mediates active transcriptional repression by mediating repression pathways in Acute Promyelocytic leukemia (APL) and Acute Myelogenous leukemia (AML). Upon binding of ligand RA to its receptor, the N-CoR–SIN3–HDAC co-repressor advanced is discharged and changed for the binding of a co-activator advanced like TIF2–CBP HAT and factor transcription is activated and is covert within the absence of ligand [9].

Objective

The objective of the study has been coming up with of the small molecular modulators that inhibit protein – protein interfaces or interaction site in HDAC complexes. One protein combines with another protein and forms protein-protein interactions and produce protein complexes. Our aim is to produce (mimic) a small molecule about protein interfaces and reacting the molecule with macromolecule (protein) complexes wherever by inhibiting the protein-protein interactions. The main focus is on the mimicking or forming of tiny molecule wherever by inhibiting the protein-protein interactions in specifically HDAC protein complexes. 

Experimentation

The discovery of small molecules that regulate protein–protein binding interactions in designing a new molecule and its practical importance. Accordingly, the prevailing approaches have been structure-based design and combinatorial methods (selection or screening of libraries). Often, design is aimed at mimicking peptide or protein structural elements in a smaller form. A successful design may then become the scaffold for a combinatorial library. Combinatorial method, on the other hand, allows quick evaluation of many possible ligands and frequently yields unexpected solutions. These may be structurally characterized and used as templates for further design. In addition, previous reports have appeared describing particular protein–protein interactions that can be inhibited by small molecules or by very short peptides.

Prediction of protein complexes

Patch Dock is an algorithm for molecular protein-protein docking. The input is two molecules of any type: proteins, , peptides, drugs. The output is a list of potential complexes sorted by shape complementarily criteria. In order to mimic the SIN3A-SMRT complex as well as SIN3A-NcoR complexes, the 3D crystal structures of proteins: NCoR (PDB ID: 1xc5), SIN3A (PDB ID: 1G1E) and SMRT (PDB ID: 2ODD) were retrieved from the Protein Data Bank (PDB). First, the SIN3A-NcoR complex was mimicked by docking SIN3A and NcoR using PatchDock (10-12). Similarly, the SIN3A-SMRT complex was mimicked by docking with the SIN3A and SMRT to obtain a mimicked two protein complex structure SIN3A-NcoR and SIN3A-SMRT complexes).

Table 1: Proteins interacting with Sin3 have a wide range of functions. For those characterized, most commonly interact with anα-helix in PAH pocket.

Factor Functions SIN3 Interacting domain SIN3 Interfaces
N-CoR Co-repressor for nuclear hormone receptors DALA alpha helix

Motif

 PAH1; PAH3-HID,

30
SMRT Co-repressor for nuclear hormone receptors Extensive (SIN3A only)

The above table has shown the Sin3 complex interfaces such as PAH1, mSIN3A, mSIN3B and PAH2 which are scaffolding proteins and are interacting with seven factors. Among the seven factors which are mention in that table no-1, only two factors have been selected for protein-protein docking or prediction of protein complexes. And this protein-protein interactions to carry out with patchdock.

Fig. 1: Docked Structure of SIN3A PAH2 domain complexed with SMRT viewed by Argus lab


Fig. 2: Docked structure of mSin3A PAH1 domain complexed with N-CoR viewed by arguslab

Prediction of protein-protein interfaces: mimics and ligand design

Protein-Protein interfaces or interaction sites were predicted the small molecules were designed which has similar structure as protein-protein interfaces by using the online tool metappisp (A Meta server for protein-protein interaction site prediction). The obtained 3D complexes SIN3A- SMRT and SIN3A-NcoR was introduced to the online metta-PPISP server for the prediction of protein-protein interface region.

The interface region were interpreted and analyzed by Swiss PDB Viewer. The possible small molecule structures that mimic the interface region were identified and obtained structures were drawn by using Marvin Sketch. Marvin Sketch has advanced chemical editor for drawing chemical structures and it has a rich list of editing features.

Table 2: Selected protein-protein interface region

S. No Interface ID Interface Region
1. Ligand -1 PHE, GLU.
2. Ligand -2 TYR, GLN.
3. Ligand -3 PRO, ASP,
4. Ligand -4 TYR, TYR, LEU.
5. Ligand -5 LYS, LYS.

Chemical substituent analog of the novel lead molecules

The five newly designed lead molecules of the drugs were created by replacing the hydrophilic region on the target molecule with other functional groups (considered at random). Five analogues of the small molecule of the newly designed lead molecules has been studied by performing various molecular properties calculation, ADME as well as protein-ligand interactions.

Fig. 3: Novel Lead Molecules of Mimicked Protein-protein Interfaces


Table 3: Chemical substituted analog

S. No. Small molecule id Possible position Chemical subsittuted analog
1. Ligand-1 CHO

NH2

 NH2OH (R1)

CH3COCH2Br (R2)
2. Ligand-2 NH2

NH2

 CH3-CO-CH2-Br(R1)

NH-OH(R2)
3. Ligand-3 NH

COOH

NH

 CH3COCH2Cl(R1)

OCH3 (R2)

N-OH(R3)
5. Ligand-4 COOH NH-OH-CO-CH2-Cl (R1)
6. Ligand-5 NH2

NH2

 (R1)

NH-OH (R2)

RESULT AND DISCUSSION

Molecular docking study

A molecular docking study by Schrodinger-Maestro 9.3.5 Version was performed to calculate of binding energy and H-bond interaction, H-bond donor & acceptors, hydrobhobicity and lipobobicity at the active site for all the newly designed small molecules and its chemical substituted analogues, totally 10 molecular structures were separately docked with HDAC2 PDB Id 3MAX were downloaded from protein data bank.

Fig. 4: Chemical substituted Analogues Of the Novel Lead Molecules


Table 3: Docking Result

S. No. Ligand(small molecule) Docking Score Ebind (MM)
1. Ligand - 2 -10.9
2. Ligand - 5 -10.6
3. Ligand - 4 -10.0
4. Ligand – 4 a -9.8
5. Ligand - 5a -9.6
6. Ligand -1 -9.5
7. Ligand - 3 -8.3

Fig. A Fig. B
Fig. C Fig. D
Fig. E Fig. F

Fig. 5: Fig-a, b, c, d, e, f Showing Most active Protein –Ligand interactions of Ligand No-1,2,4,4a,5,5a.

ADME analysis

Present study identify through docking and binding interactions of various novel lead molecules (As similar to Protein-Protein Interfaces) and its analogues for anti-cancerous histone deacetylase inhibitors. After analyzing of the molecular docking and ADME (By using qikprop command in Maestro) the ligand no 1, 2, 4, 4a, 5, 5a showing most active binding energy of the targeted protein and its obeying good Lipinski rule of five and ADME test and results are shown. and its showing more than seven hydrogen bond interactions and its including π - π stacking, hydrogen bond (side chain and backbone) and metal ion (zing binding motif) interactions.

Table 9: ADME analysis

S. No Ligand Mol. wt VD (L) FA(%) Coca2-permeability(cm/s) Log p Rotable bond
1. Ligand-1 278.307 955.142 128.831 7E-05 1.2 11
2. Ligand-2 349.386 1158.789 220.261 2E-06 -1 14
3. Ligand-3 293.322 974.936 142.448 9E-08 -2.7 12
4. Ligand-4 365.385 1179.198 231.229 2E-05 0.8 18
5. Ligand-5 301.299 953.796 288.915 3E-05 2.6 18
6. Ligand-1a 387.389 1192.889 424.07 4E-06 -1.3 18
7. Ligand-2a 457.525 1460.759 240.084 2E-05 0.5 10
8. Ligand-3a 530.577 1631.824 274.511 9E-08 -2.6 14
9. Ligand-4a 274.362 988.378 284.704 6E-08 -3.3 18
10 Ligand-5a 374.364 1245.106 404.902 4E-07 -1.7 20

Predicted properties

Table 10: The result of analysis ADME continue

Small molecule

ID

 S. A. A

(A2)

 S. A. G

(A2)

 Vol

(A3)

 Hyd. ener

Kcal/mol

 Ref

(A3)

 Polari

(A3)

 Drs Ars
Ligand-1 526.2 632.1 1198.34 -5.67 92.43 52.23 3 5
Ligand-2 546.41 620.03 1090.59 -5.53 88.65 33.75 4 8
Ligand-3 485.3 578.8 976.6 -16.41 82.76 33.31 5 8
Ligand-4 575.8 698.7 1305.7 -17.48 136.1 51.1 8 10
Ligand-5 630.9 652.8 1057.4 -18.7 82.2 32.8 6 9
Ligand-1a 654.8 673.8 1176.4 -5.57 108.5 41.7 3 6
Ligand-2a 602.8 671.9 1126.3 -22.45 93.7 37.6 5 9
Ligand-3a 552.5 623.8 1066.4 -34.6 88.6 36.3 7 11
Ligand-4a 692.5 771.7 1377.3 -21.15 123.5 49.2 4 10
Ligand-5a 618.08 624.2 1028.6 -11.49 83.5 31.03 5 9

Predicted properties

CONCLUSION

These results clearly indicate that before synthesis and biochemical testing of new lead and its analogs; one can use molecular modeling based methods for qualitative assessment of relative binding affinities for speeding up drug discovery process by eliminating less potent compounds from synthesis. The ligand No 1,2,4,4a,5,5a is a new lead and analogue for the anti-cancerous histone deacetylase inhibitor for disruption of HDAC2.

ACKNOWLEDGEMENT

I am grateful to sincere thanks to Dr. S. Kabilan., Professor From department of chemistry Annamalai University. To gave my opportunity to using the Schrodinger Software.

CONFLICT OF INTEREST

None to declare

REFERENCES

  1. Curt B, Fang F, Daniela EM, Tim HM, Huang, Kenneth P. Nephew. Minireview: Epigenetic Changes in Ovarian Cancer. endo. endojournals. Org 2009;150(9):4003-011.
  2. Dong HK, Minjung K, Ho Jeong K. Histone Deacetylases in carcinogenesis and its inhibitors as anti cancer agents. J Biochem Mol Biol 2003;36(1):110-9.
  3. Santiago R, Manel E. The role of histone Deacetylases (HDACs) in human cancer. Mol Oncol 2007;1:19-25.
  4. Xiang-Jiao, Y Serge G. Class II Histone Deacetylases: from sequence to function, regulation and clinical implication. Mol Cell Biol 2005;25:2873-84.
  5. Donald EA. Histone Deacetylases: transcriptional repression with SINers and NuRDs. Trends Cell Biol 1999;9:193-8.
  6. Bradley RC. Emerging roles for chromatin remodeling in cancer biology. Trends Cell Biol 2001;11:515-21.
  7. Andrea GC. Protein-protein interfaces: mimics and inhibitors. Curr Opin Chem Biol 2001;5:654-9.
  8. Nurcan T, Gozde K, Ozlem K, Attila, G, Ruth N. A survey of available tools and web server for analysis of protein-protein interactions and interfaces. Briefings in Bioinform 2009;10:1-16.
  9. Manivannan P, Ganesan S, Naveenkumar S, Daniel SD, Sathiamoorthi T, Muralitharan G. Molecular modelling of ure-ase accessory interaction proteins of Helicobacter Pylori J 99 and predicting an interruption in interaction by Vigna radiata Defensins. Bioinform 2011;5(10):410-5.