[Show abstract][Hide abstract] ABSTRACT: Combination of de novo methods of drug design, virtual focused combinatorial libraries, and in silico screening as a highly efficient tool for lead generation
Pavel A. Petukhov1, Eugene A. Volpe2, Yuzhi Yin2, Robert I. Glazer2, and Alan P. Kozikowski1. (1) Drug Discovery Program, Department of Neurology, Georgetown University Medical Center, 3970 Reservoir Road, NW, New Research Building, Rm EP15, Washington, DC 20057, (2) Departments of Oncology and Pharmacology, Georgetown University Medical Center, 3970 Reservoir Rd., NW, Washington, DC 20057
Efficient, fast drug discovery using in silico methods is one of the challenges in modern computational chemistry/molecular modeling. We have developed and successfully applied a novel methodology of in silico drug discovery based on a combination of in silico methods including de novo/rational design, virtual focused combinatorial libraries, lead-like and drug-like filtering, docking, and scoring. This methodology has proven to be effective and has led to the discovery of new peroxisome proliferators-activated receptor (PPAR) inhibitors with at least 5 % hit rate. The details of the approach and its application for the design of these and other inhibitors will be presented.
Structure Based Drug Design in Signal Transduction and Cell Cycle
1:30 PM-5:15 PM, Wednesday, September 10, 2003 Javits Convention Center -- 1E13, Oral
Division of Computers in Chemistry
The 226th ACS National Meeting, New York, NY, September 7-11, 2003.
[Show abstract][Hide abstract] ABSTRACT: Histone deacetylase 3 (HDAC3) is a promising epigenetic drug target for multiple therapeutic applications. Direct interaction between the Deacetylase Activating Domain of the silencing mediator for retinoid or thyroid-hormone receptors (SMRT-DAD) is required for activation of enzymatic activity of HDAC3. The structure of this complex and the nature of interactions with HDAC inhibitors in solution are unknown. Using novel photoreactive HDAC probes - "nanorulers", we determined the distance between the catalytic site of the full-length HDAC3 and SMRT-DAD in solution at physiologically relevant conditions and found it to be substantially different from that predicted by the X-ray model with a Δ379-428aa truncated HDAC3. Further experiments indicated that in solution this distance might change in response to chemical stimuli, while the enzymatic activity remained unaffected. These observations were further validated by Saturation Transfer Difference (STD) NMR experiments. We propose that the observed changes in the distance are an important part of the histone code that remains to be explored. Mapping direct interactions and distances between macromolecules with such "nanorulers" as a function of cellular events facilitates better understanding of basic biology and ways for its manipulation in cell and tissue specific manner.
ACS Chemical Biology 09/2013; · 5.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Breast cancer remains a significant cause of death in women, and few therapeutic options exist for estrogen receptor negative (ER (-)) cancers. Epigenetic reactivation of target genes using histone deacetylase (HDAC) inhibitors has been proposed in ER (-) cancers to resensitize to therapy using selective estrogen receptor modulators (SERMs) that are effective in ER (+) cancer treatment. Based upon preliminary studies in ER (+) and ER (-) breast cancer cells treated with combinations of HDAC inhibitors and SERMs, hybrid drugs, termed SERMostats, were designed with computational guidance. Assay for inhibition of four type I HDAC isoforms and antagonism of estrogenic activity in two cell lines yielded a SERMostat with 1-3 μM potency across all targets. The superior hybrid caused significant cell death in ER (-) human breast cancer cells and elicited cell death at the same concentration as the parent SERM in combination treatment and at an earlier time point.
[Show abstract][Hide abstract] ABSTRACT: Hyperactivation of the calcium-dependent cysteine protease, calpain-1 (Cal1), is implicated as a primary or secondary pathological event in a wide range of illnesses, and in neurodegenerative states, including Alzheimer's disease (AD). E-64 is an epoxide-containing natural product identified as a potent non-selective, calpain inhibitor, with demonstrated efficacy in animal models of AD. Using E-64 as a lead, three successive generations of calpain inhibitors were developed using computationally assisted design to increase selectivity for Cal1. First generation analogs were potent inhibitors, effecting covalent modification of recombinant Cal1 catalytic domain (Cal1cat), demonstrated using LC-MS/MS. Refinement yielded 2nd generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1 inhibitors, one of which was designed as an activity-based probe. These were determined to be irreversible and selective inhibitors by kinetic studies comparing full length Cal1 with the general cysteine protease, papain.
Journal of Medicinal Chemistry 07/2013; · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel series of HDAC8 inhibitors without a zinc-chelating hydroxamic acid moiety is reported. Photoaffinity labeling and molecular modeling studies suggest that these ligands are likely to bind in an 'upside-down' fashion in a secondary binding site proximal to the main catalytic site. The most potent ligand in the series exhibits an IC(50) of 28μM for HDAC8 and is found to inhibit the deacetylation of H4 but not α-tubulin in SH-SY5Y cell line.
[Show abstract][Hide abstract] ABSTRACT: With 2-3% of the worldwide population chronically infected, hepatitis C virus (HCV) infection continues to be a major health care burden. Unfortunately, current interferon-based treatment options are not effective in all patients and are associated with significant side effects. Consequently, there is an ongoing need to identify and develop new anti-HCV therapies. Towards this goal, we previously developed a cell-based HCV infection assay for antiviral compound screening based on a low multiplicity of infection approach that uniquely allows for the identification of antivirals compounds that target cell culture-derived HCV (HCVcc) at any step of the viral infection cycle. Using this assay, here we report the screening of the NCI Diversity Set II library, containing 1974 synthesized chemical compounds, and the identification of compounds with specific anti-HCV activity. In combination with toxicity counter screening, we identified 30 hits from the compound library, 13 of which showed reproducible and dose-dependent inhibition of HCV with mean therapeutic indices (CC(50)/EC(50)) of greater than 6. Using HCV pseudotype and replicon systems of multiple HCV genotypes, as well as infectious HCVcc-based assembly and secretion analysis, we determined that different compounds within this group of candidate inhibitors target different steps of viral infection. The compounds identified will not only serve as biological probes to study and further dissect the biology of viral infection but should also facilitate the development of new anti-HCV therapeutic treatments.
Antimicrobial Agents and Chemotherapy 09/2012; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A one-pot tandem direct reductive amination of aldehydes with primary amines resulting in N-Boc secondary amines using a (Boc)(2)O/sodium triacetoxyborohydride (STAB) system is reported. The tandem procedure is efficient, selective, and versatile, giving excellent yields of N-Boc protected secondary amines even in those cases where the products are prone to intramolecular lactamization.
[Show abstract][Hide abstract] ABSTRACT: The design, modeling, synthesis, biological evaluation of a novel series of photoreactive benzamide probes for class I HDAC isoforms is reported. The probes are potent and selective for HDAC1 and 2 and are efficient in crosslinking to HDAC2 as demonstrated by photolabeling experiments. The probes exhibit a time-dependent inhibition of class I HDACs. The inhibitory activities of the probes were influenced by the positioning of the aryl and alkyl azido groups necessary for photocrosslinking and attachment of the biotin tag. The probes inhibited the deacetylation of H4 in MDA-MB-231 cell line, indicating that they are cell permeable and target the nuclear HDACs.
[Show abstract][Hide abstract] ABSTRACT: A systematic investigation of the available crystal structures of HDAC8 and of the influence of different receptor structures and docking protocols is presented. The study shows that the open conformation of HDAC8 may be preferred by ligands with flexible surface binding groups, as such a conformation allows the ligands to minimize their exposure to solvent upon binding. This observation allowed us to rationalize the excellent potency of pyrazole-based inhibitors compared to that of isoxazole-based inhibitors.
Journal of Molecular Modeling 03/2012; 18(8):3927-39. · 1.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pyoderma gangrenosum (PG) is a rare, noninfectious form of skin ulceration, typically accompanied by neutrophilic infiltration. Several familial cases have been reported, suggesting the involvement of genetic factors in the aetiology of PG. Two mutations (A230T and E250Q) in the PSTPIP1 gene, encoding proline-serine-threonine phosphatase-interacting protein (PSTPIP)1 have been identified in patients with PAPA (pyogenic sterile arthritis with PG and acne) syndrome, a rare autoinflammatory disorder with autosomal dominant inheritance.
The aim of this study was to sequence PSTPIP1 complementary cDNA and genomic DNA for mutations, and to identify genetic polymorphisms in the promoter region of PSTPIP1 in patients with PG.
The genomic region and cDNA of the PSTPIP1 gene were sequenced from peripheral blood leucocytes of 14 patients with PG and 20 healthy controls.
One patient (PG1) had aberrant splicing variants of the PSTPIP1 transcript with deletions of exons 9, 11 and 12 and of exons 9-12 together, and all other patients with PG carried deletions of exon 11 and of 11-12. We also identified a novel mutation (G258A) in patient PG3, and novel polymorphisms [(CCTG)(6) and (CCTG)(8) tandem repeats] in the promoter region of the PSTPIP1 gene.
All combinations of aberrant splicing variants had frame shifts and premature stop codons leading to truncated proteins and loss of function of PSTPIP1. The (CCTG)(n) tandem repeats in the promoter region of PSTPIP1 had no association with PG. The mutations G258A and R52Q are predicted by the improved prediction algorithm to have a possibly damaging effect on PSTPIP1 function.
Clinical and Experimental Dermatology 07/2011; 36(8):889-95. · 1.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The design, synthesis, docking, and biological evaluation of novel potent HDAC3 and HDAC8 isoxazole- and pyrazole-based diazide probes suitable for binding ensemble profiling with photoaffinity labeling (BEProFL) experiments in cells is described. Both the isoxazole- and pyrazole-based probes exhibit low nanomolar inhibitory activity against HDAC3 and HDAC8, respectively. The pyrazole-based probe 3f appears to be one of the most active HDAC8 inhibitors reported in the literature with an IC(50) of 17 nM. Our docking studies suggest that unlike the isoxazole-based ligands the pyrazole-based ligands are flexible enough to occupy the second binding site of HDAC8. Probes/inhibitors 2b, 3a, 3c, and 3f exerted the antiproliferative and neuroprotective activities at micromolar concentrations through inhibition of nuclear HDACs, indicating that they are cell permeable and the presence of an azide or a diazide group does not interfere with the neuroprotection properties, or enhance cellular cytotoxicity, or affect cell permeability.
Journal of Medicinal Chemistry 06/2011; 54(13):4350-64. · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Angiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE.
HEK and CHO cells expressing mutant (Tyr465Asp) ACE demonstrate a 3- and 8-fold increase, respectively, in the rate of ACE shedding compared to wild-type ACE. Conformational fingerprinting of mutant ACE demonstrated dramatic changes in ACE conformation in several different epitopes of ACE. Cell ELISA carried out on CHO-ACE cells also demonstrated significant changes in local ACE conformation, particularly proximal to the stalk region. However, the cleavage site of the mutant ACE--between Arg1203 and Ser1204--was the same as that of WT ACE. The Y465D substitution is localized in the interface of the N-domain dimer (from the crystal structure) and abolishes a hydrogen bond between Tyr465 in one monomer and Asp462 in another.
The Y465D substitution results in dramatic increase in the rate of ACE shedding and is associated with significant local conformational changes in ACE. These changes could result in increased ACE dimerization and accessibility of the stalk region or the entire sACE, thus increasing the rate of cleavage by the putative ACE secretase (sheddase).
PLoS ONE 01/2011; 6(10):e25952. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background. Pyoderma gangrenosum (PG) is a rare, noninfectious form of skin ulceration, typically accompanied by neutrophilic infiltration. Several familial cases have been reported, suggesting the involvement of genetic factors in the aetiology of PG. Two mutations (A230T and E250Q) in the PSTPIP1 gene, encoding proline–serine–threonine phosphatase‐interacting protein (PSTPIP)1 have been identified in patients with PAPA (pyogenic sterile arthritis with PG and acne) syndrome, a rare autoinflammatory disorder with autosomal dominant inheritance.Aim. The aim of this study was to sequence PSTPIP1 complementary cDNA and genomic DNA for mutations, and to identify genetic polymorphisms in the promoter region of PSTPIP1 in patients with PG.Methods. The genomic region and cDNA of the PSTPIP1 gene were sequenced from peripheral blood leucocytes of 14 patients with PG and 20 healthy controls.Results. One patient (PG1) had aberrant splicing variants of the PSTPIP1 transcript with deletions of exons 9, 11 and 12 and of exons 9–12 together, and all other patients with PG carried deletions of exon 11 and of 11–12. We also identified a novel mutation (G258A) in patient PG3, and novel polymorphisms [(CCTG)6 and (CCTG)8 tandem repeats] in the promoter region of the PSTPIP1 gene.Conclusion. All combinations of aberrant splicing variants had frame shifts and premature stop codons leading to truncated proteins and loss of function of PSTPIP1. The (CCTG)n tandem repeats in the promoter region of PSTPIP1 had no association with PG. The mutations G258A and R52Q are predicted by the improved prediction algorithm to have a possibly damaging effect on PSTPIP1 function.
Clinical and Experimental Dermatology 01/2011; 36(8). · 1.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
[Show abstract][Hide abstract] ABSTRACT: A binding ensemble profiling with (f)photoaffinity labeling (BEProFL) approach that utilizes photolabeling of HDAC8 with a probe containing a UV-activated aromatic azide, mapping of the covalent modifications by liquid chromatography-tandem mass spectrometry, and a computational method to characterize the multiple binding poses of the probe is described. By use of the BEProFL approach, two distinct binding poses of the HDAC8 probe were identified. The data also suggest that an "upside-down" pose with the surface binding group of the probe bound in an alternative pocket near the catalytic site may contribute to the binding.
Journal of Medicinal Chemistry 11/2009; 52(22):7003-13. · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development of isoform-selective histone deacetylase (HDAC) inhibitors would be a significant step in reducing side effects and fine-tuning inhibitors for different diseases, including cancer. The problem of identifying selective inhibitors is far from being solved primarily because our current understanding of the binding modes available to the highly solvent exposed surface binding group of HDAC inhibitors is limited, and methods capable of capturing this type of information are lacking. Here, we present an approach that utilizes a selective photolabeling of HDAC8 isoform, the characterization of the binding poses adopted by a photolabeling probe by proteomics, and a computational method to identify the ensemble of the binding modes available to the photolabeling probe in the binding site of HDAC8. A photolabeling probe 1 was designed so that it would covalently attach itself to the HDAC binding site upon activation of the aromatic azide group by UV light. The probe also contains a second, less reactive, aliphatic azide group that could be linked to a biotin or a fluorescent tag. HDAC8 was subjected to photolabeling with probe 1, characterized by Western blot, affinity-purified, trypsinized, and then the tryptic peptides were mapped and sequenced using liquid chromatography-tandem mass-spectrometry (LCT-MS) to identify the sites of covalent modification. Further analysis of the LCT-MS and molecular dynamics data resulted in identification of the two major distinct poses (41% and 22%) of the probe 1 in the binding site of HDAC8. ACKNOWLEDGMENT. This study was in part funded by the Breast Cancer Congressionally Directed Research Program of Department of Defense Idea Award BC051554 and by the National Cancer Institute/NIH grant 1R01 CA131970-01A1 (PAP). We also thank Chicago Biomedical Consortium and Searle family for the support of FT ICR MS (RBvB) and OpenEye Scientific Software for providing academic license for modeling software used in this study (PAP).
38th American Chemical Society Great Lakes Regional Meeting; 05/2009
[Show abstract][Hide abstract] ABSTRACT: The accuracy of molecular dynamics (MD) simulations is limited by the availability of parameters for the molecular system of interest. In most force fields, parameters of common chemical groups are already present. With the development of novel small organic molecules as probes to study biological systems, more chemical groups require parameterization. An azide group is often used in studies of biological systems but computational studies are still impeded by the lack of parameters. In this paper, we present a set of molecular mechanics (MM) parameters for aromatic and aliphatic azido groups, and their application in MD simulations of a photoaffinity probe currently used in our laboratory for mapping binding modes available in the active site of histone deacetylases. The parameters were developed for the generalized Amber force field (GAFF) using density functional theory (DFT) calculations at B3LYP 6-311G(d) level. The parameters were validated by geometry optimization and MD simulations.
Journal of Molecular Modeling 04/2009; 15(11):1291-7. · 1.98 Impact Factor