Strategy for discovering chemical inhibitors of human cyclophilin a: focused library design, virtual screening, chemical synthesis and bioassay

The discovery of cyclophilin A (CypA) inhibitor is now of special interest in the treatment of immunological disorders. In this work, using a strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and bioassay, a series of novel small molecular CypA inhibitors have been discovered. First, using the fragments taken from our previously discovered CypA inhibitors (Bioorg. Med. Chem. 2006, 14, 2209-2224) as building blocks, we designed a focused combinatorial library containing 255 molecules employing the LD1.0 program (J. Comb. Chem. 2005, 7, 398-406) developed by us. Sixteen compounds (1a-e, 2a-b, 3a-b, and 4a-g) were selected by using virtual screening against the X-ray crystal structure of CypA as well as druglike analysis for further synthesis and bioassay. All these sixteen molecules are CypA binders with binding affinities (K(D) values) ranging from 0.076 to 41.0 microM, and five of them (4a, 4c, and 4e-g) are potent CypA inhibitors with PPIase inhibitory activities (IC(50) values) of 0.25-6.43 microM. The hit rates for binders and inhibitors are as high as 100% and 31.25%, respectively. Remarkably, both the binding affinity and inhibitory activity of the most potent compound increase approximately 10 times than that of the most active compound discovered previously. The high hit rate and the high potency of the new CypA inhibitors demonstrated the efficiency of the strategy for focused library design and screening. In addition, the novel chemical entities reported in this study could be leads for discovering new therapies against the CypA pathway.     

Synthesis of heteroannulated cyclopent[b]indoles: Exploration of in vitro cytotoxicity and molecular docking studies

A series of novel cyclopent[b]indole analogues that hold isoxazolo-, pyrido-templates were designed and synthesized in good yields. The in vitro cytotoxicity was concerned for all the newly synthesized compounds by MTT assay against HeLa (cervix adeno carcinoma) and MCF-7 (breast cancer). These synthesized compounds were further compared with the standard drug ellipticine, 5-fluorouracil, cisplatin, and methotrexate. The synthesized heteroannulated cyclopent[b]indole compounds were found to show better cytotoxic activity against HeLa and MCF-7 with primary structure activity relationship studies. To identify with the nature of interactions of these molecules, we performed molecular docking studies using the protein kinase CK2 inhibitors. The docking results afforded some valuable information for the future design of more potent inhibitors.     

Ligand-Based Pharmacophore Screening Strategy: a Pragmatic Approach for Targeting HER Proteins

Targeting ErbB family of receptors is an important therapeutic option, because of its essential role in the broad spectrum of human cancers, including non-small cell lung cancer (NSCLC). Therefore, in the present work, considerable effort has been made to develop an inhibitor against HER family proteins, by combining the use of pharmacophore modelling, docking scoring functions, and ADME property analysis. Initially, a five-point pharmacophore model was developed using known HER family inhibitors. The generated model was then used as a query to screen a total of 468,880 compounds of three databases namely ZINC, ASINEX, and DrugBank. Subsequently, docking analysis was carried out to obtain hit molecules that could inhibit the HER receptors. Further, analysis of GLIDE scores and ADME properties resulted in one hit namely BAS01025917 with higher glide scores, increased CNS involvement, and good pharmaceutically relevant properties than reference ligand, afatinib. Furthermore, the inhibitory activity of the lead compounds was validated by performing molecular dynamic simulations. Of note, BAS01025917 was found to possess scaffolds with a broad spectrum of antitumor activity. We believe that this novel hit molecule can be further exploited for the development of a pan-HER inhibitor with low toxicity and greater potential.     

A quantum mechanics/molecular mechanics study of the protein-ligand interaction of two potent inhibitors of human O-GlcNAcase: PUGNAc and NAG-thiazoline

O-glycoprotein 2-acetamino-2-deoxy-beta- d-glucopyranosidase ( O-GlcNAcase) hydrolyzes 2-acetamido-2-deoxy-beta- d-glucopyranose ( O-GlcNAc) residues of serine/threonine residues of modified proteins. O-GlcNAc is present in many intracellular proteins and appears to have a role in the etiology of several diseases including cancer, Alzheimer's disease, and type II diabetes. In this work, we have carried out molecular dynamics simulations using a hybrid quantum mechanics/molecular mechanics approach to determine the binding of two potent inhibitors, PUGNAc and NAG, with a bacterial O-GlcNAcase. The results of these simulations show that Asp-401, Asp-298, and Asp-297 residues play an important role in the protein-inhibitor interactions. These results might be useful to design compounds with more interesting inhibitory activity on the basis of its three-dimensional structure.     

Design,Synthesis, and Anticancer Activity Studies of Novel Quinoline-Chalcone Derivatives

The chalcone and quinoline scaffolds are frequently utilized to design novel anticancer agents. As the continuation of our work on effective anticancer agents, we assumed that linking chalcone fragment to the quinoline scaffold through the principle of molecular hybridization strategy could produce novel compounds with potential anticancer activity. Therefore, quinoline-chalcone derivatives were designed and synthesized, and we explored their antiproliferative activity against MGC-803, HCT-116, and MCF-7 cells. Among these compounds, compound 12e exhibited a most excellent inhibitory potency against MGC-803, HCT-116, and MCF-7 cells with IC₅₀ values of 1.38, 5.34, and 5.21 µM, respectively. The structure–activity relationship of quinoline-chalcone derivatives was preliminarily explored in this report. Further mechanism studies suggested that compound 12e inhibited MGC-803 cells in a dose-dependent manner and the cell colony formation activity of MGC-803 cells, arrested MGC-803 cells at the G2/M phase and significantly upregulated the levels of apoptosis-related proteins (Caspase3/9 and cleaved-PARP) in MGC-803 cells. In addition, compound 12e could significantly induce ROS generation, and was dependent on ROS production to exert inhibitory effects on gastric cancer cells. Taken together, all the results suggested that directly linking chalcone fragment to the quinoline scaffold could produce novel anticancer molecules, and compound 12e might be a valuable lead compound for the development of anticancer agents.     

Novel diphenylamine 2,4'-dicarboxamide based azoles as potential epidermal growth factor receptor inhibitors: synthesis and biological activity

Several hybrid molecules of diphenylamine-2,4'-dicarboxamide with various azolidinones and related heterocyclic rings have been synthesized and explored as epidermal growth factor receptor (EGFR) kinase inhibitors. Most of them displayed promising in vitro tyrosine kinase inhibition as well as potent cellular antiproliferative activity in the EGFR over-expressing breast cancer cell line (MCF-7). Compounds 12b and 13b that exhibited the highest inhibition in the kinase assay (89, 81% inhibition at 10 μM, respectively), showed potent antiproliferative effect against MCF-7 tumor cell line (IC(50) 1.04, 0.91 μM respectively). Molecular docking studies revealed that these compounds can bind to ATP binding site of the EGFR kinase domain and were involved in H-bonding with Met 793, in analogy to the known EGFR tyrosine kinase inhibitors. Moreover, compounds 15a-c possessed profound antitumor activity (IC(50) 0.59-0.73 μM) and significant EGFR-TK inhibition, making them of particular interest. In summary, the newly synthesized compounds provide promising new lead for the future design and development of anticancer agents of potential EGFR-TK inhibitory activity.     

Modeling heme proteins using atomistic simulations

Heme proteins are found in all living organisms, and perform a wide variety of tasks ranging from electron transport, to the oxidation of organic compounds, to the sensing and transport of small molecules. In this work we review the application of classical and quantum-mechanical atomistic simulation tools to the investigation of several relevant issues in heme proteins chemistry: (i) conformational analysis, ligand migration, and solvation effects studied using classical molecular dynamics simulations; (ii) electronic structure and spin state energetics of the active sites explored using quantum-mechanics (QM) methods; (iii) the interaction of heme proteins with small ligands studied through hybrid quantum mechanics-molecular mechanics (QM-MM) techniques; (iv) and finally chemical reactivity and catalysis tackled by a combination of quantum and classical tools.     

Discovering potent inhibitors against c-Met kinase: molecular design, organic synthesis and bioassay

The receptor tyrosine kinase c-Met is an attractive target for therapeutic treatment of cancers nowadays. The discovery of small molecule inhibitors is of special interest in the blockade of the c-Met kinase pathway. Here, we initiated our study from compound 1a, a novel inhibitor against c-Met kinase. A substructure similarity search against the SPECS database and chemical synthesis methods were performed to obtain a series of pyrazolidine-3,5-dione derivatives. Through the enzyme-based assay against c-Met kinase, 4 compounds (1c, 1e, 1m and 1o) showed potential inhibitory activity, with IC(50) values mostly less than 10 μM. Based on the structure-activity relationship (SAR) and binding mode analysis, a focused combinatorial library was designed by the LD1.0 program. Taking into account ADMET properties and synthesis accessibility, seven candidate compounds (5a-g) were successfully synthesized. The activity of the most potent compounds 5b (IC(50) = 0.46 μM) was 20 fold higher than that of the lead 1a. Taken together, our findings identified the pyrazolidine-3,5-dione derivatives as potent inhibitors against c-Met kinase and demonstrated the efficiency of the strategy in the development of small molecules against c-Met kinase.     

Development of predictive pharmacophore model for in silico screening,and 3D QSAR CoMFA and CoMSIA studies for lead optimization,for designing of potent tumor necrosis factor alpha converting enzyme inhibitors

A chemical feature-based pharmacophore model was developed for Tumor Necrosis Factor-α converting enzyme (TACE) inhibitors. A five point pharmacophore model having two hydrogen bond acceptors (A), one hydrogen bond donor (D) and two aromatic rings (R) with discrete geometries as pharmacophoric features was developed. The pharmacophore model so generated was then utilized for in silico screening of a database. The pharmacophore model so developed was validated by using four compounds having proven TACE inhibitory activity which were grafted into the database. These compounds mapped well onto the five listed pharmacophoric features. This validated pharmacophore model was also used for alignment of molecules in CoMFA and CoMSIA analysis. The contour maps of the CoMFA/CoMSIA models were utilized to provide structural insight for activity improvement of potential novel TACE inhibitors. The pharmacophore model so developed could be used for in silico screening of any commercial/in house database for identification of TACE inhibiting lead compounds, and the leads so identified could be optimized using the developed CoMSIA model. The present work highlights the tremendous potential of the two mutually complementary ligand-based drug designing techniques (i.e. pharmacophore mapping and 3D-QSAR analysis) using TACE inhibitors as prototype biologically active molecules.     

1,3-substituted imidazolidine-2,4,5-triones: synthesis and inhibition of cholinergic enzymes

A series of novel and highly active acetylcholinesterase and butyrylcholinesterase inhibitors derived from substituted benzothiazoles containing an imidazolidine-2,4,5-trione moiety were synthesized and characterized. The molecular structure of 1-(2,6-diisopropyl-phenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-imidazolidine-2,4,5-trione (3g) was determined by single-crystal X-ray diffraction. Both optical isomers are present as two independent molecules in the triclinic crystal system. The lipophilicity of the compounds was determined as the partition coefficient log K(ow) using the traditional shake-flask method. The in vitro inhibitory activity on acetylcholinesterase from electric eel and butyrylcholinesterase isolated from equine serum was determined. The inhibitory activity on acetylcholinesterase was significantly higher than that of the standard drug rivastigmine. The discussed compounds are also promising inhibitors of butyrylcholinesterase, as some of the prepared compounds inhibit butyrylcholinesterase better than the internal standards rivastigmine and galanthamine. The highest inhibitory activity (IC?? = 1.66 μmol/L) corresponds to the compound 1-(4-isopropylphenyl)-3-[(R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethyl]imidazolidine-2,4,5-trione (3d). For all the studied compounds, the relationships between the lipophilicity and the chemical structure as well as their structure-activity relationships are discussed.     

Design,Synthesis, and In Vitro Evaluation of Novel Indolyl DiHydropyrazole Derivatives as Potential Anticancer Agents

Indoles derived from both natural sources or artificial synthetic methods have been known to interact with aryl hydrocarbon receptors (AhR), and exhibit anticancer activity. In light of these attractive properties, a series of hybrid molecules with structural features of indoles, i.e., those bearing a pyrazoline nucleus, were evaluated for their enhanced anticancer activity. The designed molecules were subjected to molecular docking in order to screen for potential AhR interacting compounds, and the identified indolyl dihydropyrazole derivatives were synthesized. The synthesized compounds were characterized, and their cytotoxicity was evaluated against four human cancer cell lines using the MTT assay. Based on the Glide g-score, H-bonding interactions and bonding energy of 20 candidate molecules were selected for further analysis from the 64 initially designed molecules. These candidate molecules have shown promising anti-proliferative activity against the cell lines tested. Among these candidate molecules, the compounds with hydroxy phenyl substitution on the pyrazoline ring have shown potent activity across all the tested cell lines. The designed scaffold was proven effective for screening potential candidate molecules with anticancer properties, and may be further optimized structurally for yielding the ideal anti-tumorigenic compound for the treatment of various cancers.     

Molecular dynamics, density functional, ADMET predictions, virtual screening, and molecular interaction field studies for identification and evaluation of novel potential CDK2 inhibitors in cancer therapy

In this work, we have used molecular dynamics, density functional theory, virtual screening, ADMET predictions, and molecular interaction field studies to design and propose eight novel potential inhibitors of CDK2. The eight molecules proposed showed interesting structural characteristics that are required for inhibiting the CDK2 activity and show potential as drug candidates for the treatment of cancer. The parameters related to the Rule of Five were calculated, and only one of the molecules violated more than one parameter. One of the proposals and one of the drug-like compounds selected by virtual screening indicated to be promising candidates for CDK2-based cancer therapy.     

Discovery of novel IDO1 inhibitors via structure-based virtual screening and biological assays

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the first and rate-limiting step in catabolism of tryptophan via the kynurenine pathway, which plays a pivotal role in the proliferation and differentiation of T cells. IDO1 has been proven to be an attractive target for many diseases, such as breast cancer, lung cancer, colon cancer, prostate cancer, etc. In this study, docking-based virtual screening and bioassays were conducted to identify novel inhibitors of IDO1. The cellular assay demonstrated that 24 compounds exhibited potent inhibitory activity against IDO1 at micromolar level, including 8 compounds with IC₅₀ values below 10 μM and the most potent one (compound 1) with IC₅₀ of 1.18?±?0.04 μM. Further lead optimization based on similarity searching strategy led to the discovery of compound 28 as an excellent inhibitor with IC₅₀ of 0.27?±?0.02 μM. Then, the structure–activity relationship of compounds 1, 2, 8 and 14 analogues is discussed. The interaction modes of two compounds against IDO1 were further explored through a Python Based Metal Center Parameter Builder (MCPB.py) molecular dynamics simulation, binding free energy calculation and electrostatic potential analysis. The novel IDO1 inhibitors of compound 1 and its analogues could be considered as promising scaffold for further development of IDO1 inhibitors.

     

Lead validation and SAR development via chemical similarity searching; application to compounds targeting the pY+3 site of the SH2 domain of p56lck

Compound selection based on chemical similarity has been used to validate active "parent" compounds identified via database searching as viable lead compounds and to obtain initial structure-activity relationships for those leads. Twelve parent compounds that have inhibitory activity against the SH2 domain of the p56 T-cell tyrosine kinase (Lck) are the focus of this study. Lck is involved in the T-cell mediated immune response, and inhibitors of Lck protein-protein interactions could potentially be used to develop novel immunosuppressants. Similarity searches for each parent compound were performed using 2D structural fingerprints on a database containing 1,300,000 commercially available compounds. The inhibitory activity of the selected compounds was assessed using enzyme immunoassay (EIA). In general, the most active parent compounds yield the most high activity similar compounds; however, in two cases low activity parent compounds (i.e. inhibitory activity < 25% at 100 microM) yielded multiple similar compounds with activities > 60%. Such compounds may, therefore, be considered as viable lead compounds for optimization. Structure-activity relationships were explored by examining both ligand structures and their computed bound conformations to the protein. Functional groups common to the active compounds as well as key amino acid residues that form hydrogen bonds with the active compounds were identified. This information will act as the basis for the rational optimization of the lead compounds.     

Exploring sets of molecules from patents and relationships to other active compounds in chemical space networks

Patents from medicinal chemistry represent a rich source of novel compounds and activity data that appear only infrequently in the scientific literature. Moreover, patent information provides a primary focal point for drug discovery. Accordingly, text mining and image extraction approaches have become hot topics in patent analysis and repositories of patent data are being established. In this work, we have generated network representations using alternative similarity measures to systematically compare molecules from patents with other bioactive compounds, visualize similarity relationships, explore the chemical neighbourhood of patent molecules, and identify closely related compounds with different activities. The design of network representations that combine patent molecules and other bioactive compounds and view patent information in the context of current bioactive chemical space aids in the analysis of patents and further extends the use of molecular networks to explore structure–activity relationships.     

Bivalent inhibitors for disrupting protein surface-substrate interactions and for dual inhibition of protein prenyltransferases

Low-molecular-weight compounds that disrupt protein?protein interactions (PPIs) have tremendous potential applications as clinical agents and as chemical probes for investigating intracellular PPI networks. However, disrupting PPIs is extremely difficult due to the large, flat interfaces of many proteins, which often lack structurally defined cavities to which drug-like molecules could bind in a thermodynamically favorable manner. Here, we describe a series of bivalent compounds that anchor to the enzyme active site to deliver a minimally sized surface-binding module to the targeted surface involved in transient PPI with a substrate. These compounds are capable of significantly inhibiting enzymatic reactions involving protein surface?substrate interaction in the single-digit nanomole range. Inhibitors of farnesyltransferase (FTase), which possesses a negatively charged local area on its α-subunit, were designed by attaching a module derived from a branched monoamine-containing gallate to a conventional active-site-directed CVIM tetrapeptide using an alkyl spacer. A significant improvement in inhibitory activity (>200-fold) against farnesylation of the K-Ras4B peptide was observed when the gallate module was attached to the CVIM tetrapeptide. Furthermore, the bivalent compounds had submicromolar inhibitory activity against geranylgeranylation of the K-Ras4B peptide catalyzed by GGTase I, which has an α-subunit identical to that of FTase. The anchoring strategy we describe would be useful for designing a new class of PPI inhibitors as well as dual enzyme inhibitors targeting common surface structures.     

Multiple receptor-ligand based pharmacophore modeling and molecular docking to screen the selective inhibitors of matrix metalloproteinase-9 from natural products

Matrix metalloproteinase-9 (MMP-9) is an attractive target for cancer therapy. In this study, the pharmacophore model of MMP-9 inhibitors is built based on the experimental binding structures of multiple receptor-ligand complexes. It is found that the pharmacophore model consists of six chemical features, including two hydrogen bond acceptors, one hydrogen bond donor, one ring aromatic regions, and two hydrophobic (HY) features. Among them, the two HY features are especially important because they can enter the S1′ pocket of MMP-9 which determines the selectivity of MMP-9 inhibitors. The reliability of pharmacophore model is validated based on the two different decoy sets and relevant experimental data. The virtual screening, combining pharmacophore model with molecular docking, is performed to identify the selective MMP-9 inhibitors from a database of natural products. The four novel MMP-9 inhibitors of natural products, NP-000686, NP-001752, NP-014331, and NP-015905, are found; one of them, NP-000686, is used to perform the experiment of in vitro bioassay inhibiting MMP-9, and the IC₅₀ value was estimated to be only 13.4 µM, showing the strongly inhibitory activity of NP-000686 against MMP-9, which suggests that our screening results should be reliable. The binding modes of screened inhibitors with MMP-9 active sites were discussed. In addition, the ADMET properties and physicochemical properties of screened four compounds were assessed. The found MMP-9 inhibitors of natural products could serve as the lead compounds for designing the new MMP-9 inhibitors by carrying out structural modifications in the future.     

Molecular modeling studies to discover novel mIDH2 inhibitors with high selectivity for the primary and secondary mutants

Mutant isocitrate dehydrogenase 2 (mIDH2) is an emerging target for the treatment of cancer. AG-221 is the first mIDH2 inhibitor approved by the FDA for acute myeloid leukemia treatment, but its acquired resistance has recently been observed, necessitating the development of new inhibitor. In this study, a multi-step virtual screening protocol was employed for the analysis of a large database of compounds to identify potential mIDH2 inhibitors. To this end, we firstly utilized molecular dynamics (MD) simulations and binding free energy calculations to elucidate the key factors affecting ligand binding and drug resistance. Based on these findings, the receptor-ligand interaction-based pharmacophore (IBP) model and hierarchical docking-based virtual screening were sequentially carried out to assess 212,736 compounds from the Specs database. The resulting hits were finally ranked by PAINS filter and ADME prediction and the top compounds were obtained. Among them, six molecules were identified as mIDH2 putative inhibitors with high selectivity by interacting with the capping residue Asp312. Furthermore, subsequent docking and MD experiments demonstrated that compound V2 might have potential inhibitory activity against the AG-221-resistant mutants, thereby making it a promising lead for the development of novel mIDH2 inhibitors.