Santosh A. Khedkar

Pharmacophore Modeling in Drug Discovery and Development: An Overview

Pharmacophore mapping is one of the major elements of drug design in the absence of structural data of the target receptor. The tool initially applied to discovery of lead molecules now extends to lead optimization. Pharmacophores can be used as queries for retrieving potential leads from structural databases (lead discovery), for designing molecules with specific desired attributes (lead optimization), and for assessing similarity and diversity of molecules using pharmacophore fingerprints. It can also be used to align molecules based on the 3D arrangement of chemical features or to develop predictive 3D QSAR models. This review begins with a brief historical overview of the pharmacophore evolution followed by a coverage of the developments in methodologies for pharmacophore identification over the period from inception of the pharmacophore concept to recent developments of the more sophisticated tools such as Catalyst, GASP, and DISCO. In addition, we present some very recent successes of the widely used pharmacophore generation methods in drug discovery.

A comprehensive analysis of the thermodynamic events involved in ligand–receptor binding using CoRIA and its variants

Quantitative Structure-Activity Relationships (QSAR) are being used since decades for prediction of biological activity, lead optimization, classification, identification and explanation of the mechanisms of drug action, and prediction of novel structural leads in drug discovery. Though the technique has lived up to its expectations in many aspects, much work still needs to be done in relation to problems related to the rational design of peptides. Peptides are the drugs of choice in many situations, however, designing them rationally is a complicated task and the complexity increases with the length of their sequence. In order to deal with the problem of peptide optimization, one of our recently developed QSAR formalisms CoRIA (Comparative Residue Interaction Analysis) is being expanded and modified as: reverse-CoRIA (rCoRIA) and mixed-CoRIA (mCoRIA) approaches. In these methodologies, the peptide is fragmented into individual units and the interaction energies (van der Waals, Coulombic and hydrophobic) of each amino acid in the peptide with the receptor as a whole (rCoRIA) and with individual active site residues in the receptor (mCoRIA) are calculated, which along with other thermodynamic descriptors, are used as independent variables that are correlated to the biological activity by chemometric methods. As a test case, the three CoRIA methodologies have been validated on a dataset of diverse nonamer peptides that bind to the Class I major histocompatibility complex molecule HLA-A*0201, and for which some structure activity relationships have already been reported. The different models developed, and validated both internally as well as externally, were found to be robust with statistically significant values of r2 (correlation coefficient) and r2pred (predictive r2). These models were able to identify all the structure activity relationships known for this class of peptides, as well uncover some new relationships. This means that these methodologies will perform well for other peptide datasets too. The major advantage of these approaches is that they explicitly utilize the 3D structures of small molecules or peptides as well as their macromolecular targets, to extract position-specific information about important interactions between the ligand and receptor, which can assist the medicinal and computational chemists in designing new molecules, and biologists in studying the influence of mutations in the target receptor on ligand binding.

In silico screening of ligand databases: Methods and applications

Pressure is mounting on the pharmaceutical industry to reduce both the cost of drugs and the time to market. The large number of targets made available in the last decade has created a new area for technologies that can rapidly identify quality lead candidates. Virtual screening is one such technology that is gaining increasing importance in the drug discovery process. Virtual screening is a reliable and inexpensive method currently being employed as a complementary approach to high-throughput screening. Virtual screening can be adopted irrespective of the structural information of the target receptor. In the absence of structural data, virtual screening using pharmacophore-based search is a major in silico tool. However, when the structure of the receptor is available, virtual screening using both pharmacophore-based and docking techniques can be employed. Both of these methods can be synergistically integrated to improve the drug design and development process. In this article, we provide an overview of methods for virtual screening - in particular, docking and pharmacophore-based - along with commercial algorithms implementing these methods, and a successful example in this arena. Further, we enumerate the potential for patenting such kind of studies.

Comparative Occupancy Analysis (CoOAn) – A Straightforward and Directly Applicable 3D‐QSAR Formalism to Extract Molecular Features Obligatory for Designing Potent Leads

A simple and directly applicable 3D‐QSAR method, termed Comparative Occupancy Analysis (CoOAn), has been developed. The method is based on the comparison of local occupancies of fragments of an aligned set of molecules in a 3D‐grid space. The formalism commendably extracts the crucial position‐specific molecular features and correlates them quantitatively to their biological endpoints. The method has been effectively applied and efficaciously validated on three large and diverse datasetsthrombin, glycogen phosphorylase b (GPB), and thermolysin inhibitors. Several robust and statistically significant predictive 3D‐QSAR models were developed while simultaneously considering the influence of grid spacing on the accuracy of the results. The models, generated by the G/PLS chemometric method, not only unswervingly identified the obligatory chemical features but advantageously detected those that are unfavourable or detrimental for the molecular activity. The CoOAn models can profitably be used to optimize existing molecules as well as to design new leads with more desirable (and/or less detrimental) features. The activity‐modulating features (together with their distance‐constraints) extracted by the methodology can also be incorporated into a pharmacophore‐type query to search a chemical database for novel leads.

Abstract B40: Nucleotide analogs as novel STING agonists for immuno-oncology

Immunotherapy has recently emerged as a transformative approach for the treatment of cancer; nevertheless, many patients remain unresponsive to treatment. Recent evidence suggests that the activation of Stimulator of Interferon Genes (STING) pathway in tumor cells and/or antigen presenting cells (APCs) within the tumor microenvironment (TME) can induce type I Interferon production leading to apoptosis of tumor cells, as well as, induction of adaptive immune response (through priming of CD8+ T cells to tumor-associated antigens) thereby providing a powerful anti-cancer strategy. Therefore, therapeutic agents that activate STING signaling pathway in tumor cells and APCs in the TME are urgently needed. Herein, we describe the discovery of highly potent and selective first-in-class STING agonists for application in immuno-oncology. Methods: Using structure-guided drug design, in conjunction with published crystal structures of different cyclic dinucleotides bound to STING, a focused library of nucleotide compounds was prepared using standard phosphoramidite chemistry. The compounds were screened for induction of Interferon regulatory factor (IRF), Interferon-stimulated gene 54 (ISG54), and NF-KB using reporter assays. We used HEK293 cell line stably expressing ISG54 (ISRE)-promoter-driven firefly luciferase reporter gene for initial hit discovery and the actives were further characterized in PBMCs and THP1 cells. The IRF and NF-kB induction was calculated from % fold-change in luminescence compared to DMSO-treated cells and EC50 of the compounds were ascertained using Xlfit. Lead STING agonists were further evaluated for: (a) Binding affinity: Binding assays were conducted by Differential Scanning Fluorimetry (DSF) and Tm was calculated using Thermal Shift software, (b) Induction of pathogen recognition receptors (PRRs), ISGs and Programmed Death Ligands 1 & 2 (PDL1, PDL2) genes: THP1 cells and PBMCs were treated with various concentrations of lead compounds or 2,939-cGAMP or DMSO and the gene expression of different PRRs, ISGs, PDL1, and PDL2 was determined by quantitative RT-PCR using ΔΔct method, (c) Apoptosis-inducing activity: PBMCs and THP1 cells were treated with various concentrations of lead compounds, 29,39-cGAMP, or DMSO control and the apoptotic activity was evaluated using Caspase-Glo® 3/7 Assay (Promega), and (d) In vitro anti-tumor activity: STING-dependent anti-tumor activity of lead compounds in various tumor cell lines was assessed by either high-content imaging or through Cell titer Glo® Cytotoxicity Assay (Promega). Cell survival was calculated based upon % reduction of live cells compared to DMSO control. CC50 of the compounds were generated by curve fit in Xlfit. Results: Through in vitro assays in conjunction with Structure Activity Relationship (SAR) studies, we have identified several highly potent and selective first-in-class STING agonists. A promising lead nucleotide compound SB 11285 caused STING-dependent induction of: (a) IRF with an EC50 of 2 nM that is 1000-fold more potent than the natural STING agonist 29,39-cGAMP, (b) NF-kB with an EC50 of 200 nM that is >200-fold more potent than 29,39-cGAMP, (c) selective apoptosis of human monocyte leukemic cell lines (CC50, 500 nM) as compared to normal PBMCs through induction of IFN, and NF-kB signaling, and (d) expression of various PRRs and ISGs including RIG-I, MDA-5, LGP2, ISG54 and OAS-1, as well as, PDL1 and PDL2. Finally, SB 11285 showed potent in vitro anti-tumor activity in multiple tumor cell lines. Conclusion: We have discovered highly potent first-in-class STING agonists that show excellent selectivity in induction of IFN, NF-KB, ISGs, and PRRs, and apoptosis of tumor-derived cell lines. The lead STING agonist SB 11285 has potent immune-modulating, as well as, anti-tumor activities and is being advanced for additional preclinical studies for application in immuno-oncology. Citation Format: Sreerupa Challa, Shenghua Zhou, Anjaneyulu Sheri, Seetharamaiyer Padmanabhan, Samantha Delaney, Geeta Meher, Dillon Cleary, Vishal Nair, Rayomand Gimi, Santosh Khedkar, Radhakrishnan Iyer. Nucleotide analogs as novel STING agonists for immuno-oncology. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B40.

Abstract B39: Novel dinucleotides that activate STING signaling for immuno-oncology

Background: Immunotherapy has recently emerged as a transformative approach for the treatment of cancer; nevertheless, many patients remain unresponsive to treatment. It is being recognized that induction of type I interferons (IFN) and interferon-stimulated genes (ISGs) in tumor cells and within the tumor microenvironment (TME) is essential for modulating the host-immune response and inducing apoptosis of tumor cells. Furthermore, the antigen-presenting cells within TME can cause induction of adaptive immune response, through priming of CD8+ T cells and tumor killing. Importantly, the DNA released from damaged cells and cancer cells can be sensed by cyclic GMP-AMP synthase (cGAS) leading to the synthesis of cyclic-GMP-AMP (29,39-cGAMP), a second messenger that activates Stimulator of Interferon Genes (STING) pathway resulting in the production of type I IFN and ISGs. The cumulative effects of activation of innate and adaptive immune response can result in potent anti-cancer effects. Therefore, therapeutic agents that activate the cGAS-STING signaling pathway in tumor cells and TME are urgently needed. Herein, we describe the discovery of novel potent, first-in-class small molecules for application in immuno-oncology. Methods: Using structure-guided drug design, in conjunction with published crystal structures of cyclic dinucleotides bound to STING, a focused library of dinucleotide compounds was synthesized using phosphoramidite chemistry and evaluated for: (a) Induction of IFN signaling: The compounds were screened for the induction of Interferon regulatory factor (IRF), ISG54, and NF-κB using reporter assays. We used HEK293 cell line (SZ14) stably expressing ISG54 (ISRE)-promoter-driven firefly luciferase reporter gene for screening and the active compounds were further characterized in THP1 cells and human primary PBMCs. The IRF, ISG54, and NF-κB induction was calculated from % fold-change in luminescence compared to DMSO-treated cells and EC50s of the compounds were ascertained to identify active compounds, (b) Expression of IFN-β and IRF7 in THP1 cells: THP1 cells were treated with active compounds or controls for 22hrs. RNA was extracted and the expression of IFN-β, IRF7, was ascertained using semi-quantitative RT-PCR, (c) Induction of pathogen recognition receptors (PRRs) including RIG-I, MDA5, LGP2, and OAS-1 and ISG54: THP1 cells and PBMCs were treated with active compounds, 29,39-cGAMP (control), or DMSO and the gene expression of different PRRs, ISGs, was determined by quantitative RT-PCR using ΔΔct method, (d) Induction of cGAS-STING signaling using reporter assays: HEK293 cells stably expressing ISG54 were transfected with plasmids encoding human cGAS (wild-type, or K384A, K400A, or K411A mutants) and treated with active compounds, poly (dA:dT) (positive control), or DMSO for 21 hrs. ISG54 induction was calculated as fold-change in luminescence compared to DMSO-treated controls. (e) Cytotoxicity assays: THP1 cells were treated with active compounds or DMSO control with Lipofectamine and cytotoxicity assessed using the CellTiter-Glo® Luminescent assays. Cytotoxicity was calculated from %-fold change in luminescence compared to DMSO-treated sample. Results: Through in vitro assays in conjunction with Structure Activity Relationship studies, we have identified potent compounds that activate cGAS-STING signaling pathway for induction of IRF, IFN, and NF-κB. These compounds also cause induction of expression of PRRs, including RIG-I, MDA5, LGP2, as well as, ISG54 and OAS-1. Conclusion: We have discovered potent, first-in-class agents that cause induction of IFN, NF-κB, ISGs, and PRRs. Further optimization and preclinical evaluation of the compounds for application in immuno-oncology is underway. Citation Format: Shenghua Zhou, Sreerupa Challa, Seetharamaiyer Padmanabhan, Anjaneyulu Sheri, Samantha Delaney, Geeta Meher, Dillon Cleary, Rayomand Gimi, Santosh Khedkar, Radhakrishnan Iyer. Novel dinucleotides that activate STING signaling for immuno-oncology. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B39.