Radhakrishnan P. Iyer

Ensemble QSAR: A QSAR method based on conformational ensembles and metric descriptors

Quantitative structure–activity relationship (QSAR) is the most versatile tool in computer‐assisted molecular design. One conceptual drawback seen in QSAR approaches is the “one chemical–one structure–one parameter value” dogma where the model development is based on physicochemical description for a single molecular conformation, while ignoring the rest of the conformational space. It is well known that molecules have several low‐energy conformations populated at physiological temperature, and each conformer makes a significant impact on associated properties such as biological activity. At the level of molecular interaction, the dynamics around the molecular structure is of prime essence rather than the average structure. As a step toward understanding the role of these discrete microscopic states in biological activity, we have put together a theoretically rigorous and computationally tractable formalism coined as eQSAR. In this approach, the biological activity is modeled as a function of physicochemical description for a selected set of low‐energy conformers, rather than thats for a single lowest energy conformation. Eigenvalues derived from the “Physicochemical property integrated distance matrices” (PD‐matrices) that encompass both 3D structure and physicochemical properties, have been used as descriptors; is a novel addition. eQSAR is validated on three peptide datasets and explicitly elaborated for bradykinin‐potentiating peptides. The conformational ensembles were generated by a simple molecular dynamics and consensus dynamics approaches. The eQSAR models are statistically significant and possess the ability to select the most biologically relevant conformation(s) with the relevant physicochemical attributes that have the greatest meaning for description of the biological activity.

Diversity-oriented, one-pot, multi-component synthesis of substituted uracil derivatives

With the emergence of high throughput screening of bioactive molecules, there is constant need for the development of new strategies for diversity-oriented synthesis. We describe here a novel one-pot multicomponent reaction for the synthesis of uracil derivatives using easily available starting materials. This new synthetic strategy provides easy access to diverse uracil derivatives in moderate to good yields.Graphical Abstract

Local indices for similarity analysis (LISA)-a 3D-QSAR formalism based on local molecular similarity.

A simple quantitative structure activity relationship (QSAR) approach termed local indices for similarity analysis (LISA) has been developed. In this technique, the global molecular similarity is broken up as local similarity at each grid point surrounding the molecules and is used as a QSAR descriptor. In this way, a view of the molecular sites permitting favorable and rational changes to enhance activity is obtained. The local similarity index, calculated on the basis of Petkes formula, segregates the regions into equivalent, favored similar, and disfavored similar (alternatively favored dissimilar) potentials with respect to a reference molecule in the data set. The method has been tested on three large and diverse data sets-thrombin, glycogen phosphorylase b, and thermolysin inhibitors. The QSAR models derived using genetic algorithm incorporated partial least square analysis statistics are found to be comparable to the ones obtained by the standard three-dimensional (3D)-QSAR methods, such as comparative molecular field analysis and comparative molecular similarity indices analysis. The graphical interpretation of the LISA models is straightforward, and the outcome of the models corroborates well with literature data. The LISA models give insight into the binding mechanisms of the ligand with the enzyme and allow fine-tuning of the molecules at the local level to improve their activity.

In silico optimization of pharmacokinetic properties and receptor binding affinity simultaneously: a ‘parallel progression approach to drug design’ applied to β-blockers

The present work exploits the potential of in silico approaches for minimizing attrition of leads in the later stages of drug development. We propose a theoretical approach, wherein ‘parallel’ information is generated to simultaneously optimize the pharmacokinetics (PK) and pharmacodynamics (PD) of lead candidates. β-blockers, though in use for many years, have suboptimal PKs; hence are an ideal test series for the ‘parallel progression approach’. This approach utilizes molecular modeling tools viz. hologram quantitative structure activity relationships, homology modeling, docking, predictive metabolism, and toxicity models. Validated models have been developed for PK parameters such as volume of distribution (log Vd) and clearance (log Cl), which together influence the half-life (t1/2) of a drug. Simultaneously, models for PD in terms of inhibition constant pKi have been developed. Thus, PK and PD properties of β-blockers were concurrently analyzed and after iterative cycling, modifications were proposed that lead to compounds with optimized PK and PD. We report some of the resultant re-engineered β-blockers with improved half-lives and pKi values comparable with marketed β-blockers. These were further analyzed by the docking studies to evaluate their binding poses. Finally, metabolic and toxicological assessment of these molecules was done through in silico methods. The strategy proposed herein has potential universal applicability, and can be used in any drug discovery scenario; provided that the data used is consistent in terms of experimental conditions, endpoints, and methods employed. Thus the ‘parallel progression approach’ helps to simultaneously fine-tune various properties of the drug and would be an invaluable tool during the drug development process.

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.