Krishnapriya Chinnaswamy

CSAR Data Set Release 2012: Ligands, Affinities, Complexes,and Docking Decoys

A major goal in drug design is the improvement of computational methods for docking and scoring. The Community Structure Activity Resource (CSAR) has collected several data sets from industry and added in-house data sets that may be used for this purpose (www.csardock.org). CSAR has currently obtained data from Abbott, GlaxoSmithKline, and Vertex and is working on obtaining data from several others. Combined with our in-house projects, we are providing a data set consisting of 6 protein targets, 647 compounds with biological affinities, and 82 crystal structures. Multiple congeneric series are available for several targets with a few representative crystal structures of each of the series. These series generally contain a few inactive compounds, usually not available in the literature, to provide an upper bound to the affinity range. The affinity ranges are typically 3–4 orders of magnitude per series. For our in-house projects, we have had compounds synthesized for biological testing. Affinities were measured by Thermofluor, Octet RED, and isothermal titration calorimetry for the most soluble. This allows the direct comparison of the biological affinities for those compounds, providing a measure of the variance in the experimental affinity. It appears that there can be considerable variance in the absolute value of the affinity, making the prediction of the absolute value ill-defined. However, the relative rankings within the methods are much better, and this fits with the observation that predicting relative ranking is a more tractable problem computationally. For those in-house compounds, we also have measured the following physical properties: logD, logP, thermodynamic solubility, and pKa. This data set also provides a substantial decoy set for each target consisting of diverse conformations covering the entire active site for all of the 58 CSAR-quality crystal structures. The CSAR data sets (CSAR-NRC HiQ and the 2012 release) provide substantial, publically available, curated data sets for use in parametrizing and validating docking and scoring methods.

Structure-Based Design of γ-Carboline Analogues as Potent and Specific BET Bromodomain Inhibitors.

Small-molecule inhibitors of bromodomain and extra terminal proteins (BET), including BRD2, BRD3, and BRD4 proteins have therapeutic potential for the treatment of human cancers and other diseases and conditions. In this paper, we report the design, synthesis, and evaluation of γ-carboline-containing compounds as a new class of small-molecule BET inhibitors. The most potent inhibitor (compound 18, RX-37) obtained from this study binds to BET bromodomain proteins (BRD2, BRD3, and BRD4) with Ki values of 3.2-24.7 nM and demonstrates high selectivity over other non-BET bromodomain-containing proteins. Compound 18 potently and selectively inhibits cell growth in human acute leukemia cell lines harboring the rearranged mixed lineage leukemia 1 gene. We have determined a cocrystal structure of 18 in complex with BRD4 BD2 at 1.4 Å resolution, which provides a solid structural basis for the compounds high binding affinity and for its further structure-based optimization. Compound 18 represents a promising lead compound for the development of a new class of therapeutics for the treatment of human cancer and other conditions.

CSAR Benchmark Exercise 2013: Evaluation of Results from a Combined Computational Protein Design, Docking, and Scoring/Ranking Challenge

Community Structure-Activity Resource (CSAR) conducted a benchmark exercise to evaluate the current computational methods for protein design, ligand docking, and scoring/ranking. The exercise consisted of three phases. The first phase required the participants to identify and rank order which designed sequences were able to bind the small molecule digoxigenin. The second phase challenged the community to select a near-native pose of digoxigenin from a set of decoy poses for two of the designed proteins. The third phase investigated the ability of current methods to rank/score the binding affinity of 10 related steroids to one of the designed proteins (pKd = 4.1 to 6.7). We found that 11 of 13 groups were able to correctly select the sequence that bound digoxigenin, with most groups providing the correct three-dimensional structure for the backbone of the protein as well as all atoms of the active-site residues. Eleven of the 14 groups were able to select the appropriate pose from a set of plausible decoy poses. The ability to predict absolute binding affinities is still a difficult task, as 8 of 14 groups were able to correlate scores to affinity (Pearson-r > 0.7) of the designed protein for congeneric steroids and only 5 of 14 groups were able to correlate the ranks of the 10 related ligands (Spearman-ρ > 0.7).

A potent small-molecule inhibitor of the DCN1-UBC12 interaction that selectively blocks cullin 3 neddylation

The Cullin-RING E3 ubiquitin ligases (CRLs) regulate homeostasis of ~20% of cellular proteins and their activation require neddylation of their cullin subunit. Cullin neddylation is modulated by a scaffolding DCN protein through interactions with both the cullin protein and an E2 enzyme such as UBC12. Here we report the development of DI-591 as a high-affinity, cell-permeable small-molecule inhibitor of the DCN1–UBC12 interaction. DI-591 binds to purified recombinant human DCN1 and DCN2 proteins with Ki values of 10–12 nM, and disrupts the DCN1–UBC12 interaction in cells. Treatment with DI-591 selectively converts cellular cullin 3 into an un-neddylated inactive form with no or minimum effect on other cullin members. Our data firmly establish a previously unrecognized specific role of the DCN1–UBC12 interaction for cellular neddylation of cullin 3. DI-591 is an excellent probe compound to investigate the role of the cullin 3 CRL ligase in biological processes and human diseases.Cullins are central components of the ubiquitin-proteosome system and are activated via a neddylation process mediated by the DCN1–UBC12 complex. Here, the authors develop a small molecule inhibitor of the DCN1–UBC12 interaction that specifically blocks cullin 3 neddylation and can be used to probe the cellular function of cullin 3.

Conformational Sampling and Binding Site Assessment of Suppression of Tumorigenicity 2 Ectodomain

Suppression of Tumorigenicity 2 (ST2), a member of the interleukin-1 receptor (IL-1R) family, activates type 2 immune responses to pathogens and tissue damage via binding to IL-33. Dysregulated responses contribute to asthma, graft-versus-host and autoinflammatory diseases and disorders. To study ST2 structure for inhibitor development, we performed the principal component (PC) analysis on the crystal structures of IL1-1R1, IL1-1R2, ST2 and the refined ST2 ectodomain (ST2ECD) models, constructed from previously reported small-angle X-ray scattering data. The analysis facilitates mapping of the ST2ECD conformations to PC subspace for characterizing structural changes. Extensive coverage of ST2ECD conformations was then obtained using the accelerated molecular dynamics simulations started with the IL-33 bound ST2ECD structure as instructed by their projected locations on the PC subspace. Cluster analysis of all conformations further determined representative conformations of ST2ECD ensemble in solution. Alignment of the representative conformations with the ST2/IL-33 structure showed that the D3 domain of ST2ECD (containing D1-D3 domains) in most conformations exhibits no clashes with IL-33 in the crystal structure. Our experimental binding data informed that the D1-D2 domain of ST2ECD contributes predominantly to the interaction between ST2ECD and IL-33 underscoring the importance of the D1-D2 domain in binding. Computational binding site assessment revealed one third of the total detected binding sites in the representative conformations may be suitable for binding to potent small molecules. Locations of these sites include the D1-D2 domain ST2ECD and modulation sites conformed to ST2ECD conformations. Our study provides structural models and analyses of ST2ECD that could be useful for inhibitor discovery.

Mechanistic evaluation and transcriptional signature of a glutathione S-transferase omega 1 inhibitor

Glutathione S-transferase omega 1 (GSTO1) is an atypical GST isoform that is overexpressed in several cancers and has been implicated in drug resistance. Currently, no small-molecule drug targeting GSTO1 is under clinical development. Here we show that silencing of GSTO1 with siRNA significantly impairs cancer cell viability, validating GSTO1 as a potential new target in oncology. We report on the development and characterization of a series of chloroacetamide-containing potent GSTO1 inhibitors. Co-crystal structures of GSTO1 with our inhibitors demonstrate covalent binding to the active site cysteine. These potent GSTO1 inhibitors suppress cancer cell growth, enhance the cytotoxic effects of cisplatin and inhibit tumour growth in colon cancer models as single agent. Bru-seq-based transcription profiling unravelled novel roles for GSTO1 in cholesterol metabolism, oxidative and endoplasmic stress responses, cytoskeleton and cell migration. Our findings demonstrate the therapeutic utility of GSTO1 inhibitors as anticancer agents and identify the novel cellular pathways under GSTO1 regulation in colorectal cancer.

From proteomics to discovery of first-in-class ST2 inhibitors active in vivo

Soluble cytokine receptors function as decoy receptors to attenuate cytokine-mediated signaling and modulate downstream cellular responses. Dysregulated overproduction of soluble receptors can be pathological, such as soluble ST2 (sST2), a prognostic biomarker in cardiovascular diseases, ulcerative colitis, and graft-versus-host disease (GVHD). Although intervention using an ST2 antibody improves survival in murine GVHD models, sST2 is a challenging target for drug development because it binds to IL-33 via an extensive interaction interface. Here, we report the discovery of small-molecule ST2 inhibitors through a combination of high-throughput screening and computational analysis. After in vitro and in vivo toxicity assessment, 3 compounds were selected for evaluation in 2 experimental GVHD models. We show that the most effective compound, iST2-1, reduces plasma sST2 levels, alleviates disease symptoms, improves survival, and maintains graft-versus-leukemia activity. Our data suggest that iST2-1 warrants further optimization to develop treatment for inflammatory diseases mediated by sST2.

Effects of age and retinal degeneration on the expression of proprotein convertases in the visual cortex

Proprotein convertases (PCs) comprise a large family of subtilisin-like, eukaryotic, serine endoproteases that process substrates important in the development, homeostasis, and pathology of the nervous system. Despite important interactions with these substrates, including neurotrophins, PC expression throughout normal postnatal development and disease progression in the brain remains unknown. The primary objective of this study was to determine whether the expression profiles of widely expressed and tissue-specific PCs varied during normal brain development or neurological disorders. We examined the expression of mRNAs for seven PCs in the visual cortex of normal and visually impaired mice at 10 postnatal developmental time points between Week 1 and Week 35. Widely expressed PCs (furin, PACE4, PC5, and PC7) all exhibited a similar expression profile. High mRNA levels were seen at Week 1 with levels generally lower over the next 5-6 weeks. In visually impaired mice, widely expressed PCs again all exhibited a similar expression profile, but it was dramatically different than observed in normal mice. The temporal expression of tissue-specific PCs varied in wild-type mice. Interestingly, this variability was sharply reduced in visually impaired mice. Overall, these data suggest a timetable of altered PC expression that corresponds closely with the formation of functional visual maps in the visual cortex. The implications of these findings are discussed in the context of neurotrophin processing and synaptogenesis in the developing visual cortex.

High-Affinity Peptidomimetic Inhibitors of the DCN1-UBC12 Protein-Protein Interaction

The Cullin-RING ligases (CRLs) regulate the turnover of approximately 20% of the proteins in mammalian cells and are emerging therapeutic targets in human diseases. The activation of CRLs requires the neddylation of their cullin subunit, which is controlled by an activation complex consisting of Cullin-RBX1-UBC12-NEDD8-DCN1. Herein, we describe the design, synthesis, and evaluation of peptidomimetics targeting the DCN1-UBC12 protein-protein interaction. Starting from a 12-residue UBC12 peptide, we have successfully obtained a series of peptidomimetic compounds that bind to DCN1 protein with KD values of <10 nM. Determination of a cocrystal structure of a potent peptidomimetic inhibitor complexed with DCN1 provides the structural basis for their high-affinity interaction. Cellular investigation of one potent DCN1 inhibitor, compound 36 (DI-404), reveals that it effectively and selectively inhibits the neddylation of cullin 3 over other cullin members. Further optimization of DI-404 may yield a new class of therapeutics for the treatment of human diseases in which cullin 3 CRL plays a key role.

Abstract LB-058: Novel interactions of the RAS oncoprotein

Approximately 30% of all cancers harbor activating mutations in the RAS family of small GTPase proteins, making it one of the most common oncogenic aberrations in humans. Normal RAS proteins (H, K or N-RAS) localize to the inner cell membrane and transduce extracellular growth signals by cycling between an “active” GTP-bound state and “inactive” GDP-bound state, through interactions with various “GTPase activating proteins” (GAPs) that promote RAS mediated GTP hydrolysis. Oncogenic mutants of RAS lose their catalytic activity or association with the GAP proteins, resulting in constitutively active GTP-bound state that signals through direct interactions with effector kinases like RAF and PI3K and activate the MEK/ERK and/or Akt, leading to activation of hallmark cancer pathways including growth factor independence, uncontrolled cell proliferation, evasion of apoptosis and immune responses, increased metabolism as well as metastases. Although RAS is the most frequently mutated gene driving multifarious pathways of oncogenesis, our knowledge of protein interactions involving RAS proteins have been largely limited to RAS binding domains in RAF/PI3K/RalGDS. Targeting mutant RAS proteins or its direct effectors, or pathways activated by RAS effectors remains a challenging endeavor for treating RAS driven cancers. Towards the goal of a thorough understanding of RAS biology through a comprehensive identification of its interactors, we performed IP-Mass Spectrometric analysis of pan-RAS immunoprecipitates from multiple cell lines. Interestingly in our experiments, apart from the well-known interactor RAF, we found evidence of several novel RAS interacting proteins, including many with DNA and RNA binding motifs. Our study validates these findings through cell-free protein interaction analyses and explores the possible biological effects of these novel RAS interactions in mutant KRAS driven cellular transformation. Note: This abstract was not presented at the meeting. Citation Format: Sunita Shankar, Rohit Malik, Vishal Kothari, Yasuyuki Hosono, Sethuramasundaram Pitchiaya, Shanker Kalyana-Sundaram, Anastasia Yocum, June Escara-Wilke, Harika Gundlapalli, Krishnapriya Chinnaswamy, Matthew Shuler, Anton Poliakov, Xiaoju Wang, Vishalakshi Krishnan, Yasmine White, Ari Firestone, Xuhong Cao, Saravana M. Dhanasekaran, Jeanne Stuckey, Gideon Bollag, Kevin Shannon, Nils G. Walter, Chandan Kumar-Sinha, Arul Chinnaiyan. Novel interactions of the RAS oncoprotein. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-058. doi:10.1158/1538-7445.AM2015-LB-058