Anshuman Dixit

Elucidation of the Hsp90 C-terminal Inhibitor Binding Site

The Hsp90 chaperone machine is required for the folding, activation, and/or stabilization of more than 50 proteins directly related to malignant progression. Hsp90 contains small molecule binding sites at both its N- and C-terminal domains; however, limited structural and biochemical data regarding the C-terminal binding site is available. In this report, the small molecule binding site in the Hsp90 C-terminal domain was revealed by protease fingerprinting and photoaffinity labeling utilizing LC-MS/MS. The identified site was characterized by generation of a homology model for hHsp90α using the SAXS open structure of HtpG and docking the bioactive conformation of NB into the generated model. The resulting model for the bioactive conformation of NB bound to Hsp90α is presented herein.

Computational Modeling of Allosteric Communication Reveals Organizing Principles of Mutation-Induced Signaling in ABL and EGFR Kinases

The emerging structural information about allosteric kinase complexes and the growing number of allosteric inhibitors call for a systematic strategy to delineate and classify mechanisms of allosteric regulation and long-range communication that control kinase activity. In this work, we have investigated mechanistic aspects of long-range communications in ABL and EGFR kinases based on the results of multiscale simulations of regulatory complexes and computational modeling of signal propagation in proteins. These approaches have been systematically employed to elucidate organizing molecular principles of allosteric signaling in the ABL and EGFR multi-domain regulatory complexes and analyze allosteric signatures of the gate-keeper cancer mutations. We have presented evidence that mechanisms of allosteric activation may have universally evolved in the ABL and EGFR regulatory complexes as a product of a functional cross-talk between the organizing αF-helix and conformationally adaptive αI-helix and αC-helix. These structural elements form a dynamic network of efficiently communicated clusters that may control the long-range interdomain coupling and allosteric activation. The results of this study have unveiled a unifying effect of the gate-keeper cancer mutations as catalysts of kinase activation, leading to the enhanced long-range communication among allosterically coupled segments and stabilization of the active kinase form. The results of this study can reconcile recent experimental studies of allosteric inhibition and long-range cooperativity between binding sites in protein kinases. The presented study offers a novel molecular insight into mechanistic aspects of allosteric kinase signaling and provides a quantitative picture of activation mechanisms in protein kinases at the atomic level.

Hierarchical Modeling of Activation Mechanisms in the ABL and EGFR Kinase Domains: Thermodynamic and Mechanistic Catalysts of Kinase Activation by Cancer Mutations

Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type) and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition and the enhanced stabilization of the active kinase form. The results of this study reconcile current experimental data with insights from theoretical approaches, pointing to general mechanistic aspects of activating transitions in protein kinases.

A Systematic Protocol for the Characterization of Hsp90 Modulators

Several Hsp90 modulators have been identified including the N-terminal ligand geldanamycin (GDA), the C-terminal ligand novobiocin (NB), and the co-chaperone disruptor celastrol. Other Hsp90 modulators elicit a mechanism of action that remains unknown. For example, the natural product gedunin and the synthetic anti-spermatogenic agent H2-gamendazole, recently identified Hsp90 modulators, manifest biological activity through undefined mechanisms. Herein, we report a series of biochemical techniques used to classify such modulators into identifiable categories. Such studies provided evidence that gedunin and H2-gamendazole both modulate Hsp90 via a mechanism similar to celastrol, and unlike NB or GDA.

Sequence and Structure Signatures of Cancer Mutation Hotspots in Protein Kinases

Protein kinases are the most common protein domains implicated in cancer, where somatically acquired mutations are known to be functionally linked to a variety of cancers. Resequencing studies of protein kinase coding regions have emphasized the importance of sequence and structure determinants of cancer-causing kinase mutations in understanding of the mutation-dependent activation process. We have developed an integrated bioinformatics resource, which consolidated and mapped all currently available information on genetic modifications in protein kinase genes with sequence, structure and functional data. The integration of diverse data types provided a convenient framework for kinome-wide study of sequence-based and structure-based signatures of cancer mutations. The database-driven analysis has revealed a differential enrichment of SNPs categories in functional regions of the kinase domain, demonstrating that a significant number of cancer mutations could fall at structurally equivalent positions (mutational hotspots) within the catalytic core. We have also found that structurally conserved mutational hotspots can be shared by multiple kinase genes and are often enriched by cancer driver mutations with high oncogenic activity. Structural modeling and energetic analysis of the mutational hotspots have suggested a common molecular mechanism of kinase activation by cancer mutations, and have allowed to reconcile the experimental data. According to a proposed mechanism, structural effect of kinase mutations with a high oncogenic potential may manifest in a significant destabilization of the autoinhibited kinase form, which is likely to drive tumorigenesis at some level. Structure-based functional annotation and prediction of cancer mutation effects in protein kinases can facilitate an understanding of the mutation-dependent activation process and inform experimental studies exploring molecular pathology of tumorigenesis.

Computational modeling of structurally conserved cancer mutations in the RET and MET kinases: the impact on protein structure, dynamics, and stability.

Structural and biochemical characterization of protein kinases that confer oncogene addiction and harbor a large number of disease-associated mutations, including RET and MET kinases, have provided insights into molecular mechanisms associated with the protein kinase activation in human cancer. In this article, structural modeling, molecular dynamics, and free energy simulations of a structurally conserved mutational hotspot, shared by M918T in RET and M1250T in MET kinases, are undertaken to quantify the molecular mechanism of activation and the functional role of cancer mutations in altering protein kinase structure, dynamics, and stability. The mechanistic basis of the activating RET and MET cancer mutations may be driven by an appreciable free energy destabilization of the inactive kinase state in the mutational forms. According to our results, the locally enhanced mobility of the cancer mutants and a higher conformational entropy are counterbalanced by a larger enthalpy loss and result in the decreased thermodynamic stability. The computed protein stability differences between the wild-type and cancer kinase mutants are consistent with circular dichroism spectroscopy and differential scanning calorimetry experiments. These results support the molecular mechanism of activation, which causes a detrimental imbalance in the dynamic equilibrium shifted toward the active form of the enzyme. Furthermore, computer simulations of the inhibitor binding with the oncogenic and drug-resistant RET mutations have also provided a plausible molecular rationale for the observed differences in the inhibition profiles, which is consistent with the experimental data. Finally, structural mapping of RET and MET cancer mutations and the computed protein stability changes suggest a similar mechanism of activation, whereby the cancer mutations which display the higher oncogenic activity tend to have the greatest destabilization effect on the inactive kinase structure.

An investigation of structurally diverse carbamates for acetylcholinesterase (AChE) inhibition using 3D-QSAR analysis.

In order to identify the essential structural features and physicochemical properties for acetylcholinesterase (AChE) inhibitory activity in some carbamate derivatives, the systematic QSAR (Quantitative Structure Activity Relationship) studies (CoMFA, advance CoMFA and CoMSIA) have been carried out on a series of (total 78 molecules) taking 52 and 26 molecules in training and test set, respectively. Statistically significant 3D-QSAR (three-dimensional Quantitative Structure Activity Relationship) models were developed on training set molecules using CoMFA and CoMSIA and validated against test set compounds. The highly predictive models (CoMFA q(2)=0.733, r(2)=0.967, predictive r(2)=0.732, CoMSIA q(2)=0.641, r(2)=0.936, predictive r(2)=0.812) well explained the variance in binding affinities both for the training and the test set compounds. The generated models suggest that steric, electrostatic and hydrophobic interactions play an important role in describing the variation in binding affinity. In particular the carbamoyl nitrogen should be more electropositive; substitutions on this nitrogen should have high steric bulk and hydrophobicity while the amino nitrogen should be electronegative in order to have better activity. These studies may provide important insights into structural variations leading to the development of novel AChE inhibitors which may be useful in the development of novel molecules for the treatment of Alzheimers disease.

Design of Combretastatin A-4 Analogs as Tubulin Targeted Vascular Disrupting Agent with Special Emphasis on Their Cis-Restricted Isomers

Tubulin protein is a highly imperative and feasible goal for anticancer drug discovery. Hundreds of naturally occurring, semi synthetic and synthetic antitubulin agents have been reported till now. Among these, Combretastatin A - 4 (CA - 4) is effective antimitotic agent possessing potent cytotoxicity against a panel of cancer cells, including multi-drug resistant cancer cell lines. The inadequate water solubility and inactivation of these analogs during storage limit their use as clinical anticancer agents. To overcome these shortcomings, numerous water soluble amino analogs, amino acid derivative, phosphate prodrug (CA - 4P) and cis-locked CA - 4 have been developed with distinctive attributes of antitubulin and antivascular properties in a wide variety of preclinical tumor models. Subsequently, several heterocycle based cis restricted CA - 4 analogs are being reported for antitumor activity against collection of cancer cell lines. This review recapitulates the rational design, structure activity relationship, pharmacokinetic and pharmacodynamic profile of synthesized cis restricted CA - 4 analogs.

Exquisite selectivity for human toll-like receptor 8 in substituted furo[2,3-c]quinolines.

Toll-like receptor (TLR)-8 agonists activate adaptive immune responses by inducing robust production of T helper 1-polarizing cytokines, suggesting that TLR8-active compounds may be promising candidate adjuvants. We synthesized and evaluated hitherto unexplored furo[2,3-c]quinolines and regioisomeric furo[3,2-c]quinolines derived via a tandem, one-pot Sonogashira coupling and intramolecular 5-endo-dig cyclization strategy in a panel of primary screens. We observed a pure TLR8-agonistic activity profile in select furo[2,3-c]quinolines, with maximal potency conferred by a C2-butyl group (EC50 = 1.6 μM); shorter, longer, or substituted homologues as well as compounds bearing C1 substitutions were inactive, which was rationalized by docking studies using the recently described crystal structure of human TLR8. The best-in-class compound displayed prominent proinflammatory cytokine induction (including interleukin-12 and interleukin-18), but was bereft of interferon-α inducing properties, confirming its high selectivity for human TLR8.

Probing Molecular Mechanisms of the Hsp90 Chaperone: Biophysical Modeling Identifies Key Regulators of Functional Dynamics

Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based “conformational selection” of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected residue clusters may be a rather general functional requirement encoded across molecular chaperones. The obtained insights may be useful in guiding discovery of allosteric Hsp90 inhibitors targeting protein interfaces with co-chaperones and protein binding clients.