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Predicting Ligand Selectivity Across Bromodomain Families
Wednesday, March 2, 2016 sampling (REUS) molecular dynamics simulations[2]. REUS is a powerful conformational sampling method to overcome energy barriers. We applied this method to the HDAC3-T247 system and HDAC2-T247 system. These have very similar amino-acid sequences and similar structures. From results of these simulations, we obtained free energy landscapes. The results of these calculations show that the docked state is the most stable in the HDAC3-T247 system but in the HDAC2-T247 system, the docked state is less stable than undocked states. These results agree with experimental data. In this poster, we present these results. References: [1] T. Suzuki, Y. Kasuya, Y. Itoh, Y. Ota, P. Zhan, K. Asamitsu, H. Nakagawa, T. Okamoto, and N. Miyata, PLoS ONE 8, e68669 (2013). [2] Y. Sugita, A. Kitao, and Y. Okamoto, J. Chem. Phys. 113, 6042 (2000). 2687-Pos Board B64 Predicting Ligand Selectivity Across Bromodomain Families Matteo Aldeghi1, Alexander Heifetz2, Michael J. Bodkin2, Stefan Knapp3, Philip C. Biggin1. 1 Department of Biochemistry, University of Oxford, Oxford, United Kingdom, 2Evotec (U.K) Ltd., Abingdon, United Kingdom, 3Structural Genomics Consortium and Target Discovery Institute, University of Oxford, Oxford, United Kingdom. Binding selectivity is considered a requirement for the development of a safe drug, with large differences in binding affinities between the intended target and similar proteins being sought in order to avoid unwanted side effects. Selectivity is also critical for chemical probes used for investigation of the cellular function of proteins and for preclinical target validation. Nonetheless, identification of selective scaffolds is a difficult task, in particular for protein families with similar binding pocket features. Engineering selectivity adds considerable complexity to the rational design of new drugs, as it involves the optimization of multiple binding affinities. However, thanks to important advances in theory and computing in the last decades, predictions of binding affinities using physics-based simulations are gaining popularity and might be able to contribute to the efficient design of selective ligands. In particular, binding free energy estimates based on alchemical pathways have been shown to be a rigorous approach for the affinity prediction problem and hold the promise to be able to guide lead optimization. Here, we present the results of a study where we evaluated the performance of alchemical free energy estimates in predicting the selectivity profile of the broad-spectrum inhibitor bromosporine against multiple bromodomains. Bromodomains are epigenetic mark readers that bind to acetylation motifs and regulate gene transcription, and have established therapeutic potential in oncology. However, due to the structural similarity of the acetyl-lysine binding pocket, the design of selective ligands is often challenging. This study evaluates the performance of the calculations from a pseudo-prospective standpoint, as no information of the protein-ligand complexes or their affinities was used, and compares the theoretical results with high-quality isothermal titration calorimetry data. 2688-Pos Board B65 A Molecular Dynamics Study of Michaelis Complex for Designing Selective Transition State Analog Inhibitors for Cysteine Protease calpain-2 Payal Chatterjee1, Abdelaziz Alsamarah1, David Kent2, Li Qian1, David Wych3, Christine N. Pham1, Alla Avetisyan1, Steven Standley4, Michel Baudry4, Yun Luo1. 1 Department of Pharmaceutical Sciences, Western University of Health Sciences, Pomona, CA, USA, 2Department of Biological Sciences, Cal Poly, Pomona, CA, USA, 3Keck Science Department of the Claremont Colleges, Pomona, CA, USA, 4Graduate College of Biomedical Sciences, Western University of Health Sciences, Pomona, CA, USA. Calpains constitute a family of intracellular cysteine proteases, which catalyze the cleavage of target proteins in response to Ca2þ signaling. We recently discovered that, calpain-1 induces long-term potentiation, while calpain-2 limits the extent of potentiation and also is involved in neurodegeneration by NMDA activation. There is therefore a strong rationale to develop calpain-2 selective inhibitors, as such compounds could enhance learning and memory and offer neuroprotection. Among several reversible inhibitors that have been developed to target calpains, peptidyl a-ketoamide analogs carry the most stable electrophilic warhead. Although several cocrystal structures of calpain-1 and a-ketoamide are available, it is not clear how to design calpain-2 specific inhibitors based on the calpain-1 model, as the protease core is highly conserved. Structural alignment reveals that the major
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difference between calpain-1 and calpain-2 is located at the flexible gating loop. Molecular dynamics simulation of ligand-protease Michaelis complex is therefore necessary to capture the difference in the noncovalent binding interaction between the inhibitor and two isoforms and thus to guide the structure-based design. The Michaelis complex model of calpain-2 with a ketoamide compound was built from a cocrystal structure of a potent calpain-1 inhibitor. Effects of catalytic triad protonation states on the stability of the complex were investigated. The model was validated using existing calpain-2 inhibitors. Based on the simulation results, we modified the prime side (C-terminal) of the a-ketoamide peptidomimetics to favor calpain-2 over calpain-1. The dynamic of the calpain gating loop was explored using long-time scale simulation. The results show that molecular dynamics simulation of Michaelis complex can be a critical tool to explore the molecular bases of calpain activation and to design calpain-2 selective inhibitors for clinical therapeutics. 2689-Pos Board B66 Mechanism of Urea Conduction through H. Pylori UreI Ugur Akgun. Physics, Coe College, Cedar Rapids, IA, USA. The carcinogenic bacterium Helicobacter Pylori is found within the stomach of over 75% of the human population, and directly contributes to ailments such as gastritis, stomach ulcers, and stomach cancer. This neutrophile relies on the cytoplasmic hydrolysis of gastric urea into ammonia, which buffers the bacteria from the acidic environment. Urea uptake is a crucial mechanism of survival, and the protein HpUreI regulates and enhances this process. HpUreI is part of the urease operon in many bacteria. There are 7 genes in the urease operon, 2 of which are structural proteins, and 5 of which are accessory proteins. All of these proteins contribute directly to H. Pylori thriving in a low pH environment. The buffering action of the converted urea into ammonia and carbon dioxide boosts the local pH to 5.5 near the membrane. The structure of HpUreI protein shows two constriction sites in all six pores that allow water and urea to pass into the cytoplasm. This study reports results from various Molecular Dynamics simulations, including a high-concentration equilibration model saturated with urea, a steered molecular dynamics (SMD) simulation directing six ligands through each segment of the hexameric structure, and umbrella sampling simulations through the length of the pore. We report the energy profile identifying specific points of constriction and binding with both the ligand and free water saturating the structure. 2690-Pos Board B67 Dissecting the Contribution of Kinase Conformational Reorganization Energies to Inhibitor Selectivity Sonya M. Hanson1, Lucelenie Rodriguez1, Julie M. Behr1, Andrea Rizzi1, Daniel L. Parton1, Kyle A. Beauchamp1, Joshua H. Fass1, Jan-Hendrik Prinz1, Sarah E. Boyce1, Markus A. Seeliger2, Nicholas M. Levinson3, John D. Chodera1. 1 Computational Biology, MSKCC, New York, NY, USA, 2Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA, 3Pharmacology, University of Minnesota, Minneapolis, MN, USA. Kinases are an important target for many cancer therapies. However, they can be difficult to selectively target without hitting one of the other ~500 kinases in the human genome. Recent experimental and computational evidence suggests that the cost of confining the kinase to the binding-competent conformation could play a large role in determining inhibitor selectivity, specifically in the case of Src and Abl kinase and the ligand imatinib (also known as the paradigm shifting targeted cancer drug ‘Gleevec’). To test this hypothesis on a larger scale, and to dissect the contribution of this reorganization energy to kinase inhibitor affinity and selectivity, we have developed an approach that combines massively distributed molecular simulations with automated experimental biophysical measurements. Atomistic molecular simulations are run on the Folding@home worldwide distributed computing platform to collect ~ms of simulation data. Markov state models are then constructed of this data to determine the accessible conformations and relative free energies. Alchemical binding free energy calculations are used to assess the ligand binding free energies of selective kinase inhibitors to individual kinetically metastable conformations, and automated biophysical experiments using a novel fluorescence-based assay are used to directly measure binding affinities of kinase inhibitors to a variety of kinases. By analyzing this data together, we believe this work provides a more global picture of the role that kinase conformational reorganization energies play in selectivity, pushing us toward a better understanding of the requirements for selective inhibitor design.
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difference between calpain-1 and calpain-2 is located at the flexible gating loop. Molecular dynamics simulation of ligand-protease Michaelis complex is therefore necessary to capture the difference in the noncovalent binding interaction between the inhibitor and two isoforms and thus to guide the structure-based design. The Michaelis complex model of calpain-2 with a ketoamide compound was built from a cocrystal structure of a potent calpain-1 inhibitor. Effects of catalytic triad protonation states on the stability of the complex were investigated. The model was validated using existing calpain-2 inhibitors. Based on the simulation results, we modified the prime side (C-terminal) of the a-ketoamide peptidomimetics to favor calpain-2 over calpain-1. The dynamic of the calpain gating loop was explored using long-time scale simulation. The results show that molecular dynamics simulation of Michaelis complex can be a critical tool to explore the molecular bases of calpain activation and to design calpain-2 selective inhibitors for clinical therapeutics. 2689-Pos Board B66 Mechanism of Urea Conduction through H. Pylori UreI Ugur Akgun. Physics, Coe College, Cedar Rapids, IA, USA. The carcinogenic bacterium Helicobacter Pylori is found within the stomach of over 75% of the human population, and directly contributes to ailments such as gastritis, stomach ulcers, and stomach cancer. This neutrophile relies on the cytoplasmic hydrolysis of gastric urea into ammonia, which buffers the bacteria from the acidic environment. Urea uptake is a crucial mechanism of survival, and the protein HpUreI regulates and enhances this process. HpUreI is part of the urease operon in many bacteria. There are 7 genes in the urease operon, 2 of which are structural proteins, and 5 of which are accessory proteins. All of these proteins contribute directly to H. Pylori thriving in a low pH environment. The buffering action of the converted urea into ammonia and carbon dioxide boosts the local pH to 5.5 near the membrane. The structure of HpUreI protein shows two constriction sites in all six pores that allow water and urea to pass into the cytoplasm. This study reports results from various Molecular Dynamics simulations, including a high-concentration equilibration model saturated with urea, a steered molecular dynamics (SMD) simulation directing six ligands through each segment of the hexameric structure, and umbrella sampling simulations through the length of the pore. We report the energy profile identifying specific points of constriction and binding with both the ligand and free water saturating the structure. 2690-Pos Board B67 Dissecting the Contribution of Kinase Conformational Reorganization Energies to Inhibitor Selectivity Sonya M. Hanson1, Lucelenie Rodriguez1, Julie M. Behr1, Andrea Rizzi1, Daniel L. Parton1, Kyle A. Beauchamp1, Joshua H. Fass1, Jan-Hendrik Prinz1, Sarah E. Boyce1, Markus A. Seeliger2, Nicholas M. Levinson3, John D. Chodera1. 1 Computational Biology, MSKCC, New York, NY, USA, 2Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA, 3Pharmacology, University of Minnesota, Minneapolis, MN, USA. Kinases are an important target for many cancer therapies. However, they can be difficult to selectively target without hitting one of the other ~500 kinases in the human genome. Recent experimental and computational evidence suggests that the cost of confining the kinase to the binding-competent conformation could play a large role in determining inhibitor selectivity, specifically in the case of Src and Abl kinase and the ligand imatinib (also known as the paradigm shifting targeted cancer drug ‘Gleevec’). To test this hypothesis on a larger scale, and to dissect the contribution of this reorganization energy to kinase inhibitor affinity and selectivity, we have developed an approach that combines massively distributed molecular simulations with automated experimental biophysical measurements. Atomistic molecular simulations are run on the Folding@home worldwide distributed computing platform to collect ~ms of simulation data. Markov state models are then constructed of this data to determine the accessible conformations and relative free energies. Alchemical binding free energy calculations are used to assess the ligand binding free energies of selective kinase inhibitors to individual kinetically metastable conformations, and automated biophysical experiments using a novel fluorescence-based assay are used to directly measure binding affinities of kinase inhibitors to a variety of kinases. By analyzing this data together, we believe this work provides a more global picture of the role that kinase conformational reorganization energies play in selectivity, pushing us toward a better understanding of the requirements for selective inhibitor design.