計算機支援によるペプチド設計の理論と応用

学位の種別: 課程博士審査委員会委員 : (主査)東京大学客員准教授 富井 健太郎, 東京大学教授 菅野 純夫, 東京大学教授 浅井 潔, 東京大学准教授 木立 尚孝, 東京大学客員准教授 KamY. Zhang, 東京大学客員教授 泰地 真弘人University of Tokyo(東京大学

Quantifying Intramolecular Binding in Multivalent Interactions: A Structure-Based Synergistic Study on Grb2-Sos1 Complex

Numerous signaling proteins use multivalent binding to increase the specificity and affinity of their interactions within the cell. Enhancement arises because the effective binding constant for multivalent binding is larger than the binding constants for each individual interaction. We seek to gain both qualitative and quantitative understanding of the multivalent interactions of an adaptor protein, growth factor receptor bound protein-2 (Grb2), containing two SH3 domains interacting with the nucleotide exchange factor son-of-sevenless 1 (Sos1) containing multiple polyproline motifs separated by flexible unstructured regions. Grb2 mediates the recruitment of Sos1 from the cytosol to the plasma membrane where it activates Ras by inducing the exchange of GDP for GTP. First, using a combination of evolutionary information and binding energy calculations, we predict an additional polyproline motif in Sos1 that binds to the SH3 domains of Grb2. This gives rise to a total of five polyproline motifs in Sos1 that are capable of binding to the two SH3 domains of Grb2. Then, using a hybrid method combining molecular dynamics simulations and polymer models, we estimate the enhancement in local concentration of a polyproline motif on Sos1 near an unbound SH3 domain of Grb2 when its other SH3 domain is bound to a different polyproline motif on Sos1. We show that the local concentration of the Sos1 motifs that a Grb2 SH3 domain experiences is approximately 1000 times greater than the cellular concentration of Sos1. Finally, we calculate the intramolecular equilibrium constants for the crosslinking of Grb2 on Sos1 and use thermodynamic modeling to calculate the stoichiometry. With these equilibrium constants, we are able to predict the distribution of complexes that form at physiological concentrations. We believe this is the first systematic analysis that combines sequence, structure, and thermodynamic analyses to determine the stoichiometry of the complexes that are dominant in the cellular environment

Understanding Molecular Mechanisms of Protein Kinases Regulation and Inhibition

Protein kinases (PKs) play a key role in regulating cellular processes. Kinase dysfunction can lead to disease, thus kinases are important targets for drug design and a fundamental class of pharmacological targets for anti-cancer therapy. Among protein kinases, B-Raf and c-Src are remarkably interesting as anticancer drug targets because of their important role in cancer onset (B-Raf) and progression (c-Src). This thesis is mainly focused on the characterization of the molecular mechanism at the basis of the regulation and inhibition of these remarkable PKs. By using nuclear magnetic resonance (NMR) and molecular dynamics simulations (MD) we have studied in great details their activation dynamics, their inhibition and the effect of clinically-relevant oncogenic mutations on their structure and dynamics. C-Scr was the first viral oncogenic protein discovered, is involved in metastasis and is mutated in 50% of colon, liver, lung, breast and pancreas tumours. Upon phosphorylation, various conserved structural elements, including the activation loop, switch from an inactive to an active form able to bind ATP and phosphorylate a substrate in a cellular signalling process leading to cell replication. In this thesis, we will discuss how phosphorylation drastically changes the dynamics of the C-lobe in c-Src by NMR analysis, a phenomenon not easily accessible by static crystallographic studies. The second part of the thesis will be focused on B-Raf, a protein serine/threonine kinase. B-Raf kinase is a key target for the treatment of melanoma, since a single mutation (V600E) is found in more than 50% of all malignant melanomas. Despite their importance, the molecular mechanisms explaining the increased kinase activity in this mutant remains elusive. As kinase activity is often tightly regulated by one or more conformational transitions between an active and an inactive state, which are difficult to be observed experimentally, molecular dynamics simulations are often useful to interpret the experimental results. In this project, we will examine the mechanism by which the V600E mutation enhances the activity of the B-Raf monomer. We will also employ a combination of MD techniques with NMR experiments to fully map the effects of the mutation on the conformational landscape of B-Raf. An understanding at the atomic level of the mechanisms leading to their activation and inhibition is an extremely important goal in anti-cancer drug discovery. A better understanding of these proteins' mechanisms might lead to more potent and less toxic drugs. Finally, I report on the studies of a much small domain often associated with PKs in regulatory pathways: the WW domain. By using a combination of MD simulations and NMR, we have characterized the effect of a pathogenic mutation on its folding landscape

UNIQUE ALLOSTERIC MECHANISM REGULATING PROTEIN-PROTEIN INTERACTION THROUGH PHOSPHORYLATION: A CASE STUDY OF THE CONFORMATIONAL CHANGES IN THE SYK TANDEM SH2 PROTEIN

Spleen tYrosine Kinase (Syk) is one of the crucial signaling proteins involved in the development of immune cells and the initiation of inflammatory responses. Syk is a 72-kDa kinase comprising three folded domains: two SH2 domains and a catalytic domain. The tandem SH2 domains connected by linker A are key to the regulation of Syk activity. Immune signaling through Syk is initiated by the binding of the tandem SH2 to the dpITAMs found on immune cell receptors. The high-affinity, bifunctional binding of Syk tandem SH2 to the immunoreceptor requires that the two phosphotyrosines (pTyr) of the dpITAM fit the spacing and orientation of the two SH2 domains. Phosphorylation at Y130, which introduces a negative charge in the linker A, disrupts the domain-domain coupling and causes the binding affinity of each SH2 domain to the pTyr of the dpITAM to differ; the optimal binding to dpITAM seen with the unphosphorylated form is therefore no longer possible. Because Y130 is far from the dpITAM binding sites, phosphorylation of Y130, therefore, negatively regulates the association of Syk with immunoreceptors through an allosteric mechanism. In this work, the molecular detail of this allosteric mechanism was investigated using molecular dynamics simulations. The use of μs-trajectories enabled us to define the perturbation caused by Y130 phosphorylation to the domain-domain dynamics and the conformational ensemble of the tandem SH2. A picture in which the Syk-immunoreceptor interaction is regulated by a mechanism of dynamic allostery emerged

Protein-carbohydrate and protein-protein interactions: using models to better understand and predict specific molecular recognition

Any molecular recognition event results in a change in the free energy of the system. The extent of this change is related to the association constant, such that the more negative the free energy change is, the tighter the interaction between receptor and ligand. Protein-carbohydrate interactions play a critical role in signal transduction, innate immunity, and metabolism. Modeling these interactions is somewhat complicated by the inherent flexibility of carbohydrates as well as their relatively large number of functional groups. An empirical scoring function for docking carbohydrates to proteins, specifically tailored to predict both the correct binding orientation and free energy of binding of the carbohydrate-ligand/protein-receptor complex, will be presented. This new scoring function can predict free energies of binding to within 1.1 kcal/mol residual standard error, a definite improvement over existing scoring functions that result in standard errors well over 2 kcal/mol. Application of automated docking methodology to determine carbohydrate recognition specificity of the C-type lectin, human surfactant protein D, will also be presented. In the second part of the thesis, the role of pi-stacking interactions (e.g. between Tyr side chains) in stabilizing protein folds will be discussed. A 17-residue peptide derived from the naturally occurring anti-microbial peptide tachyplesin I was investigated using NMR spectroscopy. NOE cross-peaks were observed, confirming the existence of this interaction in solution. In the final part of the thesis, a quantitative NMR investigation into the self-association behavior of the regulatory domains of several Tec family member kinases will be presented. Of particular interest, self-association within Bruton\u27s tyrosine kinase (Btk) regulatory domains occurs through the formation of an asymmetric homodimer. Together this work demonstrates the importance of rigorous biophysical characterization of biomolecular recognition events and the interdependence of computational modeling and experimentation

IMPROVING RATIONAL DRUG DESIGN BY INCORPORATING NOVEL BIOPHYSICAL INSIGHT

Computer-aided drug design is a valuable and effective complement to conventional experimental drug discovery methods. In this thesis, we will discuss our contributions to advancing a number of outstanding challenges in computational drug discovery: understanding protein flexibility and dynamics, the role of water in small molecule binding and using and understanding large amounts of data. First, we describe the molecular steps involved in the induced-fit binding mechanism of p53 and MDM2. We use molecular dynamics simulations to understand the key chemistry responsible for the dynamic transition between the apo and holo structures of MDM2. This chemistry involves not only the indole side chain of the anchor residue of p53, Trp23, but surprisingly, the beta-carbon as well. We demonstrate that this chemistry plays a key role in opening the binding site by coordinating the position and orientation of MDM2 residues, Val93 and His96, through a previously undescribed transition state. We confirm these findings by observing that this chemistry is preserved in all available inhibitor-bound MDM2 co-crystal structures. Second, we discuss our advances in understanding water molecules in ligand binding sites by data mining the structural information of water molecules found in X-ray crystal structures. We examine a large set of paired bound and unbound proteins and compare the water molecules found in the binding site of the unbound structure to the functional groups on the ligand that displace them upon binding. We identify a number of generalized functional groups that are associated with characteristic clusters of water molecules. This information has been utilized in several successful and ongoing virtual screens. Third, we discuss software that we have developed that allows for very efficient exploration and selection of virtual screening results. Implemented as a PyMOL plugin, ClusterMols clusters compounds based on a user-defined level of chemical similarity. The software also provides advanced visualization tools and a number of controls for quickly navigating and selecting compounds of interest, as well as the ability to check online for available vendors. Finally, we present several published examples of modeling protein-lipid and protein-small molecules interactions for a number of important targets including ABL, c-Src and 5-LOX

Synthesis and evaluation of conformationally constrained peptide replacements and studies toward the total synthesis of kidamycin

textA series of conformationally constrained pseudopeptides derivative of the tripeptide pYVN were designed and synthesized. The conformationally restricted compounds contained either trans- or cis-cyclopropanes as replacements to enforce locally extended and reverse turn peptide conformations, respectively. In addition, the proper flexible control molecules were prepared. All compounds were evaluated for the ability to bind to the Grb2-SH2 domain in order to determine the energetic consequences of introducing a conformational constraint into peptide ligands. No difference in the ∆Gbinding between the trans-cyclopropane and its control partner was observed. Surprisingly, there was an entropic disadvantage when comparing the binding energetics of the constrained and flexible pseudopeptides. Therefore, the introduction of the cyclopropane constraint was associated with an entropic disadvantage in the system, which is the opposite of conventional wisdom. An X-ray crystal structure of the trans- cyclopropane containing ligand bound to the Grb2-SH2 domain was obtained and vii discussed. On the other hand, cis-cyclopropane containing pseudopeptides do not seem to enforce the desired turn conformation of the ligand. A method to allow access to unsymmetrical C-aryl glycoside natural products was developed through employing a disposal tether to enforce the desired regioselectivity in a [4+2] cycloaddition between benzyne and glycosyl-substituted furan. Application of this novel strategy toward the synthesis of kidamycin is discussed. Additional synthetic routes, including utilizing Suzuki’s O → C glycoside rearrangement are also provided. Studies toward the synthesis of sugar ring E and F are illustrated.Chemistry and BiochemistryChemistr

Quantitative Analysis of EGFR Phosphorylation and SH2 Domain Binding in vivo

The work presented in the following thesis dissertation examines the regulation of phosphotyrosine (pY) signaling, an essential cellular process that relies on the activity of three major protein classes: Tyrosine kinases (TKs) which induce pY signaling by phosphorylating tyrosine residues on substrate proteins, Protein tyrosine phosphatases (PTPs) which suppress pY signaling by removing phosphate moieties from tyrosine phosphorylated proteins and Src-Homology 2 (SH2) containing proteins which bind to tyrosine phosphorylated proteins and connect them to downstream signaling pathways. The effects of kinase localization, temporal changes in kinase activation, SH2 protein concentration, and negative feedback from downstream signaling pathways are all examined by the research presented here. This is accomplished by exploiting the Epidermal Growth Factor Receptor (EGFR), a clinically important transmembrane TK, and its SH2 protein mediated downstream pathways. Using EGFR signaling as a tool, this dissertation research attempts to define innate properties of pY signaling systems which are broadly applicable and advance our understanding of the field

Computational Modeling of Protein Kinases: Molecular Basis for Inhibition and Catalysis

Protein kinases catalyze protein phosphorylation reactions, i.e. the transfer of the γ-phosphoryl group of ATP to tyrosine, serine and threonine residues of protein substrates. This phosphorylation plays an important role in regulating various cellular processes. Deregulation of many kinases is directly linked to cancer development and the protein kinase family is one of the most important targets in current cancer therapy regimens. This relevance to disease has stimulated intensive efforts in the biomedical research community to understand their catalytic mechanisms, discern their cellular functions, and discover inhibitors. With the advantage of being able to simultaneously define structural as well as dynamic properties for complex systems, computational studies at the atomic level has been recognized as a powerful complement to experimental studies. In this work, we employed a suite of computational and molecular simulation methods to (1) explore the catalytic mechanism of a particular protein kinase, namely, epidermal growth factor receptor (EGFR); (2) study the interaction between EGFR and one of its inhibitors, namely erlotinib (Tarceva); (3) discern the effects of molecular alterations (somatic mutations) of EGFR to differential downstream signaling response; and (4) model the interactions of a novel class of kinase inhibitors with a common ruthenium based organometallic scaffold with different protein kinases. Our simulations established some important molecular rules in operation in the contexts of inhibitor-binding, substrate-recognition, catalytic landscapes, and signaling in the EGFR tyrosine kinase. Our results also shed insights on the mechanisms of inhibition and phosphorylation commonly employed by many kinases

Molecular dynamics simulations and drug discovery

This review discusses the many roles atomistic computer simulations of macromolecular (for example, protein) receptors and their associated small-molecule ligands can play in drug discovery, including the identification of cryptic or allosteric binding sites, the enhancement of traditional virtual-screening methodologies, and the direct prediction of small-molecule binding energies. The limitations of current simulation methodologies, including the high computational costs and approximations of molecular forces required, are also discussed. With constant improvements in both computer power and algorithm design, the future of computer-aided drug design is promising; molecular dynamics simulations are likely to play an increasingly important role