A dual origin for Bcr-Abl gene translocation/fusion as dynamics of synergism of the hematopoietic stem cell and hemangioblast in chronic myeloid leukemia

Contextual BCR-ABL tyrosine kinase over-activity determines in formulated fashion the emergence of proliferation and anti-apoptosis that arise largely as derived phenomena of otherwise homeostatic mechanisms of the c-ABL gene within hematopoietic stem cells and hemangioblasts in the bone marrow. The ability to suppress almost completely, both in terms of phenotype and cytogenetically, the myeloid cell line expansion by imatinib mesylate is indicative of a phenomenon that depends strictly on the transformed status of the cell of origin in the chronic myeloid leukemia process. It is with relevance to complex participation of the dynamics of the fused BCR- ABL protein product that contextual conditioning of the cells of origin of the gene translocation further motivates the dimensional expansion of the transformed myeloid cell clones to increasing proliferative rates, thus leading to blast crisis as eventual loss of differentiating potential.peer-reviewe

Modeling the effect of pathogenic mutations on the conformational landscape of protein kinases

Most proteins assume different conformations to perform their cellular functions. This conformational dynamics is physiologically regulated by binding events and post-translational modifications, but can also be affected by pathogenic mutations. Atomistic molecular dynamics simulations complemented by enhanced sampling approaches are increasingly used to probe the effect of mutations on the conformational dynamics and on the underlying conformational free energy landscape of proteins. In this short review we discuss recent successful examples of simulations used to understand the molecular mechanism underlying the deregulation of physiological conformational dynamics due to non-synonymous single point mutations. Our examples are mostly drawn from the protein kinase family

Molecular Determinants Underlying Binding Specificities of the Abl Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots With Network Analysis of Residue Interactions and Coevolution

Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating residues. This study has outlined mechanisms by which inhibitor binding could modulate resilience and efficiency of allosteric interactions in the kinase structures, while preserving structural topology required for catalytic activity and regulation

Exploring the role of conformational dynamics in the regulation of tyrosine kinases.

Tyrosine kinases (TKs) are a family of signalling proteins of great pharmaceutical im- portance, as they are involved in the regulation of most cellular pathways. TKs catalytic activity is strictly regulated by conformational changes and post-translational modifi- cations, and their deregulation is involved in numerous human diseases, ranging from cancer to autoimmune diseases. Among tyrosine kinases, Abl and Src are of particular interest for cancer research. The Abl domain in the BCR-Abl fusion protein is the main cause of chronic myeloid leukemia, and it was the target of the first successful anti- leukemic therapy, the powerful kinase inhibitor imatinib. We now know that imatinib effectively inhibits BCR-Abl, as well as Kit and Lck kinases, by binding to a specific inactive state, in which the conserved Asp-Phe-Gly motif (DFG) assumes a peculiar "out" conformation. Still, there are many questions on its mode of action. For instance, other TKs with an extended identity with Abl (such as Src, which has 45% sequence identity) bind much less strongly to imatinib, in spite of very similar binding mode. Moreover, the mode of action of drug-resistant mutations that induce imatinib resis- tance and cause an increasing number of relapses in patients under treatment, is still poorly understood. Understanding the molecular mechanisms responsible for the ob- served differences in imatinib activity, is essential for the development of new selective anticancer drugs. In this thesis, by using computational and experimental approaches, I have investigated the reasons leading to drug resistance and the differential binding affinity in homologous TKs. A combination of enhanced sampling molecular dynam- ics simulations (such as parallel tempering metadynamics or PTmetaD) were used to reconstruct and compare the free energy landscape associated with the relevant con- formational changes. Mutagenesis and isothermal titration calorimetry were used to validate the computational results

A Src-Like Inactive Conformation in the Abl Tyrosine Kinase Domain

The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML). The recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG) motif is flipped by approximately 180° with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-Src. We present a structure of the kinase domain of Abl, determined in complex with an ATP–peptide conjugate, in which the protein adopts an inactive conformation that resembles closely that of the Src kinases. An interesting aspect of the Src-like inactive structure, suggested by molecular dynamics simulations and additional crystal structures, is the presence of features that might facilitate the flip of the DFG motif by providing room for the phenylalanine to move and by coordinating the aspartate side chain as it leaves the active site. One class of mutations in BCR–Abl that confers resistance to imatinib appears more likely to destabilize the inactive Src-like conformation than the active or imatinib-bound conformations. Our results suggest that interconversion between distinctly different inactive conformations is a characteristic feature of the Abl kinase domain

Molecular Matchmaking: A Computational Study of the Electrostatic Interaction Between Chronic Myeloid Leukemia Drugs and Bcr-Abl Oncoprotein

In this project, we systematically use several computational techniques such as charge optimization and component analysis to study molecular recognition and binding in the chronic myeloid leukemia (CML) drug systems. Using CML drugs and their biological target, the Bcr-Abl oncoprotein, we systematically conduct a comparative analysis on five CML drugs bound to both the wild-type (WT) and T315I mutant Abl kinase. While early generation drugs (imatinib, nilotinib, and dasatinib) interact with Thr315 via a hydrogen bond, novel drugs ponatinib and PPY-A bypass interacting with Thr315 altogether. With the mutation to Ile at position 315, early generation drugs may experience a significant loss in favorable binding due to loss of electrostatic interaction and introduction of steric hindrance. To investigate the differential binding of these drugs to the WT and mutant, we optimize each of the drugs to the Abl kinase, allowing us to study how each drug binds to the native form. We also optimize PPY-A and ponatinib to the mutant T315I, comparing this charge distribution with the one generated from optimizing to the native form. Using component analysis, we identify chemical moieties of each drug that contribute favorably or unfavorably to the electrostatic free energy of binding. Taken together, we hope that by studying CML drugs, we will gain some insight into the larger picture of electrostatic binding interaction and potentially provide future direction for rational drug design and battling drug resistance

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

Mutation D816V Alters the Internal Structure and Dynamics of c-KIT Receptor Cytoplasmic Region: Implications for Dimerization and Activation Mechanisms

The type III receptor tyrosine kinase (RTK) KIT plays a crucial role in the transmission of cellular signals through phosphorylation events that are associated with a switching of the protein conformation between inactive and active states. D816V KIT mutation is associated with various pathologies including mastocytosis and cancers. D816V-mutated KIT is constitutively active, and resistant to treatment with the anti-cancer drug Imatinib. To elucidate the activating molecular mechanism of this mutation, we applied a multi-approach procedure combining molecular dynamics (MD) simulations, normal modes analysis (NMA) and binding site prediction. Multiple 50-ns MD simulations of wild-type KIT and its mutant D816V were recorded using the inactive auto-inhibited structure of the protein, characteristic of type III RTKs. Computed free energy differences enabled us to quantify the impact of D816V on protein stability in the inactive state. We evidenced a local structural alteration of the activation loop (A-loop) upon mutation, and a long-range structural re-organization of the juxta-membrane region (JMR) followed by a weakening of the interaction network with the kinase domain. A thorough normal mode analysis of several MD conformations led to a plausible molecular rationale to propose that JMR is able to depart its auto-inhibitory position more easily in the mutant than in wild-type KIT and is thus able to promote kinase mutant dimerization without the need for extra-cellular ligand binding. Pocket detection at the surface of NMA-displaced conformations finally revealed that detachment of JMR from the kinase domain in the mutant was sufficient to open an access to the catalytic and substrate binding sites

INVESTIGATING ALLOSTERIC REGULATION OF ABL AND BCR-ABL KINASES: IMPLICATIONS FOR SMALL MOLECULE INHIBITORS

BCR-ABL is the oncogenic protein-tyrosine kinase responsible for the pathogenesis of chronic myelogenous leukemia (CML). Clinical management of CML has been revolutionized by imatinib, a selective ATP-competitive inhibitor of BCR-ABL kinase activity. Despite this clinical success, imatinib is less effective in advanced disease due to the emergence of drug resistant BCR-ABL mutants. Resistant mutations often arise in the drug binding site and include the most recalcitrant gatekeeper mutation, T315I. Other mutations arise outside the active site and allosterically reduce imatinib binding by promoting the active kinase conformation. Recently, a new class of allosteric BCR-ABL inhibitors, of which GNF-2 is the prototype, has been reported that targets the myristate-binding pocket of ABL. These compounds stabilize the inactive conformation of ABL and work in concert with ATP-competitive inhibitors to overcome imatinib resistance. Mounting evidence supports a regulatory influence of the non-catalytic SH3 and SH2 domains on BCR-ABL kinase domain. The major focus of this study was to exploit the intramolecular SH3:linker interaction, to stabilize the downregulated kinase domain conformation of BCR-ABL and sensitize the kinase to both imatinib and GNF-2. To achieve this goal, I engineered High Affinity Linker (HAL) variants of both ABL and BCR-ABL in which SH3:linker interaction was tightened through sequential addition of proline residues to the linker. Enhanced SH3:linker interaction induced long-range suppressive effects on the kinase activity in c-ABL, allosterically stabilized both the active site and the myristate-binding pocket, and sensitized BCR-ABL to small molecule inhibitors. Src family kinases (SFKs) are important mediators of BCR-ABL signal transduction and oncogenesis in CML. SFKs also play important roles in clinical resistance to imatinib in the absence of BCR-ABL mutations. In the second part of my project, I explored the effect of SFK- selective inhibitor, pyrazolopyrimidine A-419259, on myeloid cells transformed with clinically relevant imatinib resistant BCR-ABL mutants. While proliferation of cells expressing BCR-ABL E255V and Y253H was inhibited by A-419259, BCR-ABL T315I cells were not. Surprisingly, cells expressing BCR-ABL-T315I maintained SFK activity in the presence of the inhibitor. This observation suggests that BCR-ABL-T315I induces cross-resistance to drugs that inhibit SFKs in CML through direct phosphorylation of the SFKs