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

Towards a Molecular Understanding of the Link between Imatinib Resistance and Kinase Conformational Dynamics.

Due to its inhibition of the Abl kinase domain in the BCR-ABL fusion protein, imatinib is strikingly effective in the initial stage of chronic myeloid leukemia with more than 90% of the patients showing complete remission. However, as in the case of most targeted anti-cancer therapies, the emergence of drug resistance is a serious concern. Several drug-resistant mutations affecting the catalytic domain of Abl and other tyrosine kinases are now known. But, despite their importance and the adverse effect that they have on the prognosis of the cancer patients harboring them, the molecular mechanism of these mutations is still debated. Here by using long molecular dynamics simulations and large-scale free energy calculations complemented by in vitro mutagenesis and microcalorimetry experiments, we model the effect of several widespread drug-resistant mutations of Abl. By comparing the conformational free energy landscape of the mutants with those of the wild-type tyrosine kinases we clarify their mode of action. It involves significant and complex changes in the inactive-to-active dynamics and entropy/enthalpy balance of two functional elements: the activation-loop and the conserved DFG motif. What is more the T315I gatekeeper mutant has a significant impact on the binding mechanism itself and on the binding kinetics

RMSF of the A-loop region and IC₅₀ values of imatinib for the studied tyrosine kinases and the designed triple mutants of Src.

<p>RMSF of the A-loop region and IC₅₀ values of imatinib for the studied tyrosine kinases and the designed triple mutants of Src.</p

Entropy and Enthalpy contributions to the DFG-in DFG-out flip as obtained from the linear regression of the Free Energy as a function of temperature.

<p>Entropy and Enthalpy contributions to the DFG-in DFG-out flip as obtained from the linear regression of the Free Energy as a function of temperature.</p

K_d for the Src mutants, Abl and Src WT.

<p>The RMSF values have been averaged from MD simulations.</p

Root mean square fluctuation analysis of TKs and Abl resistant mutants.

<p>(a) RMSF of Abl and Src. Fluctuations of the N-lobe (left) and of the A-loop (right) for the Abl mutants (b). and the TKs (c). Shades of red and blue identify strong and weak binders, respectively. Dotted lines are used for clarity.</p

Free energy of the DFG flip transition.

<p>Free energy surfaces of Abl, Src (adapted from Ref. [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004578#pcbi.1004578.ref029" target="_blank">29</a>]), and Abl drug-resistant mutants projected on the distances between DFG Asp₄₀₄ and Lys₂₉₅ (CV1) and DFG Phe₄₀₅ and Ile₂₉₃ (Leu₁₃₇ in Src) (CV2). The free energy minima corresponding to DFG-in conformations are labeled “IN”, while “OUT” correspond to DFG-out conformations. The contour lines are drawn every 1 kcal/mol.</p

Free energy of the A-loop opening.

<p>Free energy surfaces of Abl, Src, and drug-resistant mutants projected on the optimal path describing the conformational change of the A-loop from open to closed in Src (CV1) and Abl (CV2). The free energy minima corresponding to an extended A-loop active-like conformation are labeled “A”, “B” is used for A-loop semi-closed (inactive) conformations and “C” for fully closed A-loop conformations. The contour lines are drawn every 1 kcal/mol.</p

Free energy of imatinib (un-)binding to Abl and to the T315I ‘gatekeeper’ mutant.

<p>Free energy surfaces associated to the binding of imatinib to WT Abl (top panel) and the T315I Abl “gatekeeper” mutant (bottom panel). The deepest energy minima correspond to the crystallographic binding pose and are labeled A. On the way out, B and B’ correspond to an intermediate state (metastable in WT Abl) where imatinib is in between the DFG and the <i>α</i>C helix. States C and C’ correspond to the “external binding pose”. Interestingly in Abl T315I there are two exit channels and both have an higher barrier than in the WT. The contour lines are drawn every 2 kcal/mol.</p