Mechanism of Dimer Selectivity and Binding Cooperativity of BRAF inhibitors

Reviewer #1 (Public Review):

This manuscript from Clayton and co-authors, entitled "Mechanism of dimer selectivity and binding cooperativity of BRAF inhibitors", aims to clarify the molecular mechanism of BRAF dimer selectivity. Indeed, first-generation BRAF inhibitors, targeting monomeric BRAFV600E, are ineffective in treating resistant dimeric BRAF isoforms. Here, the authors employed molecular dynamics simulations to study the conformational dynamics of monomeric and dimeric BRAF, in the presence and absence of inhibitors. Multi-microsecond MD simulations showed an inward shift of the αC helix in the BRAFV600E mutant dimer. This helped in identifying a hydrogen bond between the inhibitors and the BRAF residue Glu501 as critical for dimer compatibility. The stability of the aforementioned interaction seems to be important to distinguish between dimer-selective and equipotent inhibitors.

The study is overall valuable and robust. The authors used the recently developed particle mesh Ewald constant pH molecular dynamics, a state-of-the-art method, to investigate the correct histidine protonation considering the dynamics of the protein. Then, multi-microsecond simulations showed differences in the flexibility of the αC helix and DFG motif. The dimerization restricts the αC position in the inward conformation, in agreement with the result that dimer-compatible inhibitors can stabilize the αC-in state. Noteworthy, the MD simulations were used to study the interactions between the inhibitors and the protein, suggesting a critical role for a hydrogen bond with Glu501. Finally, simulations of a mixed state of BRAF (one protomer bound to the inhibitor and the other apo) indicate that the ability to stabilize the inward αC state of the apo protomer could be at the basis of the positive cooperativity of PHI1.

One potential weakness in the manuscript is the lack of reported uncertainties related to the analyzed quantities. Providing this information would significantly enhance the clarity regarding the reliability of the analyses and the confidence in the claims presented.

Reviewer #2 (Public Review):

The authors employ molecular dynamics simulations to understand the selectivity of FDA-approved inhibitors within dimeric and monomeric BRAF species. Through these comprehensive simulations, they shed light on the selectivity of BRAF inhibitors by delineating the main structural changes occurring during dimerization and inhibitor action. Notably, they identify the two pivotal elements in this process: the movement and conformational changes involving the alpha-C helix and the formation of a hydrogen bond involving the Glu-501 residue. These findings find support in the analyses of various structures crystallized from dimers and co-crystallized monomers in the presence of inhibitors. The elucidation of this mechanism holds significant potential for advancing our understanding of kinase signaling and the development of future BRAF inhibitor drugs.

The authors employ a diverse array of computational techniques to characterize the binding sites and interactions between inhibitors and the active site of BRAF in both dimeric and monomeric forms. They combine traditional and advanced molecular dynamics simulation techniques such as CpHMD (all-atom continuous constant pH molecular dynamics) to provide mechanistic explanations. Additionally, the paper introduces methods for identifying and characterizing the formation of the hydrogen bond involving the Glu501 residue without the need for extensive molecular dynamics simulations. This approach facilitates the rapid identification of future BRAF inhibitor candidates.

The use of molecular dynamics yields crucial structural insights and outlines a mechanism to elucidate dimer selectivity and cooperativity in these systems. However, the authors could consider the adoption of free energy methods to estimate the values of hydrogen bond energies and hydrophobic interactions, thereby enhancing the depth of their analysis.

Author Response

We thank both reviewers for the positive evaluation of our work and suggestions on how to improve it.

We agree with Reviewer #1 that reporting uncertainties will both clarify and strengthen our arguments. Where applicable, uncertainties will be added in a revised version.

To Reviewer #2’s suggestion of including free energy calculations to estimate the free energies of hydrogen bond and hydrophobic interactions, the current free energy methods are capable of given accurate estimates of the relative binding free energies of similar ligands; however, accurate calculations of the absolute free energies of hydrogen bond and hydrophobic interactions are not feasible yet.

Again, we thank the reviewers for their assessment and suggestions. We will update the manuscript as we have outlined above.