Effect of Acetylation on the Nanofibril Formation of Chitosan from All-Atom De Novo Self-Assembly Simulations.

Chitosan-based materials have broad applications, from biotechnology to pharmaceutics. Recent experiments showed that the degree and pattern of acetylation along the chitosan chain modulate its biological and physicochemical properties; however, the molecular mechanism remains unknown. Here, we report, to the best of our knowledge, the first de novo all-atom molecular dynamics (MD) simulations to investigate chitosan's self-assembly process at different degrees and patterns of acetylation. Simulations revealed that 10 mer chitosan chains with 50% acetylation in either block or alternating patterns associate to form ordered nanofibrils comprised of mainly antiparallel chains in agreement with the fiber diffraction data of deacetylated chitosan. Surprisingly, regardless of the acetylation pattern, the same intermolecular hydrogen bonds mediate fibril sheet formation while water-mediated interactions stabilize sheet-sheet stacking. Moreover, acetylated units are involved in forming strong intermolecular hydrogen bonds (NH-O6 and O6H-O7), which offers an explanation for the experimental observation that increased acetylation lowers chitosan's solubility. Taken together, the present study provides atomic-level understanding the role of acetylation plays in modulating chitosan's physiochemical properties, contributing to the rational design of chitosan-based materials with the ability to tune by its degree and pattern of acetylation. Additionally, we disseminate the improved molecular mechanics parameters that can be applied in MD studies to further understand chitosan-based materials.

Mechanism of Dimer Selectivity and Binding Cooperativity of BRAF Inhibitors.

Aberrant signaling of BRAF V600E is a major cancer driver. Current FDA-approved RAF inhibitors selectively inhibit the monomeric BRAF V600E and suffer from tumor resistance. Recently, dimer-selective and equipotent RAF inhibitors have been developed; however, the mechanism of dimer selectivity is poorly understood. Here, we report extensive molecular dynamics (MD) simulations of the monomeric and dimeric BRAF V600E in the apo form or in complex with one or two dimer-selective (PHI1) or equipotent (LY3009120) inhibitor(s). The simulations uncovered the unprecedented details of the remarkable allostery in BRAF V600E dimerization and inhibitor binding. Specifically, dimerization retrains and shifts the α C helix inward and increases the flexibility of the DFG motif; dimer compatibility is due to the promotion of the α C-in conformation, which is stabilized by a hydrogen bond formation between the inhibitor and the α C Glu501. A more stable hydrogen bond further restrains and shifts the α C helix inward, which incurs a larger entropic penalty that disfavors monomer binding. This mechanism led us to propose an empirical way based on the co-crystal structure to assess the dimer selectivity of a BRAF V600E inhibitor. Simulations also revealed that the positive cooperativity of PHI1 is due to its ability to preorganize the α C and DFG conformation in the opposite protomer, priming it for binding the second inhibitor. The atomically detailed view of the interplay between BRAF dimerization and inhibitor allostery as well as cooperativity has implications for understanding kinase signaling and contributes to the design of protomer selective RAF inhibitors.

Analysis of the ERK Pathway Cysteinome for Targeted Covalent Inhibition of RAF and MEK Kinases.

The ERK pathway is one of the most important signaling cascades involved in tumorigenesis. So far, eight noncovalent inhibitors of RAF and MEK kinases in the ERK pathway have been approved by the FDA for the treatment of cancers; however, their efficacies are limited due to various resistance mechanisms. There is an urgent need to develop novel targeted covalent inhibitors. Here we report a systematic study of the covalent ligandabilities of the ERK pathway kinases (ARAF, BRAF, CRAF, KSR1, KSR2, MEK1, MEK2, ERK1, and ERK2) using constant pH molecular dynamics titration and pocket analysis. Our data revealed that the hinge GK (gate keeper)+3 cysteine in RAF family kinases (ARAF, BRAF, CRAF, KSR1, and KSR2) and the back loop cysteine in MEK1 and MEK2 are reactive and ligandable. Structure analysis suggests that the type II inhibitors belvarafenib and GW5074 may be used as scaffolds for designing pan-RAF or CRAF-selective covalent inhibitors directed at the GK+3 cysteine, while the type III inhibitor cobimetinib may be modified to label the back loop cysteine in MEK1/2. The reactivities and ligandabilities of the remote cysteine in MEK1/2 and the DFG-1 cysteine in MEK1/2 and ERK1/2 are also discussed. Our work provides a starting point for medicinal chemists to design novel covalent inhibitors of the ERK pathway kinases. The computational protocol is general and can be applied to the systematic evaluation of covalent ligandabilities of the human cysteinome.