Mechanism of Dimer Selectivity and Binding Cooperativity of BRAF Inhibitors

Aberrant signaling of BRAFV600E is a major cancer driver. Current FDA-approved RAF inhibitors selectively inhibit the monomeric BRAFV600E 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 BRAFV600E 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 BRAFV600E dimerization and inhibitor binding. Specifically, dimerization retrains and shifts the αC helix inward and increases the flexibility of the DFG motif; dimer compatibility is due to the promotion of the αC-in conformation, which is stabilized by a hydrogen bond formation between the inhibitor and the αC Glu501. A more stable hydrogen bond further restrains and shifts the αC 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 BRAFV600E inhibitor. Simulations also revealed that the positive cooperativity of PHI1 is due to its ability to preorganize the αC 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.


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2FB8 and Ref (Tkacik et al., 2023) (Tovorafenib).Note, the PDB entries indicated by an astrisk are co-crystal structures in complex with the wild type BRAF (the BRAF V600E forms are unavailable).All structures contain an inhibitor in each protomer, with the exception of PLX7904; in the PDB entry 4XV1, PLX7904 is only present in protomer A. The C helix position (in Å) is defined in the main text.Two values refer to the two protomers.
The back pockets (BPs) occupied was calculated by KLIFS (Kooistra et al., 2016) based on the definition of Liao (Liao, 2007).K-E refers to the distance (in Å) between the amine nitrogen of Lys483 and the nearest carboxylate oxygen of Glu501.
System, protomer C position (Å) K-E distance (Å) DFG dihedral (     (2022), the charged HIP state was defined by  < 0.2, while the neutral states were defined by  > 0.8 and  < 0.2 (HID) or  < 0.8 (HIE).The last 5 ns of the replica runs were used for the calculation. .RMSD time series for the dimer simulations.Time series plots of the RMSD of the protomer in each dimer simulation.The RMSD was calculated using heavy atoms of the protomer backbone, excluding residues on the a-loop and C-helix.Each of the three replicas are represented as a separate line; for simplicity, a stride of 10 ns was used while plotting.

Figure 1 .
Figure 1.Chemical structures of the monomer-selective, equipotent, and dimer-selective BRAF V600E inhibitors in Supplementary Table1.The chemical group responsible for forming the hydrogen bonds with E501 and/or D594 are circled in red.
Figure 1.Chemical structures of the monomer-selective, equipotent, and dimer-selective BRAF V600E inhibitors in Supplementary Table1.The chemical group responsible for forming the hydrogen bonds with E501 and/or D594 are circled in red.

Figure 2 .Figure 3 .
Figure 1.Chemical structures of the monomer-selective, equipotent, and dimer-selective BRAF V600E inhibitors in Supplementary Table1.The chemical group responsible for forming the hydrogen bonds with E501 and/or D594 are circled in red.

Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 9 .Figure 10 .
Figure 4. Convergence of the protonation/tautomer states of H477 and H510 at pH 7.5.Running average probability of each protonation/tautomer state for H477 and H510 at pH 7.5 as a function of simulation time (top plots for protomer A and bottom plots for protomer B).Convergence of the probability occurs after roughly 2 ns, with little change after 5 ns.

Table 1 .
List of the BRAF V600E inhibitors and the structure features of the co-crystal structures in the PDB.The monomer and dimer selectivities of inhibitors in black are based on the experimental data in Ref(Adamopoulos *

Table 2 .
Average value and standard deviation of reported quantities from out dimeric BRAF simulations, separated by system and protomer.Each quantity was calculated for each replica after removing the first 2 s for equilibration.