Novel Allosteric Sites on Ras for Lead Generation

Abstract

Aberrant Ras activity is a hallmark of diverse cancers and developmental diseases. Unfortunately, conventional efforts to develop effective small molecule Ras inhibitors have met with limited success. We have developed a novel multi-level computational approach to discover potential inhibitors of previously uncharacterized allosteric sites. Our approach couples bioinformatics analysis, advanced molecular simulations, ensemble docking and initial experimental testing of potential inhibitors. Molecular dynamics simulation highlighted conserved allosteric coupling of the nucleotide-binding switch region with distal regions, including loop 7 and helix 5. Bioinformatics methods identified novel transient small molecule binding pockets close to these regions and in the vicinity of the conformationally responsive switch region. Candidate binders for these pockets were selected through ensemble docking of ZINC and NCI compound libraries. Finally, cell-based assays confirmed our hypothesis that the chosen binders can inhibit the downstream signaling activity of Ras. We thus propose that the predicted allosteric sites are viable targets for the development and optimization of new drugs.

Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function

“…The C-terminal pocket 3 is bounded by α-helix 5 plus the N-terminal and preceding loop residues of β5 and β6. Remarkably, each of these pockets is very similar to those we have previously characterized using a different approach and a different set of probes (13). Mapping the energetic preference of small-molecule fragments by FTMap (33) identified the same binding hotspots, with binding preference for each site modulated by protein motion (Fig.…”

“…4B). To test whether the ligands stabilize a particular Ras conformation, we used a principal component (PC)-based analysis developed previously (13,(34)(35)(36) to map conformers from K-Ras-SRJ23 trajectories onto a PC plane defined by crystal structures. Fig.…”

“…Although the potential therapeutic value and mechanism of action of these compounds are still under investigation, it is clear that they do not directly bind to Ras. Recent efforts by us (13) and others (14)(15)(16) toward direct inhibition of Ras have yielded promising initial results. For instance, using fragment screening, crystallography, and other methods, two groups reported ligands that directly bind Ras and inhibit GEF-dependent nucleotide exchange (15,16).…”

Discovery of multiple hidden allosteric sites by combining Markov state models and experiments

“…For example, there are a variety of methods for understanding how information flows from one region of a protein to another (15)(16)(17)(18)(19), as well as for identifying potential binding pockets (20)(21)(22)(23)(24). More recent work has accounted for both of these ingredients (25,26). Although these methods are important developments, many are only applicable to small proteins operating on fast timescales.…”

The Ras–Membrane Interface: Isoform-Specific Differences in the Catalytic Domain

“…Codon mutation bias among the isoforms at 100% conserved effector lobe sites indicates that the allosteric features of Ras cannot be ignored. Although allosteric lobe targeting has only recently been considered a viable option, disrupting the structure-function correlation between the allosteric and effector lobe could be the key to unlocking new Ras inhibitors (75). Detailed understanding of the structural and functional impacts of isoform-specific differences in the allosteric lobe will open new venues for targeting Ras, translating to treatment of specific cancers uniquely associated with each of the isoforms.…”