Therapeutic Targeting the Allosteric Cysteinome of RAS and Kinase Families

Abstract

Allosteric mechanisms are pervasive in nature, but human-designed allosteric perturbagens are rare. The history of KRAS^G12C inhibitor development suggests that covalent chemistry may be a key to expanding the armamentarium of allosteric inhibitors. In that effort, irreversible targeting of a cysteine converted a non-deal allosteric binding pocket and low affinity ligands into a tractable drugging strategy. Here we examine the feasibility of expanding this approach to other allosteric pockets of RAS and kinase family members, given that both protein families are regulators of vital cellular processes that are often dysregulated in cancer and other human diseases. Moreover, these heavily studied families are the subject of numerous drug development campaigns that have resulted, sometimes serendipitously, in the discovery of allosteric inhibitors. We consequently conducted a comprehensive search for cysteines, a commonly targeted amino acid for covalent drugs, using AlphaFold-generated structures of those families. This new analysis presents potential opportunities for allosteric targeting of validated and understudied drug targets, with an emphasis on cancer therapy.

Keywords: KRAS inhibitor; allosteric inhibitor; covalent inhibitor; cysteinome; kinase inhibitor.

Figure 1.

Allosteric regulation is common in biology and can be targeted with drugs. (A) Hemoglobin in the T (Tense, left) and R (Relaxed, right) conformations. The T conformation is stabilized in the absence of oxygen and has low binding affinity. Binding of oxygen transitions the tetramer to the R state, increasing oxygen binding affinity. PDB: 2HHB, 1HHO. α subunit in white, β in grey, heme in red, oxygen in blue. (B) GK changes conformation to become active when the allosteric site is bound. Glucose in blue, allosteric ligand in red. PDB: 1V4T, 1V4S. (C) Binding of F2,6BP to the allosteric site of the homotetrameric phosphofructokinase-1 (PFK1) complex causes rotation of the protomers relative to each other to stimulate enzymatic activity. ATP, ADP and F6P in red, F1,6BP in blue. PDB: 4XYJ, 4XZ2. (D) Citrate allosterically activates human acetyl-CoA carboxylase (ACC) by stabilizing filament formation. PDB: 6G2D,6G2I.

Figure 2.

RAS is allosterically targetable because of mobility of the switches. (A) Superposition of 190 experimentally derived KRAS protein structures show the switches are the primary mobile elements. GTP in red, SWI in yellow and SWII in green. (B) Superposition of 91 experimentally derived structures of KRAS proteins bound to inhibitors demonstrates the location of major allosteric pockets. After the alignment, only ligands are displayed relative to a prototypical SW2-bound KRAS structure (PDB: 6OIM). G pocket binders in red, SW2 binders in orange, and SW1/2 binders in blue. (C) KRAS^G12C inhibitors are structurally related. Structurally similar components are highlighted in color for emphasis. (D) Simulated docking of SCH-54292 (orange) shows binding to the SW2 pocket of KRAS.

Figure 3.

Kinases are allosterically targetable at multiple sites. Representative binders of allosteric sites are shown relative to a prototypical kinase fold (PDB 2G2F). The conformation of the mobile αC helix is substituted from PDB 7JUY to illustrate the typical conformation seen with type III inhibitors. ATP in red (from PDB 1S9J), Compound 38 in orange (from PDB 3F9N), CHEMBL1230164 in pink (from PDB 3NEW), Compound 3 in chocolate (from PDB 3O2M), RS1 in dark blue (from PDB 4RQK), GNF2 in sky blue (from PDB 3K5V), and cobimetinib in cyan (from PDB 7JUY).

Figure 4.

Allosteric sites for RAS and kinase families are not conserved. Protein sequences for kinases or RAS families were aligned and relative conservation scores for each amino acid were calculated using the ConSurf server and plotted on the surface of a prototypical family member fold. (A) Conservation scores for the RAS family are plotted on the surface of KRAS PDB 4LDJ. Representative compounds are ARS1620 (orange from PDB 5V9U), XY-02-075 (red from PDB 5KYK) and CH-2 (blue from PDB 6GQX). (B) Conservation scores for 493 kinases are plotted on the 3D structure of Aurora A (PDB 3E5A). Representative compounds: Compound 38 in orange (from PDB 3F9N), GNF2 in cyan (from PDB 3K5V), Compound 3 in chocolate (from PDB 3O2M), RS1 in blue (from PDB 4RQK), CHEMBL1230164 in pink (from PDB 3NEW) and ATP in red (from PDB 1S9J).

Figure 5.

Potentially accessible cysteines in allosteric RAS pockets. (A, C, E) Spheres highlight αC corresponding to potentially accessible cysteines in indicated allosteric binding sites. Table 2 provides a list of specific family members. RAS protein in white, prototypical compound in yellow. (A, 5KYK; C, 6GQX; and E, 5V9U). (B, D, F) Dendrograms of the RAS family provide a high-level view of which members have targetable cysteines for the indicated pockets. Dendrograms generated using the Interactive Tree of Life (iTOL).

Figure 6.

Potentially accessible cysteines in allosteric kinase pockets. (A, C, E, G, I, K) Similar to Fig 5, spheres highlight α C corresponding to potentially accessible cysteines in indicated allosteric binding sites. Table 4 provides a list of specific family members. Kinase in white, and prototypical compounds in yellow (A, 4AW1; C, 3K5V; E, 3LW0; G, 3F9M; I, 3NEW; and K, 3O2M). (B, D, F, H, J, L) Dendrograms of the kinase family provide a high level view of which members have targetable cysteines for the indicated pockets. Dendrograms generated using KinMap.