Inhibition of RAS: proven and potential vulnerabilities

Abstract

RAS is a membrane localized small GTPase frequently mutated in human cancer. As such, RAS has been a focal target for developing cancer therapeutics since its discovery nearly four decades ago. However, efforts to directly target RAS have been challenging due to the apparent lack of readily discernable deep pockets for binding small molecule inhibitors leading many to consider RAS as undruggable. An important milestone in direct RAS inhibition was achieved recently with the groundbreaking discovery of covalent inhibitors that target the mutant Cys residue in KRAS(G12C). Surprisingly, these G12C-reactive compounds only target mutant RAS in the GDP-bound state thereby locking it in the inactive conformation and blocking its ability to couple with downstream effector pathways. Building on this success, several groups have developed similar compounds that selectively target KRAS(G12C), with AMG510 and MRTX849 the first to advance to clinical trials. Both have shown early promising results. Though the success with these compounds has reignited the possibility of direct pharmacological inhibition of RAS, these covalent inhibitors are limited to treating KRAS(G12C) tumors which account for <15% of all RAS mutants in human tumors. Thus, there remains an unmet need to identify more broadly efficacious RAS inhibitors. Here, we will discuss the current state of RAS(G12C) inhibitors and the potential for inhibiting additional RAS mutants through targeting RAS dimerization which has emerged as an important step in the allosteric regulation of RAS function.

Keywords: RAS GTPase; allosteric inhibitor; cancer; chemotherapy; dimerization; monobody.

Figure 1.. RAS mutations in cancer.

A. Presence of RAS mutations in human cancers. Data was taken from the AACR Project GENIE (7). B. Percentages of tumors possessing KRAS(G12C) mutation. PAAC, pancreatic adenocarcinoma; PAC, pancreatic carcinoma; CAC, colorectal adenocarcinoma; CC, colorectal cancer: RC, rectal cancer; LAC, lung adenocarcinoma; NSCLC, non-small cell lung carcinoma; MST, malignant solid tumor; SCLC, squamous cell lung carcinoma; AML, acute myeloid leukemia.

Figure 2.. RAS GTPase cycle.

In normal cells, RAS resides predominantly in the inactive GDP-bound state. However, activation of upstream growth factor receptors such as receptor tyrosine kinases results in recruitment of GEFs to the plasma membrane and subsequent release of GDP from RAS and formation of nucleotide-free RAS (apoRAS). Given the higher cellular levels of GTP vs GDP coupled with the picomolar affinity of RAS for nucleotides, RAS binds GTP shifting it to the active or “ON” state. RAS-GTP binds a variety of effectors to activate of a plethora of signaling pathways, e.g. RAF. The intrinsic GTPase activity cleaves GTP to GDP to terminate signaling. Due to the relative poor intrinsic enzymatic activity of RAS, this step is enhanced by GAPs. Oncogenic mutations impair the intrinsic GTPase activity and decrease GAP interaction shifting RAS to the GTP-bound active state. A subset of RAS mutants, i.e.,“fast exchange” mutants, e.g., G13D and Q61L, possess high intrinsic nucleotide release activity suggesting these mutants cycle through the apo state independent of GEFs. Once nucleotide-free in cells, mutant apoRAS will re-bind GTP (most likely, thick dashed arrow) or GDP (less likely, thin dashed arrow) due to their relative concentrations. Alternatively, apoRAS may interact with potential targets such as PI3KC2β, which is negatively regulated by apoRAS (36).

Figure 3.. KRAS(G12C)-specific inhibitors.

A. Structures of various G12C covalent inhibitors. The reactive warheads of each inhibitor are indicated by the dashed circle. B-E. Structures of KRAS bound to selected G12C inhibitors (shown in blue). SW1 is shown in orange, SW2 in yellow. The allosteric lobe is highlighted in grey and the effector lobe in white. The reactive Cys at position 12 is highlighted red. GDP is shown in green. B. KRAS bound to compound 16 (PDB: 4M22). C. KRAS bound to ARS-1620 (PDB: 5V9U). D. KRAS bound to AMG510 (PDB: 6OIM). E. KRAS bound to MRTX849 (PDB: 6UT0). His95, which is unique to KRAS and forms specific contacts with the new generation compounds (AMG510 and MRTX849), is highlighted in pink.

Figure 4.. RAS biologics targeting dimerization.

Shown are the x-ray crystal structures of NS1 Mb bound to HRAS (PDB: 5E95) and K13 DARPin bound to KRAS (PDB: 6H46). Both structures are shown in similar orientations relative to RAS. Coloring on RAS is the same as in Fig. 3. Both biologics target the ⍺4 helix and inhibit RAS dimerization (70, 81).