In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras–effector interaction

Abstract

Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Ras⋅GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Ras⋅GTP carrying an H-Ras–type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Ras⋅GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H- ras G12V –transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras G12V gene by oral administration. The NMR structure of a complex of the compound with H-Ras⋅GTP T35S , exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Ras⋅GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Ras⋅GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.

Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices

“…An elegant screening strategy based on covalent engagement of the G12C thiol identified promising inhibitors that target a new allosteric site adjacent to the nucleotide exchange region (5). These compounds not only switched the nucleotide-binding preference of KRAS G12C to GDP over GTP (5), favoring the inactive state, but may also diminish effector interactions, a distinct and complementary RAS-inhibitory activity exploited by another series of small molecules recently identified by in silico screening (6). Compounds that emerged from these molecular tethering and in silico strategies have demonstrated micromolar and submicromolar range antitumor responses in cancer cells driven by KRAS G12C and KRAS G12V, respectively (5,6).…”

Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange

“…Although a number of small molecules have been reported to bind directly to Ras (7)(8)(9)(10)(11)(12), these compounds have relatively poor binding affinities, and none have advanced to the clinic to date.…”

Inhibition of KRAS codon 12 mutants using a novel DNA-alkylating pyrrole–imidazole polyamide conjugate

“…Recently, a G12C KRAS mutation has been successfully targeted in vitro by a small molecule that allosterically controls GTP affinity and effector interactions, representing a promising approach to therapy for this specific subset of KRAS mutant tumours 6 . In the face of the challenges in developing small-molecule drugs that directly target KRAS mutant proteins 7 , we have developed a novel approach that directly targets the mutant DNA. Hairpin pyrrole (Py)-imidazole (Im) (PI) polyamides can be designed to bind with high affinity to the minor groove of specific DNA sequences 8 : Py moieties preferentially bind T, A and C bases, but not G, whereas Im is a G-reader.…”