Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices

Abstract

Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequencespecific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85D V12 activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.R AS signaling is a critical control point for a host of cellular functions ranging from cellular survival and proliferation to cellular endocytosis and motility (1). The on or off state of RAS is dictated by nucleotide exchange. GTP-bound RAS is the activated form that engages its downstream effectors with high avidity. The endogenous GTPase activity of RAS hydrolyzes GTP to GDP and inactivates signaling. This biochemical process is further regulated by GTPase-activating proteins (GAPs) that impair RAS signaling through increasing endogenous GTPase activity and guanine-nucleotide exchange factors (GEFs) that enhance RAS signaling by facilitating GDP release and, thus, GTP association. Given the central roles of RAS in cellular growth and metabolism, it is not surprising that cancer cells usurp its prosurvival activities to achieve immortality.Activating mutations in KRAS represent the most frequent oncogenic driving force among the RAS homologs K-, N-, and H-RAS, and are associated with poor prognosis and chemoresistance (2). KRAS mutations are present in ∼30% of human tumors and at even higher frequencies in cancers of the pancreas, lung, thyroid gland, colon, and liver. For example, in pancreatic ductal adenocarcinomas (PDAC) that carry a 5-y survival rate of less than 5%, activating KRAS mutations are present in more than 90% of tumors (3). Thus, therapeutic inhibition of RAS is among the highest priority goals of the cancer field. Because oncogenic forms of KRAS typically harbor single-point mutants that stabilize its active GTP-bound form, a host of recent small molecule and peptide development efforts have been aimed at disarming this pathologic biochemical state. The extremely high affinity of KRAS for its GTP substrate has hampered the development of competitive GTP inhibitors. However, a GDP mimetic that covalently modifies the mutant cysteine of KRAS G12C represents a promising approach to plugging the nucleotid...

Inhibition of Ral GTPases Using a Stapled Peptide Approach

“…This technique has been successfully applied to several different protein targets (36,37), and the first stapled peptide-based therapy, a long acting growth hormone releasing hormone (GHRH) agonist, has passed Phase I clinical trials, whereas the first anti-cancer stapled peptide, targeting the reactivation of p53, has also now entered Phase I trials. 5 Peptides based on a helix from the Ras exchange factor SOS have been used to target Ras (39,40), suggesting that small G proteins are amenable to such approaches. The Ras-binding peptides designed so far are not selective for the GTP-bound form of the Ras protein, although the stapled versions bind with a high affinity to wildtype K-Ras and several oncogenic mutants (40).…”

“…5 Peptides based on a helix from the Ras exchange factor SOS have been used to target Ras (39,40), suggesting that small G proteins are amenable to such approaches. The Ras-binding peptides designed so far are not selective for the GTP-bound form of the Ras protein, although the stapled versions bind with a high affinity to wildtype K-Ras and several oncogenic mutants (40).…”

The small GTPases Ras and Rheb studied by multidimensional NMR spectroscopy: structure and function

“…Overall, numerous groups were able to show that low molecular weight compounds can selectively bind to Ras and interfere with the Sos-mediated nucleotide exchange in H-and K-Ras (Taveras et al, 1997;Spoerner et al, 2001;Gorfe et al, 2008;Araki et al, 2011;Patgiri et al, 2011;Maurer et al, 2012;Sun et al, 2012;Hocker et al, 2013;Ostrem et al, 2013;Shima et al, 2013;Min Lim et al, 2014;Leshchiner et al, 2015;Winter et al, 2015). Apparently, the activity of small GTPases can be indirectly modulated by small molecule through disrupting the Ras/SOS guanine nucleotide exchange complex.…”

Oncogenic G12D mutation alters local conformations and dynamics of K-Ras

“…G12 is the most frequently mutated residue (89%), which most prevalently mutates to aspartate (G12D, 36%) followed by valine (G12V, 23%) and cysteine (G12C, 14%) 3,10 . This residue is located at the protein active site, which consists of a phosphate binding loop (P-loop, residues 10-17) and switch I (SI, residues [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] and II (SII, residues 60-74) regions. The active site residues are bound to the phosphate groups of GTP and are responsible for the GTPase function of K-Ras.…”

“…Although the effects of G12D mutation on the structure, conformation and flexibility of K-Ras have been studied [21][22][23][24] , the relationship between its conformational and dynamical changes still remains to be understood. At the same time, there is increasing evidence that suggests that crystal structure studies alone may miss drugbinding pockets on mutant K-Ras surface 18,[25][26][27][28][29][30][31][32][33] . Studies that include dynamics information have recently achieved promising results, however, these are limited to K-Ras G12C mutant, and the mechanisms that regulate its dynamics remain unknown.…”