Small Molecule Binding Sites on the Ras:SOS Complex Can Be Exploited for Inhibition of Ras Activation

Abstract

Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPaseactivating protein-catalysed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilising or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilisation hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf, and is effective at inhibiting the exchange of labelled GDP in both mutant (G12C and G12V) and wild type Ras.

Direct Modulation of Small GTPase Activity and Function

“…[157] Binding fragments were confirmed by TROSY-HSQC NMR titrations with the stable isotope-labeled H-Ras-SOS complex and displayed dissociation constants in the low millimolar range.Apocket identified on SOS matches abinding site previously described for 19,a nd some fragments such as compound 46 show corresponding binding modes (Figure 13 a,b). [93] Theb inding of one or two fragments to this relatively flexible cavity occurs with limited movement of protein side chains compared to the H-Ras-SOS cat structure.I nc ontrast to the earlier study,t he fragments do not show any increase in the rate of SOSmediated nucleotide exchange.…”

“…[93] Theb inding of one or two fragments to this relatively flexible cavity occurs with limited movement of protein side chains compared to the H-Ras-SOS cat structure.I nc ontrast to the earlier study,t he fragments do not show any increase in the rate of SOSmediated nucleotide exchange. [157] Another fragment binding site comprises alargely solvent-inaccessible pocket located at the interface of H-Ras and SOS cat. which corresponds to asite described in studies of uncomplexed Ras proteins.…”

“…However,s everal rounds of structure optimization did not result in compounds that could significantly stabilize the protein complex or show functional activity. [157] Therefore, aset of covalent inhibitors was designed, targeting Cys 118 of H-Ras in proximity to the GDP binding site,w hich becomes solvent accessible in the Ras-SOS complex as ar esult of reorganization of the nucleotide-binding pocket upon SOS binding.Alibrary of 400 fragments with potentially cysteine- . .…”

RAS signaling and anti-RAS therapy: lessons learned from genetically engineered mouse models, human cancer cells, and patient-related studies

“…Thus, extensive efforts have been made to develop therapeutic agents that modulate posttranscriptional modification and/or plasma membrane localization of RAS proteins [29,30], that intervene in downstream signal transductions, and that induce synthetic lethality in RAS mutant cancer cells [31]. Recently, small molecules have been reported to bind irreversibly to the mutant KRAS (G12C) protein [32], or to interfere with RAS/SOS [33,34] or RAS-effector protein interactions [35]. Nevertheless, effective anti-RAS treatment is not yet available clinically.…”

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.…”