(and alleles are less dependent on their exchange factors than wild-type cells. Such mutations reduce the ability of the small GTPase RAS to hydrolyze GTP, keeping this molecular switch in a constitutively active GTP-bound form that drives, unchecked, oncogenic downstream signaling. One strategy to reduce the levels of active RAS is to target guanine nucleotide exchange factors, which allow RAS to cycle from the inactive GDP-bound state to the active GTP-bound form. Here, we describe the identification of potent and cell-active small-molecule inhibitors which efficiently disrupt the interaction between KRAS and its exchange factor SOS1, a mode of action confirmed by a series of biophysical techniques. The binding sites, mode of action, and selectivity were elucidated using crystal structures of KRASG12CCSOS1, SOS1, and SOS2. By preventing formation of the KRASCSOS1 complex, these inhibitors block reloading of KRAS with GTP, leading to antiproliferative activity. The final compound 23 (BAY-293) selectively inhibits the KRASCSOS1 interaction with an IC50 of 21 nM and is a valuable chemical probe for future investigations. First linked to human cancer in 1982 (1C3), members of the RAS family of GTPases (which comprises is the area in the yellow box enlarged, showing hydrogen bonds as thin dashed lines and cationC interaction as a thick dashed line. (= 4). Normalization: 100% HTRF signal, DMSO control; 0% HTRF signal, without SOS1cat. Crystals of the KRASG12CCSOS1cat complex were obtained using KRASG12C_SB, a KRASG12C construct containing the mutation C118S to increase stability (26), as well as a triple mutation (D126E-T127S-K128R) identified in a surface mutation screen (and for further details on the fragment hit prioritization and fragment binding modes). F1 interacts with SOS1 via a C interaction with Phe890 in its new Phe-out position and forms two hydrogen bonds to Tyr884 and Asp887 (Fig. 1= 4). (of 2.5 C. (shows thermodynamic values obtained from fitting a Wiseman isotherm to the measured calorimetric data. (= 4). Normalization as in Fig. 1and view (and and and and and for a detailed analysis of the observed SAR of this hybrid series). Compound 23 was initially tested as a racemate (compound 22), and later separated into the active (and are indicated in gray. Data points in represent mean SD (= 4). The IC50 values of 22 to 24 for these assays are summarized in = 4. (= 3). (and alleles DprE1-IN-2 are less dependent on their exchange factors than wild-type cells. To directly test this not-yet-fully explored hypothesis with our SOS1 inhibitors, we chose Calu-1 cells, which carry two and alleles, chemical SOS1 inhibition resulted in a reduction of pERK activity by 50% (Fig. 5 em D /em ). We investigated whether this still-limited downstream effect could be further improved by co-inhibition of additional targets. Indeed, covalent KRASG12C inhibitors are known to require GDP-bound inactive KRASG12C for binding, and potential combination therapies by upstream inhibition of RAS activation (e.g., by inhibition of receptor tyrosine kinase or RASGEF activity) have been discussed (11C13). We have shown that the combination of our SOS1 inhibitor with ARS-853, a covalent inhibitor of KRASG12C, results in synergistic antiproliferative activity in a KRASG12C-mutated cell line (Fig. 5 em F /em ). We therefore present compound 23 (BAY-293) as a tool for the further investigation of RASCSOS1 biology in vitro. Improvements in the bioavailability of DprE1-IN-2 the inhibitor series will be required for in vivo experiments. Together, the data presented here indicate that inhibition of GEFs DprE1-IN-2 may represent a viable approach for targeting RAS-driven tumors. Of particular note is the synergistic effect between our inhibitors and ARS-853 Rabbit polyclonal to GRF-1.GRF-1 the human glucocorticoid receptor DNA binding factor, which associates with the promoter region of the glucocorticoid receptor gene (hGR gene), is a repressor of glucocorticoid receptor transcription. observed in a KRASG12C-mutated cancer cell line, which highlights the potential for combination therapy between a direct KRAS and a SOS1 inhibitor. Materials and Methods DNA sequences for the recombinant proteins used in this study were optimized for expression in em Escherichia coli /em , synthesized by GeneArt technology at Life Technologies, expressed in em E. coli /em , and purified via affinity chromatography and size exclusion chromatography. All details of the cloning, expression, and purification steps are described in em SI Appendix /em , em Supplementary Materials and Methods /em . All DprE1-IN-2 expression constructs are listed in em SI Appendix /em , Table S7. Quantification of SOS1cat-mediated loading of human KRASG12CCGDP with a fluorescent GTP analog was carried out by measuring energy transfer from anti-GST-terbium (FRET donor) bound to GST-KRASG12C after binding of a DprE1-IN-2 fluorescent GTP analog (FRET acceptor). Details of this assay and.