KRASG12D, the most common oncogenic KRAS mutation, is a promising target for the treatment of solid tumors. However, when compared to KRASG12C, selective inhibition of KRASG12D presents a significant challenge due to the requirement of inhibitors to bind KRASG12D with high enough affinity to obviate the need for covalent interactions with the mutant KRAS protein. Here, we report the discovery and characterization of the first noncovalent, potent, and selective KRASG12D inhibitor, MRTX1133, which was discovered through an extensive structure-based activity improvement and shown to be efficacious in a KRASG12D mutant xenograft mouse tumor model.

The ability to effectively target mutated KRAS has remained elusive despite decades of research. The recent identification of KRAS G12C inhibitors has provided an effective treatment option for patients harboring this particular mutation and has also provided insight toward targeting other KRAS mutants, including KRAS G12D . MRTX1133 was identified via a structure-based drug design (SBDD) strategy as a potent, selective, and non-covalent KRAS G12D inhibitor directed at the switch II binding pocket. MRTX1133 demonstrated a high-affinity interaction with KRAS G12D with KD or IC50 values each determined at ~0.2 pM or <2 nM using SPR direct binding or HTRF competition assays, respectively. MRTX1133 also demonstrated ~700-fold selectivity for KRAS G12D vs KRAS WT binding utilizing SPR. Interestingly, MRTX1133 demonstrated potent inhibition of active KRAS G12D using an HTRF effector interaction assay with a IC50 value of 9 nM. Insight toward the structural basis of binding of MRTX1133 to both the inactive GDP-bound and active GMPPCP-bound conformations of KRAS G12D is also provided by co-crystal structures. MRTX1133 demonstrated potent inhibition of ERK1/2 phosphorylation and cell viability in KRAS G12D -mutant cell lines with median IC50 values of ~5 nM. Consistent with binding affinity determination in cell-free systems, MRTX1133 demonstrated >1000-fold selectivity for inhibition of ERK1/2 phosphorylation in KRAS G12Dmutant cell lines compared with KRAS WT cell lines. Dose-dependent inhibition of KRASmediated signal transduction was also observed in multiple KRAS G12D -mutant tumor models in vivo. MRTX1133 demonstrated marked tumor regression (>30%) in a subset of KRAS G12Dmutant cell line-and patient-derived xenograft (PDX) models, including 8 out of 11 (73%) pancreatic ductal adenocarcinoma (PDAC) models evaluated. Pharmacological studies and CRISPR-based screens demonstrated co-targeting KRAS G12D in concert with putative feedback or bypass pathways including EGFR and PI3Kα led to enhanced anti-tumor activity relative to targeting each individual protein. Together, these data indicate the feasibility of utilizing SBDD approaches to selectively target alternative KRAS mutant variants with non-covalent, highaffinity small molecules targeting either the active or inactive state of KRAS. In addition, these data illustrate the therapeutic susceptibility and broad dependence of KRAS G12D mutationpositive tumors, including PDAC, on KRAS for tumor cell growth and survival. SignificanceThe development of clinically active KRAS G12C -selective inhibitors represents a milestone achievement for the treatment of cancer; however, the discovery of additional KRAS-mutant selective inhibitors has remained elusive. MRTX1133 is a potent KRAS G12D -selective small molecule inhibitor, is active in vitro and in vivo, induces regression in multiple xenograft tumor models and demonstrates increased anti-tumor activity in rationally designed combinations. These data confirm KRAS G12D functions as an oncogenic driver, including in pancreat...

Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α-and β-amino acid residues ("α/β-peptides") that mimic several peptides derived from the three-helix bundle "Z-domain" scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF 165 -induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain-mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because wellestablished selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.α/β-peptides | foldamers | protein-protein interactions | inhibitors | molecular recognition D esigned molecules that bind selectively to specific sites on proteins may serve as inhibitors of medically important macromolecular interactions or diagnostic tools for biomarker detection. Small molecules often fail for these applications because of the relatively large and irregularly shaped target surfaces (1-3). In contrast, large polypeptides (e.g., antibodies) can frequently be developed to recognize a protein surface with high affinity and selectivity and represent the state of the art for engineering ligands for specific biomacromolecular targets. Large polypeptides, however, suffer several disadvantages for in vivo applications, including costly production, low storage stability, and/ or low bioavailability because of rapid proteolytic degradation (4, 5).Backbone-modified peptides, an underexplored class of molecules, are proving to be a fruitful source of tight-binding and specific protein ligands. Peptidic oligomers that contain β-amino acid residues interspersed among α-residues ("α/β-peptides") can effectively mimic the recognition surface projected by an α-helix and thereby disrupt or augment protein-protein interactions in which one partner contributes a single helix to the interface (6, 7). The unnatural backbone diminishes α/β-peptide susceptibility to proteolytic degradation relative to conventional peptides (α-residues only, "α-peptides"). As a result, α/β-peptides can exhibit improved pharmacokinetic properties in vivo relative to analogous α-peptides (8, 9). To date, however, the α/β-peptide strategy has been restricted to mimicry of isolated α-helices, ...

The PRMT5•MTA complex has recently emerged as a new synthetically lethal drug target for the treatment of MTAP-deleted cancers. Here, we report the discovery of development candidate MRTX1719. MRTX1719 is a potent and selective binder to the PRMT5•MTA complex and selectively inhibits PRMT5 activity in MTAP-deleted cells compared to MTAP-wild-type cells. Daily oral administration of MRTX1719 to tumor xenograft-bearing mice demonstrated dose-dependent inhibition of PRMT5-dependent symmetric dimethylarginine protein modification in MTAP-deleted tumors that correlated with antitumor activity. A 4-(aminomethyl)­phthalazin-1­(2H)-one hit was identified through a fragment-based screen, followed by X-ray crystallography, to confirm binding to the PRMT5•MTA complex. Fragment growth supported by structural insights from X-ray crystallography coupled with optimization of pharmacokinetic properties aided the discovery of development candidate MRTX1719.

SOS1 is one of the major guanine nucleotide exchange factors that regulates the ability of KRAS to cycle through its “on” and “off” states. Disrupting the SOS1:KRAS G12C protein–protein interaction (PPI) can increase the proportion of GDP-loaded KRAS G12C , providing a strong mechanistic rationale for combining inhibitors of the SOS1:KRAS complex with inhibitors like MRTX849 that target GDP-loaded KRAS G12C . In this report, we detail the design and discovery of MRTX0902—a potent, selective, brain-penetrant, and orally bioavailable SOS1 binder that disrupts the SOS1:KRAS G12C PPI. Oral administration of MRTX0902 in combination with MRTX849 results in a significant increase in antitumor activity relative to that of either single agent, including tumor regressions in a subset of animals in the MIA PaCa-2 tumor mouse xenograft model.