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...

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Proteolytic processing of capsid assembly protein precursors by herpesvirus proteases is essential for virion maturation. A 2.5 A crystal structure of the human cytomegalovirus protease catalytic domain has been determined by X-ray diffraction. The structure defines a new class of serine protease with respect to global-fold topology and has a catalytic triad consisting of Ser-132, His-63, and His-157 in contrast with the Ser-His-Asp triads found in other serine proteases. However, catalytic machinery for activating the serine nucleophile and stabilizing a tetrahedral transition state is oriented similarly to that for members of the trypsin-like and subtilisin-like serine protease families. Formation of the active dimer is mediated primarily by burying a helix of one protomer into a deep cleft in the protein surface of the other.

The single-chain 28 kDa human cytomegalovirus (HCMV) protease catalytic domain containing the A143Q mutation has been kinetically and conformationally characterized. The specific activity of the HCMV A143Q protease (HCMVp) increases as the protease concentration increases, suggesting that this protease oligomerizes at high protein concentration to form a more active species. Both cross-linking and light-scattering studies of HCMVp show the existence of a homodimer with an apparent molecular mass of 56 kDa under low ionic strength and high protein concentration. The cosolvent and solute effects of glycerol, trisodium citrate, and NaCl as well as the temperature effects on the HCMVp activity and quaternary structure were investigated. The effects induced by cosolvents and temperature can largely be explained by their influences in the dimerization or oligomerization state of HCMVp. The dissociation constant (Kd) for the HCMVp homodimer was determined to be 8 +/- 1 microM with all activity attributed to the dimeric form. Monomeric HCMVp is inactive. This report demonstrates that in vitro, HCMV A143Q protease exists as an obligate catalytic homodimer. This protease dimerization may have regulatory significance during viral replication.

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.

We report here the production of active recombinant single-chain human cytomegalovirus protease in Escherichia coli and development of a continuous assay for this protease. In order to produce the human cytomegalovirus (HCMV) protease for structural studies and accurate kinetic analysis, mutation of alanine 143 at an internal cleavage site was introduced to prevent autoproteolysis. The resulting soluble 29-kDa A143Q protease was purified to homogeneity as a stable single-chain protein by hydrophobic interaction and ionic-exchange chromatography. The in vivo protein substrate, assembly protein precursor, was also expressed and purified for activity studies. To develop a continuous protease assay, fluorescent synthetic peptide substrates similar to the cleavage sequence P5 to P5 of the maturation site containing anthranilic acid and nitrotyrosine as a resonance energy transfer donor-acceptor pair were designed. Purified HCMV A143Q protease cleaved the recombinant assembly protein precursor with K m and k cat values of 3.0 ؎ 1.0 M and 13.3 ؎ 1.6 min ؊1. The K m for peptide substrates is at least 45-fold higher than for the natural protein substrate, but the k cat values are similar. A sensitive assay was developed using fluorescent peptide substrates, which can detect nM HCMV protease activity.The Herpesviridae family includes several human pathogenic species such as herpes simplex virus 1 and 2 (HSV-1 and -2), 1 cytomegalovirus (CMV), Epstein-Barr virus, and varicellazoster virus. Viral infection by HCMV is very common, and 40 -80% of population becomes infected by HCMV before adulthood (1). HCMV is a serious pathogen in immunocompromised individuals, especially those patients with AIDS, receiving organ or bone marrow transplants, or undergoing cancer chemotherapy or steroid therapy. CMV can cause damage in many organs, including the lung, retina, liver, and gastrointestinal tract. Ganciclovir and foscarnet are inhibitors of viral DNA polymerase and have been used to treat HCMV infections; however, they have the undesired side effects of nucleotide analogs (2).All members of the Herpesviridae family are similar at both the morphological and genomic levels. Herpesviruses contain a DNA genome of over 100 kilobases, an icosahedral capsid, and a lipoprotein envelope. The viral genome replicates inside the nucleus of infected cells and is then packaged into an intermediate capsid and followed by the acquisition of a nuclear membrane envelope and release of the virion from the infected cell. The HSV-1 assembly protein precursor, ICP35, is a major component of the intermediate capsid but is absent in mature virions (3-5). During virion maturation, ICP35 undergoes proteolytic processing to generate the mature assembly protein that lacks approximately 20 amino acids from the carboxyl terminus. An HSV-1 temperature-sensitive mutant (ts1201) that it is defective in the processing of ICP35 has been reported by Preston et al. (6). This mutant virus fails to package progeny viral DNA into virions at the nonpermissive temperatur...

Methionine aminopeptidase-2 (MetAP2) is an enzyme that cleaves an N-terminal methionine residue from a number of newly synthesized proteins. This step is required before they will fold or function correctly. Pre-clinical and clinical studies with a MetAP2 inhibitor suggest that they could be used as a novel treatment for obesity. Herein we describe the discovery of a series of pyrazolo[4,3-b]indoles as reversible MetAP2 inhibitors. A fragment-based drug discovery (FBDD) approach was used, beginning with the screening of fragment libraries to generate hits with high ligand-efficiency (LE). An indazole core was selected for further elaboration, guided by structural information. SAR from the indazole series led to the design of a pyrazolo[4,3-b]indole core and accelerated knowledge-based fragment growth resulted in potent and efficient MetAP2 inhibitors, which have shown robust and sustainable body weight loss in DIO mice when dosed orally.