The SOS1 Inhibitor MRTX0902 Blocks KRAS Activation and Demonstrates Antitumor Activity in Cancers Dependent on KRAS Nucleotide Loading.

KRAS is the most frequently mutated oncogene in human cancer and facilitates uncontrolled growth through hyperactivation of the RTK/MAPK pathway. The Son of Sevenless homolog 1 (SOS1) protein functions as a guanine nucleotide exchange factor (GEF) for the RAS subfamily of small GTPases and represents a druggable target in the pathway. Using a structure-based drug discovery approach, MRTX0902 was identified as a selective and potent SOS1 inhibitor that disrupts the KRAS:SOS1 protein-protein interaction to prevent SOS1-mediated nucleotide exchange on KRAS and translates into an anti-proliferative effect in cancer cell lines with genetic alterations of the KRAS-MAPK pathway. MRTX0902 augmented the antitumor activity of the KRAS G12C inhibitor adagrasib when dosed in combination in eight out of twelve KRAS G12C-mutant human non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) xenograft models. Pharmacogenomic profiling in preclinical models identified cell cycle genes and the SOS2 homolog as genetic co-dependencies and implicated tumor suppressor genes (NF1, PTEN) in resistance following combination treatment. Lastly, combined vertical inhibition of RTK/MAPK pathway signaling by MRTX0902 with inhibitors of EGFR or RAF/MEK led to greater downregulation of pathway signaling and improved antitumor responses in KRAS-MAPK pathway-mutant models. These studies demonstrate the potential clinical application of dual inhibition of SOS1 and KRAS G12C and additional SOS1 combination strategies that will aide in the understanding of SOS1 and RTK/MAPK biology in targeted cancer therapy.

Discovery of Pyridopyrimidinones that Selectively Inhibit the H1047R PI3Kα Mutant Protein.

The H1047R mutation of PIK3CA is highly prevalent in breast cancers and other solid tumors. Selectively targeting PI3KαH1047R over PI3KαWT is crucial due to the role that PI3KαWT plays in normal cellular processes, including glucose homeostasis. Currently, only one PI3KαH1047R-selective inhibitor has progressed into clinical trials, while three pan mutant (H1047R, H1047L, H1047Y, E542K, and E545K) selective PI3Kα inhibitors have also reached the clinical stage. Herein, we report the design and discovery of a series of pyridopyrimidinones that inhibit PI3KαH1047R with high selectivity over PI3KαWT, resulting in the discovery of compound 17. When dosed in the HCC1954 tumor model in mice, 17 provided tumor regressions and a clear pharmacodynamic response. X-ray cocrystal structures from several PI3Kα inhibitors were obtained, revealing three distinct binding modes within PI3KαH1047R including a previously reported cryptic pocket in the C-terminus of the kinase domain wherein we observe a ligand-induced interaction with Arg1047.

Discovery of Five SOS2 Fragment Hits with Binding Modes Determined by SOS2 X-Ray Cocrystallography.

SOS1 and SOS2 are guanine nucleotide exchange factors that mediate RTK-stimulated RAS activation. Selective SOS1:KRAS PPI inhibitors are currently under clinical investigation, whereas there are no reports to date of SOS2:KRAS PPI inhibitors. SOS2 activity is implicated in MAPK rebound when divergent SOS1 mutant cell lines are treated with the SOS1 inhibitor BI-3406; therefore, SOS2:KRAS inhibitors are of therapeutic interest. In this report, we detail a fragment-based screening strategy to identify X-ray cocrystal structures of five diverse fragment hits bound to SOS2.

MRTX1719 Is an MTA-Cooperative PRMT5 Inhibitor That Exhibits Synthetic Lethality in Preclinical Models and Patients with MTAP-Deleted Cancer.

Previous studies implicated protein arginine methyltransferase 5 (PRMT5) as a synthetic lethal target for MTAP-deleted (MTAP del) cancers; however, the pharmacologic characterization of small-molecule inhibitors that recapitulate the synthetic lethal phenotype has not been described. MRTX1719 selectively inhibited PRMT5 in the presence of MTA, which is elevated in MTAP del cancers, and inhibited PRMT5-dependent activity and cell viability with >70-fold selecti-vity in HCT116 MTAP del compared with HCT116 MTAP wild-type (WT) cells. MRTX1719 demonstrated dose-dependent antitumor activity and inhibition of PRMT5-dependent SDMA modification in MTAP del tumors. In contrast, MRTX1719 demonstrated minimal effects on SDMA and viability in MTAP WT tumor xenografts or hematopoietic cells. MRTX1719 demonstrated marked antitumor activity across a panel of xenograft models at well-tolerated doses. Early signs of clinical activity were observed including objective responses in patients with MTAP del melanoma, gallbladder adenocarcinoma, mesothelioma, non-small cell lung cancer, and malignant peripheral nerve sheath tumors from the phase I/II study. SIGNIFICANCE: PRMT5 was identified as a synthetic lethal target for MTAP del cancers; however, previous PRMT5 inhibitors do not selectively target this genotype. The differentiated binding mode of MRTX1719 leverages the elevated MTA in MTAP del cancers and represents a promising therapy for the ∼10% of patients with cancer with this biomarker. See related commentary by Mulvaney, p. 2310. This article is featured in Selected Articles from This Issue, p. 2293.

Selected Approaches to Disrupting Protein-Protein Interactions within the MAPK/RAS Pathway.

Within the MAPK/RAS pathway, there exists a plethora of protein-protein interactions (PPIs). For many years, scientists have focused efforts on drugging KRAS and its effectors in hopes to provide much needed therapies for patients with KRAS-mutant driven cancers. In this review, we focus on recent strategies to inhibit RAS-signaling via disrupting PPIs associated with SOS1, RAF, PDEδ, Grb2, and RAS.

Fragment optimization and elaboration strategies - the discovery of two lead series of PRMT5/MTA inhibitors from five fragment hits.

Here we describe the early stages of a fragment-based lead discovery (FBLD) project for a recently elucidated synthetic lethal target, the PRMT5/MTA complex, for the treatment of MTAP-deleted cancers. Starting with five fragment/PRMT5/MTA X-ray co-crystal structures, we employed a two-phase fragment elaboration process encompassing optimization of fragment hits and subsequent fragment growth to increase potency, assess synthetic tractability, and enable structure-based drug design. Two lead series were identified, one of which led to the discovery of the clinical candidate MRTX1719.

Design and Discovery of MRTX0902, a Potent, Selective, Brain-Penetrant, and Orally Bioavailable Inhibitor of the SOS1:KRAS Protein-Protein Interaction.

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:KRASG12C protein-protein interaction (PPI) can increase the proportion of GDP-loaded KRASG12C, providing a strong mechanistic rationale for combining inhibitors of the SOS1:KRAS complex with inhibitors like MRTX849 that target GDP-loaded KRASG12C. 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:KRASG12C 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.

Fragment-Based Discovery of MRTX1719, a Synthetic Lethal Inhibitor of the PRMT5•MTA Complex for the Treatment of MTAP-Deleted Cancers.

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.

Identification of MRTX1133, a Noncovalent, Potent, and Selective KRASG12D Inhibitor.

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.

Discovery of the Bruton's Tyrosine Kinase Inhibitor Clinical Candidate TAK-020 (S)-5-(1-((1-Acryloylpyrrolidin-3-yl)oxy)isoquinolin-3-yl)-2,4-dihydro-3H-1,2,4-triazol-3-one, by Fragment-Based Drug Design.

This publication details the successful use of FBDD (fragment-based drug discovery) principles in the invention of a novel covalent Bruton's tyrosine kinase inhibitor, which ultimately became the Takeda Pharmaceuticals clinical candidate TAK-020. Described herein are the discovery of the fragment 5-phenyl-2,4-dihydro-3H-1,2,4-triazol-3-one, the subsequent optimization of this hit molecule to the candidate, and synthesis and performance in pharmacodynamic and efficacy models along with direct biophysical comparison of TAK-020 with other clinical-level assets and the marketed drug Ibrutinib.

Acquired Resistance to KRASG12C Inhibition in Cancer.

BACKGROUND: Clinical trials of the KRAS inhibitors adagrasib and sotorasib have shown promising activity in cancers harboring KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRASG12C). The mechanisms of acquired resistance to these therapies are currently unknown. METHODS: Among patients with KRASG12C -mutant cancers treated with adagrasib monotherapy, we performed genomic and histologic analyses that compared pretreatment samples with those obtained after the development of resistance. Cell-based experiments were conducted to study mutations that confer resistance to KRASG12C inhibitors. RESULTS: A total of 38 patients were included in this study: 27 with non-small-cell lung cancer, 10 with colorectal cancer, and 1 with appendiceal cancer. Putative mechanisms of resistance to adagrasib were detected in 17 patients (45% of the cohort), of whom 7 (18% of the cohort) had multiple coincident mechanisms. Acquired KRAS alterations included G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, and high-level amplification of the KRASG12C allele. Acquired bypass mechanisms of resistance included MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3; and loss-of-function mutations in NF1 and PTEN. In two of nine patients with lung adenocarcinoma for whom paired tissue-biopsy samples were available, histologic transformation to squamous-cell carcinoma was observed without identification of any other resistance mechanisms. Using an in vitro deep mutational scanning screen, we systematically defined the landscape of KRAS mutations that confer resistance to KRASG12C inhibitors. CONCLUSIONS: Diverse genomic and histologic mechanisms impart resistance to covalent KRASG12C inhibitors, and new therapeutic strategies are required to delay and overcome this drug resistance in patients with cancer. (Funded by Mirati Therapeutics and others; ClinicalTrials.gov number, NCT03785249.).

Discovery of Potent, Selective, and Brain-Penetrant 1 H-Pyrazol-5-yl-1 H-pyrrolo[2,3- b]pyridines as Anaplastic Lymphoma Kinase (ALK) Inhibitors.

Anaplastic lymphoma kinase (ALK), a member of the receptor tyrosine kinase family, is predominantly expressed in the brain and implicated in neuronal development and cognition. However, the detailed function of ALK in the central nervous system (CNS) is still unclear. To elucidate the role of ALK in the CNS, it was necessary to discover a potent, selective, and brain-penetrant ALK inhibitor. Scaffold hopping and lead optimization of N-(2,4-difluorobenzyl)-3-(1 H-pyrazol-5-yl)imidazo[1,2- b]pyridazin-6-amine 1 guided by a cocrystal structure of compound 1 bound to ALK resulted in the identification of (6-(1-(5-fluoropyridin-2-yl)ethoxy)-1-(5-methyl-1 H-pyrazol-3-yl)-1 H-pyrrolo[2,3- b]pyridin-3-yl)((2 S)-2-methylmorpholin-4-yl)methanone 13 as a highly potent, selective, and brain-penetrable compound. Intraperitoneal administration of compound 13 significantly decreased the phosphorylated-ALK (p-ALK) levels in the hippocampus and prefrontal cortex in the mouse brain. These results suggest that compound 13 could serve as a useful chemical probe to elucidate the mechanism of ALK-mediated brain functions and the therapeutic potential of ALK inhibition.

Identification of AHCY inhibitors using novel high-throughput mass spectrometry.

S-adenosylhomocysteine hydrolase (AHCY) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and l-homocysteine. This enzyme is frequently overexpressed in many tumor types and is considered to be a validated anti-tumor target. In order to enable the development of small molecule AHCY inhibitors as targeted cancer therapeutics we developed an assay based on a RapidFire high-throughput mass spectrometry detection system, which allows the direct measurement of AHCY enzymatic activity. This technique avoids many of the problems associate with the previously reported method of using a thiol-reactive fluorescence probes to measure AHCY activity. Screening of a ∼500,000 compound library using this technique identified multiple SAH competitive hits. Co-crystal structures of the hit compounds complexed with AHCY were obtained showing that the compounds indeed bind in the SAH site of the enzyme. In addition, some hit compounds increased the SAH levels in HCT116 cells and showed growth inhibition. These compounds could be promising starting points for the optimization of cancer treatments.

Design, synthesis and optimization of 7-substituted-pyrazolo[4,3-b]pyridine ALK5 (activin receptor-like kinase 5) inhibitors.

A series of potent ALK5 inhibitors were designed using a SBDD approach and subsequently optimized to improve drug likeness. Starting with a 4-substituted quinoline screening hit, SAR was conducted using a ALK5 binding model to understand the binding site and optimize activity. The resulting inhibitors displayed excellent potency but were limited by high in vitro clearance in rat and human microsomes. Using a scaffold morphing strategy, these analogs were transformed into a related pyrazolo[4,3-b]pyridine series with improved ADME properties.

MET Tyrosine Kinase Inhibition Enhances the Antitumor Efficacy of an HGF Antibody.

Receptor tyrosine kinase therapies have proven to be efficacious in specific cancer patient populations; however, a significant limitation of tyrosine kinase inhibitor (TKI) treatment is the emergence of resistance mechanisms leading to a transient, partial, or complete lack of response. Combination therapies using agents with synergistic activity have potential to improve response and reduce acquired resistance. Chemoreagent or TKI treatment can lead to increased expression of hepatocyte growth factor (HGF) and/or MET, and this effect correlates with increased metastasis and poor prognosis. Despite MET's role in resistance and cancer biology, MET TKI monotherapy has yielded disappointing clinical responses. In this study, we describe the biological activity of a selective, oral MET TKI with slow off-rate and its synergistic antitumor effects when combined with an anti-HGF antibody. We evaluated the combined action of simultaneously neutralizing HGF ligand and inhibiting MET kinase activity in two cancer xenograft models that exhibit autocrine HGF/MET activation. The combination therapy results in additive antitumor activity in KP4 pancreatic tumors and synergistic activity in U-87MG glioblastoma tumors. Pharmacodynamic characterization of biomarkers that correlate with combination synergy reveal that monotherapies induce an increase in the total MET protein, whereas combination therapy significantly reduces total MET protein levels and phosphorylation of 4E-BP1. These results hold promise that dual targeting of HGF and MET by combining extracellular ligand inhibitors with intracellular MET TKIs could be an effective intervention strategy for cancer patients who have acquired resistance that is dependent on total MET protein. Mol Cancer Ther; 16(7); 1269-78. ©2017 AACR.

Structure-based optimization of 1H-imidazole-2-carboxamides as Axl kinase inhibitors utilizing a Mer mutant surrogate.

Axl has been a target of interest in the oncology field for several years based on its role in various oncogenic processes. To date, no wild-type Axl crystal structure has been reported. Herein, we describe the structure-based optimization of a novel chemotype of Axl inhibitors, 1H-imidazole-2-carboxamide, using a mutated kinase homolog, Mer(I650M), as a crystallographic surrogate. Iterative optimization of the initial lead compound (1) led to compound (21), a selective and potent inhibitor of wild-type Axl. Compound (21) will serve as a useful compound for further in vivo studies.

Design, synthesis and optimization of novel Alk5 (activin-like kinase 5) inhibitors.

Using SBDD, a series of 4-amino-7-azaindoles were discovered as a novel class of Alk5 inhibitors that are potent in both Alk5 enzymatic and cellular assays. Subsequently a ring cyclization strategy was utilized to improve ADME properties leading to the discovery of a series of 1H-imidazo[4,5-c]pyridin-2(3H)-one drug like Alk5 inhibitors.

Discovery of potent, reversible MetAP2 inhibitors via fragment based drug discovery and structure based drug design-Part 2.

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.

Discovery of potent, reversible MetAP2 inhibitors via fragment based drug discovery and structure based drug design-Part 1.

Methionine aminopeptidase 2 (MetAP2) is an enzyme that cleaves an N-terminal methionine residue from a number of newly synthesized proteins. Pre-clinical and clinical studies suggest that MetAP2 inhibitors could be used as a novel treatment for obesity. Herein we describe our use of fragment screening methods and structural biology to quickly identify and elaborate an indazole fragment into a series of reversible MetAP2 inhibitors with <10nM potency, excellent selectivity, and favorable in vitro safety profiles.