Identification of GDC-1971 (RLY-1971), a SHP2 Inhibitor Designed for the Treatment of Solid Tumors.

Protein tyrosine phosphatase SHP2 mediates RAS-driven MAPK signaling and has emerged in recent years as a target of interest in oncology, both for treating with a single agent and in combination with a KRAS inhibitor. We were drawn to the pharmacological potential of SHP2 inhibition, especially following the initial observation that drug-like compounds could bind an allosteric site and enforce a closed, inactive state of the enzyme. Here, we describe the identification and characterization of GDC-1971 (formerly RLY-1971), a SHP2 inhibitor currently in clinical trials in combination with KRAS G12C inhibitor divarasib (GDC-6036) for the treatment of solid tumors driven by a KRAS G12C mutation.

Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to dependence on PRMT5.

5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.

The novolactone natural product disrupts the allosteric regulation of Hsp70.

The highly conserved 70 kDa heat shock proteins (Hsp70) play an integral role in proteostasis such that dysregulation has been implicated in numerous diseases. Elucidating the precise role of Hsp70 family members in the cellular context, however, has been hampered by the redundancy and intricate regulation of the chaperone network, and relatively few selective and potent tools. We have characterized a natural product, novolactone, that targets cytosolic and ER-localized isoforms of Hsp70 through a highly conserved covalent interaction at the interface between the substrate-binding and ATPase domains. Biochemical and structural analyses indicate that novolactone disrupts interdomain communication by allosterically inducing a conformational change in the Hsp70 protein to block ATP-induced substrate release and inhibit refolding activities. Thus, novolactone is a valuable tool for exploring the requirements of Hsp70 chaperones in diverse cellular contexts.

Enzyme-dependent lysine deprotonation in EZH2 catalysis.

Protein lysine methyltransferases (PKMTs) are key players in epigenetic regulation and have been associated with a variety of diseases, including cancers. The catalytic subunit of Polycomb Repressive Complex 2, EZH2 (EC 2.1.1.43), is a PKMT and a member of a family of SET domain lysine methyltransferases that catalyze the transfer of a methyl group from S-adenosyl-l-methionine to lysine 27 of histone 3 (H3K27). Wild-type (WT) EZH2 primarily catalyzes the mono- and dimethylation of H3K27; however, a clinically relevant active site mutation (Y641F) has been shown to alter the reaction specificity, dominantly catalyzing trimethylation of H3K27, and has been linked to tumor genesis and maintenance. Herein, we explore the chemical mechanism of methyl transfer by EZH2 and its Y641F mutant with pH-rate profiles and solvent kinetic isotope effects (sKIEs) using a short peptide derived from histone H3 [H3(21-44)]. A key component of the chemical reaction is the essential deprotonation of the ε-NH3(+) group of lysine to accommodate subsequent methylation. This deprotonation has been suggested by independent studies (1) to occur prior to binding to the enzyme (by bulk solvent) or (2) to be facilitated within the active site following binding, either (a) by the enzyme itself or (b) by a water molecule with access to the binding pocket. Our pH-rate and sKIE data best support a model in which lysine deprotonation is enzyme-dependent and at least partially rate-limiting. Furthermore, our experimental data are in agreement with prior computational models involving enzyme-dependent solvent deprotonation through a channel providing bulk solvent access to the active site. The mechanism of deprotonation and the rate-limiting catalytic steps appear to be unchanged between the WT and Y641F mutant enzymes, despite their activities being highly dependent on different substrate methylation states, suggesting determinants of substrate and product specificity in EZH2 are independent of catalytic events limiting the steady-state rate.