Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor–positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor–positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. Significance: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors

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 <i>S</i>-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

Constrained Bonding Environment in the Michaelis Complex of <i>Trypanosoma cruzi</i> Uridine Phosphorylase

The transition state for the <i>Trypanosoma cruzi</i> uridine phosphorylase (TcUP) reaction has an expanded S_N2 character. We used binding isotope effects (BIE's) to probe uridine distortion in the complex with TcUP and sulfate to mimic the Michaelis complex. Inverse 1′-³H and 5′-³H BIE's indicate a constrained bonding environment of these groups in the complex. Quantum chemical modeling identified a uridine conformer whose calculated BIE's match the experimental values. This conformer differs in sugar pucker and uracil orientation from the unbound conformer and the transition-state structure. These results support ground-state stabilization in the Michaelis complex

Figure S6 from Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia

Varied levels of PI3Kα-dependency across cell lines</p

Supplemental Text from Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia

Synthesis of mutant-selective allosteric PI3Kα inhibitors</p

Figure S1 from Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia

Activation loop residues 937-954 are more disordered in mutant vs. wildtype PI3Kα</p

Figure S5 from Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia

Isoform selectivity of RLY-2608</p