Inhibition Of Wild-type Enzyme Research Articles

Abstract PO2-18-04: STX-478 is a potentially best-in-class mutant-selective PI3Kα inhibitor that demonstrates robust efficacy in ER+ breast cancer models as monotherapy and in combination with standard of care agents

Abstract Activating mutations in PI3Kα are among the most prevalent genetic aberrations in breast cancer. Constitutive signaling of PI3Kα is an established oncogenic driver in these cancers, as these mutations are predictors of response to alpelisib, an approved PI3Kα inhibitor, which inhibits both wild type and mutant PI3Kα. While inhibiting PI3Kα has proven to be efficacious, the concomitant inhibition of wild type enzyme in normal tissue results in metabolic dysfunction and ultimately dose-limiting toxicities. Selectively targeting the mutant PI3Kα enzyme is expected to inhibit tumor growth while sparing normal tissues, thus increasing the therapeutic index compared to non-mutant selective PI3Kα inhibitors. Therefore, we have developed STX-478, an allosteric, CNS penetrant, mutant-selective PI3Kα inhibitor. In a high-throughput viability screen, STX-478 selectively inhibited growth of cell lines with kinase domain and helical domain mutations in PI3Kα. In a panel of PI3Kα mutant CDX and PDX models, STX-478 provided efficacy that was similar or superior to higher than clinically achievable doses of alpelisib. Robust suppression of pharmacodynamic (PD) markers, pAKT, was observed in tumors, but not in skeletal muscle. Importantly, the efficacy achieved with STX-478 occurred at doses that did not cause metabolic dysfunction, in contrast to alpelisib which caused insulin release and suppressed glucose uptake in skeletal muscle in a 13C glucose tolerance test. Collectively, these data indicate that STX-478 has an expanded therapeutic window to selectively target mutant PI3Kα in vivo. In efficacy studies using human tumor xenograft models, STX-478 caused tumor regressions as a single agent. In multiple advanced ER+ breast cancer PDX models including models that are insensitive to the approved CDK4/6 inhibitor palbociclib, STX-478 combined with standards of care was highly efficacious and well tolerated. In a representative PDX model, STX-478 combined with the ER degrader fulvestrant and palbociclib was well tolerated for > 90 days of dosing in mice and resulted in durable tumor regressions that were superior to STX-478 alone. In conclusion, these data demonstrate STX-478 is a highly efficacious, well tolerated, mutant-specific PI3Kα inhibitor that can be combined safely with standards of care in breast cancer and provide the therapeutic benefits of PI3Kα inhibition without the side effects associated with inhibiting WT PI3Kα. These properties combined with the potential for CNS penetration and excellent pharmacokinetic profile in higher species make STX-478 a potentially best-in-class mutant-selective PI3Kα inhibitor. STX-478 is currently being evaluated in a Phase I clinical trial (NCT05768139). Citation Format: Trang Tieu, Leonard Buckbinder, David St. Jean, Samantha Manimala, Gregory Dowdell, Michael Huff, Jacob Alltucker, Erica Jackson, Angel Guzman-Perez, Darrin Stuart. STX-478 is a potentially best-in-class mutant-selective PI3Kα inhibitor that demonstrates robust efficacy in ER+ breast cancer models as monotherapy and in combination with standard of care agents [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-04.

Functional importance of αAsp-350 in the catalytic sites of Escherichia coli ATP synthase

Negatively charged residue αAsp-350 of the highly conserved VISIT-DG sequence is required for Pi binding and maintenance of the phosphate-binding subdomain in the catalytic sites of Escherichia coli F1Fo ATP synthase. αAsp-350 is situated in close proximity, 2.88 Å and 3.5 Å, to the conserved known phosphate-binding residues αR376 and βR182. αD350 is also in close proximity, 1.3 Å, to another functionally important residue αG351. Mutation of αAsp-350 to Ala, Gln, or Arg resulted in substantial loss of oxidative phosphorylation and reduction in ATPase activity by 6- to 16-fold. The loss of the acidic side chain in the form of αD350A, αD350Q, and αD350R caused loss of Pi binding. While removal of Arg in the form of αR376D resulted in the loss of Pi binding, the addition of Arg in the form of αG351R did not affect Pi binding. Our data demonstrates that αD350R helps in the proper orientation of αR376 and βR182 for Pi binding. Fluoroaluminate, fluoroscandium, and sodium azide caused almost complete inhibition of wild type enzyme and caused variable inhibition of αD350 mutant enzymes. NBD-Cl (4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole) caused complete inhibition of wild type enzyme while some residual activity was left in mutant enzymes. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites. Phosphate protected against NBD-Cl inhibition of wild type and αG351R mutant enzymes but not inhibition of αD350A, αD350Q, αD350R, or αR376D mutant enzymes. These results demonstrate that αAsp-350 is an essential residue required for phosphate binding, through its interaction with αR376 and βR182, for normal function of phosphate binding subdomain and for transition state stabilization in ATP synthase catalytic sites.

Identification of a membrane-localized cysteine cluster near the substrate-binding sites of the Streptococcus equisimilis hyaluronan synthase

The membrane-bound hyaluronan synthase (HAS) from Streptococcus equisimilis (seHAS), which is the smallest Class I HAS, has four cysteine residues (positions 226, 262, 281, and 367) that are generally conserved within this family. Although Cys-null seHAS is still active, chemical modification of cysteine residues causes inhibition of wild-type enzyme. Here we studied the effects of N-ethylmaleimide (NEM) treatment on a panel of seHAS Cys-mutants to examine the structural and functional roles of the four cysteine residues in the activity of the enzyme. We found that Cys226, Cys262, and Cys281 are reactive with NEM, but Cys367 is not. Substrate protection studies of wild-type seHAS and a variety of Cys-mutants revealed that binding of UDP-GlcUA, UDP-GlcNAc, or UDP can protect Cys226 and Cys262 from NEM inhibition. Inhibition of the six double Cys-mutants of seHAS by sodium arsenite, which can cross-link vicinyl sulfhydryl groups, also supported the conclusion that Cys262 and Cys281 are close enough to be cross-linked. Similar results indicated that Cys281 and Cys367 are also very close in the active enzyme. We conclude that three of the four Cys residues in seHAS (Cys262, Cys281, and Cys367) are clustered very close together, that these Cys residues and Cys226 are located at the inner surface of the cell membrane, and that Cys226 and Cys262 are located in or near a UDP binding site.

Identification of a Phosphate Regulatory Site and a Low Affinity Binding Site for Glucose 6-Phosphate in the N-terminal Half of Human Brain Hexokinase

Crystal structures of human hexokinase I reveal identical binding sites for phosphate and the 6-phosphoryl group of glucose 6-phosphate in proximity to Gly87, Ser88, Thr232, and Ser415, a binding site for the pyranose moiety of glucose 6-phosphate in proximity to Asp84, Asp413, and Ser449, and a single salt link involving Arg801 between the N- and C-terminal halves. Purified wild-type and mutant enzymes (Asp84 --> Ala, Gly87 --> Tyr, Ser88 --> Ala, Thr232 --> Ala, Asp413 --> Ala, Ser415 --> Ala, Ser449 --> Ala, and Arg801 --> Ala) were studied by kinetics and circular dichroism spectroscopy. All eight mutant hexokinases have kcat and Km values for substrates similar to those of wild-type hexokinase I. Inhibition of wild-type enzyme by 1,5-anhydroglucitol 6-phosphate is consistent with a high affinity binding site (Ki = 50 microM) and a second, low affinity binding site (Kii = 0.7 mM). The mutations of Asp84, Gly87, and Thr232 listed above eliminate inhibition because of the low affinity site, but none of the eight mutations influence Ki of the high affinity site. Relief of 1,5-anhydroglucitol 6-phosphate inhibition by phosphate for Asp84 --> Ala, Ser88 --> Ala, Ser415 --> Ala, Ser449 --> Ala and Arg801 --> Ala mutant enzymes is substantially less than that of wild-type hexokinase and completely absent in the Gly87 --> Tyr and Thr232 --> Ala mutants. The results support several conclusions. (i) The phosphate regulatory site is at the N-terminal domain as identified in crystal structures. (ii) The glucose 6-phosphate binding site at the N-terminal domain is a low affinity site and not the high affinity site associated with potent product inhibition. (iii) Arg801 participates in the regulatory mechanism of hexokinase I.