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The S. cerevisiae plasma membrane H+-ATPase, Pma1, is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma1 (MCP). Like other plasma membrane H+-ATPases, Pma1 assembles and functions as a hexamer, a property unique to this subfamily among the larger family of P-type ATPases. It has been unclear how Pma1 organizes the yeast membrane into MCP microdomains, or why it is that Pma1 needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. Here we report a high-resolution cryo-EM study of native Pma1 hexamers embedded in endogenous lipids. Remarkably, we found that the Pma1 hexamer encircles a liquid-crystalline membrane domain composed of 57 ordered lipid molecules. The Pma1-encircled lipid patch structure likely serves as the building block of the MCP. At pH 7.4, the carboxyl-terminal regulatory α-helix binds to the phosphorylation domains of two neighboring Pma1 subunits, locking the hexamer in the autoinhibited state. The regulatory helix becomes disordered at lower pH, leading to activation of the Pma1 hexamer. The activation process is accompanied by a 6.7 Å downward shift and a 40° rotation of transmembrane helices 1 and 2 that line the proton translocation path. The conformational changes have enabled us to propose a detailed mechanism for ATP-hydrolysis-driven proton pumping across the plasma membrane. Our structures will facilitate the development of antifungal drugs that target this essential protein., The plasma membrane H+ -ATPase is responsible for maintenance of the plasma membrane potential, which provides energy for the transport of nutrients, and the plasma membrane H+ -ATPase in S. cerevisiae (Pma1) is a P3A-type ATPase that assembles and functions as a hexamer. Here, the authors present the cryo-EM structures of autoinhibited and activated native Pma1 hexamers purified with endogenous lipids and they propose a mechanism for proton pumping across the membrane by this family of H+ -ATPases.

Abdominal aortic aneurysm (AAA) is a lethal cardiovascular disease, and there is no proven drug treatment for this condition. In this study, by using the Connectivity Map (CMap) approach, we explored naringenin, a naturally occurring citrus flavonoid, as a putative agent for inhibiting AAA. We then validated the prediction with two independent mouse models of AAA, calcium phosphate (CaPO4)-induced C57BL/6J mice and angiotensin II-infused ApoE−/− mice. Naringenin effectively blocked the formation of AAAs and the progression of established AAAs. Transcription factor EB (TFEB) is the master regulator of lysosome biogenesis. Intriguingly, the protective role of naringenin on AAA was abolished by macrophage-specific TFEB depletion in mice. Unbiased interactomics, combined with isothermal titration calorimetry (ITC) and cellular thermal shift assays (CETSAs), further revealed that naringenin is directly bound to 14-3-3 epsilon blocked the TFEB-14-3-3 epsilon interaction, and therefore promoted TFEB nuclear translocation and activation. On one hand, naringenin activated lysosome-dependent inhibition of the NLRP3 inflammasome and repressed aneurysmal inflammation. On the other hand, naringenin induced TFEB-dependent transcriptional activation of GATA3, IRF4, and STAT6 and therefore promoted reparative M2 macrophage polarization. In summary, naturally derived naringenin or macrophage TFEB activation shows promising efficacy for the treatment of AAA.

BACKGROUND: Resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a major challenge in non-small cell lung cancer (NSCLC) treatment. EGFR activating mutations, such as exon 19 deletions (Del19) and L858R substitution, have been reported in 10-50% of patients with NSCLC. Although these patients respond well to early generations of TKIs, acquired resistance by mechanisms such as T790M mutation compromises their efficacy. Recently, the C797S mutation in combination with Del19 or L858R, and with T790M, has emerged as one of the most common mechanisms underlying on-target resistance to a third generation EGFR TKI, osimertinib. We are developing a fourth-generation EGFR inhibitor, H002, with a wide spectrum and high selectivity to address the growing unmet needs in NSCLC patients harboring C797S mutation. METHODS: H002’s selectivity was assessed by KINOMEscan against more than 400 human kinases. A panel of 44 kinases was further evaluated to enable early identification of off-target effect. In vitro and in vivo anti-tumor efficacy was determined in drug-resistant mutant cell lines, CDX and PDX models. Single mutants (Del or L858R), double mutants (Del/T790M or L858R/T790M) and triple mutants (Del/T790M/C797S or L858R/T790M/C797S) as well as wild type EGFR were assessed. Drug metabolism and pharmacokinetic (DMPK) properties were further assessed. H002’s safety profile was evaluated in rats and beagle dogs, and by hERG potassium channel assay. Acute toxicity, and repeated dose GLP toxicity studies were also conducted in IND-enabling studies. RESULTS: High kinase selectivity and very low off-target effects were demonstrated by kinome-wide profiling and safety screening. H002 was highly active against all forementioned single, double or triple EGFR mutants (IC50 < 5 nM), and highly selective against wild type EGFR. H002 also exhibited potent anti-tumor effects in a dose-dependent manner in various CDX and PDX models. Excellent PK properties were observed in rats and beagle dogs with bioavailability over 70%. H002 was shown to cross the blood-brain barrier post i.g. administration in rats. No adverse effects of respiratory, cardiovascular and central nervous system were observed in safety studies. Repeated-dose toxicity studies only showed EGFR-related adverse reactions, such as gastrointestinal and liver toxicity, with high safety margin over 10-folds. CONCLUSION: H002 has been demonstrated as a promising next generation EGFR inhibitor, with high selectivity, a wide spectrum and potent anti-tumor activity against various EGFR activating mutations, favorable DMPK and safety profiles. It will be further developed as a fourth-generation EGFR inhibitor to overcome drug resistance in NSCLC. Citation Format: Wei Huang, Lin Zhu, Xiaoe Yan, Xin Huang, Jia Hao, Shan Li, Xiangyu Li, Zhiming Chen, Yunchuan Jia, Haibo Li, Jianming Zhang, Xianming Deng, Caihong Yun. H002: A wide spectrum, highly selective fourth-generation EGFR inhibitor overcoming resistance harboring C797S mutation in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5461.

Structural optimization of pyrido[2,3-d]pyrimidin-7-ones was conducted to yield a series of new selective EGFR