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Two Inhibitors of Yeast Plasma Membrane Atpase 1 (Scpma1p): Toward the Development of Novel Antifungal Therapies Sabine Ottilie1†, Gregory M

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Two Inhibitors of Yeast Plasma Membrane Atpase 1 (Scpma1p): Toward the Development of Novel Antifungal Therapies Sabine Ottilie1†, Gregory M View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by D-Scholarship@Pitt Ottilie et al. J Cheminform (2018) 10:6 https://doi.org/10.1186/s13321-018-0261-3 RESEARCH ARTICLE Open Access Two inhibitors of yeast plasma membrane ATPase 1 (ScPma1p): toward the development of novel antifungal therapies Sabine Ottilie1†, Gregory M. Goldgof1,4†, Andrea L. Cheung1, Jennifer L. Walker2, Edgar Vigil1, Kenneth E. Allen3, Yevgeniya Antonova‑Koch1, Carolyn W. Slayman3^, Yo Suzuki4 and Jacob D. Durrant2* Abstract Given that many antifungal medications are susceptible to evolved resistance, there is a need for novel drugs with unique mechanisms of action. Inhibiting the essential proton pump Pma1p, a P-type ATPase, is a potentially efective therapeutic approach that is orthogonal to existing treatments. We identify NSC11668 and hitachimycin as structur‑ ally distinct antifungals that inhibit yeast ScPma1p. These compounds provide new opportunities for drug discovery aimed at this important target. Keywords: Antifungal, PMA1, P-type ATPase, Computer modeling, Saccharomyces cerevisiae, In vitro evolution, Drug resistance Background sterol-C-24-methyltransferase and the fungal cell mem- Antifungal medications are in high demand, but low brane directly [8]. efcacy, host toxicity, and emerging resistance among Only a few approved antimycotics have mecha- clinical strains [1, 2] complicate their use. Tere is an nisms that are unrelated to ergosterol biosynthesis. urgent need for novel antimycotic therapeutics with For example, the highly efective echinocandins inhibit unique mechanisms of action. Te purpose of the cur- 1,3-β-glucan synthase, hindering production of the criti- rent work is to describe two novel antifungals: 4-N,6- cal cell-wall component β-glucan [9, 10]; and the terato- N-bis(3-chlorophenyl)-1-methylpyrazolo[3,4-d] genic compound fucytosine interferes with eukaryotic pyrimidine-4,6-diamine (NSC11668), and hitachimycin RNA/DNA synthesis [11, 12]. As these compounds act (also known as stubomycin, or NSC343256). through pharmacologically distinct mechanisms, they Most antifungals in clinical use target ergosterol, a can in principle complement anti-ergosterol interven- sterol present in fungal membranes but largely absent tions (see, for example, Ref. [13]). from human cells [3]. Polyene antimycotics bind directly Recognizing the need for additional antifungals with to ergosterol, thereby destabilizing the membrane [4]. mechanisms of action unrelated to ergosterol biosynthe- Allylamines inhibit squalene monooxygenase [5], the sis, we turned our attention to the essential proton pump frst enzyme in the ergosterol biosynthetic pathway of the P-type ATPase class ­(H+-ATPase), which is con- [6]. Most azole antifungals inhibit the downstream served in both plants and fungi [14]. In the model organ- enzyme lanosterol 14 α-demethylase [7], with the pos- ism S. cerevisiae, ScPma1p generates a proton gradient sible exception of abafungin, which may instead afect that is essential for both pH homeostasis and nutrient transport via ­H+-symport. Te resulting electrochemi- *Correspondence: [email protected] cal gradient drives further nutrient uptake via uniporters †Sabine Ottilie and Gregory M. Goldgof contributed equally to this work [15]. A ScPMA1 null mutation is lethal in haploid cells, ^ Deceased suggesting that the protein is essential for yeast survival 2 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA [14]. Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ottilie et al. J Cheminform (2018) 10:6 Page 2 of 9 ScPma1p inhibitors are not necessarily toxic to humans is an excellent model organism representing this king- [16, 17]. A BLASTP search using ScPma1p as the query dom, but its multiple drug efux pumps often reduce the sequence (UniProt P05030) revealed that the closest cytotoxicity of otherwise potent compounds. Tis yeast human homologs (calcium-transporting ATPases, e.g., defense mechanism can lead researchers to discard mol- UniProt O75185, A0A0A0MSP0, B7ZA13) share ~ 27% ecules that might otherwise be potential leads if chemi- sequence identity. An antimalarial compound known to cally optimized and/or coupled with adjuvants to prevent inhibit ScPma1p is also advancing through clinical trials, export. We therefore used the ABC16-Monster yeast demonstrating that it is possible to develop ScPma1p- strain, which lacks 16 genes encoding ATP-binding cas- specifc small-molecule inhibitors with low host toxicity. sette (ABC) transporters [31] and so is more susceptible ScPma1p is therefore an attractive target. to cytotoxic compounds. ScPma1p has at least three druggable pockets. Te We experimentally screened the ~ 1500 compounds primary, orthosteric pocket binds ATP [18] and deca- of the NCI Diversity Set IV, a repository of structur- vanadate [19]. A second pocket—which binds the drug ally diverse, freely available small molecules, for activ- + + digoxin in the homologous Na , K -ATPase [20] —lies ity against a whole-cell ABC16-Monster culture. Of between the TM1 and TM4 transmembrane helices [21, the ~ 1500 compounds tested, 36 inhibited yeast growth 22]. A third, cytoplasm-accessible pocket within the by at least 97% at 100 μM (Additional fle 1: Table S1). membrane-spanning domain binds spiroindolone [17] and tetrahydrocarbazole [23] inhibitors. Specifc binding Confrming ScPma1p inhibition in a cell‑free assay sites have not yet been validated for most ScPma1p inhib- Using a computational protocol designed to predict itors, including DMM-11 [24]; ebselen [25]; the natural small-molecule binding to the cytoplasm-accessible products chebulagic acid and tellimagrandin II [26]; and spiroindolone pocket, we selected seven of the whole- the inhibitors found in a recent high-throughput screen cell inhibitors for further study. Tese compounds were [27]. Other molecules, such as the carbazole inhibitors tested for specifc activity against ScPma1p in a vesicle- [28] and demethoxycurcumin [29], do not compete with based (cell-free) assay. In brief, we used a yeast strain ATP binding and so must bind elsewhere. Whether these that is prone to vesicle production due to an engineered compounds bind the digoxin- or spiroindolone pockets defect in secretory-vesicle/plasma-membrane fusion. We remains unknown. Mutations near both these pockets transformed these yeast with a ScPMA1 overexpression also confer resistance to omeprazole, a covalent inhibitor plasmid, so the harvested vesicles bore high levels of ScP- with modest activity [30]. ma1p. ScPma1p inhibition was measured by monitoring We recently discovered that KAE609, an antimalarial ATP hydrolysis in the presence of the vesicles. Small- compound currently in Phase II clinical trials [16], is molecule ScPma1p inhibitors prevented ATP hydrolysis, cytotoxic to S. cerevisiae and inhibits ScPma1p by bind- reducing the measured concentration of inorganic phos- ing to the cytoplasm-accessible pocket [17]. As part of phate. See Ref. [17] for full details. a subsequent search for additional, structurally distinct Two active compounds, NSC11668 and hitachimy- ScPma1p inhibitors that bind to the same pocket, we cin, had IC50 values of 4.4 μM and 7.8 μM in the cell- used two diferent experimental assays (vesicular ScP- free assay, respectively (Additional fle 1: Figure S1A). ma1p and whole-cell yeast) to evaluate compounds avail- Tese same compounds had IC50 values of 14.8 ± 1.24 able from the National Cancer Institute (NCI). Tese (s.e.m) and 0.87 ± 0.11 μM against the whole-cell eforts unexpectedly identifed two low-micromolar ABC16-Monster strain (see Additional fle 1: Figure S2, ScPma1p inhibitors, NSC11668 and hitachimycin, that Rows A and B). apparently act via binding to one of the other ScPma1p pockets. We hypothesize that they target the nucleotide The compounds are unlikely to bind the (ATP) binding site rather than the expected cytoplasm- cytoplasm‑accessible spiroindolone pocket accessible pocket. We hope these leads will be useful in We tested both ScPma1p inhibitors (in duplicate) against ongoing eforts to identify and optimize novel ScPma1p the unmodifed ABC16-Monster strain, as well as against inhibitors. two ABC16-Monster strains that each contained distinct spiroindolone-pocket ScPMA1 mutations: L290S and Results and discussion P399T (Additional fle 1: Figure S3) [17]. As expected, the Identifying whole‑cell inhibitors IC50 values of NSC11668 and hitachimycin against the Motivated by the need for novel antimycotics that act unmodifed ABC16-Monster strain were roughly equal orthogonally to existing antiergosterol therapeutics, to those found previously. As a positive control, we also we frst sought to identify chemical compounds with tested KAE609, a known ScPma1p inhibitor that binds whole-cell biological efects against fungi. S. cerevisiae the cytoplasm-accessible (spiroindolone-binding) pocket. Ottilie
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