{"paper_id":"3895bb06-6429-4ec9-a47c-5e7156ec46e5","body_text":"Discovery and mechanism of a highly selective, antifungal acetyl CoA synthetase inhibitor | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Discovery and mechanism of a highly selective, antifungal acetyl CoA synthetase inhibitor Damian Krysan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5619443/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Oct, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Acetyl CoA synthetases (ACS) have emerged as drug targets for the treatment of cancer, metabolic diseases as well as fungal and parasitic infections. Although a variety of small molecule ACS inhibitors have been discovered, the systematic optimization of these molecules has been slowed by a lack of structural information regarding their mechanism of inhibition. Through a chemical genetic-based, synthetic lethal screen of the human fungal pathogen Cryptococcus neoformans, we identified an isoxazole-based ACS inhibitor with antifungal activity and exquisite selectivity for the C. neoformans Acs1 relative to human ACSS2 as well as other fungal ACSs. Xray crystallographic characterization of the isoxazole-CnAcs1 complex revealed that the isoxazole functions as an acetyl CoA mimic and occupies both the acetyl- and CoA-binding sites of CnAcs1. Consistent with this novel mode of inhibition, the isoxazoles display uncompetitive inhibition kinetics that are similar to antimalarial ACS inhibitors also proposed to target the CoA binding site. Consequently, these data provide structural and mechanistic insights into the remarkable selectivity of Acetyl CoA pocket-targeting ACS inhibitors. In addition, these data provide strong proof-of-principle that targeting fungal and parasitic ACSs for the development of novel anti-infectives can be achieved with high selectivity and, thereby, low host toxicity. Biological sciences/Chemical biology/Mechanism of action Biological sciences/Microbiology/Fungi/Fungal biology Biological sciences/Structural biology/X-ray crystallography Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Full Text Additional Declarations Yes there is potential Competing Interest. The University of Iowa and Northern Illinois University have filed a patent disclosure related to the inhibitors described in the manuscript Supplementary Files 808.Table.1.legends.pdf Table 1 808manuscriptsupplementarymaterial.combined.pdf Supplementary Materials 9CD8fullvalidationreport.pdf Structure Validation Report for PDB 9CD8 8g0tfullvalidation.pdf Structure Validation Report for PDB 8g0t Cite Share Download PDF Status: Published Journal Publication published 14 Oct, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-5619443\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":396153101,\"identity\":\"c7a170f3-6c86-4fea-a49e-e92eb53e514d\",\"order_by\":0,\"name\":\"Damian Krysan\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYDCCA2DEYADhVTDwQBhsRGs5Q6QWBrgWxjaYMB4tfMdPJx74uYPB2HxG+sPPhfMOy/BLJD9g+FB2GKcWyTO5Gw72nmEwk7mRYyw9c9thHskZaQaMM87h1mJwIHfDAd62/zYSEjkM0rxALQa3EwyYedvwaDn/dsPBv20MQC3pj3/zzgFpSf/A/Beflhu5Gw7ztjGYSUgkmEnzNoC05BgwM+LRInnj7YbDsm0MxhI8b8yseY6l80jOf1NwsOdcOk4tfOdzN39828ZgOIM9/fFtnhpre36e4xsf/CizxqkFAQQSEOwDRKgHAn4i1Y2CUTAKRsHIAwC0TVlpY4dkugAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"University of Iowa\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Damian\",\"middleName\":\"\",\"lastName\":\"Krysan\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-12-10 21:05:27\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-5619443/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-5619443/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1038/s41467-025-64183-7\",\"type\":\"published\",\"date\":\"2025-10-14T04:00:00+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":72728522,\"identity\":\"a7a15304-b2f6-4638-ac47-b1c3b39b7c2d\",\"added_by\":\"auto\",\"created_at\":\"2025-01-01 05:09:33\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":135719,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eOutline of CnAcs1 inhibitor screen and validation strategy leading to identification of isoxazole 1. The number of compounds evaluated in the primary screen, hit validation, and counter screening steps of the screening campaign are shown. The structure of the hit isoxazole 1 is provided with the functionally distinct regions of the molecule highlighted. The initial IC50 of isoxazole 1 towards CnAcs1 is indicated; the curve is representative of two independent experiments with the IC50 and error shown.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1/3dc271beda840b4cc5efbce1.png\"},{\"id\":72728526,\"identity\":\"3aa345ff-0aaf-4bb6-88f2-60652598c85a\",\"added_by\":\"auto\",\"created_at\":\"2025-01-01 05:09:33\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":346407,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAntifungal activity of isoxazole 1. A. The minimum inhibitory concentration (MIC) of 1 against C. neoformans reference strain H99 and acl1Δ mutant strains in YPD, YNB+2%acetate, and RPMI-MOPS buffer at 37oC. The values were identical for three independent experiments performed in technical duplicate. B. MIC values against C. albicans reference strain SC5314 and C. glabrata CBS138. Fractional inhibitor concentrations against H99 for fluconazole (C) and rapamycin (D). Growth in wells is indicated by tan fill while empty wells indicate no growth. The wells with red outline indicate the fractional inhibitory concentration (FIC). Fractional inhibitor concentration index (FICI) ≤ 0.5 indicates synergy.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1/a4aec95534ac91daa535268f.png\"},{\"id\":72728529,\"identity\":\"bc760976-ac55-4db4-8c92-e7f5747f2686\",\"added_by\":\"auto\",\"created_at\":\"2025-01-01 05:09:33\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":133907,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eIn vitro cytotoxicity and microsome stability of isoxazoles. A. HepG2 cells were exposed to the indicated concentrations of isoxazole 1 for 24hr. The release of lactate dehydrogenase (LDH) into the medium was determined as described in materials and methods and normalized to detergent-induced lysis (100% lysis). Data are means of two independent experiments performed in technical triplicate with error bars indicating standard deviation. B. Structures of isoxazole 1 and 2. C. In vitro stability of isoxazole 1 in liver and mouse microsomes. The time indicate t1/2 in minutes. D. In vitro stability of isoxazole 2 in the presence and absence of pan-cytochrome inhibitor 1-amino-benzotriazole (ABT).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1/60995d46190215620f957019.png\"},{\"id\":72728533,\"identity\":\"1bec7fc5-fa0e-4b97-8ac0-f57eded81ed3\",\"added_by\":\"auto\",\"created_at\":\"2025-01-01 05:09:33\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":889907,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSGF29 mutations in isoxazole 1-resistant strains. Alignment of the Sgf29 coding region for parental H99 and the isoxazole 1-resistant isolates. Isolates H99R (derived from H99) and G4 (derived from the acl1Δ kbc1Δ double mutant) have insertion mutations leading to frame-shift truncation of Sgf29 at amino acid 110 and 74, respectively. Red boxes indicate the location of mutations in the full-length proteins for resistant isolates E3 and B4 (both derived from acl1Δ mutant). The mutations at 294 and 295 are in the Tudor domain of the protein. See Supplemental Table 2 for identify for base changes corresponding to each mutation.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1/1aaad191178133be4973aedd.png\"},{\"id\":72728532,\"identity\":\"a03dfee8-5b63-4613-83bb-0746599018bb\",\"added_by\":\"auto\",\"created_at\":\"2025-01-01 05:09:33\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":178230,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSGF29 deletion mutant is resistant to isoxazole 1. A. Homology model of Sgf29 showing predicted location of sgf29Y294S mutation. B. Summary of SIFT analysis of sgf29Y294S mutation indicating that it is predicted to be intolerable. C. Competitive growth assay between mNEON-tagged H99 and sgf29Δ mutant at the indicated concentrations of isoxazole 1. The ratio of the two strains was determined by flow cytometry with data represented as mean and standard deviation of three independent replicates.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1/29245c6e56684638b5f5349f.png\"},{\"id\":72729114,\"identity\":\"64cb9aac-d4ab-4784-a17f-4bf81a0413c6\",\"added_by\":\"auto\",\"created_at\":\"2025-01-01 05:33:34\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":147340,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eIsoxazole 1 is highly selectivity for CnAcs1 relative to other fungal ACS and human ACSS2 enzymes. A. The activity of isoxazole 1 against purified Saccharomyces cerevisiae Acs1 (ScAcs1), Candida albicans Acs2 (CaAcs2), Aspergillus fumigatus Acs1 (AfAcs1), Coccidioides immitis Acs1 (CiAcs1), and Cryptococcus neoformans Acs1 (CnAcs1). The inhibition curves are representative of three independent experiments showing similar results. B. Isoxazole 1 has minimal activity toward human ACSS2. C. Single dose experiment assessing the activity of the human ACSS2 inhibitor MTB-9655 against CnAcs1 at the maximum soluble concentration. Bar indicates mean of two independent experiments with error bars showing standard deviation. D. Antimalarial, PfAcAS inhibitor MMV084978 inhibits CnAcs1 with IC50 = 2.8 μM. The IC50 toward PfAcAS is indicated by arrow. E. PfAcAS inhibitor MMV019721 does not inhibit CnAcs1 at the maximum soluble concentration. F. Isoxazole 1 does not inhibit the C. neoformans aceto-acetyl CoA synthetase CnKbc1.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1/f87a24554f27b0342d37cb6b.png\"},{\"id\":72729113,\"identity\":\"8d7139d8-e587-43b8-977f-b791053a5e6f\",\"added_by\":\"auto\",\"created_at\":\"2025-01-01 05:33:33\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":173683,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eIsoxazole 1 is an uncompetitive inhibitor of CnAcs1. A. Schematic of the two-step reaction catalyzed by ACS and the conformational changes that occur during the reaction. APO indicates enzyme without substrate or product bound. AD indicates conformation associated with the adenylation reaction that generates the Ac-AMP intermediate. TE indicates the conformation associated with the thio-esterification reaction of Ac-AMP with CoA to yield AcCoA. CTD indicates the C-terminal domain of the protein that undergoes rearrangement through the course of the reaction. B-D. Determination of isoxazole 1 Ki values for the three substrates and goodness-of-fit (r2) values for an uncompetitive model of inhibition. The color scheme indicates the color for each of the different concentrations for the reaction plots.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1/632e110bed927da1560e71c8.png\"},{\"id\":72728531,\"identity\":\"e34ca8f7-543f-40a1-ae68-0830cb69384e\",\"added_by\":\"auto\",\"created_at\":\"2025-01-01 05:09:33\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":851349,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eX-ray crystal structure of CnAcs1-isoxazole complex. A. Overall structure of the trimeric CnAcs1-isoxazole 1 complex with the C-terminal domains (CTD) shown in ribbon format. B. Mass photometry showing that CnAcs1 is most consistent with a trimer in solution to low concentrations and that the addition of supra-inhibitory of isoxazole 1 does not change the apparent size of the CnAcs1 protein complex. C. Schematic comparing “open-TE” (tan) conformation of the CTD observed in the CnAcs1-isoxazole 1 complex to the CTD conformations in the uninhibited APO (blue), AD (green), and TE (orange) forms of the protein. D. The region of the protein bound by isoxazole 1 (green) and its position within that pocket. E. Overlay of isoxazole 1 (grey) with the bound pose of Coenzyme A (turquoise) and an ethyl-AMP inhibitor (yellow) in a previously reported structure of CnAcs1 (ref. 23) showing at I interacts with both the Coenzyme A pocket and the acetyl portion of the AcAMP binding pocket. F. The W334 residue functions to open the CoA tunnel by rotating upon CoA binding. The position of W334 CnAcs1-isoxazole 1 (grey) complex overlaps with that observed in CnAcs1 structures with CoA bound.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1/62ebe3757e0db73e1a4711ad.png\"},{\"id\":72728534,\"identity\":\"ebca633f-83ec-455c-9759-f908f7d84e57\",\"added_by\":\"auto\",\"created_at\":\"2025-01-01 05:09:33\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":278177,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMolecular dynamics and structure-activity relationship data provide insights into interactions contributing to isoxazole 1/2-CnAcs1 binding. A. Schematic represents details of the isoxazole 1 interactions with each protein residue. Interactions that occur more than 11% of the simulation time in the selected trajectory (0.00 through 1000.00 nsec), are shown. Dotted arrows indicate direct interactions between protein and ligand. Solid arrows represent interactions mediated by a water molecule. The majority of residues predicted to contribute to binding participate in hydrophobic interactions (green). A key H-bonding interaction between Thr336 and the amide carbonyl of isoxazole 1 was identified in 81% of simulations. B. IC50 curves for isoxazoles 2-7 indicating that the amide carbonyl (isoxazole 3/4) and isoxazole cyclopropyl moiety (isoxazoles 5-7) are key drivers of the CnAcs1 potency of isoxazoles 1 and 2.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1/9e1df64eb44d7458612bf0fa.png\"},{\"id\":93558489,\"identity\":\"32c7f488-df61-4a85-806c-7edc8c335a65\",\"added_by\":\"auto\",\"created_at\":\"2025-10-15 07:06:01\",\"extension\":\"pdf\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1418967,\"visible\":true,\"origin\":\"\",\"legend\":\"Article File\",\"description\":\"\",\"filename\":\"808.manuscript.sub.12.24.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1_covered_bd83c12e-b222-478d-bfaf-a7e312a80ba1.pdf\"},{\"id\":72728523,\"identity\":\"25abf7a6-09fb-4817-b79f-71518e6b40a2\",\"added_by\":\"auto\",\"created_at\":\"2025-01-01 05:09:33\",\"extension\":\"pdf\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":112608,\"visible\":true,\"origin\":\"\",\"legend\":\"Table 1\",\"description\":\"\",\"filename\":\"808.Table.1.legends.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1/573e6c5e0c51f13cf2465f24.pdf\"},{\"id\":72728537,\"identity\":\"96e00fdb-1722-4a1c-9759-6545e9fe9aab\",\"added_by\":\"auto\",\"created_at\":\"2025-01-01 05:09:33\",\"extension\":\"pdf\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":3611750,\"visible\":true,\"origin\":\"\",\"legend\":\"Supplementary Materials\",\"description\":\"\",\"filename\":\"808manuscriptsupplementarymaterial.combined.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1/9e9db5fa62cd142c5a92cebd.pdf\"},{\"id\":72728538,\"identity\":\"888fafff-a681-46bf-970e-4891b2eb4a36\",\"added_by\":\"auto\",\"created_at\":\"2025-01-01 05:09:33\",\"extension\":\"pdf\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1361931,\"visible\":true,\"origin\":\"\",\"legend\":\"Structure Validation Report for PDB 9CD8\",\"description\":\"\",\"filename\":\"9CD8fullvalidationreport.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1/2520005638fcfcd66cd1b42e.pdf\"},{\"id\":72728536,\"identity\":\"feb334ec-fcad-475b-add5-716ca520e72c\",\"added_by\":\"auto\",\"created_at\":\"2025-01-01 05:09:33\",\"extension\":\"pdf\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1664917,\"visible\":true,\"origin\":\"\",\"legend\":\"Structure Validation Report for PDB 8g0t\",\"description\":\"\",\"filename\":\"8g0tfullvalidation.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5619443/v1/17b30b5372a662a1f34a8219.pdf\"}],\"financialInterests\":\"\\u003cb\\u003eYes\\u003c/b\\u003e there is potential Competing Interest.\\nThe University of Iowa and Northern Illinois University have filed a patent disclosure related to the inhibitors described in the manuscript\",\"formattedTitle\":\"Discovery and mechanism of a highly selective, antifungal acetyl CoA synthetase inhibitor\",\"fulltext\":[],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":false,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":true,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":true,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-portfolio\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Nature Portfolio\",\"twitterHandle\":\"\",\"acdcEnabled\":false,\"dfaEnabled\":false,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5619443/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5619443/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"Acetyl CoA synthetases (ACS) have emerged as drug targets for the treatment of cancer, metabolic diseases as well as fungal and parasitic infections. Although a variety of small molecule ACS inhibitors have been discovered, the systematic optimization of these molecules has been slowed by a lack of structural information regarding their mechanism of inhibition. Through a chemical genetic-based, synthetic lethal screen of the human fungal pathogen Cryptococcus neoformans, we identified an isoxazole-based ACS inhibitor with antifungal activity and exquisite selectivity for the C. neoformans Acs1 relative to human ACSS2 as well as other fungal ACSs. Xray crystallographic characterization of the isoxazole-CnAcs1 complex revealed that the isoxazole functions as an acetyl CoA mimic and occupies both the acetyl- and CoA-binding sites of CnAcs1. Consistent with this novel mode of inhibition, the isoxazoles display uncompetitive inhibition kinetics that are similar to antimalarial ACS inhibitors also proposed to target the CoA binding site. Consequently, these data provide structural and mechanistic insights into the remarkable selectivity of Acetyl CoA pocket-targeting ACS inhibitors. In addition, these data provide strong proof-of-principle that targeting fungal and parasitic ACSs for the development of novel anti-infectives can be achieved with high selectivity and, thereby, low host toxicity.\",\"manuscriptTitle\":\"Discovery and mechanism of a highly selective, antifungal acetyl CoA synthetase inhibitor\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-01-01 05:09:28\",\"doi\":\"10.21203/rs.3.rs-5619443/v1\",\"editorialEvents\":[],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-communications\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"NCOMMS\",\"sideBox\":\"Learn more about [Nature Communications](http://www.nature.com/ncomms/)\",\"snPcode\":\"\",\"submissionUrl\":\"https://mts-ncomms.nature.com/\",\"title\":\"Nature Communications\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Nature Communications\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"763c919f-a6ee-43bc-bbd7-0b31effaebdb\",\"owner\":[],\"postedDate\":\"January 1st, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":42205038,\"name\":\"Biological sciences/Chemical biology/Mechanism of action\"},{\"id\":42205039,\"name\":\"Biological sciences/Microbiology/Fungi/Fungal biology\"},{\"id\":42205040,\"name\":\"Biological sciences/Structural biology/X-ray crystallography\"}],\"tags\":[],\"updatedAt\":\"2025-10-15T07:05:50+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-5619443\",\"link\":\"https://doi.org/10.1038/s41467-025-64183-7\",\"journal\":{\"identity\":\"nature-communications\",\"isVorOnly\":false,\"title\":\"Nature Communications\"},\"publishedOn\":\"2025-10-14 04:00:00\",\"publishedOnDateReadable\":\"October 14th, 2025\"},\"versionCreatedAt\":\"2025-01-01 05:09:28\",\"video\":\"\",\"vorDoi\":\"10.1038/s41467-025-64183-7\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41467-025-64183-7\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5619443\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5619443\",\"identity\":\"rs-5619443\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}