RESUMO
Since the discovery of the caspase-2 (Casp2)-mediated ∆tau314 cleavage product and its associated impact on tauopathies such as Alzheimer's disease, the design of selective Casp2 inhibitors has become a focus in medicinal chemistry research. In the search for new lead structures with respect to Casp2 selectivity and drug-likeness, we have taken an approach by looking more closely at the specific sites of Casp2-mediated proteolysis. Using seven selected protein cleavage sequences, we synthesized a peptide series of 53 novel molecules and studied them using in vitro pharmacology, molecular modeling, and crystallography. Regarding Casp2 selectivity, AcITV(Dab)D-CHO (23) and AcITV(Dap)D-CHO (26) demonstrated the best selectivity (1-6-fold), although these trends were only moderate. However, some analogous tetrapeptides, most notably AcDKVD-CHO (45), showed significantly increased Casp3 selectivities (>100-fold). Tetra- and tripeptides display decreased or no Casp2 affinity, supporting the assumption that a motif of five amino acids is required for efficient Casp2 inhibition. Overall, the results provide a reasonable basis for the development of both selective Casp2 and Casp3 inhibitors.
Assuntos
Caspase 2 , Caspase 2/metabolismo , Caspase 3/metabolismo , Inibidores de Caspase/farmacologia , Proteólise , Relação Estrutura-AtividadeRESUMO
Synaptic and cognitive deficits mediated by a severe reduction in excitatory neurotransmission caused by a disproportionate accumulation of the neuronal protein tau in dendritic spines is a fundamental mechanism that has been found repeatedly in models of tauopathies, including Alzheimer's disease, Lewy body dementia, frontotemporal dementia, and traumatic brain injury. Synapses thus damaged may contribute to dementia, among the most feared cause of debilitation in the elderly, and currently there are no treatments to repair them. Caspase-2 (Casp2) is an essential component of this pathological cascade. Although it is believed that Casp2 exerts its effects by hydrolyzing tau at aspartate-314, forming Δtau314, it is also possible that a noncatalytic mechanism is involved because catalytically dead Casp2 is biologically active in at least one relevant cellular pathway, that is, autophagy. To decipher whether the pathological effects of Casp2 on synaptic function are due to its catalytic or noncatalytic properties, we discovered and characterized a new Casp2 inhibitor, compound 1 [pKi (Casp2) = 8.12], which is 123-fold selective versus Casp3 and >2000-fold selective versus Casp1, Casp6, Casp7, and Casp9. In an in vitro assay based on Casp2-mediated cleavage of tau, compound 1 blocked the production of Δtau314. Importantly, compound 1 prevented tau from accumulating excessively in dendritic spines and rescued excitatory neurotransmission in cultured primary rat hippocampal neurons expressing the P301S tau variant linked to FTDP-17, a familial tauopathy. These results support the further development of small-molecule Casp2 inhibitors to treat synaptic deficits in tauopathies.
Assuntos
Demência Frontotemporal , Tauopatias , Animais , Caspase 2/metabolismo , Modelos Animais de Doenças , Demência Frontotemporal/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Ratos , Transmissão Sináptica , Tauopatias/metabolismo , Proteínas tau/metabolismoRESUMO
Alzheimer's disease (AD) was first described by Alois Alzheimer over 100 years ago, but there is still no overarching theory that can explain its cause in detail. There are also no effective therapies to treat either the cause or the associated symptoms of this devastating disease. A potential approach to better understand the pathogenesis of AD could be the development of selective caspase-2 (Casp2) probes, as we have shown that a Casp2-mediated cleavage product of tau (Δtau314) reversibly impairs cognitive and synaptic function in animal models of tauopathies. In this article, we map out the Casp2 binding site through the preparation and assay of a series of 35 pentapeptide inhibitors with the goal of gaining selectivity against caspase-3 (Casp3). We also employed computational docking methods to understand the key interactions in the binding pocket of Casp2 and the differences predicted for binding at Casp3. Moreover, we crystallographically characterized the binding of selected pentapeptides with Casp3. Furthermore, we engineered and expressed a series of recombinant tau mutants and investigated them in an in vitro cleavage assay. These studies resulted in simple peptidic inhibitors with nanomolar affinity, for example, AcVDV(Dab)D-CHO (24) with up to 27.7-fold selectivity against Casp3. Our findings provide a good basis for the future development of selective Casp2 probes and inhibitors that can serve as pharmacological tools in planned in vivo studies and as lead compounds for the design of bioavailable and more drug-like small molecules.
RESUMO
A high-throughput screen designed to discover new inhibitors of histone acetyltransferase KAT6A uncovered CTX-0124143 (1), a unique aryl acylsulfonohydrazide with an IC50 of 1.0 µM. Using this acylsulfonohydrazide as a template, we herein disclose the results of our extensive structure-activity relationship investigations, which resulted in the discovery of advanced compounds such as 55 and 80. These two compounds represent significant improvements on our recently reported prototypical lead WM-8014 (3) as they are not only equivalently potent as inhibitors of KAT6A but are less lipophilic and significantly more stable to microsomal degradation. Furthermore, during this process, we discovered a distinct structural subclass that contains key 2-fluorobenzenesulfonyl and phenylpyridine motifs, culminating in the discovery of WM-1119 (4). This compound is a highly potent KAT6A inhibitor (IC50 = 6.3 nM; KD = 0.002 µM), competes with Ac-CoA by binding to the Ac-CoA binding site, and has an oral bioavailability of 56% in rats.
Assuntos
Antineoplásicos/farmacologia , Histona Acetiltransferases/antagonistas & inibidores , Hidrazinas/farmacologia , Sulfonamidas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Descoberta de Drogas , Estabilidade de Medicamentos , Humanos , Hidrazinas/síntese química , Hidrazinas/química , Hidrazinas/farmacocinética , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacocinéticaRESUMO
Glutathione transferase omega-1 (GSTO1-1) is an enzyme whose function supports the activation of interleukin (IL)-1ß and IL-18 that are implicated in a variety of inflammatory disease states for which small-molecule inhibitors are sought. The potent reactivity of the active-site cysteine has resulted in reported inhibitors that act by covalent labeling. In this study, structure-activity relationship (SAR) elaboration of the reported GSTO1-1 inhibitor C1-27 was undertaken. Compounds were evaluated for inhibitory activity toward purified recombinant GSTO1-1 and for indicators of target engagement in cell-based assays. As covalent inhibitors, the kinact/KI values of selected compounds were determined, as well as in vivo pharmacokinetics analysis. Cocrystal structures of key novel compounds in complex with GSTO1-1 were also solved. This study represents the first application of a biochemical assay for GSTO1-1 to determine kinact/KI values for tested inhibitors and the most extensive set of cell-based data for a GSTO1-1 inhibitor SAR series reported to date. Our research culminated in the discovery of 25, which we propose as the preferred biochemical tool to interrogate cellular responses to GSTO1-1 inhibition.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/antagonistas & inibidores , Sulfonamidas/química , Sulfonamidas/farmacologia , Animais , Desenvolvimento de Medicamentos , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Humanos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , BenzenossulfonamidasRESUMO
A high-throughput screen for inhibitors of the histone acetyltransferase, KAT6A, led to identification of an aryl sulfonohydrazide derivative (CTX-0124143) that inhibited KAT6A with an IC50 of 1.0 µM. Elaboration of the structure-activity relationship and medicinal chemistry optimization led to the discovery of WM-8014 (97), a highly potent inhibitor of KAT6A (IC50 = 0.008 µM). WM-8014 competes with acetyl-CoA (Ac-CoA), and X-ray crystallographic analysis demonstrated binding to the Ac-CoA binding site. Through inhibition of KAT6A activity, WM-8014 induces cellular senescence and represents a unique pharmacological tool.
Assuntos
Benzenossulfonatos/química , Descoberta de Drogas/métodos , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Hidrazinas/química , Animais , Benzenossulfonatos/farmacologia , Células CACO-2 , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Hidrazinas/farmacologia , Camundongos , Estrutura Secundária de ProteínaRESUMO
A series of 3,3'-disubstituted 5,5'-bi(1,2,4-triazine) derivatives was synthesized and screened against the erythrocytic stage of Plasmodium falciparum 3D7 line. The most potent dimer, 6k, with an IC50 (50% inhibitory concentration) of 0.008 µM, had high in vitro potency against P. falciparum lines resistant to chloroquine (W2, IC50 = 0.0047 ± 0.0011 µM) and artemisinin (MRA1240, IC50 = 0.0086 ± 0.0010 µM). Excellent ex vivo potency of 6k was shown against clinical field isolates of both P. falciparum (IC50 = 0.022-0.034 µM) and Plasmodium vivax (IC50 = 0.0093-0.031 µM) from the blood of outpatients with uncomplicated malaria. Despite 6k being cleared relatively rapidly in mice, it suppressed parasitemia in the Peters 4-day test, with a mean ED50 value (50% effective dose) of 1.47 mg kg-1 day-1 following oral administration. The disubstituted triazine dimer 6k represents a new class of orally available antimalarial compounds of considerable interest for further development.
Assuntos
Antimaláricos/farmacologia , Triazinas/farmacologia , Animais , Antimaláricos/química , Antimaláricos/farmacocinética , Cloroquina/farmacologia , Resistência a Medicamentos , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Estrutura Molecular , Plasmodium/classificação , Plasmodium/efeitos dos fármacos , Especificidade da Espécie , Relação Estrutura-Atividade , Triazinas/química , Triazinas/farmacocinéticaRESUMO
Nonspecific target engagement by test compounds and purported chemical probes is a significant source of assay interference and promiscuous bioactivity in high-throughput screening (HTS) and chemical biology. Most counter-screens for thiol-reactive compounds utilize mass spectrometry or fluorescence detection, and non-proteinaceous reporters like glutathione that may not always approximate the reactivity of protein side-chains. By contrast, a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) is an industry-developed protein-based [1H-13C]-heteronuclear multiple quantum coherence (HMQC) NMR counter-screen to identify nonspecific protein interactions by test compounds by reporting their tendencies to modulate the human La antigen conformation. This Current Protocol is a users-guide to the production of the 13C-labeled La antigen reporter protein, the reaction of test compounds with this reporter protein, as well as the collection and analysis of characteristic NMR spectra. Combined with other assay interference counter-screens, this assay will enhance chemical biology by helping researchers better prioritize chemical matter and which will increase the number of tractable HTS screening actives and aid in the development of better chemical probes.
Assuntos
Ensaios de Triagem em Larga Escala , Sondas Moleculares/química , Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Humanos , Antígenos Comuns de Leucócito , Proteínas/síntese química , Reprodutibilidade dos TestesRESUMO
Spinocerebellar ataxia type 1 (SCA1) is a polyglutamine (polyQ) repeat neurodegenerative disease in which a primary site of pathogenesis are cerebellar Purkinje cells. In addition to polyQ expansion of ataxin-1 protein (ATXN1), phosphorylation of ATXN1 at the serine 776 residue (ATXN1-pS776) plays a significant role in protein toxicity. Utilizing a biochemical approach, pharmacological agents and cell-based assays, including SCA1 patient iPSC-derived neurons, we examine the role of Protein Kinase A (PKA) as an effector of ATXN1-S776 phosphorylation. We further examine the implications of PKA-mediated phosphorylation at ATXN1-S776 on SCA1 through genetic manipulation of the PKA catalytic subunit Cα in Pcp2-ATXN1[82Q] mice. Here we show that pharmacologic inhibition of S776 phosphorylation in transfected cells and SCA1 patient iPSC-derived neuronal cells lead to a decrease in ATXN1. In vivo, reduction of PKA-mediated ATXN1-pS776 results in enhanced degradation of ATXN1 and improved cerebellar-dependent motor performance. These results provide evidence that PKA is a biologically important kinase for ATXN1-pS776 in cerebellar Purkinje cells.
Assuntos
Ataxia/metabolismo , Ataxina-1/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células de Purkinje/metabolismo , Serina/metabolismo , Animais , Ataxia/genética , Ataxia/patologia , Ataxina-1/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fosforilação/fisiologia , Células de Purkinje/patologia , Serina/genéticaRESUMO
Glutathione transferase Omega 1 (GSTO1-1) is an atypical GST reported to play a pro-inflammatory role in response to LPS. Here we show that genetic knockout of Gsto1 alters the response of mice to three distinct inflammatory disease models. GSTO1-1 deficiency ameliorates the inflammatory response stimulated by LPS and attenuates the inflammatory impact of a high fat diet on glucose tolerance and insulin resistance. In contrast, GSTO1-1 deficient mice show a more severe inflammatory response and increased escape of bacteria from the colon into the lymphatic system in a dextran sodium sulfate mediated model of inflammatory bowel disease. These responses are similar to those of TLR4 and MyD88 deficient mice in these models and confirm that GSTO1-1 is critical for a TLR4-like pro-inflammatory response in vivo. In wild-type mice, we show that a small molecule inhibitor that covalently binds in the active site of GSTO1-1 can be used to ameliorate the inflammatory response to LPS. Our findings demonstrate the potential therapeutic utility of GSTO1-1 inhibitors in the modulation of inflammation and suggest their possible application in the treatment of a range of inflammatory conditions.
Assuntos
Proteínas de Transporte/metabolismo , Colite/metabolismo , Glutationa Transferase/metabolismo , Inflamação/metabolismo , Obesidade/metabolismo , Animais , Proteínas de Transporte/genética , Colite/tratamento farmacológico , Colite/genética , Glutationa Transferase/genética , Inflamação/tratamento farmacológico , Inflamação/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Obesidade/tratamento farmacológico , Obesidade/genética , Bibliotecas de Moléculas Pequenas/uso terapêutico , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Many compounds with potentially reactive chemical motifs and poor physicochemical properties are published as selective modulators of biomolecules without sufficient validation and then propagated in the scientific literature as useful chemical probes. Several histone acetyltransferase (HAT) inhibitors with these liabilities are now routinely used to probe epigenetic pathways. We profile the most commonly used HAT inhibitors and confirm that the majority of them are nonselective interference compounds. Most (15 out of 23, 65%) of the inhibitors are flagged by ALARM NMR, an industry-developed counter-screen for promiscuous compounds. Biochemical counter-screens confirm that most of these compounds are either thiol-reactive or aggregators. Selectivity panels show many of these compounds modulate unrelated targets in vitro, while several also demonstrate nonspecific effects in cell assays. These data demonstrate the usefulness of performing counter-screens for bioassay promiscuity and assay interference, and raise caution about the utility of many widely used, but insufficiently validated, compounds employed in chemical biology.
Assuntos
Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala/métodos , Histona Acetiltransferases/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Células HEK293 , Histona Acetiltransferases/antagonistas & inibidores , Humanos , Células MCF-7 , Estrutura Molecular , Compostos de Sulfidrila/químicaRESUMO
Facioscapulohumeral muscular dystrophy is a genetically dominant, currently untreatable muscular dystrophy. It is caused by mutations that enable expression of the normally silent DUX4 gene, which encodes a pathogenic transcription factor. A screen based on Tet-on DUX4-induced mouse myoblast death previously uncovered compounds from a 44,000-compound library that protect against DUX4 toxicity. Many of those compounds acted downstream of DUX4 in an oxidative stress pathway. Here, we extend this screen to an additional 160,000 compounds and, using greater stringency, identify a new set of DUX4-protective compounds. From 640 hits, we performed secondary screens, repurchased 46 of the most desirable, confirmed activity, and tested each for activity against other cell death-inducing insults. The majority of these compounds also protected against oxidative stress. Of the 100 repurchased compounds identified through both screens, only SHC40, 75, and 98 inhibited DUX4 target genes, but they also inhibited dox-mediated DUX4 expression. Using a target gene readout on the 640-compound hit set, we discovered three overlooked compounds, SHC351, 540, and 572, that inhibit DUX4 target gene upregulation without nonspecific effects on the Tet-on system. These novel inhibitors of DUX4 transcriptional activity may thus act on pathways or cofactors needed by DUX4 for transcriptional activation in these cells.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Proteínas de Homeodomínio/genética , Distrofia Muscular Facioescapuloumeral/tratamento farmacológico , Transcrição Gênica/efeitos dos fármacos , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/antagonistas & inibidores , Humanos , Camundongos , Distrofia Muscular Facioescapuloumeral/genética , Mutação , Estresse Oxidativo/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêutico , Ativação Transcricional/efeitos dos fármacosRESUMO
Despite its wide use, not every high-throughput screen (HTS) yields chemical matter suitable for drug development campaigns, and seldom are 'go/no-go' decisions in drug discovery described in detail. This case report describes the follow-up of a 4-aroyl-1,5-disubstituted-3-hydroxy-2H-pyrrol-2-one active from a cell-free HTS to identify small-molecule inhibitors of Rtt109-catalyzed histone acetylation. While this compound and structural analogs inhibited Rtt109-catalyzed histone acetylation in vitro, further work on this series was halted after several risk mitigation strategies were performed. Compounds with this chemotype had a poor structure-activity relationship, exhibited poor selectivity among other histone acetyltransferases, and tested positive in a ß-lactamase counter-screen for chemical aggregates. Furthermore, ALARM NMR demonstrated compounds with this chemotype grossly perturbed the conformation of the La protein. In retrospect, this chemotype was flagged as a 'frequent hitter' in an analysis of a large corporate screening deck, yet similar compounds have been published as screening actives or chemical probes versus unrelated biological targets. This report-including the decision-making process behind the 'no-go' decision-should be informative for groups engaged in post-HTS triage and highlight the importance of considering physicochemical properties in early drug discovery.
Assuntos
Ensaios de Triagem em Larga Escala , Pirróis/química , Bibliotecas de Moléculas Pequenas/química , Relação Dose-Resposta a Droga , Descoberta de Drogas , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Pirróis/farmacologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
Significant resources in early drug discovery are spent unknowingly pursuing artifacts and promiscuous bioactive compounds, while understanding the chemical basis for these adverse behaviors often goes unexplored in pursuit of lead compounds. Nearly all the hits from our recent sulfhydryl-scavenging high-throughput screen (HTS) targeting the histone acetyltransferase Rtt109 were such compounds. Herein, we characterize the chemical basis for assay interference and promiscuous enzymatic inhibition for several prominent chemotypes identified by this HTS, including some pan-assay interference compounds (PAINS). Protein mass spectrometry and ALARM NMR confirmed these compounds react covalently with cysteines on multiple proteins. Unfortunately, compounds containing these chemotypes have been published as screening actives in reputable journals and even touted as chemical probes or preclinical candidates. Our detailed characterization and identification of such thiol-reactive chemotypes should accelerate triage of nuisance compounds, guide screening library design, and prevent follow-up on undesirable chemical matter.
Assuntos
Bioensaio/normas , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Histona Acetiltransferases/antagonistas & inibidores , Compostos de Sulfidrila/química , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Reações Falso-Positivas , Humanos , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic alterations at the D4Z4 macrosatellite repeat locus on chromosome 4, resulting in inappropriate expression of the DUX4 protein. The DUX4 protein is therefore the primary molecular target for therapeutic intervention. METHODS: We have developed a high-throughput screen based on the toxicity of DUX4 when overexpressed in C2C12 myoblasts, and identified inhibitors of DUX4-induced toxicity from within a diverse set of 44,000 small, drug-like molecules. A total of 1,280 hits were then subjected to secondary screening for activity against DUX4 expressed by 3T3 fibroblasts, for absence of activity against the tet-on system used to conditionally express DUX4, and for potential effects on cellular proliferation rate. RESULTS: This allowed us to define a panel of 52 compounds to use as probes to identify essential pathways of DUX4 activity. We tested these compounds for their ability to protect wild-type cells from other types of cell death-inducing insults. Remarkably, we found that 60% of the DUX4 toxicity inhibitors that we identified also protected cells from tert-butyl hydrogen peroxide, an oxidative stress-inducing compound. Compounds did not protect against death induced by caspase activation, DNA damage, protein misfolding, or ER stress. Encouragingly, many of these compounds are also protective against DUX4 expression in human cells. CONCLUSION: These data suggest that oxidative stress is a dominant pathway through which DUX4-provoked toxicity is mediated in this system, and we speculate that enhancing the oxidative stress response pathway might be clinically beneficial in FSHD.
RESUMO
The lysine acetyltransferase (KAT) Rtt109 forms a complex with Vps75 and catalyzes the acetylation of histone H3 lysine 56 (H3K56ac) in the Asf1-H3-H4 complex. Rtt109 and H3K56ac are vital for replication-coupled nucleosome assembly and genotoxic resistance in yeast and pathogenic fungal species such as Candida albicans. Remarkably, sequence homologs of Rtt109 are absent in humans. Therefore, inhibitors of Rtt109 are hypothesized as potential and minimally toxic antifungal agents. Herein, we report the development and optimization of a cell-free fluorometric high-throughput screen (HTS) for small-molecule inhibitors of Rtt109-catalyzed histone acetylation. The KAT component of the assay consists of the yeast Rtt109-Vps75 complex, while the histone substrate complex consists of full-length Drosophila histone H3-H4 bound to yeast Asf1. Duplicated assay runs of the LOPAC demonstrated day-to-day and plate-to-plate reproducibility. Approximately 225,000 compounds were assayed in a 384-well plate format with an average Z' factor of 0.71. Based on a 3σ cut-off criterion, 1,587 actives (0.7%) were identified in the primary screen. The assay method is capable of identifying previously reported KAT inhibitors such as garcinol. We also observed several prominent active classes of pan-assay interference compounds such as Mannich bases, catechols and p-hydroxyarylsulfonamides. The majority of the primary active compounds showed assay signal interference, though most assay artifacts can be efficiently removed by a series of straightforward counter-screens and orthogonal assays. Post-HTS triage demonstrated a comparatively small number of confirmed actives with IC50 values in the low micromolar range. This assay, which utilizes five label-free proteins involved in H3K56 acetylation in vivo, can in principle identify compounds that inhibit Rtt109-catalyzed H3K56 acetylation via different mechanisms. Compounds discovered via this assay or adaptations thereof could serve as chemical probes or leads for a new class of antifungals targeting an epigenetic enzyme.