Compound Collection Research Articles

Identification of Compounds for Butyrylcholinesterase Inhibition.

Butyrylcholinesterase (BChE) is a nonspecific cholinesterase enzyme that hydrolyzes choline-based esters. BChE plays a critical role in maintaining normal cholinergic function like acetylcholinesterase (AChE) through hydrolyzing acetylcholine (ACh). Selective BChE inhibition has been regarded as a viable therapeutic approach in Alzheimer’s disease. As of now, a limited number of selective BChE inhibitors are available. To identify BChE inhibitors rapidly and efficiently, we have screened 8998 compounds from several annotated libraries against an enzyme-based BChE inhibition assay in a quantitative high-throughput screening (qHTS) format. From the primary screening, we identified a group of 125 compounds that were further confirmed to inhibit BChE activity, including previously reported BChE inhibitors (e.g., bambuterol and rivastigmine) and potential novel BChE inhibitors (e.g., pancuronium bromide and NNC 756), representing diverse structural classes. These BChE inhibitors were also tested for their selectivity by comparing their IC50 values in BChE and AChE inhibition assays. The binding modes of these compounds were further studied using molecular docking analyses to identify the differences between the interactions of these BChE inhibitors within the active sites of AChE and BChE. Our qHTS approach allowed us to establish a robust and reliable process to screen large compound collections for potential BChE inhibitors.

Inhibition of Indigoidine Synthesis as a High-Throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT.

A recently-validated and underexplored drug target in Mycobacterium tuberculosis is PptT, an essential phosphopantetheinyl transferase (PPTase) that plays a critical role in activating enzymes for both primary and secondary metabolism. PptT possesses a deep binding pocket that does not readily accept labelled coenzyme A analogues that have previously been used to screen for PPTase inhibitors. Here we report on the development of a high throughput, colourimetric screen that monitors the PptT-mediated activation of the non-ribosomal peptide synthetase BpsA to a blue pigment (indigoidine) synthesising form in vitro. This screen uses unadulterated coenzyme A, avoiding analogues that may interfere with inhibitor binding, and requires only a single-endpoint measurement. We benchmark the screen using the well-characterised Library of Pharmaceutically Active Compounds (LOPAC1280) collection and show that it is both sensitive and able to distinguish weak from strong inhibitors. We further show that the BpsA assay can be applied to quantify the level of inhibition and generate consistent EC50 data. We anticipate these tools will facilitate both the screening of established chemical collections to identify new anti-mycobacterial drug leads and to guide the exploration of structure-activity landscapes to improve existing PPTase inhibitors.

Feiyanning Formula Induces Apoptosis of Lung Adenocarcinoma Cells by Activating the Mitochondrial Pathway.

Feiyanning formula (FYN) is a traditional Chinese medicine (TCM) prescription used for more than 20 years in the treatment of lung cancer. FYN is composed of Astragalus membranaceus, Polygonatum sibiricum, Atractylodes macrocephala, Cornus officinalis, Paris polyphylla, and Polistes olivaceous, etc. All of them have been proved to have anti-tumor effect. In this study, we used the TCM network pharmacological analysis to perform the collection of compound and disease target, the prediction of compound target and biological signal and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. It was found that the activation of mitochondrial pathway might be the molecular mechanism of the anti-lung cancer effect of FYN. The experimental results showed that FYN had an inhibitory effect on the growth of lung cancer cells in a dose-dependent and time-dependent manner. Moreover, FYN induced G2/M cell cycle arrest and apoptotic cell death as early as 6 h after treatment. In addition, FYN significantly induced mitochondrial membrane depolarization and increased calreticulin expression. Metabolomics analysis showed the increase of ATP utilization (assessed by a significant increase of the AMP/ATP and ADP/ATP ratio, necessary for apoptosis induction) and decrease of polyamines (that reflects growth potential). Taken together, our study suggested that FYN induced apoptosis of lung adenocarcinoma cells by promoting metabolism and changing the mitochondrial membrane potential, further supporting the validity of network pharmacological prediction.

Antimicrobial screening of a historical collection of over 140 000 small molecules

Most of the chemical compound collection (currently about 200 000), assembled at the N. D. Zelinsky Institute of Organic Chemistry over the past 30 years, has been screened for antimicrobial activity against five ESKAPE pathogens and two fungi at the University of Queensland on a charitable basis. A total of 2517 active molecules (MIC ≤ 32 μg ml–1) were found, of which about 10% are active at very low concentrations (MIC ≤ 1 μg ml–1). Structures of 142 012 compounds and experimental data on their antimicrobial activity are publicly available through the demo version of the CheD software and the public database of the Community for Open Antimicrobial Drug Discovery.

Abstract 1482: M4645 is a potent and highly selective inhibitor of disease relevant cKIT mutations for the treatment of metastatic or recurrent GIST

Abstract Oncogenic mutations in cKIT in exons 9 and 11 are the main driver of gastrointestinal stromal tumors (GIST). Imatinib is standard of care in 1L in unresectable metastatic or recurrent GIST, yet secondary resistance mutations in exons 13/14 and 17/18 of the cKIT kinase domain frequently arise limiting efficacy. Currently approved 2L and 3L therapies sunitinib and regorafenib target these mutations only to some extent and show limited clinical benefit. Even with the recent approval of ripretinib as 4L treatment, a high unmet medical need remains for patients with imatinib resistance mutations in exon13/14. M4645 was designed to particularly inhibit the frequent V654A resistance mutation in exon 13 of cKIT. Based on cKIT inhibitor scaffolds identified within the Merck compound collection, a 3,5,6-substituted indazole-lead was discovered. Substituent optimization in the 5- and 6-positions of the core scaffold resulted in highly selective kinase inhibitors. Through variation at the 3-position, potency and physicochemical properties were optimized, resulting in the candidate molecule M4645 which combined potency, kinase selectivity, and solubility with a good safety profile. The excellent kinase selectivity could be rationalized by the crystal structure of cKIT V654A mutant kinase domain in complex with M4645. M4645 showed potent inhibition of cKIT autophosphorylation and of downstream P-ERK1/2 and P-AKT signaling in cell lines bearing activating and resistance mutations of cKIT. Dose-dependent inhibition of cKIT activation was demonstrated in a GIST xenograft model expressing the cKIT resistance mutation V654A in exon 13. Strong antitumor activity was observed in cell line and patient-derived GIST xenograft models with this imatinib resistance mutation. Compared to imatinib, M4645 induced prolonged anti-tumor response in a GIST model with a primary activating mutation of cKIT. In summary, the favorable profile and strong antitumor activity, particularly in GIST models expressing the V654A resistance mutation, suggests superiority of M4645 compared to currently available > 2L treatment options for unresectable metastatic or recurrent GIST patients. Citation Format: Nina Linde, Andreas Blum, Dieter Dorsch, Ulrich Graedler, Michael Busch, Nina Glaser, Carl Petersson, Aaron Ruff, Eva Sherbetjian, Christina Esdar. M4645 is a potent and highly selective inhibitor of disease relevant cKIT mutations for the treatment of metastatic or recurrent GIST [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1482.

Discovery of Novel Chemical Series of OXA-48 β-Lactamase Inhibitors by High-Throughput Screening.

The major cause of bacterial resistance to β-lactams is the production of hydrolytic β-lactamase enzymes. Nowadays, the combination of β-lactam antibiotics with β-lactamase inhibitors (BLIs) is the main strategy for overcoming such issues. Nevertheless, particularly challenging β-lactamases, such as OXA-48, pose the need for novel and effective treatments. Herein, we describe the screening of a proprietary compound collection against Klebsiella pneumoniae OXA-48, leading to the identification of several chemotypes, like the 4-ideneamino-4H-1,2,4-triazole (SC_2) and pyrazolo[3,4-b]pyridine (SC_7) cores as potential inhibitors. Importantly, the most potent representative of the latter series (ID2, AC50 = 0.99 μM) inhibited OXA-48 via a reversible and competitive mechanism of action, as demonstrated by biochemical and X-ray studies; furthermore, it slightly improved imipenem’s activity in Escherichia coli ATCC BAA-2523 β-lactam resistant strain. Also, ID2 showed good solubility and no sign of toxicity up to the highest tested concentration, resulting in a promising starting point for further optimization programs toward novel and effective non-β-lactam BLIs.

Maintaining a High-Quality Screening Collection: The GSK Experience.

The storage of screening collections in DMSO is commonplace in the pharmaceutical industry. To ensure a high-quality screening collection, and hence effective and efficient high-throughput screening, all compounds entering the GlaxoSmithKline (GSK) screening collection undergo a liquid chromatography-mass spectrometry (LC-MS) quality control (QC). It is generally accepted that even under optimal conditions, a small percentage of these compounds are unstable after prolonged storage in DMSO. This article presents how these QC data can be mined using a data-driven clustering algorithm to identify chemical substructures likely to cause degradation in DMSO. This knowledge provides new structural filters for use in excluding compounds with these undesirable substructures from the collection. This information also suggests an efficient, targeted approach to compound collection clean-up initiatives. Stability studies are also designed to maintain a high-quality screening collection. To define the best practice for the storage and handling of solution samples, GSK has undertaken stability experiments for two decades, initially to support the implementation of new automated liquid stores and, subsequently, to enhance storage and use of compounds in solution through an understanding of compound degradation under storage and assay conditions.

The Use of Informer Sets in Screening: Perspectives on an Efficient Strategy to Identify New Probes.

Small-molecule discovery typically involves large-scale screening campaigns, spanning multiple compound collections. However, such activities can be cost- or time-prohibitive, especially when using complex assay systems, limiting the number of compounds tested. Further, low hit rates can make the process inefficient. Sparse coverage of chemical structure or biological activity space can lead to limited success in a primary screen and represents a missed opportunity by virtue of selecting the "wrong" compounds to test. Thus, the choice of screening collections becomes of paramount importance. In this perspective, we discuss the utility of generating "informer sets" for small-molecule discovery, and how this strategy can be leveraged to prioritize probe candidates. While many researchers may assume that informer sets are focused on particular targets (e.g., kinases) or processes (e.g., autophagy), efforts to assemble informer sets based on historical bioactivity or successful human exposure (e.g., repurposing collections) have shown promise as well. Another method for generating informer sets is based on chemical structure, particularly when the compounds have unknown activities and targets. We describe our efforts to screen an informer set representing a collection of 100,000 small molecules synthesized through diversity-oriented synthesis (DOS). This process enables researchers to identify activity early and more extensively screen only a few chemical scaffolds, rather than the entire collection. This elegant and economic outcome is a goal of the informer set approach. Here, we aim not only to shed light on this process, but also to promote the use of informer sets more widely in small-molecule discovery projects.

Novel pharmacological approach for the prevention of multidrug resistance (MDR) in a human leukemia cell line

BackgroundDrug resistance mechanisms are the regulatory factors associated with drug metabolism and drug transport to inward and outward of the target cells. Maybridge fragment (MBF) library is a collection of pharmacophore rich compounds having affinity with membrane transporters. This study has been designed to evaluate the efficacy of MBFs in overcoming the leukemic cells’ resistance to imatinib. MethodsImatinib resistant cells (K562-R) were prepared using myelogenous leukemia cell line (K562) by titration method. The four MBFs were prioritized for determining their effect on imatinib resistance. The cells were treated with imatinib and MBFs and the MTT assay was performed to evaluate the efficacy of MBFs in enhancing the imatinib mediated cell death. The transcript levels of Bcr-Abl1 gene and efflux transporter genes were determined by RT-qPCR analysis. ResultsThe MBFs enhanced the imatinib mediated cell death of K562-R cells. There was also a significant decrease in the mRNA levels of the major drug efflux genes (ABCB1, ABCB10, ABCC1 and ABCG2) when treated with a combination of imatinib and MBF in comparison to imatinib treatment alone. ConclusionThe drug efflux is one of the mechanisms of multidrug resistance in cancer cells and the MBFs used in this study were all found to significantly overcome the imatinib resistance by limiting the expression of efflux genes. This study, therefore, highlights the potential of Maybridge compounds in treating the drug resistant leukemia.

Limonin as a Starting Point for the Construction of Compounds with High Scaffold Diversity

AbstractStructurally complex natural products have been a fruitful source for the discovery and development of new drugs. In an effort to construct a compound collection populated by architecturally complex members with unique scaffolds, we have used the natural product limonin as a starting point. Limonin is an abundant triterpenoid natural product and, through alteration of its heptacyclic core ring system using short synthetic sequences, a collection of 98 compounds was created, including multiple members with novel ring systems. The reactions leveraged in the construction of these compounds include novel ring cleavage, rearrangements, and cyclizations, and this work is highlighted by the discovery of a novel B‐ring cleavage reaction, a unique B/C‐ring rearrangement, an atypical D‐ring cyclization, among others. Computational analysis shows that 52 different scaffolds/ring systems were produced during the course of this work, of which 36 are unprecedented. Phenotypic screening and structure–activity relationships identified compounds with activity against a panel of cancer cell lines.

Limonin as a Starting Point for the Construction of Compounds with High Scaffold Diversity.

Structurally complex natural products have been a fruitful source for the discovery and development of new drugs. In an effort to construct a compound collection populated by architecturally complex members with unique scaffolds, we have used the natural product limonin as a starting point. Limonin is an abundant triterpenoid natural product and, through alteration of its heptacyclic core ring system using short synthetic sequences, a collection of 98 compounds was created, including multiple members with novel ring systems. The reactions leveraged in the construction of these compounds include novel ring cleavage, rearrangements, and cyclizations, and this work is highlighted by the discovery of a novel B-ring cleavage reaction, a unique B/C-ring rearrangement, an atypical D-ring cyclization, among others. Computational analysis shows that 52 different scaffolds/ring systems were produced during the course of this work, of which 36 are unprecedented. Phenotypic screening and structure-activity relationships identified compounds with activity against a panel of cancer cell lines.

Ontogenetic Changes in the Chemical Profiles of Piper Species.

The chemical composition of seedlings and adult plants of several Piper species were analyzed by 1H NMR spectroscopy combined with principal component analysis (PCA) and HPLC-DAD, HPLC-HRESIMS and GC-MS data. The chromatographic profile of crude extracts from leaves of Piper species showed remarkable differences between seedlings and adult plants. Adult leaves of P. regnellii accumulate dihydrobenzofuran neolignans, P. solmsianum contain tetrahydrofuran lignans, and prenylated benzoic acids are found in adult leaves of P. hemmendorffii and P. caldense. Seedlings produced an entirely different collection of compounds. Piper gaudichaudianum and P. solmsianum seedlings contain the phenylpropanoid dillapiole. Piper regnellii and P. hemmendorffii produce another phenylpropanoid, apiol, while isoasarone is found in P. caldense. Piper richadiaefolium and P. permucronatum contain dibenzylbutyrolactones lignans or flavonoids in adult leaves. Seedlings of P. richardiaefolium produce multiple amides, while P. permucronatum seedlings contain a new long chain ester. Piper tuberculatum, P. reticulatum and P. amalago produce amides, and their chemistry changes less during ontogeny. The chemical variation we documented opens questions about changes in herbivore pressure across ontogeny.

Predictive Models to Identify Small Molecule Activators and Inhibitors of Opioid Receptors.

Opioid receptors (OPRs) are the main targets for the treatment of pain and related disorders. The opiate compounds that activate these receptors are effective analgesics but their use leads to adverse effects, and they often are highly addictive drugs of abuse. There is an urgent need for alternative chemicals that are analgesics and to reduce/avoid the unwanted effects in order to relieve the public health crisis of opioid addiction. Here, we aim to develop computational models to predict the OPR activity of small molecule compounds based on chemical structures and apply these models to identify novel OPR active compounds. We used four different machine learning algorithms to build models based on quantitative high throughput screening (qHTS) data sets of three OPRs in both agonist and antagonist modes. The best performing models were applied to virtually screen a large collection of compounds. The model predicted active compounds were experimentally validated using the same qHTS assays that generated the training data. Random forest was the best classifier with the highest performance metrics, and the mu OPR (OPRM)-agonist model achieved the best performance measured by AUC-ROC (0.88) and MCC (0.7) values. The model predicted actives resulted in hit rates ranging from 2.3% (delta OPR-agonist) to 15.8% (OPRM-agonist) after experimental confirmation. Compared to the original assay hit rate, all models enriched the hit rate by ≥2-fold. Our approach produced robust OPR prediction models that can be applied to prioritize compounds from large libraries for further experimental validation. The models identified several novel potent compounds as activators/inhibitors of OPRs that were confirmed experimentally. The potent hits were further investigated using molecular docking to find the interactions of the novel ligands in the active site of the corresponding OPR.

Development and Pilot Screen of Novel High Content Assay for Down Regulators of Expression of Heterogenous Nuclear Ribonuclear Protein H2.

Despite recent advances in melanoma drug discovery, the average overall survival of patients with late-stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al., Cell Physiol Biochem 2019;53:656-686). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H2 that can be used for melanoma therapy and research. We established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. All plate-based QC parameters were acceptable: %CV = 6.7±0.3, S/B = 21±2.1, Z' = 0.75±0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis. We developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNPH1 and 2 in a large compound collection and, therefore, is amenable to a large-scale screening effort.

Piperidine-4-Carboxamides Target DNA Gyrase in Mycobacterium abscessus.

ABSTRACTNew, more-effective drugs for the treatment of lung disease caused by nontuberculous mycobacteria (NTM) are needed. Among NTM opportunistic pathogens, Mycobacterium abscessus is the most difficult to cure and intrinsically multidrug resistant. In a whole-cell screen of a compound collection active against Mycobacterium tuberculosis, we previously identified the piperidine-4-carboxamide (P4C) MMV688844 (844) as a hit against M. abscessus. Here, we identified a more potent analog of 844 and showed that both the parent and improved analog retain activity against strains representing all three subspecies of the M. abscessus complex. Furthermore, P4Cs showed bactericidal and antibiofilm activity. Spontaneous resistance against the P4Cs emerged at a frequency of 10−8/CFU and mapped to gyrA and gyrB encoding the subunits of DNA gyrase. Biochemical studies with recombinant M. abscessus DNA gyrase showed that P4Cs inhibit the wild-type enzyme but not the P4C-resistant mutant. P4C-resistant strains showed limited cross-resistance to the fluoroquinolone moxifloxacin, which is in clinical use for the treatment of macrolide-resistant M. abscessus disease, and no cross-resistance to the benzimidazole SPR719, a novel DNA gyrase inhibitor in clinical development for the treatment of mycobacterial diseases. Analyses of P4Cs in recA promoter-based DNA damage reporter strains showed induction of recA promoter activity in the wild type but not in the P4C-resistant mutant background. This indicates that P4Cs, similar to fluoroquinolones, cause DNA gyrase-mediated DNA damage. Together, our results show that P4Cs present a novel class of mycobacterial DNA gyrase inhibitors with attractive antimicrobial activities against the M. abscessus complex.

Diversity-oriented synthesis as a tool to expand the chemical space of DNA-encoded libraries

DNA-encoded libraries (DEL) represent a powerful technology for generating compound collections for drug discovery campaigns, that have allowed for the selection of many hit compounds over last three decades. However, the application of split-and-pool combinatorial methodologies, as well as the limitation imposed by DNA-compatible chemistry, has often brought to a limited exploration of the chemical space, with an over-representation of flat aromatic or peptide-like structures, whereas a higher scaffold complexity is generally associated with a more successful biological activity of the library. In this context, the application of Diversity-Oriented Synthesis, capable of creating sp3-rich molecular entities even starting from simple flat building blocks, can represent an efficient strategy to significantly broaden the chemical space explored by DELs. In this review, we present selected examples of DNA-compatible complexity-generating reactions that can be applied for the generation of DNA-encoded DOS libraries, including: (i) multicomponent reactions; (ii) C-H/C-X functionalization; (iii) tandem approaches; (iv) cycloadditions; (v) reactions introducing privileged elements. Also, selected case studies on the generation of DELs with high scaffold diversity are discussed, reporting their application in drug discovery programs.

Steady-state enzyme kinetics

Steady-state enzyme kinetics is a cornerstone technique of biochemistry and related sciences since it allows the characterization and quantification of enzyme behaviour. Enzyme kinetics is widely used to investigate the physiological role of enzymes, determine the effects of mutations and characterize enzyme inhibitors. Well-known examples of enzyme inhibitors used to treat diseases include anti-infectives (e.g., penicillin, clavulanic acid and HIV protease inhibitors); anti-inflammatories (e.g., aspirin and ibuprofen); cholesterol-lowering statins; tyrosine kinase inhibitors used to treat cancer; and Viagra. Commonly, new disease treatments are discovered by using enzyme kinetics to identify the few active compounds residing within a large compound collection (‘high-throughput screening’). The subject of enzyme kinetics is typically introduced to first-year undergraduates with a mathematical description of behaviour. This Beginners Guide will give a brief overview of experimental enzyme kinetics and the characterization of enzyme inhibitors. Colorimetric assays using a microtitre plate will be considered, although most principles also apply to other assays.

Nanocomposite Trimethylsilyl-Modified Nafion/SBA-15 for Extraction and Analysis of the Volatile Compounds from Nepeta haussknechtii Bornm

Headspace solid-phase microextraction (HS-SPME) using a type of SBA-15 was made more hydrophobic by modification with ethoxy trimethyl silane to obtain a hybrid organicinorganic mesoporous nanocomposite, which then was impregnated with Nafion for the collection and detection of the volatile compounds of Nepeta haussknechtii Bornm. Experimental parameters, such as sample weight, extraction temperature, humidity effect, desorption time, and desorption temperature, were examined and optimized. Using HS-SPME followed by GC–MS, 42 compounds were separated and identified. HS-SPME has the benefits of small sample size, timesaving, cheapness, and simplicity, compared to hydrodistillation (HD). As we know, this is the first report on utilizing Nafion/SBA-15 fiber within solid-phase microextraction attached to headspace for assessing medicinal plant’s volatile fraction.

Dual-target compounds for Alzheimer's disease: Natural and synthetic AChE and BACE-1 dual-inhibitors and their structure-activity relationship (SAR)

Alzheimer's disease (AD) is a chronic neurodegenerative disease and represents the major cause of dementia worldwide. Currently, there are no available treatments capable to deliver disease-modifying effects, and the available drugs can only alleviate the symptoms. The exact pathology of AD is not yet fully understood and several hallmarks such as the presence of amyloid-β (Aβ) senile plaques, neurofibrillary tangles (NFTs) as well as the loss of cholinergic function have been associated to AD. Distinct pharmacological targets have been validated to address AD, with acetylcholinesterase (AChE) and β-secretase-1 (BACE-1) being two of the most explored ones. A great deal of research has been devoted to the development of new AChE and BACE-1 effective inhibitors, tackled separately or in combination of both. The multi-factorial nature of AD conducted to the development of multi-target directed ligands (MTDLs), defined as single molecules capable to modulate more than one biological target, as an alternative approach to the old paradigm one-target one-drug. In this context, this review describes a collection of natural and synthetic compounds with dual-inhibitory properties towards both AChE and BACE-1 in the MTDLs context. Furthermore, this review also provides a critical comprehensive analysis of structure-activity relationships (SAR) of the synthetic compounds.

A fungus-derived purpactin A as an inhibitor of TMEM16A chloride channels and mucin secretion in airway epithelial cells

TMEM16A is a Ca2+-activated Cl- channel involved in mucus secretion in inflamed airways and proposed as a drug target for diseases associated with mucus hypersecretion including asthma. This study aimed to identify novel inhibitors of TMEM16A-mediated Cl- secretion in airway epithelial cells from a collection of compounds isolated from fungi indigenous in Thailand and examine its potential utility in mitigating airway mucus secretion using Calu-3 cells as a study model. Screening of > 400 fungal metabolites revealed purpactin A isolated from a soil-derived fungus Penicillium aculeatum PSU-RSPG105 as an inhibitor of TMEM16A-mediated Cl- transport with an IC50 value of ~2 µM. A consistent inhibitory effect of purpactin A on TMEM16A were observed regardless of TMEM16A activators or in the presence of an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a negative regulator of TMEM16A. In addition, purpactin A did not affect cell viability, epithelial barrier integrity and activities of membrane transport proteins essential for maintaining airway hydration including CFTR Cl- channels and apical BK K+ channels. Intriguingly, purpactin A prevented a Ca2+-induced mucin release in cytokine-treated airway cells. Taken together, purpactin A represents the first class of TMEM16A inhibitor derived from fungus, which may be beneficial for the treatment of diseases associated with mucus hypersecretion.