Stephen Johnston

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States

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Publications (10)6.99 Total impact

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    ABSTRACT: We report a new series of 8-membered benzo-fused lactams that inhibit cellular lipid uptake from HDL particles mediated by Scavenger Receptor, Class B, Type I (SR-BI). The series was identified via a high-throughput screen of the National Institutes of Health Molecular Libraries Small Molecule Repository (NIH MLSMR), measuring the transfer of the fluorescent lipid DiI from HDL particles to CHO cells overexpressing SR-BI. The series is part of a previously reported diversity-oriented synthesis (DOS) library prepared via a build-couple-pair approach. Detailed structure-activity relationship (SAR) studies were performed with a selection of the original library, as well as additional analogs prepared via solution phase synthesis. These studies demonstrate that the orientation of the substituents on the aliphatic ring have a critical effect on activity. Additionally, a lipophilic group is required at the western end of the molecule, and a northern hydroxyl group and a southern sulfonamide substituent also proved to be optimal. Compound 2p was found to possess a superior combination of potency (av IC50=0.10μM) and solubility (79μM in PBS), and it was designated as probe ML312. Copyright © 2015. Published by Elsevier Ltd.
    Bioorganic & medicinal chemistry letters 04/2015; 271(10). DOI:10.1016/j.bmcl.2015.03.073 · 2.33 Impact Factor
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    ABSTRACT: We report the outcome of a high-throughput small-molecule screen to identify novel, nontoxic, inhibitors of Trypansoma cruzi, as potential starting points for therapeutics to treat for both the acute and chronic stages of Chagas disease. Two compounds were identified that displayed nanomolar inhibition of T. cruzi and an absence of activity against host cells at the highest tested dose. These compounds have been registered with NIH Molecular Libraries Program (probes ML157 and ML158).
    Bioorganic & medicinal chemistry letters 12/2011; 21(23):7197-200. DOI:10.1016/j.bmcl.2011.09.057 · 2.33 Impact Factor
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    ABSTRACT: The effectiveness of the potent antifungal drug fluconazole is being compromised by the rise of drug-resistant fungal pathogens. While inhibition of Hsp90 or calcineurin can reverse drug resistance in Candida, such inhibitors also impair the homologous human host protein and fungal-selective chemosensitizers remain rare. The MLPCN library was screened to identify compounds that selectively reverse fluconazole resistance in a Candida albicans clinical isolate, while having no antifungal activity when administered as a single agent. A piperazinyl quinoline was identified as a new small-molecule probe (ML189) satisfying these criteria.
    Bioorganic & medicinal chemistry letters 09/2011; 21(18):5502-5. DOI:10.1016/j.bmcl.2011.06.105 · 2.33 Impact Factor
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    ABSTRACT: Micropthalmia-associated transcription factor (MITF) is a lineage restricted basic helix-loop-helix leucine zipper transcription factor that is essential for melanocyte development, function and survival. 15% of human melanomas have MITF gene amplification (1). In addition, a vast majority of melanomas are dependent upon MITF for survival. We set out to identify small molecule inhibitors of MITF activity that would allow for better molecular characterization of MITF’s role in melanoma. Using an MITF-dependent melanoma cell line, SK-MEL-5, in a cell-based luminescence assay, we measured the promoter activity of a MITF target gene, melastatin (TRPM-1), in a high throughput screen (HTS). 331,578 compounds from the NIH MLPCN compound library were screened. Of these, 3,206 compounds were active (a hit rate of 0.96%). A chloronaphthoquinone (CID 1716436/SID 22416871) was identified in the primary HTS as an inhibitor of TRPM-1 promoter activity. It had potent activity upon retesting in the primary assay, as did several closely related analogs. Structure activity relationship (SAR) studies were performed to improve potency and to minimize deleterious properties. These efforts generated a probe (CID 12387471/ML329) with improved chemical properties and selectivity. In particular, ML329 was not prone to nucleophilic glutathione addition, whereas the initial hit underwent adduct formation. ML329 was tested in two MITF-dependent melanoma cell viability assays, SK-MEL-5 and MALME-3M plus a MITF-independent cell line, A375. ML329 showed specific activity against the MITF-dependent cells, primary melanocytes but no effect on the viability in A375 cells. ML329 reduced the expression of multiple MITF target genes, including pigment-related genes and the cell cycle regulator CDK2. As a tool compound, ML329 will be useful in elucidating the role of MITF in melanocyte lineage development and in melanoma disease progression.
    Probe Reports from the NIH Molecular Libraries Program, National Center for Biotechnology Information (US).
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    ABSTRACT: Quorum sensing (QS) is a process of bacterial cell-to-cell communication that relies upon recognition of extracellular signaling molecules called autoinducers. QS allows bacteria to synchronize their behavior in response to changes in the population density and species composition of the proximal bacterial community. Known behaviors regulated by QS include bioluminescence, sporulation, virulence factor production, and biofilm formation. We carried out a high throughput screen (HTS) to identify small molecules that modulate QS in a modified V. cholerae strain carrying a luciferase operon; activation of the quorum pathway is accompanied by light production. 352,083 compounds from the NIH-MLPCN compound library were evaluated. Potential QS modulators were characterized via additional bacterium-based epistatic assays to elucidate their mode of action. We report the discovery and development of two, structurally distinct, small-molecule probes (ML343 and ML344) shown to be agonists of Vibrio cholerae CqsS, a transmembrane receptor. ML343 and ML344 should greatly expand the general understanding of how QS membrane receptors productively interact with ligands and how they relay information from the external environment. In addition these compounds could lead to the development of antibacterial drugs designed to interfere with QS which could have enormous ramifications for improving human health.
    Probe Reports from the NIH Molecular Libraries Program, National Center for Biotechnology Information (US).
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    ABSTRACT: The effectiveness of the potent antifungal drug fluconazole has been compromised by the rise of drug-resistant fungal pathogens. It has been observed that inhibition of Hsp90 can reverse drug resistance in Candida; however, it is challenging to find fungal-specific inhibitors of Hsp90 that do not also impair the human host protein. The Molecular Libraries Probe Production Centers Network (MLPCN) library was screened in duplicate dosings to identify compounds that selectively reverse fluconazole resistance in a Candida albicans clinical isolate, while having no antifungal activity when administered as a single agent. An indazole compound (CID3243873) was identified as meeting most of the probe criteria, and subsequent SAR identified a more potent analog as a new probe compound (ML212).
    Probe Reports from the NIH Molecular Libraries Program, National Center for Biotechnology Information (US).
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    ABSTRACT: The problem of tuberculosis continues to take a tremendous toll on global health, accounting for almost 2 million deaths per year, despite the discovery of antitubercular chemotherapy more than half a century ago. In fact, the crisis is growing due to the alarming increase in multi-drug resistant, and even totally-drug resistant strains, coupled with the extremely little progress made in discovering new TB drugs. One of the major barriers to discovering new, potentially more effective agents has been the lack of a fundamental understanding of the physiology of the M. tuberculosis bacilli as they exist within the infected human host. This physiology contributes to their ability to survive for decades within an infected individual despite host immunity, and to persist even in the face of what should otherwise be effective chemotherapy thus dictating the extremely long treatment courses that are required for cure. Toward the goal of developing chemical tools to help understand the M. tuberculosis bacterial state during infection and to identify essential functions that represent vulnerabilities in this state that could be targeted by chemotherapy, this report describes the discovery and development of the probe ML338. ML338 is the first small molecule which selectively targets dormant non-replicating M. tuberculosis bacilli in which a completely drug tolerant state has been adopted in the presence of nutrient deprivation. This feature distinguishes it from all current TB drugs and other molecules that have been previously described to target the M. tuberculosis bacillus. As studies have suggested the relevance of the bacterial physiologic state adopted under nutrient deprivation during TB infection, ML338 represents an invaluable tool both for identifying essential functions and vulnerabilities of the M. tuberculosis bacilli in this state and for studying the relevance of this state in vivo during infection. Thus, studies with ML338 will have the potential to provide insight into new targets but also new models for identifying more effective TB chemotherapy.
    Probe Reports from the NIH Molecular Libraries Program, National Center for Biotechnology Information (US).
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    ABSTRACT: Quorum sensing (QS) is a process of bacterial cell-to-cell communication that relies upon recognition of extracellular signaling molecules called autoinducers. QS allows bacteria to synchronize their behavior in response to changes in the population density and species composition of the proximal bacterial community. Known behaviors regulated by QS include bioluminescence, sporulation, virulence factor production, and biofilm formation. We carried out a high throughput screen (HTS) to identify small molecules that modulate QS in a modified V. cholerae strain carrying a luciferase operon; activation of the quorum pathway is accompanied by light production. 352,083 compounds from the NIH-MLPCN compound library were evaluated. Potential QS modulators were characterized via additional bacterial epistatic assays to elucidate the mode of action. We report the discovery and development of a substituted 2-amino-oxadiazole (ML366) as an inhibitor of Vibrio cholerae LuxO, a response regulator and intracellular kinase. ML366 acts directly on LuxO by inhibiting ATPase activity. ML366 should greatly expand the general understanding of how QS response regulators relay information from upstream signals that lead to modified gene expression. In addition, these compounds could lead to the development of antibacterial drugs designed to interfere with QS which could have enormous ramifications for improving human health.
    Probe Reports from the NIH Molecular Libraries Program, National Center for Biotechnology Information (US).
  • [Show abstract] [Hide abstract]
    ABSTRACT: Quorum sensing (QS) is a process of bacterial cell-to-cell communication that relies upon recognition of extracellular signaling molecules called autoinducers. QS allows bacteria to synchronize their behavior in response to changes in the population density and species composition of the proximal bacterial community. Known behaviors regulated by QS include bioluminescence, sporulation, virulence factor production, and biofilm formation. We carried out a high throughput screen (HTS) to identify small molecules that modulate QS in a modified V. cholerae strain carrying a luciferase operon; activation of the quorum pathway is accompanied by light production. 352,083 compounds from the NIH-MLPCN compound library were evaluated. Potential QS modulators were characterized via additional bacterial epistatic assays to elucidate the mode of action. We report the discovery and medicinal chemistry development of a substituted pyrazoloquinoline (ML370) shown to be an inhibitor of Vibrio cholerae LuxO, a response regulator and intracellular kinase. The probe acts directly on LuxO by inhibiting the ATPase activity. ML370 should greatly expand the general understanding of how QS response regulators relay information from upstream signals that lead to modified gene expression. In addition ML370 and compound analogues could lead to the development of antibacterial drugs designed to interfere with QS that could have enormous ramifications for improving human health.
    Probe Reports from the NIH Molecular Libraries Program, National Center for Biotechnology Information (US).
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    ABSTRACT: The role of glycolytic versus mitochondrial respiratory metabolism in supporting fungal virulence and drug-resistance have not been fully investigated due in large part to the lack of appropriate small molecule tool compounds that can reliably discriminate between these two metabolic pathways. This project seeks to identify drug-like molecules that selectively inhibit the growth of fungi when they are cultured under conditions requiring mitochondrial respiration to support metabolic needs. A prior Molecular Libraries Probe Production Centers Network (MLPCN) project designed to identify compounds capable of chemosensitizing drug-resistant C. albicans clinical isolates to fluconazole also uncovered compounds possessing potent single-agent activity against a range of opportunistic human fungal pathogens. Further experimentation determined their activity is dependent upon culture conditions that require mitochondrial respiratory metabolism for growth and survival. The current project investigated this subset of compounds as selective inhibitors of fungal respiration, and we now report the development of a thiohydantoin probe (ML316, CID 56604860) exhibiting potent antifungal activity. ML316 will be of great value in probing the metabolic requirements for fungal virulence and may provide essential leads for the development of new antifungal drugs that operate in a completely unexploited target space.
    Probe Reports from the NIH Molecular Libraries Program, National Center for Biotechnology Information (US).