Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP
ABSTRACT Cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) are intracellular mediators of innate immune responses to cytosolic pathogen-derived and host DNA. STING agonists designed to mimic the natural host STING ligand, 2′,3′-cyclic GMP-AMP (cGAMP), are promising immunother...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2024-01-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02012-23 |
| _version_ | 1797357411908976640 |
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| author | Akshay Rohilla Alok Kumar Singh Benjamin Koleske Geetha Srikrishna William R. Bishai |
| author_facet | Akshay Rohilla Alok Kumar Singh Benjamin Koleske Geetha Srikrishna William R. Bishai |
| author_sort | Akshay Rohilla |
| collection | DOAJ |
| description | ABSTRACT Cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) are intracellular mediators of innate immune responses to cytosolic pathogen-derived and host DNA. STING agonists designed to mimic the natural host STING ligand, 2′,3′-cyclic GMP-AMP (cGAMP), are promising immunotherapeutic tools for infectious diseases and solid tumor immunotherapy. We previously characterized CdnP (Rv2837c), a specific phosphodiesterase (PDE) deployed by Mycobacterium tuberculosis (M.tb), as an enzyme that blunts host immunity by directly cleaving bacterial-derived c-di-AMP and host-derived 2′,3′-cGAMP. We hypothesized that small molecule inhibitors of bacterial and host cyclic dinucleotide PDEs, namely CdnP and the endogenous host PDE, ENPP1, might potentiate the STING pathway and act as host-directed therapies (HDTs) for tuberculosis. To this end, we employed virtual screening of an NCI compound library customized for improved oral drug properties to identify potential inhibitors of CdnP and ENPP1. Compounds identified in silico were tested for their inhibitory activity against purified CdnP and ENPP1. Using biochemical and cell-based assays, we identified compounds with low IC50 values against both PDEs. We validated increased cGAS-STING signaling in primary human macrophages exposed to 2′,3′-cGAMP in the presence of a lead ENPP1 inhibitor, E-3 (NCI-14465). Our studies provide a framework for novel HDTs that target the cGAS-STING pathway to promote M.tb containment and anti-tumor immunity. IMPORTANCE In this paper, we describe novel inhibitors of cyclic dinucleotide phosphodiesterase enzymes from Mycobacterium tuberculosis (M.tb) (CdnP) and mammals (ENPP1). The phosphodiesterase enzymes hydrolyze cyclic dinucleotides, such as 2′,3′-cyclic GMP-AMP and c-di-AMP, which are stimulator of interferon gene (STING) agonists. By blocking the hydrolysis of STING agonists, the cyclic GMP-AMP synthase (cGAS)-STING-IRF3 pathway is potentiated. There is strong evidence in tuberculosis and in cancer biology that potentiation of the cGAS-STING-IRF3 pathway leads to improved M.tb clearance and also improved antitumor responses in cancer. In addition to the identification of novel inhibitors and their biochemical characterization, we provide proof-of-concept evidence that our E-3 inhibitor potentiates the cGAS-STING-IRF3 pathway in both macrophage cell lines and also in primary human monocyte-derived macrophages. |
| first_indexed | 2024-03-08T14:43:39Z |
| format | Article |
| id | doaj.art-254ced2f2c2f4d24acdaa19798d3e639 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-03-08T14:43:39Z |
| publishDate | 2024-01-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-254ced2f2c2f4d24acdaa19798d3e6392024-01-11T14:04:37ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972024-01-0112110.1128/spectrum.02012-23Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnPAkshay Rohilla0Alok Kumar Singh1Benjamin Koleske2Geetha Srikrishna3William R. Bishai4Department of Medicine, Johns Hopkins University School of Medicine , Baltimore, Maryland, USADepartment of Medicine, Johns Hopkins University School of Medicine , Baltimore, Maryland, USADepartment of Medicine, Johns Hopkins University School of Medicine , Baltimore, Maryland, USADepartment of Medicine, Johns Hopkins University School of Medicine , Baltimore, Maryland, USADepartment of Medicine, Johns Hopkins University School of Medicine , Baltimore, Maryland, USAABSTRACT Cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) are intracellular mediators of innate immune responses to cytosolic pathogen-derived and host DNA. STING agonists designed to mimic the natural host STING ligand, 2′,3′-cyclic GMP-AMP (cGAMP), are promising immunotherapeutic tools for infectious diseases and solid tumor immunotherapy. We previously characterized CdnP (Rv2837c), a specific phosphodiesterase (PDE) deployed by Mycobacterium tuberculosis (M.tb), as an enzyme that blunts host immunity by directly cleaving bacterial-derived c-di-AMP and host-derived 2′,3′-cGAMP. We hypothesized that small molecule inhibitors of bacterial and host cyclic dinucleotide PDEs, namely CdnP and the endogenous host PDE, ENPP1, might potentiate the STING pathway and act as host-directed therapies (HDTs) for tuberculosis. To this end, we employed virtual screening of an NCI compound library customized for improved oral drug properties to identify potential inhibitors of CdnP and ENPP1. Compounds identified in silico were tested for their inhibitory activity against purified CdnP and ENPP1. Using biochemical and cell-based assays, we identified compounds with low IC50 values against both PDEs. We validated increased cGAS-STING signaling in primary human macrophages exposed to 2′,3′-cGAMP in the presence of a lead ENPP1 inhibitor, E-3 (NCI-14465). Our studies provide a framework for novel HDTs that target the cGAS-STING pathway to promote M.tb containment and anti-tumor immunity. IMPORTANCE In this paper, we describe novel inhibitors of cyclic dinucleotide phosphodiesterase enzymes from Mycobacterium tuberculosis (M.tb) (CdnP) and mammals (ENPP1). The phosphodiesterase enzymes hydrolyze cyclic dinucleotides, such as 2′,3′-cyclic GMP-AMP and c-di-AMP, which are stimulator of interferon gene (STING) agonists. By blocking the hydrolysis of STING agonists, the cyclic GMP-AMP synthase (cGAS)-STING-IRF3 pathway is potentiated. There is strong evidence in tuberculosis and in cancer biology that potentiation of the cGAS-STING-IRF3 pathway leads to improved M.tb clearance and also improved antitumor responses in cancer. In addition to the identification of novel inhibitors and their biochemical characterization, we provide proof-of-concept evidence that our E-3 inhibitor potentiates the cGAS-STING-IRF3 pathway in both macrophage cell lines and also in primary human monocyte-derived macrophages.https://journals.asm.org/doi/10.1128/spectrum.02012-23Mycobacterium tuberculosiscyclic dinucleotide phosphodiesteraseimmune evasionhost-directed therapy |
| spellingShingle | Akshay Rohilla Alok Kumar Singh Benjamin Koleske Geetha Srikrishna William R. Bishai Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP Microbiology Spectrum Mycobacterium tuberculosis cyclic dinucleotide phosphodiesterase immune evasion host-directed therapy |
| title | Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP |
| title_full | Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP |
| title_fullStr | Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP |
| title_full_unstemmed | Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP |
| title_short | Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP |
| title_sort | structure based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases enpp1 and cdnp |
| topic | Mycobacterium tuberculosis cyclic dinucleotide phosphodiesterase immune evasion host-directed therapy |
| url | https://journals.asm.org/doi/10.1128/spectrum.02012-23 |
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