A Fragment-based approach to assess the ligandability of ArgB, ArgC, ArgD and ArgF in the L-arginine biosynthetic pathway of Mycobacterium tuberculosis

AbstractThe L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L-glutamate to L-arginine, appears to be attractive target for anti-Tuberculosis (TB) drug discovery. Starvation of M. tuberculosis deleted for either argB or argF genes led to rapid sterilization of these strains in mice while Chemical inhibition of ArgJ with Pranlukast was also found to clear chronic M. tuberculosis infection in animal models. In this work, the ligandability of four enzymes of the pathway ArgB, ArgC, ArgD and ArgF is explored using a fragment-based approach. We reveal several hits for these enzymes validated with biochemical and biophysical assays, and X-ray crystallographic data, which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. These results demonstrate the potential of more enzymes in this pathway to be targeted with dedicated drug discovery programmes.

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Arginine-deprivation–induced oxidative damage sterilizes Mycobacterium tuberculosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1808874115 ◽

2018 ◽

Vol 115 (39) ◽

pp. 9779-9784 ◽

Cited By ~ 22

Author(s):

Sangeeta Tiwari ◽

Andries J. van Tonder ◽

Catherine Vilchèze ◽

Vitor Mendes ◽

Sherine E. Thomas ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Mycobacterium Tuberculosis ◽

De Novo ◽

Target Space ◽

Biosynthesis Pathway ◽

Arginine Biosynthesis ◽

Arginine Deprivation

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.

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Chemical validation of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase using fragment linking and CRISPR interference

10.1101/2020.09.04.280388 ◽

2020 ◽

Author(s):

Jamal El Bakali ◽

Michal Blaszczyk ◽

Joanna C. Evans ◽

Jennifer A. Boland ◽

William J. McCarthy ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Crystal Structures ◽

Active Site ◽

Drug Target ◽

Potential Drug Target ◽

Biosynthesis Pathway ◽

Potential Drug ◽

Antimicrobial Drugs ◽

X Ray ◽

Crispr Interference

AbstractThe coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify the first inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD < 20 μM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.Abstract Figure

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Structure-guided enhancement of selectivity of chemical probe inhibitors targeting bacterial seryl-tRNA synthetase

10.1101/586255 ◽

2019 ◽

Author(s):

Ricky Cain ◽

Ramya Salimraj ◽

Avinash S. Punekar ◽

Dom Bellini ◽

Colin W. G. Fishwick ◽

...

Keyword(s):

Escherichia Coli ◽

Drug Discovery ◽

Bacterial Species ◽

Trna Synthetase ◽

Human Homologue ◽

X Ray ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases ◽

Chemical Inhibition ◽

Selective Chemical

AbstractAminoacyl-tRNA synthetases are ubiquitous and essential enzymes for protein synthesis and also a variety of other metabolic processes, especially in bacterial species. Bacterial aminoacyl-tRNA synthetases represent attractive and validated targets for antimicrobial drug discovery if issues of prokaryotic versus eukaryotic selectivity and antibiotic resistance generation can be addressed. We have determined high resolution X-ray crystal structures of the Escherichia coli and Staphylococcus aureus seryl-tRNA synthetases in complex with aminoacyl adenylate analogues and applied a structure-based drug discovery approach to explore and identify a series of small molecule inhibitors that selectively inhibit bacterial seryl-tRNA synthetases with greater than two orders of magnitude compared to their human homologue, demonstrating a route to selective chemical inhibition of these bacterial targets.

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Ligand binding mode determination in fragment-based drug discovery using X-ray crystallographic data coupled with QM/MM based refinement: further applications of XModeScore

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s0108767318098513 ◽

2018 ◽

Vol 74 (a1) ◽

pp. a148-a148

Author(s):

Oleg Y. Borbulevych ◽

Lance M. Westerhoff

Keyword(s):

Drug Discovery ◽

Ligand Binding ◽

Binding Mode ◽

Crystallographic Data ◽

X Ray ◽

Fragment Based Drug Discovery

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Emerging hiPSC Models for Drug Discovery in Neurodegenerative Diseases

International Journal of Molecular Sciences ◽

10.3390/ijms22158196 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8196

Author(s):

Dorit Trudler ◽

Swagata Ghatak ◽

Stuart A. Lipton

Keyword(s):

Drug Discovery ◽

Animal Models ◽

Neurodegenerative Diseases ◽

Disease Modeling ◽

Induced Pluripotent Stem Cell ◽

Neural Function ◽

Neural Cells ◽

Human Samples ◽

Healthy Donors ◽

Induced Pluripotent

Neurodegenerative diseases affect millions of people worldwide and are characterized by the chronic and progressive deterioration of neural function. Neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD), represent a huge social and economic burden due to increasing prevalence in our aging society, severity of symptoms, and lack of effective disease-modifying therapies. This lack of effective treatments is partly due to a lack of reliable models. Modeling neurodegenerative diseases is difficult because of poor access to human samples (restricted in general to postmortem tissue) and limited knowledge of disease mechanisms in a human context. Animal models play an instrumental role in understanding these diseases but fail to comprehensively represent the full extent of disease due to critical differences between humans and other mammals. The advent of human-induced pluripotent stem cell (hiPSC) technology presents an advantageous system that complements animal models of neurodegenerative diseases. Coupled with advances in gene-editing technologies, hiPSC-derived neural cells from patients and healthy donors now allow disease modeling using human samples that can be used for drug discovery.

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Preliminary X-ray crystallographic data on mouse submaxillary gland renin and renin-inhibitor complexes.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)90804-5 ◽

1984 ◽

Vol 259 (20) ◽

pp. 12714-12717

Author(s):

M A Navia ◽

J P Springer ◽

M Poe ◽

J Boger ◽

K Hoogsteen

Keyword(s):

Submaxillary Gland ◽

Crystallographic Data ◽

Renin Inhibitor ◽

X Ray ◽

Mouse Submaxillary Gland

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Crystallization and preliminary x-ray crystallographic data of dienelactone hydrolase from Pseudomonas sp. B13.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)39308-0 ◽

1985 ◽

Vol 260 (17) ◽

pp. 9818-9819

Author(s):

D L Ollis ◽

K L Ngai

Keyword(s):

Crystallographic Data ◽

Pseudomonas Sp ◽

X Ray ◽

Dienelactone Hydrolase

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Mycobacterium tuberculosis Infection Up-Regulates Sialyl Lewis X Expression in the Lung Epithelium

Microorganisms ◽

10.3390/microorganisms9010099 ◽

2021 ◽

Vol 9 (1) ◽

pp. 99

Author(s):

Rita Matos ◽

Kaori L. Fonseca ◽

Stefan Mereiter ◽

Ana Raquel Maceiras ◽

Joana Gomes ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Biosynthetic Pathway ◽

Molecular Mechanisms ◽

Tuberculosis Infection ◽

Host Susceptibility ◽

Sialyl Lewis X ◽

Lung Pathology ◽

Lung Epithelium ◽

Lewis X ◽

Sialyl Lewis

Glycans display increasingly recognized roles in pathological contexts, however, their impact in the host-pathogen interplay in many infectious diseases remains largely unknown. This is the case for tuberculosis (TB), one of the ten most fatal diseases worldwide, caused by infection of the bacteria Mycobacterium tuberculosis. We have recently reported that perturbing the core-2 O-glycans biosynthetic pathway increases the host susceptibility to M. tuberculosis infection, by disrupting the neutrophil homeostasis and enhancing lung pathology. In the present study, we show an increased expression of the sialylated glycan structure Sialyl-Lewis X (SLeX) in the lung epithelium upon M. tuberculosis infection. This increase in SLeX glycan epitope is accompanied by an altered lung tissue transcriptomic signature, with up-regulation of genes codifying enzymes that are involved in the SLeX core-2 O-glycans biosynthetic pathway. This study provides novel insights into previously unappreciated molecular mechanisms involving glycosylation, which modulate the host response to M. tuberculosis infection, possibly contributing to shape TB disease outcome.

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