Publications by authors named "Tanya Parish"

158 Publications

Deletion of Rv2571c confers resistance to arylamide compounds in .

Antimicrob Agents Chemother 2021 Feb 22. Epub 2021 Feb 22.

Infectious Disease Research Institute, Seattle, WA 98102, USA

Tuberculosis, caused by , is an urgent global health problem requiring new drugs, new drug targets and an increased understanding of antibiotic resistance. We have determined the mode of resistance to a series of arylamide compounds in We isolated resistant mutants to two arylamide compounds which are inhibitory to growth under host-relevant conditions (butyrate as a sole carbon source). Thirteen mutants were characterized, and all had mutations in Rv2571c; mutations included a premature stop codon and frameshifts as well as non-synonymous polymorphisms. We isolated a further ten strains with mutations in Rv2571c with resistance. Complementation with a wild-type copy of Rv2571c restored arylamide sensitivity. Over-expression of Rv2571c was toxic in both wild-type and mutant backgrounds. We constructed with an unmarked deletion of the entire Rv2571c gene by homologous recombination and confirmed that these were resistant to the arylamide series. Rv2571c is a member of the aromatic amino acid transport family and has a fusaric acid resistance domain which is associated with compound transport. Since loss or inactivation of Rv2571c leads to resistance, we propose that Rv2571c is involved in the import of arylamide compounds.
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http://dx.doi.org/10.1128/AAC.02334-20DOI Listing
February 2021

Spirocycle MmpL3 Inhibitors with Improved hERG and Cytotoxicity Profiles as Inhibitors of Growth.

ACS Omega 2021 Jan 13;6(3):2284-2311. Epub 2021 Jan 13.

TB Discovery Research, Infectious Disease Research Institute, 1616 Eastlake Avenue East, Suite 400, Seattle, Washington 98102, United States.

With the emergence of multi-drug-resistant strains of there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles () are described, which were identified following phenotypic screening of the Eli Lilly corporate library against . The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead , with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to , and under replicating conditions, demonstrated bactericidal activity against . Unfortunately, compound had no efficacy in an acute model of TB infection; this was most likely due to the exposure remaining above the minimal inhibitory concentration for only a limited time.
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http://dx.doi.org/10.1021/acsomega.0c05589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7841955PMC
January 2021

Phenoxyalkylimidazoles with an oxadiazole moiety are subject to efflux in Mycobacterium tuberculosis.

PLoS One 2021 22;16(1):e0239353. Epub 2021 Jan 22.

TB Discovery Research, Infectious Disease Research Institute, Seattle, WA, United States of America.

The phenoxyalkylimidazoles (PAI) are an attractive chemical series with potent anti-tubercular activity targeting Mycobacterium tuberculosis respiration. Our aim was to determine if the PAI compounds are subject to efflux. Two analogs containing an oxadiazole had improved potency in the presence of the efflux inhibitors reserpine and carbonyl cyanide m-chlorophenylhydrazine, whereas the potency of analogs with a diazole was not affected.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239353PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822546PMC
January 2021

Antitubercular 2-Pyrazolylpyrimidinones: Structure-Activity Relationship and Mode-of-Action Studies.

J Med Chem 2021 01 4;64(1):719-740. Epub 2021 Jan 4.

Drug Discovery and Development Centre (H3D), Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa.

Phenotypic screening of a Medicines for Malaria Venture compound library against () identified a cluster of pan-active 2-pyrazolylpyrimidinones. The biology triage of these actives using various tool strains of suggested a novel mechanism of action. The compounds were bactericidal against replicating and retained potency against clinical isolates of . Although selected MmpL3 mutant strains of showed resistance to these compounds, there was no shift in the minimum inhibitory concentration (MIC) against a mmpL3 hypomorph, suggesting mutations in MmpL3 as a possible resistance mechanism for the compounds but not necessarily as the target. RNA transcriptional profiling and the checkerboard board 2D-MIC assay in the presence of varying concentrations of ferrous salt indicated perturbation of the Fe-homeostasis by the compounds. Structure-activity relationship studies identified potent compounds with good physicochemical properties and microsomal metabolic stability with moderate selectivity over cytotoxicity against mammalian cell lines.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816196PMC
January 2021

Design, synthesis and SAR of antitubercular benzylpiperazine ureas.

Mol Divers 2021 Jan 1. Epub 2021 Jan 1.

Ambernath Organics Pvt. Ltd., 222, The Summit Business Bay, Andheri (E), Mumbai, 400 093, India.

N-furfuryl piperazine ureas disclosed by scientists at GSK Tres Cantos were chosen as antimycobacterial hits from a phenotypic whole-cell screen. Bioisosteric replacement of the furan ring in the GSK Tres Cantos molecules with a phenyl ring led to molecule (I) with an MIC of 1 μM against Mtb H37Rv, low cellular toxicity (HepG2 IC ~ 80 μM), good DMPK properties and specificity for Mtb. With the aim of delineating the SAR associated with (I), fifty-five analogs were synthesized and screened against Mtb. The SAR suggests that the piperazine ring, benzyl urea and piperonyl moieties are essential signatures of this series. Active compounds in this series are metabolically stable, have low cellular toxicity and are valuable leads for optimization. Molecular docking suggests these molecules occupy the Q0 site of QcrB like Q203. Bioisosteric replacement of N-furfuryl piperazine-1-carboxamides yielded molecule (I) a novel lead with satisfactory PD, metabolism, and toxicity profiles.
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http://dx.doi.org/10.1007/s11030-020-10158-3DOI Listing
January 2021

InhA inhibitors have activity against non-replicating Mycobacterium tuberculosis.

PLoS One 2020 17;15(11):e0239354. Epub 2020 Nov 17.

Infectious Disease Research Institute, Seattle, Washington, United States of America.

We previously identified a diazaborine series with potential for development as a new tuberculosis drug. This series has activity in vitro and in vivo and targets cell wall biosynthesis via inhibition of InhA. The overall aim of this study was to determine whether InhA inhibitors have activity against non-replicating Mycobacterium tuberculosis. We tested the ability of two molecules of the diazaborine series to kill non-replicating M. tuberculosis in the nutrient starvation model; both molecules were bactericidal, reducing viability by >3 logs in 21 days. Activity showed similar kill rates to other InhA inhibitors (isoniazid and NITD-916). We conclude that inhibition of InhA is bactericidal against nutrient-starved non-replicating M. tuberculosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239354PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671525PMC
December 2020

Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors.

mSphere 2020 10 14;5(5). Epub 2020 Oct 14.

TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington, USA

The protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.
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http://dx.doi.org/10.1128/mSphere.00985-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565900PMC
October 2020

Mutations in the anti-sigma H factor RshA confer resistance to econazole and clotrimazole in .

Access Microbiol 2019 29;1(10):e000070. Epub 2019 Oct 29.

TB Discovery Research, Infectious Disease Research Institute, Seattle, WA, USA.

Azole drugs such as econazole, are active on and ; however, the identification of their target(s) is still pending. It has been reported that mutations in the non-essential system L5-S5 conferred resistance to econazole in . We herein report that an azole-resistant mutant screen in rendered mutations in A, encoding a non-essential anti-sigma H protein.
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http://dx.doi.org/10.1099/acmi.0.000070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491931PMC
October 2019

Structure-Guided Optimization of Inhibitors of Acetyltransferase Eis from .

ACS Chem Biol 2020 06 18;15(6):1581-1594. Epub 2020 May 18.

Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky 40536-0596, United States.

The enhanced intracellular survival (Eis) protein of () is a versatile acetyltransferase that multiacetylates aminoglycoside antibiotics abolishing their binding to the bacterial ribosome. When overexpressed as a result of promoter mutations, Eis causes drug resistance. In an attempt to overcome the Eis-mediated kanamycin resistance of , we designed and optimized structurally unique thieno[2,3-]pyrimidine Eis inhibitors toward effective kanamycin adjuvant combination therapy. We obtained 12 crystal structures of enzyme-inhibitor complexes, which guided our rational structure-based design of 72 thieno[2,3-]pyrimidine analogues divided into three families. We evaluated the potency of these inhibitors as well as their ability to restore the activity of kanamycin in a resistant strain of , in which Eis was upregulated. Furthermore, we evaluated the metabolic stability of 11 compounds . This study showcases how structural information can guide Eis inhibitor design.
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http://dx.doi.org/10.1021/acschembio.0c00184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385556PMC
June 2020

Diterpenoids isolated from the Samoan marine sponge Chelonaplysilla sp. inhibit Mycobacterium tuberculosis growth.

J Antibiot (Tokyo) 2020 08 13;73(8):568-573. Epub 2020 May 13.

Departments of Chemistry and Earth, Ocean & Atmospheric Sciences, University of British Columbia, 2016 Main Mall, Vancouver, BC, V6T 1Z1, Canada.

Crude extracts of the marine sponge Chelonaplysilla sp. collected in Samoa, that were obtained from the NCI Open Repository (NCS 21903), inhibited Mycobacterium tuberculosis growth. Assay-guided fractionation of the extract led to the isolation and structural elucidation of the known diterpenoid macfarlandin D (1) and three new diterpenoids macfarlandins F (2), G (3), and H (4). Macfarlandin D (1) exhibited potent antimicrobial activity against M. tuberculosis with an MIC of 1.2 ± 0.4 µg mL. Macfarlandins F (2), G (3), and H (4) exhibited significantly weaker antitubercular activities, revealing SAR for the macfarlandin antitubercular pharmacophore. The structures of compounds 2, 3, and 4 were elucidated via detailed analysis of NMR and MS data.
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http://dx.doi.org/10.1038/s41429-020-0315-4DOI Listing
August 2020

Discovery of a novel dehydratase of the fatty acid synthase type II critical for ketomycolic acid biosynthesis and virulence of Mycobacterium tuberculosis.

Sci Rep 2020 02 7;10(1):2112. Epub 2020 Feb 7.

Département Tuberculose & Biologie des Infections, Institut de Pharmacologie et de Biologie Structurale, UMR5089, Université de Toulouse, CNRS, UPS, 31077, Toulouse, Cedex 04, France.

The fatty acid synthase type II (FAS-II) multienzyme system builds the main chain of mycolic acids (MAs), important lipid pathogenicity factors of Mycobacterium tuberculosis (Mtb). Due to their original structure, the identification of the (3 R)-hydroxyacyl-ACP dehydratases, HadAB and HadBC, of Mtb FAS-II complex required in-depth work. Here, we report the discovery of a third dehydratase protein, HadD (Rv0504c), whose gene is non-essential and sits upstream of cmaA2 encoding a cyclopropane synthase dedicated to keto- and methoxy-MAs. HadD deletion triggered a marked change in Mtb keto-MA content and size distribution, deeply impacting the production of full-size molecules. Furthermore, abnormal MAs, likely generated from 3-hydroxylated intermediates, accumulated. These data strongly suggest that HadD catalyzes the 3-hydroxyacyl dehydratation step of late FAS-II elongation cycles during keto-MA biosynthesis. Phenotyping of Mtb hadD deletion mutant revealed the influence of HadD on the planktonic growth, colony morphology and biofilm structuration, as well as on low temperature tolerance. Importantly, HadD has a strong impact on Mtb virulence in the mouse model of infection. The effects of the lack of HadD observed both in vitro and in vivo designate this protein as a bona fide target for the development of novel anti-TB intervention strategies.
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http://dx.doi.org/10.1038/s41598-020-58967-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005898PMC
February 2020

An interview with Dr Tanya Parish and her perspectives on women in drug discovery.

Expert Opin Drug Discov 2020 03 18;15(3):277-278. Epub 2020 Feb 18.

Expert Opinion Series, Taylor & Francis, London, UK.

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http://dx.doi.org/10.1080/17460441.2020.1723971DOI Listing
March 2020

drug discovery models for relevant for host infection.

Authors:
Tanya Parish

Expert Opin Drug Discov 2020 03 3;15(3):349-358. Epub 2020 Jan 3.

Seattle Children's Research Institute, Center for Global Infectious Disease Research, Seattle, WA, USA.

: Tuberculosis is the leading cause of death from infectious disease. Current drug therapy requires a combination of antibiotics taken over >6 months. An urgent need for new agents that can shorten therapy is required. In order to develop new drugs, simple assays are required that can identify efficacious compounds rapidly and predict activity in the human.: This review focusses on the most relevant assays that can be utilized in a drug discovery program and which mimic different aspects of infection or disease. The focus is largely on assays used to test >1000s of compounds reliably and robustly. However, some assays used for 10s to 100s of compounds are included where the utility outweighs the low capacity. Literature searches for high throughput screening, models and assays were undertaken.: Drug discovery and development in tuberculosis is extremely challenging due to the requirement for predicting drug efficacy in a disease with complex pathology in which bacteria exist in heterogeneous states in inaccesible locations. A combination of assays can be used to determine profiles against replicating, non-replicating, intracellular and tolerant bacteria.
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http://dx.doi.org/10.1080/17460441.2020.1707801DOI Listing
March 2020

Microbiology revamps its scope.

Authors:
Tanya Parish

Microbiology (Reading) 2019 12;165(12):1251

Infectious Disease Research Institute, 1616 Eastlake Ave. E, Suite 400, Seattle, WA 98102, USA.

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http://dx.doi.org/10.1099/mic.0.000871DOI Listing
December 2019

A rapid, low pH, nutrient stress, assay to determine the bactericidal activity of compounds against non-replicating Mycobacterium tuberculosis.

PLoS One 2019 7;14(10):e0222970. Epub 2019 Oct 7.

TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington, United States of America.

There is an urgent need for new anti-tubercular agents which can lead to a shortened treatment time by targeting persistent or non-replicating bacilli. In order to assess compound activity against non-replicating Mycobacterium tuberculosis, we developed a method to detect the bactericidal activity of novel compounds within 7 days. Our method uses incubation at low pH in order to induce a non-replicating state. We used a strain of M. tuberculosis expressing luciferase; we first confirmed the linear relationship between luminescence and viable bacteria (determined by colony forming units) under our assay conditions. We optimized the assay parameters in 96-well plates in order to achieve a reproducible assay. Our final assay used M. tuberculosis in phosphate-citrate buffer, pH 4.5 exposed to compounds for 7 days; viable bacteria were determined by luminescence. We recorded the minimum bactericidal concentration at pH 4.5 (MBC4.5) representing >2 logs of kill. We confirmed the utility of the assay with control compounds. The ionophores monensin, niclosamide, and carbonyl cyanide 3-chlorophenylhydrazone and the anti-tubercular drugs pretomanid and rifampicin were active, while several other drugs such as isoniazid, ethambutol, and linezolid were not.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0222970PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779252PMC
March 2020

Integrated Target-Based and Phenotypic Screening Approaches for the Identification of Anti-Tubercular Agents That Bind to the Mycobacterial Adenylating Enzyme MbtA.

ChemMedChem 2019 10 23;14(19):1735-1741. Epub 2019 Sep 23.

Leicester School of Pharmacy, De Montfort University, Leicester, LE1 9BH, UK.

Iron is essential for the pathogenicity and virulence of Mycobacterium tuberculosis, which synthesises salicyl-capped siderophores (mycobactins) to acquire this element from the host. MbtA is the adenylating enzyme that catalyses the initial reaction of mycobactin biosynthesis and is solely expressed by mycobacteria. A 3200-member library comprised of lead-like, structurally diverse compounds was screened against M. tuberculosis for whole-cell inhibitory activity. A set of 846 compounds that inhibited the tubercle bacilli growth were then tested for their ability to bind to MbtA using a fluorescence-based thermal shift assay and NMR-based Water-LOGSY and saturation transfer difference (STD) experiments. We identified an attractive hit molecule, 5-hydroxyindol-3-ethylamino-(2-nitro-4-trifluoromethyl)benzene (5), that bound with high affinity to MbtA and produced a MIC value of 13 μm. The ligand was docked into the MbtA crystal structure and displayed an excellent fit within the MbtA active pocket, adopting a binding mode different from that of the established MbtA inhibitor Sal-AMS.
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http://dx.doi.org/10.1002/cmdc.201900217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800809PMC
October 2019

Anthranilic amide and imidazobenzothiadiazole compounds disrupt membrane potential.

Medchemcomm 2019 Jun 3;10(6):934-945. Epub 2019 May 3.

TB Discovery Research , Infectious Disease Research Institute , 1616 Eastlake Ave E, Suite 400 , Seattle , Washington 98102 , USA . Email:

A family of compounds typified by an anthranilic amide was identified from a whole-cell screening effort targeted at identifying compounds that disrupt pH homeostasis in . demonstrated bactericidal activity against non-replicating in pH 4.5 buffer (MBC = 6.3 μM). Exploration of the structure-activity relations failed to simplify the scaffold. The antitubercular activity proved dependent on the lipophilicity and planarity of the molecule and directly correlated with mammalian cytotoxicity. Further studies revealed a pH-dependent correlation between the family's disruption of membrane potential and antitubercular activity, with active compounds causing a drop in membrane potential at concentrations below their MBC. A second compound family, identified in the same screening effort and typified by imidazo(4,5-)(2,1,3)benzothiadiazole , provided a contrasting profile. As with , structure-activity profiling of (MBC = 25 μM) failed to minimize the initial scaffold, mammalian cytotoxicity was observed for a majority of the active compounds, and many of the active compounds disrupted membrane potential. However, unlike the anthranilic amide compounds, the benzothiadiazole compounds disrupted membrane potential primarily at concentrations above the MBC in a pH-independent fashion. These differences suggest an alternative mechanism of action for the benzothiadiazole compounds. As a result, while the cytotoxicity of the anthranilic amides limits their utility to tool compounds, benzothiadiazole presents an attractive target for more focused SAR exploration.
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http://dx.doi.org/10.1039/c9md00088gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6596218PMC
June 2019

Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB) inhibitors: A polypharmacology strategy for treating Mycobacterium tuberculosis infections.

Bioorg Med Chem Lett 2019 07 24;29(13):1665-1672. Epub 2019 Apr 24.

Indiana University School of Medicine, Department of Biochemistry and Molecular Biology, 635 Barnhill Dr., Indianapolis, IN 46202, United States. Electronic address:

Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis - actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease - which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.
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http://dx.doi.org/10.1016/j.bmcl.2019.04.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531345PMC
July 2019

Structure-Guided Drug Design of 6-Substituted Adenosine Analogues as Potent Inhibitors of Mycobacterium tuberculosis Adenosine Kinase.

J Med Chem 2019 05 19;62(9):4483-4499. Epub 2019 Apr 19.

Department of Biochemistry and Biophysics , Texas A&M University , College Station , Texas 77843 , United States.

Mycobacterium tuberculosis adenosine kinase (MtbAdoK) is an essential enzyme of Mtb and forms part of the purine salvage pathway within mycobacteria. Evidence suggests that the purine salvage pathway might play a crucial role in Mtb survival and persistence during its latent phase of infection. In these studies, we adopted a structural approach to the discovery, structure-guided design, and synthesis of a series of adenosine analogues that displayed inhibition constants ranging from 5 to 120 nM against the enzyme. Two of these compounds exhibited low micromolar activity against Mtb with half maximal effective inhibitory concentrations of 1.7 and 4.0 μM. Our selectivity and preliminary pharmacokinetic studies showed that the compounds possess a higher degree of specificity against MtbAdoK when compared with the human counterpart and are well tolerated in rodents, respectively. Finally, crystallographic studies showed the molecular basis of inhibition, potency, and selectivity and revealed the presence of a potentially therapeutically relevant cavity unique to the MtbAdoK homodimer.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511943PMC
May 2019

Characterization of MenA (isoprenyl diphosphate:1,4-dihydroxy-2-naphthoate isoprenyltransferase) from Mycobacterium tuberculosis.

PLoS One 2019 12;14(4):e0214958. Epub 2019 Apr 12.

Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, United States of America.

The menaquinone biosynthetic pathway presents a promising drug target against Mycobacterium tuberculosis and potentially other Gram-positive pathogens. In the present study, the essentiality, steady state kinetics of MenA from M. tuberculosis and the mechanism of MenA inhibition by Ro 48-8071 were characterized. MenA [isoprenyl diphosphate:1,4-dihydroxy-2-naphthoate (DHNA) isoprenyltransferase] catalyzes a critical reaction in menaquinone biosynthesis that involves the conversion of cytosolic DHNA, to membrane bound demethylmenaquinone by transferring a hydrophobic 45-carbon isoprenoid chain (in the case of mycobacteria) to the ring nucleus of DHNA. Rv0534c previously identified as the gene encoding MenA in M. tuberculosis complemented a menA deletion in E. coli and an E. coli host expressing Rv0534c exhibited an eight-fold increase in MenA specific activity over the control strain harboring empty vector under similar assay conditions. Expression of Rv0534c is essential for mycobacterial survival and the native enzyme from M. tuberculosis H37Rv was characterized using membrane preparations as it was not possible to solubilize and purify the recombinant enzyme. The enzyme is absolutely dependent on the presence of a divalent cation for optimal activity with Mg+2 being the most effective and is active over a wide pH range, with pH 8.5 being optimal. The apparent Km values for DHNA and farnesyl diphosphate were found to be 8.2 and 4.3 μM, respectively. Ro 48-8071, a compound previously reported to inhibit mycobacterial MenA activity, is non-competitive with regard to DHNA and competitive with regard to the isoprenyldiphosphate substrate.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214958PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461227PMC
December 2019

Novel MenA Inhibitors Are Bactericidal against and Synergize with Electron Transport Chain Inhibitors.

Antimicrob Agents Chemother 2019 06 24;63(6). Epub 2019 May 24.

TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington, USA

is the leading cause of morbidity and death resulting from infectious disease worldwide. The incredible disease burden, combined with the long course of drug treatment and an increasing incidence of antimicrobial resistance among isolates, necessitates novel drugs and drug targets for treatment of this deadly pathogen. Recent work has produced several promising clinical candidates targeting components of the electron transport chain (ETC) of , highlighting this pathway's potential as a drug target. Menaquinone is an essential component of the ETC, as it functions to shuttle electrons through the ETC to produce the electrochemical gradient required for ATP production for the cell. We show that inhibitors of MenA, a component of the menaquinone biosynthetic pathway, are highly active against MenA inhibitors are bactericidal against under both replicating and nonreplicating conditions, with 10-fold higher bactericidal activity against nutrient-starved bacteria than against replicating cultures. MenA inhibitors have enhanced activity in combination with bedaquiline, clofazimine, and inhibitors of QcrB, a component of the cytochrome oxidase. Together, these data support MenA as a viable target for drug treatment against MenA inhibitors not only kill in a variety of physiological states but also show enhanced activity in combination with ETC inhibitors in various stages of clinical trial testing.
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http://dx.doi.org/10.1128/AAC.02661-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535543PMC
June 2019

8-Hydroxyquinolines are bactericidal against Mycobacterium tuberculosis.

Drug Dev Res 2019 08 20;80(5):566-572. Epub 2019 Mar 20.

TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington.

There is an urgent need for new treatments effective against Mycobacterium tuberculosis, the causative agent of tuberculosis. The 8-hydroxyquinoline series is a privileged scaffold with anticancer, antifungal, and antibacterial activities. We conducted a structure-activity relationship study of the series regarding its antitubercular activity using 26 analogs. The 8-hydroxyquinolines showed good activity against M. tuberculosis, with minimum inhibitory concentrations (MIC90) of <5 μM for some analogs. Small substitutions at C5 resulted in the most potent activity. Substitutions at C2 generally decreased potency, although a sub-family of 2-styryl-substituted analogs retained activity. Representative compounds demonstrated bactericidal activity against replicating M. tuberculosis with >4 log kill at 10× MIC over 14 days. The majority of the compounds demonstrated cytotoxicity (IC of <100 μM). Further development of this series as antitubercular agents should address the cytotoxicity liability. However, the 8-hydroxyquinoline series represents a useful tool for chemical genomics to identify novel targets in M. tuberculosis.
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http://dx.doi.org/10.1002/ddr.21531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767403PMC
August 2019

Cell wall inhibitors increase the accumulation of rifampicin in .

Access Microbiol 2019 20;1(1):e000006. Epub 2019 Mar 20.

TB Discovery Research, Infectious Disease Research Institute, Seattle, WA, USA.

There is a need for new combination regimens for tuberculosis. Identifying synergistic drug combinations can avoid toxic side effects and reduce treatment times. Using a fluorescent rifampicin conjugate, we demonstrated that synergy between cell wall inhibitors and rifampicin was associated with increased accumulation of rifampicin. Increased accumulation was also associated with increased cellular permeability.
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http://dx.doi.org/10.1099/acmi.0.000006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470358PMC
March 2019

The relevance of persisters in tuberculosis drug discovery.

Microbiology (Reading) 2019 05 18;165(5):492-499. Epub 2019 Feb 18.

TB Discovery Research, Infectious Disease Research Institute, 1616 Eastlake Ave. E, Suite 400, Seattle, WA 98102, USA.

Bacterial persisters are a subpopulation of cells that exhibit phenotypic resistance during exposure to a lethal dose of antibiotics. They are difficult to target and thought to contribute to the long treatment duration required for tuberculosis. Understanding the molecular and cellular biology of persisters is critical to finding new tuberculosis drugs that shorten treatment. This review focuses on mycobacterial persisters and describes the challenges they pose in tuberculosis therapy, their characteristics and formation, how persistence leads to resistance, and the current approaches being used to target persisters within mycobacterial drug discovery.
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http://dx.doi.org/10.1099/mic.0.000760DOI Listing
May 2019

Efficacy and Improved Resistance Potential of a Cofactor-Independent InhA Inhibitor of Mycobacterium tuberculosis in the C3HeB/FeJ Mouse Model.

Antimicrob Agents Chemother 2019 04 27;63(4). Epub 2019 Mar 27.

Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA.

AN12855 is a direct, cofactor-independent inhibitor of InhA in In the C3HeB/FeJ mouse model with caseous necrotic lung lesions, AN12855 proved efficacious with a significantly lower resistance frequency than isoniazid. AN12855 drug levels were better retained in necrotic lesions and caseum where the majority of hard to treat, extracellular bacilli reside. Owing to these combined attributes, AN12855 represents a promising alternative to the frontline antituberculosis agent isoniazid.
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http://dx.doi.org/10.1128/AAC.02071-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6496157PMC
April 2019

A high-throughput whole cell screen to identify inhibitors of Mycobacterium tuberculosis.

PLoS One 2019 16;14(1):e0205479. Epub 2019 Jan 16.

Infectious Disease Research Institute, Seattle, Washington, United States of America.

Tuberculosis is a disease of global importance for which novel drugs are urgently required. We developed a whole-cell phenotypic screen which can be used to identify inhibitors of Mycobacterium tuberculosis growth. We used recombinant strains of virulent M. tuberculosis which express far-red fluorescent reporters and used fluorescence to monitor growth in vitro. We optimized our high throughput assays using both 96-well and 384-well plates; both formats gave assays which met stringent reproducibility and robustness tests. We screened a compound set of 1105 chemically diverse compounds previously shown to be active against M. tuberculosis and identified primary hits which showed ≥ 90% growth inhibition. We ranked hits and identified three chemical classes of interest-the phenoxyalkylbenzamidazoles, the benzothiophene 1-1 dioxides, and the piperidinamines. These new compound classes may serve as starting points for the development of new series of inhibitors that prevent the growth of M. tuberculosis. This assay can be used for further screening, or could easily be adapted to other strains of M. tuberculosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205479PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334966PMC
September 2019

Identification of Compounds with pH-Dependent Bactericidal Activity against Mycobacterium tuberculosis.

ACS Infect Dis 2019 02 17;5(2):272-280. Epub 2018 Dec 17.

TB Discovery Research , Infectious Disease Research Institute , 1616 Eastlake Avenue E , Suite 400, Seattle , Washington 98102 , United States.

To find new inhibitors of Mycobacterium tuberculosis that have novel mechanisms of action, we miniaturized a high throughput screen to identify compounds that disrupt pH homeostasis. We adapted and validated a 384-well format assay to determine intrabacterial pH using a ratiometric green fluorescent protein. We screened 89000 small molecules under nonreplicating conditions and confirmed 556 hits that reduced intrabacterial pH (below pH 6.5). We selected five compounds that disrupt intrabacterial pH homeostasis and also showed some activity against nonreplicating bacteria in a 4-stress model, but with no (or greatly reduced) activity against replicating bacteria. The compounds selected were two benzamide sulfonamides, a benzothiadiazole, a bissulfone, and a thiadiazole, none of which are known antibacterial agents. All of these five compounds demonstrated bactericidal activity against nonreplicating bacteria in buffer. Four of the five compounds demonstrated increased activity under low pH conditions. None of the five compounds acted as ionophores or as general disrupters of membrane potential. These compounds are useful starting points for work to elucidate their mechanism of action and their utility for drug discovery.
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http://dx.doi.org/10.1021/acsinfecdis.8b00256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371205PMC
February 2019

DORA Editorial.

Microbiology (Reading) 2019 02 5;165(2):125-126. Epub 2018 Dec 5.

6​Wellcome Sanger Institute, Cambridge, UK.

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http://dx.doi.org/10.1099/mic.0.000751DOI Listing
February 2019

DORA Editorial.

J Gen Virol 2019 01 5;100(1):1-2. Epub 2018 Dec 5.

6​Wellcome Sanger Institute, Cambridge, UK.

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http://dx.doi.org/10.1099/jgv.0.001191DOI Listing
January 2019

DORA Editorial.

J Med Microbiol 2019 Feb 5;68(2):117-118. Epub 2018 Dec 5.

6​Wellcome Sanger Institute, Cambridge, UK.

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http://dx.doi.org/10.1099/jmm.0.000887DOI Listing
February 2019