Publications by authors named "Shichun Lun"

42 Publications

Facile synthesis and antimycobacterial activity of isoniazid, pyrazinamide and ciprofloxacin derivatives.

Chem Biol Drug Des 2021 Feb 26. Epub 2021 Feb 26.

School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Perth, WA, Australia.

Several rationally designed isoniazid (INH), pyrazinamide (PZA) and ciprofloxacin (CPF) derivatives were conveniently synthesized and evaluated in vitro against H37Rv Mycobacterium tuberculosis (M. tb) strain. CPF derivative 16 displayed a modest activity (MIC = 16 µg/ml) and was docked into the M. tb DNA gyrase. Isoniazid-pyrazinoic acid (INH-POA) hybrid 21a showed the highest potency in our study (MIC = 2 µg/ml). It also retained its high activity against the other tested M. tb drug-sensitive strain (DS) V4207 (MIC = 4 µg/ml) and demonstrated negligible cytotoxicity against Vero cells (IC  ≥ 64 µg/ml). Four tested drug-resistant (DR) M. tb strains were refractory to 21a, similar to INH, whilst being sensitive to CPF. Compound 21a was also inactive against two non-tuberculous mycobacterial (NTM) strains, suggesting its selective activity against M. tb. The noteworthy activity of 21a against DS strains and its low cytotoxicity highlights its potential to treat DS M. tb.
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http://dx.doi.org/10.1111/cbdd.13836DOI Listing
February 2021

Therapeutic potential of coumestan Pks13 inhibitors for tuberculosis.

Antimicrob Agents Chemother 2021 Feb 8. Epub 2021 Feb 8.

Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Polyketide synthase 13 (Pks13) is an important enzyme found in () that condenses two fatty acyl chains to produce α-alkyl β-ketoesters, which in turn serve as the precursors for the synthesis of mycolic acids that are essential building blocks for maintaining the cell wall integrity of Coumestan derivatives have recently been identified in our group as a new chemotype that exert their antitubercular effects via targeting of Pks13. These compounds were active on both drug-susceptible and drug-resistant strains of as well as showing low cytotoxicity to healthy cells and a promising selectivity profile. No cross-resistance was found between the coumestan derivatives and first-line TB drugs. Here we report that treatment of bacilli with 15 times the MIC of compound , an optimized lead coumestan compound, resulted in a colony forming unit (CFU) reduction from 6.0 log units to below the limit of detection (1.0 log units) per mL culture, demonstrating a bactericidal mechanism of action. Single dose (10 mg/kg) pharmacokinetic studies revealed favorable parameters with a relative bioavailability of 19.4%. In a mouse infection and chemotherapy model, treatment with showed dose-dependent mono-therapeutic activity, whereas treatment with in combination with rifampin showed clear synergistic effects. Together these data suggest that coumestan derivatives are promising agents for further TB drug development.
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http://dx.doi.org/10.1128/AAC.02190-20DOI Listing
February 2021

Design and synthesis of mycobacterial pks13 inhibitors: Conformationally rigid tetracyclic molecules.

Eur J Med Chem 2021 Mar 21;213:113202. Epub 2021 Jan 21.

Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai, 200062, China. Electronic address:

We previously reported a series of coumestans-a naturally occurring tetracyclic scaffold containing a δ-lactone-that effectively target the thioesterase domain of polyketide synthase 13 (Pks13) in Mycobacterium tuberculosis (Mtb), resulting in superior anti-tuberculosis (TB) activity. Compared to the corresponding 'open-form' ethyl benzofuran-3-carboxylates, the enhanced anti-TB effects seen with the conformationally restricted coumestan series could be attributed to the extra π-π stacking interactions between the benzene ring of coumestans and the phenyl ring of F1670 residue located in the Pks13-TE binding domain. To further probe this binding feature, novel tetracyclic analogues were synthesized and evaluated for their anti-TB activity against the Mtb strain HRv. Initial comparison of the 'open-form' analogueues against the tetracyclic counterparts again showed that the latter is superior in terms of anti-TB activity. In particular, the δ-lactam-containing 5H-benzofuro [3,2-c]quinolin-6-ones gave the most promising results. Compound 65 demonstrated potent activity against Mtb HRv with MIC value between 0.0313 and 0.0625 μg/mL, with high selectivity to Vero cells (64-128 fold). The thermal stability analysis supports the notion that the tetracyclic compounds bind to the Pks13-TE domain as measured by nano DSF, consistent with the observed SAR trends. Compound 65 also showed excellent selectivity against actinobacteria and therefore unlikely to develop potential drug resistance to nonpathogenic bacteria.
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http://dx.doi.org/10.1016/j.ejmech.2021.113202DOI Listing
March 2021

The integrated stress response mediates necrosis in murine Mycobacterium tuberculosis granulomas.

J Clin Invest 2021 Feb;131(3)

The National Emerging Infectious Diseases Laboratory, Boston University School of Medicine, Boston, Massachusetts, USA.

The mechanism by which only some individuals infected with Mycobacterium tuberculosis develop necrotic granulomas with progressive disease while others form controlled granulomas that contain the infection remains poorly defined. Mice carrying the sst1-suscepible (sst1S) genotype develop necrotic inflammatory lung lesions, similar to human tuberculosis (TB) granulomas, which are linked to macrophage dysfunction, while their congenic counterpart (B6) mice do not. In this study we report that (a) sst1S macrophages developed aberrant, biphasic responses to TNF characterized by superinduction of stress and type I interferon pathways after prolonged TNF stimulation; (b) the late-stage TNF response was driven via a JNK/IFN-β/protein kinase R (PKR) circuit; and (c) induced the integrated stress response (ISR) via PKR-mediated eIF2α phosphorylation and the subsequent hyperinduction of ATF3 and ISR-target genes Chac1, Trib3, and Ddit4. The administration of ISRIB, a small-molecule inhibitor of the ISR, blocked the development of necrosis in lung granulomas of M. tuberculosis-infected sst1S mice and concomitantly reduced the bacterial burden. Hence, induction of the ISR and the locked-in state of escalating stress driven by the type I IFN pathway in sst1S macrophages play a causal role in the development of necrosis in TB granulomas. Interruption of the aberrant stress response with inhibitors such as ISRIB may offer novel host-directed therapy strategies.
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http://dx.doi.org/10.1172/JCI130319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843230PMC
February 2021

Design, synthesis and antimycobacterial evaluation of novel adamantane and adamantanol analogues effective against drug-resistant tuberculosis.

Bioorg Chem 2021 Jan 19;106:104486. Epub 2020 Nov 19.

School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley, Perth, WA 6102, Australia. Electronic address:

The treacherous nature of tuberculosis (TB) combined with the ubiquitous presence of the drug-resistant (DR) forms pose this disease as a growing public health menace. Therefore, it is imperative to develop new chemotherapeutic agents with a novel mechanism of action to circumvent the cross-resistance problems. The unique architecture of the Mycobacterium tuberculosis (M. tb) outer envelope plays a predominant role in its pathogenesis, contributing to its intrinsic resistance against available therapeutic agents. The mycobacterial membrane protein large 3 (MmpL3), which is a key player in forging the M. tb rigid cell wall, represents an emerging target for TB drug development. Several indole-2-carboxamides were previously identified in our group as potent anti-TB agents that act as inhibitor of MmpL3 transporter protein. Despite their highly potent in vitro activities, the lingering Achilles heel of these indoleamides can be ascribed to their high lipophilicity as well as low water solubility. In this study, we report our attempt to improve the aqueous solubility of these indole-2-carboxamides while maintaining an adequate lipophilicity to allow effective M. tb cell wall penetration. A more polar adamantanol moiety was incorporated into the framework of several indole-2-carboxamides, whereupon the corresponding analogues were tested for their anti-TB activity against drug-sensitive (DS) M. tb H37Rv strain. Three adamantanol derivatives 8i, 8j and 8l showed nearly 2- and 4-fold higher activity (MIC = 1.32 - 2.89 µM) than ethambutol (MIC = 4.89 µM). Remarkably, the most potent adamantanol analogue 8j demonstrated high selectivity towards DS and DR M. tb strains over mammalian cells [IC (Vero cells) ≥ 169 µM], evincing its lack of cytotoxicity. The top eight active compounds 8b, 8d, 8f, 8i, 8j, 8k, 8l and 10a retained their in vitro potency against DR M. tb strains and were docked into the MmpL3 active site. The most potent adamantanol/adamantane-based indoleamides 8j/8k displayed a two-fold surge in potency against extensively DR (XDR) M. tb strains with MIC values of 0.66 and 0.012 µM, respectively. The adamantanol-containing indole-2-carboxamides exhibited improved water solubility both in silico and experimentally, relative to the adamantane counterparts. Overall, the observed antimycobacterial and physicochemical profiles support the notion that adamantanol moiety is a suitable replacement to the adamantane scaffold within the series of indole-2-carboxamide-based MmpL3 inhibitors.
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http://dx.doi.org/10.1016/j.bioorg.2020.104486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775894PMC
January 2021

Design, synthesis, and biological evaluation of novel arylcarboxamide derivatives as anti-tubercular agents.

RSC Adv 2020 Feb 19;10(13):7523-7540. Epub 2020 Feb 19.

School of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, Bentley, Perth, WA 6102, Australia. Email:

Our group has previously reported several indolecarboxamides exhibiting potent antitubercular activity. Herein, we rationally designed several arylcarboxamides based on our previously reported homology model and the recently published crystal structure of the mycobacterial membrane protein large 3 (MmpL3). Many analogues showed considerable anti-TB activity against drug-sensitive (DS) () strain. Naphthamide derivatives and were the most active compounds in our study (MIC: 6.55, 7.11 μM, respectively), showing comparable potency to the first line anti-tuberculosis (anti-TB) drug ethambutol (MIC: 4.89 μM). In addition to the naphthamide derivatives, we also identified the quinolone-2-carboxamides and 4-arylthiazole-2-carboxamides as potential MmpL3 inhibitors in which compounds and had MIC values of 9.97 and 9.82 μM, respectively. All four compounds retained their high activity against multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. It is worth noting that the two most active compounds and also exhibited the highest selective activity towards DS, MDR and XDR strains over mammalian cells [IC (Vero cells) ≥ 227 μM], indicating their potential lack of cytotoxicity. The four compounds were docked into the MmpL3 active site and were studied for their drug-likeness using Lipinski's rule of five.
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http://dx.doi.org/10.1039/c9ra10663dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497412PMC
February 2020

Design, synthesis, and biological evaluation of novel imidazo[1,2-a]pyridinecarboxamides as potent anti-tuberculosis agents.

Chem Biol Drug Des 2020 Dec 9;96(6):1362-1371. Epub 2020 Jul 9.

Division of Infectious Disease, Department of Medicine, Center for Tuberculosis Research, Johns Hopkins School of Medicine, Baltimore, MD, USA.

Tuberculosis (TB) is a highly infectious disease that has been plaguing the human race for centuries. The emergence of multidrug-resistant strains of TB has been detrimental to the fight against tuberculosis with very few safe therapeutic options available. As part of an ongoing effort to identify potent anti-tuberculosis agents, we synthesized and screened a series of novel imidazo[1,2-a]pyridinecarboxamide derivatives for their anti-tuberculosis properties. These compounds were designed based on reported anti-tuberculosis properties of the indolecarboxamides (I2Cs) and imidazo[1,2-a]pyridinecarboxamides (IPAs). In this series, we identified compounds 15 and 16 with excellent anti-TB activity against H37Rv strain of tuberculosis (MIC = 0.10-0.19 μM); these compounds were further screened against selected clinical isolates of Mtb. Compounds 15 and 16 showed excellent activities against multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of TB (MIC range: 0.05-1.5 μM) with excellent selectivity indices. In addition, preliminary ADME studies on compound 16 showed favorable pharmacokinetic properties.
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http://dx.doi.org/10.1111/cbdd.13739DOI Listing
December 2020

Revisiting the β-Lactams for Tuberculosis Therapy with a Compound-Compound Synthetic Lethality Approach.

Antimicrob Agents Chemother 2019 11 22;63(11). Epub 2019 Oct 22.

Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

The suboptimal effectiveness of β-lactam antibiotics against has hindered the utility of this compound class for tuberculosis treatment. However, the results of treatment with a second-line regimen containing meropenem plus a β-lactamase inhibitor were found to be encouraging in a case study of extensively drug-resistant tuberculosis (M. C. Payen, S. De Wit, C. Martin, R. Sergysels, et al., Int J Tuberc Lung Dis 16:558-560, 2012, https://doi.org/10.5588/ijtld.11.0414). We hypothesized that the innate resistance of to β-lactams is mediated in part by noncanonical accessory proteins that are not considered the classic targets of β-lactams and that small-molecule inhibitors of those accessory targets might sensitize to β-lactams. In this study, we screened an NIH small-molecule library for the ability to sensitize to meropenem. We identified six hit compounds, belonging to either the -arylindole or benzothiophene chemotype. Verification studies confirmed the synthetic lethality phenotype for three of the -arylindoles and one benzothiophene derivative. The latter was demonstrated to be partially bioavailable via oral administration in mice. Structure-activity relationship studies of both structural classes identified analogs with potent antitubercular activity, alone or in combination with meropenem. Transcriptional profiling revealed that oxidoreductases, MmpL family proteins, and a 27-kDa benzoquinone methyltransferase could be the targets of the -arylindole potentiator. In conclusion, our compound-compound synthetic lethality screening revealed novel small molecules that were capable of potentiating the action of meropenem, presumably via inhibition of the innate resistance conferred by β-lactam accessory proteins. β-Lactam compound-compound synthetic lethality may be an alternative approach for drug-resistant tuberculosis.
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http://dx.doi.org/10.1128/AAC.01319-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6811440PMC
November 2019

Correction for Mir et al., "Mycobacterial Gene Is Required for Optimal Nutrient Utilization and Virulence".

Infect Immun 2019 Aug 23;87(8). Epub 2019 Jul 23.

Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA

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http://dx.doi.org/10.1128/IAI.00414-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6652758PMC
August 2019

The preclinical candidate indole-2-carboxamide improves immune responses to Mycobacterium tuberculosis infection in healthy subjects and individuals with type 2 diabetes.

Int Microbiol 2020 May 19;23(2):161-170. Epub 2019 Jun 19.

Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, CA, 91766-1854, USA.

A novel group of agents known as the indole-2-carboxamides (often referred to as indoleamides) have been shown to demonstrate high antimycobacterial activity. Studies have demonstrated that the best indoleamides possess desirable ADME/Tox properties, with less adverse effects and increased efficacy against both MDR-TB (multi-drug resistant TB) and XDR-TB (extensively drug-resistant TB). The primary mechanism of killing Mycobacterium tuberculosis (Mtb) by indoleamides is by disrupting the function of the essential mycolic acid transporter MmpL3 protein (Mycobacterial membrane protein Large 3). Therefore, targeting this essential mycobacterial transporter by small molecules opens new possibility for the development of novel and effective anti-TB agents. In the present study, we characterized the effects of indoleamides in altering the viability of Mtb in an in vitro granuloma model using immune cells derived from healthy subjects and those with type 2 diabetes mellitus (T2DM). Our results indicate that treatment with the best indoleamide 3 resulted in a significant reduction in the viability of Mtb in both THP-1 macrophages as well as in granulomas derived from healthy individuals and subjects with T2DM. Graphical Abstract.
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http://dx.doi.org/10.1007/s10123-019-00086-0DOI Listing
May 2020

Advancing the Therapeutic Potential of Indoleamides for Tuberculosis.

Antimicrob Agents Chemother 2019 07 24;63(7). Epub 2019 Jun 24.

Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Indole-2-carboxamide derivatives are inhibitors of MmpL3, the cell wall-associated mycolic acid transporter of In the present study, we characterized indoleamide effects on bacterial cell morphology and reevaluated pharmacokinetics and efficacy using an optimized oral formulation. Morphologically, indoleamide-treated cells demonstrated significantly higher numbers of dimples near the poles or septum, which may serve as the mechanism of cell death for this bactericidal scaffold. Using the optimized formulation, an expanded-spectrum indoleamide, compound 2, showed significantly improved pharmacokinetic (PK) parameters and efficacy in mouse infection models. In a comparative study, compound 2 showed superior efficacy over compound 3 (NITD-304) in a high-dose aerosol mouse infection model. Since indoleamides are equally active on drug-resistant , these findings demonstrate the therapeutic potential of this novel scaffold for the treatment of both drug-susceptible and drug-resistant tuberculosis.
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http://dx.doi.org/10.1128/AAC.00343-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591635PMC
July 2019

Identification of Novel Coumestan Derivatives as Polyketide Synthase 13 Inhibitors against Mycobacterium tuberculosis. Part II.

J Med Chem 2019 04 27;62(7):3575-3589. Epub 2019 Mar 27.

Center for Tuberculosis Research, Department of Medicine, Division of Infectious Disease , Johns Hopkins School of Medicine , Baltimore , Maryland 21231-1044 , United States.

Our group recently reported the identification of novel coumestan derivatives as Mycobacterium tuberculosis ( Mtb) Pks13-thioesterase (TE) domain inhibitors, with mutations observed (D1644G and N1640K) in the generated coumestan-resistant Mtb colonies. Herein, we report a further structure-activity relationships exploration exploiting the available Pks13-TE X-ray co-crystal structure that resulted in the discovery of extremely potent coumestan analogues 48 and 50. These molecules possess excellent anti-tuberculosis activity against both the drug-susceptible (MIC = 0.0039 μg/mL) and drug-resistant Mtb strains (MIC = 0.0078 μg/mL). Moreover, the excellent in vitro activity is translated to the in vivo mouse serum inhibitory titration assay, with administration of coumestan 48 at 100 mg/kg showing an 8-fold higher activity than that of isoniazid or TAM16 given at 10 or 100 mg/kg, respectively. Preliminary ADME-Tox data for the coumestans were promising and, coupled with the practicality of synthesis, warrant further in vivo efficacy assessments of the coumestan derivatives.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00010DOI Listing
April 2019

Identification of Novel Coumestan Derivatives as Polyketide Synthase 13 Inhibitors against Mycobacterium tuberculosis.

J Med Chem 2018 02 29;61(3):791-803. Epub 2018 Jan 29.

Center for Tuberculosis Research, Department of Medicine, Division of Infectious Disease, Johns Hopkins School of Medicine , Baltimore, Maryland 21231-1044, United States.

Inhibition of the mycolic acid pathway has proven a viable strategy in antitubercular drug discovery. The AccA3/AccD4/FadD32/Pks13 complex of Mycobacterium tuberculosis constitutes an essential biosynthetic mechanism for mycolic acids. Small molecules targeting the thioesterase domain of Pks13 have been reported, including a benzofuran-based compound whose X-ray cocrystal structure has been very recently solved. Its initial inactivity in a serum inhibition titration (SIT) assay led us to further probe other structurally related benzofurans with the aim to improve their potency and bioavailability. Herein, we report our preliminary structure-activity relationship studies around this scaffold, highlighting a natural product-inspired cyclization strategy to form coumestans that are shown to be active in SIT. Whole genome deep sequencing of the coumestan-resistant mutants confirmed a single nucleotide polymorphism in the pks13 gene responsible for the resistance phenotype, demonstrating the druggability of this target for the development of new antitubercular agents.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01319DOI Listing
February 2018

Targeting Mycolic Acid Transport by Indole-2-carboxamides for the Treatment of Mycobacterium abscessus Infections.

J Med Chem 2017 07 15;60(13):5876-5888. Epub 2017 Jun 15.

Institut de Recherche en Infectiologie (IRIM), CNRS, UMR 9004, Université de Montpellier , Montpellier Cedex 5 34 293, France.

Mycobacterium abscessus is a fast-growing, multidrug-resistant organism that has emerged as a clinically significant pathogen in cystic fibrosis (CF) patients. The intrinsic resistance of M. abscessus to most commonly available antibiotics seriously restricts chemotherapeutic options. Herein, we report the potent activity of a series of indolecarboxamides against M. abscessus. The lead compounds, 6 and 12, exhibited strong activity in vitro against a wide panel of M. abscessus isolates and in infected macrophages. High resistance levels to the indolecarboxamides appear to be associated with an A309P mutation in the mycolic acid transporter MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis remained unaffected, the indolecarboxamides strongly inhibited the transport of trehalose monomycolate, resulting in the loss of trehalose dimycolate production and abrogating mycolylation of arabinogalactan. Our data introduce a hereto unexploited chemical structure class active against M. abscessus infections with promising translational development possibilities for the treatment of CF patients.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00582DOI Listing
July 2017

Indole-2-carboxamide-based MmpL3 Inhibitors Show Exceptional Antitubercular Activity in an Animal Model of Tuberculosis Infection.

J Med Chem 2016 07 22;59(13):6232-47. Epub 2016 Jun 22.

Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago , 833 South Wood Street, Chicago, Illinois 60612, United States.

Our team had previously identified certain indolecarboxamides that represented a new chemical scaffold that showed promising anti-TB activity at both an in vitro and in vivo level. Based on mutational analysis using bacteria found resistant to one of these indolecarboxamides, we identified the trehalose monomycolate transporter MmpL3 as the likely target of these compounds. In the present work, we now further elaborate on the SAR of these compounds, which has led in turn to the identification of a new analog, 4,6-difluoro-N-((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)-1H-indole-2-carboxamide (26), that shows excellent activity against drug-sensitive (MIC = 0.012 μM; SI ≥ 16000), multidrug-resistant (MDR), and extensively drug-resistant (XDR) Mycobacterium tuberculosis strains, has superior ADMET properties, and shows excellent activity in the TB aerosol lung infection model. Compound 26 is also shown to work in synergy with rifampin. Because of these properties, we believe that indolecarboxamide 26 is a possible candidate for advancement to human clinical trials.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00415DOI Listing
July 2016

Erratum to "Efficacy of Adjunctive Tofacitinib Therapy in Mouse Models of Tuberculosis" [EBioMedicine 2 (8), August 2015, 868-873].

EBioMedicine 2015 Nov 19;2(11):1834. Epub 2015 Oct 19.

Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA.

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http://dx.doi.org/10.1016/j.ebiom.2015.10.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740325PMC
November 2015

Efficacy of Adjunctive Tofacitinib Therapy in Mouse Models of Tuberculosis.

EBioMedicine 2015 Aug 14;2(8):868-73. Epub 2015 Jul 14.

Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, MD, USA ; Howard Hughes Medical Institute, Chevy Chase, MD, USA.

The global tuberculosis (TB) epidemic and the spread of multi- and extensively-drug resistant strains of Mycobacterium tuberculosis (M.tb) have been fueled by low adherence to following lengthy treatment protocols, and the rapid spread of HIV (Human Immunodeficiency Virus). Persistence of the infection in immunocompetent individuals follows from the ability of M.tb to subvert host immune responses in favor of survival within macrophages. Alternative host-directed strategies are therefore being currently sought to improve treatment efficacy and duration. In this study, we evaluated tofacitinib, a new oral Janus kinase (JAK) blocker with anti-inflammatory properties, in shortening tuberculosis treatment. BALB/c mice, which are immunocompetent, showed acceleration of M.tb clearance achieving apparent sterilization after 16 weeks of adjunctive tofacitinib therapy at average exposures higher than recommended in humans, while mice receiving standard treatment alone did not achieve clearance until 24 weeks. True sterilization with tofacitinib was not achieved until five months. C3HeB/FeJ mice, which show reduced pro-inflammatory cytokines during M.tb infection, did not show improved clearance with adjunctive tofacitinib therapy, indicating that the nature of granulomatous lesions and host immunity may influence responsiveness to tofacitinib. Our findings suggest that the JAK pathway could be explored further for host-directed therapy in immunocompetent individuals.
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http://dx.doi.org/10.1016/j.ebiom.2015.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4563140PMC
August 2015

Mutation of Rv2887, a marR-like gene, confers Mycobacterium tuberculosis resistance to an imidazopyridine-based agent.

Antimicrob Agents Chemother 2015 Nov 24;59(11):6873-81. Epub 2015 Aug 24.

Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University, Baltimore, Maryland, USA

Drug resistance is a major problem in Mycobacterium tuberculosis control, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity against M. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independent M. tuberculosis mutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations in Rv2887 were common to all three MP-III-71-resistant mutants, and we confirmed the role of Rv2887 as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified in Escherichia coli to negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation of Rv2887 abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations of Rv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance of M. tuberculosis Rv2887 mutants may involve efflux pump upregulation and also drug methylation.
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http://dx.doi.org/10.1128/AAC.01341-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604386PMC
November 2015

Synthetic lethality reveals mechanisms of Mycobacterium tuberculosis resistance to β-lactams.

mBio 2014 Sep 16;5(5):e01767-14. Epub 2014 Sep 16.

Unlabelled: Most β-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe's innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate β-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem β-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in β-lactam resistance. The global transcriptional response of the bacterium to β-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to β-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for β-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis.

Importance: The global emergence of multidrug-resistant and extensively drug-resistant M. tuberculosis strains has threatened public health worldwide, yet the pipeline of new tuberculosis drugs under development remains limited. One strategy to cope with the urgent need for new antituberculosis agents is to repurpose existing, approved antibiotics. The carbapenem class of β-lactam antibiotics has been proposed as one such class of drugs. Our study identifies molecular determinants of innate resistance to β-lactam drugs in M. tuberculosis, and we demonstrate that functional loss of one of these genes enables successful treatment of M. tuberculosis with β-lactams in the mouse model.
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http://dx.doi.org/10.1128/mBio.01767-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172077PMC
September 2014

Biological evaluation of potent triclosan-derived inhibitors of the enoyl-acyl carrier protein reductase InhA in drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis.

ChemMedChem 2014 Nov 27;9(11):2528-37. Epub 2014 Aug 27.

Drug Discovery Program, Department of Medicinal Chemistry & Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612 (USA); Department of Pharmaceutical Sciences, College of Pharmacy, Chicago State University, 9501 South King Drive, Chicago, IL 60628 (USA).

New triclosan (TRC) analogues were evaluated for their activity against the enoyl-acyl carrier protein reductase InhA in Mycobacterium tuberculosis (Mtb). TRC is a well-known inhibitor of InhA, and specific modifications to its positions 5 and 4' afforded 27 derivatives; of these compounds, seven derivatives showed improved potency over that of TRC. These analogues were active against both drug-susceptible and drug-resistant Mtb strains. The most active compound in this series, 4-(n-butyl)-1,2,3-triazolyl TRC derivative 3, had an MIC value of 0.6 μg mL(-1) (1.5 μM) against wild-type Mtb. At a concentration equal to its MIC, this compound inhibited purified InhA by 98 %, and showed an IC50 value of 90 nM. Compound 3 and the 5-methylisoxazole-modified TRC 14 were able to inhibit the biosynthesis of mycolic acids. Furthermore, mc(2) 4914, an Mtb strain overexpressing inhA, was found to be less susceptible to compounds 3 and 14, supporting the notion that InhA is the likely molecular target of the TRC derivatives presented herein.
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http://dx.doi.org/10.1002/cmdc.201402255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4213240PMC
November 2014

Mycobacterial gene cuvA is required for optimal nutrient utilization and virulence.

Infect Immun 2014 Oct 21;82(10):4104-17. Epub 2014 Jul 21.

Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA

To persist and cause disease in the host, Mycobacterium tuberculosis must adapt to its environment during infection. Adaptations include changes in nutrient utilization and alterations in growth rate. M. tuberculosis Rv1422 is a conserved gene of unknown function that was found in a genetic screen to interact with the mce4 cholesterol uptake locus. The Rv1422 protein is phosphorylated by the M. tuberculosis Ser/Thr kinases PknA and PknB, which regulate cell growth and cell wall synthesis. Bacillus subtilis strains lacking the Rv1422 homologue yvcK grow poorly on several carbon sources, and yvcK is required for proper localization of peptidoglycan synthesis. Here we show that Mycobacterium smegmatis and M. tuberculosis strains lacking Rv1422 have growth defects in minimal medium containing limiting amounts of several different carbon sources. These strains also have morphological abnormalities, including shortened and bulging cells, suggesting a cell wall defect. In both mycobacterial species, the Rv1422 protein localizes uniquely to the growing cell pole, the site of peptidoglycan synthesis in mycobacteria. An M. tuberculosis ΔRv1422 strain is markedly attenuated for virulence in a mouse infection model, where it elicits decreased inflammation in the lungs and shows impaired bacterial persistence. These findings led us to name this gene cuvA (carbon utilization and virulence protein A) and to suggest a model in which deletion of cuvA leads to changes in nutrient uptake and/or metabolism that affect cell wall structure, morphology, and virulence. Its role in virulence suggests that CuvA may be a useful target for novel inhibitors of M. tuberculosis during infection.
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http://dx.doi.org/10.1128/IAI.02207-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187881PMC
October 2014

Indoleamides are active against drug-resistant Mycobacterium tuberculosis.

Nat Commun 2013 ;4:2907

1] Center for Tuberculosis Research, Johns Hopkins University School of Medicine, 1550 Orleans Street, CRB2 Suite 103, Baltimore, Maryland 21231, USA [2] KwaZulu-Natal Research Institute for Tuberculosis and HIV, 719 Umbilo Road, Durban 4001, South Africa [3] Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, Maryland 20815, USA.

Responsible for nearly two million deaths each year, the infectious disease tuberculosis remains a serious global health challenge. The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis confounds control efforts, and new drugs with novel molecular targets are desperately needed. Here we describe lead compounds, the indoleamides, with potent activity against both drug-susceptible and drug-resistant strains of M. tuberculosis by targeting the mycolic acid transporter MmpL3. We identify a single mutation in mmpL3, which confers high resistance to the indoleamide class while remaining susceptible to currently used first- and second-line tuberculosis drugs, indicating a lack of cross-resistance. Importantly, an indoleamide derivative exhibits dose-dependent antimycobacterial activity when orally administered to M. tuberculosis-infected mice. The bioavailability of the indoleamides, combined with their ability to kill tubercle bacilli, indicates great potential for translational developments of this structure class for the treatment of drug-resistant tuberculosis.
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http://dx.doi.org/10.1038/ncomms3907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3909880PMC
July 2014

Pharmacokinetic and in vivo efficacy studies of the mycobactin biosynthesis inhibitor salicyl-AMS in mice.

Antimicrob Agents Chemother 2013 Oct 15;57(10):5138-40. Epub 2013 Jul 15.

Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Mycobactin biosynthesis in Mycobacterium tuberculosis facilitates iron acquisition, which is required for growth and virulence. The mycobactin biosynthesis inhibitor salicyl-AMS [5'-O-(N-salicylsulfamoyl)adenosine] inhibits M. tuberculosis growth in vitro under iron-limited conditions. Here, we conducted a single-dose pharmacokinetic study and a monotherapy study of salicyl-AMS with mice. Intraperitoneal injection yielded much better pharmacokinetic parameter values than oral administration did. Monotherapy of salicyl-AMS at 5.6 or 16.7 mg/kg significantly inhibited M. tuberculosis growth in the mouse lung, providing the first in vivo proof of concept for this novel antibacterial strategy.
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http://dx.doi.org/10.1128/AAC.00918-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811451PMC
October 2013

Detection of inhibitors of phenotypically drug-tolerant Mycobacterium tuberculosis using an in vitro bactericidal screen.

J Microbiol 2013 Oct 25;51(5):651-8. Epub 2013 Jun 25.

School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.

Many whole cell screens of chemical libraries currently in use are based on inhibition of bacterial growth. The goal of this study was to develop a chemical library screening model that enabled detection of compounds that are active against drug-tolerant non-growing cultures of Mycobacterium tuberculosis. An in vitro model of low metabolically active mycobacteria was established with 8 and 30 day old cultures of M. smegmatis and M. tuberculosis, respectively. Reduction of resazurin was used as a measure of viability and the assay was applied in screens of chemical libraries for bactericidal compounds. The model provided cells that were phenotypically-resilient to killing by first and second-line clinical drugs including rifampicin. Screening against chemical libraries identified proteasome inhibitors, NSC310551 and NSC321206, and a structurally-related series of thiosemicarbazones, as having potent killing activity towards aged cultures. The inhibitors were confirmed as active against virulent M. tuberculosis strains including multi- and extensively-drug resistant clinical isolates. Our library screen enabled detection of compounds with a potent level of bactericidal activity towards phenotypically drug-tolerant cultures of M. tuberculosis.
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http://dx.doi.org/10.1007/s12275-013-3099-4DOI Listing
October 2013

Preliminary structure-activity relationships and biological evaluation of novel antitubercular indolecarboxamide derivatives against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains.

J Med Chem 2013 May 3;56(10):4093-103. Epub 2013 May 3.

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, USA.

Tuberculosis (TB) remains one of the leading causes of mortality and morbidity worldwide, with approximately one-third of the world's population infected with latent TB. This is further aggravated by HIV coinfection and the emergence of multidrug- and extensively drug-resistant (MDR and XDR, respectively) TB; hence the quest for highly effective antitubercular drugs with novel modes of action is imperative. We report herein the discovery of an indole-2-carboxamide analogue, 3, as a highly potent antitubercular agent, and the subsequent chemical modifications aimed at establishing a preliminary body of structure-activity relationships (SARs). These efforts led to the identification of three molecules (12-14) possessing an exceptional activity in the low nanomolar range against actively replicating Mycobacterium tuberculosis , with minimum inhibitory concentration (MIC) values lower than those of the most prominent antitubercular agents currently in use. These compounds were also devoid of apparent toxicity to Vero cells. Importantly, compound 12 was found to be active against the tested XDR-TB strains and orally active in the serum inhibition titration assay.
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http://dx.doi.org/10.1021/jm4003878DOI Listing
May 2013

Rv2190c, an NlpC/P60 family protein, is required for full virulence of Mycobacterium tuberculosis.

PLoS One 2012 31;7(8):e43429. Epub 2012 Aug 31.

Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) possesses at least five genes predicted to encode proteins with NlpC/P60 hydrolase domains, including the relatively uncharacterized Rv2190c. As NlpC/P60 domain-containing proteins are associated with diverse roles in bacterial physiology, our objective was to characterize Rv2190c in M. tuberculosis growth and virulence. Our data indicate that lack of Rv2190c is associated with impaired growth, both in vitro and during an in vivo mouse model of TB. These growth defects are associated with altered colony morphology and phthiocerol dimycocerosate levels, indicating that Rv2190c is involved in cell wall maintenance and composition. In addition, we have demonstrated that Rv2190c is expressed during active growth phase and that its protein product is immunogenic during infection. Our findings have significant implications, both for better understanding the role of Rv2190c in M. tuberculosis biology and also for translational developments.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043429PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3432046PMC
February 2013

Risk of tuberculosis reactivation with tofacitinib (CP-690550).

J Infect Dis 2012 Jun 3;205(11):1705-8. Epub 2012 Apr 3.

Center for Tuberculosis Research, Johns Hopkins School of Medicine, Baltimore, MD, USA.

Individuals with latent tuberculosis infection (LTBI) live with a risk of reactivation, and several treatments for chronic inflammatory conditions are highly associated with such reactivation. A new Janus kinase inhibitor, tofacitinib (CP-690550), has shown promising results for treatment of inflammatory disorders, thus raising concerns of risk of active tuberculosis. Our goal was to characterize the impact of tofacitinib on LTBI using a mouse model of contained tuberculosis. Our data indicate that tofacitinib reduces host containment of Mycobacterium tuberculosis and promotes bacterial replication in the lungs, suggesting tuberculosis reactivation. Tofacitinib may carry a significant risk for LTBI reactivation in humans.
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http://dx.doi.org/10.1093/infdis/jis269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3415851PMC
June 2012

Isoniazid resistance without a loss of fitness in Mycobacterium tuberculosis.

Nat Commun 2012 Mar 20;3:753. Epub 2012 Mar 20.

Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.

The emergence of multi- and extensively drug-resistant tuberculosis (MDR-TB and XDR-TB, respectively) has intensified the critical public health implications of this global disease. The fitness of Mycobacterium tuberculosis (M.tb.) strains exhibiting MDR and XDR phenotypes is of fundamental importance in predicting whether the MDR-/XDR-TB epidemic will be sustained across the human population. Here we describe a potential mechanism of M.tb. resistance to the TB drug isoniazid (INH) conferred by loss of a sigma factor, SigI. We demonstrate that the gain of INH resistance in the M.tb. ΔsigI mutant might not diminish the organism's fitness for causing disease. These findings have significant implications when considering the ability of drug-resistant M.tb. strains to initiate untreatable TB epidemics, as it is possible that loss or alteration of SigI function could have a role in the generation of MDR and XDR M.tb. strains of suitable fitness to spread in a community setting.
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http://dx.doi.org/10.1038/ncomms1724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3648870PMC
March 2012

Successful shortening of tuberculosis treatment using adjuvant host-directed therapy with FDA-approved phosphodiesterase inhibitors in the mouse model.

PLoS One 2012 3;7(2):e30749. Epub 2012 Feb 3.

Center for Tuberculosis Research, School of Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America.

Global control of tuberculosis (TB), an infectious disease that claims nearly 2 million lives annually, is hindered by the long duration of chemotherapy required for curative treatment. Lack of adherence to this intense treatment regimen leads to poor patient outcomes, development of new or additional drug resistance, and continued spread of M.tb. within communities. Hence, shortening the duration of TB therapy could increase drug adherence and cure in TB patients. Here, we report that addition of the United Stated Food and Drug Administration-approved phosphodiesterase inhibitors (PDE-Is) cilostazol and sildenafil to the standard TB treatment regimen reduces tissue pathology, leads to faster bacterial clearance and shortens the time to lung sterilization by one month, compared to standard treatment alone, in a murine model of TB. Our data suggest that these PDE-Is could be repurposed for use as adjunctive drugs to shorten TB treatment in humans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030749PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272040PMC
September 2012