Publications by authors named "William R Bishai"

213 Publications

Therapeutic targeting with DABIL-4 depletes myeloid suppressor cells in 4T1 triple-negative breast cancer model.

Mol Oncol 2021 Mar 7. Epub 2021 Mar 7.

Department of Medicine, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

In many solid tumors including triple-negative breast cancer (TNBC), upregulation of the interleukin-4 receptor (IL-4R) has been shown to promote cancer cell proliferation, apoptotic resistance, metastatic potential, and a Th2 response in the tumor microenvironment (TME). Since immunosuppressive cells in the TME and spleen including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) also express the IL-4R, we hypothesized that selective depletion of IL-4R-bearing cells in TNBC would result in the direct killing of tumor cells and the depletion of immunosuppressive cells and lead to an enhanced antitumor response. To selectively target IL-4R cells, we employed DABIL-4, a fusion protein toxin consisting of the catalytic and translocation domains of diphtheria toxin fused to murine IL-4. As anticipated, DABIL-4 has potent cytotoxic activity against TNBC cells both in vitro and in vivo. We demonstrate in the murine 4T1 TNBC model that DABIL-4 significantly reduces tumor growth, splenomegaly, and lung metastases. Importantly, we also show that the administration of DABIL-4 results in the selective depletion of MDSCs, TAMs, and regulatory T cells in treated mice, with a concomitant increase in IFN-γ CD8 effector T cells in the TME. Since the 4T1 antitumor activity of DABIL-4 was largely diminished in IL-4R knockout mice, we postulate that DABIL-4 functions primarily as an immunotherapeutic by the depletion of MDSCs, TAMs, and regulatory T cells. NanoString analysis of control and treated tumors confirmed and extended these observations by showing a marked decline of mRNA transcripts that are associated with tumorigenesis and metastasis. In conclusion, we demonstrate that DABIL-4 targeting of both tumor and immunosuppressive host cells likely represents a novel and effective treatment strategy for 4T1 TNBC and warrants further study.
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http://dx.doi.org/10.1002/1878-0261.12938DOI Listing
March 2021

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

Immunologic goalposts for TB vaccine development.

Cell Host Microbe 2021 02;29(2):158-159

Center for Tuberculosis Research, Johns Hopkins School of Medicine, 1550 Orleans St., Rm 108, Baltimore, MD 21287, USA. Electronic address:

A limiting factor in identifying effective tuberculosis (TB) vaccines is our incomplete understanding of correlates of protection. In this issue of Cell Host & Microbe, Esaulova et al. reveal cell types associated with TB containment and disease progression in non-human primates, which may provide immunologic goalposts for vaccine design.
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http://dx.doi.org/10.1016/j.chom.2021.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874915PMC
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

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

Post translational modifications in tuberculosis: ubiquitination paradox.

Autophagy 2021 Mar 24;17(3):814-817. Epub 2020 Nov 24.

JH-Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India.

Innate immune signaling and xenophagy are crucial innate defense strategies exploited by the host to counteract intracellular pathogens with ubiquitination as a critical regulator of these processes. These pathogens, including (), co-opt the host ubiquitin machinery by utilizing secreted or cell surface effectors to dampen innate host defenses. Inversely, the host utilizes ubiquitin ligase-mediated ubiquitination of intracellular pathogens and recruits autophagy receptors to induce xenophagy. In the current article, we discuss the co-option of the ubiquitin pathway by the virulence effectors.: ANAPC2: anaphase promoting complex subunit 2; IL: interleukin; Lys: lysine (K); MAPK: mitogen-activated protein kinase; MAP3K7/TAK1; mitogen-activated protein kinase kinase kinase 7; ; NFKB/NF-κB: nuclear factor kappa B subunit; PtpA: protein tyrosine phosphatase; SQSTM1/p62: sequestosome 1; V-ATPase: vacuolar-type H+-ATPase; UBA: a eukaryotic-like ubiquitin-associated domain.
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http://dx.doi.org/10.1080/15548627.2020.1850009DOI Listing
March 2021

Short Communication: Genetic Variation in Human Proximal Promoter and Susceptibility to HIV-1 Infection in Mali, West Africa.

AIDS Res Hum Retroviruses 2021 01 23;37(1):57-61. Epub 2020 Nov 23.

Division of Infectious Diseases and Institute for Global Health, Feinberg School of Medicine, Northwestern University (NU), Chicago, Illinois, USA.

It is now recognized that to fully understand the role of host genetic variation on susceptibility to HIV-1 infection, investigations must be extended to African populations. We sought to determine if genetic variation in are associated with HIV-1 infection in a West African cohort in Mali. HIV-infected and -uninfected individuals were genotyped for three common single nucleotide polymorphisms (SNPs) located at positions -592 (C/A), -819 (C/T), and -1082 (G/A) of the promoter. We found that the ATA haplotype, which has been previously associated with low IL-10 expression, was the most represented in the cohort. Although we observed a trend toward an increased frequency of ATA/ATA carriage in HIV-infected compared with -uninfected individuals, the difference was not statistically significant. Similarly, individual SNPs were not significantly enriched in the HIV-infected group, suggesting that genetic variants are not associated with HIV-1 in this West African cohort from Mali.
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http://dx.doi.org/10.1089/AID.2020.0140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864090PMC
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

Getting to the point in point-of-care diagnostics for tuberculosis.

J Clin Invest 2020 11;130(11):5671-5673

Division of Infectious Diseases.

Tuberculosis (TB) continues to affect over 10 million people per year worldwide. Despite advances in diagnosis, smear microscopy insufficiently detects pulmonary disease, with test result reporting taking longer than a day. While urine assays to detect the lipopolysaccharide lipoarabinomannan (LAM), present in mycobacterial cell walls, can provide results within minutes, the currently available assay has low sensitivity and its application is limited to patients with HIV suspected of having TB. In this issue of the JCI, Broger and Nicol et al. investigated 3 rapid urine tests in 372 ambulatory HIV-negative individuals suspected of having TB in South Africa and Peru. FujiLAM emerged as a rapid test to confirm TB diagnosis in the HIV-seronegative population. This study shows that FujiLAM has considerable potential to reshape the TB diagnostics landscape, making diagnosis and treatment in one office visit a reality for TB.
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http://dx.doi.org/10.1172/JCI142497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598045PMC
November 2020

Infectability of human BrainSphere neurons suggests neurotropism of SARS-CoV-2

ALTEX 2020 26;37(4):665-671. Epub 2020 Jun 26.

Johns Hopkins University, Bloomberg School of Public Health, Center for Alternatives to Animal Testing (CAAT), Baltimore, MD, USA.

Reports from Wuhan suggest that 36% of COVID-19 patients show neurological symptoms, and cases of viral encephalitis have been reported, suggesting that the virus is neurotropic under unknown circumstances. This is well established for other coronaviruses. In order to understand why some patients develop such symptoms and others do not, we address herein the infectability of the central nervous system (CNS). Reports that the ACE2 receptor – critical for virus entry into lung cells – is found in different neurons support this expectation. We employed a human induced pluripotent stem cell (iPSC)- derived BrainSphere model, which we used earlier for Zika, Dengue, HIV and John Cunningham virus infection studies. We detected the expression of the ACE2 receptor, but not TMPRSS2, in the model. Incubating the BrainSpheres for 6 hours with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 led to infection of a fraction of neural cells with replication of the virus evident at 72 hpi. Virus particles were found in the neuronal cell body extending into apparent neurite structures. PCR measurements corroborated the replication of the virus, suggesting at least a tenfold increase in virus copies per total RNA. Leveraging state-of-the-art 3D organotypic cell culture, which has been shown to allow both virus infection and modeling of (developmental) neurotoxicity but is at the same time simple enough to be transferred and used in a BSL-3 environment, we demonstrate, for the first time, the potential critically important neurotropism of SARS-CoV-2.
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http://dx.doi.org/10.14573/altex.2006111DOI Listing
October 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

Cavitary tuberculosis: the gateway of disease transmission.

Lancet Infect Dis 2020 06 5;20(6):e117-e128. Epub 2020 May 5.

Center for Tuberculosis Research, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Center for Infection and Inflammation Imaging Research, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address:

Tuberculosis continues to be a major threat to global health. Cavitation is a dangerous consequence of pulmonary tuberculosis associated with poor outcomes, treatment relapse, higher transmission rates, and development of drug resistance. However, in the antibiotic era, cavities are often identified as the most extreme outcome of treatment failure and are one of the least-studied aspects of tuberculosis. We review the epidemiology, clinical features, and concurrent standards of care for individuals with cavitary tuberculosis. We also discuss developments in the understanding of tuberculosis cavities as dynamic physical and biochemical structures that interface the host response with a unique mycobacterial niche to drive tuberculosis-associated morbidity and transmission. Advances in preclinical models and non-invasive imaging can provide valuable insights into the drivers of cavitation. These insights will guide the development of specific pharmacological interventions to prevent cavitation and improve lung function for individuals with tuberculosis.
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http://dx.doi.org/10.1016/S1473-3099(20)30148-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357333PMC
June 2020

Relative and Quantitative Phosphoproteome Analysis of Macrophages in Response to Infection by Virulent and Avirulent Reveals a Distinct Role of the Cytosolic RNA Sensor RIG-I in Pathogenesis.

J Proteome Res 2020 06 14;19(6):2316-2336. Epub 2020 May 14.

Laboratory of Mycobacterial Genetics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad 121001, Haryana, India.

Comparative phosphoproteomics of (Mtb)- and BCG (BCG)-infected macrophages could be instrumental in understanding the characteristic post-translational modifications of host proteins and their subsequent involvement in determining Mtb pathogenesis. To identify proteins acquiring a distinct phosphorylation status, herein, we compared the phosphorylation profile of macrophages upon exposure to Mtb and BCG. We observed a significant dephosphorylation of proteins following Mtb infection relative to those with uninfected or BCG-infected cells. A comprehensive tandem mass tag mass spectrometry (MS) approach detected ∼10% phosphosites on a variety of host proteins that are modulated in response to infection. Interestingly, the innate immune-enhancing interferon (IFN)-stimulated genes were identified as a class of proteins differentially phosphorylated during infection, including the cytosolic RNA sensor RIG-I, which has been implicated in the immune response to bacterial infection. We show that Mtb infection results in the activation of RIG-I in primary human macrophages. Studies using RIG-I knockout macrophages reveal that the Mtb-mediated activation of RIG-I promotes IFN-β, IL-1α, and IL-1β levels, dampens autophagy, and facilitates intracellular Mtb survival. To our knowledge, this is the first study providing exhaustive information on relative and quantitative changes in the global phosphoproteome profile of host macrophages that can be further explored in designing novel anti-TB drug targets. The peptide identification and MS/MS spectra have been deposited to the ProteomeXchange Consortium the PRIDE partner repository with the dataset identifier PXD013171.
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http://dx.doi.org/10.1021/acs.jproteome.9b00895DOI Listing
June 2020

Acyldepsipeptide Antibiotics and a Bioactive Fragment Thereof Differentially Perturb Mycobacterium tuberculosis ClpXP1P2 Activity in vitro.

ACS Chem Biol 2020 Feb 21. Epub 2020 Feb 21.

Proteolytic complexes in Mycobacterium tuberculosis (Mtb), the deadliest bacterial pathogen, are major foci in tuberculosis drug development programs. The Clp proteases, which are essential for Mtb viability, are high priority targets. These proteases function through the collaboration of ClpP1P2, a barrel-shaped heteromeric peptidase, with associated ATP-dependent chaperones like ClpX and ClpC1 that recognize and unfold specific substrates in an ATP-dependent fashion. The critical interaction of the peptidase and its unfoldase partners is blocked by the competitive binding of acyldepsipeptide antibiotics (ADEPs) to the interfaces of the ClpP2 subunits. The resulting inhibition of Clp protease activity is lethal to Mtb. Here, we report the surprising discovery that a fragment of the ADEPs retains anti-Mtb activity, yet stimulates rather than inhibits the ClpXP1P2-catalyzed degradation of proteins. Our data further suggest that the fragment stabilizes the ClpXP1P2 complex and binds ClpP1P2 in a fashion distinct from the intact ADEPs. A structure-activity relationship study of the bioactive fragment defines the pharmacophore and points the way towards the development of new drug leads for the treatment of tuberculosis.
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http://dx.doi.org/10.1021/acschembio.9b00454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842861PMC
February 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

: Diphtheria Toxin, the Operon, and Its Regulation by Fe2 Activation of apo-DtxR.

Microbiol Spectr 2019 07;7(4)

Department of Medicine, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD 21231.

Diphtheria is one of the most well studied of all the bacterial infectious diseases. These milestone studies of toxigenic along with its primary virulence determinant, diphtheria toxin, have established the paradigm for the study of other related bacterial protein toxins. This review highlights those studies that have contributed to our current understanding of the structure-function relationships of diphtheria toxin, the molecular mechanism of its entry into the eukaryotic cell cytosol, the regulation of diphtheria expression by holo-DtxR, and the molecular basis of transition metal ion activation of apo-DtxR itself. These seminal studies have laid the foundation for the protein engineering of diphtheria toxin and the development of highly potent eukaryotic cell-surface receptor-targeted fusion protein toxins for the treatment of human diseases that range from T cell malignancies to steroid-resistant graft-versus-host disease to metastatic melanoma. This deeper scientific understanding of diphtheria toxin and the regulation of its expression have metamorphosed the third-most-potent bacterial toxin known into a life-saving targeted protein therapeutic, thereby at least partially fulfilling Paul Erlich's concept of a magic bullet-"a chemical that binds to and specifically kills microbes or tumor cells."
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http://dx.doi.org/10.1128/microbiolspec.GPP3-0063-2019DOI Listing
July 2019

TRAV1-2 CD8 T-cells including oligoconal expansions of MAIT cells are enriched in the airways in human tuberculosis.

Commun Biol 2019 5;2:203. Epub 2019 Jun 5.

5Department of Pulmonary & Critical Care Medicine, Oregon Health & Science University, Portland, OR USA.

Mucosal-associated invariant T (MAIT) cells typically express a TRAV1-2 semi-invariant TCRα that enables recognition of bacterial, mycobacterial, and fungal riboflavin metabolites presented by MR1. MAIT cells are associated with immune control of bacterial and mycobacterial infections in murine models. Here, we report that a population of pro-inflammatory TRAV1-2 CD8 T cells are present in the airways and lungs of healthy individuals and are enriched in bronchoalveolar fluid of patients with active pulmonary tuberculosis (TB). High-throughput T cell receptor analysis reveals oligoclonal expansions of canonical and donor-unique TRAV1-2 MAIT-consistent TCRα sequences within this population. Some of these cells demonstrate MR1-restricted mycobacterial reactivity and phenotypes suggestive of MAIT cell identity. These findings demonstrate enrichment of TRAV1-2 CD8 T cells with MAIT or MAIT-like features in the airways during active TB and suggest a role for these cells in the human pulmonary immune response to .
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http://dx.doi.org/10.1038/s42003-019-0442-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6549148PMC
May 2020

Potential for breath test diagnosis of urease positive pathogens in lung infections.

J Breath Res 2019 06 4;13(3):032002. Epub 2019 Jun 4.

Department of Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America.

Roles for urease in virulence are accepted for Helicobacter pylori and urinary tract pathogens. However, urease is widely expressed by bacterial and fungal lung pathogens causing emerging and opportunistic lung infections, organisms causing acute exacerbations of chronic bronchitis, mycobacterial lung diseases such as tuberculosis (TB), and ventilator associated pneumonia and health care associated pneumonia. Detection of urease provides a method for rapid in vivo detection of these lung pathogens by inhaled C- breath test, and this review discusses the range of lung pathogens that might be amenable to rapid diagnosis.
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http://dx.doi.org/10.1088/1752-7163/ab2225DOI Listing
June 2019

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

Second-generation IL-2 receptor-targeted diphtheria fusion toxin exhibits antitumor activity and synergy with anti-PD-1 in melanoma.

Proc Natl Acad Sci U S A 2019 02 4;116(8):3100-3105. Epub 2019 Feb 4.

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21231;

Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3.7-fold less lethal to mice by LD analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin using that secretes fully folded, biologically active, monomeric s-DAB-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-IL-2(V6A) could provide a superior activity window. In a sequential dual-therapy study in tumors that have progressed for 10 days, both s-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti-programmed cell death-1 (anti-PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria-toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.
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http://dx.doi.org/10.1073/pnas.1815087116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386727PMC
February 2019

Differential HLA allele frequency in Mycobacterium africanum vs Mycobacterium tuberculosis in Mali.

HLA 2019 01 2;93(1):24-31. Epub 2019 Jan 2.

University of Sciences, Techniques and Technologies of Bamako (USTTB), Bamako, Mali.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis complex (MTBC), however, the distribution and frequency of MTBC lineages and sublineages vary in different parts of the globe. Mycobacterium africanum, a member of MTBC is responsible for a large percentage of TB cases in West Africa, however, it is rarely identified outside of this part of the World. Whether or not differential HLA polymorphism (an important host factor) is contributing to the geographic restriction of M. africanum to West Africa is unknown. Here, we conducted a cohort study in Mali of newly diagnosed individuals with active pulmonary TB and normal healthy controls. The MTBC isolates were spoligotyped to determine the TB study groups (M. tuberculosis sensu stricto LAM10 and M. africanum), and HLA typing was performed on peripheral blood. Unlike previous reports on other populations, we found that HLA class-I alleles were significantly associated with active TB disease in this population. HLA-B alleles (B*07:02, B*08:01, B*14:02, B*15:03, B*15:10, B*18:01, B*42:01, B*42:02, B*51:01 and B*81:01) were significantly associated with M. africanum (40%-45%) and M. tuberculosis (75%) compared with healthy controls. Many HLA-A alleles (A*02:05, A*34:02, A*66:01 and A*68:02) were also associated with both TB groups (65%-70%). However, many class II HLA-DR variants were found to be associated with M. tuberculosis but not M. africanum with the exception of the DRB1*03:01, which was associated with both groups. The differential HLA distribution observed in this study might be at least partially responsible for the geographical restriction of M. africanum infections to West Africa.
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http://dx.doi.org/10.1111/tan.13448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333481PMC
January 2019

Lysosomal Cathepsin Release Is Required for NLRP3-Inflammasome Activation by in Infected Macrophages.

Front Immunol 2018 21;9:1427. Epub 2018 Jun 21.

Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States.

Lysosomal cathepsin B (CTSB) has been proposed to play a role in the induction of acute inflammation. We hypothesised that the presence of active CTSB in the cytosol is crucial for NLRP3-inflammasome assembly and, consequently, for mature IL-1β generation after mycobacterial infection . Elevated levels of CTSB was observed in the lungs of mice and rabbits following infection with (Mtb) H37Rv as well as in plasma from acute tuberculosis patients. H37Rv-infected murine bone marrow-derived macrophages (BMDMs) displayed both lysosomal leakage, with release of CTSB into the cytosol, as well as increased levels of mature IL-1β. These responses were diminished in BMDM infected with a mutant H37Rv deficient in ESAT-6 expression. Pharmacological inhibition of cathepsin activity with CA074-Me resulted in a substantial reduction of both mature IL-1β production and caspase-1 activation in infected macrophages. Moreover, cathepsin inhibition abolished the interaction between NLRP3 and ASC, measured by immunofluorescence imaging in H37Rv-infected macrophages, demonstrating a critical role of the enzyme in NLRP3-inflammasome activation. These observations suggest that during Mtb infection, lysosomal release of activated CTSB and possibly other cathepsins inhibitable by CA07-Me is critical for the induction of inflammasome-mediated IL-1β processing by regulating NLRP3-inflammasome assembly in the cytosol.
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http://dx.doi.org/10.3389/fimmu.2018.01427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021483PMC
June 2018

Matrix Metalloproteinase Inhibition in a Murine Model of Cavitary Tuberculosis Paradoxically Worsens Pathology.

J Infect Dis 2019 01;219(4):633-636

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

Matrix metalloproteinases (MMPs) degrade extracellular matrix and are implicated in tuberculosis pathogenesis and cavitation. In particular, MMP-7 is induced by hypoxia and highly expressed around pulmonary cavities of Mycobacterium tuberculosis-infected C3HeB/FeJ mice. In this study, we evaluated whether administration of cipemastat, an orally available potent inhibitor of MMP-7, could reduce pulmonary cavitation in M. tuberculosis-infected C3HeB/FeJ mice. We demonstrate that, compared with untreated controls, cipemastat treatment paradoxically increases the frequency of cavitation (32% vs 7%; P = .029), immunopathology, and mortality. Further studies are needed to understand the role of MMP inhibitors as adjunctive treatments for pulmonary tuberculosis.
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http://dx.doi.org/10.1093/infdis/jiy373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601699PMC
January 2019

Diverse Cavity Types and Evidence that Mechanical Action on the Necrotic Granuloma Drives Tuberculous Cavitation.

Am J Pathol 2018 07 23;188(7):1666-1675. Epub 2018 May 23.

Center for Tuberculosis Research, Department of Medicine, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland. Electronic address:

Effacement of normal lung parenchyma by cavities is an important sequela of pulmonary tuberculosis. Despite its clinical significance, the pathogenesis of tuberculous cavitation is poorly understood, with controversy as to whether the fundamental mechanism involves matrix depletion, lipid pneumonia, or mechanical factors. In this study, a repetitive aerosol infection model using Mycobacterium tuberculosis was used to generate cavities in 20 New Zealand white rabbits. Serial computed tomography was performed to monitor cavity progression over 14 weeks. Three-dimensional reconstructions were compiled for each time point, allowing comprehensive four-dimensional cavity mapping. Terminally, cavities were processed for histopathology. Cavities progressed rapidly from areas of consolidation, and often showed a pattern of explosive growth followed by gradual contraction. Cavities formed preferentially in the caudodorsal lung fields, and frequently were subpleural. Cavitation was associated invariably with necrosis. Histomorphology showed four distinct cavity types that provide mechanistic clues and insight on early cavity development. Our study shows that cavitation is a highly dynamic process with preferential formation at sites of high mechanical stress. These findings suggest a model for the pathogenesis of tuberculous cavitation in which mechanical stress acts on the necrotic granuloma to produce acute tears in structurally weakened tissue, with subsequent air trapping and cavity expansion.
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http://dx.doi.org/10.1016/j.ajpath.2018.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109696PMC
July 2018

Organoid culture of bladder cancer cells.

Investig Clin Urol 2018 05 30;59(3):149-151. Epub 2018 Apr 30.

The James Buchanan Brady Urological Institute and Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA.

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http://dx.doi.org/10.4111/icu.2018.59.3.149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934275PMC
May 2018

Pharmacokinetics of rifapentine and rifampin in a rabbit model of tuberculosis and correlation with clinical trial data.

Sci Transl Med 2018 04;10(435)

Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

In clinical trials of two rifamycin antibiotics (rifampin and rifapentine) for treating tuberculosis (TB), patients with cavitary lung lesions did not appear to derive benefit from rifapentine. Rifapentine was found not to outperform rifampin, despite a lower minimum inhibitory concentration against in mouse models of TB. To understand these findings, we have developed a rabbit model of TB that reliably develops lung cavities with features similar to those of patients with pulmonary cavitary TB. After single or multiple doses of rifampin or rifapentine that produced human-equivalent plasma exposures, rabbits were sacrificed at different time points after dosing. We measured site-of-disease drug pharmacokinetics and tissue drug distribution. We used pharmacokinetic-pharmacodynamic (PK/PD) modeling to estimate drug penetration into different types of tubercular lesions. Both drugs penetrated rabbit lung cellular lesions, as well as the fibrotic cavity wall of cavitary lesions (penetration coefficients ≥1 compared to plasma). For the necrotic liquefied material inside cavitary lesions known as caseum (which contains high numbers of bacteria), the penetration coefficient was 1.0 for rifampin but only 0.25 for rifapentine. When estimates of site-of-disease drug PK were substituted into clinical PK/PD models, the relationship between site-of-action exposure and sputum culture conversion was significant ( < 10). We propose that poor penetration of rifapentine into lung cavitary lesions explains, in part, why rifapentine doses required to improve treatment outcomes in two phase 2 clinical trials were four times higher in TB patients with large cavities compared to TB patients without cavitary lung disease.
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http://dx.doi.org/10.1126/scitranslmed.aai7786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969904PMC
April 2018

Repetitive Aerosol Exposure Promotes Cavitary Tuberculosis and Enables Screening for Targeted Inhibitors of Extensive Lung Destruction.

J Infect Dis 2018 06;218(1):53-63

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

Background: Cavitation is a serious consequence of tuberculosis. We tested the hypothesis that repetitive exposure to the same total bacterial burden of Mycobacterium tuberculosis drives greater lung destruction than a single exposure. We also tested whether inhibition of endogenous matrix metalloproteinase-1 (MMP-1) may inhibit cavitation during tuberculosis.

Methods: Over a 3-week interval, we infected rabbits with either 5 aerosols of 500 colony-forming units (CFU) of M. tuberculosis or a single aerosol of 2500 CFU plus 4 sham aerosols. We administered the MMP-1 inhibitor cipemastat (100 mg/kg daily) during weeks 5-10 to a subset of the animals.

Results: Repetitive aerosol infection produced greater lung inflammation and more cavities than a single aerosol infection of the same bacterial burden (75% of animals vs 25%). Necropsies confirmed greater lung pathology in repetitively exposed animals. For cipemastat-treated animals, there was no significant difference in cavity counts, cavity volume, or disease severity compared to controls.

Conclusions: Our data show that repetitive aerosol exposure with M. tuberculosis drives greater lung damage and cavitation than a single exposure. This suggests that human lung destruction due to tuberculosis may be exacerbated in settings where individuals are repeatedly exposed. MMP-1 inhibition with cipemastat did not prevent the development of cavitation in our model.
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http://dx.doi.org/10.1093/infdis/jiy127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989619PMC
June 2018