Anti-Tuberculosis Potential of OJT008 against Active and Multi-Drug-Resistant Mycobacterium Tuberculosis: In Silico and In Vitro Inhibition of Methionine Aminopeptidase.

Despite the recent progress in the diagnosis of tuberculosis (TB), the chemotherapeutic management of TB continues to be challenging. Mycobacterium tuberculosis (Mtb), the etiological agent of TB, is classified as the 13th leading cause of death globally. In addition, 450,000 people were reported to develop multi-drug-resistant TB globally. The current project focuses on targeting methionine aminopeptidase (MetAP), an essential protein for the viability of Mtb. MetAP is a metalloprotease that catalyzes the excision of the N-terminal methionine (NME) during protein synthesis, allowing the enzyme to be an auspicious target for the development of novel therapeutic agents for the treatment of TB. Mtb possesses two MetAP1 isoforms, MtMetAP1a and MtMetAP1c, which are vital for Mtb viability and, hence, a promising chemotherapeutic target for Mtb therapy. In this study, we cloned and overexpressed recombinant MtMetAP1c. We investigated the in vitro inhibitory effect of the novel MetAP inhibitor, OJT008, on the cobalt ion- and nickel ion-activated MtMetAP1c, and the mechanism of action was elucidated through an in silico approach. The compound's potency against replicating and multi-drug-resistant (MDR) Mtb strains was also investigated. The induction of the overexpressed recombinant MtMetAP1c was optimized at 8 h with a final concentration of 1 mM Isopropyl ß-D-1-thiogalactopyranoside. The average yield from 1 L of Escherichia coli culture for MtMetAP1c was 4.65 mg. A preliminary MtMetAP1c metal dependency screen showed optimum activation with nickel and cobalt ions occurred at 100 µM. The half-maximal inhibitory concentration (IC50) values of OJT008 against MtMetAP1c activated with CoCl2 and NiCl2 were 11 µM and 40 µM, respectively. The in silico study showed OJT008 strongly binds to both metal-activated MtMetAP1c, as evidenced by strong molecular interactions and a higher binding score, thereby corroborating our result. This in silico study validated the pharmacophore's metal specificity. The potency of OJT008 against both active and MDR Mtb was <0.063 µg/mL. Our study reports OJT008 as an inhibitor of MtMetAP1c, which is potent at low micromolar concentrations against both active susceptible and MDR Mtb. These results suggest OJT008 is a potential lead compound for the development of novel small molecules for the therapeutic management of TB.

Thioaptamer targeted discoidal microparticles increase self immunity and reduce Mycobacterium tuberculosis burden in mice.

Worldwide, tuberculosis (TB) remains one of the most prevalent infectious diseases causing morbidity and death in >1.5 million patients annually. Mycobacterium tuberculosis (Mtb), the etiologic agent of TB, usually resides in the alveolar macrophages. Current tuberculosis treatment methods require more than six months, and low compliance often leads to therapeutic failure and multidrug resistant strain development. Critical to improving TB-therapy is shortening treatment duration and increasing therapeutic efficacy. In this study, we sought to determine if lung hemodynamics and pathological changes in Mtb infected cells can be used for the selective targeting of microparticles to infected tissue(s). Thioaptamers (TA) with CD44 (CD44TA) targeting moiety were conjugated to discoidal silicon mesoporous microparticles (SMP) to enhance accumulation of these agents/carriers in the infected macrophages in the lungs. In vitro, CD44TA-SMP accumulated in macrophages infected with mycobacteria efficiently killing the infected cells and decreasing survival of mycobacteria. In vivo, increased accumulations of CD44TA-SMP were recorded in the lung of M. tuberculosis infected mice as compared to controls. TA-targeted carriers significantly diminished bacterial load in the lungs and caused recruitment of T lymphocytes. Proposed mechanism of action of the designed vector accounts for a combination of increased uptake of particles that leads to infected macrophage death, as well as, activation of cellular immunity by the TA, causing increased T-cell accumulation in the treated lungs. Based on our data with CD44TA-SMP, we anticipate that this drug carrier can open new avenues in TB management.

Characterization of 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone as a novel inhibitor of methionine aminopeptidases from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) and the Human Immunodeficiency Virus (HIV) pose a major public health threat. The 2015 World Health Organization (WHO) report estimates that one in three HIV deaths is due to Mtb, the causative agent of Tuberculosis (TB). The lethal synergy between these two pathogens leads to a decline in the immune function of infected individuals as well as a rise in morbidity and mortality rates. The deadly interaction between TB and HIV, along with the heightened emergence of drug resistance, drug-drug interactions, reduced drug efficacy and increased drug toxicity, has made the therapeutic management of co-infected individuals a major challenge. Hence, the development of new drug targets and/or new drug leads are imperative for the effective therapeutic management of co-infected patients. Here, we report the characterization of 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (311), a known inhibitor of HIV-1 replication and transcription as a new inhibitor of methionine aminopeptidases (MetAPs) from Mycobacterium tuberculosis: MtMetAP1a and MtMetAP1c. MetAP is a metalloprotease that removes the N-terminal methionine during protein synthesis. The essential role of MetAP in microbes makes it a promising chemotherapeutic target. We demonstrated that 311 is a potent and selective inhibitor of MtMetAP1a and MtMetAP1c. Furthermore, we found that 311 is active against replicating and aged non-growing Mtb at low micromolar concentrations. These results suggest that 311 is a promising lead for the development of novel class of therapeutic agents with dual inhibition of TB and HIV for the treatment of TB-HIV co-infection.

Differentiating between live and dead Mycobacterium smegmatis using autofluorescence.

While there have been research efforts to find faster and more efficient diagnostic techniques for tuberculosis (TB), it is equally important to monitor a patient's response to treatment over time, especially with the increasing prevalence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) TB. Between sputum smear microscopy, culture, and GeneXpert, only culture can verify viability of mycobacteria. However, it may take up to six weeks to grow Mycobacterium tuberculosis (Mtb), during which time the patient may have responded to treatment or the mycobacteria are still viable because the patient has MDR or XDR TB. In both situations, treatment incurs increased patient costs and makes them more susceptible to host-drug effects such as liver damage. Coenzyme Factor 420 (F420) is a fluorescent coenzyme found naturally in mycobacteria, with an excitation peak around 420 nm and an emission peak around 470 nm. Using Mycobacterium smegmatis, we show that live and dead mycobacteria undergo different rates of photobleaching over a period of 2 min. These preliminary experiments suggest that the different photobleaching rates could be used to help monitor a patient's response to TB treatment. In future studies, we propose to describe these experiments with Mtb as both M. smegmatis and Mtb use F420.

Molecular epidemiology of Mycobacterium tuberculosis among South African gold miners.

RATIONALE: HIV-associated tuberculosis remains a major health problem among the gold-mining workforce in South Africa. We postulate that high levels of recent transmission, indicated by strain clustering, are fueling the tuberculosis epidemic among gold miners. OBJECTIVES: To combine molecular and epidemiologic data to describe Mycobacterium tuberculosis genetic diversity, estimate levels of transmission, and examine risk factors for clustering. METHODS: We conducted a cross-sectional study of culture-positive M. tuberculosis isolates in 15 gold mine shafts across three provinces in South Africa. All isolates were subject IS6110-based restriction fragment length polymorphisms, and we performed spoligotyping analysis and combined it with basic demographic and clinical information. MEASUREMENTS AND MAIN RESULTS: Of the 1,602 M. tuberculosis patient isolates, 1,240 (78%) had genotyping data available for analysis. A highly diverse bacillary population was identified, comprising a total of 730 discrete genotypes. Four genotypic families (Latin American Mediterranean spoligotype family; W-Beijing; AH or X; and T1-T4) accounted for over 50% of all strains. Overall, 45% (560/1,240) of strains were genotypically clustered. The minimum estimate for recent transmission (n - 1 method) was 32% (range, 27-34%). There were no individual-level risk factors for clustering, apart from borderline evidence for being non-South African and having self-reported HIV infection. CONCLUSIONS: The high M. tuberculosis genetic diversity and lack of risk factors for clustering are indicative of a universal risk for disease among gold miners and likely mixing with nonmining populations. Our results underscore the urgent need to intensify interventions to interrupt transmission across the entire gold-mining workforce in South Africa.

Tuberculosis screening by tuberculosis skin test or QuantiFERON-TB Gold In-Tube Assay among an immigrant population with a high prevalence of tuberculosis and BCG vaccination.

RATIONALE: Each year 1 million persons acquire permanent U.S. residency visas after tuberculosis (TB) screening. Most applicants undergo a 2-stage screening with tuberculin skin test (TST) followed by CXR only if TST-positive at > 5 mm. Due to cross reaction with bacillus Calmette-Guérin (BCG), TST may yield false positive results in BCG-vaccinated persons. Interferon gamma release assays exclude antigens found in BCG. In Vietnam, like most high TB-prevalence countries, there is universal BCG vaccination at birth. OBJECTIVES: 1. Compare the sensitivity of QuantiFERON-TB Gold In-Tube Assay (QFT) and TST for culture-positive pulmonary TB. 2. Compare the age-specific and overall prevalence of positive TST and QFT among applicants with normal and abnormal CXR. METHODS: We obtained TST and QFT results on 996 applicants with abnormal CXR, of whom 132 had TB, and 479 with normal CXR. RESULTS: The sensitivity for tuberculosis was 86.4% for QFT; 89.4%, 81.1%, and 52.3% for TST at 5, 10, and 15 mm. The estimated prevalence of positive results at age 15-19 years was 22% and 42% for QFT and TST at 10 mm, respectively. The prevalence increased thereafter by 0.7% year of age for TST and 2.1% for QFT, the latter being more consistent with the increase in TB among applicants. CONCLUSIONS: During 2-stage screening, QFT is as sensitive as TST in detecting TB with fewer requiring CXR and being diagnosed with LTBI. These data support the use of QFT over TST in this population.

Evaluation of large genotypic Mycobacterium tuberculosis clusters: contributions from remote and recent transmission.

Tuberculosis genotypic clustering is used as a proxy for recent transmission. The association between clustering and recent transmission becomes problematic when the genotyping method lacks specificity in defining a cluster, as well as for clusters with extensive jurisdictional histories and/or common genotypes. We investigated the four largest spoligotype/12 loci MIRU-VNTR-defined clusters in Harris County, Texas from 2006-2012 to determine their historical contribution to tuberculosis morbidity, estimate the contributions from recent and remote transmission, and determine the impact of secondary genotyping on cluster definition. The clusters contained 189, 64, 51 and 38 cases. Each cluster was linked to cluster(s) previously identified by Houston Tuberculosis Initiative; 3 since 1995 and the fourth in 2002. Among cases for which timing of Mycobacterium tuberculosis transmission relative to tuberculosis disease could be ascertained, nearly equal proportions were associated with recent and remote transmission. The extent to which genotyping with an additional 12 MIRU-VNTR loci modified the cluster definition varied from little or no impact for the two smaller clusters to moderate impact for the larger clusters. Tuberculosis control measures to reduce morbidity associated with large clusters must involve strategies to identify and treat individuals who recently acquired infection, as well as persons infected for years.

Test variability of the QuantiFERON-TB gold in-tube assay in clinical practice.

RATIONALE: Although IFN-γ release assays (IGRAs) are widely used to screen for Mycobacterium tuberculosis infection in high-income countries, published data on repeatability are limited. OBJECTIVES: To determine IGRA repeatability. METHODS: The study population included consecutive patients referred to The Methodist Hospital (Houston, TX) between August 1, 2010 and July 31, 2011 for latent tuberculosis (TB) infection screening with an IGRA (QuantiFERON-TB Gold In-Tube; Cellestis, Carnegie, Australia). We performed multiple IGRA tests using leftover stimulated plasma according to a prospectively formulated quality control protocol. We analyzed agreement in interpretation of test results classified according to manufacturer-recommended criteria and repeatability of quantitative TB response. MEASUREMENTS AND MAIN RESULTS: During the study period, 1,086 test results were obtained from 543 subjects. Per the manufacturer's cut-point, the result of the second test was discordant from that of the first in 28 (8%) of 366 patients with valid test results, including 13 with an initial negative result and 15 with an initial positive result. Although agreement between repeat test results was high (κ = 0.84; 95% confidence interval, 0.79-0.90), the normal expected range of within-subject variability in TB response on retesting included differences of ± 0.60 IU/ml for all individuals (coefficient of variation, 14%), and ± 0.24 IU/ml (coefficient of variation, 27%) for individuals whose initial TB response was between 0.25 and 0.80 IU/ml. CONCLUSIONS: There is substantial variability in TB response when IGRAs are repeated using the same patient sample. IGRA results should be interpreted cautiously when TB response is near interpretation cut-points.

Polymorphic allele of human IRGM1 is associated with susceptibility to tuberculosis in African Americans.

An ancestral polymorphic allele of the human autophagy-related gene IRGM1 is associated with altered gene expression and a genetic risk for Crohn's Disease (CD). We used the single nucleotide polymorphism rs10065172C/T as a marker of this polymorphic allele and genotyped 370 African American and 177 Caucasian tuberculosis (TB) cases and 180 African American and 110 Caucasian controls. Among African Americans, the TB cases were more likely to carry the CD-related T allele of rs10065172 (odds ratio of 1.54; 95% confidence interval, 1.17-2.02; P<0.01) compared to controls. Our finding suggests that this CD-related IRGM1 polymorphic allele is also associated with human susceptibility to TB disease among African Americans.

Rapid attenuation of circadian clock gene oscillations in the rat heart following ischemia-reperfusion.

The intracellular circadian clock consists of a series of transcriptional modulators that together allow the cell to perceive the time of day. Circadian clocks have been identified within various components of the cardiovascular system (e.g. cardiomyocytes, vascular smooth muscle cells) and possess the potential to regulate numerous aspects of cardiovascular physiology and pathophysiology. The present study tested the hypothesis that ischemia/reperfusion (I/R; 30 min occlusion of the rat left main coronary artery in vivo) alters the circadian clock within the ischemic, versus non-ischemic, region of the heart. Left ventricular anterior (ischemic) and posterior (non-ischemic) regions were isolated from I/R, sham-operated, and naïve rats over a 24-h period, after which mRNAs encoding for both circadian clock components and known clock-controlled genes were quantified. Circadian clock gene oscillations (i.e. peak-to-trough fold differences) were rapidly attenuated in the I/R, versus the non-ischemic, region. Consistent with decreased circadian clock output, we observe a rapid induction of E4BP4 in the ischemic region of the heart at both the mRNA and protein levels. In contrast with I/R, chronic (1 week) hypobaric chamber-induced hypoxia did not attenuate oscillations in circadian clock genes in either the left or right ventricle of the rat heart. In conclusion, these data show that in a rodent model of myocardial I/R, circadian clocks within the ischemic region become rapidly impaired, through a mechanism that appears to be independent of hypoxia.

Circadian rhythms in myocardial metabolism and contractile function: influence of workload and oleate.

Multiple extracardiac stimuli, such as workload and circulating nutrients (e.g., fatty acids), known to influence myocardial metabolism and contractile function exhibit marked circadian rhythms. The aim of the present study was to investigate whether the rat heart exhibits circadian rhythms in its responsiveness to changes in workload and/or fatty acid (oleate) availability. Thus, hearts were isolated from male Wistar rats (housed during a 12:12-h light-dark cycle: lights on at 9 AM) at 9 AM, 3 PM, 9 PM, and 3 AM and perfused in the working mode ex vivo with 5 mM glucose plus either 0.4 or 0.8 mM oleate. Following 20-min perfusion at normal workload (i.e., 100 cm H(2)O afterload), hearts were challenged with increased workload (140 cm H(2)O afterload plus 1 microM epinephrine). In the presence of 0.4 mM oleate, myocardial metabolism exhibited a marked circadian rhythm, with decreased rates of glucose oxidation, increased rates of lactate release, decreased glycogenolysis capacity, and increased channeling of oleate into nonoxidative pathways during the light phase. Rat hearts also exhibited a modest circadian rhythm in responsiveness to the workload challenge when perfused in the presence of 0.4 mM oleate, with increased myocardial oxygen consumption at the dark-to-light phase transition. However, rat hearts perfused in the presence of 0.8 mM oleate exhibited a markedly blunted contractile function response to the workload challenge during the light phase. In conclusion, these studies expose marked circadian rhythmicities in myocardial oxidative and nonoxidative metabolism as well as responsiveness of the rat heart to changes in workload and fatty acid availability.