Development Of anti-TB Drugs Research Articles

Nuclear Magnetic Resonance Fingerprinting and Principal Component Analysis Strategies Lead to Anti-Tuberculosis Natural Product Discovery from Actinomycetes.

The increasing prevalence of drug-resistant tuberculosis (TB) underscores the urgent need for novel antimicrobial agents. This study integrates cultivation optimization, nuclear magnetic resonance (NMR) fingerprinting, and principal component analysis (PCA) to explore microbial secondary metabolites as potential anti-TB agents. Using the combined approach, 11 bioactive compounds were isolated and identified, all exhibiting anti-Mycobacterium bovis BCG activity. Notable findings include borrelidin, a potent threonyl-tRNA synthetase inhibitor with broad biological activities, and L-O-Lac-L-Val-D-O-Hiv-D-Val, a peptide isolated for the first time from a plant endophyte, demonstrating broad-spectrum antimicrobial activity. Additionally, elaiophylin and polycyclic tetramate macrolactams (PTMs) displayed significant bactericidal effects, with elaiophylin achieving complete BCG inhibition at 72 h and PTMs marking their first reported anti-TB activity. The study also identified bafilomycins as potent scaffolds for anti-TB drug development, showcasing rapid bactericidal activity at low MIC values. These findings emphasize the value of microbial metabolites as a reservoir of bioactive compounds and provide new avenues for developing next-generation anti-TB therapies.

Toward Mycobacterium tuberculosis Virulence Inhibition: Beyond Cell Wall.

Mycobacterium tuberculosis (Mtb) is one of the most successful bacterial pathogens in human history. Even in the antibiotic era, Mtb is widespread and causes millions of new cases of tuberculosis each year. The ability to disrupt the host's innate and adaptive immunity, as well as natural persistence, complicates disease control. Tuberculosis traditional therapy involves the long-term use of several antibiotics. Treatment failures are often associated with the development of resistance to one or more drugs. The development of medicines that act on new targets will expand treatment options for tuberculosis caused by multidrug-resistant or extensively drug-resistant Mtb. Therefore, the development of drugs that target virulence factors is an attractive strategy. Such medicines do not have a direct bacteriostatic or bactericidal effect, but can disarm the pathogen so that the host immune system becomes able to eliminate it. Although cell wall-associated targets are being actively studied for anti-TB drug development, other virulence factors important for adaptation and host interaction are also worth comprehensive analysis. In this review, specific Mtb virulence factors (such as secreted phosphatases, regulatory systems, and the ESX-1 secretion system) are identified as promising targets for novel anti-virulence drug development. Additionally, models for the search of virulence inhibitors are discussed, such as virtual screening in silico, in vitro enzyme inhibition assay, the use of recombinant Mtb strains with reporter constructs, phenotypic analysis using in vitro cell infection models and specific environments.

An exacerbated phosphate starvation response triggers Mycobacterium tuberculosis glycerol utilization at acidic pH.

The mechanisms controlling Mycobacterium tuberculosis (Mtb) replication and survival inside its human host remain ill-defined. Phagosome acidification and nutrient deprivation are common mechanisms used by macrophages to restrict the replication of intracellular bacteria. Mtb stops replicating at mildly acidic pH (<pH5.8), an adaptive process called "acid growth arrest," which is thought to play an important role in Mtb virulence. Using a genome-wide mutagenesis screen, we identified 95 genes whose disruption either decreases or increases Mtb fitness during acid growth arrest. We show that the virulence-associated inorganic phosphate (Pi) uptake system (Pst-1) regulates the ability of Mtb to replicate in acidic conditions. Deletion of pstA1, a gene coding for a subunit of the Pst-1 system, results in the overexpression of the Pi starvation regulator regX3, which is sufficient to restore Mtb growth in acid conditions. Our data further support the role of limited glycerol uptake and ROS-mediated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition in causing Mtb acid growth arrest. This study reveals an unexpected role of the Pi starvation response in regulating acid growth arrest and highlights the intricacy of the mechanisms regulating redox homeostasis, acid stress response, and nutrient utilization in Mtb.IMPORTANCEDespite the availability of antibiotic treatment, M. tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains a major infectious disease killer worldwide. A better understanding of the environments that Mtb faces during infection and the mechanisms Mtb employs to respond and adapt may help identify currently unexplored pathways and targets for the development of novel anti-TB drugs. Here, we demonstrate that Mtb growth in acid can be restored by the over-expression of the Pi starvation response regulator regX3. This work paves the way toward a better understanding of the mechanisms controlling Mtb growth at acidic pH and highlights the role of inorganic phosphate in this process.

The progress of Mycobacterium tuberculosis drug targets.

Tuberculosis (TB) has been troubling humans for hundreds of years, is a highly infectious disease caused by Mycobacterium tuberculosis (Mtb) infection, Mtb can infect almost all organs of the body and is one of the deadly infectious diseases in the world. At present, the first-line treatment regimen has a long treatment cycle and is prone to multiple drug resistance. Anti-tuberculosis drugs and latent tuberculosis infection (LTBI) resistance are increasing year by year, and new targets and new bioactive compounds are urgently needed to treat this disease. This review focuses on the latest reported anti-TB drug targets and related compounds in recent years, reviews the current TB drug regimen and major defects, outlines the key drug targets developed to date in Mtb, and the current situation of newly discovered anti-TB resistant forms of drugs. To provide a reference for the research and development of new anti-TB drugs and bring new treatment strategies for TB patients.

Mycobacterial Targets for Thiourea Derivatives: Opportunities for Virtual Screening in Tuberculosis Drug Discovery.

Tuberculosis (TB) remains a primary global health concern, necessitating the discovery and development of new anti-TB drugs, mainly to combat drug-resistant strains. In this context, thiourea derivatives have emerged as promising candidates in TB drug discovery due to their diverse chemical structures and pharmacological properties. This review aimed to explore this potential, identifying and exploring molecular targets for thiourea derivatives in Mycobacterium tuberculosis (Mtb) and the potential application of virtual screening techniques in drug discovery. We have compiled a comprehensive list of possible molecular targets of thiourea derivatives in Mtb. The enzymes are primarily involved in the biosynthesis of various cell wall components, including mycolic acids, peptidoglycans, and arabinans, or targets in the branched-chain amino acid biosynthesis (BCAA) pathway and detoxification mechanisms. We discuss the potential of these targets as critical constituents for the design of novel anti-TB drugs. Besides, we highlight the opportunities that virtual screening methodologies present in identifying potential thiourea derivatives that can interact with these molecular targets. The presented findings contribute to the ongoing efforts in TB drug discovery and lay the foundation for further research in designing and developing more effective treatments against this devastating disease.

Identification of Novel Antimicrobial Compounds Targeting Mycobacterium tuberculosis S-Adenosyl-L-Homocysteine Hydrolase Using Dual Hierarchical In Silico Structure-Based Drug Screening.

The emergence of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis (M. tuberculosis) has become a major medical problem. S-adenosyl-L-homocysteine hydrolase (MtSAHH) was selected as the target protein for the identification of novel anti-TB drugs. Dual hierarchical in silico Structure-Based Drug Screening was performed using a 3D compound structure library (with over 150 thousand synthetic chemicals) to identify compounds that bind to MtSAHH's active site. In vitro experiments were conducted to verify whether the nine compounds selected as new drug candidates exhibited growth-inhibitory effects against mycobacteria. Eight of the nine compounds that were predicted by dual hierarchical screening showed growth-inhibitory effects against Mycobacterium smegmatis (M. smegmatis), a model organism for M. tuberculosis. Compound 7 showed the strongest antibacterial activity, with an IC50 value of 30.2 µM. Compound 7 did not inhibit the growth of Gram-negative bacteria or exert toxic effects on human cells. Molecular dynamics simulations of 40 ns using the MtSAHH-Compound 7 complex structure suggested that Compound 7 interacts stably with the MtSAHH active site. These in silico and in vitro results suggested that Compound 7 is a promising lead compound for the development of new anti-TB drugs.

Evaluation of the multiple doses thiozonide’s pharmacokinetics in patients diagnosed with pulmonary tuberculosis with multidrug-resistant or extensively drug-resistant &lt;i&gt;Mycobacterium tuberculosis&lt;/i&gt;

Relevance. The increase in the number of cases of multidrug-resistant tuberculosis (MDR-TB) determines the relevance of the development of new anti-TB drugs (ATDs). Determining the pharmacokinetics of a drug in the context of MDR-TB therapy is an integral part of the study of the original drug. Objective. Evaluation of the pharmacokinetic properties of a new original drug, thiozonide, in patients with MDR-TB with multiple doses of the drug. Methods. Thiozonide was used in patients with a verified diagnosis of MDR-TB as part of a clinical trial according to protocol No. THIO22 "Multicenter 12-week doubleblind, randomized, placebo-controlled clinical trial on the selection of optimal dosages of the drug thiozonide, capsules (CJSC Pharm-Sintez") for against the background of standard anti-tuberculosis chemotherapy in patients diagnosed with pulmonary tuberculosis with multidrug-resistant or extensively drugresistant Mycobacterium tuberculosis” (permission of the Ministry of Health of the Russian Federation No. 661 dated 11/24/2014). Results. The pharmacokinetic characteristics of thiozonide were determined during its repeated use in patients diagnosed with pulmonary tuberculosis with multidrug-resistant or extensively drug-resistant mycobacterium tuberculosis, receiving standard anti-tuberculosis chemotherapy, by HPLC with mass spectrometric detection. The time to reach the maximum concentration T max of thiozonide with repeated administration at a dose of 200 mg, 400 mg and 600 mg was 4.21 ± 1.23 hours, 4.9 ± 1.08 and 5.29 ± 0.91, respectively; the half-life T1/2 for the same dosages of the drug was 7.84 ± 1.86, 7.56 ± 1.92 and 6.3 ± 2.12 hours, respectively. The maximum concentration of thiozonide after taking the drug thiozoinide by volunteers was Cmax was observed at the level of 1386.89 ± 533.68 ng / ml in the group taking 200 mg of thiozonide per day, 2684.48 ± 712.40 when taking the drug at a dosage of 400 mg and 5558, 99 ± 2143.81 – at a dosage of 600 mg. Conclusion. A linear dependence of the maximum concentration and the area under the pharmacokinetic curve on the dose taken and also the average concentration of thiozonide in the blood plasma of patients with pulmonary tuberculosis with multidrug resistance of the causative agent of the disease was revealed.

Recent advances in drug therapy for tuberculosis

Despite global efforts to control and eradicate tuberculosis (TB), it remains a significant source of morbidity and mortality worldwide. Drug-resistant TB (DRTB) is a significant public health threat with implications for global health security, economic burden, and access to quality care. To address this challenge, continued investment in research, surveillance, and innovative strategies is crucial. Collaboration among international organizations, governments, and health-care systems is crucial to tackle this global health concern effectively. Bedaquiline (BDQ), a diarylquinoline drug, is indicated for combination therapy in adult patients with pulmonary multidrug-resistant TB (MDRTB). The Food and Drug Administration (FDA) granted accelerated approval in December 2012, based on analysis of time to sputum culture conversion from two phase 2 trials. The World Health Organization (WHO) published interim policy guidance for BDQ in conjunction with WHO-recommended MDRTB treatments in June 2013. The European Medicines Agency authorized Delamanid as a first-in-class bicyclic nitroimidazole for treating MDRTB, which has been used in 89 and 54 nations due to its quick acquisition of resistance. To prevent resistance, combined therapy with additional anti-TB drugs is advised, along with proper use and drug resistance monitoring. Pretomanid received approval from the US FDA in August 2019 after a phase III trial yielded encouraging results. The bedaquiline, pretomanid, linezolid, and moxifloxacin (BPaLM) regimen, a component of the BPaLM regimen, has been approved for treating adults with extensive pulmonary DRTB or treatment-intolerant or non-responsive MDRTB. This significant advancement in management offers improved clinical outcomes and quality of life for MDRTB patients. The inclusion in the WHO guidelines is encouraging for global combat against DRTB. Researchers have identified and investigated new drug targets within the TB pathogenesis, offering promising avenues for the development of innovative anti-TB drugs. Advances in pediatric TB treatment have made significant progress, focusing on improved diagnostic methods, effective drug treatments, and global initiatives. Nanotechnology has shown promising potential in various fields, including medicine and TB treatment.

Mycobacterium tuberculosis as a resilient foe in the mechanisms of colonisation, pathogenesis and host immune responses serves as a prerequisite for the development of potential mangrove plant-derived anti-TB drugs

Mycobacterium tuberculosis as a resilient foe in the mechanisms of colonisation, pathogenesis and host immune responses serves as a prerequisite for the development of potential mangrove plant-derived anti-TB drugs

Microbial glycosylation of antitubercular agent chlorflavonin

Flavonoids have shown health-benefiting properties, such as antioxidative and anti-inflammatory activities, and are commonly used as nutraceuticals and pharmaceuticals. Although flavonoids are predominantly identified from plants, several filamentous fungal species have also been reported to produce bioactive flavonoids, including chlorflavonin from Aspergillus candidus, a novel halogenated flavonoid with potent antifungal and antitubercular (anti-TB) activities. Unfortunately, the low water-solubility of this molecule may hinder its bioavailability. Glycosylation is an effective method to enhance the polarity of natural products and alter their physicochemical properties. This work focuses on the development of novel water-soluble chlorflavonin derivatives to combat the threat of drug-resistant tuberculosis. In this study, we first increased the production titer of chlorflavonin in A.candidus NRRL 5214 by optimizing the fermentation and purification processes. Next, chlorflavonin-5-O-β-d-glucuronopyranoside (1) and chlorflavonin-7-O-4″-O-methyl-β-d-glucopyranoside (2) were produced from chlorflavonin using Streptomyces chromofuscus ATCC 49982 and Beauveria bassiana ATCC 7159, respectively. Compared to chlorflavonin (4.38 ± 0.54mg/L in water), the water solubility of the two new glycosides was determined to be 117.86 ± 4.81mg/L (1) and 124.34 ± 9.13mg/L (2), respectively. This study provides a promising method to create water-soluble glycosides of chlorflavonin for the development of novel anti-TB drugs.

Mycobacterium tuberculosis: Pathogenesis and therapeutic targets.

Tuberculosis (TB) remains a significant public health concern in the 21st century, especially due to drug resistance, coinfection with diseases like immunodeficiency syndrome (AIDS) and coronavirus disease 2019, and the lengthy and costly treatment protocols. In this review, we summarize the pathogenesis of TB infection, therapeutic targets, and corresponding modulators, including first-line medications, current clinical trial drugs and molecules in preclinical assessment. Understanding the mechanisms of Mycobacterium tuberculosis (Mtb) infection and important biological targets can lead to innovative treatments. While most antitubercular agents target pathogen-related processes, host-directed therapy (HDT) modalities addressing immune defense, survival mechanisms, and immunopathology also hold promise. Mtb's adaptation to the human host involves manipulating host cellular mechanisms, and HDT aims to disrupt this manipulation to enhance treatment effectiveness. Our review provides valuable insights for future anti-TB drug development efforts.

Metabolically distinct roles of NAD synthetase and NAD kinase define the essentiality of NAD and NADP in Mycobacterium tuberculosis.

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated derivative (NADP) are essential cofactors that participate in hundreds of biochemical reactions and have emerged as therapeutic targets in cancer, metabolic disorders, neurodegenerative diseases, and infections, including tuberculosis. The biological basis for the essentiality of NAD(P) in most settings, however, remains experimentally unexplained. Here, we report that inactivation of the terminal enzyme of NAD synthesis, NAD synthetase (NadE), elicits markedly different metabolic and microbiologic effects than those of the terminal enzyme of NADP biosynthesis, NAD kinase (PpnK), in Mycobacterium tuberculosis (Mtb). Inactivation of NadE led to parallel reductions of both NAD and NADP pools and Mtb viability, while inactivation of PpnK selectively depleted NADP pools but only arrested growth. Inactivation of each enzyme was accompanied by metabolic changes that were specific for the affected enzyme and associated microbiological phenotype. Bacteriostatic levels of NAD depletion caused a compensatory remodeling of NAD-dependent metabolic pathways in the absence of an impact on NADH/NAD ratios, while bactericidal levels of NAD depletion resulted in a disruption of NADH/NAD ratios and inhibition of oxygen respiration. These findings reveal a previously unrecognized physiologic specificity associated with the essentiality of two evolutionarily ubiquitous cofactors. IMPORTANCE The current course for cure of Mycobacterium tuberculosis (Mtb)-the etiologic agent of tuberculosis (TB)-infections is lengthy and requires multiple antibiotics. The development of shorter, simpler treatment regimens is, therefore, critical to the goal of eradicating TB. NadE, an enzyme required for the synthesis of the ubiquitous cofactor NAD, is essential for survival of Mtb and regarded as a promising drug target. However, the basis of this essentiality was not clear due to its role in the synthesis of both NAD and NADP. Here, we resolve this ambiguity through a combination of gene silencing and metabolomics. We specifically show that NADP deficiency is bacteriostatic, while NAD deficiency is bactericidal due to its role in Mtb's respiratory capacity. These results argue for a prioritization of NAD biosynthesis inhibitors in anti-TB drug development.

Identification of novel antimicrobial compounds targeting Mycobacterium tuberculosis shikimate kinase using in silico hierarchical structure-based drug screening

The development of new anti-TB drugs to prevent the spread of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains is imperative. Mtb shikimate kinase (MtSK) was selected as the target protein to screen for new anti-TB drugs. We performed hierarchical in silico screening using a library of 154,118 compounds to search for novel compounds that could bind to the active site of MtSK. The growth-inhibitory effects of the candidate compounds on Mycobacterium smegmatis were evaluated in vitro. Nine of the 11 candidate compounds exhibited inhibitory effects against mycobacteria in vitro. The inhibitory activity of Compound 2 (IC50 = 1.39 μM) was higher than that of isoniazid, the first-line drug for TB treatment. Moreover, Compound 2 did not exhibit toxicity against mammalian cells and Escherichia coli. Molecular dynamics simulations using the MtSK-Compound 2 complex structure in a timeframe of 100 ns suggested that Compound 2 could stably bind to MtSK. The binding free energy of Compound 2 was estimated to be −37.96 kcal/mol using the MM/PBSA method, demonstrating that Compound 2 can stably bind to MtSK. These in silico and in vitro results indicated that Compound 2 is a promising hit compound for the development of novel anti-TB drugs.

Tuberculosis: Pathogenesis, Current Treatment Regimens and New Drug Targets.

Mycobacterium tuberculosis (M. tb), the causative agent of TB, is a recalcitrant pathogen that is rife around the world, latently infecting approximately a quarter of the worldwide population. The asymptomatic status of the dormant bacteria escalates to the transmissible, active form when the host's immune system becomes debilitated. The current front-line treatment regimen for drug-sensitive (DS) M. tb strains is a 6-month protocol involving four different drugs that requires stringent adherence to avoid relapse and resistance. Poverty, difficulty to access proper treatment, and lack of patient compliance contributed to the emergence of more sinister drug-resistant (DR) strains, which demand a longer duration of treatment with more toxic and more expensive drugs compared to the first-line regimen. Only three new drugs, bedaquiline (BDQ) and the two nitroimidazole derivatives delamanid (DLM) and pretomanid (PMD) were approved in the last decade for treatment of TB-the first anti-TB drugs with novel mode of actions to be introduced to the market in more than 50 years-reflecting the attrition rates in the development and approval of new anti-TB drugs. Herein, we will discuss the M. tb pathogenesis, current treatment protocols and challenges to the TB control efforts. This review also aims to highlight several small molecules that have recently been identified as promising preclinical and clinical anti-TB drug candidates that inhibit new protein targets in M. tb.

Supramolecular organization and dynamics of mannosylated phosphatidylinositol lipids in the mycobacterial plasma membrane

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function.

Discovery of new inhibitors of Mycobacterium tuberculosis EPSP synthase - A computational study

Tuberculosis (TB) is a highly infectious disease caused by the pathogen Mycobacterium tuberculosis (Mtb). EPSP Synthase (MtEPSPS), the enzyme responsible for the sixth step of the shikimate pathway, is a potential target for the development of new drugs for the treatment of TB, as it is essential in mycobacteria but absent in humans. In this work, we performed virtual screening using sets of molecules from two databases and three crystallographic structures of MtEPSPS. The initial hits obtained from molecular docking were filtered based on predicted binding affinity and interactions with binding site residues. Subsequently, molecular dynamics simulations were carried out to analyze the stability of protein-ligand complexes. We have found that MtEPSPS forms stable interactions with several candidates, including already approved pharmaceutical drugs such as Conivaptan and Ribavirin monophosphate. In particular, Conivaptan had the highest estimated binding affinity with the open conformation of the enzyme. The complex formed between MtEPSPS and Ribavirin monophosphate was also energetically stable as shown by RMSD, Rg and FEL analyses, and the ligand was stabilized by hydrogen bonds with important residues of the binding site. The findings reported in this work could serve as the basis of promising scaffolds for the discovery, design, and development of new anti-TB drugs.

An efficient CRISPR interference-based prediction method for synergistic/additive effects of novel combinations of anti-tuberculosis drugs.

Tuberculosis (TB) is treated by chemotherapy with multiple anti-TB drugs for a long period, spanning 6 months even in a standard course. In perspective, to prevent the emergence of antimicrobial resistance, novel drugs that act synergistically or additively in combination with major anti-TB drugs and, if possible, shorten the duration of TB therapy are needed. However, their combinatorial effect cannot be predicted until the lead identification phase of the drug development. Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a powerful genetic tool that enables high-throughput screening of novel drug targets. The development of anti-TB drugs promises to be accelerated by CRISPRi. This study determined whether CRISPRi could be applicable for predictive screening of the combinatorial effect between major anti-TB drugs and an inhibitor of a novel target. In the checkerboard assay, isoniazid killed Mycobacterium smegmatis synergistically or additively in combinations with rifampicin or ethambutol, respectively. The susceptibility to rifampicin and ethambutol was increased by knockdown of inhA, which encodes a target molecule of isoniazid. Additionally, knockdown of rpoB, which encodes a target molecule of rifampicin, increased the susceptibility to isoniazid and ethambutol, which act synergistically with rifampicin in the checkerboard assay. Moreover, CRISPRi could successfully predict the synergistic action of cyclomarin A, a novel TB drug candidate, with isoniazid or rifampicin. These results demonstrate that CRISPRi is a useful tool not only for drug target exploration but also for screening the combinatorial effects of novel combinations of anti-TB drugs. This study provides a rationale for anti-TB drug development using CRISPRi.

Identification of potential inhibitors of Mycobacterium tuberculosis shikimate kinase: molecular docking, in silico toxicity and in vitro experiments.

Tuberculosis (TB) is one of the main causes of death from a single pathological agent, Mycobacterium tuberculosis (Mtb). In addition, the emergence of drug-resistant TB strains has exacerbated even further the treatment outcome of TB patients. It is thus needed the search for new therapeutic strategies to improve the current treatment and to circumvent the resistance mechanisms of Mtb. The shikimate kinase (SK) is the fifth enzyme of the shikimate pathway, which is essential for the survival of Mtb. The shikimate pathway is absent in humans, thereby indicating SK as an attractive target for the development of anti-TB drugs. In this work, a combination of in silico and in vitro techniques was used to identify potential inhibitors for SK from Mtb (MtSK). All compounds of our in-house database (Centro de Pesquisas em Biologia Molecular e Funcional, CPBMF) were submitted to in silico toxicity analysis to evaluate the risk of hepatotoxicity. Docking experiments were performed to identify the potential inhibitors of MtSK according to the predicted binding energy. In vitro inhibitory activity of MtSK-catalyzed chemical reaction at a single compound concentration was assessed. Minimum inhibitory concentration values for in vitro growth of pan-sensitive Mtb H37Rv strain were also determined. The mixed approach implemented in this work was able to identify five compounds that inhibit both MtSK and the in vitro growth of Mtb.

Mycobacterium Fluoroquinolone Resistance Protein D (MfpD), a GTPase-Activating Protein of GTPase MfpB, Is Involved in Fluoroquinolones Potency.

Tuberculosis (TB) caused by Mycobacterium tuberculosis infection remains one of the most serious global health problems. Fluoroquinolones (FQs) are an important component of drug regimens against multidrug-resistant tuberculosis, but challenged by the emergence of FQ-resistant strains. Mycobacterium fluoroquinolone resistance protein A (MfpA) is a pentapeptide protein that confers resistance to FQs. MfpA is the fifth gene in the mfp operon among most Mycobacterium, implying other mfp genes might regulate the activity of MfpA. To elucidate the function of this operon, we constructed deletion mutants and rescued strains and found that MfpD is a GTPase-activating protein (GAP) involved in FQs activity. We showed that the recombinant strains overexpressing mfpD became more sensitive to FQs, whereas an mfpD deletion mutant was more resistant to FQs. By using site-directed mutagenesis and mycobacterial protein fragment complementation, we genetically demonstrated that mfpD participated in FQs susceptibility via directly acting on mfpB. We further biochemically demonstrated that MfpD was a GAP capable of stimulating the GTPase activity of MfpB. Our studies suggest that MfpD, a GAP of MfpB, is involved in MfpA-mediated FQs resistance. The function of MfpD adds new insights into the role of the mfp operon in Mycobacterium fluoroquinolone resistance. IMPORTANCE Tuberculosis is one of the leading causes of morbidity and mortality worldwide largely due to increasingly prevalent drug-resistant strains. Fluoroquinolones are important antibiotics used for treating multidrug-resistant tuberculosis (MDR-TB). The resistance mechanism mediated by the Mycobacterium fluoroquinolone resistance protein (MfpA) is unique in Mycobacterium. However, the regulatory mechanism of MfpA remains largely unclear. In this study, we first report that MfpD acts as a GAP for MfpB and characterize a novel pathway that controls Mycobacterium small G proteins. Our findings provide new insights into the regulation of MfpA and inspiration for new candidate targets for the discovery and development of anti-TB drugs.

Molecular Interplay between Vitamin D and Immunity can Aid Antitubercular Treatment Vitamin D in Immunomodulation of TB

Tuberculosis (TB) causes maximum mortality and morbidity worldwide. 25 per cent of the global population harbour Mycobacterium tuberculosis (Mtb) and therefore are at risk of developing active disease. Of late, the disseminated diseases of TB are on the increase. Nearly one-third of all TB infections can be classified as extrapulmonary-TB (EPTB). TB can spread to the bone, brain, intestine, peritoneum, genitourinary system, and female genital sites leading to problems of conception. Therefore undoubtedly, TB has turned out to be a tremendous public health problem globally. The emergence of drug-resistant bacteria calls for new anti-tuberculous drugs to enhance response to antimicrobial therapy for active TB. However, discoveries of very effective anti-TB new medicines have not materialised yet. Thus, nutritional anti-TB intervention is highly important. In the pre-antibiotic era, Vitamin D was used for the treatment of TB. Its active component 1,25-dihydroxy-vitamin D3 was shown to display anti-TB activity in vitro. Vitamin D deficient humans display greater susceptibility to TB. Vitamin D deficiency induces worse disease progression in TB cases as observed in many clinical trials. The efficacy of the addition of vitamin D supplements in TB treatment has also been estimated. Thus, by now, the role of vitamin D in TB prevention and treatment is well established. Knowledge of the molecular mechanism of vitamin D is crucially vital for new anti-TB drug design. This review article discusses the recent advancement regarding the molecular mechanism of vitamin D-related anti-TB action. Further elucidation of this area may help novel anti-TB drug development.