Mycobacterial Protein Research Articles

Structure-directed identification of pyridine-2-methylamine derivatives as MmpL3 inhibitors for use as antitubercular agents

Mycobacterial membrane protein Large 3 (MmpL3), an inner membrane protein, plays a crucial role in the transport of mycolic acids that are essential for the viability of M. tuberculosis and has been a promising therapeutic target for new anti-TB agents. Herein, we report the discovery of pyridine-2-methylamine antitubercular compounds using a structure-based drug design strategy. Compound 62 stands out as the most potent compound with high activity against M. tb strain H37Rv (MIC = 0.016 μg/mL) as well as the clinically isolated strains of MDR/XDR-TB (MIC = 0.0039–0.0625 μg/mL), low Vero cell toxicity (IC50 ≥ 16 μg/mL), and moderate liver microsomal stability (CLint = 28 μL/min/mg). Furthermore, the resistant mutant of S288T due to single nucleotide polymorphism in mmpL3 was resistant to pyridine-2-methylamine 62, demonstrating compound 62 is likely target to MmpL3.

Design, synthesis, and biological evaluation of 1,2,4-triazole derivatives as potent antitubercular agents

Inhibition of mycobacterial membrane protein large 3 (MmpL3) thereby affecting the mycolic acid biosynthetic pathway has been proven to be an effective strategy for developing antitubercular drugs. Based on the X-ray crystal structure of MmpL3 inhibitor complexes, a series of novel 1,2,4-triazole derivatives were designed, synthesized and evaluated antitubercular activity against Mtb strain H37Rv. Comprehensive structure–activity relationship exploration resulted in the identification of compounds 21 and 28, which possess potent antitubercular activity against Mtb strain H37Rv [minimum inhibitory concentration (MIC) = 0.03–0.13 µg/mL] and the clinical isolates of multidrug resistance (MDR) and extensive drug resistance (XDR) tuberculosis (MIC = 0.06–1.0 µg/mL). Moreover, compounds 21 and 28 showed neglectable cytotoxicity (IC50 ≥ 32 µg/mL) to the mammalian Vero cells and favorable physicochemical and pharmacokinetic properties according to the in silico absorption, distribution, metabolism and excretion (ADME) prediction. Finally, the potential target of representative 1,2,4-triazole 28 was identified to be MmpL3 using a microscale thermophoresis (MST) assay.

OPTIMIZATION OF CULTIVATION CONDITIONS FOR RECOMBINANT INFLUENZA VIRUSES EXPRESSING IMMUNODOMINANT M. BOVIS PROTEINS

The use of recombinant viral vectors is one of the promising approaches to the creation of a new generation of vaccines against bovine tuberculosis. For the prevention of tuberculosis in cattle, two reassortant recombinant strains of influenza A virus expressing mycobacterial proteins ESAT-6 and TB-10.4 were constructed based on the method of reverse genetics. As a result of optimization of virus cultivation conditions, the cytopathic effect of the virus was recorded on the Vero cell culture. It has been established that the maximum accumulation of viruses is noted when cultivating in a Vero cell culture using a serum-free DMEM medium at a temperature of (37 ± 0.5) ºС, with access to 5% carbon dioxide. The peak of the biological activity of the recombinant strains for both samples reached at 72 hours of the study and lasted for 96 hours. The biological activity of FLU NS_ESAT 6 was 7.50 ± 0.08 lg TCD50/cm3 , and for FLU NS_TB10.4 was 7,75 ± 0,08 lg TCD50/cm3. The results obtained are the basis for the development of viral biomass for work on the creation of an experimental series of vaccines against bovine tuberculosis.

Extracellular vesicles from Mycobacterium tuberculosis-infected neutrophils induce maturation of monocyte-derived dendritic cells and activation of antigen-specific Th1 cells.

Tuberculosis remains one of the leading public health problems in the world. The mechanisms that lead to the activation of the immune response against Mycobacterium tuberculosis have been extensively studied, with a focus on the role of cytokines as the main signals for immune cell communication. However, less is known about the role of other signals, such as extracellular vesicles, in the communication between immune cells, particularly during the activation of the adaptive immune response. In this study, we determined that extracellular vesicles released by human neutrophils infected with M. tuberculosis contained several host proteins that are ectosome markers. In addition, we demonstrated that extracellular vesicles released by human neutrophils infected with M. tuberculosis released after only 30 min of infection carried mycobacterial antigens and pathogen-associated molecular patterns, and we identified 15 mycobacterial proteins that were consistently found in high concentrations in extracellular vesicles released by human neutrophils infected with M. tuberculosis; these proteins contain epitopes for CD4 T-cell activation. We found that extracellular vesicles released by human neutrophils infected with M. tuberculosis increased the expression of the costimulatory molecule CD80 and of the coinhibitory molecule PD-L1 on immature monocyte-derived dendritic cells. We also found that immature and mature dendritic cells treated with extracellular vesicles released by human neutrophils infected with M. tuberculosis were able to induce IFN-γ production by autologous M. tuberculosis antigen-specific CD4 T cells, indicating that these extracellular vesicles acted as antigen carriers and transferred mycobacterial proteins to the antigen-presenting cells. Our results provide evidence that extracellular vesicles released by human neutrophils infected with M. tuberculosis participate in the activation of the adaptive immune response against M. tuberculosis.

Immunogenicity of Chitosan-Based Non-invasive Vaccine Strategy Against Mycobacterium tuberculosis

Background: In spite of Bacillus Calmette-Guerin (BCG) vaccination, still tuberculosis caused by Mycobacterium tuberculosis remains a problematic impediment that requires perspective management. The aim of this study was to evaluate the immunogenicity of chitosan nanoparticles containing recombinant mycobacterial proteins and adjuvant in the Balb/C mice through a non-invasive nasal inhalation delivery route and measure the level of cytokines interferon gamma (IFN-γ), interleukin-4 (IL-4), and IL-17. Methods: Thirty mice in five different groups were vaccinated through inhalation with compounds set in different combinations. Two weeks after the last nasal delivery, IFN-γ, IL-4, and IL-17 were measured in spleen cell culture supernatants. Results: The IFN-γ and IL-17 concentrations were found to increase in the groups that received chitosan nanoparticles containing protein and adjuvant alone or as a BCG booster. Our study showed that the chitosan nanoparticle containing protein and adjuvant induced a Th1 response. However, the groups that first received BCG and then chitosan nanoparticles containing protein and adjuvant had the greatest Th1 response in terms of IFN-γ and IL-17 production in all the groups. Conclusion: Our findings showed that the vaccine designed to be administered through the nasal mucosa well stimulates cellular immunity and enhances the BCG vaccine’s effectiveness.

A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis

Background: Tuberculosis (TB) is among the deadliest diseases and a significant cause of illness across the globe. Several studies on mycobacterial proteins, such as proteases and transporters that are essential for survival and pathogenesis have aimed to develop an efficient anti-tubercular agent. In mycobacterium, lysine exporter (LysE) is an amino acid transporter and a probable target for an anti-tubercular agent as it is responsible for bacterial growth inhibition and is also absent in the widely used Bacillus Calmette-Guérin (BCG) vaccine. Methods: Some studies have purified LysE using different protocols. This study describes a protocol for purifying different constructs of LysE, focusing on its hydrophobic region using immobilized metal affinity chromatography (IMAC) after expressing LysE gene in a bacterial expression system. pET vector (pET28a) is used as an expression vector. Amplied LysE gene is ligated with the pET28a vector, and the resultant plasmid is then transformed into E. coli cells. The vector has a histidine tag that makes the purification process convenient. After IMAC, the samples will be subjected to size-exclusion chromatography for further purification. Results: Cloning and amplification findings will be analyzed using 1% agarose gel, and protein expression and purification outcomes will be examined using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Domain-specific constructs of LysE can be further analyzed as an anti-tubercular agent. Conclusions: Despite being a potential anti-tubercular target, research is quite limited on this protein. Therefore, we aim to purify LysE protein for further analysis. Similar protocols have already been implemented to purify several other bacterial proteins with >95% purity.

Insights into substrate transport and water permeation in the mycobacterial transporter MmpL3

Mycobacteria, such as Mycobacterium tuberculosis, are characterized by a uniquely thick and waxy cell envelope that consists of two membranes, with a variety of mycolates comprising their outer membrane (OM). The protein Mycobacterial membrane protein Large 3 (MmpL3) is responsible for the transport of a primary OM component, trehalose monomycolate (TMM), from the inner (cytoplasmic) membrane (IM) to the periplasmic space, a process driven by the proton gradient. Although multiple structures of MmpL3 with bound substrates have been solved, the exact pathway(s) for TMM or proton transport remains elusive. Here, employing molecular dynamics simulations we investigate putative pathways for either transport species. We hypothesized that MmpL3 will cycle through similar conformational states as the related transporter AcrB, which we used as targets for modeling the conformation of MmpL3. A continuous water pathway through the transmembrane region was found in one of these states, illustrating a putative pathway for protons. Additional equilibrium simulations revealed that TMM can diffuse from the membrane into a binding pocket in MmpL3 spontaneously. We also found that acetylation of TMM, which is required for transport, makes it more stable within MmpL3’s periplasmic cavity compared with the unacetylated form.

Mycobacterial Heat Shock Proteins in Sarcoidosis and Tuberculosis.

Pathological similarities between sarcoidosis (SA) and tuberculosis (TB) suggest the role of mycobacterial antigens in the etiopathogenesis of SA. The Dubaniewicz group revealed that not whole mycobacteria, but Mtb-HSP70, Mtb-HSP 65, and Mtb-HSP16 were detected in the lymph nodes, sera, and precipitated immune complexes in patients with SA and TB. In SA, the Mtb-HSP16 concentration was higher than that of Mtb-HSP70 and that of Mtb-HSP65, whereas in TB, the Mtb-HSP16 level was increased vs. Mtb-HSP70. A high Mtb-HSP16 level, induced by low dose-dependent nitrate/nitrite (NOx), may develop a mycobacterial or propionibacterial genetic dormancy program in SA. In contrast to TB, increased peroxynitrite concentration in supernatants of peripheral blood mononuclear cell cultures treated with Mtb-HSP may explain the low level of NOx detected in SA. In contrast to TB, monocytes in SA were resistant to Mtb-HSP-induced apoptosis, and CD4+T cell apoptosis was increased. Mtb-HSP-induced apoptosis of CD8+T cells was reduced in all tested groups. In Mtb-HSP-stimulated T cells, lower CD8+γδ+IL-4+T cell frequency with increased TNF-α,IL-6,IL-10 and decreased INF-γ,IL-2,IL-4 production were present in SA, as opposed to an increased presence of CD4+γδ+TCR cells with increased TNF-α,IL-6 levels in TB, vs. controls. Mtb-HSP modulating the level of co-stimulatory molecules, regulatory cells, apoptosis, clonal deletion, epitope spread, polyclonal activation and molecular mimicry between human and microbial HSPs may also participate in the induction of autoimmunity, considered in SA. In conclusion, in different genetically predisposed hosts, the same antigens, e.g., Mtb-HSP, may induce the development of TB or SA, including an autoimmune response in sarcoidosis.

Mycobacterial nucleoid-associated protein Lsr2 is required for productive mycobacteriophage infection

Mycobacteriophages are a diverse group of viruses infecting Mycobacterium with substantial therapeutic potential. However, as this potential becomes realized, the molecular details of phage infection and mechanisms of resistance remain ill-defined. Here we use live-cell fluorescence microscopy to visualize the spatiotemporal dynamics of mycobacteriophage infection in single cells and populations, showing that infection is dependent on the host nucleoid-associated Lsr2 protein. Mycobacteriophages preferentially adsorb at Mycobacterium smegmatis sites of new cell wall synthesis and following DNA injection, Lsr2 reorganizes away from host replication foci to establish zones of phage DNA replication (ZOPR). Cells lacking Lsr2 proceed through to cell lysis when infected but fail to generate consecutive phage bursts that trigger epidemic spread of phage particles to neighbouring cells. Many mycobacteriophages code for their own Lsr2-related proteins, and although their roles are unknown, they do not rescue the loss of host Lsr2.

Evaluation of the genetic stability of recombinant flu vectors encoding <i>Mycobacterium bovis</i> proteins using RT-PCR and optimization of their cultivation conditions

Prevention by immunizing cattle against tuberculosis with traditional vaccines and regular testing has long been the main method of controlling this infection. However, the non-specificity of the traditional method shows the need for alternative approaches in the creation of anti-infective vaccines. The development of recombinant vector vaccines based on influenza vectors shows great potential and advantages in providing a specific immune response.The purpose of the study is to evaluate the growth properties of the recombinant influenza virus strains expressing protective proteins of mycobacteria for further use in creating a vector vaccine against bovine tuberculosis.This article presents the results of work on the cultivation and reproduction of recombinant influenza virus strains. Using reverse genetics methods, recombinant strains of the influenza virus carrying the mycobacterial Mycobacterium bovis ESAT-6 and TB10.4 proteins in the NS gene sequence were constructed. Based on the results of the work carried out, the optimal conditions for cultivating recombinant influenza virus strains were determined. Both variants of the recombinant strain showed reproductive activity in the developing chick embryo system, under optimal cultivation conditions.The evaluation of the genetic stability of the insertion of mycobacterial proteins into the NS gene of the influenza virus was confirmed using the RT-PCR method. As a result, it was found that the NS gene segment contains an insertion of mycobacterial proteins TB10.4 and ESAT-6, which is retained throughout the studied 5 passages.

Molecular insights into anti-mycobacterial compounds targeting MmpL3.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that poses a severe threat to human health. Mycobacterial infections require long-term treatment and have a multitude of associated undesirable effects. Therefore, finding new targets and new anti-TB drugs is of great importance. One promising target is Mycobacterial membrane protein Large 3 (MmpL3), a member of the resistance-nodulation-division (RND) class of transporters located in the inner membrane (IM). MmpL3 transports trehalose monomycolates (TMMs), an abundant outer membrane lipids of Mtb, from the IM to the periplasmic space. Indole-2-carboxamides have already been demonstrated to inhibit MmpL3, but display limited bioavailability in vivo. To gain more insight into their potential for further drug development, we carried out molecular docking for 10 modified compounds based on indole-2-carboxamides but containing higher ratios of heteroatoms. The docking results show that acetamide-based compounds have high affinity, which is consistent with experiment results that they have minimum inhibitory concentration. Taking a broader view, we also used molecular dynamics simulations to investigate the behavior of TMMs, substrates of MmpL3, in the IM. We found that, unlike phospholipids, the longer of the two acyl chains of TMM can extend into the middle of the two leaflets of the IM as well as occasionally insert into the opposite leaflet.

Mycobacterial infection alters host mitochondrial activity.

The ability of Mycobacterium tuberculosis (M. tb) to hijack host mitochondria and control host immune signaling is the key to its successful infection. Infection of M. tb causes distinct changes in mitochondrial morphology, metabolism, disruption of innate signaling, and cell fate. The alterations in mitochondria are intricately linked to the immunometabolism of host immune cells such as macrophages, dendritic cells, and T cells. Different immune cells are tuned to diverse immunometabolic states that decide their immune response. These changes could be attributed to the several proteins targeted to host mitochondria by M. tb. Bioinformatic analyses and experimental evidence revealed the potential localization of secreted mycobacterial proteins in host mitochondria. Given the central role of mitochondria in the host metabolism, innate signaling, and cell fate, its manipulation by M. tb renders it susceptible to infection. Restoring mitochondrial health can override M. tb-mediated manipulation and thus clear infection. Several reviews are available on the role of different immune cells in tuberculosis infection and M. tb evasion of immune responses; in the present chapter, we discuss the mitochondrial functional alterations in the innate immune signaling of various immune cells driven by differential mitochondrial immunometabolism during M. tb infection and the role of M. tb proteins, which are directly targeted to the host mitochondria and compromise its innate signaling system. Further studies would help in uncovering the molecular mechanisms of M. tb-directed proteins in host mitochondria to conceptualize both host- directed and pathogen- directed interventions in TB disease management.

Metabolic switching and cell wall remodelling of Mycobacterium tuberculosis during bone tuberculosis

ObjectivesBone tuberculosis (TB) is the third most common types of extrapulmonary tuberculosis. It is critical to understand mycobacterial adaptive strategies within bone lesions to identify mycobacterial factors that may have role in disease pathogenesis. MethodsWhole genome microarray was used to characterize the in-vivo transcriptome of Mycobacterium tuberculosis (M.tb) within bone TB specimens. Mycobacterial virulent proteins were identified by bioinformatic software. An in vitro osteoblast cell line model was used to study the role of these proteins in bone TB pathogenesis. Results914 mycobacterial genes were significantly overexpressed and 1688 were repressed in bone TB specimens. Pathway analysis of differentially expressed genes demonstrated a non-replicative and hypometabolic state of M.tb, reinforcement of the mycobacterial cell wall and induction of DNA damage repair responses, suggesting possible survival strategies of M.tb within bone. Bioinformatics mining of microarray data led to identification of five virulence proteins. The genes encoding these proteins were also upregulated in the in vitro MC3T3 osteoblast cell line model of bone TB. Further, exposure of osteoblast cells to two of these virulence proteins (Rv1046c and Rv3663c) significantly inhibited osteoblast differentiation. ConclusionM.tb alters its transcriptome to establish infection in bone by upregulating certain virulence genes which play a key role in disturbing bone homeostasis.

Pharmacophore based virtual screening & molecular docking approach for identification of mycobacterial membrane protein large 3 (MmpL3) inhibitors

Tuberculosis (TB) disease continues to remain one of the global threats for mankind. Till date many antibacterial compounds have been identified to target mycobacterium tuberculosis (MTB). However, the mutating nature of the mycobacteria has always posed a challenge for designing newer drugs which can target both the non-mutating and mutating forms of TB. In this process, Mycobacterial membrane protein Large 3 (MmpL3) transporter was identified as one of the key targets for inhibiting tuberculosis. Herein we have made an effort to find potential inhibitors against MmpL3 by using a pharmacophore-based virtual screening workflow, followed by molecular docking studies and molecular dynamic simulations. Based on a set of 220 compounds showing anti-tubercular activity proposed to target MmpL3 transporter with MIC values ranging from 0.003 to 737 μM, a 5-point pharmacophore ADHHR_2 model possessing one hydrogen acceptor, one hydrogen donor, two hydrophobic groups and an aromatic ring system was generated. The model validated by enrichment study was used to screen Asinex and DrugBank database to identify a potential lead compound such as DrugBank_6059 that was found to show better binding affinity (−11.36) and hydrophobic interactions with target protein in comparison to standard drug SQ109. Communicated by Ramaswamy H. Sarma

Novel fluorophenyl tethered thiazole and chalcone analogues as potential anti-tubercular agents: Design, synthesis, biological and in silico evaluations

Novel analogues of fluorophenyl tethered thiazoles 7a-k and chalcones 10a-k were designed through molecular hybridization approach. All the synthesized final compounds were evaluated for their in vitro antimycobacterial activity against M. tuberculosis H37Rv strain. Among the two series, compound 10g displayed potent inhibition with MIC99 of 1.56 µM against parental and isoniazid-resistant strains of M. tuberculosis. Further, the same compound inhibited the growth of intracellular M. tuberculosis. To gain an insight into the molecular mechanism of actions, in silico molecular docking experiments were conducted using the molecular structure of the DNA gyrase enzyme, which revealed crucial interactions. This was further substantiated through molecular dynamics simulation study of the mycobacterial DNA Gyrase protein against the lead compound 10g and the reference drug (CFX-Ciprofloxacin). Furthermore, the drug-likeliness of the synthesized compounds was computed based on Lipinski's rule of five and ADME pharmacokinetic parameters. Thus, a chalcone-based lead 10g was identified in our study and showed promising antitubercular activity with drug-like properties. The identified lead molecule could act as a starting point to develop novel, potent antitubercular agents against the parental and drug-resistant tuberculosis strains.

Mycobacterium tuberculosis senses host Interferon-γ via the membrane protein MmpL10

Mycobacterium tuberculosis (Mtb) is one of the most successful human pathogens. Several cytokines are known to increase virulence of bacterial pathogens, leading us to investigate whether Interferon-γ (IFN-γ), a central regulator of the immune defense against Mtb, has a direct effect on the bacteria. We found that recombinant and T-cell derived IFN-γ rapidly induced a dose-dependent increase in the oxygen consumption rate (OCR) of Mtb, consistent with increased bacterial respiration. This was not observed in attenuated Bacillus Calmette–Guérin (BCG), and did not occur for other cytokines tested, including TNF-α. IFN-γ binds to the cell surface of intact Mtb, but not BCG. Mass spectrometry identified mycobacterial membrane protein large 10 (MmpL10) as the transmembrane binding partner of IFN-γ, supported by molecular modelling studies. IFN-γ binding and the OCR response was absent in Mtb Δmmpl10 strain and restored by complementation with wildtype mmpl10. RNA-sequencing and RT-PCR of Mtb exposed to IFN-γ revealed a distinct transcriptional profile, including genes involved in virulence. In a 3D granuloma model, IFN-γ promoted Mtb growth, which was lost in the Mtb Δmmpl10 strain and restored by complementation, supporting the involvement of MmpL10 in the response to IFN-γ. Finally, IFN-γ addition resulted in sterilization of Mtb cultures treated with isoniazid, indicating clearance of phenotypically resistant bacteria that persist in the presence of drug alone. Together our data are the first description of a mechanism allowing Mtb to respond to host immune activation that may be important in the immunopathogenesis of TB and have use in novel eradication strategies.

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.

MmpL3 Inhibition as a Promising Approach to Develop Novel Therapies against Tuberculosis: A Spotlight on SQ109, Clinical Studies, and Patents Literature.

Tuberculosis (TB) is accountable for considerable global morbidity and mortality. Effective TB therapy with multiple drugs completes in about six months. The longer duration of TB therapy challenges patient compliance and contributes to treatment collapse and drug resistance (DR) progress. Therefore, new medications with an innovative mechanism of action are desperately required to shorten the TB therapy's duration and effective TB control. The mycobacterial membrane protein Large 3 (MmpL3) is a novel, mycobacteria-conserved and recognized promiscuous drug target used in the development of better treatments for multi-drug resistance TB (MDR-TB) and extensively drug-resistant TB (XDR-TB). This article spotlights MmpL3, the clinical studies of its inhibitor (SQ109), and the patent literature. The literature on MmpL3 inhibitors was searched on PubMed and freely available patent databases (Espacenet, USPTO, and PatentScope). SQ109, an analog of ethambutol (EMB), is an established MmpL3 inhibitor and has completed Phase 2b-3 clinical trials. Infectex and Sequella are developing orally active SQ109 in partnership to treat MDR pulmonary TB. SQ109 has demonstrated activity against drug-sensitive (DS) and drug-resistant (DR) Mycobacterium tuberculosis (Mtb) and a synergistic effect with isoniazid (INH), rifampicin (RIF), clofazimine (CFZ), and bedaquiline (BNQ). The combination of SQ109, clofazimine, bedaquiline, and pyrazinamide (PZA) has been patented due to its excellent anti-TB activity against MDR-TB, XDR-TB, and latent-TB. The combinations of SQ109 with other anti-TB drugs (chloroquine, hydroxychloroquine, and sutezolid) have also been claimed in the patent literature. SQ109 is more potent than EMB and could substitute EMB in the intensive stage of TB treatment with the three- or four-drug combination. Developing MmpL3 inhibitors is a promising approach to fighting the challenges associated with DS-TB and DR-TB. The authors foresee MmpL3 inhibitors such as SQ109 as future drugs for TB treatment.

Screening of marine natural products for potential inhibitors targeting biotin biosynthesis pathway in Mycobacterium tuberculosis

Tuberculosis (TB) remains as one of the major public health concerns worldwide. A successful TB control and treatment is very challenging, due to continuing emergence of Mycobacterium tuberculosis strains resistant to known drugs. Therefore, the development of new drugs with different chemical and biological approaches is necessary to obtain more efficient anti-tubercular therapeutics. Biotin is an essential cofactor for lipid biosynthesis and gluconeogenesis in M. tuberculosis. M. tuberculosis relies on de novo biotin biosynthesis to obtain this vital cofactor since it cannot scavenge sufficient biotin from a mammalian host. In this study, comprehensive in silico methods including structure-based virtual screening, molecular docking, and molecular dynamic simulation analysis for ∼8000 marine natural products were performed against two essential enzymes involved in biotin synthesis and ligation of M. tuberculosis namely, pyridoxal 5′-phosphate-dependent transaminase (BioA) and mycobacterial biotin protein ligase (MtBPL). Two compounds; CMNPD10112 and CMNPD10113 are unveiled to bind the enzymes consistently and with high affinities. The binding pattern of compounds is further noticed in very stable binding modes as analyzed by molecular dynamics simulation and the mean RMSD of the complexes is within 4 Å. The intermolecular binding free energies validated complexes are less than −40 kcal/mol, which demonstrates strong and stable complexes formation. The identified hit compounds could be seeds for design of effective anti-mycobacterium therapeutics by inhibition of bacterial growth through blocking the biotin biosynthesis. Communicated by Ramaswamy H. Sarma

Identification of Arginine Phosphorylation in Mycolicibacterium smegmatis.

ABSTRACTTuberculosis is a leading cause of worldwide infectious mortality. The prevalence of multidrug-resistant Mycobacterium tuberculosis infections drives an urgent need to exploit new drug targets. One such target is the ATP-dependent protease ClpC1P1P2, which is strictly essential for viability. However, few proteolytic substrates of mycobacterial ClpC1P1P2 have been identified to date. Recent studies in Bacillus subtilis have shown that the orthologous ClpCP protease recognizes proteolytic substrates bearing posttranslational arginine phosphorylation. While several lines of evidence suggest that ClpC1P1P2 is similarly capable of recognizing phosphoarginine-bearing proteins, the existence of phosphoarginine modifications in mycobacteria has remained in question. Here, we confirm the presence of posttranslational phosphoarginine modifications in Mycolicibacterium smegmatis, a nonpathogenic surrogate of M. tuberculosis. Using a phosphopeptide enrichment workflow coupled with shotgun phosphoproteomics, we identified arginine phosphosites on several functionally diverse targets within the M. smegmatis proteome. Interestingly, phosphoarginine modifications are not upregulated by heat stress, suggesting divergent roles in mycobacteria and Bacillus. Our findings provide new evidence supporting the existence of phosphoarginine-mediated proteolysis by ClpC1P1P2 in mycobacteria and other actinobacterial species.IMPORTANCE Mycobacteria that cause tuberculosis infections employ proteolytic pathways that modulate cellular behavior by destroying specific proteins in a highly regulated manner. Some proteolytic enzymes have emerged as novel antibacterial targets against drug-resistant tuberculosis infections. However, we have only a limited understanding of how these enzymes function in the cell and how they select proteins for destruction. Some proteolytic enzymes are capable of recognizing proteins that carry an unusual chemical modification, arginine phosphorylation. Here, we confirm the existence of arginine phosphorylation in mycobacterial proteins. Our work expands our understanding of a promising drug target in an important global pathogen.