Indazole to 2-cyanoindole scaffold progression for mycobacterial lipoamide dehydrogenase inhibitors achieves extended target residence time and improved antibacterial activity.

Tuberculosis remains a leading cause of death from a single infection worldwide. Drug resistance to existing and even new antimycobacterials calls for research into novel targets and unexplored mechanisms of action. Recently we reported on the development of tight-binding inhibitors of Mycobacterium tuberculosis (Mtb) lipoamide dehydrogenase (Lpd), which selectively inhibit the bacterial but not the human enzyme based on a differential modality of inhibitor interaction with these targets. Here we report on the striking improvement in inhibitor residence time on the Mtb enzyme associated with scaffold progression from an indazole to 2-cyanoindole. Cryo-EM of Lpd with the bound 2-cyanoindole inhibitor 19 confirmed displacement of the buried water molecule deep in the binding channel with a cyano group. The ensuing hours-long  improvement in on-target residence time is associated with enhanced antibacterial activity in axenic culture and in primary mouse macrophages. Resistance to 2-cyanoindole inhibitors involves mutations within the inhibitor binding site that have little effect on inhibitor affinity but change the modality of inhibitor-target interaction, resulting in fast dissociation from Lpd. These findings underscore that on-target residence time is a major determinant of antibacterial activity and in vivo efficacy.

Nonredundant functions of Mycobacterium tuberculosis chaperones promote survival under stress.

Bacterial chaperones ClpB and DnaK, homologs of the respective eukaryotic heat shock proteins Hsp104 and Hsp70, are essential in the reactivation of toxic protein aggregates that occur during translation or periods of stress. In the pathogen Mycobacterium tuberculosis (Mtb), the protective effect of chaperones extends to survival in the presence of host stresses, such as protein-damaging oxidants. However, we lack a full understanding of the interplay of Hsps and other stress response genes in mycobacteria. Here, we employ genome-wide transposon mutagenesis to identify the genes that support clpB function in Mtb. In addition to validating the role of ClpB in Mtb's response to oxidants, we show that HtpG, a homolog of Hsp90, plays a distinct role from ClpB in the proteotoxic stress response. While loss of neither clpB nor htpG is lethal to the cell, loss of both through genetic depletion or small molecule inhibition impairs recovery after exposure to host-like stresses, especially reactive nitrogen species. Moreover, defects in cells lacking clpB can be complemented by overexpression of other chaperones, demonstrating that Mtb's stress response network depends upon finely tuned chaperone expression levels. These results suggest that inhibition of multiple chaperones could work in concert with host immunity to disable Mtb.

Potentiation of rifampin activity in a mouse model of tuberculosis by activation of host transcription factor EB.

Efforts at host-directed therapy of tuberculosis have produced little control of the disease in experimental animals to date. This is not surprising, given that few specific host targets have been validated, and reciprocally, many of the compounds tested potentially impact multiple targets with both beneficial and detrimental consequences. This puts a premium on identifying appropriate molecular targets and subjecting them to more selective modulation. We discovered an aminopyrimidine small molecule, 2062, that had no direct antimycobacterial activity, but synergized with rifampin to reduce bacterial burden in Mtb infected macrophages and mice and also dampened lung immunopathology. We used 2062 and its inactive congeners as tool compounds to identify host targets. By biochemical, pharmacologic, transcriptomic and genetic approaches, we found that 2062's beneficial effects on Mtb control and clearance in macrophages and in mice are associated with activation of transcription factor EB via an organellar stress response. 2062-dependent TFEB activation led to improved autophagy, lysosomal acidification and lysosomal degradation, promoting bacterial clearance in macrophages. Deletion of TFEB resulted in the loss of IFNγ-dependent control of Mtb replication in macrophages. 2062 also targeted multiple kinases, such as PIKfyve, VPS34, JAKs and Tyk2, whose inhibition likely limited 2062's efficacy in vivo. These findings support a search for selective activators of TFEB for HDT of TB.

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations.

Mycobacterium tuberculosis (Mtb) encounters stresses during the pathogenesis and treatment of tuberculosis (TB) that can suppress replication of the bacteria and render them phenotypically tolerant to most available drugs. Where studied, the majority of Mtb in the sputum of most untreated subjects with active TB have been found to be nonreplicating by the criterion that they do not grow as colony-forming units (cfus) when plated on agar. However, these cells are viable because they grow when diluted in liquid media. A method for generating such "differentially detectable" (DD) Mtb in vitro would aid studies of the biology and drug susceptibility of this population, but lack of independent confirmation of reported methods has contributed to skepticism about their existence. Here, we identified confounding artifacts that, when avoided, allowed development of a reliable method of producing cultures of ≥90% DD Mtb in starved cells. We then characterized several drugs according to whether they contribute to the generation of DD Mtb or kill them. Of the agents tested, rifamycins led to DD Mtb generation, an effect lacking in a rifampin-resistant strain with a mutation in rpoB, which encodes the canonical rifampin target, the ß subunit of RNA polymerase. In contrast, thioridazine did not generate DD Mtb from starved cells but killed those generated by rifampin.

Evidence for dispensability of protein kinase R in host control of tuberculosis.

Genetic deficiency of protein kinase R (PKR) in mice was reported to enhance macrophage activation in vitro in response to interferon-γ (IFNγ) and to reduce the burden of Mycobacterium tuberculosis (Mtb) in vivo (Wu et al. PloS One. 2012 7:e30512). Consistent with this, treatment of wild-type (WT) macrophages in vitro with a novel PKR inhibitor (Bryk et al., Bioorg. Med. Chem. Lett. 2011 21:4108-4114) also enhanced IFN-γ-dependent macrophage activation (Wu et al. PloS One. 2012 7:e30512). Here we show that co-treatment with IFN-γ and a new PKR inhibitor identified herein to be highly but not completely selective likewise induced macrophages to produce more reactive nitrogen intermediates (RNI) and tumor necrosis factor alpha (TNF-α) and less interleukin 10 (IL-10) than seen with IFN-γ alone. Unexpectedly, however, this new PKR inhibitor had a comparable effect on PKR-deficient macrophages. Retrospective investigation revealed that the PKR-deficient mice in (Wu et al. PloS One. 2012 7:e30512) had not been backcrossed. On comparing genetically matched PKR-deficient and WT mice, we saw no impact of PKR deficiency on macrophage activation in vitro or during the course of Mtb infection in vivo. In addition, although 129S1/SvImJ macrophage responses to IFN-γ were greater than those of C57BL/6J macrophages, PKR was not required to mediate the IFN-γ-dependent production of IL-10, RNI or TNF-α in either strain. Together the data cast doubt on PKR as a potential therapeutic target for tuberculosis.

Nonsteroidal anti-inflammatory drug sensitizes Mycobacterium tuberculosis to endogenous and exogenous antimicrobials.

Existing drugs are slow to eradicate Mycobacterium tuberculosis (Mtb) in patients and have failed to control tuberculosis globally. One reason may be that host conditions impair Mtb's replication, reducing its sensitivity to most antiinfectives. We devised a high-throughput screen for compounds that kill Mtb when its replication has been halted by reactive nitrogen intermediates (RNIs), acid, hypoxia, and a fatty acid carbon source. At concentrations routinely achieved in human blood, oxyphenbutazone (OPB), an inexpensive anti-inflammatory drug, was selectively mycobactericidal to nonreplicating (NR) Mtb. Its cidal activity depended on mild acid and was augmented by RNIs and fatty acid. Acid and RNIs fostered OPB's 4-hydroxylation. The resultant 4-butyl-4-hydroxy-1-(4-hydroxyphenyl)-2-phenylpyrazolidine-3,5-dione (4-OH-OPB) killed both replicating and NR Mtb, including Mtb resistant to standard drugs. 4-OH-OPB depleted flavins and formed covalent adducts with N-acetyl-cysteine and mycothiol. 4-OH-OPB killed Mtb synergistically with oxidants and several antituberculosis drugs. Thus, conditions that block Mtb's replication modify OPB and enhance its cidal action. Modified OPB kills both replicating and NR Mtb and sensitizes both to host-derived and medicinal antimycobacterial agents.

Relationships of illness perception, symptoms response and social support with acute myocardial infarction patients' prehospital delay in rural China: protocol for a cross-sectional study.

INTRODUCTION: The timely treatment of acute myocardial infarction (AMI) patients is of utmost importance, and yet, there remains a significant disparity between urban and rural areas in China due to the unequal distribution of medical resources. The manifestation of symptoms and psychosocial factors play a crucial role in shaping medical decisions for AMI patients. It is well established that minimising prehospital delay (PHD) is crucial for the successful implementation of recanalisation therapy and reducing mortality in out-of-hospital settings. However, there remains a paucity of studies investigating the correlation between illness perception, symptom response, social support, and PHD in AMI patients. AIM: The aim of this study was to analyse the relationship pathways between symptom response, illness perception, social support and PHD time in patients with AMI in rural areas of China. METHODS: A primary care-based cross-sectional study was designed to investigate the inpatients initially diagnosed with AMI in the emergency department of three tertiary care hospitals in three counties in northern Zhejiang Province by convenience sampling method from January 2023 to December 2023. A minimum of 286 patients will be enrolled (voluntary response sample). Each participant will complete a paper-based questionnaire to gather research outcomes. Statistical analyses will be performed using logistic regression and structural equation model with PHD as main outcome parameter. DISCUSSION: This is the first study of the factors influencing PHD in AMI in rural China using structural equation model. Our study will address this gap in the available research. The implementation and findings of this study may provide a reliable basis for reducing PHD in AMI patients in rural areas and establish a relevant theoretical foundation for the implementation of targeted interventions and risk prevention measures in primary care hospitals.

High-Throughput/High Content Imaging Screen Identifies Novel Small Molecule Inhibitors and Immunoproteasomes as Therapeutic Targets for Chordoma.

Chordomas account for approximately 1-4% of all malignant bone tumors and 20% of primary tumors of the spinal column. It is a rare disease, with an incidence estimated to be approximately 1 per 1,000,000 people. The underlying causative mechanism of chordoma is unknown, which makes it challenging to treat. Chordomas have been linked to the T-box transcription factor T (TBXT) gene located on chromosome 6. The TBXT gene encodes a protein transcription factor TBXT, or brachyury homolog. Currently, there is no approved targeted therapy for chordoma. Here, we performed a small molecule screening to identify small chemical molecules and therapeutic targets for treating chordoma. We screened 3730 unique compounds and selected 50 potential hits. The top three hits were Ribociclib, Ingenol-3-angelate, and Duvelisib. Among the top 10 hits, we found a novel class of small molecules, including proteasomal inhibitors, as promising molecules that reduce the proliferation of human chordoma cells. Furthermore, we discovered that proteasomal subunits PSMB5 and PSMB8 are increased in human chordoma cell lines U-CH1 and U-CH2, confirming that the proteasome may serve as a molecular target whose specific inhibition may lead to better therapeutic strategies for chordoma.

A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium.

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome-lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.

Type I interferon signaling mediates Mycobacterium tuberculosis-induced macrophage death.

Macrophages help defend the host against Mycobacterium tuberculosis (Mtb), the major cause of tuberculosis (TB). Once phagocytized, Mtb resists killing by macrophages, replicates inside them, and leads to their death, releasing Mtb that can infect other cells. We found that the death of Mtb-infected mouse macrophages in vitro does not appear to proceed by a currently known pathway. Through genome-wide CRISPR-Cas9 screening, we identified a critical role for autocrine or paracrine signaling by macrophage-derived type I IFNs in the death of Mtb-infected macrophages in vitro, and blockade of type I IFN signaling augmented the effect of rifampin, a first-line TB drug, in Mtb-infected mice. Further definition of the pathway of type I IFN-mediated macrophage death may allow for host-directed therapy of TB that is more selective than systemic blockade of type I IFN signaling.

Oxidative damage and delayed replication allow viable Mycobacterium tuberculosis to go undetected.

"Viable but nonculturable" states of bacteria pose challenges for environmental and clinical microbiology, but their biological mechanisms remain obscure. Mycobacterium tuberculosis (Mtb), the leading cause of death from infection until the coronavirus disease 2019 pandemic, affords a notable example of this phenotype. Mtb can enter into a "differentially detectable" (DD) state associated with phenotypic antimicrobial resistance. In this state, Mtb cells are viable but undetectable as colony-forming units. We found that Mtb cells enter the DD state when they undergo sublethal oxidative stress that damages their DNA, proteins, and lipids. In addition, their replication process is delayed, allowing time for repair. Mycobacterium bovis and its derivative, BCG, fail to enter the DD state under similar conditions. These findings have implications for tuberculosis latency, detection, relapse, treatment monitoring, and development of regimens that overcome phenotypic antimicrobial resistance.

Characterization of Phosphopantetheinyl Hydrolase from Mycobacterium tuberculosis.

Phosphopantetheinyl hydrolase, PptH (Rv2795c), is a recently discovered enzyme from Mycobacterium tuberculosis that removes 4'-phosphopantetheine (Ppt) from holo-carrier proteins (CPs) and thereby opposes the action of phosphopantetheinyl transferases (PPTases). PptH is the first structurally characterized enzyme of the phosphopantetheinyl hydrolase family. However, conditions for optimal activity of PptH have not been defined, and only one substrate has been identified. Here, we provide biochemical characterization of PptH and demonstrate that the enzyme hydrolyzes Ppt in vitro from more than one M. tuberculosis holo-CP as well as holo-CPs from other organisms. PptH provided the only detectable activity in mycobacterial lysates that dephosphopantetheinylated acyl carrier protein M (AcpM), suggesting that PptH is the main Ppt hydrolase in M. tuberculosis. We could not detect a role for PptH in coenzyme A (CoA) salvage, and PptH was not required for virulence of M. tuberculosis during infection of mice. It remains to be determined why mycobacteria conserve a broadly acting phosphohydrolase that removes the Ppt prosthetic group from essential CPs. We speculate that the enzyme is critical for aspects of the life cycle of M. tuberculosis that are not routinely modeled. IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis, was the leading cause of death from an infectious disease before COVID, yet the in vivo essentiality and function of many of the protein-encoding genes expressed by M. tuberculosis are not known. We biochemically characterize M. tuberculosis's phosphopantetheinyl hydrolase, PptH, a protein unique to mycobacteria that removes an essential posttranslational modification on proteins involved in synthesis of lipids important for the bacterium's cell wall and virulence. We demonstrate that the enzyme has broad substrate specificity, but it does not appear to have a role in coenzyme A (CoA) salvage or virulence in a mouse model of TB.

Whole Cell Active Inhibitors of Mycobacterial Lipoamide Dehydrogenase Afford Selectivity over the Human Enzyme through Tight Binding Interactions.

Tuberculosis remains a leading cause of death from a single bacterial infection worldwide. Efforts to develop new treatment options call for expansion into an unexplored target space to expand the drug pipeline and bypass resistance to current antibiotics. Lipoamide dehydrogenase is a metabolic and antioxidant enzyme critical for mycobacterial growth and survival in mice. Sulfonamide analogs were previously identified as potent and selective inhibitors of mycobacterial lipoamide dehydrogenase in vitro but lacked activity against whole mycobacteria. Here we present the development of analogs with improved permeability, potency, and selectivity, which inhibit the growth of Mycobacterium tuberculosis in axenic culture on carbohydrates and within mouse primary macrophages. They increase intrabacterial pyruvate levels, supporting their on-target activity within mycobacteria. Distinct modalities of binding between the mycobacterial and human enzymes contribute to improved potency and hence selectivity through induced-fit tight binding interactions within the mycobacterial but not human enzyme, as indicated by kinetic analysis and crystallography.

Macrocyclic Peptides that Selectively Inhibit the Mycobacterium tuberculosis Proteasome.

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) is phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill nonreplicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here, we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6. The cocrystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose dependently led to the accumulation of Pup-tagged GFP that is degradable but resistant to depupylation and death of nonreplicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.

Mycobacterium tuberculosis expresses methionine sulphoxide reductases A and B that protect from killing by nitrite and hypochlorite.

Methionine sulphoxide reductases (Msr) reduce methionine sulphoxide to methionine and protect bacteria against reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Many organisms express both MsrA, active against methionine-(S)-sulphoxide, and MsrB, active against methionine-(R)-sulphoxide. Mycobacterium tuberculosis (Mtb) expresses MsrA, which protects DeltamsrA-Escherichia coli from ROI and RNI. However, the function of MsrA in Mtb has not been defined, and it is unknown whether Mtb expresses MsrB. We identified MsrB as the protein encoded by Rv2674 in Mtb and confirmed the distinct stereospecificities of recombinant Mtb MsrA and MsrB. We generated strains of Mtb deficient in MsrA, MsrB or both and complemented the mutants. Lysates of singly deficient strains displayed half as much Msr activity as wild type against N-acetyl methionine sulphoxide. However, in contrast to other bacteria, single mutants were no more vulnerable than wild type to killing by ROI/RNI. Only Mtb lacking both MsrA and MsrB was more readily killed by nitrite or hypochlorite. Thus, MsrA and MsrB contribute to the enzymatic defences of Mtb against ROI and RNI.

Identification of a copper-binding metallothionein in pathogenic mycobacteria.

A screen of a genomic library from Mycobacterium tuberculosis (Mtb) identified a small, unannotated open reading frame (MT0196) that encodes a 4.9-kDa, cysteine-rich protein. Despite extensive nucleotide divergence, the amino acid sequence is highly conserved among mycobacteria that are pathogenic in vertebrate hosts. We synthesized the protein and found that it preferentially binds up to six Cu(I) ions in a solvent-shielded core. Copper, cadmium and compounds that generate nitric oxide or superoxide induced the gene's expression in Mtb up to 1,000-fold above normal expression. The native protein bound copper within Mtb and partially protected Mtb from copper toxicity. We propose that the product of the MT0196 gene be named mycobacterial metallothionein (MymT). To our knowledge, MymT is the first metallothionein of a Gram-positive bacterium with a demonstrated function.

Correction: Improved Control of Tuberculosis and Activation of Macrophages in Mice Lacking Protein Kinase R.

[This corrects the article DOI: 10.1371/journal.pone.0030512.].

Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis.

The success of Mycobacterium tuberculosis (Mtb) as a pathogen depends on the redundant and complex mechanisms it has evolved for resisting nitrosative and oxidative stresses inflicted by host immunity. Improving our understanding of these defense pathways can reveal vulnerable points in Mtb pathogenesis. In this study, we combined genetic, structural, computational, biochemical, and biophysical approaches to identify a novel enzyme class represented by Rv2466c. We show that Rv2466c is a mycothiol-dependent nitroreductase of Mtb and can reduce the nitro group of a novel mycobactericidal compound using mycothiol as a cofactor. In addition to its function as a nitroreductase, Rv2466c confers partial protection to menadione stress.

Recognition of bacteria in the cytosol of Mammalian cells by the ubiquitin system.

Recent studies have suggested the existence of innate host surveillance systems for the detection of bacteria in the cytosol of mammalian cells. The molecular details of how bacteria are recognized in the cytosol, however, remain unclear. Here we examined the fate of Salmonella typhimurium, a gram-negative bacterial pathogen that can infect a variety of hosts, in the cytosol of mammalian cells. These bacteria typically occupy a membrane bound compartment, the Salmonella-containing vacuole (SCV), in host cells. We show that some wild-type bacteria escape invasion vacuoles and are released into the cytosol. Subsequently, polyubiquitinated proteins accumulate on the bacterial surface, a response that was witnessed in several cell types. In macrophages but not epithelial cells, the proteasome was observed to undergo a dramatic subcellular relocalization and become associated with the surface of bacteria in the cytosol. Proteasome inhibition promoted replication of S. typhimurium in the cytosol of both cell types, in part through destabilization of the SCV. Surprisingly, the cytosol-adapted pathogen Listeria monocytogenes avoided recognition by the ubiquitin system by using actin-based motility. Our findings indicate that the ubiquitin system plays a major role in the recognition of bacterial pathogens in the cytosol of mammalian cells.

Salmonella impairs RILP recruitment to Rab7 during maturation of invasion vacuoles.

After invasion of epithelial cells, Salmonella enterica Typhimurium resides within membrane-bound vacuoles where it survives and replicates. Like endocytic vesicles, the Salmonella-containing vacuoles (SCVs) undergo a maturation process that involves sequential acquisition of Rab5 and Rab7 and displacement toward the microtubule-organizing center. However, SCVs fail to merge with lysosomes and instead develop subsequently into a filamentous network that extends toward the cell periphery. We found that the initial centripetal displacement of the SCV is due to recruitment by Rab7 of Rab7-interacting lysosomal protein (RILP), an effector protein that can simultaneously associate with the dynein motor complex. Unlike the early SCVs, the Salmonella-induced filaments (Sifs) formed later are devoid of RILP and dynein, despite the presence of active Rab7 on their membranes. Kinesin seems to be involved in the elongation of Sifs. SifA, a secreted effector of Salmonella, was found to be at least partly responsible for uncoupling Rab7 from RILP in Sifs and in vitro experiments suggest that SifA may exert this effect by interacting with Rab7. We propose that, by disengaging RILP from Rab7, SifA enables the centrifugal extension of tubules from the Salmonella-containing vacuoles, thereby providing additional protected space for bacterial replication.