Environmental sphagnum-associated fungi target thiol homeostasis to compete with Mycobacterium tuberculosis.

Mycobacterial species in nature are found in abundance in sphagnum peat bogs where they compete for nutrients with a variety of microorganisms including fungi. We screened a collection of fungi isolated from sphagnum bogs by co-culture with Mycobacterium tuberculosis (Mtb) to look for inducible expression of antitubercular agents and identified five fungi that produced cidal antitubercular agents upon exposure to live Mtb. Whole genome sequencing of these fungi followed by fungal RNAseq after Mtb exposure allowed us to identify biosynthetic gene clusters induced by co-culture. Three of these fungi induced expression of patulin, one induced citrinin expression and one induced the production of nidulalin A. The biosynthetic gene clusters for patulin and citrinin have been previously described but the genes involved in nidulalin A production have not been described before. All three of these potent electrophiles react with thiols and treatment of Mtb cells with these agents followed by Mtb RNAseq showed that these natural products all induce profound thiol stress suggesting a rapid depletion of mycothiol. The induction of thiol-reactive mycotoxins through three different systems in response to exposure to Mtb suggests that fungi have identified this as a highly vulnerable target in a similar microenvironment to that of the caseous human lesion.

Testing an Innovative Approach to Improve Hypertension Management at a Federally Qualified Health Center.

Despite the availability of effective treatments, hypertension control rates remain inadequate in the United States and locally in Los Angeles County. To address this health condition, QueensCare Health Centers developed and launched a team-based hypertension management program that was led by clinical pharmacists and designed to mitigate treatment barriers encountered at the system, provider, and patient levels. System- and provider-focused strategies included incorporating self-monitored blood pressure values into the electronic health record and retraining clinicians to regularly review these values; adding a community health worker to the disease management team; and utilizing clinical pharmacists to assess and titrate medications. Patient-focused strategies included tailoring education materials to reduce literacy and linguistic barriers; providing tailored one-on-one education and support; and providing blood pressure cuffs and pedometers. This multilevel intervention serves as a practical example of how team-based care can be optimized at a Federally Qualified Health Center.

Mtb-Selective 5-Aminomethyl Oxazolidinone Prodrugs: Robust Potency and Potential Liabilities.

Linezolid is a drug with proven human antitubercular activity whose use is limited to highly drug-resistant patients because of its toxicity. This toxicity is related to its mechanism of action─linezolid inhibits protein synthesis in both bacteria and eukaryotic mitochondria. A highly selective and potent series of oxazolidinones, bearing a 5-aminomethyl moiety (in place of the typical 5-acetamidomethyl moiety of linezolid), was identified. Linezolid-resistant mutants were cross-resistant to these molecules but not vice versa. Resistance to the 5-aminomethyl molecules mapped to an N-acetyl transferase (Rv0133) and these mutants remained fully linezolid susceptible. Purified Rv0133 was shown to catalyze the transformation of the 5-aminomethyl oxazolidinones to their corresponding N-acetylated metabolites, and this transformation was also observed in live cells of Mycobacterium tuberculosis. Mammalian mitochondria, which lack an appropriate N-acetyltransferase to activate these prodrugs, were not susceptible to inhibition with the 5-aminomethyl analogues. Several compounds that were more potent than linezolid were taken into C3HeB/FeJ mice and were shown to be highly efficacious, and one of these (9) was additionally taken into marmosets and found to be highly active. Penetration of these 5-aminomethyl oxazolidinone prodrugs into caseum was excellent. Unfortunately, these compounds were rapidly converted into the corresponding 5-alcohols by mammalian metabolism which retained antimycobacterial activity but resulted in substantial mitotoxicity.

The Antitubercular Activities of Natural Products with Fused-Nitrogen-Containing Heterocycles.

Tuberculosis (TB) is notorious as the leading cause of death worldwide due to a single infectious entity and its causative agent, Mycobacterium tuberculosis (Mtb), has been able to evolve resistance to all existing drugs in the treatment arsenal complicating disease management programs. In drug discovery efforts, natural products are important starting points in generating novel scaffolds that have evolved to specifically bind to vulnerable targets not only in pathogens such as Mtb, but also in mammalian targets associated with human diseases. Structural diversity is one of the most attractive features of natural products. This review provides a summary of fused-nitrogen-containing heterocycles found in the natural products reported in the literature that are known to have antitubercular activities. The structurally targeted natural products discussed in this review could provide a revealing insight into novel chemical aspects with novel biological functions for TB drug discovery efforts.

Synthesis and Structure-Activity Relationships of a New Class of Oxadiazoles Targeting DprE1 as Antitubercular Agents.

The continuing prevalence of drug-resistant tuberculosis threatens global TB control programs, highlighting the need to discover new drug candidates to feed the drug development pipeline. In this study, we describe a high-throughput screening hit (4-benzylpiperidin-1-yl)(1-(5-phenyl-1,3,4-oxadiazol-2-yl)piperidin-4-yl)methanone (P1) as a potent antitubercular agent. Structure-activity guided synthesis led to the discovery of several analogs with high in vitro potency. P1 was found to have promising potency against many drug-resistant strains, as well as drug-susceptible clinical isolates. It also showed cidality against Mtb growing in host macrophages. Whole genome sequencing of genomic DNA from resistant mutants raised to P1 revealed mutations in decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1). This novel oxadiazole scaffold expands the set of chemical tools for targeting a well-validated pathway to treat tuberculosis.

Chemical approaches to unraveling the biology of mycobacteria.

Mycobacterium tuberculosis (Mtb), perhaps more than any other organism, is intrinsically appealing to chemical biologists. Not only does the cell envelope feature one of the most complex heteropolymers found in nature1 but many of the interactions between Mtb and its primary host (we humans) rely on lipid and not protein mediators.2,3 Many of the complex lipids, glycolipids, and carbohydrates biosynthesized by the bacterium still have unknown functions, and the complexity of the pathological processes by which tuberculosis (TB) disease progress offers many opportunities for these molecules to influence the human response. Because of the importance of TB in global public health, chemical biologists have applied a wide-ranging array of techniques to better understand the disease and improve interventions.

Phenotype-based screening rediscovered benzopyran-embedded microtubule inhibitors as anti-neuroinflammatory agents by modulating the tubulin-p65 interaction.

Neuroinflammation is one of the critical processes implicated in central nervous system (CNS) diseases. Therefore, alleviating neuroinflammation has been highlighted as a therapeutic strategy for treating CNS disorders. However, the complexity of neuroinflammatory processes and poor drug transport to the brain are considerable hurdles to the efficient control of neuroinflammation using small-molecule therapeutics. Thus, there is a significant demand for new chemical entities (NCEs) targeting neuroinflammation. Herein, we rediscovered benzopyran-embedded tubulin inhibitor 1 as an anti-neuroinflammatory agent via phenotype-based screening. A competitive photoaffinity labeling study revealed that compound 1 binds to tubulin at the colchicine-binding site. Structure-activity relationship analysis of 1's analogs identified SB26019 as a lead compound with enhanced anti-neuroinflammatory efficacy. Mechanistic studies revealed that upregulation of the tubulin monomer was critical for the anti-neuroinflammatory activity of SB26019. We serendipitously found that the tubulin monomer recruits p65, inhibiting its translocation from the cytosol to the nucleus and blocking NF-κB-mediated inflammatory pathways. Further in vivo validation using a neuroinflammation mouse model demonstrated that SB26019 suppressed microglial activation by downregulating lba-1 and proinflammatory cytokines. Intraperitoneal administration of SB26019 showed its therapeutic potential as an NCE for successful anti-neuroinflammatory regulation. Along with the recent growing demands on tubulin modulators for treating various inflammatory diseases, our results suggest that colchicine-binding site-specific modulation of tubulins can be a potential strategy for preventing neuroinflammation and treating CNS diseases.

Activating Mucosal-Associated Invariant T Cells Induces a Broad Antitumor Response.

Mucosal-associated invariant T (MAIT) cells are MR1-restricted innate-like T cells that recognize non-peptide antigens including riboflavin derivates. Although in vitro-activated MAIT cells show antitumor activity, the in vivo role of MAIT cells in cancer is still unclear. Here, we have shown that MAIT cells have antitumor function in vivo when activated by a combination of the synthetic riboflavin synthesis pathway-derived antigen 5-OP-RU [5-(2-oxopropylideneamino)-6-D-ribitylaminouracil] and the Toll-like receptor 9 (TLR9) agonist CpG. Coadministration of 5-OP-RU and CpG induced strong systemic in vivo expansion and activation of MAIT cells with high CD69 expression, pronounced effector memory phenotype, and upregulated levels of effector molecules including IFNγ, granzyme B, and perforin. Activated and expanded MAITs induced a potent and broad antitumor immune response in murine models of liver metastasis and hepatocellular carcinoma, lung metastasis, and subcutaneous tumors in two different mouse strains. Such tumor inhibition was absent in MAIT-deficient Mr1 -/- mice. CRISPR/Cas9-mediated MR1 knockout in tumor cells did not affect efficacy of this MAIT-directed immunotherapy, pointing toward an indirect mechanism of action. Our findings suggest that MAIT cells are an attractive target for cancer immunotherapy.See related Spotlight by Lantz, p. 996.

Functional inactivation of pulmonary MAIT cells following 5-OP-RU treatment of non-human primates.

Targeting MAIT cells holds promise for the treatment of different diseases and infections. We previously showed that treatment of Mycobacterium tuberculosis infected mice with 5-OP-RU, a major antigen for MAIT cells, expands MAIT cells and enhances bacterial control. Here we treated M. tuberculosis infected rhesus macaques with 5-OP-RU intratracheally but found no clinical or microbiological benefit. In fact, after 5-OP-RU treatment MAIT cells did not expand, but rather upregulated PD-1 and lost the ability to produce multiple cytokines, a phenotype resembling T cell exhaustion. Furthermore, we show that vaccination of uninfected macaques with 5-OP-RU+CpG instillation into the lungs also drives MAIT cell dysfunction, and PD-1 blockade during vaccination partly prevents the loss of MAIT cell function without facilitating their expansion. Thus, in rhesus macaques MAIT cells are prone to the loss of effector functions rather than expansion after TCR stimulation in vivo, representing a significant barrier to therapeutically targeting these cells.

Tuberculosis Drug Discovery: A Decade of Hit Assessment for Defined Targets.

More than two decades have elapsed since the publication of the first genome sequence of Mycobacterium tuberculosis (Mtb) which, shortly thereafter, enabled methods to determine gene essentiality in the pathogen. Despite this, target-based approaches have not yielded drugs that have progressed to clinical testing. Whole-cell screening followed by elucidation of mechanism of action has to date been the most fruitful approach to progressing inhibitors into the tuberculosis drug discovery pipeline although target-based approaches are gaining momentum. This review discusses scaffolds that have been identified over the last decade from screens of small molecule libraries against Mtb or defined targets where mechanism of action investigation has defined target-hit couples and structure-activity relationship studies have described the pharmacophore.

Identification of Piperidine-3-carboxamide Derivatives Inducing Senescence-like Phenotype with Antimelanoma Activities.

This study evaluated the potential use of senescence-inducing small molecules in the treatment of melanoma. We screened commercially available small-molecule libraries with high-throughput screening and high-content screening image-based technology. Our findings showed an initial hit with the embedded N-arylpiperidine-3-carboxamide scaffold-induced senescence-like phenotypic changes in human melanoma A375 cells without serious cytotoxicity against normal cells. A focused library containing diversely modified analogues were constructed and examined to evaluate the structure-activity relationship of N-arylpiperidine-3-carboxamide derivatives starting from hit 1. This work identified a novel compound with remarkable antiproliferative activity in vitro and demonstrated the key structural moieties within.

Resistance of Mycobacterium tuberculosis to indole 4-carboxamides occurs through alterations in drug metabolism and tryptophan biosynthesis.

Tryptophan biosynthesis represents an important potential drug target for new anti-TB drugs. We identified a series of indole-4-carboxamides with potent antitubercular activity. In vitro, Mycobacterium tuberculosis (Mtb) acquired resistance to these compounds through three discrete mechanisms: (1) a decrease in drug metabolism via loss-of-function mutations in the amidase that hydrolyses these carboxamides, (2) an increased biosynthetic rate of tryptophan precursors via loss of allosteric feedback inhibition of anthranilate synthase (TrpE), and (3) mutation of tryptophan synthase (TrpAB) that decreased incorporation of 4-aminoindole into 4-aminotryptophan. Thus, these indole-4-carboxamides act as prodrugs of a tryptophan antimetabolite, 4-aminoindole.

Structure-Activity Relationships of Pyrazolo[1,5-a]pyrimidin-7(4H)-ones as Antitubercular Agents.

Pyrazolo[1,5-a]pyrimidin-7(4H)-one was identified through high-throughput whole-cell screening as a potential antituberculosis lead. The core of this scaffold has been identified several times previously and has been associated with various modes of action against Mycobacterium tuberculosis (Mtb). We explored this scaffold through the synthesis of a focused library of analogues and identified key features of the pharmacophore while achieving substantial improvements in antitubercular activity. Our best hits had low cytotoxicity and showed promising activity against Mtb within macrophages. The mechanism of action of these compounds was not related to cell-wall biosynthesis, isoprene biosynthesis, or iron uptake as has been found for other compounds sharing this core structure. Resistance to these compounds was conferred by mutation of a flavin adenine dinucleotide (FAD)-dependent hydroxylase (Rv1751) that promoted compound catabolism by hydroxylation from molecular oxygen. Our results highlight the risks of chemical clustering without establishing mechanistic similarity of chemically related growth inhibitors.

MAIT cell-directed therapy of Mycobacterium tuberculosis infection.

Mucosal-associated invariant T (MAIT) cells are potential targets of vaccination and host-directed therapeutics for tuberculosis, but the role of MAIT cells during Mycobacterium tuberculosis (Mtb) infection in vivo is not well understood. Here we find that following Mtb infection MAIT cells mount minimal responses, and MAIT cell-deficient MR1-/- mice display normal survival. Preinfection expansion of MAIT cells through 5-OP-RU vaccination fails to protect against subsequent Mtb challenge. In fact, 5-OP-RU vaccination delays Mtb-specific CD4 T cell priming in lung-draining lymph nodes, and conversely MR1 deficiency or blockade accelerates T cell priming. The MAIT cell-mediated delay in T cell priming is partly dependent on TGF-ß. Surprisingly, 5-OP-RU treatment during chronic infection drives MAIT cell expansion and an IL-17A-dependent reduction in bacterial loads. Thus, during early infection MAIT cells directly contribute to the notoriously slow priming of CD4 T cells, but later during infection MAIT cell stimulation may be an effective host-directed therapy for tuberculosis.

Discovery of 4H-chromeno[2,3-d]pyrimidin-4-one derivatives as senescence inducers and their senescence-associated antiproliferative activities on cancer cells using advanced phenotypic assay.

Current research suggests therapy-induced senescence (TIS) of cancer cells characterized by distinct morphological and biochemical phenotypic changes represent a novel functional target that may enhance the effectiveness of cancer therapy. In order to identify novel small-molecule inducers of cellular senescence and determine the potential to be used for the treatment of melanoma, a new method of high-throughput screening (HTS) and high-contents screening (HCS) based on the detection of morphological changes was designed. This image-based and whole cell-based technology was applied to screen and select a novel class of antiproliferative agents on cancer cells, 4H-chromeno[2,3-d]pyrimidin-4-one derivatives, which induced senescence-like phenotypic changes in human melanoma A375 cells without serious cytotoxicity against normal cells. To evaluate structure-activity relationship (SAR) study of 4H-chromeno[2,3-d]pyrimidin-4-one scaffold starting from hit 3, a focused library containing diversely modified analogues was constructed and which led to the identification of 38, a novel compound to have remarkable anti-melanoma activity in vitro with good metabolic stability.

Inhibition of CorA-Dependent Magnesium Homeostasis Is Cidal in Mycobacterium tuberculosis.

Mechanisms of magnesium homeostasis in Mycobacterium tuberculosis are poorly understood. Here, we describe the characterization of a pyrimidinetrione amide scaffold that disrupts magnesium homeostasis in the pathogen by direct binding to the CorA Mg2+/Co2+ transporter. Mutations in domains of CorA that are predicted to regulate the pore opening in response to Mg2+ ions conferred resistance to this scaffold. The pyrimidinetrione amides were cidal against the pathogen under both actively replicating and nonreplicating conditions in vitro and were efficacious against the organism during macrophage infection. However, the compound lacked efficacy in infected mice, possibly due to limited exposure. Our results indicate that inhibition of Mg2+ homeostasis by CorA is an attractive target for tuberculosis drug discovery and encourage identification of improved CorA inhibitors.

Discovery and Structure-Activity-Relationship Study of N-Alkyl-5-hydroxypyrimidinone Carboxamides as Novel Antitubercular Agents Targeting Decaprenylphosphoryl-ß-d-ribose 2'-Oxidase.

Magnesium plays an important role in infection with Mycobacterium tuberculosis ( Mtb) as a signal of the extracellular environment, as a cofactor for many enzymes, and as a structural element in important macromolecules. Raltegravir, an antiretroviral drug that inhibits HIV-1 integrase is known to derive its potency from selective sequestration of active-site magnesium ions in addition to binding to a hydrophobic pocket. In order to determine if essential Mtb-related phosphoryl transfers could be disrupted in a similar manner, a directed screen of known molecules with integrase inhibitor-like pharmacophores ( N-alkyl-5-hydroxypyrimidinone carboxamides) was performed. Initial hits afforded compounds with low-micromolar potency against Mtb, acceptable cytotoxicity and PK characteristics, and robust SAR. Elucidation of the target of these compounds revealed that they lacked magnesium dependence and instead disappointingly inhibited a known promiscuous target in Mtb, decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1, Rv3790).

Role of Chemical Biology in Tuberculosis Drug Discovery and Diagnosis.

The use of chemical techniques to study biological systems (often referred to currently as chemical biology) has become a powerful tool for both drug discovery and the development of novel diagnostic strategies. In tuberculosis, such tools have been applied to identifying drug targets from hit compounds, matching high-throughput screening hits against large numbers of isolated protein targets and identifying classes of enzymes with important functions. Metabolites unique to mycobacteria have provided important starting points for the development of innovative tools. For example, the unique biology of trehalose has provided both novel diagnostic strategies as well as probes of in vivo biological processes that are difficult to study any other way. Other mycobacterial metabolites are potentially valuable starting points and have the potential to illuminate new aspects of mycobacterial pathogenesis.

cdc-25.2, a Caenorhabditis elegans ortholog of cdc25, is required for male tail morphogenesis.

Cell division cycle 25 (Cdc25) is an evolutionarily conserved phosphatase that promotes cell cycle progression by activating cyclin-dependent kinases (Cdks) which are inactivated by Wee1/Myt1 kinases. It was previously reported that cdc-25.2 promotes oocyte maturation and intestinal cell divisions in Caenorhabditis elegans hermaphrodites. Here, we report a novel function of cdc-25.2 in male tail development which was significantly deformed by cdc-25.2 RNAi depletion and in cdc-25.2 mutant males. The deformation was also observed after RNAi depletion of other cell cycle regulators, cdk-1, cyb-3, cyd-1, and cyl-1. Furthermore, wee-1.3 counteracted cdc-25.2 in male tail development as observed in oocyte maturation and intestine development. The number of cells in ray precursor cell lineages was significantly reduced in cdc-25.2 depleted males. These results indicate that CDC-25.2 is essential for cell divisions in ray precursor cell lineages for proper male tail development.