Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase.

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.

Mycobacterium tuberculosis PptT Inhibitors Based on Heterocyclic Replacements of Amidinoureas.

4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme for Mycobacterium tuberculosis (Mtb) survival and virulence and therefore an attractive target for a tuberculosis therapeutic. In this work, two modeling-informed approaches toward the isosteric replacement of the amidinourea moiety present in the previously reported PptT inhibitor AU 8918 are reported. Although a designed 3,5-diamino imidazole unexpectedly adopted an undesired tautomeric form and was inactive, replacement of the amidinourea moiety afforded a series of active PptT inhibitors containing 2,6-diaminopyridine scaffolds.

Discovery of natural-product-derived sequanamycins as potent oral anti-tuberculosis agents.

The emergence of drug-resistant tuberculosis has created an urgent need for new anti-tubercular agents. Here, we report the discovery of a series of macrolides called sequanamycins with outstanding in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb). Sequanamycins are bacterial ribosome inhibitors that interact with the ribosome in a similar manner to classic macrolides like erythromycin and clarithromycin, but with binding characteristics that allow them to overcome the inherent macrolide resistance of Mtb. Structures of the ribosome with bound inhibitors were used to optimize sequanamycin to produce the advanced lead compound SEQ-9. SEQ-9 was efficacious in mouse models of acute and chronic TB as a single agent, and it demonstrated bactericidal activity in a murine TB infection model in combination with other TB drugs. These results support further investigation of this series as TB clinical candidates, with the potential for use in new regimens against drug-susceptible and drug-resistant TB.

"Upcycling" known molecules and targets for drug-resistant TB.

Despite reinvigorated efforts in Tuberculosis (TB) drug discovery over the past 20 years, relatively few new drugs and candidates have emerged with clear utility against drug resistant TB. Over the same period, significant technological advances and learnings around target value have taken place. This has offered opportunities to re-assess the potential for optimization of previously discovered chemical matter against Mycobacterium tuberculosis (M.tb) and for reconsideration of clinically validated targets encumbered by drug resistance. A re-assessment of discarded compounds and programs from the "golden age of antibiotics" has yielded new scaffolds and targets against TB and uncovered classes, for example beta-lactams, with previously unappreciated utility for TB. Leveraging validated classes and targets has also met with success: booster technologies and efforts to thwart efflux have improved the potential of ethionamide and spectinomycin classes. Multiple programs to rescue high value targets while avoiding cross-resistance are making progress. These attempts to make the most of known classes, drugs and targets complement efforts to discover new chemical matter against novel targets, enhancing the chances of success of discovering effective novel regimens against drug-resistant TB.

Identification of ß-Lactams Active against Mycobacterium tuberculosis by a Consortium of Pharmaceutical Companies and Academic Institutions.

Rising antimicrobial resistance challenges our ability to combat bacterial infections. The problem is acute for tuberculosis (TB), the leading cause of death from infection before COVID-19. Here, we developed a framework for multiple pharmaceutical companies to share proprietary information and compounds with multiple laboratories in the academic and government sectors for a broad examination of the ability of ß-lactams to kill Mycobacterium tuberculosis (Mtb). In the TB Drug Accelerator (TBDA), a consortium organized by the Bill & Melinda Gates Foundation, individual pharmaceutical companies collaborate with academic screening laboratories. We developed a higher order consortium within the TBDA in which four pharmaceutical companies (GlaxoSmithKline, Sanofi, MSD, and Lilly) collectively collaborated with screeners at Weill Cornell Medicine, the Infectious Disease Research Institute (IDRI), and the National Institute of Allergy and Infectious Diseases (NIAID), pharmacologists at Rutgers University, and medicinal chemists at the University of North Carolina to screen ∼8900 ß-lactams, predominantly cephalosporins, and characterize active compounds. In a striking contrast to historical expectation, 18% of ß-lactams screened were active against Mtb, many without a ß-lactamase inhibitor. One potent cephaloporin was active in Mtb-infected mice. The steps outlined here can serve as a blueprint for multiparty, intra- and intersector collaboration in the development of anti-infective agents.

In Vitro and In Vivo Inhibition of the Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT by Amidinoureas.

A newly validated target for tuberculosis treatment is phosphopantetheinyl transferase, an essential enzyme that plays a critical role in the biosynthesis of cellular lipids and virulence factors in Mycobacterium tuberculosis. The structure-activity relationships of a recently disclosed inhibitor, amidinourea (AU) 8918 (1), were explored, focusing on the biochemical potency, determination of whole-cell on-target activity for active compounds, and profiling of selective active congeners. These studies show that the AU moiety in AU 8918 is largely optimized and that potency enhancements are obtained in analogues containing a para-substituted aromatic ring. Preliminary data reveal that while some analogues, including 1, have demonstrated cardiotoxicity (e.g., changes in cardiomyocyte beat rate, amplitude, and peak width) and inhibit Cav1.2 and Nav1.5 ion channels (although not hERG channels), inhibition of the ion channels is largely diminished for some of the para-substituted analogues, such as 5k (p-benzamide) and 5n (p-phenylsulfonamide).

Cyclohexyl-griselimycin Is Active against Mycobacterium abscessus in Mice.

Cyclohexyl-griselimycin is a preclinical candidate for use against tuberculosis (TB). Here, we show that this oral cyclodepsipeptide is also active against the intrinsically drug-resistant nontuberculous mycobacterium Mycobacterium abscessus in vitro and in a mouse model of infection. This adds a novel advanced lead compound to the M. abscessus drug pipeline and supports a strategy of screening chemical matter generated in TB drug discovery efforts to fast-track the discovery of novel antibiotics against M. abscessus.

Integrative Analysis of Human Macrophage Inflammatory Response Related to Mycobacterium tuberculosis Virulence.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, kills 1.5 to 1.7 million people every year. Macrophages are Mtb's main host cells and their inflammatory response is an essential component of the host defense against Mtb. However, Mtb is able to circumvent the macrophages' defenses by triggering an inappropriate inflammatory response. The ability of Mtb to hinder phagolysosome maturation and acidification, and to escape the phagosome into the cytosol, is closely linked to its virulence. The modulation of the host inflammatory response relies on Mtb virulence factors, but remains poorly studied. Understanding macrophage interactions with Mtb is crucial to develop strategies to control tuberculosis. The present study aims to determine the inflammatory response transcriptome and miRNome of human macrophages infected with the virulent H37Rv Mtb strain, to identify macrophage genetic networks specifically modulated by Mtb virulence. Using human macrophages infected with two different live strains of mycobacteria (live or heat-inactivated Mtb H37Rv and M. marinum), we quantified and analyzed 184 inflammatory mRNAs and 765 micro(mi)RNAs. Transcripts and miRNAs differently modulated by H37Rv in comparison with the two other conditions were analyzed using in silico approaches. We identified 30 host inflammatory response genes and 37 miRNAs specific for H37Rv virulence, and highlight evidence suggesting that Mtb intracellular-linked virulence depends on the inhibition of IL-1ß-dependent pro-inflammatory response, the repression of apoptosis and the delay of the recruitment and activation of adaptive immune cells. Our findings provide new potential targets for the development of macrophage-based therapeutic strategies against TB.

Bactericidal Disruption of Magnesium Metallostasis in Mycobacterium tuberculosis Is Counteracted by Mutations in the Metal Ion Transporter CorA.

A defining characteristic of treating tuberculosis is the need for prolonged administration of multiple drugs. This may be due in part to subpopulations of slowly replicating or nonreplicating Mycobacterium tuberculosis bacilli exhibiting phenotypic tolerance to most antibiotics in the standard treatment regimen. Confounding this problem is the increasing incidence of heritable multidrug-resistant M. tuberculosis A search for new antimycobacterial chemical scaffolds that can kill phenotypically drug-tolerant mycobacteria uncovered tricyclic 4-hydroxyquinolines and a barbituric acid derivative with mycobactericidal activity against both replicating and nonreplicating M. tuberculosis Both families of compounds depleted M. tuberculosis of intrabacterial magnesium. Complete or partial resistance to both chemotypes arose from mutations in the putative mycobacterial Mg2+/Co2+ ion channel, CorA. Excess extracellular Mg2+, but not other divalent cations, diminished the compounds' cidality against replicating M. tuberculosis These findings establish depletion of intrabacterial magnesium as an antimicrobial mechanism of action and show that M. tuberculosis magnesium homeostasis is vulnerable to disruption by structurally diverse, nonchelating, drug-like compounds.IMPORTANCE Antimycobacterial agents might shorten the course of treatment by reducing the number of phenotypically tolerant bacteria if they could kill M. tuberculosis in diverse metabolic states. Here we report two chemically disparate classes of agents that kill M. tuberculosis both when it is replicating and when it is not. Under replicating conditions, the tricyclic 4-hydroxyquinolines and a barbituric acid analogue deplete intrabacterial magnesium as a mechanism of action, and for both compounds, mutations in CorA, a putative Mg2+/Co2+ transporter, conferred resistance to the compounds when M. tuberculosis was under replicating conditions but not under nonreplicating conditions, illustrating that a given compound can kill M. tuberculosis in different metabolic states by disparate mechanisms. Targeting magnesium metallostasis represents a previously undescribed antimycobacterial mode of action that might cripple M. tuberculosis in a Mg2+-deficient intraphagosomal environment of macrophages.

Dual-Pharmacophore Pyrithione-Containing Cephalosporins Kill Both Replicating and Nonreplicating Mycobacterium tuberculosis.

The historical view of ß-lactams as ineffective antimycobacterials has given way to growing interest in the activity of this class against Mycobacterium tuberculosis (Mtb) in the presence of a ß-lactamase inhibitor. However, most antimycobacterial ß-lactams kill Mtb only or best when the bacilli are replicating. Here, a screen of 1904 ß-lactams led to the identification of cephalosporins substituted with a pyrithione moiety at C3' that are active against Mtb under both replicating and nonreplicating conditions, neither activity requiring a ß-lactamase inhibitor. Studies showed that activity against nonreplicating Mtb required the in situ release of the pyrithione, independent of the known class A ß-lactamase, BlaC. In contrast, replicating Mtb could be killed both by released pyrithione and by the parent ß-lactam. Thus, the antimycobacterial activity of pyrithione-containing cephalosporins arises from two mechanisms that kill mycobacteria in different metabolic states.

Opposing reactions in coenzyme A metabolism sensitize Mycobacterium tuberculosis to enzyme inhibition.

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death in humans. Synthesis of lipids critical for Mtb's cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4'-phosphopantetheine (Ppt) from coenzyme A (CoA) to diverse acyl carrier proteins. We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss-of-function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor cocrystal structure may aid further development of antimycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.

The microglial reaction signature revealed by RNAseq from individual mice.

Microglial cells have a double life as the immune cells of the brain in times of stress but have also specific physiological functions in homeostatic conditions. In pathological contexts, microglia undergo a phenotypic switch called "reaction" that promotes the initiation and the propagation of neuro-inflammation. Reaction is complex, molecularly heterogeneous and still poorly characterized, leading to the concept that microglial reactivity might be too diverse to be molecularly defined. However, it remains unknown whether reactive microglia from different pathological contexts share a common molecular signature. Using improved flow cytometry and RNAseq approaches we studied, with higher statistical power, the remodeling of microglia transcriptome in a mouse model of sepsis. Through bioinformatic comparison of our results with published datasets, we defined the microglial reactome as a set of genes discriminating reactive from homeostatic microglia. Ultimately, we identified a subset of 86 genes deregulated in both acute and neurodegenerative conditions. Our data provide a new comprehensive resource that includes functional analysis and specific molecular markers of microglial reaction which represent new tools for its unambiguous characterization.

Delivery of miR-146a to Ly6Chigh Monocytes Inhibits Pathogenic Bone Erosion in Inflammatory Arthritis.

Rationale: Monocytes play critical roles in the pathogenesis of arthritis by contributing to the inflammatory response and bone erosion. Among genes involved in regulating monocyte functions, miR-146a negatively regulates the inflammatory response and osteoclast differentiation of monocytes. It is also the only miRNA reported to differentially regulate the cytokine response of the two classical Ly6Chigh and non-classical Ly6Clow monocyte subsets upon bacterial challenge. Although miR-146a is overexpressed in many tissues of arthritic patients, its specific role in monocyte subsets under arthritic conditions remains to be explored. Methods: We analyzed the monocyte subsets during collagen-induced arthritis (CIA) development by flow cytometry. We quantified the expression of miR-146a in classical and non-classical monocytes sorted from healthy and CIA mice, as well as patients with rheumatoid arthritis (RA). We monitored arthritis features in miR-146a-/- mice and assessed in vivo the therapeutic potential of miR-146a mimics delivery to Ly6Chigh monocytes. We performed transcriptomic and pathway enrichment analyses on both monocyte subsets sorted from wild type and miR-146a-/- mice. Results: We showed that the expression of miR-146a is reduced in the Ly6Chigh subset of CIA mice and in the analogous monocyte subset (CD14+CD16-) in humans with RA as compared with healthy controls. The ablation of miR-146a in mice worsened arthritis severity, increased osteoclast differentiation in vitro and bone erosion in vivo. In vivo delivery of miR-146a to Ly6Chigh monocytes, and not to Ly6Clow monocytes, rescues bone erosion in miR-146a-/- arthritic mice and reduces osteoclast differentiation and pathogenic bone erosion in CIA joints of miR-146a+/+ mice, with no effect on inflammation. Silencing of the non-canonical NF-κB family member RelB in miR-146a-/- Ly6Chigh monocytes uncovers a role for miR-146a as a key regulator of the differentiation of Ly6Chigh, and not Ly6Clow, monocytes into osteoclasts under arthritic conditions. Conclusion: Our results show that classical monocytes play a critical role in arthritis bone erosion. They demonstrate the theranostics potential of manipulating miR-146a expression in Ly6Chigh monocytes to prevent joint destruction while sparing inflammation in arthritis.

miR-125b controls monocyte adaptation to inflammation through mitochondrial metabolism and dynamics.

Metabolic changes drive monocyte differentiation and fate. Although abnormal mitochondria metabolism and innate immune responses participate in the pathogenesis of many inflammatory disorders, molecular events regulating mitochondrial activity to control life and death in monocytes remain poorly understood. We show here that, in human monocytes, microRNA-125b (miR-125b) attenuates the mitochondrial respiration through the silencing of the BH3-only proapoptotic protein BIK and promotes the elongation of the mitochondrial network through the targeting of the mitochondrial fission process 1 protein MTP18, leading to apoptosis. Proinflammatory activation of monocyte-derived macrophages is associated with a concomitant increase in miR-125b expression and decrease in BIK and MTP18 expression, which lead to reduced oxidative phosphorylation and enhanced mitochondrial fusion. In a chronic inflammatory systemic disorder, CD14+ blood monocytes display reduced miR-125b expression as compared with healthy controls, inversely correlated with BIK and MTP18 messenger RNA expression. Our findings not only identify BIK and MTP18 as novel targets for miR-125b that control mitochondrial metabolism and dynamics, respectively, but also reveal a novel function for miR-125b in regulating metabolic adaptation of monocytes to inflammation. Together, these data unravel new molecular mechanisms for a proapoptotic role of miR-125b in monocytes and identify potential targets for interfering with excessive inflammatory activation of monocytes in inflammatory disorders.

MicroRNA Profiling of B Cell Subsets from Systemic Lupus Erythematosus Patients Reveals Promising Novel Biomarkers.

MicroRNAs control the differentiation and function of B cells, which are considered key elements in the pathogenesis of systemic lupus erythematosus (SLE). However, a common micro(mi)RNA signature has not emerged since published data includes patients of variable ethnic background, type of disease, and organ involvement, as well as heterogeneous cell populations. Here, we aimed at identifying a miRNA signature of purified B cells from renal and non-renal severe SLE patients of Latin American background, a population known to express severe disease. Genome-wide miRNA expression analyses were performed on naive and memory B cells and revealed two categories of miRNA signatures. The first signature represents B cell subset-specific miRNAs deregulated in SLE: 11 and six miRNAs discriminating naive and memory B cells of SLE patients from healthy controls (HC), respectively. Whether the miRNA was up or down-regulated in memory B cells as compared with naive B cells in HC, this difference was abolished in SLE patients, and vice versa. The second signature identifies six miRNAs associated with specific pathologic features affecting renal outcome, providing a further understanding for SLE pathogenesis. Overall, the present work provided promising biomarkers in molecular diagnostics for disease severity as well as potential new targets for therapeutic intervention in SLE.

Estrogen and retinoic acid antagonistically regulate several microRNA genes to control aerobic glycolysis in breast cancer cells.

In addition to estrogen receptor modulators, retinoic acid and other retinoids are promising agents to prevent breast cancer. Retinoic acid and estrogen exert antagonistic regulations on the transcription of coding genes and we evaluated here whether these two compounds have similar effects on microRNAs. Using an integrative approach based on several bioinformatics resources together with experimental validations, we indeed found that retinoic acid positively regulates miR-210 and miR-23a/24-2 expressions and is counteracted by estrogen. Conversely, estrogen increased miR-17/92 and miR-424/450b expressions and is inhibited by retinoic acid. In silico functional enrichment further revealed that this combination of transcriptional/post-transcriptional regulations fully impacts on the molecular effects of estrogen and retinoic acid. Besides, we unveiled a novel effect of retinoic acid on aerobic glycolysis. We specifically showed that it increases extracellular lactate production, an effect counteracted by the miR-210 and the miR-23a/24-2, which simultaneously target lactate dehydrogenase A and B mRNAs. Together our results provide a new framework to better understand the estrogen/retinoic acid antagonism in breast cancer cells.

Expression profile and up-regulation of PRAX-1 mRNA by antidepressant treatment in the rat brain.

A protein associated with the peripheral-type benzodiazepine receptor (PRAX-1) has recently been cloned, but its regional distribution in the central nervous system and its function remain to be clarified. In situ hybridization was carried out to localize PRAX-1 mRNA in the rat brain and revealed a high expression of the transcript in limbic structures such as the CA1 region of the hippocampus, as well as the dentate gyrus, septum, amygdala, and the islands of Calleja. A dense hybridization signal was also observed in the nucleus accumbens, caudate nucleus, olfactory tubercle, pineal gland, and cerebellar cortex. PRAX-1 mRNA expression was largely neuronal; it colocalized with neuron-specific enolase but not glial fibrillary acidic protein. Long-term treatments (21 days) with the neuroleptic haloperidol increased PRAX-1 mRNA expression only in the dentate gyrus, whereas anxiolytic/anticonvulsant diazepam had no effect in any of the hippocampal region studied. Repeated electroconvulsive shock administration significantly enhanced PRAX1 expression in the CA1 subfield and dentate gyrus. Several classes of antidepressant treatment, including serotonin selective reuptake inhibitor (fluoxetine), mixed serotonin- and norepinephrine-uptake inhibitor (imipramine), and monoamine oxidase inhibitors (iproniazid and tranylcypromine), shared this effect. Furthermore, the selective nonpeptide NK2 receptor antagonist (S)-N-methyl-N-[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide (SR48968), which shows an antidepressant profile in animal studies, also enhanced PRAX-1 mRNA expression. These results point to a potential role of PRAX-1 function in the central nervous system and suggest that the up-regulation of PRAX-1 mRNA represents a common action of chronic antidepressant treatment.