Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3.

Glycosphingolipids are important components of the plasma membrane where they modulate the activities of membrane proteins including signalling receptors. Glycosphingolipid synthesis relies on competing reactions catalysed by Golgi-resident enzymes during the passage of substrates through the Golgi cisternae. The glycosphingolipid metabolic output is determined by the position and levels of the enzymes within the Golgi stack, but the mechanisms that coordinate the intra-Golgi localisation of the enzymes are poorly understood. Here, we show that a group of sequentially-acting enzymes operating at the branchpoint among glycosphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting as a component of the cisternal maturation mechanism. Through these effects, GOLPH3 controls the sub-Golgi localisation and the lysosomal degradation rate of specific enzymes. Increased GOLPH3 levels, as those observed in tumours, alter glycosphingolipid synthesis and plasma membrane composition thereby promoting mitogenic signalling and cell proliferation. These data have medical implications as they outline a novel oncogenic mechanism of action for GOLPH3 based on glycosphingolipid metabolism.

Arylvinylpiperazine Amides, a New Class of Potent Inhibitors Targeting QcrB of Mycobacterium tuberculosis.

New drugs are needed to control the current tuberculosis (TB) pandemic caused by infection with Mycobacterium tuberculosis We report here on our work with AX-35, an arylvinylpiperazine amide, and four related analogs, which are potent antitubercular agents in vitro All five compounds showed good activity against M. tuberculosisin vitro and in infected THP-1 macrophages, while displaying only mild cytotoxicity. Isolation and characterization of M. tuberculosis-resistant mutants to the arylvinylpiperazine amide derivative AX-35 revealed mutations in the qcrB gene encoding a subunit of cytochrome bc1 oxidase, one of two terminal oxidases of the electron transport chain. Cross-resistance studies, allelic exchange, transcriptomic analyses, and bioenergetic flux assays provided conclusive evidence that the cytochrome bc1-aa3 is the target of AX-35, although the compound appears to interact differently with the quinol binding pocket compared to previous QcrB inhibitors. The transcriptomic and bioenergetic profiles of M. tuberculosis treated with AX-35 were similar to those generated by other cytochrome bc1 oxidase inhibitors, including the compensatory role of the alternate terminal oxidase cytochrome bd in respiratory adaptation. In the absence of cytochrome bd oxidase, AX-35 was bactericidal against M. tuberculosis Finally, AX-35 and its analogs were active in an acute mouse model of TB infection, with two analogs displaying improved activity over the parent compound. Our findings will guide future lead optimization to produce a drug candidate for the treatment of TB and other mycobacterial diseases, including Buruli ulcer and leprosy.IMPORTANCE New drugs against Mycobacterium tuberculosis are urgently needed to deal with the current global TB pandemic. We report here on the discovery of a series of arylvinylpiperazine amides (AX-35 to AX-39) that represent a promising new family of compounds with potent in vitro and in vivo activities against M. tuberculosis AX compounds target the QcrB subunit of the cytochrome bc1 terminal oxidase with a different mode of interaction compared to those of known QcrB inhibitors. This study provides the first multifaceted validation of QcrB inhibition by recombineering-mediated allelic exchange, gene expression profiling, and bioenergetic flux studies. It also provides further evidence for the compensatory role of cytochrome bd oxidase upon QcrB inhibition. In the absence of cytochrome bd oxidase, AX compounds are bactericidal, an encouraging property for future antimycobacterial drug development.

Optimized Background Regimen for Treatment of Active Tuberculosis with the Next-Generation Benzothiazinone Macozinone (PBTZ169).

The efficacy of the standardized four-drug regimen (comprising isoniazid, rifampin, pyrazinamide, and ethambutol) for the treatment of tuberculosis (TB) is menaced by the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis Intensive efforts have been made to develop new antibiotics or to repurpose old drugs, and several of these are currently being evaluated in clinical trials for their antitubercular activity. Among the new candidate drugs is macozinone (MCZ), the piperazine-containing benzothiazinone PBTZ169, which is currently being evaluated in phase I/II clinical trials. Here, we determined the in vitro and in vivo activity of MCZ in combination with a range of anti-TB drugs in order to design a new regimen against active TB. Two-drug combinations with MCZ were tested against M. tuberculosis using checkerboard and CFU enumeration after drug exposure assays. MCZ was observed to have no interactions with all first- and second-line anti-TB drugs. At the MIC of each drug, MCZ with either bedaquiline (BDQ), clofazimine (CLO), delamanid (DMD), or sutezolid (STZ) reduced the bacterial burden by 2 logs compared to that achieved with the drugs alone, indicating synergism. MCZ also displayed synergism with clomiphene (CLM), a potential inhibitor of the undecaprenyl pyrophosphate synthase (UppS) in mycobacteria. For all the other drugs tested in combination with MCZ, no synergistic activity was observed. Neither antagonism nor increased cytotoxicity was found for most combinations, suggesting that MCZ could be added to different TB treatment regimens without any significant adverse effects.

Structure-Based Drug Design and Characterization of Sulfonyl-Piperazine Benzothiazinone Inhibitors of DprE1 from Mycobacterium tuberculosis.

Macozinone (MCZ) is a tuberculosis (TB) drug candidate that specifically targets the essential flavoenzyme DprE1, thereby blocking synthesis of the cell wall precursor decaprenyl phosphoarabinose (DPA) and provoking lysis of Mycobacterium tuberculosis As part of the MCZ backup program, we exploited structure-guided drug design to produce a new series of sulfone-containing derivatives, 2-sulfonylpiperazin 8-nitro 6-trifluoromethyl 1,3-benzothiazin-4-one, or sPBTZ. These compounds are less active than MCZ but have a better solubility profile, and some derivatives display enhanced stability in microsomal assays. DprE1 was efficiently inhibited by sPBTZ, and covalent adducts with the active-site cysteine residue (C387) were formed. However, despite the H-bonding potential of the sulfone group, no additional bonds were seen in the crystal structure of the sPBTZ-DprE1 complex with compound 11326127 compared to MCZ. Compound 11626091, the most advanced sPBTZ, displayed good antitubercular activity in the murine model of chronic TB but was less effective than MCZ. Nonetheless, further testing of this MCZ backup compound is warranted as part of combination treatment with other TB drugs.

Rv3852 (H-NS) of Mycobacterium tuberculosis Is Not Involved in Nucleoid Compaction and Virulence Regulation.

A handful of nucleoid-associated proteins (NAPs) regulate the vast majority of genes in a bacterial cell. H-NS, the histone-like nucleoid-structuring protein, is one of these NAPs and protects Escherichia coli from foreign gene expression. Though lacking any sequence similarity with E. coli H-NS, Rv3852 was annotated as the H-NS ortholog in Mycobacterium tuberculosis, as it resembles human histone H1. The role of Rv3852 was thoroughly investigated by immunoblotting, subcellular localization, construction of an unmarked rv3852 deletion in the M. tuberculosis genome, and subsequent analysis of the resulting Δrv3852 strain. We found that Rv3852 was predominantly present in the logarithmic growth phase with a decrease in protein abundance in stationary phase. Furthermore, it was strongly associated with the cell membrane and not detected in the cytosolic fraction, nor was it secreted. The Δrv3852 strain displayed no growth defect or morphological abnormalities. Quantitative measurement of nucleoid localization in the Δrv3852 mutant strain compared to that in the parental H37Rv strain showed no difference in nucleoid position or spread. Infection of macrophages as well as severe combined immunodeficient (SCID) mice demonstrated that loss of Rv3852 had no detected influence on the virulence of M. tuberculosis We thus conclude that M. tuberculosis Rv3852 is not involved in pathogenesis and is not a typical NAP. The existence of an as yet undiscovered Rv3852 ortholog cannot be excluded, although this role is likely played by the well-characterized Lsr2 protein.IMPORTANCEMycobacterium tuberculosis is the causative agent of the lung infection tuberculosis, claiming more than 1.5 million lives each year. To understand the mechanisms of latent infection, where M. tuberculosis can stay dormant inside the human host, we require deeper knowledge of the basic biology and of the regulatory networks. In our work, we show that Rv3852, previously annotated as H-NS, is not a typical nucleoid-associated protein (NAP) as expected from its initial annotation. Rv3852 from M. tuberculosis has neither influence on nucleoid shape or compaction nor a role in virulence. Our findings reduce the repertoire of identified nucleoid-associated proteins in M. tuberculosis to four transcription regulators and underline the importance of genetic studies to assign a function to bacterial genes.

New 1-hydroxy-2-thiopyridine derivatives active against both replicating and dormant Mycobacterium tuberculosis.

Tuberculosis (TB) treatment is confounded by the range of metabolic states displayed by Mycobacterium tuberculosis, by the long duration required and by the increasing prevalence of drug-resistant strains. Latent TB infection is especially difficult to treat due to the phenotypic antibiotic resistance of non-replicating M. tuberculosis. Therefore, the development of new drugs effective against both active and latent TB infection is needed. New 1-hydroxy-2-thiopyridine derivatives were synthesized and found to be highly effective in vitro against both actively growing and dormant non-culturable M. tuberculosis. Such compounds are leads for the development of new drugs for all forms of TB including latent infection.

Discovery and Biosynthesis of Gladiolin: A Burkholderia gladioli Antibiotic with Promising Activity against Mycobacterium tuberculosis.

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.

Lansoprazole is an antituberculous prodrug targeting cytochrome bc1.

Better antibiotics capable of killing multi-drug-resistant Mycobacterium tuberculosis are urgently needed. Despite extensive drug discovery efforts, only a few promising candidates are on the horizon and alternative screening protocols are required. Here, by testing a panel of FDA-approved drugs in a host cell-based assay, we show that the blockbuster drug lansoprazole (Prevacid), a gastric proton-pump inhibitor, has intracellular activity against M. tuberculosis. Ex vivo pharmacokinetics and target identification studies reveal that lansoprazole kills M. tuberculosis by targeting its cytochrome bc1 complex through intracellular sulfoxide reduction to lansoprazole sulfide. This novel class of cytochrome bc1 inhibitors is highly active against drug-resistant clinical isolates and spares the human H(+)K(+)-ATPase thus providing excellent opportunities for targeting the major pathogen M. tuberculosis. Our finding provides proof of concept for hit expansion by metabolic activation, a powerful tool for antibiotic screens.

The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1 from Mycobacterium tuberculosis.

8-Nitro-benzothiazinones (BTZs), such as BTZ043 and PBTZ169, inhibit decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1) and display nanomolar bactericidal activity against Mycobacterium tuberculosis in vitro. Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. To date, substitution of the 8-nitro group has led to extensive loss of antimycobacterial activity. Here, we report the synthesis and characterization of the pyrrole-benzothiazinones PyrBTZ01 and PyrBTZ02, non-nitro-benzothiazinones that retain significant antimycobacterial activity, with MICs of 0.16 µg/ml against M. tuberculosis. These compounds inhibit DprE1 with 50% inhibitory concentration (IC50) values of <8 µM and present favorable in vitro absorption-distribution-metabolism-excretion/toxicity (ADME/T) and in vivo pharmacokinetic profiles. The most promising compound, PyrBTZ01, did not show efficacy in a mouse model of acute tuberculosis, suggesting that BTZ-mediated killing through DprE1 inhibition requires a combination of both covalent bond formation and compound potency.

Towards a new combination therapy for tuberculosis with next generation benzothiazinones.

The benzothiazinone lead compound, BTZ043, kills Mycobacterium tuberculosis by inhibiting the essential flavo-enzyme DprE1, decaprenylphosphoryl-beta-D-ribose 2-epimerase. Here, we synthesized a new series of piperazine-containing benzothiazinones (PBTZ) and show that, like BTZ043, the preclinical candidate PBTZ169 binds covalently to DprE1. The crystal structure of the DprE1-PBTZ169 complex reveals formation of a semimercaptal adduct with Cys387 in the active site and explains the irreversible inactivation of the enzyme. Compared to BTZ043, PBTZ169 has improved potency, safety and efficacy in zebrafish and mouse models of tuberculosis (TB). When combined with other TB drugs, PBTZ169 showed additive activity against M. tuberculosis in vitro except with bedaquiline (BDQ) where synergy was observed. A new regimen comprising PBTZ169, BDQ and pyrazinamide was found to be more efficacious than the standard three drug treatment in a murine model of chronic disease. PBTZ169 is thus an attractive drug candidate to treat TB in humans.