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The broad-spectrum antibiotic d -cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of d -alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and d -Ala: d -Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5′-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria. d -Cycloserine inactivates alanine racemase by forming an adduct with the pyridoxal 5′-phosphate cofactor, but structural and spectroscopic analyses reveal that reactivation occurs on adduct hydrolysis and product rearrangement to a stable oxime.

D-cycloserine is an antibiotic which targets sequential bacterial cell wall peptidoglycan biosynthesis enzymes: alanine racemase and D-alanine:D-alanine ligase. By a combination of structural, chemical and mechanistic studies here we show that the inhibition of D-alanine:D-alanine ligase by the antibiotic D-cycloserine proceeds via a distinct phosphorylated form of the drug. This mechanistic insight reveals a bimodal mechanism of action for a single antibiotic on different enzyme targets and has significance for the design of future inhibitor molecules based on this chemical structure. The antibiotic D-cycloserine (DCS) targets the peptidoglycan biosynthesis enzyme D-Ala-D-Ala ligase (Ddl). Here the authors reveal the DCS inhibitory mechanism by determining the structure of E. coli DdlB with a phosphorylated DCS molecule in the active site that formed in crystallo and mimics the D-alanyl phosphate intermediate.

The emergence of untreatable drug‐resistant strains of Mycobacterium tuberculosis is a major public health problem worldwide, and the identification of new efficient treatments is urgently needed. Mycobacterium tuberculosis cytochrome P450 CYP121A1 is a promising drug target for the treatment of tuberculosis owing to its essential role in mycobacterial growth. Using a rational approach, which includes molecular modelling studies, three series of azole pyrazole derivatives were designed through two synthetic pathways. The synthesized compounds were biologically evaluated for their inhibitory activity towards M. tuberculosis and their protein binding affinity (KD). Series 3 biarylpyrazole imidazole derivatives were the most effective with the isobutyl (10 f) and tert‐butyl (10 g) compounds displaying optimal activity (MIC 1.562 μg/mL, KD 0.22 μM (10 f) and 4.81 μM (10 g)). The spectroscopic data showed that all the synthesised compounds produced a type II red shift of the heme Soret band indicating either direct binding to heme iron or (where less extensive Soret shifts are observed) putative indirect binding via an interstitial water molecule. Evaluation of biological and physicochemical properties identified the following as requirements for activity: LogP >4, H‐bond acceptors/H‐bond donors 4/0, number of rotatable bonds 5–6, molecular volume >340 Å3, topological polar surface area <40 Å2. Three series of azole pyrazole derivatives were synthesised and evaluated for their inhibitory activity towards M. tuberculosis and their CYP121A1 protein binding affinity (KD). Biarylpyrazole imidazole derivatives were the most effective with the isobutyl (10 f) and tert‐butyl (10 g) compounds displaying optimal activity (MIC 1.562 μg/mL, KD 0.22 μM (10 f) and 4.81 μM (10 g)). Molecular modelling, X‐ray crystallography and evaluation of biological and physicochemical properties provide useful SAR for further development.

Drug resistant infections represent one of the most challenging medical problems of our time. D -cycloserine is an antibiotic used for six decades without significant appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We therefore investigated why Mycobacterium tuberculosis fails to become resistant to D -cycloserine. To address this question, we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low rate of emergence of D-cycloserine resistance mutations is the dominant biological factor delaying the appearance of clinical resistance to this antibiotic. Furthermore, we also identified potential compensatory mechanisms able to minimize the severe fitness costs of primary D -cycloserine resistance conferring mutations. D-cycloserine (DCS) has been used for decades to treat Mycobacterium tuberculosis (Mtb) but resistance is rarely observed in clinical isolates. Here, the authors report ultra-low rate of emergence of resistance mutations as the underlying mechanism of DCS resistance evasion in Mtb.

Mycobacterium tuberculosis ’s ( Mtb ) autarkic lifestyle within the host involves rewiring its transcriptional networks to combat host-induced stresses. With the help of RNA sequencing performed under various stress conditions, we identified that genes belonging to Mtb sulfur metabolism pathways are significantly upregulated during oxidative stress. Using an integrated approach of microbial genetics, transcriptomics, metabolomics, animal experiments, chemical inhibition, and rescue studies, we investigated the biological role of non-canonical L -cysteine synthases, CysM and CysK2. While transcriptome signatures of RvΔcysM and RvΔcysK2 appear similar under regular growth conditions, we observed unique transcriptional signatures when subjected to oxidative stress. We followed pool size and labelling ( 34 S) of key downstream metabolites, viz. mycothiol and ergothioneine, to monitor L-cysteine biosynthesis and utilization. This revealed the significant role of distinct L-cysteine biosynthetic routes on redox stress and homeostasis. CysM and CysK2 independently facilitate Mtb survival by alleviating host-induced redox stress, suggesting they are not fully redundant during infection. With the help of genetic mutants and chemical inhibitors, we show that CysM and CysK2 serve as unique, attractive targets for adjunct therapy to combat mycobacterial infection.

ATP-phosphoribosyltransferase (ATP-PRT) is a hexameric enzyme in conformational equilibrium between an open and seemingly active state and a closed and presumably inhibited form. The structure-function relationship of allosteric regulation in this system is still not fully understood. Here, we develop a screening strategy for modulators of ATP-PRT and identify 3-(2-thienyl)- l- alanine (TIH) as an allosteric activator of this enzyme. Kinetic analysis reveals co-occupancy of the allosteric sites by TIH and l -histidine. Crystallographic and native ion-mobility mass spectrometry data show that the TIH-bound activated form of the enzyme closely resembles the inhibited l -histidine-bound closed conformation, revealing the uncoupling between ATP-PRT open and closed conformations and its functional state. These findings suggest that dynamic processes are responsible for ATP-PRT allosteric regulation and that similar mechanisms might also be found in other enzymes bearing a ferredoxin-like allosteric domain. Active and inactive state ATP-phosphoribosyltransferases (ATP-PRTs) are believed to have different conformations. Here the authors show that in both states, ATP-PRT has a similar structural arrangement, suggesting that dynamic alterations are involved in ATP-PRT regulation by allosteric modulators.

Antiretroviral role for SAMHD1 protein Mutations in SAMHD1 protein are associated with the human autoimmune disease Aicardi–Goutières syndrome, and SAMHD1 was recently shown to be responsible for restriction of HIV-1 replication in myeloid cells. Ian Taylor and colleagues reveal a previously unknown function of SAMHD1 that could explain its antivirus role. They provide a crystal structure of the catalytic core of SAMHD1 and show that it is a dGTP-stimulated triphosphohydrolase that hydrolyses dNTPs, the building blocks of DNA. This activity may prevent reverse transcription and viral synthesis of complementary DNA by keeping the concentration of cellular dNTPs at a low level. SAMHD1, an analogue of the murine interferon (IFN)-γ-induced gene Mg11 (ref. 1 ), has recently been identified as a human immunodeficiency virus-1 (HIV-1) restriction factor that blocks early-stage virus replication in dendritic and other myeloid cells 2 , 3 and is the target of the lentiviral protein Vpx, which can relieve HIV-1 restriction 4 , 5 , 6 , 7 . SAMHD1 is also associated with Aicardi–Goutières syndrome (AGS), an inflammatory encephalopathy characterized by chronic cerebrospinal fluid lymphocytosis and elevated levels of the antiviral cytokine IFN-α 8 . The pathology associated with AGS resembles congenital viral infection, such as transplacentally acquired HIV. Here we show that human SAMHD1 is a potent dGTP-stimulated triphosphohydrolase that converts deoxynucleoside triphosphates to the constituent deoxynucleoside and inorganic triphosphate. The crystal structure of the catalytic core of SAMHD1 reveals that the protein is dimeric and indicates a molecular basis for dGTP stimulation of catalytic activity against dNTPs. We propose that SAMHD1, which is highly expressed in dendritic cells, restricts HIV-1 replication by hydrolysing the majority of cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA (cDNA) synthesis.

The increasing prevalence of antimicrobial resistance is an important challenge that warrants new approaches to antibiotic development. Currently, all antibiotics inhibit biological processes. To explore whether activation of a biochemical pathway can elicit bactericidal effects we engineered variants of Mycobacterium tuberculosis ATP-phosphoribosyltransferase (ATP-PRT) that are resistant to allosteric inhibition by l -histidine, leading to supraphysiological activation of ATP-PRT and l -histidine overproduction. Upregulation of L-histidine biosynthesis significantly reduces the growth of M. tuberculosis in culture and causes a loss of fitness owing to nutrient and energy depletion. Moreover, the expression of allosteric variants in M. tuberculosis significantly reduced infections in human macrophages and in a mouse model of infection. Thus, metabolic activation represents a new mycobactericidal mechanism that could be applied to antimycobacterial drug discovery. Here, the authors report metabolic activation as a potential antimicrobial strategy against Mycobacterium tuberculosis . They show that supra-physiological activation of L-histidine biosynthesis reduces bacterial growth and causes loss of fitness due to nutrient and energy depletion.

Tuberculosis (TB) is the biggest cause of death from infectious disease worldwide. The causative agent, Mycobacterium tuberculosis (Mtb), possesses a complex cell envelope comprised of multiple classes of unique lipids. The macrophage phagosome is a key reservoir of infection in pulmonary TB and multiple studies have shown that inorganic phosphate (Pi) is limiting in this environment. Here, we show that during Pi restriction the Mtb lipidome markedly remodels such that phospholipids are replaced with multiple classes of phosphorus-free lipids. This envelope lipidome remodelling suggests that standard Mtb culture conditions that use media with high concentrations of Pi do not reflect the physiologic environment during infection, thereby undermining vaccine and drug development for tuberculosis. Further, we discover that Mtb can metabolise phospholipid polar heads abundant in host pulmonary surfactant as an alternative phosphate source. Therefore, we present two mechanisms where Mtb manipulates lipid metabolism to overcome host restriction. Here, the authors show that Mycobacterium tuberculosis manipulates lipid metabolism to overcome host restriction, by remodelling its lipidome and utilising host lipids as an alternative phosphate source.

Persistent TH2 cytokine responses following chronic helminth infections can often lead to the development of tissue pathology and fibrotic scarring. Despite a good understanding of the cellular mechanisms involved in fibrogenesis, there are very few therapeutic options available, highlighting a significant medical need and gap in our understanding of the molecular mechanisms of TH2-mediated immunopathology. In this study, we found that the Map3 kinase, TPL-2 (Map3k8; Cot) regulated TH2-mediated intestinal, hepatic and pulmonary immunopathology following Schistosoma mansoni infection or S. mansoni egg injection. Elevated inflammation, TH2 cell responses and exacerbated fibrosis in Map3k8-/-mice was observed in mice with myeloid cell-specific (LysM) deletion of Map3k8, but not CD4 cell-specific deletion of Map3k8, indicating that TPL-2 regulated myeloid cell function to limit TH2-mediated immunopathology. Transcriptional and metabolic assays of Map3k8-/-M2 macrophages identified that TPL-2 was required for lipolysis, M2 macrophage activation and the expression of a variety of genes involved in immuno-regulatory and pro-fibrotic pathways. Taken together this study identified that TPL-2 regulated TH2-mediated inflammation by supporting lipolysis and M2 macrophage activation, preventing TH2 cell expansion and downstream immunopathology and fibrosis.