Journal: Chemistry & biology
5-Aminoimidazole-4-carboxamide ribonucleotide (known as ZMP) is a metabolite produced in de novo purine biosynthesis and histidine biosynthesis, but only utilized in the cell by a homodimeric bifunctional enzyme (called ATIC) that catalyzes the last two steps of de novo purine biosynthesis. ZMP is known to act as an allosteric activator of the cellular energy sensor adenosine monophosphate-activated protein kinase (AMPK), when exogenously administered as the corresponding cell-permeable ribonucleoside. Here, we demonstrate that endogenous ZMP, produced by the aforementioned metabolic pathways, is also capable of activating AMPK. Using an inhibitor of ATIC homodimerization to block the ninth step of de novo purine biosynthesis, we demonstrate that the subsequent increase in endogenous ZMP activates AMPK and its downstream signaling pathways. We go on to illustrate the viability of using this approach to AMPK activation as a therapeutic strategy with an in vivo mouse model for metabolic disorders.
Evidence suggests that some nonsteroidal anti-inflammatory drugs (NSAIDs) possess antibacterial properties with an unknown mechanism. We describe the in vitro antibacterial properties of the NSAIDs carprofen, bromfenac, and vedaprofen, and show that these NSAIDs inhibit the Escherichia coli DNA polymerase III β subunit, an essential interaction hub that acts as a mobile tether on DNA for many essential partner proteins in DNA replication and repair. Crystal structures show that the three NSAIDs bind to the sliding clamp at a common binding site required for partner binding. Inhibition of interaction of the clamp loader and/or the replicative polymerase α subunit with the sliding clamp is demonstrated using an in vitro DNA replication assay. NSAIDs thus present promising lead scaffolds for novel antibacterial agents targeting the sliding clamp.
Mycolic acids are major and specific lipid components of the mycobacterial cell envelope and are essential for the survival of members of the genus Mycobacterium that contains the causative agents of both tuberculosis and leprosy. In the alarming context of the emergence of multidrug-resistant, extremely drug-resistant, and totally drug-resistant tuberculosis, understanding the biosynthesis of these critical determinants of the mycobacterial physiology is an important goal to achieve, because it may open an avenue for the development of novel antimycobacterial agents. This review focuses on the chemistry, structures, and known inhibitors of mycolic acids and describes progress in deciphering the mycolic acid biosynthetic pathway. The functional and key biological roles of these molecules are also discussed, providing a historical perspective in this dynamic area.
Micacocidin is a thiazoline-containing natural product from the bacterium Ralstonia solanacearum that shows significant activity against Mycoplasma pneumoniae. The presence of a pentylphenol moiety distinguishes micacocidin from the structurally related siderophore yersiniabactin, and this residue also contributes to the potent antimycoplasma effects. The biosynthesis of the pentylphenol moiety, as deduced from bioinformatic analysis and stable isotope feeding experiments, involves an iterative type I polyketide synthase (iPKS), which generates a linear tetraketide intermediate from acyl carrier protein-tethered hexanoic acid by three consecutive, decarboxylative Claisen condensations with malonyl-coenzyme A. The final conversion into 6-pentylsalicylic acid depends on a ketoreductase domain within the iPKS, as demonstrated by heterologous expression in E. coli and subsequent site-directed mutagenesis experiments. Our results unveil the early steps in micacocidin biosynthesis and illuminate a bacterial enzyme that functionally resembles fungal polyketide synthases.
Monoacylglycerol lipase (MAGL) terminates the signaling function of the endocannabinoid, 2-arachidonoylglycerol (2-AG). During 2-AG hydrolysis, MAGL liberates arachidonic acid, feeding the principal substrate for the neuroinflammatory prostaglandins. In cancer cells, MAGL redirects lipid stores toward protumorigenic signaling lipids. Thus MAGL inhibitors may have great therapeutic potential. Although potent and increasingly selective MAGL inhibitors have been described, their number is still limited. Here, we have characterized piperazine and piperidine triazole ureas that combine the high potency attributable to the triazole leaving group together with the bulky aromatic benzodioxolyl moiety required for selectivity, culminating in compound JJKK-048 that potently (IC50 < 0.4 nM) inhibited human and rodent MAGL. JJKK-048 displayed low cross-reactivity with other endocannabinoid targets. Activity-based protein profiling of mouse brain and human melanoma cell proteomes suggested high specificity also among the metabolic serine hydrolases.
Lipid translocation from one lipid bilayer leaflet to the other, termed flip-flop, is required for the distribution of newly synthesized phospholipids during membrane biogenesis. However, a dedicated biogenic lipid flippase has not yet been identified. Here, we show that the efficiency by which model transmembrane peptides facilitate flip of reporter lipids with different headgroups critically depends on their content of helix-destabilizing residues, the charge state of polar flanking residues, and the composition of the host membrane. In particular, increased backbone dynamics of the transmembrane helix relates to its increased ability to flip lipids with phosphatidylcholine and phosphatidylserine headgroups, whereas a more rigid helix favors phosphatidylethanolamine flip. Further, the transmembrane domains of many SNARE protein subtypes share essential features with the dynamic model peptides. Indeed, recombinant SNAREs possess significant lipid flippase activity.
Rhamnolipids are multipurpose surface-active molecules produced by the bacterium Pseudomonas aeruginosa from L-rhamnose and R-3-hydroxyalkanoate (C10±2) precursors. R-3-hydroxyalkanoate precursor is believed to be synthesized de novo. We demonstrate, however, that β-oxidation is the predominant source of this precursor. Inhibition of β-oxidation sharply decreases rhamnolipids production, even when using a nonfatty acid carbon source (glycerol). Isotope tracing shows that β-oxidation intermediates are direct precursors of rhamnolipids. A mutant-based survey revealed an operon coding for enoyl-CoA hydratases/isomerases (ECH/I), named RhlYZ, implicated in rhamnolipids production via an axial role in 3-hydroxyalkanoate synthesis. In vitro, RhlZ is an R-ECH/I transforming 2-decenoyl-CoA, a β-oxidation intermediate, into R-3-hydroxydecanoyl-CoA, the potential rhamnolipids precursor. Interestingly, polyhydroxyalkanoates share with rhamnolipids the RhlYZ-generated R-3-hydroxyalkanoates pool, as demonstrated by the decrease of polyhydroxyalkanoates upon mutation of rhlYZ and the increase of rhamnolipids in a polyhydroxyalkanoates-defective mutant.
Gram-negative bacteria utilize heptoses as part of their repertoire of extracellular polysaccharide virulence determinants. Disruption of heptose biosynthesis offers an attractive target for novel antimicrobials. A critical step in the synthesis of heptoses is their 1-O phosphorylation, mediated by kinases such as HldE or WcbL. Here, we present the structure of WcbL from Burkholderia pseudomallei. We report that WcbL operates through a sequential ordered Bi-Bi mechanism, loading the heptose first and then ATP. We show that dimeric WcbL binds ATP anti-cooperatively in the absence of heptose, and cooperatively in its presence. Modeling of WcbL suggests that heptose binding causes an elegant switch in the hydrogen-bonding network, facilitating the binding of a second ATP molecule. Finally, we screened a library of drug-like fragments, identifying hits that potently inhibit WcbL. Our results provide a novel mechanism for control of substrate binding and emphasize WcbL as an attractive anti-microbial target for Gram-negative bacteria.
Pharmacological mitigation of injuries caused by high-dose ionizing radiation is an unsolved medical problem. A specific nonlipid agonist of the type 2 G protein coupled receptor for lysophosphatidic acid (LPA2) 2-[4-(1,3-dioxo-1H,3H-benzoisoquinolin-2-yl)butylsulfamoyl]benzoic acid (DBIBB) when administered with a postirradiation delay of up to 72 hr reduced mortality of C57BL/6 mice but not LPA2 knockout mice. DBIBB mitigated the gastrointestinal radiation syndrome, increased intestinal crypt survival and enterocyte proliferation, and reduced apoptosis. DBIBB enhanced DNA repair by augmenting the resolution of γ-H2AX foci, increased clonogenic survival of irradiated IEC-6 cells, attenuated the radiation-induced death of human CD34(+) hematopoietic progenitors and enhanced the survival of the granulocyte/macrophage lineage. DBIBB also increased the survival of mice suffering from the hematopoietic acute radiation syndrome after total-body irradiation. DBIBB represents a drug candidate capable of mitigating acute radiation syndrome caused by high-dose γ-radiation to the hematopoietic and gastrointestinal system.
Apoptosis is accompanied by distinct morphological changes at the plasma and organelle membrane level. Involvement of certain lipids in apoptosis has been established; however, we have limited understanding of the specific lipid structures that participate in this process. We used untargeted comparative lipidomics to study the changes in lipid composition during staurosporine-induced apoptosis in HCT-116. Our results revealed that ceramides, dihydroceramides, and sphingomyelins, with defined acyl chains, constitute the majority of changes in the lipidome. Expression levels and activities of enzymes responsible for the biosynthesis of lipids that change suggest that de novo synthesis causes these specific changes. Further analysis of the lipidome during apoptosis in other cancer and non-cancer cell lines suggested that accumulation of ceramides and dihydroceramides is specific to cancer cells. Taken together, our data propose that these molecules are regulated at the lipid-specific level during apoptosis and that this regulation differs between cancer and non-cancer cells.