2-Aminopyridine Analogs Inhibit Both Enzymes of the Glyoxylate Shunt in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents.

Anion-π Interactions in Computer-Aided Drug Design: Modeling the Inhibition of Malate Synthase by Phenyl-Diketo Acids.

Human infection by Mycobacterium tuberculosis (Mtb) continues to be a global epidemic. Computer-aided drug design (CADD) methods are used to accelerate traditional drug discovery efforts. One noncovalent interaction that is being increasingly identified in biological systems but is neglected in CADD is the anion-π interaction. The study reported herein supports the conclusion that anion-π interactions play a central role in directing the binding of phenyl-diketo acid (PDKA) inhibitors to malate synthase (GlcB), an enzyme required for Mycobacterium tuberculosis virulence. Using density functional theory methods (M06-2X/6-31+G(d)), a GlcB active site template was developed for a predictive model through a comparative analysis of PDKA-bound GlcB crystal structures. The active site model includes the PDKA molecule and the protein determinants of the electrostatic, hydrogen-bonding, and anion-π interactions involved in binding. The predictive model accurately determines the Asp 633-PDKA structural position upon binding and precisely predicts the relative binding enthalpies of a series of 2-ortho halide-PDKAs to GlcB. A screening model was also developed to efficiently assess the propensity of each PDKA analog to participate in an anion-π interaction; this method is in good agreement with both the predictive model and the experimental binding enthalpies for the 2-ortho halide-PDKAs. With the screening and predictive models in hand, we have developed an efficient method for computationally screening and evaluating the binding enthalpy of variously substituted PDKA molecules. This study serves to illustrate the contribution of this overlooked interaction to binding affinity and demonstrates the importance of integrating anion-π interactions into structure-based CADD.

The Mycobacterium tuberculosis H37Ra gene MRA_1916 causes growth defects upon down-regulation.

D-amino acid oxidases play an important role in converting D-amino acids to their corresponding α-keto acids. MRA_1916 of Mycobacterium tuberculosis H37Ra (Mtb-Ra) is annotated to be a D-amino acid oxidase (DAO). However, not much information is available about its physiological role during Mtb-Ra growth and survival. The present study was taken-up to understand the role of DAO during different stages of growth and effect of its down-regulation on growth. Recombinant Mtb-Ra strains with DAO and GlcB (malate synthase: MRA_1848) gene knockdown were developed and their growth was studied using Microtiter Alamar Blue Assay (MABA) with glycerol, acetate and glycine as a carbon source. Ethyl bromopyruvate (BrP) was used as an inhibitor of GlcB. MABA study showed inhibition of wild-type (WT) and knockdowns in the presence of BrP (2.5mM). However, growth inhibition of WT was less noticeable at lower concentrations of BrP. Mtb-Ra with DAO knockdown showed poor utilization of glycine in the presence of BrP. The DAO localization study showed its prominent distribution in cytosolic fraction and to some extent in cell wall and membrane fractions. Growth profile of WT under oxygen and nutritional stress showed changes in expression of DAO, GlcB, PckA (phosphoenolpyruvate carboxykinase: MRA_0219) and GlyA1 (serine hydroxymethyltransferase: MRA_1104).

Alkaloids as inhibitors of malate synthase from Paracoccidioides spp.: receptor-ligand interaction-based virtual screening and molecular docking studies, antifungal activity, and the adhesion process.

Paracoccidioides is the agent of paracoccidioidomycosis. Malate synthase plays a crucial role in the pathogenicity and virulence of various fungi, such as those that are human pathogens. Thus, an inhibitor of this enzyme may be used as a powerful antifungal without side effects in patients once these enzymes are absent in humans. Here, we searched for compounds with inhibitory capacity against the malate synthase of Paracoccidioides species (PbMLS). The three-dimensional (3D) structure of PbMLS was determined using the I-TASSER server. Compounds were selected from the ZINC database. Based on the mechanism underlying the interaction of the compounds with PbMLS, it was possible to identify ß-carboline moiety as a standard key structure. The compounds with ß-carboline moiety that are available in our laboratories were investigated. A total of nine alkaloid compounds were selected. The primary mechanisms of interaction of the alkaloid compounds in the binding pocket of PbMLS were identified and compared with the mechanism of interaction of acetyl coenzyme A (acetyl-CoA). We discovered that the amphipathic nature of the compounds, concomitant with the presence of ß-carboline moiety, was crucial for their stability in the binding pocket of PbMLS. In addition, the importance of a critical balance of the polar and nonpolar contacts of the compounds in this region was observed. Four ß-carboline alkaloid compounds showed the ability to inhibit recombinant PbMLS (PbMLSr) activity, Paracoccidioides species growth, and adhesion of the fungus and PbMLSr to the extracellular matrix components. The cytotoxicity of the alkaloids was also evaluated.

ß-Carboline alkaloids from Galianthe ramosa inhibit malate synthase from Paracoccidioides spp.

As part of our continuing chemical and biological analyses of Rubiaceae species from Cerrado, we isolated novel alkaloids 1 and 2, along with known compounds epicatechin, ursolic acid, and oleanolic acid, from Galianthe ramosa. Alkaloid 2 inhibited malate synthase from the pathogenic fungus Paracoccidioides spp. This enzyme is considered an important molecular target because it is not found in humans. Molecular docking simulations were used to describe the interactions between the alkaloids and malate synthase.

Long-range effects in anion-π interactions: their crucial role in the inhibition mechanism of Mycobacterium tuberculosis malate synthase.

The glyoxylate shunt is an anaplerotic bypass of the traditional Krebs cycle. It plays a prominent role in Mycobacterium tuberculosis virulence, so it can be exploited for the development of antitubercular therapeutics. The shunt involves two enzymes: isocitrate lyase (ICL) and malate synthase (GlcB). The shunt bypasses two steps of the tricarboxylic acid cycle, allowing the incorporation of carbon, and thus, refilling oxaloacetate under carbon-limiting conditions. The targeting of ICL is complicated; however, GlcB, which accommodates the pantothenate tail of acetyl-CoA in the active site, is easier to target. A catalytic Mg(2+) unit is located at the bottom of the cavity, and plays a very important role. Recently, the development of effective antituberculosis drugs based on phenyldiketo acids (PDKAs) has been reported. Interestingly, all the crystal structures of GlcB-inhibitor complexes exhibit close contact between the carboxylate of Asp633 and the face of the aromatic ring of the inhibitor. Remarkably, the replacement of the phenyl ring in PDKA by aliphatic moieties yields inactive inhibitors, suggesting that the aromatic moiety is crucial for inhibition. However, the aromatic ring of PDKA is not electron-deficient, and consequently, the anion-π interaction is expected to be very weak (dominated only by polarization effects). Herein, through a combination analysis of the recent X-ray structures of GlcB-PDKA complexes retrieved from the protein data bank (PDB) and computational ab initio studies (RI-MP2/def2-TZVP level of theory), we demonstrate the prominent role of the Mg(2+) ion in the active site, which promotes long-range enhancement of the anion-π interaction.

A systems chemical biology study of malate synthase and isocitrate lyase inhibition in Mycobacterium tuberculosis during active and NRP growth.

The ability of Mycobacterium tuberculosis (Mtb) to survive in low oxygen environments enables the bacterium to persist in a latent state within host tissues. In vitro studies of Mtb growth have identified changes in isocitrate lyase (ICL) and malate synthase (MS) that enable bacterial persistence under low oxygen and other environmentally limiting conditions. Systems chemical biology (SCB) enables us to evaluate the effects of small molecule inhibitors not only on the reaction catalyzed by malate synthase and isocitrate lyase, but the effect on the complete tricarboxylic acid cycle (TCA) by taking into account complex network relationships within that system. To study the kinetic consequences of inhibition on persistent bacilli, we implement a systems-chemical biology (SCB) platform and perform a chemistry-centric analysis of key metabolic pathways believed to impact Mtb latency. We explore consequences of disrupting the function of malate synthase (MS) and isocitrate lyase (ICL) during aerobic and hypoxic non-replicating persistence (NRP) growth by using the SCB method to identify small molecules that inhibit the function of MS and ICL, and simulating the metabolic consequence of the disruption. Results indicate variations in target and non-target reaction steps, clear differences in the normal and low oxygen models, as well as dosage dependent response. Simulation results from singular and combined enzyme inhibition strategies suggest ICL may be the more effective target for chemotherapeutic treatment against Mtb growing in a microenvironment where oxygen is slowly depleted, which may favor persistence.

Non-traditional antibacterial screening approaches for the identification of novel inhibitors of the glyoxylate shunt in gram-negative pathogens.

Antibacterial compounds that affect bacterial viability have traditionally been identified, confirmed, and characterized in standard laboratory media. The historical success of identifying new antibiotics via this route has justifiably established a traditional means of screening for new antimicrobials. The emergence of multi-drug-resistant (MDR) bacterial pathogens has expedited the need for new antibiotics, though many in the industry have questioned the source(s) of these new compounds. As many pharmaceutical companies' chemical libraries have been exhaustively screened via the traditional route, we have concluded that all compounds with any antibacterial potential have been identified. While new compound libraries and platforms are being pursued, it also seems prudent to screen the libraries we currently have in hand using alternative screening approaches. One strategy involves screening under conditions that better reflect the environment pathogens experience during an infection, and identifying in vivo essential targets and pathways that are dispensable for growth in standard laboratory media in vitro. Here we describe a novel screening strategy for identifying compounds that inhibit the glyoxylate shunt in Pseudomonas aeruginosa, a pathway that is required for bacterial survival in the pulmonary environment. We demonstrate that these compounds, which were not previously identified using traditional screening approaches, have broad-spectrum antibacterial activity when they are tested under in vivo-relevant conditions. We also show that these compounds have potent activity on both enzymes that comprise the glyoxylate shunt, a feature that was supported by computational homology modeling. By dual-targeting both enzymes in this pathway, we would expect to see a reduced propensity for resistance development to these compounds. Taken together, these data suggest that understanding the in vivo environment that bacterial pathogens must tolerate, and adjusting the antibacterial screening paradigm to reflect those conditions, could identify novel antibiotics for the treatment of serious MDR pathogens.

Structure-guided discovery of phenyl-diketo acids as potent inhibitors of M. tuberculosis malate synthase.

The glyoxylate shunt plays an important role in fatty acid metabolism and has been shown to be critical to survival of several pathogens involved in chronic infections. For Mycobacterium tuberculosis (Mtb), a strain with a defective glyoxylate shunt was previously shown to be unable to establish infection in a mouse model. We report the development of phenyl-diketo acid (PDKA) inhibitors of malate synthase (GlcB), one of two glyoxylate shunt enzymes, using structure-based methods. PDKA inhibitors were active against Mtb grown on acetate, and overexpression of GlcB ameliorated this inhibition. Crystal structures of complexes of GlcB with PDKA inhibitors guided optimization of potency. A selected PDKA compound demonstrated efficacy in a mouse model of tuberculosis. The discovery of these PDKA derivatives provides chemical validation of GlcB as an attractive target for tuberculosis therapeutics.

Atomic resolution structures of Escherichia coli and Bacillus anthracis malate synthase A: comparison with isoform G and implications for structure-based drug discovery.

Enzymes of the glyoxylate shunt are important for the virulence of pathogenic organisms such as Mycobacterium tuberculosis and Candida albicans. Two isoforms have been identified for malate synthase, the second enzyme in the pathway. Isoform A, found in fungi and plants, comprises approximately 530 residues, whereas isoform G, found only in bacteria, is larger by approximately 200 residues. Crystal structures of malate synthase isoform G from Escherichia coli and Mycobacterium tuberculosis were previously determined at moderate resolution. Here we describe crystal structures of E. coli malate synthase A (MSA) in the apo form (1.04 A resolution) and in complex with acetyl-coenzyme A and a competitive inhibitor, possibly pyruvate or oxalate (1.40 A resolution). In addition, a crystal structure for Bacillus anthracis MSA at 1.70 A resolution is reported. The increase in size between isoforms A and G can be attributed primarily to an inserted alpha/beta domain that may have regulatory function. Upon binding of inhibitor or substrate, several active site loops in MSA undergo large conformational changes. However, in the substrate bound form, the active sites of isoforms A and G from E. coli are nearly identical. Considering that inhibitors bind with very similar affinities to both isoforms, MSA is as an excellent platform for high-resolution structural studies and drug discovery efforts.

Challenges associated with current and future TB treatment.

Current tuberculosis (TB) treatment is based on a combination of drugs that were developed mostly in the central decades of the last century. Cure rates are high for drug sensitive strains of Mycobacterium tuberculosis (M. tb) when the recommended complex and lengthy treatment protocols are adhered to. However the difficulty in correctly prescribing and adhering to these protocols, the emergence of M tb strains resistant to multiple drugs, and drug-drug interactions that interfere with optimal treatment of HIV and TB coinfected patients have generated a pressing need for improved TB therapies. Together with the ominous global burden of TB, these shortcomings of current treatment have contributed to a renewed interest in the development of improved drugs and protocols for the treatment of tuberculosis. This article highlights hurdles related to the optimized use of existing drugs and challenges related to the development of novel, improved products, focusing in particular on aspects inherent in TB drug clinical development. Concluding comments propose processes for more efficient development of new TB therapies.

High throughput crystallography of TB drug targets.

Tuberculosis (TB) infects one-third of the world population. Despite 50 years of available drug treatments, TB continues to increase at a significant rate. The failure to control TB stems in part from the expense of delivering treatment to infected individuals and from complex treatment regimens. Incomplete treatment has fueled the emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (Mtb). Reducing non-compliance by reducing the duration of chemotherapy will have a great impact on TB control. The development of new drugs that either kill persisting organisms, inhibit bacilli from entering the persistent phase, or convert the persistent bacilli into actively growing cells susceptible to our current drugs will have a positive effect. We are taking a multidisciplinary approach that will identify and characterize new drug targets that are essential for persistent Mtb. Targets are exposed to a battery of analyses including microarray experiments, bioinformatics, and genetic techniques to prioritize potential drug targets from Mtb for structural analysis. Our core structural genomics pipeline works with the individual laboratories to produce diffraction quality crystals of targeted proteins, and structural analysis will be completed by the individual laboratories. We also have capabilities for functional analysis and the virtual ligand screening to identify novel inhibitors for target validation. Our overarching goals are to increase the knowledge of Mtb pathogenesis using the TB research community to drive structural genomics, particularly related to persistence, develop a central repository for TB research reagents, and discover chemical inhibitors of drug targets for future development of lead compounds.

Purification and characterization of malate synthase from the glucose-grown wood-rotting basidiomycete Fomitopsis palustris.

Malate synthase (EC 4.1.3.2), the key enzyme of the glyoxylate cycle, was purified to a homogeneous protein from the wood-rotting basidiomycete Fomitopsis palustris grown on glucose. The purified enzyme, with a molecular mass of 520 kDa, was found to consist of eight 65-kDa subunits, and to have Km of 45 and 2.2 microM for glyoxylate and acetyl-CoA, respectively. The enzyme activity was competitively inhibited by oxalate (K1, 8.5 microM) and glycolate (Ki, 17 microM), and uncompetitively by coenzyme A (Ki, 100 microM). The potent inhibition of the activity by p-chloromercuribenzoate suggests that the enzyme has a sulfhydryl group at the active center. However, the enzyme was inhibited moderately by adenine nucleotides and weakly by some of the metabolic intermediates of glycolysis and tricarboxylic acid cycle. The enzyme was completely inactive in the absence of metal ions and was maximally activated by Mg2+ (Km, 0.4 microM), which also served to significantly prevent enzyme inactivation during storage.

Lachnellins A, B, C, D, and naphthalene-1,3,8-triol, biologically active compounds from a Lachnellula species (ascomycetes).

In the course of our search for new biologically active metabolites, lachnellin A (1), a metabolite with high cytotoxic and antimicrobial activities, the structurally related lachnellins B, C and D (3, 4, 7), and naphthalene-1,3,8-triol (8), an inhibitor of malate synthase (EC 4.1.3.2), were isolated from submerged cultures of the ascomycete Lachnellula sp. A 32-89. The antimicrobial, cytotoxic and phytotoxic activities of lachnellin A depended on its reactivity and could be abolished by the addition of cysteine. The enzyme inhibiting activity of (8) was due to reactive intermediates during melanization and was no longer observed in the presence of serum albumin. In addition, rac-scytalone (9), (+)-trans-3,4-dihydro-3,4,8-trihydroxy-1 (2H)-naphthalenone (10), 2,5-dihydroxytoluene (11), and (R)-(-)-5-methylmellein (12) were obtained from the same source and biologically characterized.

X-ray induced inactivation of the sulfhydryl enzyme malate synthase in the presence of various additives. Probing the extent of primary and post-irradiation inactivation and repair by rapid screening on the microlevel.

The sulfhydryl enzyme malate synthase was inactivated by X-irradiation in air-saturated aqueous solution, in the absence or presence of a variety of additives (thiols, antioxienzymes, typical radical scavengers, inorganic salts, buffer components, substrates, products, analogues). Radiation-induced changes of enzymic activity were registered immediately after stop of irradiation and in the post-irradiation period. Repair experiments were initiated by post-irradiation addition of dithiothreitol. Additionally, post-irradiation inactivation was modulated by some further additives. Probing the extent of primary and post-irradiation inactivation and repair was accomplished effectively by screening experiments on the microlevel, and by derivation of normalized efficiency parameters which allowed quick comparisons of the various additives with respect to their protective and repair-promotive efficiencies. Correlations between the efficiency parameters were studied by means of binary and ternary diagrams. Most of the substances added before irradiation were found to protect the enzyme against primary and post-irradiation inactivation and to increase the reparability of the enzyme by dithiothreitol, the extent of the effects depending on the nature (and concentration) of the additives used. Our results indicate that both specific protection (by substrates, products, analogues, and by sulfhydryl agents) and scavenging are responsible for the radioprotective efficiencies of the additives.

Primary and post-irradiation inactivation of the sulfhydryl enzyme malate synthase: correlation of protective effects of additives.

The presence of additives during X-irradiation of malate synthase led to radioprotective effects against primary and post-irradiation inactivation. Pronounced effects were provided by typical scavengers, sulfhydryl reagents and specific ligands (substrates, products, analogues). The results show that scavenging and specific protection are responsible for the protective efficiency of additives. Scavengers delete noxious species formed during irradiation or post-radiationem. Sulfhydryl reagents may act as repair substances. Specific ligands protect the active site of the enzyme and the essential sulfhydryls; specific protection is more pronounced post-radiationem. Ligands and sulfhydryl reagents may additionally act as scavengers. A cumulative index for the protective power of additives against both sorts of inactivation was established.

Post-irradiation inactivation of the sulfhydryl enzyme malate synthase.

The sulfhydryl enzyme malate synthase was inactivated in air-saturated aqueous solution by X-irradiation (2 kGy). Changes of activity were registered up to 60 h after irradiation. Effects of specific additives (formate, superoxide dismutase, catalase), added before and/or after irradiation, revealed the role of the deleterious radical and non-radical species responsible for the radiation damage: inactivation during irradiation is mainly due to the action of OH, to a minor extent to O(2) and H2O2; post-irradiation inactivation is mainly caused by H2O2. A partial restoration of enzyme activity by dithiothreitol, added after irradiation, was found for all systems investigated; repairs were significant even when they were initiated 60 h after irradiation.

Physiological basis for meso-tartrate sensitivity in some strains of Salmonella typhimurium.

meso-Tartrate inhibited the growth of non-meso-tartrate-utilizing strains of Salmonella typhimurium in peptone water media and mineral salts media with some, but not all, carbon sources. C-R intermediates of the tricarboxylic acid cycle or compounds readily converted to them and substrates metabolized independently of the C-6 part of the cycle spared bacteria from the inhibitory effects of meso-tartrate when added to cultures along with meso-tartrate. Experiments with cell-free extracts of non-meso-tartrate-utilizing strains from batch and continuous cultures showed that meso-tartrate was a competitive inhibitor of isocitrate dehydrogenase and isocitrate lyase activities and also inhibited citrate synthase and malate synthase activities. The synthesis of these enzymes was not inhibited by meso-tartrate. The isocitrate enzymes of meso-tartrate-utilizing strains of S. typhimurium were similarly inhibited by meso-tartrate, but inhibition of the growth of meso-tartrate-utilizing strains was demonstrable only in uninduced cultures in which the intracellular concentrations of meso-tartrate were high.