Discovery of a Novel Inhibitor Structure of Mycobacterium tuberculosis Isocitrate Lyase.

Tuberculosis remains a global threat to public health, and dormant Mycobacterium tuberculosis leads to long-term medication that is harmful to the human body. M. tuberculosis isocitrate lyase (MtICL), which is absent in host cells, is a key rate-limiting enzyme of the glyoxylic acid cycle and is essential for the survival of dormant M. tuberculosis. The aim of this study was to evaluate natural compounds as potential MtICL inhibitors through docking and experimental verification. Screening of the TCMSP database library was done using Discovery Studio 2019 for molecular docking and interaction analysis, with the putative inhibitors of MtICL, 3-BP, and IA as reference ligands. Daphnetin (MOL005118), with a docking score of 94.8 and -CDOCKER interaction energy of 56 kcal/mol, was selected and verified on MtICL in vitro and M. smegmatis; daphnetin gave an IC50 of 4.34 µg/mL for the MtICL enzyme and an MIC value of 128 µg/mL against M. smegmatis, showing enhanced potential in comparison with 3-BP and IA. The interactions and essential amino acid residues of the protein were analyzed. In summary, natural daphnetin may be a promising new skeleton for the design of inhibitors of MtICL to combat dormant M. tuberculosis.

Mechanism-Based Inactivation of Mycobacterium tuberculosis Isocitrate Lyase 1 by (2R,3S)-2-Hydroxy-3-(nitromethyl)succinic acid.

The isocitrate lyase paralogs of Mycobacterium tuberculosis (ICL1 and 2) are essential for mycobacterial persistence and constitute targets for the development of antituberculosis agents. We report that (2R,3S)-2-hydroxy-3-(nitromethyl)succinic acid (5-NIC) undergoes apparent retro-aldol cleavage as catalyzed by ICL1 to produce glyoxylate and 3-nitropropionic acid (3-NP), the latter of which is a covalent-inactivating agent of ICL1. Kinetic analysis of this reaction identified that 5-NIC serves as a robust and efficient mechanism-based inactivator of ICL1 (kinact/KI = (1.3 ± 0.1) × 103 M-1 s-1) with a partition ratio <1. Using enzyme kinetics, mass spectrometry, and X-ray crystallography, we identified that the reaction of the 5-NIC-derived 3-NP with the Cys191 thiolate of ICL1 results in formation of an ICL1-thiohydroxamate adduct as predicted. One aspect of the design of 5-NIC was to lower its overall charge compared to isocitrate to assist with cell permeability. Accordingly, the absence of the third carboxylate group will simplify the synthesis of pro-drug forms of 5-NIC for characterization in cell-infection models of M. tuberculosis.

Inhibitory Effects of Epipolythiodioxopiperazine Fungal Metabolites on Isocitrate Lyase in the Glyoxylate Cycle of Candida albicans.

Four epipolythiodioxopiperazine fungal metabolites (1-4) isolated from the sponge-derived Aspergillus quadrilineatus FJJ093 were evaluated for their capacity to inhibit isocitrate lyase (ICL) in the glyoxylate cycle of Candida albicans. The structures of these compounds were elucidated using spectroscopic techniques and comparisons with previously reported data. We found secoemestrin C (1) (an epitetrathiodioxopiperazine derivative) to be a potent ICL inhibitor, with an inhibitory concentration of 4.77 ± 0.08 µM. Phenotypic analyses of ICL-deletion mutants via growth assays with acetate as the sole carbon source demonstrated that secoemestrin C (1) inhibited C. albicans ICL. Semi-quantitative reverse-transcription polymerase chain reaction analyses indicated that secoemestrin C (1) inhibits ICL mRNA expression in C. albicans under C2-assimilating conditions.

Covalent Inactivation of Mycobacterium tuberculosis Isocitrate Lyase by cis-2,3-Epoxy-Succinic Acid.

The isocitrate lyases (ICL1/2) are essential enzymes of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. At present, no ICL1/2 inhibitors have progressed to clinical evaluation, despite extensive drug discovery efforts. Herein, we surveyed succinate analogs against ICL1 and found that dicarboxylic acids constrained in their synperiplanar conformations, such as maleic acid, comprise uncompetitive inhibitors of ICL1 and inhibit more potently than their trans-isomers. From this, we identified cis-2,3 epoxysuccinic acid (cis-EpS) as a selective, irreversible covalent inactivator of Mtb ICL1 (kinact/Kinact= (5.0 ± 1.4) × 104 M-1 s-1; Kinact = 200 ± 50 nM), the most potent inactivator of ICL1 yet characterized. Crystallographic and mass spectrometric analysis demonstrated that Cys191 of ICL1 was S-malylated by cis-EpS, and a crystallographic "snapshot" of inactivation lent insight into the chemical mechanism of this inactivation. Proteomic analysis of E. coli lysates showed that cis-EpS selectively labeled plasmid-expressed Mtb ICL1. Consistently, cis-EpS, but not its trans-isomer, inhibited the growth of Mtb under conditions in which ICL function is essential. These findings encourage the development of analogs of cis-2,3-epoxysuccinate as antituberculosis agents.

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.

Octyl gallate triggers dysfunctional mitochondria leading to ROS driven membrane damage and metabolic inflexibility along with attenuated virulence in Candida albicans.

Recently the high incidence of worldwide Candida infections has substantially increased. The growing problem about toxicity of antifungal drugs and multidrug resistance aggravates the need for the development of new effective strategies. Natural compounds in this context represent promising alternatives having potential to be exploited for improving human health. The present study was therefore designed to evaluate the antifungal effect of a naturally occurring phenolic, octyl gallate (OG), on Candida albicans and to investigate the underlying mechanisms involved. We demonstrated that OG at 25 µg/ml could effectively inhibit C. albicans. Mechanistic insights revealed that OG affects mitochondrial functioning as Candida cells exposed to OG did not grow on non-fermentable carbon sources. Dysfunctional mitochondria triggered generation of reactive oxygen species (ROS), which led to membrane damage mediated by lipid peroxidation. We explored that OG inhibited glucose-induced reduction in external pH and causes decrement in ergosterol levels by 45%. Furthermore, OG impedes the metabolic flexibility of C. albicans by inhibiting the glyoxylate enzyme isocitrate lyase, which was also confirmed by docking analysis. Additionally, OG affected virulence traits such as morphological transition and cell adherence. Furthermore, we depicted that OG not only prevented biofilm formation but eliminates the preformed biofilms. In vivo studies with Caenorhabditis elegans nematode model confirmed that OG could enhance the survival of C. elegans after infection with Candida. Toxicity assay using red blood cells showed only 27.5% haemolytic activity. Taken together, OG is a potent inhibitor of C. albicans that warrants further structural optimization and pharmacological investigations.

New Peptides from The Marine-Derived Fungi Aspergillus allahabadii and Aspergillus ochraceopetaliformis.

Four new peptides were isolated from the culture broths of the marine-derived fungi Aspergillus allahabadii and A. ochraceopetaliformis. Based on the results of chemical and spectroscopic analyses, two compounds (1 and 2) from A. allahabadii were determined to be cyclopentapeptides, while those from A. ochraceopetaliformis were a structurally-related cyclodepsihexapeptide (3) and its linear analog (4). In addition to the presence of a D-amino acid residue, the almost reversed sequence of peptides in 3 and 4, relative to those of the 1 and 2, is notable. These new compounds exhibited moderate inhibition against the enzyme sortase A as well as a weak inhibition against isocitrate lyase (2).

Phenolic N-monosubstituted carbamates: Antitubercular and toxicity evaluation of multi-targeting compounds.

The research of novel antimycobacterial drugs represents a cutting-edge topic. Thirty phenolic N-monosubstituted carbamates, derivatives of salicylanilides and 4-chlorophenol, were investigated against Mycobacterium tuberculosis H37Ra, H37Rv including multidrug- and extensively drug-resistant strains, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium aurum and Mycobacterium smegmatis as representatives of nontuberculous mycobacteria (NTM) and for their cytotoxic and cytostatic properties in HepG2 cells. Since salicylanilides are multi-targeting compounds, we determined also inhibition of mycobacterial isocitrate lyase, an enzyme involved in the maintenance of persistent tuberculous infection. The minimum inhibitory concentrations were from ≤0.5 µM for both drug-susceptible and resistant M. tuberculosis and from ≤0.79 µM for NTM with no cross-resistance to established drugs. The presence of halogenated salicylanilide scaffold results into an improved activity. We have verified that isocitrate lyase is not a key target, presented carbamates showed only moderate inhibitory activity (up to 18% at a concentration of 10 µM). Most of the compounds showed no cytotoxicity for HepG2 cells and some of them were without cytostatic activity. Cytotoxicity-based selectivity indexes of several carbamates for M. tuberculosis, including resistant strains, were higher than 125, thus favouring some derivatives as promising features for future development.

Inhibitory Effects of Diketopiperazines from Marine-Derived Streptomyces puniceus on the Isocitrate Lyase of Candida albicans.

The glyoxylate cycle is a sequence of anaplerotic reactions catalyzed by the key enzymes isocitrate lyase (ICL) and malate synthase, and plays an important role in the pathogenesis of microorganisms during infection. An icl-deletion mutant of Candida albicans exhibited reduced virulence in mice compared with the wild type. Five diketopiperazines, which are small and stable cyclic peptides, isolated from the marine-derived Streptomyces puniceus Act1085, were evaluated for their inhibitory effects on C. albicans ICL. The structures of these compounds were elucidated based on spectroscopic data and comparisons with previously reported data. Cyclo(L-Phe-L-Val) was identified as a potent ICL inhibitor, with a half maximal inhibitory concentration of 27 µg/mL. Based on the growth phenotype of the icl-deletion mutants and icl expression analyses, we demonstrated that cyclo(L-Phe-L-Val) inhibits the gene transcription of ICL in C. albicans under C2-carbon-utilizing conditions.

Latent Tuberculosis Infection (LTBI) and Its Potential Targets: An Investigation into Dormant Phase Pathogens.

One-third of the world's population harbours the latent tuberculosis infection (LTBI) with a lifetime risk of reactivation. Although, the treatment of LTBI relies significantly on the first-line therapy, identification of novel drug targets and therapies are the emerging focus for researchers across the globe. The current review provides an insight into the infection, diagnostic methods and epigrammatic explanations of potential molecular targets of dormant phase bacilli. This study also includes current preclinical and clinical aspects of tubercular infections and new approaches in antitubercular drug discovery.

Screening of Anti-mycobacterial Phytochemical Compounds for Potential Inhibitors against Mycobacterium Tuberculosis Isocitrate Lyase.

Background and Introduction: Tuberculosis (TB) is a leading infectious disease caused by Mycobacterium tuberculosiswith high morbidity and mortality. Isocitrate lyase (MtbICL), a key enzyme of glyoxylate pathway has been shown to be involved in mycobacterial persistence, is attractive drug target against persistent tuberculosis. METHODS: Virtual screening, molecular docking and MD simulation study has been integrated for screening of phytochemical based anti-mycobacterial compounds. Docking study of reported MtbICL inhibitors has shown an average binding affinity score -7.30 Kcal/mol. In virtual screening, compounds exhibiting lower binding energy than calculated average binding energy were selected as top hit compounds followed by calculation of drug likeness property. Relationship between experimental IC50 value and calculated binding gibbs free energy of reported inhibitors was also calculated through regression analysis to predict IC50 value of potential inhibitors. RESULTS: Docking and MD simulation studies of top hit compounds have identified shinjudilactone (quassinoid), lecheronol A (pimarane) and caniojane (diterpene) as potential MtbICL inhibitors. CONCLUSION: Phytochemical based anti-mycobacterial compound can further developed into effective drugs against persistence tuberculosis with lesser toxicity and side effects.

Applications of Ensemble Docking in Potential Inhibitor Screening for Mycobacterium tuberculosis Isocitrate Lyase Using a Local Plant Database.

Isocitrate lyase (ICL) is a persistent factor for the survival of dormant stage Mycobacterium tuberculosis (MTB), thus a potential drug target for tuberculosis treatment. In this work, ensemble docking approach was used to screen for potential inhibitors of ICL. The ensemble conformations of ICL active site were obtained from molecular dynamics simulation on three dimer form systems, namely the apo ICL, ICL in complex with metabolites (glyoxylate and succinate), and ICL in complex with substrate (isocitrate). Together with the ensemble conformations and the X-ray crystal structures, 22 structures were used for the screening against Malaysian Natural Compound Database (NADI). The top 10 compounds for each ensemble conformation were selected. The number of compounds was then further narrowed down to 22 compounds that were within the Lipinski's Rule of Five for drug-likeliness and were also docked into more than one ensemble conformation. Theses 22 compounds were furthered evaluate using whole cell assay. Some compounds were not commercially available; therefore, plant crude extracts were used for the whole cell assay. Compared to itaconate (the known inhibitor of ICL), crude extracts from Manilkara zapota, Morinda citrifolia, Vitex negundo, and Momordica charantia showed some inhibition activity. The MIC/MBC value were 12.5/25, 12.5/25, 0.78/1.6, and 0.39/1.6 mg/mL, respectively. This work could serve as a preliminary study in order to narrow the scope for high throughput screening in the future.

Analysis of the L-malate biosynthesis pathway involved in poly(ß-L-malic acid) production in Aureobasidium melanogenum GXZ-6 by addition of metabolic intermediates and inhibitors.

Poly(ß-L-malic acid) (PMA) is a promising polyester formed from L-malate in microbial cells. Malate biosynthesis is crucial for PMA production. Previous studies have shown that the non-oxidative pathway or oxidative pathway (TCA cycle) is the main biosynthetic pathway of malate in most of PMA-producing strains, while the glyoxylate cycle is only a supplementary pathway. In this study, we investigated the effect of exogenous metabolic intermediates and inhibitors of the malate biosynthetic pathway on PMA production by Aureobasidium melanogenum GXZ-6. The results showed that PMA production was stimulated by maleic acid (a fumarase inhibitor) and sodium malonate (a succinate dehydrogenase inhibitor) but inhibited by succinic acid and fumaric acid. This indicated that the TCA cycle might not be the only biosynthetic pathway of malate. In addition, the PMA titer increased by 18.1% upon the addition of glyoxylic acid after 72 h of fermentation, but the PMA titer decreased by 7.5% when itaconic acid (an isocitrate lyase inhibitor) was added, which indicated that malate for PMA production was synthesized significantly via the glyoxylate cycle rather than the TCA cycle. Furthermore, in vitro enzyme activities of the TCA and glyoxylate cycles suggested that the glyoxylate cycle significantly contributed to the PMA production, which is contradictory to what has been reported previously in other PMA-producing A. pullulans.

Identification of a new antifungal compound against isocitrate lyase of Paracoccidioides brasiliensis.

Aim: To perform virtual screening of compounds based on natural products targeting isocitrate lyase of Paracoccidioides brasiliensis. Materials & methods: Homology modeling and molecular dynamics simulations were applied in order to obtain conformational models for virtual screening. The selected hits were tested in vitro against enzymatic activity of ICL of the dimorphic fungus P. brasiliensis and growth of the Paracoccidioides spp. The cytotoxicity and selectivity index of the compounds were defined. Results & conclusion: Carboxamide, lactone and ß-carboline moieties were identified as interesting chemical groups for the design of new antifungal compounds. The compounds inhibited ICL of the dimorphic fungus P. brasiliensis activity. The compound 4559339 presented minimum inhibitory concentration of 7.3 µg/ml in P. brasiliensis with fungicidal effect at this concentration. Thus, a new potential antifungal against P. brasiliensis is proposed.

Bromopyrrole Alkaloids from the Sponge Agelas kosrae.

Two new sceptrin derivatives (1,2) and eight structurally-related known bromopyrrole-bearing alkaloids were isolated from the tropical sponge Agelas kosrae. By a combination of spectroscopic methods, the new compounds, designated dioxysceptrin (1) and ageleste C (2), were determined to be structural analogs of each other that differ at the imidazole moiety. Dioxysceptrin was also found to exist as a mixture of α-amido epimers. The sceptrin alkaloids exhibited weak cytotoxicity against cancer cells. Compounds 1 and 2 also moderately exhibited anti-angiogenic and isocitrate lyase-inhibitory activities, respectively.

Alternate pathway to ascorbate induced inhibition of Mycobacterium tuberculosis.

Ascorbate has been demonstrated to interfere with the growth of Mycobacterium tuberculosis. It scavenges oxygen in the culture medium to induce dormancy of M. tuberculosis. It kills the mycobacteria by generating reactive oxygen intermediates via iron mediated Fenton reactions. In this study, we observed that ascorbate can inhibit M. tuberculosis isocitrate lyase (MtbICL) with an IC50 of 2.15 mΜ. MtbICL is an essential enzyme for the survival of M. tuberculosis under dormancy. We studied the effect of ascorbate on the growth of M. tuberculosis H37Rv metabolizing through citric acid cycle or glyoxylate cycle with glucose or acetate respectively as the sole carbon source. It was observed that 4 mM ascorbate inhibited ∼89% of the growth in glucose medium, which was confirmed to be mediated by Fenton reaction, as the inhibition was significantly lesser (61%) under low iron condition. On the other hand, in acetate medium, ∼97% of the growth was inhibited and the inhibition was uninfluenced by the iron levels. 3-nitropropionate, a known inhibitor of MtbICL, was seen to cause significantly higher inhibition in the acetate medium than in the glucose medium; however it was indifferent to iron levels in either medium. Molecular docking and dynamic simulation studies confirmed stable binding of ascorbate to MtbICL leading to its inhibition. These observations suggest an additional pathway for ascorbate induced inhibition of M. tuberculosis through inhibition of glyoxylate cycle. Since human immune cells can accumulate ascorbate in millimolar concentrations, the in vitro activity range (1-4 mM) of ascorbate against M. tuberculosis could be extrapolated in vivo. Our result supports the possible benefits of adding high vitamin C diet in TB-treated patients.

Gluconeogenic precursor availability regulates flux through the glyoxylate shunt in Pseudomonas aeruginosa.

The glyoxylate shunt bypasses the oxidative decarboxylation steps of the tricarboxylic acid (TCA) cycle, thereby conserving carbon skeletons for gluconeogenesis and biomass production. In Escherichia coli, carbon flux is redirected through the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), following phosphorylation and inactivation of the TCA cycle enzyme, isocitrate dehydrogenase (ICD), by the kinase/phosphatase, AceK. In contrast, mycobacterial species lack AceK and employ a phosphorylation-insensitive isocitrate dehydrogenase (IDH), which is allosterically activated by the product of ICL activity, glyoxylate. However, Pseudomonas aeruginosa expresses IDH, ICD, ICL, and AceK, raising the question of how these enzymes are regulated to ensure proper flux distribution between the competing pathways. Here, we present the structure, kinetics, and regulation of ICL, IDH, and ICD from P. aeruginosa We found that flux partitioning is coordinated through reciprocal regulation of these enzymes, linking distribution of carbon flux to the availability of the key gluconeogenic precursors, oxaloacetate and pyruvate. Specifically, a greater abundance of these metabolites activated IDH and inhibited ICL, leading to increased TCA cycle flux. Regulation was also exerted through AceK-dependent phosphorylation of ICD; high levels of acetyl-CoA (which would be expected to accumulate when oxaloacetate is limiting) stimulated the kinase activity of AceK, whereas high levels of oxaloacetate stimulated its phosphatase activity. In summary, the TCA cycle-glyoxylate shunt branch point in P. aeruginosa has a complex enzymology that is profoundly different from those in other species characterized to date. Presumably, this reflects its predilection for consuming fatty acids, especially during infection scenarios.

The Nitro Group as a Masked Electrophile in Covalent Enzyme Inhibition.

We report the unprecedented reaction between a nitroalkane and an active-site cysteine residue to yield a thiohydroximate adduct. Structural and kinetic evidence suggests the nitro group is activated by conversion to its nitronic acid tautomer within the active site. The nitro group, therefore, shows promise as a masked electrophile in the design of covalent inhibitors targeting binding pockets with appropriately placed cysteine and general acid residues.

Structure-based screening and molecular dynamics simulations offer novel natural compounds as potential inhibitors of Mycobacterium tuberculosis isocitrate lyase.

Mycobacterium tuberculosis is the etiological agent of tuberculosis in humans and is responsible for more than two million deaths annually. M. tuberculosis isocitrate lyase (MtbICL) catalyzes the first step in the glyoxylate cycle, plays a pivotal role in the persistence of M. tuberculosis, which acts as a potential target for an anti-tubercular drug. To identify the potential anti-tuberculosis compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (n = 1,67,748) against the MtbICL structure. The ligands were docked against MtbICL in three sequential docking modes that resulted in 340 ligands having better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 27 compounds were found to fit well with re-docking studies. After refinement by molecular docking and drug-likeness analyses, three potential inhibitors (ZINC1306071, ZINC2111081, and ZINC2134917) were identified. These three ligands and the reference compounds were further subjected to molecular dynamics simulation and binding energy analyses to compare the dynamic structure of protein after ligand binding and the stability of the MtbICL and bound complexes. The binding free energy analyses were calculated to validate and capture the intermolecular interactions. The results suggested that the three compounds had a negative binding energy with -96.462, -143.549, and -122.526 kJ mol-1 for compounds with IDs ZINC1306071, ZINC2111081, and ZINC2134917, respectively. These lead compounds displayed substantial pharmacological and structural properties to be drug candidates. We concluded that ZINC2111081 has a great potential to inhibit MtbICL and would add to the drug discovery process against tuberculosis.

Apparent isocitrate lyase activity in Leishmania amazonensis.

Early reports have demonstrated the occurrence of glyoxylate cycle enzymes in several Leishmania species. However, these results have been underestimated because genes for the two key enzymes of the cycle, isocitrate lyase (ICL) and malate synthase (MS), are not annotated in Leishmania genomes. We have re-examined this issue in promastigotes of Leishmania amazonensis. Enzyme activities were assayed spectrophotometrically in cellular extracts and characterized partially. A 40 kDa band displaying ICL activity was visualized on zymograms of the extracts. By immunoblotting with mouse antibodies against ICL from Bacillus stearothermophilus, a band of approximately 40 kDa was identified, coincident with the relative molecular mass of the activity band revealed on zymograms. Indirect immunofluorescence of intact promastigotes showed that the recognized antigen is distributed as a punctuated pattern, mainly distributed beneath the subpellicular microtubules, over a diffused cytoplasmic stain. These results clearly demonstrate the existence of an apparent ICL activity in L. amazonensis promastigotes, which is associated to a 40 kDa polypeptide and distributed both diffused and as punctuate aggregates in the cytoplasm. The relevance of this activity is discussed.