Bridging drug discovery through hierarchical subtractive genomics against asd, trpG, and secY of pneumonia causing MDR Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is an opportunistic gram-positive, non-motile, and non-sporulating bacteria that induces pneumonia, a provocative lung infection affecting mainly the terminal bronchioles and the small air sacs known as alveoli. Recently, it has developed antibiotic resistance to the available consortium as per the WHO reports; thereby, novel remedial targets and resilient medications to forestall and cure this illness are desperately needed. Here, using pan-genomics, a total of 1,387 core proteins were identified. Subtractive proteome analyses further identified 12 proteins that are vital for bacteria. One membrane protein (secY) and two cytoplasmic proteins (asd and trpG) were chosen as possible therapeutic targets concerning minimum % host identity, essentiality, and other cutoff values, such as high resistance in the MDR S. aureus. The UniProt AA sequences of the selected targets were modelled and docked against 3 drug-like chemical libraries. The top-ranked compounds i.e., ZINC82049692, ZINC85492658 and 3a of Isosteviol derivative for Aspartate-semialdehyde dehydrogenase (asd); ZINC38222743, ZINC70455378, and 5 m Isosteviol derivative for Anthranilate synthase component II (trpG); and finally, ZINC72292296, ZINC85632684, and 7 m Isosteviol derivative for Protein translocase subunit secY (secY), were further subjected to molecular dynamics studies for thermodynamic stability and energy calculation. Our study proposes new therapeutic targets in S. aureus, some of which have previously been reported in other pathogenic microorganisms. Owing to further experimental validation, we anticipate that the adapted methodology and the predicted results in this work could make major contributions towards novel drug discovery and their targets in S. aureus caused pneumonia.

Structural-functional analysis of drug target aspartate semialdehyde dehydrogenase.

Aspartate ß-semialdehyde dehydrogenase (ASADH) is a key enzyme in the biosynthesis of essential amino acids in microorganisms and some plants. Inhibition of ASADHs can be a potential drug target for developing novel antimicrobial and herbicidal compounds. This review covers up-to-date information about sequence diversity, ligand/inhibitor-bound 3D structures, potential inhibitors, and key pharmacophoric features of ASADH useful in designing novel and target-specific inhibitors of ASADH. Most reported ASADH inhibitors have two highly electronegative functional groups that interact with two key arginyl residues present in the active site of ASADHs. The structural information, active site binding modes, and key interactions between the enzyme and inhibitors serve as the basis for designing new and potent inhibitors against the ASADH family.

Structural characterization of aspartate-semialdehyde dehydrogenase from Pseudomonas aeruginosa and Neisseria gonorrhoeae.

Gonorrhoea infection rates and the risk of infection from opportunistic pathogens including P. aeruginosa have both risen globally, in part due to increasing broad-spectrum antibiotic resistance. Development of new antimicrobial drugs is necessary and urgent to counter infections from drug resistant bacteria. Aspartate-semialdehyde dehydrogenase (ASADH) is a key enzyme in the aspartate biosynthetic pathway, which is critical for amino acid and metabolite biosynthesis in most microorganisms including important human pathogens. Here we present the first structures of two ASADH proteins from N. gonorrhoeae and P. aeruginosa solved by X-ray crystallography. These high-resolution structures present an ideal platform for in silico drug design, offering potential targets for antimicrobial drug development as emerging multidrug resistant strains of bacteria become more prevalent.

Evaluation of a novel inhibitor of aspartate semialdehyde dehydrogenase as a potent antitubercular agent against Mycobacterium tuberculosis.

The in vitro activity of IMB-XMA0038, a novel inhibitor targeting Mycobacterial tuberculosis (Mtb) aspartate semialdehyde dehydrogenase, was evaluated. Minimum inhibitory concentrations (MICs) of IMB-XMA0038 were against 20 Mtb isolates, including H37Rv (ATCC 27294), ten clinical pan-sensitive isolates, and nine clinical multidrug-resistant (MDR) isolates. In addition, minimum bactericidal concentrations (MBCs) were also determined against the H37Rv and 6 MDR isolates (the background information is same as above in order). A model was generated to evaluate IMB-XMA0038 activity against dormant Mtb. The post-antibiotic effect (PAE), an important indicator of antimicrobial drug dosing schedules to obtain efficacy, was determined based on time required for regrowth of Mtb to 50% of the OD600max value after treatment with various concentrations of IMB-XMA0038 and INH. In addition, interactions between IMB-XMA0038 and other anti-tuberculosis drugs, measured using a checkerboard assay, revealed that IMB-XMA0038 MICs of 0.5-1 µg/mL could be achieved in combinations. Synergistic effects were observed for IMB-XMA0038 when used together with almost all other anti-tuberculosis drugs against most Mtb isolates. IMB-XMA0038 exhibited greater activity than rifampin against Mtb under hypoxic conditions, as reflected by CFU decreases of 1.1-log-unit versus 0.8-log-unit, respectively, for IMB-XMA0038 and rifampin concentrations of 4 × MIC. IMB-XMA0038-induced PAEs (9, 10, 11 days) were comparable to INH PAEs (10, 11, 12 days). These findings suggest that addition of IMB-XMA0038 to current therapeutic regimens could be useful to improve the efficacy of treatments for drug-resistant and drug-susceptible TB.

Design and testing of selective inactivators against an antifungal enzyme target.

Systemic infections from fungal organisms are becoming increasingly difficult to treat as drug resistance continues to emerge. To substantially expand the antifungal drug landscape new compounds must be identified and developed with novel modes of action against previously untested drug targets. Most drugs block the activity of their targets through reversible, noncovalent interactions. However, a significant number of drugs form irreversible, covalent bonds with their selected targets. While more challenging to develop, these irreversible inactivators offer some significant advantages as novel antifungal agents. Vinyl sulfones contain a potentially reactive functional group that could function as a selective enzyme inactivator, and members of this class of compounds are now being developed as inactivators against an antifungal drug target. The enzyme aspartate semialdehyde dehydrogenase (ASADH) catalyzes a key step in an essential microbial pathway and is essential for the survival of every microorganism examined. A series of vinyl sulfones have been designed, guided by molecular modeling and docking studies to enhance their affinity for fungal ASADHs. These newly synthesized compounds have been examined against this target enzyme from the pathogenic fungal organism Candida albicans. Vinyl sulfones containing complementary structural elements inhibit this enzyme with inhibition constants in the low-micromolar range. These inhibitors have also led to the rapid and irreversible inactivation of this enzyme, and show some initial selectivity when compared to the inactivation of a bacterial ASADH. The best inactivators will serve as lead compounds for the development of potent and selective antifungal agents.

Molecular evolution, binding site interpretation and functional divergence of aspartate semialdehyde dehydrogenase.

Aspartate Semialdehyde Dehydrogenase (ASDH) is an important enzyme essential for the viability of pathogenic microorganisms. ASDH is mainly involved in amino acid and cell wall biosynthesis of microorganisms, hence it is considered to be a promising target for drug design. This enzyme depicts similar mechanistic function in all microorganisms; although, the kinetic efficiency of an enzyme differs according to their active site residual composition. Therefore, understanding the residual variation and kinetic efficiency of the enzyme would pave new insights in structure-based drug discovery and a novel drug molecule against ASDH. Here, ASDH from Wolbachia endosymbiont of Brugia malayi is used as a prime enzyme to execute evolutionary studies. The phylogenetic analysis was opted to classify 400 sequences of ASDH enzymes based on their structure and electrostatic surfaces. Analysis resulted in 37 monophyletic clades of diverse pathogenic and non-pathogenic organisms. The representative structures of 37 ASDHs from different clades were further deciphered to structural homologues. These enzymes exhibited presence of more positively charged surfaces than negatively charged surfaces in the active site pocket which restrains evolutionary significance. Docking studies of NADP+ with 37 enzymes reveals that site-specific residual variation in the active site pocket modulates the binding affinity (ranges of -13 to -9 kcal/mol). Type-I and Type-II divergence studies show, no significant functional divergence among ASDH, but residual changes were found among the enzyme that modulates the biochemical characteristics and catalytic efficiency. The present study not only explores residual alteration and catalytic variability, it also aids in the design of species-specific inhibitors.Communicated by Ramaswamy H. Sarma.

IMB-XMA0038, a new inhibitor targeting aspartate-semialdehyde dehydrogenase of Mycobacterium tuberculosis.

The emergence of drug-resistant tuberculosis (TB) constitutes a major challenge to TB control programmes. There is an urgent need to develop effective anti-TB drugs with novel mechanisms of action. Aspartate-semialdehyde dehydrogenase (ASADH) is the second enzyme in the aspartate metabolic pathway. The absence of the pathway in humans and the absolute requirement of aspartate in bacteria make ASADH a highly attractive drug target. In this study, we used ASADH coupled with Escherichia coli type III aspartate kinase (LysC) to establish a high-throughput screening method to find new anti-TB inhibitors. IMB-XMA0038 was identified as an inhibitor of MtASADH with an IC50 value of 0.59 µg/mL through screening. The interaction between IMB-XMA0038 and MtASADH was confirmed by surface plasmon resonance (SPR) assay and molecular docking analysis. Furthermore, IMB-XMA0038 was found to inhibit various drug-resistant MTB strains potently with minimal inhibitory concentrations (MICs) of 0.25-0.5 µg/mL. The conditional mutant strain MTB::asadh cultured with different concentrations of inducer (10-5 or 10-1 µg/mL pristinamycin) resulted in a maximal 16 times difference in MICs. At the same time, IMB-XMA0038 showed low cytotoxicity in vitro and vivo. In mouse model, it encouragingly declined the MTB colony forming units (CFU) in lung by 1.67 log10 dosed at 25 mg/kg for 15 days. In conclusion, our data demonstrate that IMB-XMA0038 is a promising lead compound against drug-resistant tuberculosis.

In silico anti-fungal efficacy and the mechanism of binding of some Syzygium aromaticum ingredient compounds to aspartate semialdehyde dehydrogenase, 6C8W and 6C85, enzymes from Blastomyces dermatitidis.

This in silico work was carried out to reveal the proposed anti-fungal efficacy of some clove ingredient compounds against aspartate semialdehyde dehydrogenase, 6C8W and 6C85, enzymes from Blastomyces dermatitidis. The molecular docking simulation was implemented utilizing the Auto Dock 4.2. software. A set of 17 compounds were selected for this study, which is known to be active ingredients of Syzygium aromaticum crude and oil. The best docking scores associated with the Blastomyces dermatitidis enzymes 6C85 and 6C8W were for Maslinic acid and Oleanolic acid, followed by Stigmasterol and Campesterol. It was found that these compounds possess inhibitory potential against 6C85 and 6C8W and hence have anti-fungal efficacy. Maslinic acid and Oleanolic acid produced the strongest binding to 6C85 and 6C8W over the remaining bioactive compounds by forming H-bonds with some amino acids in these enzymes.

Aspartate semialdehyde dehydrogenase inhibition suppresses the growth of the pathogenic fungus Candida albicans.

Potent inhibitors of an essential microbial enzyme have been shown to be effective growth inhibitors of Candida albicans, a pathogenic fungus. C. albicans is the main cause of oropharyngeal candidiasis, and also causes invasive fungal infections, including systemic sepsis, leading to serious complications in immunocompromised patients. As the rates of drug-resistant fungal infections continue to rise novel antifungal treatments are desperately needed. The enzyme aspartate semialdehyde dehydrogenase (ASADH) is critical for the functioning of the aspartate biosynthetic pathway in microbes and plants. Because the aspartate pathway is absent in humans, ASADH has the potential to be a promising new target for antifungal research. Deleting the asd gene encoding for ASADH significantly decreases the survival of C. albicans, establishing this enzyme as essential for this organism. Previously developed ASADH inhibitors were tested against several strains of C. albicans to measure their possible therapeutic impact. The more potent inhibitors show a good correlation between enzyme inhibitor potency and fungal growth inhibition. Growth curves generated by incubating different C. albicans strains with varying enzyme inhibitor levels show significant slowing of fungal growth by these inhibitors against each of these strains, similar to the effect observed with a clinical antifungal drug. The most effective inhibitors also demonstrated relatively low cytotoxicity against a human epithelial cell line. Taken together, these results establish that the ASADH enzyme is a promising new target for further development as a novel antifungal treatment against C. albicans and related fungal species.

In Silico and In Vitro Anti-Helicobacter Pylori Effects of Combinations of Phytochemicals and Antibiotics.

Helicobacter pylori infection is a WHO class 1 carcinogenic factor of gastric adenocarcinoma. In the past decades, many studies have demonstrated the increasing trend of antibiotic resistance and pointed out the necessity of new effective treatment. This study was aimed at identifying phytochemicals that can inhibit H. pylori and possibly serve as adjuvant treatments. Here, in silico molecular docking and drug-like properties analyses were performed to identify potential inhibitors of urease, shikimate kinase and aspartate-semialdehyde dehydrogenase. These three enzymes are targets of the treatment of H. pylori. Susceptibility and synergistic testing were performed on the selected phytochemicals and the positive control antibiotic, amoxicillin. The in-silico study revealed that oroxindin, rosmarinic acid and verbascoside are inhibitors of urease, shikimate kinase and aspartate-semialdehyde dehydrogenase, respectively, in which, oroxindin has the highest potency against H. pylori, indicated by a minimum inhibitory concentration (MIC) value of 50 µg/mL. A combination of oroxindin and amoxicillin demonstrated additive effects against H. pylori, as indicated by a fractional inhibitory concentration (FIC) value of 0.75. This study identified phytochemicals that deserve further investigation for the development of adjuvant therapeutic agents to current antibiotics against H. pylori.

Identification of anti-filarial leads against aspartate semialdehyde dehydrogenase of Wolbachia endosymbiont of Brugia malayi: combined molecular docking and molecular dynamics approaches.

Lymphatic filariasis is a debilitating vector borne parasitic disease that infects human lymphatic system by nematode Brugia malayi. Currently available anti-filarial drugs are effective only on the larval stages of parasite. So far, no effective drugs are available for humans to treat filarial infections. In this regard, aspartate semialdehyde dehydrogenase (ASDase) in lysine biosynthetic pathway from Wolbachia endosymbiont Brugia malayi represents an attractive therapeutic target for the development of novel anti-filarial agents. In this present study, molecular modeling combined with molecular dynamics simulations and structure-based virtual screening were performed to identify potent lead molecules against ASDase. Based on Glide score, toxicity profile, binding affinity and mode of interactions with the ASDase, five potent lead molecules were selected. The molecular docking and dynamics results revealed that the amino acid residues Arg103, Asn133, Cys134, Gln161, Ser164, Lys218, Arg239, His246, and Asn321 plays a crucial role in effective binding of Top leads into the active site of ASDase. The stability of the ASDase-lead complexes was confirmed by running the 30 ns molecular dynamics simulations. The pharmacokinetic properties of the identified lead molecules are in the acceptable range. Furthermore, density functional theory and binding free energy calculations were performed to rank the lead molecules. Thus, the identified lead molecules can be used for the development of anti-filarial agents to combat the pathogenecity of Brugia malayi.

The heritable natural competency trait of Burkholderia pseudomallei in other Burkholderia species through comE and crp.

Natural competency requires uptake of exogenous DNA from the environment and the integration of that DNA into recipient bacteria can be used for DNA-repair or genetic diversification. The Burkholderia genus is unique in that only some of the species and strains are naturally competent. We identified and characterized two genes, comE and crp, from naturally competent B. pseudomallei 1026b that play a role in DNA uptake and catabolism. Single-copies of rhamnose-inducible comE and crp genes were integrated into a Tn7 attachment-site in non-naturally competent Burkholderia including pathogens B. pseudomallei K96243, B. cenocepacia K56-2, and B. mallei ATCC23344. Strains expressing comE or crp were assayed for their ability to uptake and catabolize DNA. ComE and Crp allowed non-naturally competent Burkholderia species to catabolize DNA, uptake exogenous gfp DNA and express GFP. Furthermore, we used synthetic comE and crp to expand the utility of the λ-red recombineering system for genetic manipulation of non-competent Burkholderia species. A newly constructed vector, pKaKa4, was used to mutate the aspartate semialdehyde dehydrogenase (asd) gene in four B. mallei strains, leading to the complete attenuation of these tier-1 select-agents. These strains have been excluded from select-agent regulations and will be of great interest to the field.

Structural insights into inhibitor binding to a fungal ortholog of aspartate semialdehyde dehydrogenase.

The aspartate pathway, uniquely found in plants and microorganisms, offers novel potential targets for the development of new antimicrobial drugs. Aspartate semialdehyde dehydrogenase (ASADH) catalyzes production of a key intermediate at the first branch point in this pathway. Several fungal ASADH structures have been determined, but the prior crystallization conditions had precluded complex formation with enzyme inhibitors. The first inhibitor-bound and cofactor-bound structures of ASADH from the pathogenic fungi Blastomyces dermatitidis have now been determined, along with a structural and functional comparison to other ASADH family members. The structure of this new ASADH is similar to the other fungal orthologs, but with some critical differences in the orientation of some active site functional groups and in the subunit interface region. The presence of this bound inhibitor reveals the first details about inhibitor binding interactions, and the flexible orientation of its aromatic ring provides helpful insights into the design of potentially more potent and selective antifungal compounds.

A Fragment Library Screening Approach to Identify Selective Inhibitors against an Essential Fungal Enzyme.

Pathogenic fungi represent a growing threat to human health, with an increase in the frequency of drug-resistant fungal infections. Identifying targets from among the selected metabolic pathways that are unique to microbial species presents an opportunity to develop new antifungal agents against new and untested targets to combat this growth threat. Aspartate semialdehyde dehydrogenase (ASADH) catalyzes a key step in a uniquely microbial amino acid biosynthetic pathway and is essential for microbial viability. This enzyme, purified from four pathogenic fungal organisms ( Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, and Blastomyces dermatitidis), has been screened against fragment libraries to identify initial enzyme inhibitors. The binding of structural analogs of the most promising lead compounds was measured against these fungal ASADHs to establish important structure-activity relationships among these different inhibitor classes. The most potent of these inhibitors have been docked into structures of this fungal enzyme target to identify important structural elements that serve as critical binding determinants. Several inhibitors with low micromolar inhibition constants have been identified that showed selectivity against these related enzymes from different fungal species. Subsequent screening against a library of drugs and drug candidates identified some additional inhibitors containing a consistent set of functional groups required for fungal ASADH inhibition. Additional elaboration of these core structures will likely lead to more potent and selective inhibitors.

Structure of aspartate ß-semialdehyde dehydrogenase from Francisella tularensis.

Aspartate ß-semialdehyde dehydrogenase (ASADH) is an enzyme involved in the diaminopimelate pathway of lysine biosynthesis. It is essential for the viability of many pathogenic bacteria and therefore has been the subject of considerable research for the generation of novel antibiotic compounds. This manuscript describes the first structure of ASADH from Francisella tularensis, the causative agent of tularemia and a potential bioterrorism agent. The structure was determined at 2.45 Šresolution and has a similar biological assembly to other bacterial homologs. ASADH is known to be dimeric in bacteria and have extensive interchain contacts, which are thought to create a half-sites reactivity enzyme. ASADH from higher organisms shows a tetrameric oligomerization, which also has implications for both reactivity and regulation. This work analyzes the apo form of F. tularensis ASADH, as well as the binding of the enzyme to its cofactor NADP+.

Aspartate-ß-semialdeyhyde dehydrogenase as a potential therapeutic target of Mycobacterium tuberculosis H37Rv: Evidence from in silico elementary mode analysis of biological network model.

The emergence of multi-drug resistant strains and co-occurrence of tuberculosis with HIV creates a major burden to the human health globally. Failure of primary antibacterial therapy necessitates the identification of new mycobacterial drugs. In this study, a comprehensive analysis involving bottom-up systems biology approach was applied wherein we have identified potential therapeutic targets of Mycobacterium tuberculosis infections. Our study prioritized M. tuberculosis therapeutic targets (aspartate-ß-semialdeyhde dehydrogenase [ASD], dihydrodipicolinate reductase and diaminopimelate decarboxylase) based on flux and elementary mode analysis using direct mathematical modeling of the relevant metabolic pathways. Molecular docking and simulation studies of the priority target (ie, ASD) revealed the therapeutic potential of the selected natural products (Huperzine A, Rosmarinic acid, and Curcumin) based ASD inhibitors. The study highlights the crucial role of systems biology in conjunction with molecular interaction (docking) for probing novel leads against an increasingly resistant pathogen, M. tuberculousis.

Proteome mining for the identification and in-silico characterization of putative drug targets of multi-drug resistant Clostridium difficile strain 630.

Clostridium difficile is an enteric pathogen that causes approximately 20% to 30% of antibiotic-associated diarrhea. In recent years, there has been a substantial rise in the rate of C. difficile infections as well as the emergence of virulent and antibiotic resistant C. difficile strains. So, there is an urgent need for the identification of therapeutic potential targets and development of new drugs for the treatment and prevention of C. difficile infections. In the current study, we used a hybrid approach by combining sequence similarity-based approach and protein-protein interaction network topology-based approach to identify and characterize the potential drug targets of C. difficile. A total of 155 putative drug targets of C. difficile were identified and the metabolic pathway analysis of these putative drug targets using DAVID revealed that 46 of them are involved in 9 metabolic pathways. In-silico characterization of these proteins identified seven proteins involved in pathogen-specific peptidoglycan biosynthesis pathway. Three promising targets viz. homoserine dehydrogenase, aspartate-semialdehyde dehydrogenase and aspartokinase etc. were found to be involved in multiple enzymatic pathways of the pathogen. These 3 drug targets are of particular interest as they can be used for developing effective drugs against multi-drug resistant C. difficile strain 630 in the near future.

Structure of a fungal form of aspartate-semialdehyde dehydrogenase from Aspergillus fumigatus.

Aspartate-semialdehyde dehydrogenase (ASADH) functions at a critical junction in the aspartate biosynthetic pathway and represents a validated target for antimicrobial drug design. This enzyme catalyzes the NADPH-dependent reductive dephosphorylation of ß-aspartyl phosphate to produce the key intermediate aspartate semialdehyde. The absence of this entire pathway in humans and other mammals will allow the selective targeting of pathogenic microorganisms for antimicrobial development. Here, the X-ray structure of a new form of ASADH from the pathogenic fungal species Aspergillus fumigatus has been determined. The overall structure of this enzyme is similar to those of its bacterial orthologs, but there are some critical differences both in biological assembly and in secondary-structural features that can potentially be exploited for the development of species-selective drugs with selective toxicity against infectious fungal organisms.

Biochemical and structural characterization of quinoprotein aldose sugar dehydrogenase from Thermus thermophilus HJ6: Mutational analysis of Tyr156 in the substrate-binding site.

The gene encoding a quinoprotein aldose sugar dehydrogenase (ASD) from Thermus thermophilus HJ6 (Tt_ASD) was cloned and sequenced; it comprised 1059 nucleotides encoding a protein containing 352 amino acids that had a predicted molecular mass of 38.9 kDa. The deduced amino acid sequence showed 42.9% and 33.9% identities to the ASD proteins from Pyrobaculum aerophilum and Escherichia coli, respectively. The biochemical properties of Tt_ASD were characterized. The optimum pH for the oxidation of glucose was 7.0-7.5 and the optimum temperature was 70 °C. The half-life of heat inactivation for the apoenzyme was about 25 min at 85 °C. The enzyme was highly thermostable, and the activity of the pyrroloquinoline quinone-bound holoenzyme was not lost after incubation at 85 °C for 100 min. Tt_ASD could oxidize various sugars, including hexoses, pentoses, disaccharides, and polysaccharides, in addition to alcohols. Structural analysis suggested that Tyr156 would be the substrate-binding residue. Two mutants, Y156A and Y156K, had impaired activities and affinities for all substrates and completely lost their activities for alcohols. This structural and mutational analysis of Tt_ASD demonstrates the crucial role of Tyr156 in determining substrate specificity.