Early Stage Efficacy and Toxicology Screening for Antibiotics and Enzyme Inhibitors (original) (raw)
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Bioorganic & Medicinal Chemistry, 2015
Aspartate-β-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the aspartate metabolic pathway which leads to the biosynthesis of several essential amino acids and some important metabolites. This pathway is crucial for many metabolic processes in plants and microbes like bacteria and fungi, but is absent in mammals. Therefore, the key microbial enzymes involved in this pathway are attractive potential targets for development of new antibiotics with novel modes of action. The ASADH enzyme family shares the same substrate binding and active site catalytic groups; however, the enzymes from representative bacterial and fungal species show different inhibition patterns when previously screened against low molecular weight inhibitors identified from fragment library screening. In the present study several approaches, including fragment based drug discovery (FBDD), inhibitor docking, kinetic, and structure activity relationship (SAR) studies have been used to guide ASADH inhibitor development. Elaboration of a core structure identified by FBDD has led to the synthesis of low micromolar inhibitors of the target enzyme, with high selectivity introduced between the Gram-negative and Gram-positive orthologs of ASADH. This new set of structures open a novel direction for the development of inhibitors against this validated drug-target enzyme.
Journal of Biomolecular Screening, 2004
Efficacy and tolerability are the key criteria for a successful medication in the clinic. Therefore, a new test method to obtain selective and active lead molecules has been developed. Recently, this novel screening strategy enabled a breakthrough in drug discovery in the field of herpes viruses. Here the authors report that this assay is a generally applicable screening test, which allows not only for identifying tolerable and potent antimicrobial agents in compound libraries, but also covers all potential in vitro targets of both the pathogen and the host simultaneously. The test system mimics the smallest unit of a natural infection. Host cells are incubated in the presence of the test sample and are infected with microbes, such as viruses, bacteria, or fungi. Analogous to (lethal challenge) animal models, cell survival is determined. This assay maximizes the chances of success of anti-infective drug discovery, is sensitive, robust, time-and cost-efficient, and especially effective in optimizing screening hits to lead structures and development candidates. In addition to the minimal inhibitory concentration or dose, this test system simultaneously provides the selectivity index, a measure of tolerability in vitro. The authors propose the activity selectivity assay format as a new standard in anti-infective drug discovery and clinical development. (Journal of Biomolecular Screening 2004:578-587)
Bioorganic & Medicinal Chemistry, 2012
Microbes that have gained resistance against antibiotics pose a major emerging threat to human health. New targets must be identified that will guide the development of new classes of antibiotics. The selective inhibition of key microbial enzymes that are responsible for the biosynthesis of essential metabolites can be an effective way to counter this growing threat. Aspartate semialdehyde dehydrogenases (ASADHs) produce an early branch point metabolite in a microbial biosynthetic pathway for essential amino acids and for quorum sensing molecules. In this study, molecular modeling and docking studies were performed to achieve two key objectives that are important for the identification of new selective inhibitors of ASADH. First, virtual screening of a small library of compounds was used to identify new core structures that could serve as potential inhibitors of the ASADHs. Compounds have been identified from diverse chemical classes that are predicted to bind to ASADH with high affinity. Next, molecular docking studies were used to prioritize analogs within each class for synthesis and testing against representative bacterial forms of ASADH from Streptococcus pneumoniae and Vibrio cholerae. These studies have led to new micromolar inhibitors of ASADH, demonstrating the utility of this molecular modeling and docking approach for the identification of new classes of potential enzyme inhibitors.
Differential Assay for High-Throughput Screening of Antibacterial Compounds
Journal of Biomolecular Screening, 2007
The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/ surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed β-gal in the periplasm, suggesting leakage of β-gal as the means by which this assay detects compound activities. A model is proposed according to which β-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-β-D-galactoside as a single reagent. Cell wall inhibitors release β-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielde...
Acta crystallographica. Section D, Biological crystallography, 2014
The aspartate pathway is essential for the production of the amino acids required for protein synthesis and of the metabolites needed in bacterial development. This pathway also leads to the production of several classes of quorum-sensing molecules that can trigger virulence in certain microorganisms. The second enzyme in this pathway, aspartate β-semialdehyde dehydrogenase (ASADH), is absolutely required for bacterial survival and has been targeted for the design of selective inhibitors. Fragment-library screening has identified a new set of inhibitors that, while they do not resemble the substrates for this reaction, have been shown to bind at the active site of ASADH. Structure-guided development of these lead compounds has produced moderate inhibitors of the target enzyme, with some selectivity observed between the Gram-negative and Gram-positive orthologs of ASADH. However, many of these inhibitor analogs and derivatives have not yet achieved the expected enhanced affinity. Str...
Drug Discovery of Newer Analogs of Anti-Microbials Through Enzyme-Inhibition: A Review
International Journal of Pharmacy and Pharmaceutical Sciences, 2014
There is a growing interest towards the development of new antibiotics from last decades due to emergence of newer pathogenic bacterial strains with high resistance to powerful antibiotics of last resort. This has caused decline in research for developing newer antibacterial agents. Hence, there is continuous need to develop newer antibiotics that interact with essential mechanisms in bacteria. Recently, enzymes responsible for bio synthesis of the essential amino acid lysine in bacteria have been targeted and it has augmented interest to develop novel antibiotics and to enhance lysine yields in over-producing organisms. Peptidoglycan layer consists of a beta-1,4-linked polysaccharide of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) sugar units, cross linked by short pentapeptide (muramyl residues) side chain of general structure L-Ala-g-D-Glu-X-D-Ala-D-Ala, where X is either L-Lysine or meso-DAP. Formation of the cross-links makes bacterial cell wall resistant to lysis by intracellular osmotic pressure. Compounds which inhibit lysine or DAP biosynthesis could therefore be very effective antibiotics and novel targets. Lysine is a constituent in gram-positive bacteria while meso-DAP occurs in gram negative ones. In this review, substrate-based inhibitors of enzymes in the DAP pathway and inhibitors that allow better understanding of enzymology of the targets and provide insight for design of new inhibitors have been discussed. Resistant bacterial strains can be inhibited by using synthetic enzyme inhibitors of DAP pathway that are less toxic to mammals. Newer antimicrobial drugs can be thus developed by targeting the enzymes involved in this pathway.
Aspartate-semi aldehyde dehydrogenase (ASADH) is the enzyme that occurs in the biosynthetic pathway at a very first branch point. It lies in the biosynthetic pathway of important amino acids including methionine and lysine and the cell-wall component diaminopimelate (DAP). The enzymatic reaction of ASADH is the reductive de phosphorylation of aspartyl-β-phosphate (ABP) to aspartate β-semi aldehyde (ASA). Aspartate pathway is very essential for the survival of many microbes and is absent in humans, the enzymes involved in this pathway can be considered to be potential antibacterial drug targets. In this work, the structure of ASADH from Mycobacterium tuberculosis H37Rv (Mtb-ASADH) has been determined in complex with S-methyl-L-cysteine sulfoxide (SMCS) and sulfate at 1.95 Å resolution. The overall structure of Mtb-ASADH is similar to those of its orthologues. S-methyl cysteine sulfoxide is the known covalent bond forming inhibitor of ASADH enzyme. By this virtual screening and dockin...
The rise in organisms resistant to existing drugs has added urgency to the search for new antimicrobial agents. Aspartate β-semialdehyde dehydrogenase (ASADH) catalyzes a critical step in an essential microbial pathway that is absent in mammals. Our laboratory is using fragment library screening to identify efficient and selective ASADH inhibitors. These preliminary agents are then tested to identify compounds with desired antimicrobial properties for further refinement. Toward this end, we have established a microplate-based, dual-assay approach using a single reagent to evaluate antibiotic activity and mammalian cell toxicity during early stage development. The bacterial assay uses nonpathogenic bacteria to allow efficacy testing without a dedicated microbial laboratory. Toxicity assays are performed with a panel of mammalian cells derived from representative susceptible tissues. These assays can be adapted to target other microbial systems, such as fungi and biofilms, and additional mammalian cell lines can be added as needed. Application of this screening approach to antibiotic standards demonstrates the ability of these assays to identify bacterial selectivity and potential toxicity issues. Tests with selected agents from the ASADH inhibitor fragment library show some compounds with antibiotic activity, but as expected, most of these early agents display higher than desired mammalian cell toxicity.
Role of Toxicogenomics in the Development of Safe, Efficacious and Novel Anti-microbial Therapies
Infectious Disorders - Drug Targets, 2013
Over the last two decades, occurrence of bacterial resistance to commonly used antibiotics has necessitated the development of safer and more potent anti-microbial drugs. However, the development of novel antibiotics is severely hampered by adverse side effects, such as drug-induced liver toxicity. Several antibacterial drugs are known to have the potential to cause severe liver damage. The major challenge in developing novel anti-microbial drugs is to predict, with certain amount of probability, the drug-induced toxicity during the pre-clinical stages, thus optimizing and reducing the time and cost of drug development. Toxicogenomics approach is generally used to harness the potential of genomic tools and to understand the physiological basis of drug-induced toxicity based on the in-depth analysis of Metagenomic data sets, i.e., transcriptional, translational or metabolomic profiles. Toxicogenomics, therefore, represents a new paradigm in the drug development process, and is anticipated to play an invaluable role in future to develop safe and efficacious medicines, by predicting the toxic potential of a new chemical entity (NCE) in early stages of drug discovery. This review examines the toxicogenomic approach in predicting the safety/toxicity of novel anti-microbial drugs, and analyses the promises, pitfalls and challenges of applying this powerful technology to the drug development process.
A Systematic Screen of FDA-Approved Drugs for Inhibitors of Biological Threat Agents
PLoS ONE, 2013
Background: The rapid development of effective medical countermeasures against potential biological threat agents is vital. Repurposing existing drugs that may have unanticipated activities as potential countermeasures is one way to meet this important goal, since currently approved drugs already have well-established safety and pharmacokinetic profiles in patients, as well as manufacturing and distribution networks. Therefore, approved drugs could rapidly be made available for a new indication in an emergency.