Structural characterization of inhibitors with selectivity against members of a homologous enzyme family (original) (raw)
Related papers
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.
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.
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...
Active Site Analysis of the Potential Antimicrobial Target Aspartate Semialdehyde Dehydrogenase
Biochemistry, 2001
Aspartate--semialdehyde dehydrogenase (ASADH) lies at the first branch point in the biosynthetic pathway through which bacteria, fungi, and the higher plants synthesize amino acids, including lysine and methionine and the cell wall component diaminopimelate from aspartate. Blocks in this biosynthetic pathway, which is absent in mammals, are lethal, and inhibitors of ASADH may therefore serve as useful antibacterial, fungicidal, or herbicidal agents. We have determined the structure of ASADH from Escherichia coli by crystallography in the presence of its coenzyme and a substrate analogue that acts as a covalent inhibitor. This structure is comparable to that of the covalent intermediate that forms during the reaction catalyzed by ASADH. The key catalytic residues are confirmed as cysteine 135, which is covalently linked to the intermediate during the reaction, and histidine 274, which acts as an acid/base catalyst. The substrate and coenzyme binding residues are also identified, and these active site residues are conserved throughout all of the ASADH sequences. Comparison of the previously determined apoenzyme structure [Hadfield et al. J. Mol. Biol. (1999) 289, 991-1002 and the complex presented here reveals a conformational change that occurs on binding of NADP that creates a binding site for the amino acid substrate. These results provide a structural explanation for the preferred order of substrate binding that is observed kinetically.
Biochemical and Biophysical Research Communications, 2018
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.
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...
A structural basis for the mechanism of aspartate- -semialdehyde dehydrogenase from Vibrio cholerae
Protein Science, 2003
L-Aspartate--semialdehyde dehydrogenase (ASADH) catalyzes the reductive dephosphorylation of -aspartyl phosphate to L-aspartate--semialdehyde in the aspartate biosynthetic pathway of plants and microorganisms. The aspartate pathway produces fully one-quarter of the naturally occurring amino acids, but is not found in humans or other eukaryotic organisms, making ASADH an attractive target for the development of new antibacterial, fungicidal, or herbicidal compounds. We have determined the structure of ASADH from Vibrio cholerae in two states; the apoenzyme and a complex with NADP, and a covalently bound active site inhibitor, S-methyl-L-cysteine sulfoxide. Upon binding the inhibitor undergoes an enzymecatalyzed reductive demethylation leading to a covalently bound cysteine that is observed in the complex structure. The enzyme is a functional homodimer, with extensive intersubunit contacts and a symmetrical 4-amino acid bridge linking the active site residues in adjacent subunits that could serve as a communication channel. The active site is essentially preformed, with minimal differences in active site conformation in the apoenzyme relative to the ternary inhibitor complex. The conformational changes that do occur result primarily from NADP binding, and are localized to the repositioning of two surface loops located on the rim at opposite sides of the NADP cleft.
Scientific Reports, 2016
Aspartate-β-semialdehyde dehydrogenase (ASADH) catalyzes the second reaction in the aspartate pathway, a pathway required for the biosynthesis of one fifth of the essential amino acids in plants and microorganisms. Microarray analysis of a fungal pathogen T. rubrum responsible for most human dermatophytoses identified the upregulation of ASADH (trASADH) expression when the fungus is exposed to human skin, underscoring its potential as a drug target. Here we report the crystal structure of trASADH, revealing a tetrameric ASADH with a GAPDH-like fold. The tetramerization of trASADH was confirmed by sedimentation and SAXS experiments. Native PAGE demonstrated that this ASADH tetramerization is apparently universal in fungal species, unlike the functional dimer that is observed in all bacterial ASADHs. The helical subdomain in dimeric bacteria ASADH is replaced by the cover loop in archaeal/fungal ASADHs, presenting the determinant for this altered oligomerization. Mutations that disrupt the tetramerization of trASADH also abolish the catalytic activity, suggesting that the tetrameric state is required to produce the active fungal enzyme form. Our findings provide a basis to categorize ASADHs into dimeric and tetrameric enzymes, adopting a different orientation for NADP binding and offer a structural framework for designing drugs that can specifically target the fungal pathogens. Trichophyton rubrum is the most prevalent fungal pathogen for human dermatophytoses, accounting for ~ 70% of the total dermatophyte infections 1. Recent microarray analysis revealed that the expression of a group of genes were upregulated when T. rubrum was exposed to human skin, suggesting their roles as virulence factors and the potential for drug targeting against this fungal organism. Among the upregulated genes EL785855 drew our attention because it encodes for an aspartate-β-semialdehyde dehydrogenase (ASADH) 2. This enzyme catalyzes the second reaction in the aspartate pathway that is essential in amino acid biosynthesis. ASADH converts β-aspartyl phosphate to aspartate-β-semialdehyde (ASA), which is then either converted to homoserine, a common intermediate in the biosynthesis of threonine, isoleucine, and methionine, or is condensed with pyruvate leading to the production of lysine 3. The aspartate pathway is the only source for the synthesis of one fifth of the essential amino acids for protein production in plants and microorganisms 4,5. In addition, the aspartate pathway provides the upstream source for cell-wall biosynthesis 6 , the protective dormancy process 7 and virulence factor production 8. Therefore, it is no wonder that the asd gene belongs to the minimal gene set shown to be indispensable for microorganism survival 9,10. It has been demonstrated that disruption of the asd gene will be lethal for many microbial pathogens 11-13 , and ASADH does not have homologs in mammalian cells. Therefore, inhibitors targeting ASADH are considered a promising strategy for the development of novel biocides 3. In order to assist the drug design against ASADH, high-resolution structural details and full elucidation of the catalytic mechanism are essential. A large collection of crystal structures for ASADHs have been determined to date 3,14-19. Crystallographic data demonstrates that although ASADHs from a variety of organisms exhibit significant sequence diversities (ranging from10 to 95% homology comparing to the prototype Escherichia coli ASADH, ecASADH), the overall fold,
J Biol Chem, 2006
Aspartate--semialdehyde dehydrogenase (ASADH) catalyzes a critical branch point transformation in amino acid biosynthesis. The products of the aspartate pathway are essential in microorganisms, and this entire pathway is absent in mammals, making this enzyme an attractive target for antibiotic development. The first structure of an ASADH from a Gram-positive bacterium, Streptococcus pneumoniae, has now been determined. The overall structure of the apoenzyme has a similar fold to those of the Gram-negative and archaeal ASADHs but contains some interesting structural variations that can be exploited for inhibitor design. Binding of the coenzyme NADP, as well as a truncated nucleotide analogue, into an alternative conformation from that observed in Gram-negative ASADHs causes an enzyme domain closure that precedes catalysis. The covalent acyl-enzyme intermediate was trapped by soaking the substrate into crystals of the coenzyme complex, and the structure of this elusive intermediate provides detailed insights into the catalytic mechanism.