Structural characterization of inhibitors with selectivity against members of a homologous enzyme family

The aspartate biosynthetic pathway is found only in plants and microbes. The commitment step to this pathway is the phosphorylation of aspartic acid catalyzed by aspartokinases (1). The next enzyme, aspartate-β-semialdehyde dehydrogenase (ASADH), catalyzes a reductive dephosphorylation to produce aspartate β-semialdehyde. At this point the pathway can branch to produce either homoserine as the common precursor for the synthesis of methionine, threonine and isoleucine, or go through dihydrodipicolinate and diaminopimelate to synthesize lysine (2). Thus, one-quarter of the amino acids required for protein synthesis in these organisms are linked through and synthesized by this pathway. In addition to these essential amino acids this pathway also produces several important metabolites that play crucial roles in microbial developmental processes, including cell wall biosynthesis, protective dormancy, and virulence factor production. These intermediates include dihydrodipicolinate, a precursor of dipicolinate which is the major component of bacterial spores (3), and diaminopimelate, required for cross-linking of the peptidoglycan polymers in bacterial cell wall synthesis (4). Another product of this pathway, S-adenosylmethionine, is an essential methyl group donor that also serves as a precursor for quorum sensing signaling molecules (5,6) with critical roles in triggering virulence factors in infectious organisms. Selective perturbations of the asd gene that encodes for ASADH have been shown to be lethal to microorganisms (7), and numerous studies to determine the minimal set of genes needed for organism survival have identified the asd gene as being essential (8-11). Our goal is to identify selective inhibitors of this validated target that can be developed into lead compounds for antimicrobial development. The identification of new inhibitors against a target enzyme have traditionally followed two quite different approaches, either using the known structures of substrates and products to guide the synthesis of structural analogues or by screening compound libraries to identify novel groups of inhibitory structures. Screening to identify initial hits has been driven by the search for high affinity compounds for each potential drug target. Modifications of these initial hits to further enhance target affinity and improve target selectivity are then used to develop the advanced leads that move towards clinical trials. In contrast to this traditional approach, fragment-based drug discovery is designed around the hypothesis that high affinity is not the best selection criteria with which to identify initial hits (12). Instead, ligand efficiency (L.E.) has been proposed as the selection metric, where ligand efficiency is defined as the free energy of binding (ΔG) per heavy (non-hydrogen) atom in the ligand (13). Typical fragments, developed from incorporating useful functional groups into molecular scaffolds, are in the molecular weight range of 120-250 Da and have affinities in the high micromolar to low millimolar range. These fragments are then screened to probe the essential binding sites within the active site of a target enzyme. The aim is to identify a set of minimal functional components that bind to an enzyme target with reasonable affinity and high L.E. values. We have examined both the substrate analogue and the fragment screening approaches to identify new enzyme inhibitors that show selectivity against representative ASADH enzymes isolated from different microorganisms. Kinetic studies had been used to screen fragment molecule libraries to identify new compounds that bind to ASADHs with high ligand efficiencies. Inhibitors were identified with selectivity against either Gram-negative or Gram-positive bacterial enzyme forms, or compounds that inhibit only a fungal form of ASADH (14). Different compounds from these groups of inhibitors have now been crystallized with ASADHs from a representative Gram-positive Streptococcus pneumoniae and a representative Gram-negative Vibrio cholerae bacterial species. The structures reported herein are being used to guide the design of more potent inhibitors with enhanced selectivity towards a single microbial species or a subset of bacterial or fungal species.