Structural investigation of inhibitor designs targeting 3-dehydroquinate dehydratase from the shikimate pathway of Mycobacterium tuberculosis.

The shikimate pathway is essential in Mycobacterium tuberculosis and its absence from humans makes the enzymes of this pathway potential drug targets. In the present paper, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from M. tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analogue of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active-site subpocket inducing significant structural rearrangement. The flexible extensions of inhibitors designed to form π-stacking interactions with the catalytic Tyr24 have been investigated. The high-resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19-24 in MtDHQase ligand binding and catalytic mechanism and provide a rationale for the design and efficacy of inhibitors.

Application of fragment screening and fragment linking to the discovery of novel thrombin inhibitors.

The screening of fragments is an alternative approach to high-throughput screening for the identification of leads for therapeutic targets. Fragment hits have been discovered using X-ray crystallographic screening of protein crystals of the serine protease enzyme thrombin. The fragment library was designed to avoid any well-precedented, strongly basic functionality. Screening hits included a novel ligand (3), which binds exclusively to the S2-S4 pocket, in addition to smaller fragments which bind to the S1 pocket. The structure of these protein-ligand complexes are presented. A chemistry strategy to link two such fragments together and to synthesize larger drug-sized compounds resulted in the efficient identification of hybrid inhibitors with nanomolar potency (e.g., 7, IC50 = 3.7 nM). These potent ligands occupy the same area of the active site as previously described peptidic inhibitors, while having very different chemical architecture.

Design and structural analysis of aromatic inhibitors of type II dehydroquinase from Mycobacterium tuberculosis.

3-Dehydroquinase, the third enzyme in the shikimate pathway, is a potential target for drugs against tuberculosis. Whilst a number of potent inhibitors of the Mycobacterium tuberculosis enzyme based on a 3-dehydroquinate core have been identified, they generally show little or no in vivo activity, and were synthetically complex to prepare. This report describes studies to develop tractable and drug-like aromatic analogues of the most potent inhibitors. A range of carbon-carbon linked biaryl analogues were prepared to investigate the effect of hydrogen bond acceptor and donor patterns on inhibition. These exhibited inhibitory activity in the high-micromolar range. The addition of flexible linkers in the compounds led to the identification of more potent 3-nitrobenzylgallate- and 5-aminoisophthalate-based analogues.

Discovery of Schaeffer's acid analogues as lead structures of mycobacterium tuberculosis type†II dehydroquinase using a rational drug design approach.

Rational ligand design: Schaeffer's acid analogues were identified as novel inhibitors of M. tuberculosis type II dehydroquinase, a key enzyme of the shikimate pathway. Their likely binding mode was predicted using a combination of ensemble docking and flexible active site residues. Potentially, this scaffold could provide a good starting point for the design of antitubercular agents.

Hierarchical virtual screening for the discovery of new molecular scaffolds in antibacterial hit identification.

One of the initial steps of modern drug discovery is the identification of small organic molecules able to inhibit a target macromolecule of therapeutic interest. A small proportion of these hits are further developed into lead compounds, which in turn may ultimately lead to a marketed drug. A commonly used screening protocol used for this task is high-throughput screening (HTS). However, the performance of HTS against antibacterial targets has generally been unsatisfactory, with high costs and low rates of hit identification. Here, we present a novel computational methodology that is able to identify a high proportion of structurally diverse inhibitors by searching unusually large molecular databases in a time-, cost- and resource-efficient manner. This virtual screening methodology was tested prospectively on two versions of an antibacterial target (type II dehydroquinase from Mycobacterium tuberculosis and Streptomyces coelicolor), for which HTS has not provided satisfactory results and consequently practically all known inhibitors are derivatives of the same core scaffold. Overall, our protocols identified 100 new inhibitors, with calculated K(i) ranging from 4 to 250 µM (confirmed hit rates are 60% and 62% against each version of the target). Most importantly, over 50 new active molecular scaffolds were discovered that underscore the benefits that a wide application of prospectively validated in silico screening tools is likely to bring to antibacterial hit identification.

Crystal structure of Escherichia coli enterobactin-specific isochorismate synthase (EntC) bound to its reaction product isochorismate: implications for the enzyme mechanism and differential activity of chorismate-utilizing enzymes.

EntC, one of two isochorismate synthases in Escherichia coli, is specific to the biosynthesis of the siderophore enterobactin. Here, we report the crystal structure of EntC in complex with isochorismate and Mg(2+)at 2.3 A resolution, the first structure of a chorismate-utilizing enzyme with a non-aromatic reaction product. EntC exhibits a complex alpha+beta fold like the other chorismate-utilizing enzymes, such as salicylate synthase and anthranilate synthase. Comparison of active site structures allowed the identification of several residues, not discussed previously, that might be important for the isochorismate activity of the EntC. Although EntC, MenF and Irp9 all convert chorismate to isochorismate, only Irp9 subsequently exhibits isochorismate pyruvate lyase activity resulting in the formation of salicylate and pyruvate as the reaction products. With a view to understanding the roles of these amino acid residues in the conversion of chorismate to isochorismate and to obtaining clues about the pyruvate lyase activity of Irp9, several mutants of EntC were generated in which the selected residues in EntC were substituted for those of Irp9: these included A303T, L304A, F327Y, I346L and F359Q mutations. Biochemical analysis of these mutants indicated that the side chain of A303 in EntC may be crucial in the orientation of the carbonyl to allow formation of a hydrogen bond with isochorismate. Some mutations, such as L304A and F359Q, give rise to a loss of catalytic activity, whereas others, such as F327Y and I346L, show that subtle changes in the otherwise closely similar active sites influence activity. We did not find a combination of these residues that conferred pyruvate lyase activity.

Salicylate biosynthesis: overexpression, purification, and characterization of Irp9, a bifunctional salicylate synthase from Yersinia enterocolitica.

In some bacteria, salicylate is synthesized using the enzymes isochorismate synthase and isochorismate pyruvate lyase. In contrast, gene inactivation and complementation experiments with Yersinia enterocolitica suggest the synthesis of salicylate in the biosynthesis of the siderophore yersiniabactin involves a single protein, Irp9, which converts chorismate directly into salicylate. In the present study, Irp9 was for the first time heterologously expressed in Escherichia coli as a hexahistidine fusion protein, purified to near homogeneity, and characterized biochemically. The recombinant protein was found to be a dimer, each subunit of which has a molecular mass of 50 kDa. Enzyme assays, reverse-phase high-pressure liquid chromatography and 1H nuclear magnetic resonance (NMR) spectroscopic analyses confirmed that Irp9 is a salicylate synthase and converts chorismate to salicylate with a K(m) for chorismate of 4.2 microM and a k(cat) of 8 min(-1). The reaction was shown to proceed through the intermediate isochorismate, which was detected directly using 1H NMR spectroscopy.

Synthesis and activity of 5'-uridinyl dipeptide analogues mimicking the amino terminal peptide chain of nucleoside antibiotic mureidomycin A.

A series of 5'-uridinyl dipeptides were synthesised which mimic the amino terminal chain of nucleoside antibiotic mureido omycin A. Aminoacyl-beta-alanyl- and aminoacyl-N-methyl-beta-alanyl- dipeptides were attached either via an ester linkage to the 5'-hydroxyl of uridine, or via an amide linkage to 5'-amino-5'-deoxyuridine. The most active inhibitor of Escherichia coli phospho-MurNAc-pentapeptide translocase (MraY) was 5'-O-(L-Ala-N-methyl-beta-alanyl)-uridine (13l), which also showed 97% enzyme inhibition at 2.35 mM concentration, and showed antibacterial activity at 100 microg/mL concentration against Pseudomonas putida. Both the central N-methyl amide linkage and a 5' uridine ester linkage were required for highest biological activity. Enzyme inhibition was shown to be competitive with Mg(2+). It is proposed that the primary amino terminus of the inhibitor binds in place of the Mg(2+) cofactor at the MraY active site, positioned via a cis-N-methyl amide linkage.