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.

An iron carbonyl approach to the influenza neuraminidase inhibitor oseltamivir.

A novel synthetic route towards oseltamivir, an influenza neuraminidase inhibitor, has been achieved employing a cationic iron carbonyl complex, providing an alternate pathway with the potential to access diverse analogues.

Determination of the P1', P2' and P3' subsite-specificity of factor Xa.

Factor Xa is a central protease in the coagulation cascade and the target for many anticoagulant compounds currently under development. The preferences of the enzyme for substrates incorporating residues N-terminal to the cleavage site (P1, P2, etc.) have been elucidated, but little is known of its preferences for residues C-terminal to the cleavage site (P1', P2', etc.). The preferences of bovine factor Xa for substrate residues in the P1', P2' and P3' positions were mapped using fluorescence-quenched substrates. Bovine factor Xa, often used as a model for factor Xa, was most selective for the P2' position, less selective at the P1' position and almost completely non-selective at the P3' position. It appears that while the prime side subsites of factor Xa impose some selectivity towards substrates, the influence of these sites on factor Xa cleavage specificity is relatively low in comparison to related enzymes such as thrombin.

The preparation of fluorescence-quenched probes for use in the characterization of human factor Xa substrate binding domains.

The preparation and characterization by LCMS of a library of 55 fluorescence- quenched peptides is described. The peptides bear a terminal anthranilamide fluorophore and a penultimate 2,4-dinitrophenyl-L-lysine quencher, and will be used to probe the substrate binding domain of the human blood coagulation enzyme, factor Xa.

Synthetic applications of cationic iron and cobalt carbonyl complexes.

Metal carbonyl stabilized cationic species react with a wide range of nucleophiles under mild conditions, and have thus found many synthetic applications. In this Perspective, we describe the utility of iron carbonyl dienyl cations in solution and solid phase parallel synthesis, and in the development of a new synthetic route towards oseltamivir phosphate (Tamiflu). We also discuss the solid phase version of the Nicholas reaction, employing cobalt carbonyl stabilized propargylic cations, and giving access to substituted alkynes.

Approaches to selective peptidic inhibitors of factor Xa.

Inhibitors of procoagulant enzymes, such as factor Xa (fXa) and thrombin, are important for treating thrombosis. Thrombin has complex pro- and anti-coagulant roles and thus fXa is thought to represent an ideal target. Discrete kcat and Km values for cleavage of a library of fluorescence-quenched substrates by fXa were determined. The results highlighted the low selectivity of fXa at its prime sites, and its poor efficiency compared with thrombin, creating a challenge for the design of fXa-specific peptidic inhibitors. We hypothesized that Km rather than kcat/Km values may be better indicators of inhibitor potential for a peptidic sequence, leading us to design peptide sequences for both fXa and thrombin in three forms: fluorescence-quenched substrates, standard alpha-peptides and peptides containing a beta-homoarginine at the cleavage site. Kinetic and competitive inhibition assays with both fXa and thrombin showed the fluorescence-quenched substrates to be the best inhibitors, while the inhibitory effect of the beta-homoarginine peptides varied for the two proteases. Importantly, fXa was inhibited to a much greater extent by the beta-peptides than the corresponding alpha-peptides, resulting in an increased selectivity for fXa inhibition over thrombin for those peptides containing a beta-amino acid at the cleavage site.