L. Tizon

A prodrug approach for improving antituberculosis activity of potent Mycobacterium tuberculosis type II dehydroquinase inhibitors.

The synthesis of high-affinity reversible competitive inhibitors of Mycobacterium tuberculosis type II dehydroquinase, an essential enzyme in Mycobacterium tuberculosis bacteria, is reported. The inhibitors reported here are mimics of the enol intermediate and the effect of substitution on C2 was studied. The crystal structures of Mycobacterium tuberculosis type II dehydroquinase in complex with three of the reported inhibitors are also described. The results show that an aromatic substituent on C2 prevents the closure of the active site by impeding the hydrogen-bonding interaction of Arg108 with the essential Tyr24 of the flexible loop, the residue that initiates catalysis. Chemical modifications of the reported acids were also carried out to improve internalization into Mycobacterium tuberculosis through an ester prodrug approach. Propyl esters proved to be the most efficient in achieving optimal in vitro activities.

Understanding the Key Factors that Control the Inhibition of Type II Dehydroquinase by (2R)-2- Benzyl-3-Dehydroquinic Acids.

The binding mode of several substrate analogues, (2R)‐2‐benzyl‐3‐dehydroquinic acids 4, which are potent reversible competitive inhibitors of type II dehydroquinase (DHQ2), the third enzyme of the shikimic acid pathway, has been investigated by structural and computational studies. The crystal structures of Mycobacterium tuberculosis and Helicobacter pylori DHQ2 in complex with one of the most potent inhibitor, p‐methoxybenzyl derivative 4 a, have been solved at 2.40 Å and 2.75 Å, respectively. This has allowed the resolution of the M. tuberculosis DHQ2 loop containing residues 20–25 for the first time. These structures show the key interactions of the aromatic ring in the active site of both enzymes and additionally reveal an important change in the conformation and flexibility of the loop that closes over substrate binding. The loop conformation and the binding mode of compounds 4 b–d has been also studied by molecular dynamics simulations, which suggest that the benzyl group of inhibitors 4 prevent appropriate orientation of the catalytic tyrosine of the loop for proton abstraction and disrupts its basicity.

Synthesis and biological evaluation of new nanomolar competitive inhibitors of Helicobacter pylori type II dehydroquinase. Structural details of the role of the aromatic moieties with essential residues.

The shikimic acid pathway is essential to many pathogens but absent in mammals. Enzymes in its pathway are therefore appropriate targets for the development of novel antibiotics. Dehydroquinase is the third enzyme of the pathway, catalyzing the reversible dehydratation of 3-dehydroquinic acid to form 3-dehydroshikimic acid. Here we present the synthesis of novel inhibitors with high affinity for Helicobacter pylori type II dehydroquinase and efficient inhibition characteristics. The structure of Helicobacter pylori type II dehydroquinase in complex with the most potent inhibitor shows that the aromatic functional group interacts with the catalytic Tyr22 by pi-stacking, expelling the Arg17 side chain, which is essential for catalysis, from the active site. The structure therefore explains the favorable properties of the inhibitor and will aid in design of improved antibiotics.

Tetrahydrobenzothiophene Derivatives: Conformationally Restricted Inhibitors of Type II Dehydroquinase

Restriction is good for inhibition! Tetrahydrobenzothiophene-derived rigid mimics of the type II dehydroquinase (DHQ2)-catalyzed reaction intermediate are reported. These derivatives fix the interaction with the tyrosine, the base that initiates the enzymatic reaction, in an inappropriate orientation for catalysis. Two competitive inhibitors in the series, 2-propenyl derivative 5 e and 2-cyclopropylethyl compound 5 i (shown), were crystallized in complex with DHQ2 from Helicobacter pylori, and the X-ray structures were determined at 1.95A and 1.85A, respectively.

Mechanistic Basis of the Inhibition of Type II Dehydroquinase by (2S)- and (2R)-2-Benzyl-3-Dehydroquinic Acids.

The structural changes caused by the substitution of the aromatic moiety in (2S)-2-benzyl-3-dehydroquinic acids and its epimers in C2 by electron-withdrawing or electron-donating groups in type II dehydroquinase enzyme from M. tuberculosis and H. pylori has been investigated by structural and computational studies. Both compounds are reversible competitive inhibitors of this enzyme, which is essential in these pathogenic bacteria. The crystal structures of M. tuberculosis and H. pylori in complex with (2S)-2-(4-methoxy)benzyl- and (2S)-2-perfluorobenzyl-3-dehydroquinic acids have been solved at 2.0, 2.3, 2.0, and 1.9 Å, respectively. The crystal structure of M. tuberculosis in complex with (2R)-2-(benzothiophen-5-yl)methyl-3-dehydroquinic acid is also reported at 1.55 Å. These crystal structures reveal key differences in the conformation of the flexible loop of the two enzymes, a difference that depends on the presence of electron-withdrawing or electron-donating groups in the aromatic moiety of the inhibitors. This loop closes over the active site after substrate binding, and its flexibility is essential for the function of the enzyme. These differences have also been investigated by molecular dynamics simulations in an effort to understand the significant inhibition potency differences observed between some of these compounds and also to obtain more information about the possible movements of the loop. These computational studies have also allowed us to identify key structural factors of the H. pylori loop that could explain its reduced flexibility in comparison to the M. tuberculosis loop, specifically by the formation of a key salt bridge between the side chains of residues Asp18 and Arg20.

Chemical Modification of a Dehydratase Enzyme Involved in Bacterial Virulence by an Ammonium Derivative: Evidence of its Active Site Covalent Adduct

The first example of an ammonium derivative that causes a specific modification of the active site of type I dehydroquinase (DHQ1), a dehydratase enzyme that is a promising target for antivirulence drug discovery, is described. The resolution at 1.35 Å of the crystal structure of DHQ1 from Salmonella typhi chemically modified by this ammonium derivative revealed that the ligand is covalently attached to the essential Lys170 through the formation of an amine. The detection by mass spectroscopy of the reaction intermediates, in conjunction with the results of molecular dynamics simulations, allowed us to explain the inhibition mechanism and the experimentally observed differences between S. typhi and Staphylococcus aureus enzymes. The results presented here reveal that the replacement of Phe225 in St-DHQ1 by Tyr214 in Sa-DHQ1 and its hydrogen bonding interaction with the conserved water molecule observed in several crystal structures protects the amino adduct against further dehydration/aromatization reactions. In contrast, for the St-DHQ1 enzyme, the carboxylate group of Asp114, with the assistance of this water molecule, would trigger the formation of a Schiff base that can undergo further dehydration reactions until full aromatization of the cyclohexane ring is achieved. Moreover, in vitro antivirulence studies showed that the reported compound is able to reduce the ability of Salmonella Enteritidis to kill A459 respiratory cells. These studies have identified a good scaffold for the design of irreversible inhibitors that can be used as drugs and has opened up new opportunities for the development of novel antivirulence agents by targeting the DHQ1 enzyme.