Verónica F. V. Prazeres

Competitive inhibitors of Helicobacter pylori type II dehydroquinase: synthesis, biological evaluation, and NMR studies.

Several 3‐heteroaryl analogs of the known dehydroquinase inhibitor (1R,4R,5R)‐1,4,5‐trihydroxy‐2‐cyclohexene‐1‐carboxylic acid (4) were synthesized and tested as inhibitors of Helicobacter pylori type II dehydroquinase, the third enzyme of the shikimic acid pathway. All of these compounds proved to be reversible competitive inhibitiors of this enzyme and proved to be, with the exception of nitrile 8 e, more potent than the parent inhibitor 4 (Ki=370  μM). The 2‐thienyl derivative 8 b was found to be the most potent inhibitor of the series and has a Ki value of 540 nM, which is almost seven hundred times lower than that of the parent inhibitor. The 3‐nitrothienyl derivative 8 d and 2‐furanyl derivative 8 a also had a good affinity of 1 μM. The conformation of the potent competitive inhibitor 8 b, when bound in the active site of the H. pylori enzyme, was elucidated by 1D‐selective inversion NOE, Saturation Transfer Difference (STD) and transferred NOESY NMR experiments. One of the conformations that exists in solution for the potent competitive inhibitor 2‐thienyl derivative 8 b is selected when it is bound to the active site of the enzyme. In the bound conformation derivative 8 b has the sulfur atom of its thienyl group oriented towards the double bond of the cyclohexene moiety. The large STD effects observed for the aromatic protons of 8 b show that it is the thiophene side of the ligand that makes closest contact with enzyme protons. Docking studies using GOLD3.0.1 suggest that the conformation determined by NMR allows strong lipophilic interactions with the enzyme residues Pro9, Asn10, Ile11, Gly78 and Ala 79. Competitive STD experiments carried out with high‐, medium‐ and low‐affinity ligands 8 b, 5 d and 5 f show that they all bind in the same site of Helicobacter pylori dehydroquinase.

Nanomolar Competitive Inhibitors of Mycobacterium tuberculosis and Streptomyces coelicolor Type II Dehydroquinase

Isomeric nitrophenyl and heterocyclic analogues of the known inhibitor (1S,3R,4R)‐1,3,4‐trihydroxy‐5‐cyclohexene‐1‐carboxylic acid have been synthesized and tested as inhibitors of M. tuberculosis and S. coelicolor type II dehydroquinase, the third enzyme of the shikimic acid pathway. The target compounds were synthesized by a combination of Suzuki and Sonogashira cross‐coupling and copper(I)‐catalyzed 2,3‐dipolar cycloaddition reactions from a common vinyl triflate intermediate. These studies showed that a para‐nitrophenyl derivative is almost 20‐fold more potent as a competitive inhibitor against the S. coelicolor enzyme than that of M. tuberculosis. The opposite results were obtained with the meta isomer. Five of the bicyclic analogues reported herein proved to be potent competitive inhibitors of S. coelicolor dehydroquinase, with inhibition constants in the low nanomolar range (4–30 nM). These derivatives are also competitive inhibitors of the M. tuberculosis enzyme, but with lower affinities. The most potent inhibitor against the S. coelicolor enzyme, a 6‐benzothiophenyl derivative, has a Ki value of 4 nM—over 2000‐fold more potent than the best previously known inhibitor, (1R,4R,5R)‐1,5‐dihydroxy‐4‐(2‐nitrophenyl)cyclohex‐2‐en‐1‐carboxylic acid (8 μM), making it the most potent known inhibitor against any dehydroquinase. The binding modes of the analogues in the active site of the S. coelicolor enzyme (GOLD 3.0.1), suggest a key π‐stacking interaction between the aromatic rings and Tyr 28, a residue that has been identified as essential for enzyme activity.

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.

2‐Substituted‐3‐Dehydroquinic Acids as Potent Competitive Inhibitors of Type II Dehydroquinase

The shikimic acid pathway catalyses the sequential conversion of erythrose-4-phosphate and phosphoenol pyruvate to chorismic acid, which is the precursor to aromatic compounds such as amino acids l-phenylalanine, l-tyrosine and l-tryptophan, folates, ubiquinone, and vitamins E and K. This pathway is present in bacteria, fungi, higher plants and has also been discovered in apicomplexan parasites, Plasmodium falciparum (which are the cause of malaria), Toxoplasma gondii and Cryptosporidium parvum. The absence of the shikimate pathway in mammals, combined with its essential role in certain microorganisms, makes it an attractive target for the development of new antimicrobial and antiparasitic agents. Dehydroquinase (3-dehydroquinate dehydratase, DHQ, EC 4.2.1.10) is the third enzyme in the shikimic acid pathway, and catalyses the reversible dehydration of 3-dehydroquinic acid (1) to 3-dehydroshikimic acid (2) (Scheme 1). Biochemical

Determination of the bound conformation of a competitive nanomolar inhibitor of mycobacterium tuberculosis type II dehydroquinase by NMR spectroscopy.

The synergy between tuberculosis and the AIDS epidemic, along with the surge of multidrug‐resistant isolates of M. tuberculosis, has reaffirmed tuberculosis as a primary public health threat. It is therefore necessary to discover new, safe, and more efficient antibiotics against this disease. On the other hand, mapping the dynamic interactions of inhibitors of a target protein can provide information for the development of more potent inhibitors and consequently, more potent potential drugs. In this context, the conformational binding of our previously reported nanomolar inhibitor of M. tuberculosis type II dehydroquinase, the 3‐nitrophenyl derivative 1, was studied using saturation transfer difference (STD) and transferred NOESY experiments. These studies have shown that in the bound state, one conformation of those present in solution of the competitive nanomolar inhibitor 3‐nitrophenyl derivative 1 is selected. In the bound conformation, the aromatic ring is slightly shifted from coplanarity, with the double bond and the nitro group of 1 oriented towards the double bond side.

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.