Concepción González-Bello

Mycobacterium tuberculosis shikimate kinase inhibitors: design and simulation studies of the catalytic turnover.

Shikimate kinase (SK) is an essential enzyme in several pathogenic bacteria and does not have any counterpart in human cells, thus making it an attractive target for the development of new antibiotics. The key interactions of the substrate and product binding and the enzyme movements that are essential for catalytic turnover of the Mycobacterium tuberculosis shikimate kinase enzyme (Mt-SK) have been investigated by structural and computational studies. Based on these studies several substrate analogs were designed and assayed. The crystal structure of Mt-SK in complex with ADP and one of the most potent inhibitors has been solved at 2.15 Å. These studies reveal that the fixation of the diaxial conformation of the C4 and C5 hydroxyl groups recognized by the enzyme or the replacement of the C3 hydroxyl group in the natural substrate by an amino group is a promising strategy for inhibition because it causes a dramatic reduction of the flexibility of the LID and shikimic acid binding domains. Molecular dynamics simulation studies showed that the product is expelled from the active site by three arginines (Arg117, Arg136, and Arg58). This finding represents a previously unknown key role of these conserved residues. These studies highlight the key role of the shikimic acid binding domain in the catalysis and provide guidance for future inhibitor designs.

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.

Designing Irreversible Inhibitors—Worth the Effort?

Despite the unquestionable success of numerous irreversible drugs that are currently in clinical use, such as acetylsalicylic acid (Aspirin) and penicillin, the number of such approved drugs is much lower than that of noncovalent drugs. Over the years, the potential off‐target effects of these types of compounds have been the primary concern that has hampered their development. However, their remarkable advantages over noncovalent drugs and a better analysis of the risks have decreased the widespread skepticism surrounding them. The design of irreversible inhibitors is a challenge, particularly considering that in some cases their efficacy is due to complex and unexpected mechanisms of action. In this review the main advantages of irreversible inhibition are summarized, and the complexity of certain covalent modification mechanisms is highlighted with selected examples.

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.

Design, synthesis and evaluation of bifunctional inhibitors of type II dehydroquinase

Inhibitors of type II dehydroquinase were designed to straddle the two distinct binding sites identified for the inhibitor (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid and a glycerol molecule in a crystallographic study of the Streptomyces coelicolor enzyme. A number of compounds were designed to incorporate characteristics of both ligands. These analogues were synthesized from quinic acid, and were assayed against type I (Salmonella typhi) and type II (S. coelicolor) dehydroquinases. None of the analogues showed inhibition for type I dehydroquinase. Six of the analogues were shown to have inhibition constants in the micromolar to low millimolar range against the S. coelicolor type II dehydroquinase, while two showed no inhibition. The binding modes of the analogues in the active site of the S. coelicolor enzyme were studied by molecular docking with GOLD1.2. These studies suggest a binding mode where the ring is in a similar position to (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid in the crystal structure and the side-chain occupies part of the glycerol binding-pocket.

Metal-assisted ring-closing/opening process of a chiral tetrahydroquinazoline.

The ring-chain tautomerism of a 2-aryl-1,2,3,4-tetrahydroquinazoline has been exploited to induce reversible changes in the aminal-imine equilibrium, as desired, by coordination of a suitable metal ion. This process was studied by NMR and UV-vis spectroscopies, X-ray crystallography, and molecular modeling approach. The results obtained show that the imine H(2)L(i) undergoes a selective ring-closing reaction upon complexation with Ni(2+). As a result, complexes of the type Ni(HL(a))(2) are obtained, whose chirality arises from the chiral ligand H(2)L(a) and the helicity of the structure. Hence, helical enantiomers form the following racemates: [Δ-C(R,R)N(S,S),Λ-C(S,S)N(R,R)]-Ni(HL(a))(2)·2HOAc and [Δ,Λ-C(S,R)N(R,S)]-Ni(HL(a))(2)·4MeOH. In contrast to the situation observed for Ni(2+), the cyclic tautomer of the ligand, H(2)L(a), undergoes a selective ring-opening reaction upon complex formation with Pd(2+), ultimately yielding Pd(HL(i))(2)·MeOH, in which the open-chain imine ligand is bidentate through the N,O donor set of the quinoline residue. Density functional theory calculations were conducted to provide insight into the different behavior of both coordinated metals (Ni(2+) and Pd(2+)) and to propose a mechanism for the metal-assisted opening/closing reaction of the tetrahydroquinazoline ring.

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