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J. Med. Chem. 2006, 49, 1282-1290
Crystal Structures of Helicobacter pylori Type II Dehydroquinase Inhibitor Complexes: New Directions for Inhibitor Design David A. Robinson,† Kirsty A. Stewart,‡ Nicholas C. Price,† Peter A. Chalk,§ John R. Coggins,† and Adrian J. Lapthorn*,‡ DiVision of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, and Department of Chemistry, UniVersity of Glasgow, Glasgow G12 8QQ, Scotland, U.K., and GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, SteVenage, Herts SG1 2NY, U.K. ReceiVed June 7, 2005
The crystal structures of the type II dehydroquinase (DHQase) from Helicobacter pylori in complex with three competitive inhibitors have been determined. The inhibitors are the substrate analogue 2,3anhydroquinate (FA1), citrate, and an oxoxanthene sulfonamide derivative (AH9095). Despite the very different chemical nature of the inhibitors, in each case the primary point of interaction with the enzyme is via the residues that bind the C1 functionalities of the substrate, 3-dehydroquinate, i.e., N76, H102, I103, and H104. The DHQase/AH9095 complex crystal structure shows that sulfonamides can form a scaffold for nonsubstrate-like inhibitors and identifies a large conserved hydrophobic patch at the entrance to the active site as a locus that can be exploited in the development of new ligands. Introduction The enzyme 3-dehydroquinate dehydratase (dehydroquinase, DHQase; EC 4.2.1.10) catalyses the reversible dehydration of 3-dehydroquinate to form 3-dehydroshikimate (Scheme 1), a reaction common to the biosynthetic shikimate pathway and the catabolic quinate pathway. The shikimate (pre-chorismate) pathway presents an ideal target for the development of novel antimicrobial agents, as it is present in bacteria, fungi, plants, and apicomplexan parasites but not present in mammals.1-3 The importance of the pathway in bacteria is highlighted by the use of disruption mutants of typically aroA (EPSP synthase) and aroD (type I dehydroquinase) (produced in the presence of suitable media supplements) as attenuated live vaccines in a number of pathogenic bacteria such as Salmonella typhimurium4 and Bacillus anthracis.5 The attenuation of the bacteria is thought to be the result of depletion of folic acid of which the p-aminobenzoic acid (PABA) is derived from the shikimate pathway. Disruption of aroQ (type II dehydroquinase) in Corynebacterium pseudotuberculosis was more problematic, showing reduced growth in Vitro (in complex media) and loss of virulence.6 However, in Mycobacterium tuberculosis, the construction of viable aro gene disruption mutants has proved to be unsuccessful and has been attributed in part to the slow growth of mycobacteria. Recently, Parish and Stoker have successfully disrupted aroK, the gene for shikimate kinase, and shown it to be essential for viability of the organism, even in the presence of media supplements.7 They propose, therefore, that the shikimate pathway is essential for the viability of M. tuberculosis. Two structurally and mechanistically distinct forms of dehydroquinase, the third enzyme in the shikimate pathway, have been characterized.8-10 This presents a significant opportunity for the development of selective antimicrobials. The type I enzymes are typically of monomer mass ∼25 kDa and active as homodimers, each monomer possessing an (R/β)8 fold similar * To whom correspondence should be addressed. Tel.: 0141-330-5940. Fax: 0141-330-4888. E-mail:
[email protected]. † Division of Biochemistry and Molecular Biology, University of Glasgow. ‡ Department of Chemistry, University of Glasgow. § GlaxoSmithKline.
Scheme 1. Reaction Catalyzed by Type II Dehydroquinase Together with the Structures of 1 the Transition State Analogue 2,3-Anhydroquinic Acid (FA1, 1), Citrate (2), and N-Tetrazol-5-yl-9-oxo-9H-xanthene-2-sulfonamide (AH9095, 3) Identified from Compound Library Screening
to that of triose phosphate isomerase. The reaction catalyzed by the type I enzyme is a syn elimination of water, proceeding via an imine (Schiff base) intermediate, formed with a lysine residue within the active site.11 The monomers of the type II enzymes are smaller (approximately 16 kDa) and possess a flavodoxin-like fold; the enzymes are active as dodecamers. The type II reaction has been shown to proceed via an enol/enolate intermediate, resulting in anti elimination of water.12 Type I enzymes are found in plants, fungi, archaea, and bacteria exclusively associated with the shikimate pathway, while the type II enzymes operate in both the shikimate pathway and the catabolic quinate pathway and are found in fungi and bacteria. On the basis of the proposed catalytic mechanisms, selective inhibitors of type I and II DHQase have been developed from transition state analogues.13-15 The inhibitor 2,3-anhydroquinic
10.1021/jm0505361 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/02/2006
Inhibitors of H. pylori Type II Dehydroquinase
acid (FA1, Scheme 1) 1 was identified as a generic inhibitor of type II DHQase, whereas the ligand 3-dehydroquinate-E-oxime showed selectivity between type II DHQases from different species. Both inhibitors showed low micromolar inhibition of the type II enzymes while they were millimolar inhibitors of the type I enzyme. Crystal structures of several type II inhibitors complexes with DHQases have been elucidated from Streptomyces coelicolor16,17 and M. tuberculosis (unpublished PDB codes 1H0R and 1H0S). The active site of the S. coelicolor DHQase in complex with 1 included both an ordered glycerol and tartrate molecule derived from the crystallization conditions (PDB code 1GU1). This structure, and in particular the glycerol binding site, which is 3.7 Å away from the inhibitor, has been the focus of attempts to combine these two ligand binding sites and therefore enhance the inhibition of a series of second-generation compounds.18,19 To further understand the extent of the binding pocket for design of type II DHQases inhibitors, we have turned our attention to the enzyme from Helicobacter pylori. H. pylori is a slow-growing bacterium found in the gastric mucous layer or attached to the epithelial lining of the stomach. The bacterium has been shown to be the causative agent of more than 90% of duodenal ulcers and up to 80% of gastric ulcers. A crystal structure of the type II DHQase from H. pylori has recently been reported (PDB code 1J2Y)20 showing the enzyme to have the same folded structure as the previously described type II enzymes. Disruption of dehydroquinase in H. pylori results in severely impaired growth even in complex media (C. L. Clayton, personal communication). Type II dehydroquinase inhibitors might have potential use as novel antibiotics for human therapy, as they would be expected to have a restricted spectrum of antimicrobial activity to those human pathogens expressing the type II enzyme such as H. pylori and M. tuberculosis. A high-throughput screen of some 150 000 compounds against purified recombinant type II dehydroquinase from H. pylori expressed in Escherichia coli was used as a starting point to identify novel type II dehydroquinase inhibitors. This provided approximately 100 chemical inhibitors giving more than 50% inhibition of H. pylori dehydroquinase enzyme activity at a concentration of 20 µg/ mL and with little or no structural similarity to substrate. Surprisingly, none of these compounds had equivalent activity against the recombinant type II dehydroquinase from M. tuberculosis. Furthermore, none of these compounds had potent antimicrobial activity. We report here the crystal structure of one of these compounds, N-tetrazol-5-yl-9-oxo-9H-xanthene2-sulfonamide (AH9095) (Scheme 1) 3 in complex with H. pylori DHQase to 1.5 Å resolution. This structure shows how sulfonamides can inhibit DHQases and identifies new regions of the active site that could be exploited in inhibitor design. In addition, we have solved the crystal structure of the type II DHQase from H. pylori in complex with 1 and reinterpreted the structure 1J2Y, identifying the ligand as citrate 2. Comparison of the inhibition of H. pylori, S. coelicolor, and M. tuberculosis type II DHQases by the three ligands is reported and the structural basis of ligand specificity discussed. Results Kinetic Analysis. The effects of the inhibitors 1-3 on type II DHQases are summarized in Table 2. All three ligands showed competitive inhibition toward the type II DHQases from S. coelicolor and H. pylori. The inhibition of S. coelicolor and M. tuberculosis DHQase by 1 has been characterized previously,13 with the ligand showing 7-fold selectivity toward S.
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1283 Table 1. Crystallographic Data and Refinement Statistics Details of Data Collection AH9095 FA1 0.870 1.00 F23 P31 a ) b ) c ) a ) b ) 103.9, c ) 131.37 217.5 50.0-1.5 46.5-3.1 334 701 145 663 27 302 43 824 100 90.4 4.4 9.2
citratef 1.00 P4232 a)b)c) 98.9 44.2-2.5 239 220 6146 100 8.6
Refinement Statistics resolution range (Å) 30.0-1.55 25.0-3.1 b R-factor (Rwork/Rfree) 15.6/19.3 20.7/23.7 1165/40/177 14224/144/number of atomsc rms bond length deviation (Å) 0.014 0.024 rms bond angle deviation (deg) 1.56 2.07 mean B-factor (Å)d 23/16/45 61/60/coordinate error (Å)e 0.0929 0.5105
30.0-2.5 17.00/21.16 1227/13/40 0.027 2.24 21/23/25/38 0.2847
data set wavelength (Å) space group unit cell dimensions (Å) resolution range (Å) observations unique reflections % completeness Rmergea (%)
aR b c merge ) ∑|I - 〈I〉|/∑〈I〉. R factor ) ∑|Fo - Fc|/∑Fo. Number of atoms of protein, heteroatoms and water molecules, respectively. d Mean B factor for protein, inhibitor and water atoms, respectively. e Calculated using the method of Cruickshank.38 f Statistics from ref 21; structure factors from PDB entry 1J2Y.
Table 2. Inhibition of Type II Dehydroquinases by Different Ligandsa DHQase
1 (µM)
2 (mM)
3 (µM)
phosphate (mM)b
H. pylori S. coelicolor M. tuberculosis
370( 50 30 ( 10c 200 ( 20c
2.5 ( 0.2 7.3 ( 0.9 d
20 ( 3 230 ( 25 e
9 ( 0.6 7 ( 0.6 complex pattern
a The values shown are the K values for the three inhibitors that show EI competitive inhibition with respect to the substrate, 3-dehydroquinate. The kinetic parameters for the three enzymes are as follows: H. pylori, kcat ) 1 s-1 and Km ) 205 µM; S. coelicolor, kcat ) 125 s-1 and Km ) 100 µM; and M. tuberculosis, kcat ) 5 s-1 and Km ) 24 µM, as reported in Evans et al.23 b Data from Evans et al.23 c Data from Frederickson et al.13 d
