John N. Champness

Crystal structure of the anti-bacterial sulfonamide drug target dihydropteroate synthase.

Sulfonamides were amongst the first clinically useful antibacterial agents to be discovered. The identification of sulfanilamide as the active component of the dye Prontosil rubrum led to the synthesis of clinically useful analogues. Today sulf amethoxazole (in combination with trimethoprim), is used to treat urinary tract infections caused by bacteria such as Escherichia coli and is also a first-line treatment for pneumonia caused by the fungus Pneumocystis carinii, a common condition in AIDS patients. The site of action is the de novo f olate biosynthesis enzyme dihydropteroate synthase (DHPS) where sulfonamides act as analogues of one of the substrates, para-aminobenzoic acid (pABA). We report here the crystal structure of E.coli DHPS at 2.0 Å resolution refined to an R-factor of 0.185. The single domain of 282 residues forms an eight-stranded α/β-barrel. The 7,8-dihydropterin pyrophosphate (DHPPP) substrate binds in a deep cleft in the barrel, whilst sulfanilamide binds closer to the surface. The DHPPP ligand site is highly conserved amongst prokaryotic and eukaryotic DHPSs.

Exploring the active site of herpes simplex virus type-1 thymidine kinase by X-ray crystallography of complexes with aciclovir and other ligands

Antiherpes therapies are principally targeted at viral thymidine kinases and utilize nucleoside analogs, the triphosphates of which are inhibitors of viral DNA polymerase or result in toxic effects when incorporated into DNA. The most frequently used drug, aciclovir (Zovirax), is a relatively poor substrate for thymidine kinase and high‐resolution structural information on drugs and other molecules binding to the target is therefore important for the design of novel and more potent chemotherapy, both in antiherpes treatment and in gene therapy systems where thymidine kinase is expressed. Here, we report for the first time the binary complexes of HSV‐1 thymidine kinase (TK) with the drug molecules aciclovir and penciclovir, determined by X‐ray crystallography at 2.37 Å resolution. Moreover, from new data at 2.14 Å resolution, the refined structure of the complex of TK with its substrate deoxythymidine (R = 0.209 for 96% of all data) now reveals much detail concerning substrate and solvent interactions with the enzyme. Structures of the complexes of TK with four halogen‐containing substrate analogs have also been solved, to resolutions better than 2.4 Å. The various TK inhibitors broadly fall into three groups which together probe the space of the enzyme active site in a manner that no one molecule does alone, so giving a composite picture of active site interactions that can be exploited in the design of novel compounds. Proteins 32:350–361, 1998.

The structure of mouse L1210 dihydrofolate reductase-drug complexes and the construction of a model of human enzyme

The structure of mouse L1210 dihydrofolate reductase (DHFR) complexed with NADPH and trimethoprim has been refined at 2.0 Å resolution. The analogous complex with NADPH and methotrexate has been refined at 2.5 Å resolution. These structures reveal for the first time details of drug interactions with a mammalian DHFR, which are compared with those observed from previous X‐ray investigations of DHFR/inhibitor complexes. The refined L1210 structure has been used as the basis for the construction of a model of the human enzyme. There are only twenty‐one sequence differences between mouse L1210 and human DHFRs, and all but two of these are located close to the molecular surface: a strong indication that the active sites are essentially identical in these two mammalian enzymes.

The structure of Pneumocystis carinii dihydrofolate reductase to 1.9 å resolution

BACKGROUND The fungal pathogen Pneumocystis carinii causes a pneumonia which is an opportunistic infection of AIDS patients. Current therapy includes the dihydrofolate reductase (DHFR) inhibitor trimethoprim which is selective but only a relatively weak inhibitor of the enzyme for P. carinii. Determination of the three-dimensional structure of the enzyme should form the basis for design of more potent and selective therapeutic agents for treatment of the disease. RESULTS The structure of P. carinii DHFR in complex with reduced nicotinamide adenine dinucleotide phosphate and trimethoprim has accordingly been solved by X-ray crystallography. The structure of the ternary complex has been refined at 1.86 A resolution (R = 0.181). A similar ternary complex with piritrexim (which is a tighter binding, but less selective inhibitor) has also been solved, as has the binary complex holoenzyme, both at 2.5 A resolution. CONCLUSIONS These structures show how two drugs interact with a fungal DHFR. A comparison of the three-dimensional structure of this relatively large DHFR with bacterial or mammalian enzyme-inhibitor complexes determined previously highlights some additional secondary structure elements in this particular enzyme species. These comparisons provide further insight into the principles governing DHFR-inhibitor interaction, in which the volume of the active site appears to determine the strength of inhibitor binding.

The binding of trimethoprim to bacterial dihydrofolate reductase

Dihydrofolate reductase (DHFR, EC 1.5.1.3) catalyses the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate; further metabolites of tetrahydrofolate are involved in the incorporation of singlecarbon units into purines, pyrimidines and amino acids [ 1,2]. The inhibition of DHFR can therefore lead to a deficiency of the components of nucleic acids and proteins, to eventual cessation of DNA synthesis and hence to cell death. DHFR inhibitors are used in the control of a number of disease processes including various tumours and bacterial infections. Understanding of inhibitor binding at the molecular level is of potential value in the design of drugs having greater inhibitory potential and improved selectivity towards the appropriate organism. Trimethoprim (TMP), a widely used antibacterial drug [3-61, is a potent inhibitor of bacterial DHFRs but a much weaker inhibitor of the vertebrate enzymes (e.g., ICse values against Escherichia coli and human enzyme [7] are, respectively, 5 X lo-’ M and 3 X lo4 M). To provide information on the action of this drug at the molecular level, we have determined the structure of the binary complex of E. coli (strain RTSOO) form I DHFR with TMP and compared it with that of the complex of DHFR with methotrexate (MTX), a drug which binds tightly to both bacterial and vertebrate DHFR. The structure of our TMP-enzyme complex differs from that in [8] of an MTX-enzyme complex from a different strain (MB1428) ofE. coli. The amino acid sequences of the two enzymes are currently thought to differ at 3 positions [9].

Structure to 1.9 A resolution of a complex with herpes simplex virus type-1 thymidine kinase of a novel, non-substrate inhibitor: X-ray crystallographic comparison with binding of aciclovir.

Treatment of herpes infections with nucleoside analogues requires as an initial step the activation of the compounds by thymidine kinase. As an aid to developing more effective chemotherapy, both for treatment of recurrent herpes infection and in gene therapy systems where thymidine kinase is expressed, two high‐resolution X‐ray structures of thymidine kinase have been compared: one with the relatively poor substrate aciclovir (Zovirax), the other with a synthetic inhibitor having an N 2‐substituted guanine. Both compounds have similar binding modes in spite of their size difference and apparently distinct ligand properties.

Pyrrolo[2,3-d]pyrimidines and pyrido[2,3-d]pyrimidines as conformationally restricted analogues of the antibacterial agent trimethoprim

Conformationally restricted analogues of the antibacterial agent trimethoprim (TMP) were designed to mimic the conformation of drug observed in its complex with bacterial dihydrofolate reductase (DHFR). This conformation of TMP was achieved by linking the 4-amino function to the methylene group by one- and two-carbon bridges. A pyrrolo[2,3-d]pyrimidine, a dihydro analogue, and a tetrahydropyrido[2,3-d]pyrimidine were synthesized and tested as inhibitors of DHFR. One analogue showed activity equivalent to that of TMP against DHFR from three species of bacteria. An X-ray crystal structure of this inhibitor bound to Escherichia coli DHFR was determined to evaluate the structural consequences of the conformational restriction.

Crystal structure of Escherichia coli UvrB C-terminal domain, and a model for UvrB-UvrC interaction

A crystal structure of the C‐terminal domain of Escherichia coli UvrB (UvrB′) has been solved to 3.0 Å resolution. The domain adopts a helix‐loop‐helix fold which is stabilised by the packing of hydrophobic side‐chains between helices. From the UvrB′ fold, a model for a domain of UvrC (UvrC′) that has high sequence homology with UvrB′ has been made. In the crystal, a dimerisation of UvrB′ domains is seen involving specific hydrophobic and salt bridge interactions between residues in and close to the loop region of the domain. It is proposed that a homologous mode of interaction may occur between UvrB and UvrC. This interaction is likely to be flexible, potentially spanning >50 Å.

2.0 Å X-ray structure of the ternary complex of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase from Escherichia coli with ATP and a substrate analogue

The X‐ray crystal structure of 7,8‐dihydro‐6‐hydroxymethylpterinpyrophosphokinase (PPPK) in a ternary complex with ATP and a pterin analogue has been solved to 2.0 Å resolution, giving, for the first time, detailed information of the PPPK/ATP intermolecular interactions and the accompanying conformational change. The first 100 residues of the 158 residue peptide contain a βαββαβ motif present in several other proteins including nucleoside diphosphate kinase. Comparative sequence examination of a wide range of prokaryotic and lower eukaryotic species confirms the conservation of the PPPK active site, indicating the value of this de novo folate biosynthesis pathway enzyme as a potential target for the development of novel broad‐spectrum anti‐infective agents.

Crystallographic investigation of the cooperative interaction between trimethoprim, reduced cofactor and dihydrofolate reductase

The structure of the complex between E. coli (RT500) form I dihydrofolate reductase, the antibacterial trimethoprim and NADPH has been determined by X‐ray crystallography. The inhibitor and cofactor are in mutual contact. A flexible chain segment which includes Met 20 is in contact with the inhibitor in the presence of NADPH, but more distant in its absence. By contrast, the inhibitor conformation is little changed with NADPH present. We discuss these observations with regard to the mutually cooperative binding of these ligands to the protein, and to the associated enhancement of inhibitory selectivity shown by trimethoprim for bacterial as opposed to vertebrate enzyme.