2,4-diamino-5-(3,5-dimethoxy-4-substituted)-benzyl-pyrimidines as ligands in affinity chromatography of bacterial dihydrofolate reductases.

Inhibitors of the trimethoprim-type, bearing terminal amino, hydroxyl or carboxyl groups in position 4 of the benzene ring as well as methotrexate were coupled to either CH-Sepharose 4B, epoxy-activated Sepharose 6B or AH-Sepharose 4B, respectively. In contrast to the methotrexate-affinity gel, trimethoprim-like ligands retained bacterial but not mammalian dihydrofolate reductases. The affinity gels prepared with these ligands could be used for effective purification of bacterial dihydrofolate reductases (EC 1.5.1.3), but differed in their affinity for this enzyme.

Structure and function of the dihydropteroate synthase from Staphylococcus aureus.

The gene encoding the dihydropteroate synthase of staphylococcus aureus has been cloned, sequenced and expressed in Escherichia coli. The protein has been purified for biochemical characterization and X-ray crystallographic studies. The enzyme is a dimer in solution, has a steady state kinetic mechanism that suggests random binding of the two substrates and half-site reactivity. The crystal structure of apo-enzyme and a binary complex with the substrate analogue hydroxymethylpterin pyrophosphate were determined at 2.2 A and 2.4 A resolution, respectively. The enzyme belongs to the group of "TIM-barrel" proteins and crystallizes as a non-crystallographic dimer. Only one molecule of the substrate analogue bound per dimer in the crystal. Sequencing of nine sulfonamide-resistant clinical isolates has shown that as many as 14 residues could be involved in resistance development. The residues are distributed over the surface of the protein, which defies a simple interpretation of their roles in resistance. Nevertheless, the three-dimensional structure of the substrate analogue binary complex could give important insight into the molecular mechanism of this enzyme.

A single amino acid substitution in Staphylococcus aureus dihydrofolate reductase determines trimethoprim resistance.

A single amino acid substitution, Phe98 to Tyr98, in dihydrofolate reductase (DHFR) is the molecular origin of trimethoprim (TMP) resistance in Staphylococcus aureus. This active site amino acid substitution was found in all S. aureus TMP-resistant clinical isolates tested. In order to explore the structural role of Tyr98 in TMP-resistance the ternary complexes of the chromosomal S. aureus DHFR (SaDHFR) with methotrexate (MTX) and TMP in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as well as that of mutant Phe98Tyr DHFR SaDHFR(F98Y) ternary folate-NADPH complex have been determined by X-ray crystallography. Critical evidence concerning the resistance mechanism has also been provided by NMR spectral analyses of 15N-labelled TMP in the ternary complexes of both wild-type and mutant enzyme. These studies show that the mutation results in loss of a hydrogen bond between the 4-amino group of TMP and the carbonyl oxygen of Leu5. This mechanism of resistance is predominant in both transferable plasmid-encoded and non-transferable chromosomally encoded resistance. Knowledge of the resistance mechanism at a molecular level could help in the design of antibacterials active against multi-resistant Staphylococcus aureus (MRSA), one of todays most serious problems in clinical infectology.

N-terminal amino acid sequence of the chromosomal dihydrofolate reductase purified from trimethoprim-resistant Staphylococcus aureus.

The existence of two distinct dihydrofolate reductases (DHFR) in highly trimethoprim-resistant clinical isolates has been unequivocally demonstrated. The enzymes have been characterized with regard to the affinity for substrates and sensitivity to inhibitors. The chromosomal, trimethoprim-sensitive DHFR was purified to homogeneity by a new simple two-step procedure. Its N-terminal amino acid sequence, determined up to the first 35 amino acids, showed 69% homology with the Escherichia coli DHFR.

Identical genes for trimethoprim-resistant dihydrofolate reductase from Staphylococcus aureus in Australia and central Europe.

The nucleotide sequence of a 1.25 kb BglII/EcoRI fragment from the 34 kb trimethoprim (Tp)-resistant plasmid pABU1 of Staphylococcus aureus 157/4696, isolated in Zürich, was determined. It contained the entire Tp-resistant dihydrofolate reductase gene, 197 bp of the thymidylate synthetase, 395 bp of a truncated gene and 111 bp of IS257R1. With the exception of one single base pair at position of 862 the sequence of the whole fragment was identical to nucleotides 1633 to 2885 of the Tp-resistant transposon Tn4003 in plasmid pSK1 from an Australian S. aureus isolate. This suggests the worldwide dissemination of Tn4003 in multiresistant Staphylococci.

A 1H NMR study of the interactions and conformations of rationally designed brodimoprim analogues in complexes with Lactobacillus casei dihydrofolate reductase.

A consideration of the detailed structural information available from X-ray crystallographic and NMR studies on complexes of dihydrofolate reductase with inhibitors has led to the design of trimethoprim analogues with improved binding properties. Computer graphic techniques have been used to predict which substituent groups were required at the 3'-O position of brodimoprim (2,4-diamino-5-(3,5-dimethoxy-4-bromobenzyl)pyrimidine) to make additional interactions with the enzyme. NMR spectroscopy provided a convenient method of assessing if the analogues were binding in the predicted manner. On the basis of this approach, the C4,C6-dicarboxylic acid analogue IX was designed to interact with Arg-57 and His-28 in the enzyme, and this analogue was found to bind 3 orders of magnitude more tightly than the parent brodimoprim.

6-Acetylmethylenepenicillanic acid (Ro 15-1903), a potent beta-lactamase inhibitor. I. Inhibition of chromosomally and R-factor-mediated beta-lactamases.

6-Acetylmethylenepenicillanic acid (Ro 15-1903) was seen to be a powerful inhibitor of various beta-lactamases. Nearly all of the chromosomally and R-factor-mediated beta-lactamases which were studied were inhibited at much lower concentrations than required with clavulanic acid or sulbactam. Ro 15-1903 protected beta-lactamase-labile penicillins and cephalosporins from hydrolysis. The compound itself was stable against hydrolysis and inhibition of beta-lactamase was irreversible.

Studies on 6-acetylmethylenepenicillanic acid (Ro 15-1903). III. Relationship between in vitro activity and chemical stability.

To resolve discrepancies between its enzymological activity and its in vitro or in vivo activity, 6-acetylmethylenepenicillanic acid (Ro 15-1903), a potent beta-lactamase inhibitor, was investigated for its chemical stability and its ability to penetrate the bacterial cell envelope. Although Ro 15-1903 was fairly stable in water or saline, it was found to be unstable in a rich medium, in mouse plasma and in human serum. Decomposition half-lives in Tryptic Soy Broth (TSB) and mouse plasma were determined by spectrometry to be 1.3 hours and 12 minutes respectively. These values were confirmed by a biochemical method for determination of Ro 15-1903. Furthermore, a large enhancement of the in vitro activity was noticed when the assay medium was changed from TSB to a synthetic medium in which Ro 15-1903 was more stable. The ampicillin-potentiating activity marginally increased if a permeability mutant harboring the R6K plasmid, which codes for TEM-1 beta-lactamase production, was used instead of the wild-type strain. These results prove that the chemical instability of Ro 15-1903 is the main cause of its disproportionally low activity in vitro and in vivo. Ro 15-1903 is not nonspecifically inactivated by proteins, since it did not lose its activity after incubation with bovine serum albumin (50 mg/ml) for 2 hours at 37 degrees C. It seems to react specifically with beta-lactamase.

Antimicrobial dihydrofolate reductase inhibitors--achievements and future options: review.

Despite all progress made in the fight against infections caused by bacteria, fungi, protozoa or viruses, there is a need for more and new active agents. Intensive efforts are currently directed against many new and attractive targets, and are hoped to result in new useful agents. The opportunities offered by some known and validated targets are, however, by far not exhausted. Dihydrofolate reductase (DHFR, EC 1.5.1.3) attracted much attention over several decades, which yielded several useful agents. There are excellent chances for new drugs in this field, and they are thought to increase by limiting the spectrum of activity. Whereas trimethoprim seems to present the optimum which can be achieved for a broad spectrum antibacterial agent, specific agents could probably be designed for well defined groups or specific organisms, such as staphylococci among the bacteria, or for a number of parasites, such as Plasmodium falciparum, the fungus Pneumocystis carinii, and several protozoa, such as Trypanosoma, Toxoplasma, and others. This would even extend to herbicides or specific plant pathogens. Achievements and current efforts directed against new DHFR-inhibitors are reviewed, considering only the most recent literature.

History and future of antimicrobial diaminopyrimidines.

Numerous pyrimidine and purine analogs were synthesized in the late forties in G.H. Hitchings' group as potential nucleic acid antagonists. Several key observations finally led to the selection of pyrimethamine as an antimalarial and trimethoprim (TMP) as an antibacterial agent: i) 2,4-diamino-5-substituted pyrimidines interfered with folic acid utilization rather than being thymine antagonists as expected; ii) a large degree of selectivity could be obtained by suitable substitution and non-toxic diaminopyrimidines with preferential antimicrobial activity were found; iii) the identification of dihydrofolate reductase (DHFR) as the specific target for aminopterin and methotrexate in 1958 and for TMP in 1965, and the diversity of this enzyme in different species. Although several diaminopyrimidines were initially tested as monotherapies in clinical trials, the pronounced synergism between some of these new compounds and sulfonamides seen against Plasmodium was finally also applied in the development of TMP. Its combination with sulfamethoxazole later proved one of the most successful agents ever developed. Further milestones in the application of antimicrobial DHFR inhibitors were the introduction of TMP alone in 1972, the launch of a new combination of tetroxoprim, a close TMP-analog, with sulfadiazine, and the successful clinical trials with brodimoprim, which proved clinically efficacious and safe with once-daily low dose monotherapy. Efforts to discover new antimicrobial DHFR inhibitors have recently intensified. DHFRs from important gram-positive problem organisms such as S. aureus, S. epidermidis have been cloned and sequenced, as well as DHFRs from opportunistic pathogens such as P. carinii, T. gondii, and of mycobacteria. DHFR crystal structures from several of these organisms are available to aid rational inhibitor design.(ABSTRACT TRUNCATED AT 250 WORDS)

Frequency and transferability of trimethoprim and sulfonamide resistance in methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis.

A total of 374 Staphylococcus aureus and 126 Staphylococcus epidermidis strains from 14 countries were studied for their resistance to methicillin, trimethoprim (Tp) and sulfonamides (Su), alone and combined (TpSu). The frequency of resistance to Tp, Su and TpSu was much higher in methicillin-resistant S. epidermidis (MRSE) than in methicillin-resistant S. aureus (MRSA). Considerable differences, however, existed in isolates from different countries. Resistance to Tp, Su or TpSu in MRSA was low or absent in isolates from Switzerland, Spain, Japan, Mexico, Argentina and Chile, but high in isolates from Germany and Brazil. High level Tp resistance mostly resided on large plasmids. It could be transferred in 17 out of 97 strains. Su resistance was never cotransferred. Strains cured of their large Tp resistance plasmids remained Su-resistant, which suggests a chromosomal location of Su resistance.

Ability of newer beta-lactam antibiotics to induce beta-lactamase production in Enterobacter cloacae.

The beta-lactamase inducing properties of various new beta-lactam antibiotics in two isogenic strains of Enterobacter cloacae were investigated. Beta-lactamase activity was measured two hours after addition of inducer to cells in the late logarithmic growth-phase. Beta-lactamase expression was highly dependent on the growth medium used, highest levels being obtained after induction with cefoxitin in Tryptic Soy broth, Mueller-Hinton broth and Nutrient broth. Upon induction the mutant 908 Ssi produced tenfold higher beta-lactamase levels than its parent wild type 908 Swi. Among the new antibiotics investigated, sulfoxides of several oxyimino-cephalosporins, HR 810, cefetamet, cefteram, carumonam and BRL 36650 were moderate or poor inducers. The penem FCE 22101 resembled imipenem in its strong inducing properties.

Role of thymidine for the activity of trimethoprim, sulfonamides and their combinations.

Thymidine levels in urines from 14 patients suffering from severe chronic urinary tract infection were determined microbiologically. The concentrations found were < 10 ng/ml to 33 ng/ml and these do not seem to be any different from the concentrations determined in healthy persons. Results from growth kinetic experiments support the assumption of low thymidine levels in the urines of these patients and in most cases trimethoprim or co-trimoxazole were seen to act in a bactericidal manner in these urine samples. Various aspects of the role of exogenous and endogenous thymidine for the activity and bactericidal effect of trimethoprim, sulfonamides and their combinations, their mutual synergism, the importance of thymine auxotrophs, the problem of thymidine availability and its role in testing media are discussed.

Properties of Ro 13-99041 as a substrate and inhibitor of beta-lactamases.

Ro 13-9904 is a novel semisynthetic and highly active parenteral cephalosporin. Its stability against hydrolysis by several beta-lactamases was studied. The enzymes were isolated from various Enterobacteriaceae or Staphylococcus aureus and several commercially available enzyme preparations were also included. Most of the penicillinases, cephalosporinases or the TEM-type beta-lactamase studied were unable to hydrolyze this novel cephalosporin. The cephalosporinases from Bacillus cereus 569/H9 and Proteus vulgaris 1028, however, were found to readily cleave all new cephalosporins like cefuroxime, cefotaxime and Ro 13-9904, but not cefoxitin. Ro 13-9904 was seen to be a potent inhibitor of several cephalosporinases, but had little or no affinity for penicillinases or the TEM lactamase.

Neisseriaceae, a group of bacteria with dihydrofolate reductases, moderately susceptible to trimethoprim.

Dihydrofolate reductases of five species of the family Neisseriaceae were compared by means of inhibition profiles, using several structurally different inhibitors, including trimethoprim (TMP) and pyrimethamine. All enzymes were seen to be highly susceptible to the folate analog aminopterin, but exhibited moderate susceptibility to all other inhibitors tested. Approximately 200-fold higher concentrations of TMP were needed to inhibit neisserial reductases as compared to the E. coli enzyme. Besides poor penetration this is assumed to be the main basis for the low susceptibility of neisseriae to TMP. In addition to TMP all other inhibitors were also moderately active or inactive in vitro. The enzymatic differences, as seen from inhibition profiles, were statistically significant but small among all species of the genus Neisseria. Branhamella catarrhalis on the other hand was seen to be far less related to the other neisseriae, as seen by the inhibition profile of its reductase, its dihydrofolate reductase conttent, as well as by its in vitro properties.

Mechanisms of resistance to trimethoprim, the sulfonamides, and trimethoprim-sulfamethoxazole.

A variety of different mechanisms are known to be responsible for either natural or acquired resistance to trimethoprim, the sulfonamides, or trimethoprim-sulfonamide combinations. Some mechanisms of obvious clinical importance have been studied intensively. Among these are unique bypass mechanisms such as the synthesis of drug-resistant, plasmid-coded dihydrofolate reductase or dihydropteroate synthetase; such mechanisms so far have not been encountered in studies of resistance to other drugs. This article focuses on several mechanisms of resistance that have rarely been discussed in the past, including metabolic alteration of trimethoprim or the sulfonamides and hyperproduction of p-aminobenzoic acid, and on the simultaneous presence of more than one mechanism. The role of these mechanisms in the resistance of clinical isolates requires further investigation.

Interaction of Ro 17-2301 (AMA-1080) with beta-lactamases.

Against a variety of beta-lactamases tested, mostly of chromosomal origin, Ro 17-2301 (AMA-1080) proved to be more stable than the new cephalosporins and thus resembles aztreonam. Against the beta-lactamases from Klebsiella oxytoca and Pseudomonas vulgaris, however, Ro 17-2301 proved to be much more stable than aztreonam. Enzymatic hydrolysis, performed with the K. oxytoca beta-lactamase, yielded a single compound, viz. the microbiologically inactive, ring-opened structure. Ro 17-2301 is a potent and progressive inhibitor of cephalosporinases found, e.g., in Enterobacter cloacae and other gram-negative organisms. The IC50 and Ki values, however, showed that the affinity for these enzymes is lower than that of aztreonam.