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.

In vitro and in vivo properties of Ro 63-9141, a novel broad-spectrum cephalosporin with activity against methicillin-resistant staphylococci.

Ro 63-9141 is a new member of the pyrrolidinone-3-ylidenemethyl cephem series of cephalosporins. Its antibacterial spectrum was evaluated against significant gram-positive and gram-negative pathogens in comparison with those of reference drugs, including cefotaxime, cefepime, meropenem, and ciprofloxacin. Ro 63-9141 showed high antibacterial in vitro activity against gram-positive bacteria except ampicillin-resistant enterococci, particularly vancomycin-resistant strains of Enterococcus faecium. Its MIC at which 90% of the isolates tested were inhibited (MIC(90)) for methicillin-resistant Staphylococcus aureus (MRSA) was 4 microg/ml. Ro 63-9141 was bactericidal against MRSA. Development of resistance to the new compound in MRSA was not observed. Ro 63-9141 was more potent than cefotaxime against penicillin-resistant Streptococcus pneumoniae (MIC(90) = 2 microg/ml). It was active against ceftazidime-susceptible strains of Pseudomonas aeruginosa and against Enterobacteriaceae except Proteus vulgaris and some isolates producing extended-spectrum beta-lactamases. The basis for the antibacterial spectrum of Ro 63-9141 lies in its affinity to essential penicillin-binding proteins, including PBP 2' of MRSA, and its stability towards beta-lactamases. The in vivo findings were in accordance with the in vitro susceptibilities of the pathogens. These data suggest the potential utility of Ro 63-9141 for the therapy of infections caused by susceptible pathogens, including MRSA. Since insufficient solubility of Ro 63-9141 itself precludes parenteral administration in humans, a water-soluble prodrug, Ro 65-5788, is considered for development.

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.

Antibacterial activities of epiroprim, a new dihydrofolate reductase inhibitor, alone and in combination with dapsone.

Epiroprim (EPM; Ro 11-8958) is a new selective inhibitor of microbial dihydrofolate reductase. EPM displayed excellent activity against staphylococci, enterococci, pneumococci, and streptococci which was considerably better than that of trimethoprim (TMP). EPM was also active against TMP-resistant strains, although the MICs were still relatively high. Its combination with dapsone (DDS) was synergistic and showed as in vitro activity superior to that of the TMP combination with sulfamethoxazole (SMZ). The EPM-DDS (ratio, 1:19) combination inhibited more than 90% of all important gram-positive pathogens at a concentration of 2 + 38 micrograms/ml. Only a few highly TMP-resistant staphylococci and enterococci were not inhibited. EPM was also more active than TMP against Moraxella catarrhalis, Neisseria meningitidis, and Bacteroides spp., but it was less active than TMP against all other gram-negative bacteria tested. Atypical mycobacteria were poorly susceptible to EPM, but the combination with DDS was synergistic and active at concentrations most probably achievable in biological fluids (MICs from 0.25 +/- 4.75 to 4 + 76 micrograms/ml). EPM and the EPM-DDS combination were also highly active against experimental staphylococcal infections in a mouse septicemia model. The combination EPM-DDS has previously been shown to exhibit activity in Pneumocystis carinii and Toxoplasma models and, as shown in the present study, also shows good activity against a broad range of bacteria including many strains resistant to TMP and TMP-SMZ.

Cloning and characterization of a novel, plasmid-encoded trimethoprim-resistant dihydrofolate reductase from Staphylococcus haemolyticus MUR313.

In recent years resistance to the antibacterial agent trimethoprim (Tmp) has become more widespread, and several trimethoprim-resistant (Tmpr) dihydrofolate reductases (DHFRs) have been described from gram-negative bacteria. In staphylococci, only one Tmpr DHFR has been described, the type S1 DHFR, which is encoded by the dfrA gene found on transposon Tn4003. In order to investigate the coincidence of high-level Tmp resistance and the presence of dfrA, we analyzed the DNAs from various Tmpr staphylococci for the presence of dfrA sequences by PCR with primers specific for the thyE-dfrA genes from Tn4003. We found that 30 or 33 isolates highly resistant to Tmp (MICs, > or = 512 micrograms/ml) contained dfrA sequences, whereas among the Tmpr (MICs, < or = 256 micrograms/ml) and Tmps isolates only the Staphylococcus epidermidis isolates (both Tmpr and Tmps) seemed to contain the dfrA gene. Furthermore, we have cloned and characterized a novel, plasmid-encoded Tmpr DHFR from Staphylococcus haemolyticus MUR313. The dfrD gene of plasmid pABU17 is preceded by two putative Shine-Dalgarno sequences potentially allowing for the start of translation at two triplets separated by nine nucleotides. The predicted protein of 166 amino acids, designated S2DHFR, encoded by the longer open reading frame was overproduced in Escherichia coli, purified, and characterized. The molecular size of the recombinant S2DHFR was determined by ion spray mass spectrometry to be 19,821.2 +/- 2 Da, which is in agreement with the theoretical value of 19,822 Da. In addition, the recombinant S2DHFR was shown to exhibit DHFR activity and to be highly resistant to Tmp.

Characterization of the gene for the chromosomal dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990: the origin of the trimethoprim-resistant S1 DHFR from Staphylococcus aureus?

The gene for the chromosomally encoded dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990 has been cloned and characterized. The structural gene encodes a polypeptide of 161 amino acid residues with a calculated molecular weight of 18,417. This trimethoprim-sensitive (Tmps) DHFR, SeDHFR, differs in only three amino acids (Val-31-->Ile, Gly-43-->Ala, and Phe-98-->Tyr) from the trimethoprim-resistant (Tmpr) S1 DHFR encoded by transposon Tn4003. Since in addition the S. epidermidis gene also forms part of an operon with thyE and open reading frame 140 as in Tn4003, the chromosomally located gene encoding the Tmps SeDHFR is likely to be the molecular origin of the plasmid-located gene encoding the Tmpr S1 DHFR. Site-directed mutagenesis and kinetic analysis of the purified enzymes suggest that a single Phe-->Tyr change at position 98 is the major determinant of trimethoprim resistance.

A pilot study on the efficacy of epiroprim against developmental stages of Toxoplasma gondii and Pneumocystis carinii in animal models.

The new bacteriocidal drug epiroprim (Ro-11-8958) was tested (alone or in combination with dapsone) on its efficacy against Toxoplasma gondii and Pneumocystis carinii in their hosts: laboratory mice and/or nude mice/rats, and was compared to the curative effects of the recent drugs of choice. The experiments clearly pointed out that epiroprim has significant effects on the reduction of both parasites when given alone. In combination with dapsone epiroprim led to a complete cure of toxoplasmosis in mice. This finding is of some importance for AIDS patients mostly suffering from bacteriosis and parasitosis at the same time.

Activity of epiroprim (Ro 11-8958), a dihydrofolate reductase inhibitor, alone and in combination with dapsone against Toxoplasma gondii.

We examined the effect of epiroprim (Ro 11-8958), a dihydrofolate reductase inhibitor, alone and in combination with dapsone, against Toxoplasma gondii. In vitro, the anti-T. gondii effects of epiroprim and dapsone were observed at nanogram-per-milliliter levels when a 72-h uracil assay and an infection rate of one parasite per 120 macrophages were used. In combination, these drugs exerted a synergistic effect that, however, was only parasitostatic. In a model of acute infection, mice were infected intraperitoneally with 10(4) parasites of the RH strain of T. gondii and were treated for 14 days by gavage (therapy divided into two daily dosages), starting 24 h after infection. Used alone, dapsone and epiroprim, each at a dose of 50 mg/kg of body weight per day, protected 10 and 0% of the mice, respectively. When these drugs were administered simultaneously, a 100% survival rate was observed. Pyrimethamine-sulfadiazine (4 and 250 mg/kg/day, respectively) protected 100% of the mice. A 3-week therapy of chronically infected mice with either epiroprim (50 mg/kg/day), dapsone (50 mg/kg/day), or pyrimethamine (15 mg/kg/day) reduced the numbers of T. gondii cysts and the inflammation in their brains. A combination of epiroprim and dapsone, both at 50 mg/kg/day, further reduced the number of brain cysts in comparison with the number after the corresponding monotherapies. Epiroprim may have a role in the prophylaxis or therapy of human toxoplasmosis, especially when combined with other drugs active against T. gondii, such as dapsone.

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)

Characterization of the gene for chromosomal trimethoprim-sensitive dihydrofolate reductase of Staphylococcus aureus ATCC 25923.

The gene for the trimethoprim-sensitive (Tmps) chromosomal dihydrofolate reductase (DHFR) of Staphylococcus aureus ATCC 25923 was cloned and characterized. The structural gene encodes a polypeptide of 159 amino acid residues and has a calculated molecular weight of 18,251. The amino acid sequences of this Tmps DHFR and those of the trimethoprim-resistant type S1 DHFR encoded by transposon Tn4003 are 80% identical. In contrast to the trimethoprim-resistant enzyme, the Tmps DHFR can be highly overexpressed in Escherichia coli, with most of the recombinant protein occurring in a soluble and an active form.

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.

Lipopolysaccharide alterations responsible for combined quinolone and beta-lactam resistance in Pseudomonas aeruginosa.

Resistant variants of three clinical Pseudomonas aeruginosa isolates were obtained in the presence of aztreonam. The variants exhibited a four- to eightfold increase in the minimal inhibitory concentrations to beta-lactam antibiotics (except imipenem) to quinolones, such as norfloxacin and fleroxacin, chloramphenicol and tetracycline, but not to gentamicin and polymyxin B. beta-Lactamase production was barely detectable in both wild-type strains and the resistant clones. Only ampicillin, cefoxitin and imipenem increased the production of beta-lactamase, whereas various other beta-lactams did not. Penicillin-binding proteins remained unchanged in the aztreonam-resistant clones. The analysis of the outer membrane proteins did not reveal differences in the outer membrane proteins between the wild-type strains and the aztreonam-resistant clones. Two of the three antibiotic-resistant isogenic clones contained less lipopolysaccharides (LPSs) than their corresponding wild-type strains. Moreover, it could be demonstrated that the ratio of 2-keto-3-deoxy octonate to carbohydrate of the LPS changed in any case between the wild-type strains and the aztreonam-resistant clones. These alterations were accompanied by a decrease in surface hydrophobicity of the resistant clones as compared to the wild-type strains. Therefore, quantitative as well as qualitative alterations in the LPS may provide an explanation for the resistant phenotype observed.

In vitro and in vivo evaluation of Ro 09-1428, a new parenteral cephalosporin with high antipseudomonal activity.

Ro 09-1428, a new parenteral cephalosporin with a catechol moiety attached at position 7 of the cephalosporin ring, showed high in vitro activity against Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, and Streptococcus pyogenes, with MICs for 90% of strains tested (MIC90s) of less than or equal to 0.39 micrograms/ml. Morganella morganii, Providencia rettgeri, Citrobacter freundii, Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae were inhibited with MIC90s of less than or equal to 3.13 micrograms/ml. Serratia marcescens was less susceptible to Ro 09-1428, with a MIC90 of 25 micrograms/ml. The most distinctive feature of Ro 09-1428 was its potent activity against Pseudomonas aeruginosa and Acinetobacter calcoaceticus, with MIC90s of 0.39 and 6.25 micrograms/ml, respectively. Most of the ceftazidime-resistant strains of P. aeruginosa, E. cloacae, and C. freundii were inhibited by Ro 09-1428, while those of S. marcescens were resistant at a concentration of 12.5 micrograms/ml. Ro 09-1428 was more active than ceftazidime against staphylococci. PBP 3 was the most sensitive target in E. coli and P. aeruginosa. The response to ferric iron in growth medium suggests that Ro 09-1428 may be taken up by transport mechanisms similar to those of other catechol cephalosporins. In accordance with its in vitro activity, Ro 09-1428 activity was equal to or greater than ceftazidime activity in efficacy against experimental septicemias in mice. The results indicate that Ro 09-1428 is a broad-spectrum cephalosporin with advantages over ceftazidime in its activity against P. aeruginosa, staphylococci, and ceftazidime-resistant strains of C. freundii and E. cloacae.

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.

Permeability and penicillin-binding protein alterations in Salmonella muenchen: stepwise resistance acquired during beta-lactam therapy.

A patient with Salmonella muenchen sepsis was unsuccessfully treated with ampicillin. During therapy, four strains that showed stepwise ampicillin resistance and affected other beta-lactams and unrelated antibiotics were isolated sequentially. Resistance was caused by decreased outer membrane permeability associated with diminished expression of porin OmpF. Furthermore, the most resistant isolate overproduced the PBP 3 target molecule.

In vitro antibacterial properties of cefetamet and in vivo activity of its orally absorbable ester derivative, cefetamet pivoxil.

The in vitro activity of cefetamet, the microbiologically active metabolite of the orally administered prodrug cefetamet pivoxil, was compared with that of cephalexin, cefaclor, cefuroxime and amoxicillin. Cefetamet was highly active against Enterobacteriaceae, Neisseria spp., Vibrio spp., Haemophilus influenzae and streptococci other than enterococci. Cefetamet inhibited cefaclor-resistant species such as Proteus vulgaris, Providencia stuartii, Providencia rettgeri and Serratia marcescens. Staphylococci, Pseudomonas aeruginosa and cephalosporinase-overproducing strains of Enterobacter cloacae were resistant to cefetamet. The superior activity of cefetamet compared with older oral beta-lactam antibiotics against a large number of gram-negative pathogens correlated with enhanced stability towards beta-lactamases. In accordance with the in vitro findings, cefetamet pivoxil showed good activity in experimental infections in the mouse and rat, suggesting satisfactory bioavailability in these animals after oral administration.

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.