Development of a simple homogeneous assay to screen for inhibitors of N-acetylglucosamine-6-sulfotransferases.

L-selectin, a leukocyte adhesion molecule, plays a central role in lymphocyte homing to secondary lymphoid tissue and to certain sites of inflammation. Carbohydrate sulfation was implicated in this process, when it was demonstrated that carbohydrate sulfotransferase-mediated sulfation of N-acetylglucosamine (GlcNAc) within sialyl Lewis X of cognate endothelial ligands for L-selectin was an essential modification for L-selectin binding. The recently identified GlcNAc-6-sulfotransferases GlcNAc6ST-1 and -2, which facilitate GlcNAc sulfation by catalyzing the transfer of a sulfonyl group from 3(')-phosphoadenosine 5(')-phosphosulfate (PAPS) to the 6-hydroxy group of the acceptor GlcNAc moiety, contribute to the biosynthesis of the 6-sulfosialyl Lewis X motif. Due to their pivotal role in L-selectin ligand biosynthesis, this enzyme class has recently emerged as an important and relatively unexplored class of potential targets for anti-inflammatory therapy. However, no inhibitors have been reported to date and screening for lead inhibitors has been hampered by the lack of simple assay formats suitable for high-throughput screening. Here, we report the development of a simple homogeneous in vitro sulfotransferase assay using a newly synthesized biotinylated glycoside as a substrate. The assay is based on GlcNAc6ST-2-mediated [35S]sulfate transfer from [35S]PAPS to the biotinylated glycoside and subsequent detection using streptavidin-coated SPA beads. K(m) values with partially purified GlcNAc6ST-2 for PAPS and the biotinylated glycoside were estimated to be 8.4 and 34.5 microM, respectively. The sulfotransferase reaction could be inhibited by 3('),5(')-ADP with an IC(50) of 2.1 microM. The assay can be operated in 384-well format; is characterized by a high signal-to-noise ratio, low variation, and excellent Z factors; and is highly suitable for high-throughput screening.

Efficacy of trimethoprim in murine experimental infection with a thymidine kinase-deficient mutant of Escherichia coli.

The antimicrobial activity of trimethoprim is antagonized by thymidine in in vitro susceptibility tests. The purpose of this investigation was to determine whether this antagonism also occurred during experimental infection in mice, which have high serum thymidine concentrations. We derived a mutant strain of Escherichia coli, TT-48, incapable of utilizing exogenous thymidine from parent strain E. coli KC-14 and then investigated the in vitro and in vivo antimicrobial activities of trimethoprim, sulfamethoxazole, cefdinir, and ofloxacin against these strains. E. coli TT-48 lacked the activity of thymidine kinase, which catalyzes the conversion of thymidine to thymidylate, but its growth curve remained close to that of the parent strain. The MICs of all of the antimicrobial agents tested, except cefdinir, for the mutant strain were slightly inferior to those for the parent strain. The bactericidal effect of trimethoprim against the parent strain was antagonized by thymidine at concentrations of more than 1 microg/ml, while that against the mutant strain was not affected by thymidine even at the highest concentration (10 microg/ml). The therapeutic efficacy of trimethoprim in experimental murine infections was significantly higher when the mutant rather than the parent strain was used, whereas the therapeutic efficacy of cefdinir or ofloxacin, whose antimicrobial action is independent of folic acid synthesis, was the same with both strains. Unexpectedly, sulfamethoxazole also had similar efficacy against both strains. Thus, high thymidine concentrations antagonized the antimicrobial activity of trimethoprim in vitro and in vivo.

Expression and characterization of the Trypanosoma cruzi dihydrofolate reductase domain.

We have cloned and expressed in Escherichia coli a 702-base pair gene coding for the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Trypanosoma cruzi. The DHFR domain was purified to homogeneity by methotrexate-Sepharose chromatography followed by an anion-exchange chromatography step in a mono Q column, and displayed a single 27-kDa band on SDS-PAGE. Gel filtration showed that the catalytic domain was expressed as a monomer. Kinetic parameters were similar to those reported for the wild-type bifunctional enzyme with Km values of 0.75 microM for dihydrofolate and 16 microM for NADPH and a kcat value of 16.5 s-1. T. cruzi DHFR is poorly inhibited by trimethoprim and pyrimethamine and the inhibition constants were always lower for the bifunctional enzyme. The binding of methotrexate was characteristic of a class of inhibitors that form an initial complex which isomerizes slowly to a tighter complex and are referred to as 'slow, tight-binding' inhibitors. While the slow-binding step of inhibition was apparently unaffected in the individually expressed DHFR domain, the overall inhibition constant was two-fold higher as a consequence of the superior inhibition constant value obtained for the initial inhibitory complex.

Reversal of activity of trimethoprim against gram-positive cocci by thymidine, thymine and 'folates'.

The reversal of the antibacterial activity of trimethoprim against different species of Gram-positive cocci (Staphylococcus aureus, Staph. epidermidis, Staph. saprophyticus, group B streptococci, Streptococcus faecalis and Str. faecium) by thymine, thymidine and various 'folates' (folate, folinate, dihydrofolate and tetrahydrofolate) was tested. Against group B streptococci and staphylococci only thymidine antagonized trimethoprim. However, for enterococci, thymine, thymidine, dihydrofolate, tetrahydrofolate and folinate all reversed the activity of trimethoprim, although by different amounts. Dihydrofolate was significantly more effective as a trimethoprim antagonist for Str. faecium than for Str. faecalis. While thymine and thymidine caused the MIC of trimethoprim against enterococci to increase more than 100-fold--thereby rendering them resistant--the 'folates' brought about much smaller increases in trimethoprim MIC, in the order of ten-fold only. Thus, even in the presence of 'folates' enterococci remain sensitive in vitro to trimethoprim.

Resistance of urinary tract isolates of Escherichia coli to cotrimoxazole, sulphonamide, trimethoprim and ampicillin: an 11-year survey.

The susceptibility of urinary tract Escherichia coli isolates to cotrimoxazole, sulphonamide, trimethoprim, and ampicillin was monitored over an 11-year period. A trend in increasing resistance to cotrimoxazole and trimethoprim was observed, but there was no comparable alteration in sulphonamide resistance. Ampicillin resistance was high at the beginning of the survey period and continued to rise.

Reversal of the in vitro susceptibility of enterococci to trimethoprim-sulfamethoxazole by folinic acid.

The in vitro susceptibilities of 21 clinical isolates of Streptococcus faecalis to trimethoprim (TMP) in combination with sulfamethoxazole (SMX) was evaluated in Mueller-Hinton broth (MHB) in the presence and absence of folinic acid as well as in urine. The mean MIC and MBC in MHB, expressed as the TMP concentration, was 0.13 and 0.32 micrograms of TMP-SMX per ml, respectively. In MHB supplemented with folinic acid, the mean MIC and MBC was 3.3 and 5.5 micrograms of TMP-SMX per ml, respectively. In urine the mean MIC of TMP-SMX for these isolates was 8.1 micrograms/ml (range, 1.6 to 50 micrograms/ml). All isolates were inhibited by less than 0.01 micrograms of TMP-SMX per ml when methotrexate was added to the urine.

Exogenous thymidine and reversal of the inhibitory effect of sulfamethoxazole-trimethoprim on streptococci.

The practice of using sulfamethoxazole-trimethoprim (SXT) for the selective isolation of Streptococcus pyogenes and as a taxonomic character in the presumptive identification of streptococci was applied to 17 strains of different groups of streptococci to determine their characteristic behaviour in the presence of exogenous thymidine. Streptococcus pyogenes, Streptococcus agalactiae and group D enterococci utilized thymidine, the first two species obtaining a maximum reversal of the inhibitory effect of SXT at thymidine concentrations of 1.2 micrograms/ml and 0.6 micrograms/ml or higher, respectively. For group D enterococci, the degree of reversal of the inhibitory effect was proportional to the thymidine concentration. In contrast, the four viridans species studied (Streptococcus sanguis I, Streptococcus salivarius, Streptococcus mitis and Streptococcus sanguis II) and Streptococcus pneumoniae were unable to utilize thymidine from an exogenous source and thus growth remained inhibited even at the highest concentrations of thymidine tested. For selective isolation and identification of streptococci only stable media with batch-to-batch consistency are recommended together with a known quantity of thymidine.

Effects of trimethoprim on leukaemic cells in vitro.

Trimethoprim-Sulfamethoxazole (TMP-SMZ) is a fixed combination of antibiotics which is widely used for prophylaxis and treatment of infections in patients undergoing cancer chemotherapy. TMP has been reported to inhibit growth of haemopoietic stem cells in vitro. If TMP-SMZ inhibits leukaemic cell growth, it could interfere with antileukaemic treatment, especially with S phase specific agents. TMP-SMZ at a concentration of 10 micrograms/ml (TMP) produced 50% inhibition of incorporation of 3H-deoxyuridine in DNA of L1210 and human lymphoblastic leukaemia cell. TMP-SMZ (1 g/ml TMP) produced 30% prolongation of doubling time of L1210 in vitro. Pure TMP (10 micrograms/ml) but not SMZ (50 micrograms/ml) produced the same effect as TMP-SMZ. Cell inhibitory effects could be completely reversed by folinic acid. These findings suggest that TMP produces some degree of inhibition of dihydrofolic acid reductase in mammalian cells and can potentially influence the effects of chemotherapy on tumour and/or host cells.

[The formulation of semi-defined culture media to overcome antibiotic antagonism (author's transl)]. / Die Zusammensetzung unvollständig definierter Kulturmedien zur Uberwindung des antibiotischen Antagonismus.

Semi-defined susceptibility media are a compromise between undefined and totally defined media. Antibiotic antagonism can still occur in semi-defined media. The undefined nutrients antagonizing sulphonamide/trimethroprim and the defined additives antagonizing other antibiotics. Methods used to overcome these problems are discussed.

Effect of thymidine on activity of trimethoprim and sulphamethoxazole.

Thymidine at levels as low as 0.05 mg/1 reduces the activities of sulphamethoxazole and trimethoprim and their combination in vitro. Using a biological assay procedure, levels of thymidine greater than this were interpreted as being present in urine. The addition of sulphamethoxazole and trimethoprim, singly or in combination, to urine obtained from patients with urinary tract infections showed that all the antibacterial effect towards sensitive organisms was due to the trimethoprim component. It is suggested that trimethoprim should replace the combination co-trimoxazole for the treatment of some lower urinary tract infections, and that laboratory media, if they are to resemble the clinical environment, should contain thymidine.

Failure to demonstrate an advantage in combining sulphamethoxazole with trimethoprim in an experimental model of urinary infection.

Co-trimoxazole was found to have a predominantly bacteriostatic effect upon 28 urinary isolates of Enterobacteriaceae in nutrient broth and was never bactericidal in artificially infected urine. The components of co-trimoxazole were tested individually and trimethoprim was found to be at least as effective as co-trimoxazole in nutrient broth and in urine. Trimethoprim alone produced some bactericidal effect in urine but this was antagonized by sulphamethoxazole. Laboratory tests for evaluating these drugs may give a misleading impression of their activity in vivo. Further clinical comparisons should therefore be made between trimethoprim and cotrimoxazole to determine when trimethoprim should be used in preference to the combination.

Trimethoprim action and its analogy with thymine starvation.

In a minimal medium, trimethoprim is merely bacteriostatic on the prototroph Escherichia coli 114. The drug was bactericidal when the amino acids methionine and glycine, plus a purine or purine nucleoside, were also present. This response could be reversed completely when thymine and lysine were added to the culture. Methionine, glycine, and the purine are thought to maintain the integrity of the tetrahydrofolate pool under trimethoprim treatment and prevent the thymidylate synthetase reaction. Thus, the organism behaves phenotypically as a thymineless mutant. The mechanisms by which thymine and lysine reverse the bactericidal effect of trimethoprim in a minimal medium containing methionine, glycine, and adenine is discussed.