Sordarins: in vitro activities of new antifungal derivatives against pathogenic yeasts, Pneumocystis carinii, and filamentous fungi.

GM 193663, GM 211676, GM 222712, and GM 237354 are new semisynthetic derivatives of the sordarin class. The in vitro antifungal activities of GM 193663, GM 211676, GM 222712, and GM 237354 against 111 clinical yeast isolates of Candida albicans, Candida kefyr, Candida glabrata, Candida parapsilosis, Candida krusei, and Cryptococcus neoformans were compared. The in vitro activities of some of these compounds against Pneumocystis carinii, 20 isolates each of Aspergillus fumigatus and Aspergillus flavus, and 30 isolates of emerging less-common mold pathogens and dermatophytes were also compared. The MICs of GM 193663, GM 211676, GM 222712, and GM 237354 at which 90% of the isolates were inhibited (MIC90s) were 0.03, 0.03, 0.004, and 0.015 microg/ml, respectively, for C. albicans, including strains with decreased susceptibility to fluconazole; 0.5, 0.5, 0.06, and 0.12 microg/ml, respectively, for C. tropicalis; and 0.004, 0.015, 0.008, and 0.03 microg/ml, respectively, for C. kefyr. GM 222712 and GM 237354 were the most active compounds against C. glabrata, C. parapsilosis, and Cryptococcus neoformans. Against C. glabrata and C. parapsilosis, the MIC90s of GM 222712 and GM 237354 were 0.5 and 4 microg/ml and 1 and 16 microg/ml, respectively. The MIC90s of GM 222712 and GM 237354 against Cryptococcus neoformans were 0.5 and 0.25 microg/ml, respectively. GM 193663, GM 211676, GM 222712, and GM 237354 were extremely active against P. carinii. The efficacies of sordarin derivatives against this organism were determined by measuring the inhibition of the uptake and incorporation of radiolabelled methionine into newly synthesized proteins. All compounds tested showed 50% inhibitory concentrations of <0.008 microg/ml. Against A. flavus and A. fumigatus, the MIC90s of GM 222712 and GM 237354 were 1 and 32 microg/ml and 32 and >64 microg/ml, respectively. In addition, GM 237354 was tested against the most important emerging fungal pathogens which affect immunocompromised patients. Cladosporium carrioni, Pseudallescheria boydii, and the yeast-like fungi Blastoschizomyces capitatus and Geotrichum clavatum were the most susceptible of the fungi to GM 237354, with MICs ranging from /=2 microg/ml. In summary, we concluded that some sordarin derivatives, such as GM 222712 and GM 237354, showed excellent in vitro activities against a wide range of pathogenic fungi, including Candida spp., Cryptococcus neoformans, P. carinii, and some filamentous fungi and emerging invasive fungal pathogens.

Sordarins: A new class of antifungals with selective inhibition of the protein synthesis elongation cycle in yeasts.

GR135402, a sordarin derivative, was isolated in an antifungal screening program. GR135402, sordarin, and derivatives of both compounds were evaluated for their ability to inhibit cell-free translational systems from five different pathogenic fungi (Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Cryptococcus neoformans). The activity profile of GR135402 is extended to other chemical compounds derived from sordarin. Experimental results indicate that sordarin analogs exert their antifungal effects by specifically inhibiting the protein synthesis elongation cycle in yeasts but do not affect protein synthesis machinery in mammalian systems. Intrinsically resistant strains owe their resistance to differences in the molecular target of sordarins in these strains. Preliminary studies performed to elucidate the mode of action of this new class of antifungal agents have shown that the putative target of sordarins is one of the protein synthesis elongation factors.

TEM- and SHV-derived extended-spectrum beta-lactamases: relationship between selection, structure and function.

The later-generation cephalosporins were developed to overcome beta-lactamases which conferred resistance to earlier beta-lactam drugs. Within two years of their clinical introduction, the ubiquitous TEM and SHV plasmid-encoded beta-lactamase genes underwent simple point mutations that changed key amino acids around the active site of the protein and enabled the enzyme to bind and hydrolyse these new drugs. Successive mutations interacted in concert, radically increasing the enzymes' abilities to bind and confer resistance to later-generation cephalosporins. These modified enzymes have been classified in three groups, based on activity, and altered functions have been correlated with changes in the enzyme structure.

Protein O-mannosylation in Candida albicans. Determination of the amino acid sequences of peptide acceptors for protein O-mannosyltransferase.

A protein-O-D-mannosyltransferase (PMT) assay was optimised using a microsomal membrane preparation from Candida albicans and a peptide acceptor, YNPTSV. [14C]Mannose was transferred from dolichyl phosphate [14C]mannose to the threonine or serine residues of the peptide. During the assay, the peptide was highly susceptible to proteolysis. A blocked peptide Ac-YNPTSV-NH2 was resistant to proteolysis and was apparently a better acceptor for O-mannosylation. This peptide had a Km value of 4.3 mM in the assay. A number of other peptides were tested with altered sequences. Maximum incorporation of [14C]mannose was obtained with a pentapeptide YATAV (Km 2.2 mM) which was further improved by blocking both ends: Ac-YATAV-NH2 (Km 0.25 mM). Finally, and unexpectedly, an improvement was noted if the acetyl group on the N terminus was replaced by a biotin residue. Biotin-YATAV-NH2 had a Km of 0.075 mM. The biotin residue may be important in increasing the lipophilicity of the peptide and thus aid its adhesion to the Candida membranes. The simplest peptide that could act as an efficient mannose acceptor was Ac-ATA-NH2, whilst no incorporation was observed with Ac-GTG-NH2.

No detection of characteristic fungal protein elongation factor EF-3 in Pneumocystis carinii.

The taxonomic status of Pneumocystis carinii is uncertain, and P. carinii has been categorized both as a fungus and as a protozoan. Recent comparisons of RNA sequence homologies between P. carinii and several genera of fungi and protozoa suggest that P. carinii has closer affinities with the ascomycetes than with the protozoa. The translatory systems of the fungi, however, require three soluble protein factors for peptide chain elongation rather than the two necessary in other eukaryotic systems; to date the additional protein elongation factor (EF-3) appears to be unique to fungi. Western blot analysis of cell-free extracts of P. carinii, derived from rat, was done using a polyclonal antibody raised in rabbits to Saccharomyces cerevisiae EF-3. Anti-EF-3 cross-reacting material was detected only in lysates of Candida albicans and S. cerevisiae included as fungal controls; no cross reaction was detected in lysates of P. carinii, P. carinii-infected rat lung, or a protozoan control (Trichomonas vaginalis).

Characterisation of a unique ceftazidime-hydrolysing beta-lactamase, TEM-E2.

A strain of Klebsiella oxytoca, originally isolated in Liverpool in 1982, has been found to produce a novel transferable beta-lactamase, TEM-E2. This enzyme confers resistance to ceftazidime and focused as a doublet band with an iso-electric point (pI) of 5.3. The strain also produced the TEM-1 beta-lactamase. Both TEM-1 and TEM-E2 beta-lactamases were encoded by a transferable 103 kb plasmid; these two enzymes also had similar molecular weights, were inhibited by clavulanic acid, and hydrolysed ampicillin, carbenicillin and cephaloridine at similar rates. However, unlike the TEM-1 enzyme, the TEM-E2 beta-lactamase hydrolysed ceftazidime and cefotaxime with similar efficiency, although it conferred much greater resistance to ceftazidime in the host strain. This is the earliest documented example of a TEM-like enzyme which confers transferable resistance to ceftazidime and related cephalosporins.

Separation of plasmid-mediated extended spectrum beta-lactamases by fast protein liquid chromatography (FPLC system).

We have devised a reliable procedure for the separation of three beta-lactamases of isoelectric focusing points (pI), 5.4, 6.5, and 7.9 by Fast Protein Liquid Chromatography (FPLC System). All of these enzymes were transferable and originated from a ceftazidime and cefotaxime resistant Klebsiella pneumoniae isolated in Bombay, India. The complete separation of the enzymes, achievable by this method, allowed each of the different individual beta-lactamases to be characterized biochemically. This analysis revealed that the enzymes of pI 6.5 and pI 7.9 hydrolysed ceftazidime and cefotaxime, and were responsible for the resistance of K. pneumoniae, and its Escherichia coli J53-2 transconjugant to third generation cephalosporins. The enzyme of pI 5.4 was the TEM-1 beta-lactamase. The beta-lactamase of pI 7.9 appears quite different from any previously reported third generation cephalosporin hydrolysing beta-lactamase, and consequently given the preliminary designation DJP-1. This is also the first example of extended spectrum hydrolysing beta-lactamases found in Asia.

Discovery of fluconazole, a novel antifungal agent.

Fluconazole, 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)-2-propanol, is the result of a research program aimed at the development of a broad-spectrum antifungal agent active by both the oral and the intravenous routes for the treatment of superficial and systemic infections. The program began in 1978, and azole derivatives were chosen as a starting point because they were generally well tolerated and offered the advantage of a selective mode of action--inhibition of fungal sterol C-14 demethylation. Initial investigations focused on imidazole derivatives, but attention soon switched to triazole analogues because of their reduced susceptibility to metabolic activity. Polar derivatives were emphasized in an effort to facilitate the attainment of high levels in the blood and to reduce metabolic breakdown still further. This strategy resulted in a number of novel 1,3-bis-triazole-2-arylpropan-2-ol derivatives with good activity in a range of models of fungal infection. These models included systemic fungal infections in both normal and immunosuppressed animals and superficial infections in animals with normal immune function. The 2,4-difluorophenyl analogue, fluconazole, was chosen for development on the basis of an optimal combination of antifungal efficacy, pharmacokinetic characteristics, aqueous solubility, and safety profile.

Mutants of the TEM-1 beta-lactamase conferring resistance to ceftazidime.

Spontaneous ceftazidime resistant mutants were obtained from an Escherichia coli K12 J62-2 expressing the TEM-1 beta-lactamase (mutation frequency = 10(-9). These mutants produced beta-lactamases with similar molecular weights, kinetic parameters and isoelectric points (pI) to the beta-lactamases produced by ceftazidime resistant clinical isolates which have recently been identified in this laboratory. Mutant enzyme A focused as a doublet band at pI 5.3 with an additional weak pI 5.4 band. The doublet co-focused with the TEM-E2 beta-lactamase, produced by a ceftazidime resistant Klebsiella oxytoca isolate, which was originally obtained in a Liverpool hospital. Mutant enzyme B had a pI identical to the TEM-E1 beta-lactamase produced by a ceftazidime resistant clinical isolate of E. coli found in Belgium. These results suggest that the two beta-lactamases in the clinical strains may have come from simple mutations of the TEM-1 beta-lactamase gene.

TEM-E1: a novel beta-lactamase conferring resistance to ceftazidime.

A novel beta-lactamase, conferring resistance to ceftazidime, has been identified to be encoded by a 31 kb plasmid (pUK720) in a clinical E. coli strain isolated in Belgium. The beta-lactamase, new designated TEM-E1, has a pI of approximately 5.4 and lies in between the iso-electric focused bands of the beta-lactamases TEM-1 and TEM-7. The TEM-E1 beta-lactamase has a similar molecular weight of 22,000 to the TEM-1 and it is also inhibited by clavulanic acid. However, the TEM-E1 enzyme differs from TEM-1 by its low rates and efficiency of hydrolysis for ceftazidime and cefotaxime, TEM-E1 has similar efficiency of hydrolysis values for ceftazidime and cefotaxime, but only confers resistance to ceftazidime.

1-(3-cyano-2,3-dideoxy-beta-D-erythro-pentofuranosyl)thymine (cyanothymidine): synthesis and antiviral evaluation against human immunodeficiency virus.

1-(3-Cyano-2,3-dideoxy-beta-D-erythro-pentofuranosyl)thymine (cyanothymidine) (3a) has been prepared by an unambiguous route starting from D-xylose. The relative and absolute stereochemistry of 3a and its anomeric isomer 9 have been confirmed by NOE experiments and by X-ray diffraction analysis. In antiviral tests vs HIV 3a was shown to be inactive, a surprising result in view of a preliminary disclosure claiming potent anti-HIV activity. The activity previously assigned to 3a is believed to be due to contamination of that sample with the known antiviral nucleoside analogue 5b.

Efficacy of fluconazole (UK-49,858) against experimental aspergillosis and cryptococcosis in mice.

The efficacy of fluconazole, a new bis-triazole antifungal agent, was compared with that of orally administered ketoconazole and parenterally administered amphotericin B against aspergillus and cryptococcus infections in mice. Fluconazole was 5-20-fold more active than ketoconazole against systemic aspergillosis and against systemic, intracranial and pulmonary cryptococcosis but was less active than amphotericin B.

An alteration in tubulin expression detected in SV40 transformed 3T3 cells.

Tubulin expression was analysed in normal and simian virus-40 (SV40) transformed 3T3 cells by two-dimensional polyacrylamide gel electrophoresis and immunoblotting studies using monoclonal antibodies raised to alpha- and beta-tubulin subunits. The ratio of alpha- to beta-tubulin recognised was calculated for both cell lines and found to shift from 2.50 in normal cells to 0.52 in virally transformed cells. beta-Tubulin was thereby shown to be the predominant subunit in SV40-transformed 3T3 cells in contrast to normal 3T3 cells.

Efficacy of UK-49,858 (fluconazole) against Candida albicans experimental infections in mice.

UK-49,858 (fluconazole), a new, orally absorbed bis-triazole derivative, has been evaluated against systemic infections with Candida albicans in normal and immunosuppressed mice and against an intestinal infection with C. albicans in immunosuppressed mice. Orally administered ketoconazole was used as a comparison agent throughout, and orally administered amphotericin B was included for comparative in the experimental intestinal infection. In a 10-day dosage regimen, UK-49,858 was far more active than ketoconazole against systemic infections with C. albicans in normal and immunosuppressed mice. In normal mice, extension of UK-49,858 dosing to 30 days resulted in prolongation of survival to over 90 days, and up to 60% of treated animals had no detectable C. albicans in their kidneys. In addition, over 90% of mice with intestinal candidiasis had culture-negative feces after a 3-day treatment with UK-49,858, but only 62 and 23% of mice gave this response after amphotericin B and ketoconazole therapy, respectively. These data suggest that UK-49,858 may be of value in the treatment of systemic and gastrointestinal infections due to C. albicans in humans.

Activity of UK-49,858, a bis-triazole derivative, against experimental infections with Candida albicans and Trichophyton mentagrophytes.

The therapeutic potential of UK-49,858, a difluorophenyl bis-triazole derivative, has been assessed by evaluating its activity against systemic infections with Candida albicans in normal mice and rats and in mice with impaired defence mechanisms, against vaginal infections with C. albicans in mice, and against dermal infections with Trichophyton mentagrophytes in guinea pigs. Orally administered ketoconazole was used as a comparative agent throughout, and parenterally administered amphotericin B was included in the study of C. albicans systemic infection in normal mice. The activity of UK-49,858 given orally to mice or rats infected systemically with C. albicans was far superior to that of ketoconazole. In addition, UK-49,858 showed activity comparable to that of amphotericin B when given parenterally, although the latter gave more prolonged protection. UK-49,858 was also effective orally in curing experimental candidal vaginitis in mice and trichophytosis in guinea pigs, against which it was approximately 10 times more active than ketoconazole. These data suggest that UK-49,858 may be of value in the treatment of both C. albicans and dermatophyte fungal infections in man.