Update on antifungals targeted to the cell wall: focus on beta-1,3-glucan synthase inhibitors.

Currently available antifungal drugs for serious infections are either fungistatic and vulnerable to resistance (azoles) or fungicidal but toxic to the host (polyenes). Cell wall-acting antifungals are inherently selective and fungicidal, features that make them particularly attractive for clinical development. Three classes of such compounds, targeted respectively to chitin synthase (nikkomycins), beta-1,3-glucan synthase (echinocandins) and mannoproteins (pradimicins/benanomicins), have entered clinical development. While nikkomycins and pradimicins/benanomicins are no longer in development, echinocandins have emerged as potentially clinically useful and three compounds, caspofungin (MK-991, L-743,872), micafungin (FK-463) and anidulafungin (LY-303366) are in late clinical development (Phase II and III).

Antifungals targeted to sphingolipid synthesis: focus on inositol phosphorylceramide synthase.

Currently available antifungal drugs for serious infections have essentially two molecular targets, 14alpha demethylase (azoles) and ergosterol (polyenes). The former is a fungistatic target, vulnerable to resistance development; the latter, while a fungicidal target, is not sufficiently different from the host to ensure high selectivity. Antifungals in clinical development have a third molecular target, beta-1,3-glucan synthase. Drugs aimed at totally new targets are required to increase our chemotherapeutic options and to forestall, alone or in combination chemotherapy, the emergence of drug resistance. Sphingolipids, essential membrane components in eukaryotic cells, but distinct in mammalian and fungal cells, present an attractive new target. Several natural product inhibitors of sphingolipid biosynthesis have been discovered in recent years, some of which act at a step unique to fungi and have potent and selective antifungal activity.

Inhibition of inositol phosphorylceramide synthase by aureobasidin A in Candida and Aspergillus species.

Inositol phosphorylceramide (IPC) synthase is an enzyme common to fungi and plants that catalyzes the transfer of phosphoinositol from phosphatidylinositol to ceramide to form IPC. The reaction is a key step in fungal sphingolipid biosynthesis and the target of the antibiotics galbonolide A, aureobasidin A, and khafrefungin. As a first step toward understanding the antifungal spectrum of IPC synthase inhibitors, we examined the sensitivity of IPC synthase to aureobasidin A in membrane preparations of Candida species (Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei) and Aspergillus species (Aspergillus fumigatus, A. flavus, A. niger, and A. terreus). As expected, preparations from the five Candida species, all exquisitely susceptible to aureobasidin A (MICs, <2 microgram/ml), had IPC synthase activity (specific activity, 50 to 400 pmol/min/mg of protein) sensitive to aureobasidin A (50% inhibitory concentrations [IC(50)s], 2 to 4 ng/ml). Surprisingly, preparations from the four Aspergillus species, including A. fumigatus and A. flavus, which are intrinsically resistant to aureobasidin A (MICs, >50 microgram/ml), had IPC synthase activity (specific activity, 1 to 3 pmol/min/mg of protein) also sensitive to aureobasidin A (IC(50)s, 3 to 5 ng/ml). The mammalian multidrug resistance modulators verapamil, chlorpromazine, and trifluoperazine lowered the MIC of aureobasidin A for A. fumigatus from >50 microgram/ml to 2 to 3 microgram/ml, suggesting that the resistance of this major fungal pathogen is the result of increased efflux.

Inhibition of yeast inositol phosphorylceramide synthase by aureobasidin A measured by a fluorometric assay.

Inositol phosphorylceramide synthase (IPC synthase) is an essential and unique enzyme in fungal sphingolipid biosynthesis and is the target of the cyclic nonadepsipeptide antibiotic aureobasidin A. As a first step towards understanding the mechanism of aureobasidin A inhibition, we developed a fluorometric HPLC assay for IPC synthase using the Saccharomyces cerevisiae enzyme and the fluorescent substrate analog 6-[N-(7-nitro-2,1, 3-benzoxadiazol-4-yl)amino]-hexanoyl ceramide (C(6)-NBD-cer). The kinetic parameters for C(6)-NBD-cer were comparable to those for the synthetic substrate N-acetylsphinganine used previously. Aureobasidin A acted as a tight-binding, non-competitive inhibitor with respect to C(6)-NBD-cer and had a K(i) of 0.55 nM.

Antifungals: mechanism of action and resistance, established and novel drugs.

Serious fungal infections, caused mostly by opportunistic species, are increasingly common in immunocompromised and other vulnerable patients. The use of antifungal drugs, primarily azoles and polyenes, has increased in parallel. Yet, established agents do not satisfy the medical need completely: azoles are fungistatic and vulnerable to resistance, whereas polyenes cause serious host toxicity. Drugs in clinical development include echinocandins, pneumocandins, and improved azoles. Promising novel agents in preclinical development include several inhibitors of fungal protein, lipid and cell wall syntheses. Recent advances in fungal genomics, combinatorial chemistry, and high-throughput screening may accelerate the antifungal discovery process.

Fungal diagnostics, pathogenesis and chemotherapy symposia.

This report focuses on the symposia related to fungal diagnostics, pathogenesis and antifungal chemotherapy, with emphasis on new developments of established agents and new agents in preclinical and clinical development.

Antifungals targeted to the cell wall.

Serious fungal infections are increasingly common in immunocompromised patients and existing antifungals do not completely satisfy the medical need. The latter have either considerable toxicity, e.g., amphotericin, which is, however, less toxic in lipid formulations, or have limited activity, e.g., azoles. Cell wall acting antifungals are inherently selective and fungicidal; two classes of compounds--nikkomycin Z targeted at chitin synthase, and echinocandin LY 303366 and pneumocandin L-743,872 targeted at alpha-1,3-glucan synthase--are currently in clinical development.

Mammalian microsomal and soluble Ras-processing peptidase activities are distinct.

Microsomal and soluble peptidases from bovine liver and pig brain hydrolyze the farnesylated, Ras-based CAAX peptide [3H]Ac-fCVIM-OH. However, they differ in their sensitivity to substrate-based inhibitors, sulfhydryl and chelating agents, pH and ionic strength optima, and stability. The microsomal activity was exquisitely sensitive to the substrate-based inhibitor Boc-fC[CH2]VIM-OH, moderately sensitive to the sulfhydryl agent pCMB, but insensitive to NEM and the metal-chelating agent o-phenanthroline. The soluble activity was insentive to Boc-fC[CH2]VIM-OH, but very sensitive to pCMB, NEM and o-phenanthroline, suggesting it to be the previously reported (Biochem. Biophys. Res. Commun. 198, 787-794 (1994)) zinc metallopeptidase. The microsomal activity is most likely to be a cysteine peptidase involved in the post-translational processing of Ras proteins.

Inhibitors of farnesyltransferase and Ras processing peptidase.

Four analogs of the carboxy terminus of unprocessed p21Ras protein were evaluated as inhibitors of the p21Ras processing farnesyltransferase and peptidase. While three showed no crossover of inhibitory activity between the enzymes, the fourth (a naphthyl-substituted peptide) inhibited both farnesyltransferase and peptidase, with IC50s of 16 microM and 3 microM, respectively. Such inhibition of more than one step of Ras processing may complicate assessment of the mode of action for some inhibitors of Ras processing peptidase.

The fungal cell wall as a drug target.

Fungal infections are increasingly common and, in certain vulnerable patients, can be serious and even life threatening. The fungal cell wall, a structure with no mammalian counterpart, presents an attractive therapeutic target. Inhibitors of the synthesis of one cell-wall component, beta-(1,3)-glucan, are currently under development as antifungal and antipneumocystis agents.

Low-level resistance to the cephalosporin 3'-quinolone ester Ro 23-9424 in Escherichia coli.

Four spontaneous, single-step mutants of Escherichia coli K-12 resistant to low levels of the cephalosporin 3'-quinolone ester Ro 23-9424 were isolated at a frequency of 10(-10) to 10(-11) mutants per CFU plated. The mutants were cross-resistant to both cephalosporin (cefotaxime) and quinolone (fleroxacin) components. Accordingly, they had altered porins and replicative DNA biosynthesis resistant to fleroxacin. There was no increase in beta-lactamase activity when tested with nitrocephin, and the penicillin-binding protein profiles were normal.

A radiometric assay for Ras-processing peptidase using an enzymatically radiolabeled peptide.

A simple and sensitive radiometric assay for the peptidase involved in the post-translational processing of p21ras proteins at the carboxy-terminal Cys-aliphatic-aliphatic--any amino acid (CAAX) motif is described. An isoprenylated tetrapeptide substrate, N-acetyl-S-[3H]farnesyl-Cys-Val-Ile-Ser-OH (22-27 Ci/mmol), was synthesized from N-acetyl-Cys-Val-Ile-Ser-OH and commercial [3H]farnesyl pyrophosphate via farnesyltransferase. The isoprenylated tetrapeptide was then used at a concentration (0.3 microM) well below Km (6 microM) in assays with a microsomal preparation of Ras-processing peptidase from bovine liver. Under assay conditions, the peptidase reaction followed first order kinetics with respect to the substrate, allowing the IC50 values for alternative substrates and inhibitors to approximate Km and Ki values, respectively. In a further simplification, substrate and N-acetyl-S-[3H]farnesyl-Cys-OH product were separated by thin-layer chromatography on silica gel plates using chloroform:acetic acid:methanol:acetone (60:5:10:20, v/v) as solvents. The assay does not require costly, specialized equipment and provides easy means for screening potential substrates and inhibitors of Ras-processing peptidase.

beta-Lactamase hydrolysis of cephalosporin 3'-quinolone esters, carbamates, and tertiary amines.

The beta-lactam hydrolysis of five cephalosporin 3'-quinolones (dual-action cephalosporins) by three gram-negative beta-lactamases was examined. The dual-action cephalosporins tested were the ester Ro 23-9424; the carbamates Ro 25-2016, Ro 25-4095, and Ro 25-4835; and the tertiary amine Ro 25-0534. Also tested were cephalosporins with similar side chains (cefotaxime, desacetylcefotaxime, cephalothin, cephacetrile, and Ro 09-1227 [SR 0124]) and standard beta-lactams (penicillin G, cephaloridine). The beta-lactamases used were the plasmid-mediated TEM-1 and TEM-3 enzymes and the chromosomal AmpC. The cephacetrile-related compounds Ro 25-4095 and Ro 25-4835 were hydrolyzed by all three beta-lactamases with catalytic efficiencies (relative to penicillin G) ranging from approximately 5 (TEM-1, AmpC) to approximately 25 (TEM-3). The cephalothin-related Ro 25-2016 was also hydrolyzed by all three beta-lactamases, particularly the AmpC enzyme (relative catalytic efficiency, 110). The cefotaxime-related compounds Ro 25-0534 and Ro 23-9424 were hydrolyzed to any significant extent only by the TEM-3 enzyme (relative catalytic efficiencies, 1.2 and 4.7, respectively.

Dual-action cephalosporins incorporating a 3'-tertiary-amine-linked quinolone.

We have previously reported that linking quinolones to the cephalosporin 3'-position through an ester bond, a carbamate function, or a bond through a quaternary nitrogen produced cephalosporins with a dual mode of antibacterial action. We now describe a new class of dual-action cephalosporins, with greater chemical stability than those previously reported, in which the basic nitrogen of ciprofloxacin is bonded directly to the 3'-cephalosporin position, i.e., the two moieties are linked through a tertiary amine function. These compounds have demonstrated potent activity against a broad spectrum of Gram-positive and Gram-negative bacteria, including beta-lactam-resistant strains.

Porins and lipopolysaccharide of Escherichia coli ATCC 25922 and isogenic rough mutants.

The lipopolysaccharide and porin profile of Escherichia coli ATCC 25922, a smooth strain commonly used in antibiotic susceptibility testing, and five isogenic rough mutants was examined. The lipopolysaccharide of the parent strain had the characteristic ladder pattern on polyacrylamide gels, while that of the mutants appeared similar to chemotypes Ra and Rc of Salmonella typhimurium with some changes in chemical composition. Of the porins, OmpC appeared markedly reduced in the parent strain while OmpF appeared markedly reduced in the mutants. In addition, a new outer-membrane protein of size intermediate to that of OmpC and OmpF was detected in all mutants. Neither parent nor mutants were susceptible to the LPS core-specific P1 phage or the porin-specific PA2 and K20 phages.

Mechanisms of action of cephalosporin 3'-quinolone esters, carbamates, and tertiary amines in Escherichia coli.

Cephalosporin 3'-quinolone esters, carbamates, and tertiary amines are potent antibiotics whose antibacterial activities reflect the action of both the beta-lactam and the quinolone components. The biological properties of representative compounds from each class were compared in Escherichia coli. All compounds bound to the essential PBP 3, inhibited DNA gyrase, and caused filamentation in growing cells. To distinguish between cephalosporin- and quinolone-induced filaments, nucleoid segregation was also examined, as quinolones disrupt nucleoid segregation while the beta-lactams do not (N. H. Georgopapadakou and A. Bertasso, Antimicrob. Agents Chemother. 35:2645-2648, 1991). The cephalosporin quinolone esters Ro 23-9424 and Ro 24-6392, at concentrations causing filamentation in E. coli ATCC 25922, did not affect nucleoid segregation after 1 h of incubation (cephalosporin response) but did not affect it after 2 h (quinolone response), indicating the release of free quinolone. Accordingly, only the quinolone response was produced in a strain possessing TEM-3, an expanded-spectrum beta-lactamase. The cephalosporin carbamate Ro 24-4383 and the tertiary amine Ro 24-8138 produced a quinolone response in E. coli ATCC 25922, though they produced a cephalosporin response in a quinolone-resistant strain. Carbamate and tertiary amine linkages are chemically more stable than the ester linkage, and both cephalosporin 3'-quinolone carbamates and tertiary amines are more potent inhibitors of DNA gyrase than are the corresponding esters. The results suggest that, while intact cephalosporin 3'-quinolone esters act as cephalosporins, carbamates and amines may possess both cephalosporin and quinolone activity in the intact molecule.