Nonspecific effect of Mycograb on amphotericin B MIC.

Mycograb C28Y is a recombinant human antibody fragment thought to target HSP-90 and potentiate amphotericin B (AMB). Absence of in vivo efficacy led us to reevaluate its in vitro activity. Interactions between AMB and Mycograb were investigated using a checkerboard design. Addition of Mycograb or various unrelated proteins, including human serum, resulted in similar decreases in the MIC of AMB. Potentiation of AMB by Mycograb appears to be a nonspecific protein effect.

A collaborative hit-to-lead investigation leveraging medicinal chemistry expertise with high throughput library design, synthesis and purification capabilities.

High throughput screening (HTS) campaigns, where laboratory automation is used to expose biological targets to large numbers of materials from corporate compound collections, have become commonplace within the lead generation phase of pharmaceutical discovery. Advances in genomics and related fields have afforded a wealth of targets such that screening facilities at larger organizations routinely execute over 100 hit-finding campaigns per year. Often, 10(5) or 10(6) molecules will be tested within a campaign/cycle to locate a large number of actives requiring follow-up investigation. Due to resource constraints at every organization, traditional chemistry methods for validating hits and developing structure activity relationships (SAR) become untenable when challenged with hundreds of hits in multiple chemical families per target. To compound the issue, comparison and prioritization of hits versus multiple screens, or physical chemical property criteria, is made more complex by the informatics issues associated with handling large data sets. This article describes a collaborative research project designed to simultaneously leverage the medicinal chemistry and drug development expertise of the Novartis Institutes for Biomedical Research Inc. (NIBRI) and ArQule Inc.'s high throughput library design, synthesis and purification capabilities. The work processes developed by the team to efficiently design, prepare, purify, assess and prioritize multiple chemical classes that were identified during high throughput screening, cheminformatics and molecular modeling activities will be detailed.

Antifungal drug response in an in vitro model of dermatophyte nail infection.

Despite terbinafine being fungicidal against Trichophyton rubrum in standard NCCLS assays and rapidly accumulating in nails in vivo, onychomycosis patients require prolonged terbinafine treatment to be cured. To investigate this, we developed a more clinically relevant onychomycosis in vitro test model. Human nail powder inoculated with T. rubrum and incubated in liquid RPMI 1640 salt medium, which did not support growth alone, developed extensive and invasive mycelial growth. Antifungal drugs were added at different concentrations and cultures incubated for 1 to 4 weeks. Fungal survival was determined by spreading cultures on PDA plates without drug and measuring CFU after 1 to 4 weeks incubation. Drug activity was expressed as the nail minimum fungicidal concentration (Nail-MFC) required for 99.9% elimination of viable fungus. Terbinafine Nail-MFC was 4 microg/ml after 1 week exposure, decreasing to 1 microg/ml after 4 weeks exposure, much higher than MFCs < or = 0.03 microg/ml determined in standard NCCLS MIC assays. In contrast, other clinically used drugs were unable to kill T. rubrum after 4 weeks incubation in this model. Invasive mycelial growth on nail appears to protect T. rubrum from the cidal action of systemic drugs, thus providing a rationale for the long treatment periods in onychomycosis.

Alpha-substituted hydroxamic acids as novel bacterial deformylase inhibitor-based antibacterial agents.

We report the synthesis and biological activity of analogues of VRC3375 (N-hydroxy-3-R-butyl-3-[(2-S-(tert-butoxycarbonyl)-pyrrolidin-1-ylcarbonyl]propionamide), an orally active peptide deformylase inhibitor. This study explores the structure-activity relationship of various chelator groups, alpha substituents, P(2)' and P(3)' substituents in order to achieve optimal antibacterial activity with minimal toxicity liability.

In vitro susceptibility of Microsporum canis and other dermatophyte isolates from veterinary infections during therapy with terbinafine or griseofulvin.

We investigated the in vitro activity of terbinafine against fresh veterinary isolates of Microsporum canis and the potential of this organism to develop resistance in vivo during oral therapy. Dermatophyte cultures (n = 300) were obtained from naturally infected cats and dogs undergoing oral therapy with terbinafine or griseofulvin. M. canis comprised 92% of isolates; other species included Microsporum gypseum and Trichophyton mentagrophytes. Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of terbinafine and griseofulvin were determined by broth macrodilution assay. Terbinafine was highly active against all three species with MIC90< or =0.03 microg ml(-1), in agreement with published data. However, terbinafine exhibited primary cidal activity against 66% of Microsporum isolates (n = 275) in contrast to the almost complete cidal effect in Trichophyton (n = 18). Griseofulvin was significantly less active than terbinafine (MIC90 = 4 microg ml(-1)) but had a primary cidal action on about 40% of the isolates. The data were analysed for changes in MIC and MFC during the course of therapy, which could be indicative for development of acquired resistance. Oral treatment of 37 animals with terbinafine for up to 39 weeks caused no increase in MIC or MFC of terbinafine, either in individual patients or in the whole group.

Inhibition of chitin synthetase from Saccharomyces cerevisiae by a new UDP-GlcNAc analogue.

The synthesis and biological evaluation of a new UDP-GlcNAc competitor (I), designed to mimic the transition state of the sugar donor in the enzymatic reaction catalysed by chitin synthetase, is described. Compound (I) was found to competitively inhibit chitin synthetase from Saccharomyces cerevisiae with respect to UDP-GlcNAc, but displayed minimal antifungal activity.

Synergistic interaction of terbinafine with triazoles or amphotericin B against Aspergillus species.

The in vitro activity of terbinafine alone and in combination with other antifungal agents was tested against isolates of Aspergillus fumigatus, A. flavus and A. niger. Testing was performed in a modified National Committee for Clinical Laboratory Standards (NCCLS) macrodilution broth assay, and interactions were examined using a checkerboard design. Terbinafine was highly active against Aspergillus isolates (minimum inhibitory concentration [MIC] 0.01 to 2 microg ml(-1)) with a primary fungicidal action (minimum fungicidal concentration [MFC] 0.02 to 4 microg ml(-1)). Amphotericin B was also highly active and cidal as expected (MIC 1 microg ml(-1), MFC 1 to 4 microg ml(-1)). The triazoles itraconazole and voriconazole were highly active but showed a variable degree of cidal activity against the different strains, voriconazole having the more potent cidal activity. Fluconazole had no significant activity (MIC > 128 microg ml(-1)). Drug combinations were tested in the A. fumigatus and A. niger strains. Terbinafine and amphotericin showed an additive to synergistic interaction depending on the isolate. Combinations of terbinafine with itraconazole or voriconazole displayed a potent synergistic interaction and fungicidal activity against all isolates. Surprisingly, fluconazole also potentiated the activity of terbinafine in an additive to synergistic fashion, despite its lack of activity alone. The results suggest potential clinical application of terbinafine in aspergillosis, either alone or in combination with amphotericin or triazoles.

Synthesis of new (difluoromethylphosphono)azadisaccharides designed as bisubstrate analogue inhibitors for GlcNAc:beta-1,4 glycosyltransferases.

We report here the design, synthesis and antifungal evaluation of a new model of bisubstrate analogue inhibitor for glycosyltransferases. The synthetic strategy relies on the reductive amination between the aldehyde derived from an N-allylphosphono-pyrrolidine and an aminosugar.

An evaluation of the in vitro activity of terbinafine.

Terbinafine has previously been shown to be highly active against dermatophytes and many other filamentous fungi. However, its activity against yeasts is controversial, with earlier reports suggesting that it has low activity, while more recent studies demonstrated that terbinafine is effective against yeasts. In this study, the in vitro activity of terbinafine was evaluated against a broad range of fungal isolates. We examined the susceptibility of 100 yeast strains (10 species including Candida albicans, non-C. albicans, fluconazole-susceptible and -resistant candidal strains), and 184 strains of filamentous fungi and dermatophytes (29 species including Aspergillus, Fusarium, Sporothrix, Trichophyton rubrum, T. mentagrophytes, T. tonsurans, Microsporum canis and Epidermophyton floccosum), using the NCCLS M27-A microdilution methodology for yeasts and a modified M38-P methodology for moulds. The endpoint for terbinafine was defined as 80% inhibition compared with the growth control well. The mean yeast and filamentous fungi minimum inhibitory concentration values +/- SEM (in microg ml(-1)) for terbinafine were: 6.60 +/- 0.73 and 1.04 +/- 0.28, respectively. In conclusion, our data suggest that terbinafine, in addition to its potent activity against dermatophytes, is considerably effective against a broad range of yeasts and filamentous fungi in vitro. Therefore, investigations concerning its antifungal activity in vivo against such organisms should be pursued.

In vitro comparison of terbinafine and itraconazole against Penicillium marneffei.

We evaluated terbinafine and itraconazole against 30 isolates of Penicillium marneffei using a modification of the National Committee for Clinical Laboratory Standards broth macrodilution MIC testing protocol for yeasts. The minimal fungicidal concentration (MFC) was determined by plating 100 microl from each MIC drug dilution having no growth onto Sabouraud glucose agar incubated at 30 degrees C. The MFC was the dilution at which growth was absent at 72 h of incubation. The MICs, in micrograms per milliliter, were as follows: terbinafine, 0.03 to 1.0 (geometric mean titer, 0.09); itraconazole, 0.03 to 0.5 (geometric mean titer, 0.04). The MFCs, in micrograms per milliliter, were as follows: terbinafine, 0.03 to 8 (geometric mean titer, 2.60); itraconazole, 0.03 to 8 (geometric mean titer, 2. 45). Primary fungicidal activity (MFC within 2 dilutions of MIC) was observed with terbinafine in eight isolates and with itraconazole in four isolates. The data indicate that terbinafine is active against P. marneffei in vitro and may have a previously unrealized role in the management of infections caused by this fungus.

Multiple amino acid substitutions in lanosterol 14alpha-demethylase contribute to azole resistance in Candida albicans.

Lanosterol 14alpha-demethylase (14DM) is the target of the azole antifungals, and alteration of the 14DM sequence leading to a decreased affinity of the enzyme for azoles is one of several potential mechanisms for resistance to these drugs in Candida albicans. In order to identify such alterations the authors investigated a collection of 19 C. albicans clinical isolates demonstrating either frank resistance (MICs > or = 32 microg ml(-1)) or dose-dependent resistance (MICs 8-16 microg ml(-1)) to fluconazole. In cell-free extracts from four isolates, including the Darlington strain ATCC 64124, sensitivity of sterol biosynthesis to inhibition by fluconazole was greatly reduced, suggesting that alterations in the activity or affinity of the 14DM could contribute to resistance. Cloning and sequencing of the 14DM gene from these isolates revealed 12 different alterations (two to four per isolate) leading to changes in the deduced amino acid sequence. Five of these mutations have not previously been reported. To demonstrate that these alterations could affect fungal susceptibility to azoles, the 14DM genes from one sensitive and three resistant C. albicans strains were tagged at the carboxyl terminus with a c-myc epitope and expressed in Saccharomyces cerevisiae under control of the endogenous promoter. Transformants receiving 14DM genes from resistant strains had fluconazole MICs up to 32-fold higher than those of transformants receiving 14DM from a sensitive strain, although Western blot analysis indicated that the level of expressed 14DM was similar in all transformants. Amino acid substitutions in the 14DM gene from the Darlington strain also conferred a strong cross-resistance to ketoconazole. In conclusion, multiple genetic alterations in C. albicans 14DM, including several not previously reported, can affect the affinity of the enzyme for azoles and contribute to resistance of clinical isolates.

Activity of terbinafine against serious fungal pathogens.

Although primarily indicated for dermatophyte infections, the allylamine terbinafine is active in vitro against a broad spectrum of filamentous and dimorphic fungi, in most cases with a primary fungicidal action. Using the standard NCCLS M27-A assay, recent studies confirmed the high activity of terbinafine against dematiaceous fungi and other medically important moulds such as Aspergillus and Penicillium marneffei. Terbinafine displayed a geometric mean MIC of 1.4 micrograms/ml against Candida albicans (n = 259) and has significant in vitro activity against other species of Candida, Cryptococcus, Trichosporon and Blastoschizomyces. As an approach to treatment of refractory infections, interactions of terbinafine with azoles and other agents are being investigated. Terbinafine was synergistic with azoles (and in some cases amphotericin B) against Candida species, Trichosporon beigelii, Aspergillus species, Pseudallescheria boydii and Scopulariopsis brevicaulis, some of which were unresponsive to any drug used singly. Terbinafine combined with fluconazole showed potent synergy against fluconazole- and multidrug-resistant C. albicans isolates. In conclusion, recent in vitro data suggest that terbinafine, either alone or in combination with other antifungal drugs, has potential in the therapy of a range of more severe fungal infections, in addition to its current widespread use against dermatomycoses.

Antifungal agents.

At this year's ICAAC Meeting, new data on approximately 20 different antifungal agents were presented, while no new agents were disclosed. Drugs in late development include the triazoles, voriconazole (Pfizer Ltd) and Sch-56592 (Schering-Plough Corp), and the echinocandins, caspofungin (Merck & Co Inc) and FK-463 (Fujisawa Pharmaceutical Co Ltd). In contrast to previous years, presentations on these and earlier developmental compounds were relatively modest in scope, with few significant new data. Little new information appeared on the most recent novel class of agents, the sordarins (Glaxo Wellcome plc). Early clinical results were presented for FK-463, showing acceptable tolerability and dose-dependent efficacy in AIDS-associated esophageal candidiasis. A new liposomal formulation of nystatin (Nyotran; Aronex Pharmaceuticals Inc) was shown to be equivalent to conventional amphotericin B in empiric therapy of presumed fungal infection in neutropenic patients, but with reduced toxicity. Intravenous itraconazole (Janssen Pharmaceutica NV) was an effective prophylactic therapy in invasive pulmonary aspergillosis, while oral itraconazole was discussed as a treatment for fungal infection in heart and liver transplant patients. The allylamine compound, terbinafine (Novartis AG), showed good clinical efficacy against fungal mycetoma, a serious tropical infection. A major highlight was the first presentation of inhibitors of fungal efflux pumps as a strategy for overcoming resistance. MC-510027 (milbemycin alpha-9; Microcide Pharmaceuticals Inc) and its derivatives, potentiated the antifungal activity of triazoles and terbinafine in a number of Candida spp. Another pump inhibitor, MC-005172 (Microcide Pharmaceuticals Inc) showed in vivo potentiation of fluconazole in a mouse kidney infection model. Microcide Pharmaceuticals Inc also presented inhibitors of bacterial efflux pumps.

Activity of terbinafine against serious fungal pathogens.

Although primarily indicated for dermatophyte infections, the allylamine terbinafine is active in vitro against a broad spectrum of filamentous and dimorphic fungi, in most cases with a primary fungicidal action. Using the standard NCCLS M27-A assay, recent studies confirmed the high activity of terbinafine against dematiaceous fungi and other medically important moulds such as Aspergillus and Penicillium marneffei. Terbinafine displayed a geometric mean MIC of 1.4 µg/ml against Candida albicans (n = 259) and has significant in vitro activity against other species of Candida, Cryptococcus, Trichosporon and Blastoschizomyces. As an approach to treatment of refractory infections, interactions of terbinafine with azoles and other agents are being investigated. Terbinafine was synergistic with azoles (and in some cases amphotericin B) against Candida species, Trichosporon beigelii, Aspergillus species, Pseudallescheria boydii and Scopulariopsis brevicaulis, some of which were unresponsive to any drug used singly. Terbinafine combined with fluconazole showed potent synergy against fluconazole- and multidrug-resistant C. albicans isolates. In conclusion, recent in vitro data suggest that terbinafine, either alone or in combination with other antifungal drugs, has potential in the therapy of a range of more severe fungal infections, in addition to its current widespread use against dermatomycoses.

In vitro activities of terbinafine against cutaneous isolates of Candida albicans and other pathogenic yeasts.

Terbinafine is active in vitro against a wide range of pathogenic fungi, including dermatophytes, molds, dimorphic fungi, and some yeasts, but earlier studies indicated that the drug had little activity against Candida albicans. In contrast, clinical studies have shown topical and oral terbinafine to be active in cutaneous candidiasis and Candida nail infections. In order to define the anti-Candida activity of terbinafine, we tested the drug against 350 fresh clinical isolates and additional strains by using a broth dilution assay standardized according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) M27-A assay. Terbinafine was found to have an MIC of 1 microg/ml for reference C. albicans strains. For 259 clinical isolates, the MIC at which 50% of the isolates are inhibited (MIC50) of terbinafine was 1 microg/ml (fluconazole, 0.5 microg/ml), and the MIC90 was 4 microg/ml (fluconazole, 1 microg/ml). Terbinafine was highly active against Candida parapsilosis (MIC90, 0.125 microg/ml) and showed potentially interesting activity against isolates of Candida dubliniensis, Candida guilliermondii, Candida humicola, and Candida lusitaniae. It was not active against the Candida glabrata, Candida krusei, and Candida tropicalis isolates in this assay. Cryptococcus laurentii and Cryptococcus neoformans were highly susceptible to terbinafine, with MICs of 0.06 to 0.25 microg/ml. The NCCLS macrodilution assay provides reproducible in vitro data for terbinafine against Candida and other yeasts. The MICs for C. albicans and C. parapsilosis are compatible with the known clinical efficacy of terbinafine in cutaneous infections, while the clinical relevance of its activities against the other species has yet to be determined.

Fluvastatin enhances receptor-stimulated intracellular Ca2+ release in human keratinocytes.

We analyzed the effect of isoprenoid depletion by fluvastatin on bradykinin (BK)- and epidermal growth factor (EGF)-mediated Ca2+ mobilization and prostaglandin E2 production, in the human keratinocyte cell line HaCaT. BK and EGF stimulated Ca2+ mobilization in an agonist-dependent manner. The synthesis of prostaglandin E2 paralleled the level of Ca2+ mobilization induced by BK and EGF. Treatment with fluvastatin increased the EGF-promoted but not the BK-promoted Ca2+ mobilization and prostaglandin E2 production in Ca(2+)-containing medium. In the absence of extracellular Ca2+, fluvastatin treatment led to an increase in intracellular Ca2+ release by both agonists. This effect was abolished by depleting the intracellular pool of Ca2+ with thapsigargin. Our findings showed that the intracellular Ca2+ release was dependent on the metabolism of mevalonate and that the Ca2+ mobilization modulated prostaglandin E2 synthesis in human keratinocytes.

Bradykinin induces actin reorganization and enhances cell motility in HaCaT keratinocytes.

In HaCaT keratinocytes bradykinin-triggered actin reorganization was inhibited by quinacrine, a phospholipase A2 inhibitor, and restored by addition of arachidonic acid. Bradykinin-induced actin breakdown and cortical actin formation were respectively prevented by indomethacin, a cyclooxygenase inhibitor, and nordihydroguaiaretic acid, a lipoxygenase inhibitor. Addition of prostaglandins or leukotrienes, respectively, reversed the effects of inhibitors. This suggested a crucial role for a cyclooxygenase product in actin depolymerization and for a lipoxygenase product in cortical actin formation. Furthermore, we found that bradykinin stimulated HaCaT keratinocyte migration. This event was blocked by quinacrine, indomethacin or nordihydroguaiaretic acid, and restored by addition of prostaglandins or leukotrienes, respectively. We also showed that genistein, a tyrosine kinase inhibitor, inhibited HaCaT cell locomotion. In conclusion, bradykinin modulated actin reorganization and cell motility in keratinocytes, probably by a mechanism involving arachidonic acid metabolites and a tyrosine kinase activity.

Cloning and expression of squalene epoxidase from the pathogenic yeast Candida albicans.

The allylamine antimycotic terbinafine prevents the formation of sterols by specifically inhibiting squalene epoxidase (SE). The biological and biochemical action of terbinafine on fungal pathogens has been well investigated, but little is known at the molecular level. Here we report the cloning, sequencing and expression of the target of terbinafine from the major pathogen Candida albicans. A C. albicans genomic DNA library was constructed in gamma ZAP Express and screened with a DNA fragment obtained by polymerase chain reaction with two primers designed from sequences common to Saccharomyces cerevisiae and rodent SEs. Two types of clone, approximately 3.9 kbp and 4.1 kbp, were isolated. Both contained an identical open reading frame of 1488 nucleotides, while a few sequence differences were found in the flanking regions, suggesting an allelic heterogeneity. The deduced protein sequence of C. albicans SE, 496 amino acids (55324 Da), is 54% and 41% identical to those of S. cerevisiae and rat, respectively. A 1.8-kb transcript was observed on Northern blots of C. albicans mRNA. Polyclonal antibodies, raised against an internal peptide of C. albicans SE, recognized a protein associated with the particulate fraction of M(r) 55000 on Western blots of C. albicans extracts. C. albicans SE was overexpressed in S. cerevisiae with the expression vector pYES2. In homogenates from S. cerevisiae overexpressing the C. albicans protein SE activity was 10-fold higher than the endogenous activity from controls.