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Over the past two decades, the occurrence of life-threatening fungal infections in immunocompromised patients such as cancer patients has drastically increased. Although Aspergillus and Candida spp. represent the most common causes of these infections, an emerging number of other organisms including Cryptococcus neoformans and species of Trichosporon have been implicated (14, 30, 32). Existing therapies include flucytosine and polyene and azole antifungals such as amphotericin B and fluconazole (28). However, these antifungals are limited by host toxicities (30) and the emergence of drug resistance (3, 9, 13, 33). Thus, the development of new antifungal agents with potent fungicidal activities and new cellular targets is critical.

Dolastatin 10 (dolavaline-valine-dolaisoleuine-dolaproine-dolaphenine) (19), a unique linear peptide (Fig. 1A), was originally isolated from the Indian Ocean sea hare (Dolabella auricularia) (18). The development of an efficient synthetic route for this peptide (20, 22) facilitated investigation of its remarkable cytostatic and antineoplastic activities (23), as well as the synthesis of numerous antitumor-active structural modifications (21, 22). Dolastatin 10 is undergoing phase I and II cancer clinical trials, and its mammalian tubulin-binding activity has been described in detail (for a review, see reference 24). Briefly, the peptide inhibits mammalian tubulin polymerization and the associated GTP hydrolysis (1) and acts as a noncompetitive inhibitor of vincristine and vinblastine (2).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Structures of dolastatin 10 (A) and auristatin PHE (B).

Recently, the antifungal spectrum of dolastatin 10 was reported (27). Against a limited number of yeasts and filamentous fungi, the compound had selective activity against C. neoformans. Included in the previous report (27) was the structural modification dolavaline-valine-dolaisoleuine-dolaproine phenylalanine-methyl ester (auristatin PHE) (25, 26) (Fig. 1B), which, in addition to extremely low MICs for C. neoformans, was fungicidal (as determined by minimal fungicidal concentration [MFC]/MIC ratios and time-kill experiments), was largely unaffected by pH variations, and had enhanced activity in the presence of human serum. Auristatin PHE was thus selected for further study. We report here on its spectrum of activity, the duration of its postantifungal effect (PAFE), and its effect on cryptococcal budding.

Dolastatin 10 and auristatin PHE were synthesized as described previously (20, 22, 25, 26) and were stored desiccated in the dark. Prior to each experiment, the peptides were reconstituted in a small volume of sterile dimethyl sulfoxide (DMSO) and were then diluted in growth medium to the appropriate concentration. Antifungal susceptibility testing of yeasts was performed by the reference broth microdilution assay (BMA) outlined by NCCLS (17). Susceptibility testing of filamentous fungi was performed by BMA according to a proposed standardized procedure (8), with minor modifications. Filamentous fungi were grown on potato dextrose agar (PDA) slants at 35°C (Paecilomyces lilacinus at 30°C) for 6 days to induce conidium and sporangiospore formation. Fungal slants were covered with 1 ml of sterile 0.85% NaCl (Aspergillus flavus, Aspergillus niger, P. lilacinus, Rhizopus nigricans, Rhizopus oligosporus) or 0.05% Tween 80 (Aspergillus fumigatus, Aspergillus nidulans) and probed with a sterile Pasteur pipette. After the mixture was transferred to a sterile microcentrifuge tube, heavy particles were allowed to settle for 10 min. The upper homogeneous suspension was removed, adjusted spectrophotometrically, and diluted in morpholine propanesulfonic acid (MOPS)-buffered RPMI 1640 medium to yield final inocula of 0.5 × 10³ to 2.5 × 10³ CFU/ml. Susceptibility testing of yeasts and filamentous fungi was performed in microtiter plates containing twofold dilutions of the antifungal compounds in RPMI 1640 medium buffered to pH 7.0 with 0.165 M MOPS. For susceptibility testing of Malassezia furfur, MOPS-buffered RPMI 1640 medium was supplemented with 2% olive oil. One drug-free well containing an equivalent volume of DMSO served as a turbidity control, and one well containing medium only served as a sterility control. The microtiter plates were incubated without agitation in a moist chamber at 25°C (Bulleromyces albus, Cryptococcus albidus ATCC 10666, Cryptococcus ater), 30°C (Blastoschizomyces capitatus, C. albidus ATCC 34140 and ATCC 66030, Cryptococcus humicolus, Cryptococcus laurentii, Cryptococcus uniguttulatus, Filobasidium uniguttulatum, Kluyveromyces spp., M. furfur, Rhodotorula spp., Trichosporon spp.), or 35°C (Candida spp., C. neoformans, Geotrichum candidum, Pichia anomala, Saccharomyces cerevisiae, Aspergillus spp., P. lilacinus, Rhizopus spp.). MICs were read after 72 h for B. albus, B. capitatus, Cryptococcus spp., F. uniguttulatum, M. furfur, P. anomala, Rhodotorula spp., and Trichosporon spp. and after 48 h for all genera. The MIC was defined as the lowest concentration of drug that inhibited all visible growth of the test organism (optically clear). No trailing was observed. MFCs were determined by subculture of 100 µl from each negative well and from the positive growth control well of the BMA series onto drug-free plates (PDA for all filamentous fungi, Emmon's Sabouraud dextrose agar [SDA] for B. capitatus and Trichosporon spp., SDA supplemented with 2% olive oil for M. furfur, and SDA for all other genera). The plates were incubated at the appropriate temperature (see above) for 48 h before MFCs were read. The MFC was defined as the lowest drug concentration that completely inhibited growth on plates.

BMAs with a large panel of fungi, with an emphasis on basidiomycetes, revealed that auristatin PHE had narrow-spectrum antifungal activity against C. neoformans and several species of Trichosporon (Table 1). The peptide was very active against fluconazole-resistant clinical isolates of C. neoformans, with MICs ranging from 0.0156 to 0.5 µg/ml. MICs were considerably higher for Trichosporon spp., ranging from 4 to >64 µg/ml. MFC/MIC ratios indicated that auristatin PHE was fungicidal for C. neoformans but not Trichosporon spp. Interestingly, species phylogenetically more closely related to C. neoformans than Trichosporon, for example, B. albus and C. laurentii (10, 11), were not susceptible to auristatin PHE in BMAs. In tests with a limited number of species, dolastatin 10 was previously reported to have selective activity against C. neoformans (27). With a large number of species closely related to C. neoformans, we have now confirmed this result for dolastatin 10 (Table 1).

Pharmacodynamic parameters such as the PAFE, or the suppression of fungal growth that persists after a short exposure to an antifungal agent (4), yield information that contributes to the determination of optimal dosing regimens. The mechanism by which antibiotics suppress growth after their removal is likely the result of drug-induced nonlethal damage and/or persistence at the target site (5, 31). The duration of the PAFE presumably reflects the time required for repair or regeneration of drug-induced sublethal damage (e.g., resynthesis of nucleic acids, cellular proteins, and cell wall material) or the time necessary for a drug to dissociate and diffuse from its target (if reversible). The PAFEs for auristatin PHE and dolastatin 10 were compared using C. neoformans ATCC 90112. Early-log-phase cultures were exposed to four and eight times the broth microdilution MIC of auristatin PHE for 30 min, 45 min, 1 h, and 2 h and to four and eight times the broth microdilution MIC of dolastatin 10 for 1 h at 35°C in a shaking incubator. Control cultures contained an equivalent volume of DMSO. Following exposure, the peptides were removed by three cycles of centrifugation (5 min at 12,000 × g), followed by washing in 0.85% NaCl. After the final centrifugation, the yeast pellets were resuspended in fresh, warm RPMI 1640 medium to yield a 10¹ dilution (washing plus dilution was previously shown to remove compound [data not shown]). Nonexposed control cultures (containing DMSO) were processed identically. Cultures were returned to the 35°C shaking incubator, and samples were removed every 2 h for dilution plating.

In studies that have examined the influence of human serum on the PAFE of auristatin PHE, both drug exposure and reincubation after drug removal were performed in RPMI 1640 medium supplemented with 10% fresh or 10% heat-inactivated (30 min at 56°C) normal human serum (Lampire). Exposure to auristatin PHE was for 1 h at four and eight times the MIC determined by BMA in the presence of 10% serum. Control cultures also contained 10% serum. The samples were vortexed vigorously before dilution plating and were examined microscopically throughout the experiments to ensure that serum was not promoting clumping of cells. No clumping was found, confirming previous studies with C. neoformans (CDC 9759) in the presence of 5% non-heat-inactivated and heat-inactivated human serum (16). Higher serum concentrations were not tested, as they inhibited the growth of C. neoformans. Such inhibitory effects of human serum on the multiplication of C. neoformans have been shown to be due to a donor-independent and heat-stable macromolecular component but not to albumin and globulin (16). PAFEs for all experiments were determined by the formula PAE = T  C, where PAE is the postantibiotic effect, T is the time required to achieve 1 log₁₀ growth after drug removal for the antifungal-exposed sample, and C is the corresponding time for the unexposed control sample (4). The time for cultures to increase 1 log was determined on enlarged graphs containing grid lines, and standard errors of the means were calculated from at least two experiments.

The PAFE plateaued after 1 h of exposure to auristatin PHE, with a relatively long, dose-independent PAFE (Table 2). A 2-h exposure yielded similar values. At 1 h of exposure, saturation of a target molecule or a rate-limiting enzyme may occur. C. neoformans exposed to dolastatin 10 for 1 h did not have a measurable PAFE (Table 2). In the presence of 10% human serum, auristatin PHE PAFEs were concentration dependent and prolonged (Table 2). Prolonged PAEs and PAFEs of antimicrobial agents in the presence of human serum have been described (7, 15, 16). Human serum has been reported to increase considerably the PAEs of fluoroquinolones against Staphylococcus aureus, possibly due to the interaction of some serum component with the fluoroquinolones (7). A low-molecular-weight serum component that enhances the anticryptococcal activity of fluconazole has been described (16). Similarly, there may be a synergistic interaction between auristatin PHE and a human serum component(s) that lowers the MIC and that prolongs the PAFE at eight times the MIC. The duration of the PAFE after 1 h of exposure was concentration dependent in the presence of serum only, suggesting an additional mechanism that may relate to binding of serum components.

To investigate possible morphological changes, auristatin PHE-treated C. neoformans cells were analyzed microscopically over time. Early-log-phase cultures of C. neoformans ATCC 90112 in RPMI 1640 medium were exposed to auristatin PHE at four and eight times the broth microdilution MIC. Control cells were exposed to an equivalent volume of DMSO. Cultures were incubated for 6 h at 35°C in a shaking incubator, and samples were aseptically removed every 30 min for microscopic observation. To determine the proportion of cells arrested in the budding stage, a minimum of 200 cell arrangements (single cells and cells with a bud were each counted as one cell arrangement) were counted and described for each sample at each time point. Standard errors of the means were calculated from at least two experiments. Light microscopy was performed with a Nikon inverted microscope equipped with differential interference contrast enhancement and with a Plan-Neofluar ×100/1.4 (oil immersion) objective. Approximately 40% of the cells in early-log-phase cultures exposed to DMSO only (control cultures) were in the early to late budding stages over a 6-h period (Fig. 2). Among the cells treated with auristatin PHE a gradual increase in the percentage of cells found in the budding stage was demonstrated, reaching a peak of 95 to 97% after 240 min of drug exposure. From 240 to 300 min, cells were arrested in the budding stage. The budding stage-arrested cell population consisted almost entirely (~97%) of slightly swollen, large budded cells.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Percentage of C. neoformans ATCC 90112 cells in the budding stage: control cells containing DMSO () and cells treated with auristatin PHE at four times () and eight times () the MIC.

Very little is known about chromosome segregation, nuclear division, or cytokinesis in C. neoformans. The arrest of auristatin PHE-treated cells in the large budded stage may be a consequence of the binding or inhibition of intranuclear and cytoplasmic microtubules. All known inhibitors of nuclear division in yeast also block cell division (12, 29). Nocodazole, for example, completely disassembles cytoplasmic and intranuclear microtubules in S. cerevisiae, leading to inhibition of nuclear and cellular division (12). We are investigating the interaction of auristatin PHE with cryptococcal tubulin using fluorescent antitubulin antibodies and 2,6-diamidinophenylindole (DAPI). Preliminary experiments with DAPI-stained nuclei indicate blocking of nuclear migration and subsequent division in auristatin PHE-treated C. neoformans cells. At present, it is not known if the specificity of auristatin PHE for C. neoformans and Trichosporon spp. is due to similarities in tubulin sequence, enhanced drug uptake, or other mechanisms. Two C. neoformans -tubulin genes, TUB1 and TUB2, have been sequenced; TUB1 encodes the primary tubulin for microtubule assembly (6). The sequence homologies of TUB1 to -tubulins from Schizophyllum commune, A. nidulans, and humans are 84, 81, and 82% respectively, demonstrating that the gene sequence is relatively conserved (6).

In summary, auristatin PHE exhibits properties in vitro which make it an attractive candidate for development as an anticryptococcal agent. The peptide is active at low doses, is fungicidal, has a prolonged PAFE in human serum, and may have a novel fungal target. Protection studies in murine models of cryptococcosis are ongoing. In addition to potential clinical use, auristatin PHE may aid in the description of nuclear and cellular division in Cryptococcus and Trichosporon.