In Vitro Activities and Postantifungal Effects of the Potent Dolastatin 10 Derivative Auristatin PHE

Abstract

The pentapeptide dolavaline-valine-dolaisoleuine-dolaproine-phenylalanine-methyl ester (auristatin PHE) is a derivative of the anticancer drug dolastatin 10 (dolavaline-valine-dolaisoleuine-dolaproine-dolaphenine). Broth microdilution assays with a wide variety of yeast and filamentous fungal species demonstrated the specificity of auristatin PHE for Cryptococcus neoformans and several species of Trichosporon. The duration of the postantifungal effect (PAFE) for C. neoformans was determined for exposure times ranging from 30 min to 2 h. For the derivative, a PAFE was detectable after 45 min of exposure. The effect plateaued after 1 h of exposure, with a PAFE of approximately 6.5 h at four or eight times the auristatin PHE MIC. In contrast, there was no measurable PAFE after 1 h of exposure to dolastatin 10. Human serum greatly prolonged the PAFE of auristatin PHE at eight times the MIC. Auristatin PHE arrested C. neoformans in the budding stage, possibly due to a tubulin-inhibitory action. Auristatin PHE has potential as a narrow-spectrum fungicidal agent and as a probe that can be used to study cryptococcal cell division.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-dolaproinedolaphenine) (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).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...

Peptides: Production, bioactivity, functionality, and applications

“…Several studies have shown that peptides from different sources such as sea hare Turner et al, 1998;Madden et al, 2000;Vaishampayan et al, 2000;Woyke et al, 2001), fish sauce (Thang and Zhao, 2015;Bah et al, 2016), and cod (Xhindoli et al, 2016) were able to block proliferation of carcinogenic cells. However, it seems there is still a long way in order to make commercial use of these potentially potent anticancer peptides to treat different kinds of cancer.…”

Effect of Auristatin PHE on Microtubule Integrity and Nuclear Localization in Cryptococcus neoformans

“…In the fungus Uromyces appendiculatus, dolastatin 10 disrupts microtubules (41). We recently reported that a 4-h exposure to Ն2.6 M auristatin PHE (4 times the MIC, as determined in RPMI medium) resulted in a C. neoformans population consisting almost entirely (ϳ97%) of large-budded cells (50). These results led us to evaluate the effects of auristatin PHE on the microtubule organization of C. neoformans and to compare them to those of a compound known to interfere with the microtubule cytoskeleton in yeast (17,21).…”

“…Auristatin PHE has fungicidal activity against C. neoformans and fungicidal or fungistatic activity against various species of Trichosporon (39,50). Auristatin PHE caused C. neoformans cells to arrest in a large-budded stage (50), suggesting that it might interfere with the microtubule dynamics in a manner similar to that of nocodazole in Saccharomyces cerevisiae (17).…”

Differential Gene Expression in Auristatin PHE-Treated Cryptococcus neoformans

“…Broth microdilution assays (BMAs) with a large panel of fungi, with an emphasis on basidiomycetes, have shown that dolastatin 10 has selective antifungal activity against C. neoformans, and auristatin PHE has selective antifungal activity against C. neoformans and some species of Trichosporon (43,61). Other species in the genus Cryptococcus are not inhibited by high concentrations of these peptides.…”

“…Furthermore, the susceptible organisms may share a unique target that enhances binding and/or interaction with the drug(s). Compared to dolastatin 10, auristatin PHE had lower MICs (43,61), exhibited concentration-dependent killing between the MIC and four times the MIC (43), and demonstrated increased activity as well as prolonged postantifungal effects in the presence of human serum (43,61). Iterative deconvolution in conjunction with an optimized C. neoformans microtubule immunolabeling procedure enabled us to investigate the mechanism of action of auristatin PHE (62).…”