Identification of compounds having fungal antimitotic activity.
A library of 4,464 compounds that inhibit the growth of at least one of four fungal species (S. cerevisiae, C. albicans, A. fumigatus, or Cryptococcus neoformans) was screened for fungal antimitotic activity. An S. cerevisiae strain that is selectively hypersensitive to the benzimidazole class of tubulin-depolymerizing compounds was used to perform the antimitotic screen. SC1712, which carries a cold-sensitive α-tubulin mutation (tub1-1712), was found to be 8-fold and 16-fold supersensitive to benomyl and nocodazole, respectively, but was not hypersensitive to a variety of other antifungal agents.
The SC1712 and the wild-type parent were exposed in parallel to 4, 8, 16, and 32 μg of each compound screened/ml. Seven compounds (0.16% of the library) inhibited growth of the mutant, but not an isogenic wild-type strain, at two or more of the concentrations tested and were selected for further analysis. One of these seven compounds, MC-291,734, is a benzimidazole carbamate with a structure similar to the known antifungal agent carbendazim (Fig. ). The other six compounds were not structurally related to known tubulin-binding compounds and thus were considered potentially novel antimitotic agents.
In order to further evaluate the compounds for putative antimitotic activity, their ability to arrest the tubulin-dependent process of nuclear division was examined. Table shows the percentages of cells treated with growth-arresting concentrations of the compounds that accumulate with a single nucleus and large-budded morphology indicative of nuclear division arrest. All seven compounds identified by tub1-1712 hypersensitivity screening were capable of effectively arresting the nuclear division cycle of the tub1-1712 strain, while in contrast, 80 control compounds with no known effect on tubulin did not arrest nuclear division. Only one of the compounds identified in the screen, MC-305,904, was able to arrest the cell cycle of the wild-type parent strain. The lack of wild-type cell cycle arrest for the other compounds was attributed to non-tubulin-related modes of action that predominate at the high compound concentrations required to inhibit wild-type growth; poor solubility of the compounds in aqueous media may underlie this phenomenon. Alternatively, some compounds may interact more effectively with the mutant tubulin than with the normal protein.
tub1-1712 mutant hypersensitivity and cell cycle arrest of compound-treated S. cerevisiae
Several observations served to focus attention on MC-305,904. First, it is a relatively potent inhibitor of yeast cell growth. Second, it can arrest nuclear division of the wild-type strain. Third, the collection of screening hits contains a close structural analog, MC-253,166. Both MC-305,904 and MC-253,166 were poorly soluble in aqueous solution. A number of close structural analogs were synthesized that overcame this liability (e.g., solubility of MC-305,904 was 5 μg/ml in 50 mM phosphate buffer at pH 7 compared with 262 μg/ml for MC-06,341) and allowed more accurate measurements in further studies described below.
MC-305,904 series mode of action and fungal tubulin specificity.
Compounds that disrupt mammalian tubulin function are typically highly cytotoxic, particularly against sensitive cell lines such as the human lymphoma line K562 (18
). Evaluating the fungal specificity of compounds in this study was begun by comparing the concentrations of each compound resulting in fungal growth inhibition, K562 cytotoxicity, and inhibition of bovine brain tubulin polymerization in vitro (Table ). Two compounds with relatively poor antifungal activity, MC-226,728 and MC-291,734, are highly toxic to the human cell line (<0.2 μM) and are potent inhibitors of mammalian tubulin polymerization (<10 μM). In contrast, two members of the MC-305,904 series (MC-305,904 and MC-06,341) were relatively potent in inhibiting fungal growth, were relatively noncytotoxic toward cultured human cells, and lacked measurable activity against mammalian tubulin in vitro. These compounds were selected for further evaluation. Other compounds, such as MC-239,300, MC-247,136, or MC-253,637, were not studied further due to relatively poor antifungal activity.
Comparative antifungal, cytotoxic, and mammalian tubulin polymerization inhibitory concentrations for tub1-1712 mutant-hypersensitive compounds
A set of experiments further demonstrated the fungal specificity of compounds in the MC-305,904 series and showed that the compounds act by disrupting the polymerization of fungal tubulin. Flow cytometric results displayed in Fig. to D showed that yeast cells exposed to a soluble MC-305,904 analog were arrested with an increased nuclear DNA content identical to that caused by the benzimidazole class tubulin depolymerizer nocodazole, establishing that the nuclear division arrest occurred during the mitotic phase. Flow cytometric analysis of human cells treated with cytotoxic concentrations of MC-305,904 analogs, including MC-06,341, did not cause increase in nuclear DNA content (Fig. to G), confirming the lack of mammalian tubulin-related activity seen in vitro. Cultured cell toxicity among MC-305,904 analogs varies but not in proportion to antifungal potency.
FIG. 2. Representative results of flow cytometric analysis of cell ploidy upon exposure to soluble MC-305,904 analogs. (A to D) Propidium iodide staining fluorescence intensity from log-phase S. cerevisiae strain F760 in the absence of compound (A) or after 6 (more ...)
Tubulin polymerization assays using purified S. cerevisiae as well as mammalian tubulin further characterized the fungal specificity of these compounds and established that fungal tubulin is the molecular target for the series. None of the six MC-305,904 analogs tested were effective inhibitors of bovine brain tubulin assembly in vitro, as judged by light-scattering measurements under polymerization-inducing conditions (Table ). In contrast, the compounds with more potent antifungal activity, namely, MC-06,341 and MC-06,307, completely prevented the in vitro polymerization of purified S. cerevisiae tubulin at 50 μM compound concentrations. Overall, the ability of compounds tested to inhibit yeast tubulin polymerization (Table ) paralleled the compounds' yeast growth-inhibitory potency. Representative results of the electron microscopic assay used are shown in Fig. . The lower limit of the MC-06,341 concentration required to inhibit yeast tubulin polymerization in vitro has not been determined, but the data in Table demonstrate greater-than-20-fold-higher selectivity of the compound for yeast tubulin over that for mammalian tubulin.
FIG. 3. MC-305,904 series inhibition of S. cerevisiae tubulin polymerization in vitro. Electron micrographs of 12.5 μM tubulin assembled in the absence of compound (A) or in the presence of 50 μM nocodazole (B), MC-06,341 (C), or MC-05,905 (D). (more ...) MC-305,904-resistant mutants.
In order to understand the interaction of MC-305,904 series compounds with tubulin, we generated mutant S. cerevisiae isolates capable of growth in medium containing 64 μg of MC-305,904/ml. Genetic crosses showed that compound resistance in eight independent isolates was recessive and attributable to a single locus closely linked to the TUB2 gene. TUB2 encodes the sole β-tubulin gene in S. cerevisiae, and mutations in β-tubulin genes have been previously associated with resistance to benzimidazole class tubulin depolymerizing agents in both fungal and nematode species (see Discussion). We sequenced the TUB2 genes from each of the resistant isolates and found that each carried one or more distinct point mutations. Each of the sequenced tub2 mutant alleles was transferred into a naïve genetic background (see Materials and Methods) and was shown to confer MC-305,904 resistance on the resultant strain, confirming the role of the characterized mutations in the compound resistance phenomenon (Tables and , MC-305,904 selection). None of the mutant tub2 genes alters susceptibility to compounds, such as fluconazole, that act by mechanisms unrelated to tubulin. Since the solubility limit of MC-305,904 is near the concentration required to inhibit wild-type cell growth, a concern was that limitations on the maximum achievable compound concentration could limit the ability to distinguish various degrees of compound resistance. Therefore, resistance to soluble analogs, including MC-06,341, was also examined, and differing degrees of resistance for different mutant strains were apparent.
In examining the locations of the affected amino acids with respect to the available crystal structure of bovine brain tubulin (14
), one region of the protein in particular prompted further investigation (Fig. ). The most robust resistance to MC-06,341 was conferred by two mutations affecting closely localized amino acids, A165 and F167. A third MC-305,904 resistance mutation affected both D161 and F200, also in close three-dimensional proximity to A165 and F167. Amino acids at all of these positions are conserved among fungal species, but the counterparts of A165 and F200 are conserved as N and Y, respectively, in known mammalian β-tubulins. The effects of changing these two residues to their mammalian tubulin counterparts on resistance to MC-305,904 analogs were tested. The A165N mutant alone or in combination with F200Y conferred >16-fold-greater resistance to MC-06,341, while an F200Y mutation caused 4-fold-greater resistance (Table ). An A165V mutation resulted in MC-06,341 resistance nearly equal to that of less conservative perturbations. Thus, primary amino acid sequence differences between fungal and mammalian β-tubulins in the vicinity of A165 are sufficient to explain the fungal specificity of the MC-305,904 series. The specific yet dramatic effects of conservative β-tubulin mutations on MC-305,904 series sensitivity suggest that these mutations may alter a binding site for the compounds. This possibility was further investigated by expanding the analysis of mutations that affect amino acids near A165 as well as mutations that affect other parts of the β-tubulin protein.
FIG. 4. Proposed binding site of MC-305,904 series compounds on tubulin based on analysis of compound sensitivity-altering mutations. (A) Homology model of yeast tubulin E198 region. The solvent-exposed side chain of E198 is shown at the center of a hydrophobic (more ...)
A prominent feature of the spatial region surrounding β-tubulin residue A165 is a clustered group of acidic amino acids, including D197 and E198 (Fig. ). The possibility that D197 and/or E198 plays a role in determining MC-305,904 sensitivity was probed by constructing mutations that affect the two residues. A D197A mutation had little effect on sensitivity to MC-305,904 or MC-06,341, while an E198A mutation caused compound resistance. The importance of the acidic side chain of E198 is further illustrated by the robust MC-305,904 resistance of an E198Q mutant. The acidic side chain of E198 may participate in high energy binding interactions with MC-305,904 and perhaps with other classes of compounds as well (see Discussion). Two additional mutations affecting residues within 8 Å of the E198 carboxyl carbon, namely, M163I and L253V, rendered mutant strains specifically supersensitive to MC-305,904 series compounds. The effects of other β-tubulin mutations are shown in Table and Fig. and are discussed below.
Antifungal spectrum of MC-305,904 analogs.
While MC-305,904 lacks whole cell activity against the major fungal pathogens C. albicans and Aspergillus fumigatus, a subset of (more soluble) analogs bearing a methyl substituent in place of the MC-305904 phenyl ring trifluormethyl group (Table , inset) does inhibit the growth of one or both pathogens (a detailed account of observed structure-activity relationships in a library of 650 analogs will be the focus of a subsequent report). Table illustrates that among these compounds, substitutions on the C4 position of the thiazoline ring strongly influence the antifungal species spectrum. A set of MC-305,904 analogs having increasingly large, hydrophobic C4 substituents trends toward diminishing activity against the yeast pathogen C. albicans, while the same compounds trend toward increasing activity against the mold pathogen A. fumigatus. Differential growth sensitivity assays were used to evaluate whether these opposing trends are due to differences in the tubulin proteins or, alternatively, due to differences in compound access or efflux.
Two S. cerevisiae strains were generated—one in which the TUB2 gene encodes a protein essentially identical to C. albicans TUB2 (SC2257) and one in which the TUB2 gene was modified to reflect a conserved amino acid sequence difference between yeast and mold species (SC2255). Alignment of predicted β-tubulin protein sequences from the yeasts S. cerevisiae and C. albicans and the mold species A. fumigatus, A. nidulans, A. flavus, and N. crassa was performed, and the positions of amino acid sequence differences between the species were analyzed with respect to the location of the presumed MC-305,904 series binding site near S. cerevisiae residue 198. A conserved difference between yeast and mold β-tubulins affects the helix 8 region of the tubulin structure proposed to form one face of the MC-305,904-binding site; leucine 257 of yeast species is replaced by methionine in mold tubulins. No amino acid sequence differences between S. cerevisiae and C. albicans β-tubulins affect residues in the E198 region. Based on these observations, a tub2-L257M mutant strain of S. cerevisiae (SC2255) was constructed.
Table compares growth inhibition sensitivities of the S. cerevisiae strains bearing the endogenous, Candida-like, and Aspergillus-like TUB2 genes when exposed to compounds with various activities toward the wild-type organisms. In all cases, the sensitivities of the wild-type strain (SC2256) and the strain bearing the Candida-like TUB2 gene (SC2257) were identical, as might be predicted based on the identity of amino acids in the MC-305,904-binding pocket. The Aspergillus-like tub2-L257M mutant strain (SC2255), in contrast, was selectively hypersensitive to compounds that had growth-inhibitory activity against wild-type A. fumigatus. The magnitude of tub2-L257M hypersensitivity varied independently of wild-type S. cerevisiae activity but in approximate proportion to wild-type A. fumigatus activity. Hypersensitivity of the tub2-L257M mutant to the soluble benzimidazole thiabendazole was also observed. Overall, these results suggest that the potency of analogs toward C. albicans is strongly influenced by compound access or efflux issues, while differences in Aspergillus potencies more closely reflect differential interactions with an MC-305,904-binding site on the β-tubulin protein.