Kiso Tetsuo, Fujita Ken-Ichi, Ping Xu, Tanaka Toshio, Taniguchi Makoto
Department of Bio- and Geoscience, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan.
Antimicrob Agents Chemother. 2004 May;48(5):1739-48. doi: 10.1128/AAC.48.5.1739-1748.2004.
The microtubule, which is one of the major targets of anthelmintics, anticancer drugs, and fungicides, is composed mainly of alpha- and beta-tubulins. We focused on a unique characteristic of an Aspergillus nidulans benA33 mutant to screen for microtubule-disrupting antifungal agents. This mutant, which has a beta-tubulin with a mutation of a single amino acid, undergoes mitotic arrest due to the formation of hyperstable microtubules at 37 degrees C. The heat sensitivity of the mutant is remedied by some antimicrotubule agents. We found that an agar plate assay with the mutant was able to distinguish three types of microtubule inhibitors. The growth recovery zones of the mutant were formed around paper disks containing microtubule inhibitors, including four benzimidazoles, ansamitocin P-3, griseofulvin, and rhizoxin, on the agar plate at 37 degrees C. Nocodazole, thiabendazole, and griseofulvin reversed the mitotic arrest of the mutant and promoted its hyphal growth. Ansamitocin P-3 and rhizoxin showed growth recovery zones around the growth-inhibitory zones. Benomyl and carbendazim also reversed mitotic arrest but produced weaker growth recovery than the aforementioned drugs. Other microtubule inhibitors, such as colchicine, Colcemid, paclitaxel, podophyllotoxin, TN-16, vinblastine, and vincristine, as well as some cytoskeletal inhibitors tested, did not show such activity. In our screening, we newly identified two mycotoxins, citrinin and patulin, two sesquiterpene dialdehydes, polygodial and warburganal, and four phenylalanine derivatives, arphamenine A, L-2,5-dihydrophenylalanine (DHPA), N-tosyl-L-phenylalanine chloromethylketone, and N-carbobenzoxy-L-phenylalanine chloromethyl ketone. In a wild-type strain of A. nidulans, DHPA caused selective losses of microtubules, as determined by fluorescence microscopy, and of both alpha- and beta-tubulins, as determined by Western blot analysis. This screening method involving the benA33 mutant of A. nidulans is useful, convenient, and highly selective. The phenylalanine derivatives tested are of a novel type of microtubule-disrupting antifungal agents, producing an accompanying loss of tubulins, and are different from well-known tubulin inhibitors affecting the assembly of tubulin dimers into microtubules.
微管是驱虫药、抗癌药和杀真菌剂的主要作用靶点之一,主要由α-微管蛋白和β-微管蛋白组成。我们聚焦构巢曲霉benA33突变体的一个独特特性,以筛选破坏微管的抗真菌剂。该突变体的β-微管蛋白有一个单氨基酸突变,在37℃时由于形成超稳定微管而发生有丝分裂停滞。一些抗微管药物可纠正该突变体的热敏感性。我们发现用该突变体进行的琼脂平板试验能够区分三种类型的微管抑制剂。在37℃的琼脂平板上,在含有微管抑制剂(包括四种苯并咪唑、安丝菌素P-3、灰黄霉素和根霉素)的滤纸片周围形成了该突变体的生长恢复区。诺考达唑、噻苯达唑和灰黄霉素逆转了该突变体的有丝分裂停滞并促进其菌丝生长。安丝菌素P-3和根霉素在生长抑制区周围显示出生长恢复区。苯菌灵和多菌灵也逆转了有丝分裂停滞,但产生的生长恢复作用比上述药物弱。其他微管抑制剂,如秋水仙碱、秋水仙酰胺、紫杉醇、鬼臼毒素、TN-16、长春碱和长春新碱,以及一些测试的细胞骨架抑制剂,均未显示出这种活性。在我们的筛选中,我们新鉴定出两种霉菌毒素,橘霉素和展青霉素,两种倍半萜二醛,聚癸二醛和warburganal,以及四种苯丙氨酸衍生物,阿弗门汀A、L-2,5-二氢苯丙氨酸(DHPA)、N-甲苯磺酰-L-苯丙氨酸氯甲基酮和N-苄氧羰基-L-苯丙氨酸氯甲基酮。在构巢曲霉的野生型菌株中,通过荧光显微镜观察,DHPA导致微管选择性丧失,通过蛋白质免疫印迹分析确定,α-微管蛋白和β-微管蛋白均丧失。这种涉及构巢曲霉benA33突变体的筛选方法有用、便捷且具有高度选择性。所测试的苯丙氨酸衍生物是一类新型的破坏微管的抗真菌剂,会导致微管蛋白伴随丧失,与影响微管蛋白二聚体组装成微管的知名微管蛋白抑制剂不同。