Screening for Microtubule-Disrupting Antifungal Agents by Using a Mitotic-Arrest Mutant of Aspergillus nidulans and Novel Action of Phenylalanine Derivatives Accompanying Tubulin Loss
Screening for Microtubule-Disrupting Antifungal Agents by Using a Mitotic-Arrest Mutant of Aspergillus nidulans and Novel Action of Phenylalanine Derivatives Accompanying Tubulin Loss
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DOI：
10.1128/aac.48.5.1739-1748.2004
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发表时间：
2004-05
影响因子：
4.9
通讯作者：
T. Kiso;Ken-Ichi Fujita;X. Ping;Toshio Tanaka;M. Taniguchi
中科院分区：
文献类型：
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作者：
T. Kiso;Ken-Ichi Fujita;X. Ping;Toshio Tanaka;M. Taniguchi
摘要微管主要由α-微管蛋白和β-微管蛋白组成，是驱虫药、抗癌药和杀菌剂的主要作用靶点之一。我们专注于一个独特的特性构巢曲霉benA 33突变筛选微管破坏抗真菌剂。该突变体具有一个单一氨基酸突变的β-微管蛋白，由于在37°C下形成超稳定微管而经历有丝分裂停滞。突变株的热敏感性可以通过一些抗微管剂来补救。我们发现，与突变体的琼脂平板试验是能够区分三种类型的微管抑制剂。在37°C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）的纸片周围形成突变体的生长恢复区。诺考达唑、噻苯达唑和灰黄霉素可逆转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，但比上述药物产生更弱的生长恢复。其他微管抑制剂，如秋水仙碱，秋水仙胺，紫杉醇，鬼臼毒素，TN-16，长春碱，长春新碱，以及一些细胞骨架抑制剂测试，没有显示这样的活动。在我们的筛选中，我们新鉴定了两种真菌毒素，桔霉素和棒曲霉素，两种倍半萜二醛，远志醛和warburganal，以及四种苯丙氨酸衍生物，arphamenine A，1 - 2，5-二氢苯丙氨酸（DHPA），N-甲苯磺酰基-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A. DHPA引起微管的选择性损失，如通过荧光显微镜测定的，以及α-和β-微管蛋白的选择性损失，如通过Western印迹分析测定的。本研究以A. nidulans是有用的、方便的和高选择性的。所测试的苯丙氨酸衍生物是一种新型的微管破坏抗真菌剂，产生伴随的微管蛋白的损失，并且不同于影响微管蛋白二聚体组装成微管的众所周知的微管蛋白抑制剂。
ABSTRACT The microtubule, which is one of the major targets of anthelmintics, anticancer drugs, and fungicides, is composed mainly of α- and β-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 β-tubulin with a mutation of a single amino acid, undergoes mitotic arrest due to the formation of hyperstable microtubules at 37°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°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 α- and β-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.