Natural organic compounds that affect to microtubule functions.

Microtubules (MT), composed of a protein tubulin (TN) alpha,beta-heterodimer with concomitant other proteins, microtubule associated proteins (MAPs and tau), are known to be the main component of spindles in a mitotic apparatus of eucaryotic cells, and are also involved in many other basic and essential cell functions. There are a number of natural and synthetic compounds that interfere with MT function to cause the mitotic arrest of eucaryotic cells. Such antimitotic agents show a broad biological activity, and can be used for medicinal and agrochemical purposes. On the other hand, they are also important as the biochemical tools for understanding the dynamics of MT network. Most of such antimitotic agents, with a few exceptions, bind to beta-TN. Among them, colchicine (CLC), vinblastine (VLB) and taxol have been of major importance in biochemical studies of MT and in studies of their intracellular functions. The former two both inhibit MT assembly but their binding sites on beta-TN are different; CLC-site and VLB-site, and many MT inhibitors bind to either sites. Taxol bind to TN at a site other than CLC-site and VLB-site, and promote MT assembly. We have worked on a variety of antimitotic agents that bind to CLC, VLB or taxol-site, in discoveries, structures, biological actions and/or interactions with TN. In this paper, I summarized the results of our studies on VLB-site ligands; (1) rhizoxin (RZX), isolated as a phytotoxin produced by a plant pathogenic fungus, and its related compounds, (2) derivatives of ansamitocin P-3 (ASMP3) (maytansinoid: MAY), isolated as a cytotoxic metabolite of an Actinomycete, (3) phomopsin A (PMSA), isolated as a mycotoxin produced by a plant parasitic fungus, (4) dolastatin 10 (DLS10), isolated as a cytotoxic metabolite of a see animal, (5) ustiloxins (USL) A-F, isolated as a mycotoxin produced by a plant pathogenic fungus, (6) arenastation A (ARSA), isolated as a cytotoxic metabolite of a sponge, and its synthetic analogs. From our studies on interactions of these VLB-site ligands with TN, we showed that the presence of a distinct RZX/MAY-binding site which only partially overlap with VLB-site, and that PMSA, DLS10, USLs and ARSA bind to the RZX/MAY site. RZX, ASMP3 and ARSA inhibit the growth of a variety of fungi, including Aspergillus nidulans. In order to obtain information as to the drug-TN interaction at the RZX/MAY site, RZX-resistant beta-TN gene mutants were isolated from RZX-sensitive wild-type A. nidulans. In all the beta-TN gene mutants, single amino acid (100th) alteration, asparagine-to-isoleucine, was observed. Sequence displacement experiments confirmed that this alteration conferred resistance to RZX and ASMP3, and also to ARSA. This resistance mechanism was further verified with yeasts Schizosaccharomyces pombe and Saccharomyces serevisiae. All the natural ligands mentioned above show potent cytotoxicity against human and murine tumor cells, but VLB, PMSA, DLS10 and USLA are inactive to both RZX-sensitive and -resistant fungal strains.