ansamitocins and rhizoxin

The trypanosomatid cytoskeleton is responsible for the parasite's shape and it is modulated throughout the different stages of the parasite's life cycle. When parasites are exposed to media with reduced osmolarity, they initially swell, but subsequently undergo compensatory shrinking referred to as regulatory volume decrease (RVD). We studied the effects of anti-microtubule (Mt) drugs on the proliferation of Leishmania mexicana promastigotes and their capacity to undergo RVD. All of the drugs tested exerted antiproliferative effects of varying magnitudes [ansamitocin P3 (AP3)> trifluoperazine > taxol > rhizoxin > chlorpromazine]. No direct relationship was found between antiproliferative drug treatment and RVD. Similarly, Mt stability was not affected by drug treatment. Ansamitocin P3, which is effective at nanomolar concentrations, blocked amastigote-promastigote differentiation and was the only drug that impeded RVD, as measured by light dispersion. AP3 induced 2 kinetoplasts (Kt) 1 nucleus cells that had numerous flagella-associated Kts throughout the cell. These results suggest that the dramatic morphological changes induced by AP3 alter the spatial organisation and directionality of the Mts that are necessary for the parasite's hypotonic stress-induced shape change, as well as its recovery.

Topics: Animals; Chlorpromazine; Cytoskeleton; Leishmania mexicana; Macrolides; Maytansine; Mice; Paclitaxel; Trifluoperazine; Tubulin Modulators

The binding of four potent antimitotic agents, rhizoxin (RZX), phomopsin A (PMS-A), ansamitocin P-3 (ASMP-3), and vinblastine (VLB), to tubulins from RZX-sensitive and -resistant strains of Aspergillus nidulans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae was investigated. Mycelial extracts to which RZX could bind contained beta-tubulin with Asn as the 100th amino acid residue (Asn-100) in all cases, and those without affinity for RZX contained beta-tubulins with either Ile-100 or Val-100. Though PMS-A shares the same binding site as RZX and ASMP-3 on porcine brain tubulin (Asn-100), only ASMP-3 bound Asn-100 fungal tubulins in a competitive manner with respect to RZX. PMS-A and VLB, which strongly bind to porcine brain tubulin, did not bind to any of the fungal mycelial extracts examined. The results indicate differential interactions of these antimitotic agents with brain and fungal tubulins.

Topics: Animals; Antineoplastic Agents; Aspergillus nidulans; Binding Sites; Binding, Competitive; Brain; Brain Chemistry; Fungi; Lactones; Macrolides; Maytansine; Mycotoxins; Saccharomyces cerevisiae; Schizosaccharomyces; Swine; Tubulin; Vinblastine

Phomopsin A is an antimitotic cyclic peptide containing a 13-member ring including an ether linkage. It was isolated from the fungus Phomopsis leptostromiformis as the causal agent of lupinosis. Phomopsin A strongly inhibited microtubule assembly (IC50: 2.4 microM). Our study using radiolabeled phomopsin A, prepared biosynthetically by feeding L-[U-14C]isoleucine to the culture of P. leptostromiformis, indicated that at least two binding sites of phomopsin A exist on tubulin on the basis of a Scatchard analysis; i.e. the dissociation constants of a high affinity site (Kd1) and a low affinity site (Kd2) at 37 degrees were determined to be 1 x 10(-8) and 3 x 10(-7) M, respectively. Phomopsin A inhibited the binding of radiolabeled rhizoxin to tubulin with an inhibition constant (Ki) of 0.8 x 10(-8) M. This showed that the high affinity site of phomopsin A is identical to the rhizoxin binding site. The binding of the radiolabeled phomopsin A was also inhibited by rhizoxin and ansamitocin P-3, with an inhibition constant of 10(-7) M, and to a lesser extent by vinblastine. Phomopsin A had no inhibitory effect on colchicine binding to tubulin.

Topics: Animals; Binding Sites; Binding, Competitive; Brain; Colchicine; Kinetics; Lactones; Macrolides; Maytansine; Microtubule Proteins; Mycotoxins; Polymers; Protein Conformation; Structure-Activity Relationship; Swine; Tubulin; Tubulin Modulators; Vinblastine

The binding of rhizoxin, a potent inhibitor of mitosis and in vitro microtubule assembly, to porcine brain tubulin was studied. Tubulin possesses one binding site for rhizoxin per molecule with a dissociation constant (Kd) of 1.7.10(-7) M. Ansamitocin P-3, a homologue of maytansine, was a competitive inhibitor of rhizoxin binding, with an inhibition constant of 1.3.10(-7) M. Vinblastine also inhibited rhizoxin binding, but was not fully competitive, and the inhibition constant was 2.9.10(-6) M. In contrast, both rhizoxin and ansamitocin P-3 were potent inhibitors of vinblastine binding. Rhizoxin inhibited tau-promoted tubulin assembly, but it, differing from vinblastine, did not induce tubulin aggregation into spirals, even at a concentration as high as 2.10(-5) M. In addition, rhizoxin strongly inhibited vinblastine-induced tau-dependent tubulin aggregation. Rhizoxin binding to tubulin was completely independent from colchicine binding. These effects resemble those of maytansine. The results suggested that rhizoxin binds to the maytansine-binding site and that the binding sites of rhizoxin and vinblastine are not the same.

Topics: Animals; Binding Sites; Binding, Competitive; Colchicine; Kinetics; Lactones; Macrolides; Maytansine; Microscopy, Electron; Microtubule-Associated Proteins; Microtubules; Oxazines; Swine; tau Proteins; Tubulin; Vinblastine