RPR112378 and RPR115781: Two Representatives of a New Family of Microtubule Assembly Inhibitors

Abstract

A screening program aimed at the discovery of new antimicrotubule agents yielded RPR112378 and RPR115781, two natural compounds extracted from the Indian plant Ottelia alismoides. We report their isolation, structural determination, and mechanisms of action. RPR112378 is an efficient inhibitor of tubulin polymerization (IC(50) = 1.2 microM) and is able to disassemble preformed microtubules. Regarding tubulin activity, RPR115781 is 5-fold less active than RPR112378. Tubulin-RPR112378 complexes, when isolated by gel filtration, were able to block further tubulin addition to growing microtubules, a mechanism that accounts for the substoichiometric effect of the drug. RPR112378 was found to prevent colchicine binding but not vinblastine binding to tubulin. Although colchicine binding is known to induce an increase of tubulin GTPase activity, no such increase was observed with RPR112378. We show that RPR112378 is a highly cytotoxic compound and that RPR115781 is 10, 000-fold less active as an inhibitor of KB cell growth. Part of the cytotoxicity of RPR112378 is probably caused by a reaction of addition with sulfhydryl groups, an observation that has not been made with RPR115781. In conclusion, these molecules represent a new class of inhibitors of microtubule assembly with potential therapeutic value.

2-Cyano-3,12-dioxooleana-1,9(11)-diene-28-oic Acid Disrupts Microtubule Polymerization: A Possible Mechanism Contributing to Apoptosis

“…6 and 7). Thus, like many other sulfhydryl-binding compounds known to inhibit tubulin polymerization (Kuriyama and Sakai, 1974;Mellon and Rebhun, 1976;Deinum et al, 1981;Lee et al, 1981;Luduena and Roach, 1981;Roach and Luduena, 1984;Bai et al, 1989;Li and Broome, 1999;Shan et al, 1999;Combeau et al, 2000), CDDO may function in part by disrupting the regulatory role of a sulfhydryl group in the formation of the mitotic spindle . Furthermore, because the binding site of CDDO on ␤-tubulin was localized to a region also bound by bis-ANS, which is thought to reside in a flexible area distal from the ␣␤-interface (Ward et al, 1994), CDDO may also inhibit tubulin polymerization in a manner analogous to bis-ANS (Mazumdar et al, 1992).…”

Novel combretastatin A-4 derivative XN0502 induces cell cycle arrest and apoptosis in A549 cells

“…Our results show that XN0502-caused downregulation of procaspase-9 and procaspase-3, which consequently induces the formation of caspase-3 and PARP cleavage. Furthermore, the downregulation of XIAP, one of the inhibitors of apoptosis proteins (IAPs) family capable of hampering caspase-3 activation to protect cells from apoptosis [2,32], was detected in XN0502-treated A549 cells. Therefore, we claim that XN0502 triggers apoptosis by activating caspase cascade and blocking XIAP.…”

“…Western blot analysis Cell lysates were prepared as described before [2]. Proteins were fractionated on 8-15% SDS-PAGE and transferred to PVDF membrane and probed with specific primary antibodies and then HRP-labeled secondary antibodies.…”

“…Microtubules are composed of heterodimers of two similar protein subunits, α-and β-tubulin [1,2]. They are critical elements of the cellular cytoskeleton in eukaryotic cells and involved in cellular processes such as cell movement, organelle localization, intracellular material transportation and cell division regulation [3,4].…”

Characterization of the Covalent Binding ofN-Phenyl-N′-(2-chloroethyl)ureas to β-Tubulin: Importance of Glu198 in Microtubule Stability

“…In addition, mutations, such as ␤F270V and ␤T274I, in the paclitaxel (Taxol)-binding site inhibit the binding of paclitaxel (Giannakakou et al, 2000), thus leading to chemoresistance to the drug. Moreover, modification of ␤Cys239 and ␤Cys354 by alkylating drugs, such as 2-chloroacetyl-2-demethylthiocolchicine (Bai et al, 2000), ottelione A (RPR112378) (Combeau et al, 2000), and 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (T138067) (Shan et al, 1999), inhibits the polymerization of tubulins into microtubules. In contrast, modification of ␤Thr220 and ␤Asn228 by cyclostreptin decreases the dissociation of tubulin-GDP at the microtubule ends (Buey et al, 2007).…”