Diazonamide toxins reveal an unexpected function for ornithine δ-amino transferase in mitotic cell division

Abstract

We have studied a naturally occurring small-molecule antimitotic called diazonamide A. Diazonamide A is highly effective at blocking spindle assembly in mammalian cell culture and does so through a unique mechanism. A biotinylated form of diazonamide A affinity purifies ornithine ␦-amino transferase (OAT), a mitochondrial enzyme, from HeLa cell and Xenopus egg extracts. In the latter system, the interaction between diazonamide A and OAT is regulated by RanGTP. We find that specific OAT knockdown in human cervical carcinoma and osteosarcoma cells by RNA interference blocks cell division and causes cell death, the effects largely phenocopying diazonamide A treatment in these cell lines. Our experiments reveal an unanticipated, paradoxical role for OAT in mitotic cell division and identify the protein as a target for chemotherapeutic drug development.A variety of small molecules block progression through M phase of the cell cycle. The most common are tubulin ligands. Tubulin-binding toxins have helped elucidate the structure and organization of the mitotic spindle, and certain of these toxins are clinically effective as cancer chemotherapy. However, systemic disruption of the tubulin cytoskeleton has drawbacks. Microtubule poisons disturb nonmitotic functions of the cytoskeleton in both replicating cells and differentiated nondividing cells. Wasting, neutropenia, and peripheral neuropathy are severe dose-limiting toxicities common to this family of drugs in vivo (1). As a result, considerable effort has been made to identify alternative antineoplastics that target mitotic regulatory factors or components of the spindle other than tubulin (2). The development of specific inhibitors of the aurora kinases (3) and the kinesin motor protein Eg5 (2) are notable recent examples.

Affinity purification in target identification: the specificity challenge

“…7a) (Oda et al 2003;Wang et al 2007;Ong et al 2009). However, besides matrix binding, a large number of nonspecific proteins are from low affinity ligand binding or indirect binding to target proteins.…”

“…In order to control for these interactions, one may use a ''negative ligand'', an analog structurally similar but is inactive in the corresponding cellular assay (Fig. 7a) (Oda et al 2003;Wang et al 2007;Li et al 2014a). However, caution should be taken when choosing the ''negative ligand'', since it is possible that an inactive analog may be still able to physically bind the target but with reduced affinity so that it does not exhibit the same cellular activity as the positive compound.…”

Therapeutic anticancer efficacy of a synthetic diazonamide analog in the absence of overt toxicity

“…Although AB-5 functions as an antimitotic, both in vitro and in vivo, Wang et al (17) have shown that it does so through a unique mode of action involving a heretofore unrecognized role for OAT in cell division (17). Mice deficient in OAT The AB-5 metabolite was generated by incubation of AB-5 with human S9 protein for 4 h and then fractionated by HPLC on a C18 column.…”

“…Notably, however, studies by CruzMonserrate et al (15) indicated that diazonamide A does not compete with vinblastine or colchicine for tubulin binding. In fact, our own studies have shown that neither biotinylated nor radiolabeled congeners of the natural product interact specifically with purified tubulin in vitro, raising the possibility that the compound does not act by binding directly to tubulin (17).…”

“…These experiments have shown conclusively that tubulin binding does not underpin the antimitotic effects of this compound. Rather, diazonamide A interacts with a specifically proteolyzed form of ornithine ␦-amino transferase (OAT) critically involved in a heretofore novel spindle assembly pathway controlling mitosis (17). Paradoxically, the OAT-mediated spindle assemble pathway appears not to be essential for normal cell division.…”