Leishmania donovani Polyamine Biosynthetic Enzyme Overproducers as Tools To Investigate the Mode of Action of Cytotoxic Polyamine Analogs

Abstract

A number of anticancer and antiparasitic drugs are postulated to target the polyamine biosynthetic pathway and polyamine function, but the exact mode of action of these compounds is still being elucidated. To establish whether polyamine analogs specifically target enzymes of the polyamine pathway, a model was developed using strains of the protozoan parasite Leishmania donovani that overproduce each of the polyamine biosynthetic enzymes. Promastigotes overexpressing episomal constructs encoding ornithine decarboxylase (ODC), Sadenosylmethionine decarboxylase (ADOMETDC), or spermidine synthase (SPDSYN) revealed robust overproduction of the corresponding polyamine biosynthetic enzyme. Polyamine pools, however, were either unchanged or only marginally affected, implying that regulatory mechanisms must exist. The ODC, ADOMETDC, and SPDSYN overproducer strains exhibited a high level of resistance to difluoromethylornithine, 5-{[(Z)-4-amino-2-butenyl]methylamino}-5-deoxyadenosine, and n-butylamine, respectively, confirming previous observations that these agents specifically target polyamine enzymes. Conversely, augmented levels of polyamine biosynthetic enzymes did not affect the sensitivity of L. donovani promastigotes to pentamidine, berenil, and mitoguazone, drugs that were postulated to target the polyamine pathway, implying alternative and/or additional targets for these agents. The sensitivities of wild-type and overproducing parasites to a variety of polyamine analogs were also tested. The polyamine enzyme-overproducing lines offer a rapid cell-based screen for assessing whether synthetic polyamine analogs exert their mechanism of action predominantly on the polyamine biosynthetic pathway in L. donovani. Furthermore, the drug resistance engendered by the amplification of target genes and the overproduction of the encoded protein offers a general strategy for evaluating and developing therapeutic agents that target specific proteins in Leishmania.Polyamines are ubiquitous organic cations that play critical but still not completely defined roles in key cellular processes such as cell proliferation, differentiation, and nucleic acid synthesis (6,17,19,34,35,59,65). Since polyamines are especially important to rapidly growing cells, the polyamine pathway has been targeted in a multiplicity of antineoplastic and antiparasitic drug regimens. Most notably, DL-␣-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), the first enzyme in the polyamine biosynthesis pathway, is effectively curative against late-stage African sleeping sickness caused by the protozoan parasite Trypanosoma brucei gambiense (3,14,57). Interestingly, the selectivity of DFMO for the metabolic machinery of the parasite is not brought about by differential sensitivities of the parasite and human ODC enzymes to inactivation by DFMO but is rather due to a novel mechanism involving disparities in ODC turnover rates between T. brucei and the mammalian host (22,23). DFMO is also active against other trypanosome spe...

Spermidine Synthase Is Required for Virulence of Leishmania donovani

“…The membranes were probed with monospecific polyclonal antibody raised to the purified L. donovani SPDSYN (50,53) or with commercially available anti-␣-tubulin mouse monoclonal antibody (DM1A) (Calbiochem).…”

Leishmania donovani Ornithine Decarboxylase Is Indispensable for Parasite Survival in the Mammalian Host

“…Parasite lysates from exponentially growing cell populations were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (35) and blotted onto either nitrocellulose or Nytran membranes (Schleicher and Schuell, Keene, NH), and Western blot analysis was performed according to standardized procedures (53). The membranes were probed with polyclonal antibodies raised to the L. donovani ODC, ADOMETDC, and SPDSYN (31,(46)(47)(48), with commercially available anti-␣-tubulin mouse monoclonal antibody (DM1A) (Calbiochem., La Jolla, CA), or with antisera to the L. donovani A2 protein (59), which was generously furnished to the Ullman laboratory by Greg Matlashewski (McGill University, Montreal, CA).…”

“…DFMO was not tested against either of the two ⌬odc knockout lines, because these strains do not replicate efficiently in macrophages in the absence of putrescine (Fig. 4) and lack the pharmacologic target of DFMO, or the "add-back" lines, which are refractory to DFMO because of ODC overproduction (46).…”

The genetic toolbox forLeishmaniaparasites

“…The episomes contain drug resistance markers to facilitate selection and raising the drug concentration increases the copy number of the episomes thus yielding higher protein expression. 51 The overproducer strains can be used as tools to investigate the mode of action of cytotoxic analogs in Leishmania because they will be more resistant to a drug specifically targeting the overproduced protein. 51,52 Overexpression of certain genes can also help to understand their biological function as the phenotypic effects caused by the investigated protein will be amplified.…”

“…51 The overproducer strains can be used as tools to investigate the mode of action of cytotoxic analogs in Leishmania because they will be more resistant to a drug specifically targeting the overproduced protein. 51,52 Overexpression of certain genes can also help to understand their biological function as the phenotypic effects caused by the investigated protein will be amplified. 53,54 A novel system utilizing artificial linear episome-based structures shows promise for expressing large quantities of target proteins in L. tarentolae for the purpose of protein purification.…”