Metabolism of an Alkyl Polyamine Analog by a Polyamine Oxidase from the Microsporidian Encephalitozoon cuniculi

Abstract

Encephalitozoon cuniculi is a microsporidium responsible for systemic illness in mammals. In the course of developing leads to new therapy for microsporidiosis, we found that a bis(phenylbenzyl)3-7-3 analog of spermine, 1,15-bis{N-[o-(phenyl)benzylamino}-4,12-diazapentadecane (BW-1), was a substrate for an E. cuniculi amine oxidase activity. The primary natural substrate for this oxidase activity was N-acetylspermine, but BW-1 had activity comparable to that of the substrate. As the sole substrate, BW-1 gave linear reaction rates over 15 min and K m of 2 M. In the presence of N-acetylspermine, BW-1 acted as a competitive inhibitor of oxidase activity and may be a subversive substrate, resulting in increased peroxide production. By use of 13 C-labeled BW-1 as a substrate and nuclear magnetic resonance analysis, two products were determined to be oxidative metabolites, a hydrated aldehyde or dicarboxylate and 2(phenyl)benzylamine. These products were detected after exposure of 13 C-labeled BW-1 to E. cuniculi preemergent spore preparations and to uninfected host cells. In previous studies, BW-1 was curative in a rodent model of infection with E. cuniculi. The results in this study demonstrate competitive inhibition of oxidase activity by BW-1 and support further studies of this oxidase activity by the parasite and host.

Metabolism of N-alkylated spermine analogues by polyamine and spermine oxidases

“…However, other methods are valid for testing substrate properties of tested compounds and can be used in inhibition studies. A recent application of using 13 C NMR for the detection of oxidase-mediated catabolism of labelled tracer drug in vitro offers means to characterize different reaction products (Bacchi et al 2009). NMR is a feasible method but lacks sensitivity, and mixtures of polyamine analogues and reaction products are sometimes hard to interpret accordingly.…”

“…Further studies are clearly needed in order to assess the role of APAO and SMO in polyamine analogue-mediated drug response. Large versatility in their substrate properties and flexible cleavage of their substrates could implicate that SMO and APAO may have other natural substrates in addition to natural polyamines (Bacchi et al 2009; Lentini et al 2007). It should be noted that the studied analogues resembled SPM (3-4-3) carbon backbone.…”

Polyamines and transglutaminases: biological, clinical, and biotechnological perspectives

“…Recently, the significance of polyamines in cell proliferation and carcinogenesis has stimulated further research interest in their biochemical and metabolic regulation at genomic level (Zhu et al 2012;Cervelli et al 2013;Murray-Stewart et al 2013;Pasini et al 2013). Inhibition of polyamine biosynthesis as an antitumor strategy has been demonstrated to be generally ineffective in clinical trials, but there is some potential in human diseases like cancer (Raj et al 2013) and parasitic diseases (Bacchi et al 2009;Preeti et al 2013;von Koschitzky and Kaiser 2013). It is clear that questions on polyamine action should be unraveled at molecular level to clarify the significance of polyamines in cell homeostasis related to various biological conditions.…”

Antiprotozoal/Antiparasitic Agents

“…Importantly, compound 60 inhibited growth in the K 243 As-10-3 arsenic resistant strain (IC 50 ¼ 165 nM), against which melarsoprol is inactive. Recent studies indicate that 60 has a broad spectrum of antiparasitic activity, and was shown to produce cures in mice infected with Microsporidia [157]. In these initial studies, analogs with a 3-3-3 carbon backbone had poor activity as antiparasitic agents, but were effective antitumor compounds.…”