In search of selective inhibitors of polyamine and spermine oxidase: a preliminary kinetic study

Polyamine catabolism plays an important role in cellular homeostasis and its dysregulation may contribute to the development of several pathological states, including cancer (Nowotarski et al 2013). On this basis, polyamine metabolic pathway has been considered a rational target for antineoplastic therapy. To develop efficient therapies, the role of each enzyme of the polyamine catabolic pathway should be well-defined. For this purpose selective inhibitors of acetylpolyamine oxidase (APAO) and spermine oxidase (SMO) are needed, considering that these enzymes are also involved in the catabolism of some antitumor polyamine analogues (Casero and Pegg, 2009, Haikkinen et al, 2010, Cervelli et al., 2010). APAO and SMO inhibitors developed so far are generally polyamine analogues, such as MDL 72527(N1,N4—bis(2,3-butadienyl)-1,4-butanediamine), but they lack of a good selectivity. The discovery of selective inhibitors of APAO and SMO could represent an important tool for the development of novel drugs and therapies. In search of novel and selective APAO and SMO inhibitors, we investigated the structural features required to improve the selective interactions of a polyamine scaffold with these enzymes. As probes, we assayed fifteen polyamine analogues, most of which are spermine and methoctramine analogues, unsymmetrically and symmetrically substituted with different aromatic moieties on the terminal nitrogens of the polyamine chain, and varying in the number of amine functions and length of the polymethylene chains linking the nitrogen atoms. By a kinetic approach, these analogues were tested both as potential inhibitors and substrates of murine SMO and APAO. From the kinetic studies carried out so far the main result is the finding of a competitive and selective inhibitor for APAO, methoctramine, a well known muscarinic M2 receptor antagonist (Minarini et al., 2010). Methoctramine inhibits APAO with a selectivity of 1:120 respect to SMO (KI= 10 nM for APAO vs KI=1.2 M for SMO). In addition, the spermine analogue mono-substituted with a thiophene ring on one of the primary amine functions (Bonaiuto et al., 2013) was found to be a good substrate, endowed with a very high affinity for APAO (Km= 44 nM for APAO vs Km = 10 microM for SMO, in physiological conditions). Docking simulations onto the three dimensional models of APAO and SMO have been carried out to obtain information about the structural parameters controlling the binding mode of the molecules with the active site of the enzymes. Further studies are necessary to evaluate the inhibitory behaviour of methoctramine on APAO and SMO in a cellular system.