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

Abstract

SUMMARYN-alkylated polyamine analogues have potential as anticancer and antiparasitic drugs. However, their metabolism in the host has remained incompletely defined thus potentially limiting their utility. Here, we have studied the degradation of three different spermine analogues N, N′-bis-(3-ethylaminopropyl)butane-1,4-diamine (DESPM), N-(3-benzyl-aminopropyl)-N'-(3-ethylaminopropyl)butane-1,4-diamine (BnEtSPM) and N,N′-bis-(3-benzylaminopropyl)butane-1,4-diamine (DBSPM) and related mono-alkylated derivatives as substrates of recombinant human polyamine oxidase (APAO) and spermine oxidase (SMO). APAO and SMO metabolized DESPM to EtSPD (K m(APAO) =10μM, k cat(APAO) =1.1s −1 and K m(SMO) =28μM, k cat(SMO) =0.8s −1 , respectively), metabolized BnEtSPM to EtSPD (K m(APAO) =0.9 μM, k cat(APAO) =1.1s −1 and K m(SMO) =51μM, k cat(SMO) =0.4s −1 , respectively), and metabolized DBSPM to BnSPD (K m(APAO) =5.4μM, k cat(APAO) = 2.0s −1 and K m(SMO) =33μM, k cat(SMO) =0.3s −1 , respectively). Interestingly, mono-alkylated spermine derivatives were metabolized by APAO and SMO to SPD (EtSPM K m(APAO) =16μM, k cat(APAO) =1.5s −1 ; K m(SMO) =25μM, k cat(SMO) =8.2s −1 ; BnSPM K m(APAO) =6.0μM, k cat(APAO) =2.8s −1 ; K m(SMO) =19μM, k cat(SMO) =0.8s −1 , respectively). Surprisingly, E t S P D ( K m(APAO) =37μM, k cat(APAO) =0.1s −1 ; K m(SMO) =48μM, k cat(SMO) =0.05s −1 ) and BnSPD (K m(APAO) =2.5μM, k cat(APAO) =3.5s −1 ; K m(SMO) =60μM, k cat(SMO) =0.54s −1 ) were metabolized to SPD by both the oxidases. Furthermore, we studied the degradation of DESPM, BnEtSPM or DBSPM in the DU145 prostate carcinoma cell line. The same major metabolites EtSPD and/or BnSPD were detected both in the culture medium and intracellularly after 48 hours of culture. Moreover, EtSPM and BnSPM were detected from cell samples. Present data shows that inducible SMO parallel with APAO could play an important role in polyamine based drug action, i.e. degradation of parent drug and its metabolites, having significant impact on efficiency of these drugs, and hence for the development of novel N-alkylated polyamine analogues.

Development of Irreversible Inactivators of Spermine Oxidase and <i>N</i>¹-Acetylpolyamine Oxidase

“…This is consistent with previous observations reported in the literature. 3,11,18) Based on these results, N The rate of spermidine production rapidly decreased with time, indicating that the enzyme became progressively more inactivated. The apparent molar proportions of spermidine to APAO were approximately 60 and 80 for N 1 -and N 8 -butadienyl Spd, respectively, which were calculated by extrapolating to the point of complete inactivation.…”

Current Status of the Polyamine Research Field

“…They may act by directly inducing SMO, which is highly responsive to many analogs, and thus reduce spermine and increase ROS (48, 60, 229), by metabolism generating toxic metabolites via SMO or the SSAT/APAO pathways (60, 237) or by additional mechanisms such as blocking binding of polyamines to key sites for normal effects on growth, interfering with polyamine-mediated cell signaling (238) or by acting on the cytoskeleton (230). It should also be noted that the potential of polyamine analogs for therapeutic use is not limited to cancer therapy.…”

Controlling ofN-alkylpolyamine analogue metabolism by selective deuteration

“…More importantly, the stereospecificity of APAO is changed by supplementing either benzaldehyde (R-activating) or pyridoxal (S-activating) with racemic 1methylspermidine as a substrate [12]. Furthermore, we have characterized earlier the preferred catabolic pathways of symmetrically substituted Spm analogues DESpm and N 1 ,N 12 -dibenzylspermine (DBSpm, N,N'-bis-(3-benzyl-aminopropyl)butane-1,4-diamine) (Figure 1), and unsymmetrically substituted N 1 -benzyl-N 12 -ethylspermine (BnEtSpm, N-(3-benzyl-aminopropyl)-N'-(3-ethylaminopropyl)butane-1,4-diamine) by APAO and SMOX [14]. As protium-deuterium exchange could be used to redirect oxidative metabolism mediated via hydrogen abstraction [1], we synthesized a set of selectively deuterated polyamine analogues ( Figure 1) for a proof-of-concept study to alter the catabolic pathways of synthesized analogues and to study the impact of targeted deuteration on the analogues' biological efficacy.…”