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

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Abstract

N-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 [Km(APAO) = 10 μM, kcat(APAO) = 1.1 s−1 and Km(SMO) = 28 μM, kcat(SMO) = 0.8 s−1, respectively], metabolized BnEtSPM to EtSPD [Km(APAO) = 0.9 μM, kcat(APAO) = 1.1 s−1 and Km(SMO) = 51 μM, kcat(SMO) = 0.4 s−1, respectively], and metabolized DBSPM to BnSPD [Km(APAO) = 5.4 μM, kcat(APAO) = 2.0 s−1 and Km(SMO) = 33 μM, kcat(SMO) = 0.3 s−1, respectively]. Interestingly, mono-alkylated spermine derivatives were metabolized by APAO and SMO to SPD [EtSPM Km(APAO) = 16 μM, kcat(APAO) = 1.5 s−1; Km(SMO) = 25 μM, kcat(SMO) = 8.2 s−1; BnSPM Km(APAO) = 6.0 μM, kcat(APAO) = 2.8 s−1; Km(SMO) = 19 μM, kcat(SMO) = 0.8 s−1, respectively]. Surprisingly, EtSPD [Km(APAO) = 37 μM, kcat(APAO) = 0.1 s−1; Km(SMO) = 48 μM, kcat(SMO) = 0.05 s−1] and BnSPD [Km(APAO) = 2.5 μM, kcat(APAO) = 3.5 s−1; Km(SMO) = 60 μM, kcat(SMO) = 0.54 s−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 h 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.

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Abbreviations

ACN:

Acetonitrile

APAO:

(Acetyl)polyamine oxidase (exo-N4-amino) [EC 1.5.3.11]

BnDAP:

N1-benzyl-propane-1,3-diamine

BnDAP-4D:

N1-benzyl-2,2,3,3-2H4-propane-1,3-diamine

BnEtSPM:

N-(3-benzylaminopropyl)-N′-(3-ethylaminopropyl)butane-1,4-diamine

BnEtSPM-8D:

N-(3-benzylamino-1,1,2,2-2H4-propyl)-N′-(3-ethylamino-1,1,2,2-2H4-propyl)butane-1,4-diamine

BnNH2 :

Benzylamine

BnNH2-2D:

α,α-2H2-benzylamine

BnSPD:

N1-(3-benzylaminopropyl)butane-1,4-diamine

BnSPD-4D:

N1-(3-benzylamino-1,1,2,2-2H4-propyl)butane-1,4-diamine

BnSPM:

N-(3-aminopropyl)-N′-(3-benzylaminopropyl)butane-1,4-diamine

BnSPM-4D:

N-(3-aminopropyl)-N′-(3-benzylamino-1,1,2,2-2H4-propyl)butane-1,4-diamine

CID:

Collision energy

DAP:

Propane-1,3-diamine

DAP-2D:

1,1-2H2-propane-1,3-diamine

DBSPM:

N,N′-bis-(3-benzylaminopropyl)butane-1,4-diamine

DBSPM-8D:

N,N′-bis-(3-benzylamino-1,1,2,2-2H4-propyl)butane-1,4-diamine

DENSPM:

Diethylnorspermine, N,N′-bis-(3-ethylaminopropyl)-propane-1,3-diamine

DESPM:

N,N′-bis-(3-ethylaminopropyl)butane-1,4-diamine

DESPM-4D:

N,N′-bis-(3-ethylamino-1,1-2H2-propyl)butane-1,4-diamine

EtDAP:

N1-ethylpropane-1,3-diamine

EtDAP-2D:

N1-ethyl-3,3-2H2-propane-1,3-diamine

EtSPD:

N1-(3-ethylaminopropyl)butane-1,4-diamine trihydrochloride

EtSPD-2D:

N1-(3-ethylamino-1,1-2H2-propyl)butane-1,4-diamine

EtSPM:

N-(3-aminopropyl)-N′-(3-ethylaminopropyl)butane-1,4-diamine

EtSPM-2D:

N-(3-aminopropyl)-N′-(3-ethylamino-1,1-2H2-propyl)butane-1,4-diamine

FDA:

US, Food and Drug Administration, USA

HFBA:

Heptafluorobutyric acid

HPLC:

High pressure liquid chromatography

IS:

Internal standard

LC-MS/MS:

Liquid chromatography-electrospray ionization-tandem mass spectrometry

MDL 72527:

N,N′-bis-(2,3-butadienyl)-1,4-butanediamine

NMR:

Nuclear magnetic resonance

N1-AcSPD:

N1,N12-diacetylspermine

N1-AcSPM:

N1,N12-diacetylspermidine

N1,N12-DiAcSPM:

N1,N12-Diacetylspermine

PA:

Polyamine

PUT:

Putrescine, butane-1,4-diamine

PUT-8D:

1,1,2,2,3,3,4,4-2H8-butane-1,4-diamine

SMO:

(PAOh1), Spermine oxidase [EC 1.5.3.-]

SPD:

Spermidine, N 1-(3-aminopropyl)butane-1,4-diamine

SPD-2D:

N1-(3-amino-1,1-2H2-propyl)butane-1,4-diamine

SPM:

Spermine, N,N′-bis-(3-aminopropyl)butane-1,4-diamine

SPM-4D:

N-(3-amino-1,1,2,2-2H4-propyl)-N′-(3-aminopropyl)butane-1,4-diamine

SRM:

Selected reaction monitoring

SSAT:

Spermidine/spermine N 1-acetyltransferase [EC 2.3.1.57]

STD:

Standard

QC:

Quality control

QL:

Qualifier ion

QT:

Quantifier ion

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Acknowledgements

We thank Ms. Helena Vepsäläinen and Ms. Maritta Salminkoski, Department of Biosciences, Laboratory of Chemistry, University of Kuopio, for their help with LC-MS/MS sample preparation and in the synthesis work. Ms. Anne Karppinen and Ms. Tuula Reponen at A. I. Virtanen Institute, University of Kuopio, are acknowledged for their help with enzyme and cell experiments. This work was supported by Academy of Finland (projects 1241851 and 1287022), NIH (USA) CA984544, and the Russian Foundation for Basic Research (project 08-04-917775).

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Authors and Affiliations

  1. Laboratory of Chemistry, Department of Biosciences, Biocenter Kuopio, University of Kuopio, P.O.Box 1627, 70211, Kuopio, Finland

    Merja R. Häkkinen & Jouko Vepsäläinen

  2. Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, Biocenter Kuopio, University of Kuopio, P.O.Box 1627, 70211, Kuopio, Finland

    Mervi T. Hyvönen, Leena Alhonen & Tuomo A. Keinänen

  3. Department of Pharmaceutical Chemistry, Biocenter Kuopio, University of Kuopio, P.O.Box 1627, 70211, Kuopio, Finland

    Seppo Auriola

  4. Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, 21231, USA

    Robert A. Casero Jr

  5. Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, 21231, USA

    Robert A. Casero Jr

  6. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, Moscow, 119991, Russia

    Alex R. Khomutov

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  1. Merja R. Häkkinen
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  2. Mervi T. Hyvönen
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  8. Tuomo A. Keinänen
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Correspondence to Merja R. Häkkinen.

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Häkkinen, M.R., Hyvönen, M.T., Auriola, S. et al. Metabolism of N-alkylated spermine analogues by polyamine and spermine oxidases. Amino Acids 38, 369–381 (2010). https://doi.org/10.1007/s00726-009-0429-2

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