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A rapid and robust assay for the determination of the amino acid hypusine as a possible biomarker for a high-throughput screening of antimalarials and for the diagnosis and therapy of different diseases

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Abstract

Eukaryotic initiation factor 5A (eIF5A) has recently been identified as a biomarker of prognostic significance and therapeutic potential for the treatment in hepatocellular carcinoma. This prompted us to establish a rapid and robust assay to determine deoxyhypusine and hypusine formed with the purified enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) from Plasmodium to develop a rapid screening assay for antimalarial drugs. The peptide hydrolysate obtained from hypusinylated eIF5A was analyzed by ultra performance liquid chromatography (UPLC) with retention times for deoxyhypusine of 7.44 min and for hypusine of 7.30 min, respectively. The limit of detection for both compounds was 0.144 ng/μl. Determination of the specific activity of Plasmodium DOHH resulted in a twofold higher specific activity than its human counterpart. Following the iron-complexing strategy of the ferrous iron which is present in the active site of Plasmodium DOHH, a series of iron chelating compounds was tested. 2,2′-Dipyridyl and mimosine abolished DOHH activity completely while 4-oxo-piperidine-carboxylates i.e. the nitrophenylether JK8-2 and EHW 437, the oxime ether of the piperidine aldehyde, showed no inhibition although they were highly active in in vitro cultures of Plasmodium and in vivo in a rodent mouse model. The method allows a high-throughput screening (HPTS) of antimalarial drugs and the evaluation of eIF5A as a biomarker.

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Abbreviations

eIF5A:

Eukaryotic initiation factor 5A

UPLC:

Ultra performance liquid chromatography

HPTS:

High-throughput screening

DHS:

Deoxyhypusine synthase

DOHH:

Deoxyhypusine hydroxylase

AccQ-Tag:

6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate

2ME:

2-Mercaptoethanol

OPA:

ortho-Phthalaldehyde

PDA:

Photodiode-array detector

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Acknowledgments

The authors thank Dr. T. Langrock from Waters, Eschborn, Germany for the support and advice. Drugs EHW 437 and JK8-2 were kindly provided by Professor Dr. U. Holzgrabe and are described within (Goebel et al. 2008).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Institut für Technische Chemie und Pharmazeutische Chemie, University of Applied Sciences, Cologne, Betzdorfer Str.2, 50679, Cologne, Germany

    Annette Kaiser

  2. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilow Street 32, 119991, Moscow, Russia

    Alex R. Khomutov & Alina Simonian

  3. Department of Biochemical Sciences ‘A. Rossi Fanelli’, SAPIENZA University of Rome and CNR, Biology and Molecular Pathology Institutes, P.le Aldo Moro 5, 00185, Rome, Italy

    Enzo Agostinelli

Authors
  1. Annette Kaiser
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  2. Alex R. Khomutov
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  4. Enzo Agostinelli
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Correspondence to Annette Kaiser.

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Kaiser, A., Khomutov, A.R., Simonian, A. et al. A rapid and robust assay for the determination of the amino acid hypusine as a possible biomarker for a high-throughput screening of antimalarials and for the diagnosis and therapy of different diseases. Amino Acids 42, 1651–1659 (2012). https://doi.org/10.1007/s00726-011-0859-5

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