Monoclonal Antibodies That Recognize the Alkylation Signature of Antimalarial Ozonides OZ277 (Arterolane) and OZ439 (Artefenomel)

Joëlle Jourdan, Hugues Matile, Ellen Reift, Oliver Biehlmaier, Yuxiang Dong, Xiaofang Wang, Pascal Mäser, Jonathan L. Vennerstrom, Sergio Wittlin

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

The singular structure of artemisinin, with its embedded 1,2,4-trioxane heterocycle, has inspired the discovery of numerous semisynthetic artemisinin and structurally diverse synthetic peroxide antimalarials, including ozonides OZ277 (arterolane) and OZ439 (artefenomel). Despite the critical importance of artemisinin combination therapies (ACTs), the precise mode of action of peroxidic antimalarials is not fully understood. However, it has long been proposed that the peroxide bond in artemisinin and other antimalarial peroxides undergoes reductive activation by ferrous heme released during hemoglobin digestion to produce carbon-centered radicals that alkylate heme and parasite proteins. To probe the mode of action of OZ277 and OZ439, this paper now describes initial studies with monoclonal antibodies that recognize the alkylation signature (sum of heme and protein alkylation) of these synthetic peroxides. Immunofluorescence experiments conducted with ozonide-treated parasite cultures showed that ozonide alkylation is restricted to the parasite, as no signal was found in the erythrocyte or its membrane. In Western blot experiments with ozonide-treated Plasmodium falciparum malaria parasites, distinct protein bands were observed. Significantly, no protein bands were detected in parallel Western blot experiments performed with lysates from ozonide-treated Babesia divergens, parasites that also proliferate inside erythrocytes but, in contrast to P. falciparum, do not catabolize hemoglobin. However, subsequent immunoprecipitation experiments with these antibodies failed to identify the P. falciparum proteins alkylated by OZ277 and OZ439. To the best of the authors' knowledge, this shows for the first time that antimalarial ozonides, such as the artemisinins, alkylate proteins in P. falciparum.

Original languageEnglish (US)
Pages (from-to)54-61
Number of pages8
JournalACS infectious diseases
Volume2
Issue number1
DOIs
StatePublished - Jan 8 2016

Fingerprint

Antimalarials
Alkylation
Monoclonal Antibodies
Peroxides
Parasites
Plasmodium falciparum
Proteins
Heme
Artemisinins
Hemoglobins
Erythrocytes
Western Blotting
Hemeproteins
Babesia
Falciparum Malaria
arterolane
1,2,4-trioxane
Immunoprecipitation
Fluorescent Antibody Technique
Digestion

Keywords

  • Plasmodium falciparum
  • alkylation
  • artemisinin
  • immunofluorescence
  • monoclonal antibody
  • ozonide

ASJC Scopus subject areas

  • Infectious Diseases

Cite this

Monoclonal Antibodies That Recognize the Alkylation Signature of Antimalarial Ozonides OZ277 (Arterolane) and OZ439 (Artefenomel). / Jourdan, Joëlle; Matile, Hugues; Reift, Ellen; Biehlmaier, Oliver; Dong, Yuxiang; Wang, Xiaofang; Mäser, Pascal; Vennerstrom, Jonathan L.; Wittlin, Sergio.

In: ACS infectious diseases, Vol. 2, No. 1, 08.01.2016, p. 54-61.

Research output: Contribution to journalArticle

Jourdan, Joëlle ; Matile, Hugues ; Reift, Ellen ; Biehlmaier, Oliver ; Dong, Yuxiang ; Wang, Xiaofang ; Mäser, Pascal ; Vennerstrom, Jonathan L. ; Wittlin, Sergio. / Monoclonal Antibodies That Recognize the Alkylation Signature of Antimalarial Ozonides OZ277 (Arterolane) and OZ439 (Artefenomel). In: ACS infectious diseases. 2016 ; Vol. 2, No. 1. pp. 54-61.
@article{02a21bdc5d1d42a0b86dec328b345833,
title = "Monoclonal Antibodies That Recognize the Alkylation Signature of Antimalarial Ozonides OZ277 (Arterolane) and OZ439 (Artefenomel)",
abstract = "The singular structure of artemisinin, with its embedded 1,2,4-trioxane heterocycle, has inspired the discovery of numerous semisynthetic artemisinin and structurally diverse synthetic peroxide antimalarials, including ozonides OZ277 (arterolane) and OZ439 (artefenomel). Despite the critical importance of artemisinin combination therapies (ACTs), the precise mode of action of peroxidic antimalarials is not fully understood. However, it has long been proposed that the peroxide bond in artemisinin and other antimalarial peroxides undergoes reductive activation by ferrous heme released during hemoglobin digestion to produce carbon-centered radicals that alkylate heme and parasite proteins. To probe the mode of action of OZ277 and OZ439, this paper now describes initial studies with monoclonal antibodies that recognize the alkylation signature (sum of heme and protein alkylation) of these synthetic peroxides. Immunofluorescence experiments conducted with ozonide-treated parasite cultures showed that ozonide alkylation is restricted to the parasite, as no signal was found in the erythrocyte or its membrane. In Western blot experiments with ozonide-treated Plasmodium falciparum malaria parasites, distinct protein bands were observed. Significantly, no protein bands were detected in parallel Western blot experiments performed with lysates from ozonide-treated Babesia divergens, parasites that also proliferate inside erythrocytes but, in contrast to P. falciparum, do not catabolize hemoglobin. However, subsequent immunoprecipitation experiments with these antibodies failed to identify the P. falciparum proteins alkylated by OZ277 and OZ439. To the best of the authors' knowledge, this shows for the first time that antimalarial ozonides, such as the artemisinins, alkylate proteins in P. falciparum.",
keywords = "Plasmodium falciparum, alkylation, artemisinin, immunofluorescence, monoclonal antibody, ozonide",
author = "Jo{\"e}lle Jourdan and Hugues Matile and Ellen Reift and Oliver Biehlmaier and Yuxiang Dong and Xiaofang Wang and Pascal M{\"a}ser and Vennerstrom, {Jonathan L.} and Sergio Wittlin",
year = "2016",
month = "1",
day = "8",
doi = "10.1021/acsinfecdis.5b00090",
language = "English (US)",
volume = "2",
pages = "54--61",
journal = "ACS Infectious Diseases",
issn = "2373-8227",
publisher = "American Chemical Society",
number = "1",

}

TY - JOUR

T1 - Monoclonal Antibodies That Recognize the Alkylation Signature of Antimalarial Ozonides OZ277 (Arterolane) and OZ439 (Artefenomel)

AU - Jourdan, Joëlle

AU - Matile, Hugues

AU - Reift, Ellen

AU - Biehlmaier, Oliver

AU - Dong, Yuxiang

AU - Wang, Xiaofang

AU - Mäser, Pascal

AU - Vennerstrom, Jonathan L.

AU - Wittlin, Sergio

PY - 2016/1/8

Y1 - 2016/1/8

N2 - The singular structure of artemisinin, with its embedded 1,2,4-trioxane heterocycle, has inspired the discovery of numerous semisynthetic artemisinin and structurally diverse synthetic peroxide antimalarials, including ozonides OZ277 (arterolane) and OZ439 (artefenomel). Despite the critical importance of artemisinin combination therapies (ACTs), the precise mode of action of peroxidic antimalarials is not fully understood. However, it has long been proposed that the peroxide bond in artemisinin and other antimalarial peroxides undergoes reductive activation by ferrous heme released during hemoglobin digestion to produce carbon-centered radicals that alkylate heme and parasite proteins. To probe the mode of action of OZ277 and OZ439, this paper now describes initial studies with monoclonal antibodies that recognize the alkylation signature (sum of heme and protein alkylation) of these synthetic peroxides. Immunofluorescence experiments conducted with ozonide-treated parasite cultures showed that ozonide alkylation is restricted to the parasite, as no signal was found in the erythrocyte or its membrane. In Western blot experiments with ozonide-treated Plasmodium falciparum malaria parasites, distinct protein bands were observed. Significantly, no protein bands were detected in parallel Western blot experiments performed with lysates from ozonide-treated Babesia divergens, parasites that also proliferate inside erythrocytes but, in contrast to P. falciparum, do not catabolize hemoglobin. However, subsequent immunoprecipitation experiments with these antibodies failed to identify the P. falciparum proteins alkylated by OZ277 and OZ439. To the best of the authors' knowledge, this shows for the first time that antimalarial ozonides, such as the artemisinins, alkylate proteins in P. falciparum.

AB - The singular structure of artemisinin, with its embedded 1,2,4-trioxane heterocycle, has inspired the discovery of numerous semisynthetic artemisinin and structurally diverse synthetic peroxide antimalarials, including ozonides OZ277 (arterolane) and OZ439 (artefenomel). Despite the critical importance of artemisinin combination therapies (ACTs), the precise mode of action of peroxidic antimalarials is not fully understood. However, it has long been proposed that the peroxide bond in artemisinin and other antimalarial peroxides undergoes reductive activation by ferrous heme released during hemoglobin digestion to produce carbon-centered radicals that alkylate heme and parasite proteins. To probe the mode of action of OZ277 and OZ439, this paper now describes initial studies with monoclonal antibodies that recognize the alkylation signature (sum of heme and protein alkylation) of these synthetic peroxides. Immunofluorescence experiments conducted with ozonide-treated parasite cultures showed that ozonide alkylation is restricted to the parasite, as no signal was found in the erythrocyte or its membrane. In Western blot experiments with ozonide-treated Plasmodium falciparum malaria parasites, distinct protein bands were observed. Significantly, no protein bands were detected in parallel Western blot experiments performed with lysates from ozonide-treated Babesia divergens, parasites that also proliferate inside erythrocytes but, in contrast to P. falciparum, do not catabolize hemoglobin. However, subsequent immunoprecipitation experiments with these antibodies failed to identify the P. falciparum proteins alkylated by OZ277 and OZ439. To the best of the authors' knowledge, this shows for the first time that antimalarial ozonides, such as the artemisinins, alkylate proteins in P. falciparum.

KW - Plasmodium falciparum

KW - alkylation

KW - artemisinin

KW - immunofluorescence

KW - monoclonal antibody

KW - ozonide

UR - http://www.scopus.com/inward/record.url?scp=84969247696&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84969247696&partnerID=8YFLogxK

U2 - 10.1021/acsinfecdis.5b00090

DO - 10.1021/acsinfecdis.5b00090

M3 - Article

C2 - 26819968

AN - SCOPUS:84969247696

VL - 2

SP - 54

EP - 61

JO - ACS Infectious Diseases

JF - ACS Infectious Diseases

SN - 2373-8227

IS - 1

ER -