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. 2016 May 23;55(22):6401-5.
doi: 10.1002/anie.201512062. Epub 2016 Apr 18.

A Click Chemistry-Based Proteomic Approach Reveals that 1,2,4-Trioxolane and Artemisinin Antimalarials Share a Common Protein Alkylation Profile

Hanafy M Ismail  1 , Victoria E Barton  2 , Matthew Panchana  1 , Sitthivut Charoensutthivarakul  2 , Giancarlo A Biagini  1 , Stephen A Ward  1 , Paul M O'Neill  3
Affiliations

A Click Chemistry-Based Proteomic Approach Reveals that 1,2,4-Trioxolane and Artemisinin Antimalarials Share a Common Protein Alkylation Profile

Hanafy M Ismail et al. Angew Chem Int Ed Engl. .

Erratum in

Abstract

In spite of the recent increase in endoperoxide antimalarials under development, it remains unclear if all these chemotypes share a common mechanism of action. This is important since it will influence cross-resistance risks between the different classes. Here we investigate this proposition using novel clickable 1,2,4-trioxolane activity based protein-profiling probes (ABPPs). ABPPs with potent antimalarial activity were able to alkylate protein target(s) within the asexual erythrocytic stage of Plasmodium falciparum (3D7). Importantly, comparison of the alkylation fingerprint with that generated from an artemisinin ABPP equivalent confirms a highly conserved alkylation profile, with both endoperoxide classes targeting proteins in the glycolytic, hemoglobin degradation, antioxidant defence, protein synthesis and protein stress pathways, essential biological processes for plasmodial survival. The alkylation signatures of the two chemotypes show significant overlap (ca. 90 %) both qualitatively and semi-quantitatively, suggesting a common mechanism of action that raises concerns about potential cross-resistance liabilities.

Keywords: antimalarial; artemisinin; chemical biology; probes.

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Figures

Figure 1
Figure 1
Artemisinin 1 a, semi‐synthetics 1 b and 1 c with structures of frontline synthetic peroxide based antimalarials OZ277 (2 b) and OZ439 (2 c).
Scheme 1
Scheme 1
Iron‐mediated fragmentation of endoperoxides to reactive intermediates capable of reacting with parasite proteins. (Only the secondary carbon‐centered radical derived from artemisinin is depicted.)
Figure 2
Figure 2
Rational design of endoperoxide activity based probes P1 (6 a) and P2 (7 a) with structures of control non‐peroxidic derivatives CP1 (6 b) and CP2 (7 b).
Scheme 2
Scheme 2
Synthesis of probes and control molecules used in chemical proteomic interrogation of drug activation.
Figure 3
Figure 3
Labeling of parasite proteins (P. falciparum, 3D7 strain) using 7 a. a) Chemical structure of ozonide azide probe (P2 (7 a)) and deoxyether analogue (CP2 (7 b)) and their antimalarial activity against P. falciparum 3D7. b) General workflows of copper‐free click methodology for in situ parasite protein identification using azide trioxolanes probes as detailed mentioned in methodology section. c) Fluorescence image of 1D gel for proteins labeled in situ with alkyne probes (P1 (6 a) and CP1 (6 b)) vs. azide probes (P2 (7 a) and CP2 (7 b)), note that no labeling occurs with negative control alkyne (CP1 (6 b)) and azide control (CP2 (7 b)). d) Arbitrary fluorescence intensity measurements of the major protein bands labeled and identified with 20 μm Alexa flour 488 azide for parasite proteins tagged with 1 μm of alkyne probe (P1 (6 a)) vs. proteins tagged with 1 μm of azide probe (P2 (7 a)) identified with 20 μm Click‐IT Alexa Fluor 488 DIBO Alkyne. Fluorescence arbitrary units reveal higher sensitivity in case of bio‐orthogonal copper free click reaction, that is, P2 (7 a) treatment. e) Gel image of P2 (7 a) treatment vs. control, pre and post coomassie stain with equal protein loading. f) Fluorescence image representing probe titration from 1 to 0.1 μm P2 (7 a) probe; proteins identified via copper free click reaction with Click‐IT Alexa Fluor 488 DIBO Alkyne. No changes were observed in labeling profiles of the trioxolane‐tagged proteins with concentrations relevant to pharmacological concentration of the drug (100 nm). g) Titration of DIBO dye at various concentrations up to 20 μm for parasite treated in situ with 1 μm P2 (7 a). h) Time dependent increase of fluorescence signal for proteins tagged with 1 μm P2 (7 a) and 20 μm Click‐IT Alexa Fluor 488 DIBO Alkyne indicating that the maximum band intensities could be achieved after 1‐hour of click reaction incubation.
Figure 4
Figure 4
Mass spectrometry experiments with azide trioxolane azide probe (P2 (7 a)) vs. artemsinin azide probe (P3 (11 a)). a) Venn diagram demonstrating overlap between proteins identified with the endoperoxide probes, P2 (7 a) and P3 (11 a) respectively. (b) Percentage of the glutathionylated proteins, which contains the GSH binding motif that was identified with endoperoxides probes P2 (7 a) and P3 (11 a) in light of Kehr et al.11 (c) Head to head comparison between proteins identified with P2 (7 a) vs. P3 (11 a). Proteins sorted according to their molecular weight from high to low. Errors bars represented the standard deviation for protein quantity in each treatment calculated by dividing the exponentially modified protein abundance index (emPAI)16 for each protein by the total emPAI values (each treatment contain two replicate, for accuracy each replicate is the average of four injections into the Orbitrap LC‐MS/MS instrument).
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