A Chemical Proteomics Approach for the Search of Pharmacological Targets of the Antimalarial Clinical Candidate Albitiazolium in Plasmodium falciparum Using Photocrosslinking and Click Chemistry

Abstract

Plasmodium falciparum is responsible for severe malaria which is one of the most prevalent and deadly infectious diseases in the world. The antimalarial therapeutic arsenal is hampered by the onset of resistance to all known pharmacological classes of compounds, so new drugs with novel mechanisms of action are critically needed. Albitiazolium is a clinical antimalarial candidate from a series of choline analogs designed to inhibit plasmodial phospholipid metabolism. Here we developed an original chemical proteomic approach to identify parasite proteins targeted by albitiazolium during their native interaction in living parasites. We designed a bifunctional albitiazolium-derived compound (photoactivable and clickable) to covalently crosslink drug–interacting parasite proteins in situ followed by their isolation via click chemistry reactions. Mass spectrometry analysis of drug–interacting proteins and subsequent clustering on gene ontology terms revealed parasite proteins involved in lipid metabolic activities and, interestingly, also in lipid binding, transport, and vesicular transport functions. In accordance with this, the albitiazolium-derivative was localized in the endoplasmic reticulum and trans-Golgi network of P. falciparum. Importantly, during competitive assays with albitiazolium, the binding of choline/ethanolamine phosphotransferase (the enzyme involved in the last step of phosphatidylcholine synthesis) was substantially displaced, thus confirming the efficiency of this strategy for searching albitiazolium targets.

Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

“…Using alkyne/azide-coupling partners through "click chemistry," we identified several cytochrome P450 enzymes that metabolized deltamethrin in rat liver microsomes (10). More recently, a chemical proteomic approach was developed to identify parasite proteins targeted by an albitiazolium antimalarial drug candidate in situ using a photoactivation cross-linking approach (11). However, this generic approach can introduce significant promiscuity in the proteins tagged based on the intracompartmental distribution of drug independent of actual mechanisms.…”

Muddled mechanisms: recent progress towards antimalarial target identification

“…A typical chemical strategy is synthesis of compound analogs to incorporate a “click” handle to facilitate drug target identification and validation in P. falciparum 95 . For example, a bifunctional compound based on the clinical candidate albitiazolium was synthesized that was photoactivatable and taggable 96 . MS identified a discrete list of potential drug targets in P. falciparum , while bioinformatic and interactome analyses were used to predict protein functions 96 .…”

“…For example, a bifunctional compound based on the clinical candidate albitiazolium was synthesized that was photoactivatable and taggable 96 . MS identified a discrete list of potential drug targets in P. falciparum , while bioinformatic and interactome analyses were used to predict protein functions 96 . As albitiazolium inhibits phospholipid metabolism, most of the target proteins are involved in lipid metabolic activities 96 .…”

“…MS identified a discrete list of potential drug targets in P. falciparum , while bioinformatic and interactome analyses were used to predict protein functions 96 . As albitiazolium inhibits phospholipid metabolism, most of the target proteins are involved in lipid metabolic activities 96 . A number of surprising targets were also uncovered, such as proteins involved in vesicular budding and transport functions, thereby demonstrating the utility of the method 96 .…”

Chloroquine-containing compounds: a patent review (2010 – 2014)

“…The IC 50 values against 3D7 for CQ and 64 were 42.7 and 349.3 nM, respectively [35]. This approach has recently been successfully employed by the Vial group in Montpellier to search for pharmacological targets of the antimalarial clinical candidate albitiazolium by integrating photo-crosslinking and click chemistry [36]. In this study, the authors identified proteins involved in phospholipid metabolism, lipid binding and transport, as well as in vesicular transport functions through whole cell imagery, in-gel detection with fluorescent reporters and affinity purification.…”