The efficacy of therapeutics is dependent on a drug binding to its cognate target. Optimization of target engagement by drugs in cells is often challenging, because drug binding cannot be monitored inside cells. We have developed a method for evaluating drug binding to target proteins in cells and tissue samples. This cellular thermal shift assay (CETSA) is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. Using this assay, we validated drug binding for a set of important clinical targets and monitored processes of drug transport and activation, off-target effects and drug resistance in cancer cell lines, as well as drug distribution in tissues. CETSA is likely to become a valuable tool for the validation and optimization of drug target engagement.

Thermal shift assays are used to study thermal stabilization of proteins upon ligand binding. Such assays have been used extensively on purified proteins in the drug discovery industry and in academia to detect interactions. Recently, we published a proof-of-principle study describing the implementation of thermal shift assays in a cellular format, which we call the cellular thermal shift assay (CETSA). The method allows studies of target engagement of drug candidates in a cellular context, herein exemplified with experimental data on the human kinases p38α and ERK1/2. The assay involves treatment of cells with a compound of interest, heating to denature and precipitate proteins, cell lysis, and the separation of cell debris and aggregates from the soluble protein fraction. Whereas unbound proteins denature and precipitate at elevated temperatures, ligand-bound proteins remain in solution. We describe two procedures for detecting the stabilized protein in the soluble fraction of the samples. One approach involves sample workup and detection using quantitative western blotting, whereas the second is performed directly in solution and relies on the induced proximity of two target-directed antibodies upon binding to soluble protein. The latter protocol has been optimized to allow an increased throughput, as potential applications require large numbers of samples. Both approaches can be completed in a day.

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We recently demonstrated that 2,3,4,5,6-pentahydroxyxanthone (X5) inhibits the in vitro growth of both chloroquine-sensitive and multidrug-resistant strains of P. falcipamm.To study the molecular basis of its antimalarial action, we tested X5 and selected hydroxyxanthone analogs as inhibitors of in vitro heme polymerization in a low ionic strength phosphate solution at mildly acidic pH. We found that addition of 1 Eq. of X5 resulted in complete inhibition of polymerization in this system whereas addition of up to 40 Eqs. of standard antimalarial compounds (chloroquine, primaquine, quinacrine, artemisinin and méthylène blue) had no such effect although these compounds did co-precipitate with heme. The antimalarial potency of the hydroxyxanthones correlated well with their ability to inhibit in vitro heme polymerization in our assay, suggesting that these compounds exert their antimalarial action by preventing hemozoin formation. Based on the observed structure-activity relationships, we propose a model displaying possible interactions between hydroxyxanthones and heme.

Abstract. The erythrocytic development of Plasmodium falciparum is divided into the ring, trophozoite, and schizont stages based on morphologic assessment. Using highly synchronous ring and trophozoite cultures of P. falciparum, we observed considerable differences in their sensitivity to hydroxyxanthones: trophozoites were much more sensitive to the drugs than ring-stage parasites. Trophozoites treated with a prototypic xanthone, the 2,3,4,5,6-pentahydroxy derivative (X5), were arrested in their development and became degenerate in appearance within 24 hr of drug exposure. These morphologic changes appeared to reflect the cytotoxic nature of the action of the drug against the parasite, since daughter ring-stage forms were not observed following addition of the drug. That X5 was more active against parasites in the later stages of intraerythrocytic development is consistent with the proposed mode of action, inhibition of heme polymerization. Knowledge of the structure-activity relationships for xanthones as antimalarial agents has also been expanded. Xanthones with a hydroxyl group in the peri-position exhibited decreased antimalarial activity, possibly due to intramolecular hydrogen bonding with the carbonyl and consequent reduced affinity for heme. Paired hydroxyls attached to the lower half of the xanthone greatly enhanced drug potency.We were led to investigate the antiparasitic action of xanthones by the discovery of a remarkable antimalarial synergy between exifone (a hexahydroxybenzophenone) and two oxidant drugs (rufigallol and ascorbic acid). [1][2][3] We speculated that free radical hydroxylation of exifone followed by cyclodehydration inside parasitized red blood cells results in the formation of 2,3,4,5,6-pentahydroxyxanthone (X5), and that this xanthone is responsible for the enhanced antimalarial effect. 3 Synthetic X5 was shown to possess an impressive inhibitory activity in vitro against both chloroquinesensitive and multidrug-resistant strains of Plasmodium falciparum. 3As a direct result of hemoglobin digestion, a vast quantity of heme is liberated in the food vacuole of the parasite. 4 To avoid accumulation of toxic heme during this catabolic process, the parasite has evolved a mechanism of heme polymerization that results in the formation of an insoluble substance commonly referred to as hemozoin. We have demonstrated the ability of X5 to form soluble complexes with heme and to prevent the polymerization of heme in vitro. 5 Figure 1 shows the results of one such experiment in which heme was incubated under mildly acidic conditions in the presence and absence of X5. These findings support the hypothesis that the antimalarial activity of X5 is due to inhibition of heme polymerization in the parasite digestive vacuole. Structure-activity profiling of a limited number of xanthones pointed to the 4,5-dihydroxy derivative as the minimal structural unit of X5 that retained activity in the heme polymerization assay and against malarial parasites.In this report we describe the stage-specific action of...