UCSF

Activity-based protein profiling with chemical probes derived from cysteine-reactive small molecule inhibitors provides a mechanism for prioritizing target lists for further studies and lead optimization. Thousands of proteins remain uncharacterized for the parasitic organisms that are responsible for a variety of tropical diseases. This makes target selection for the development of new, safer therapeutic agents difficult. The current deficit in the understanding of parasitic protein function has contributed to the slow progress toward the eradication of these diseases. Chemoproteomic profiling of cysteine-reactive small molecules with anti-parasitic activity can help identify and validate potential drug targets, while also providing useful tools to study the biological roles of these proteins in cells.

Protein kinases have emerged as potential therapeutic targets for many diseases, including African trypanosomiasis caused by Trypanosoma brucei. However, identifying relevant kinase drug targets is difficult because most of the 182 T. brucei protein kinases are still uncharacterized. Hypothemycin, an electrophilic kinase inhibitor shown to react selectively with kinases bearing a "CDXG" motif, was found to potently kill T. brucei parasites in tissue culture and in infected mice. Using quantitative chemoproteomics with propargyl-hypothemycin, eight CDXG kinases inhibited by hypothemycin were identified; four of these exhibited greater than 50% inhibition by 200 nM hypothemycin in vitro. Of those four, only TbCLK1 was both essential by RNAi and inhibited in the cells at concentrations of hypothemycin sufficient to kill the parasite in tissue culture. We later found that expression of an allele of TbCLK1 resistant to hypothemycin was insufficient to rescue the parasites in a cellular proliferation assay. This result suggests that the inhibition of other cellular targets may contribute to hypothemycin's trypanocidal activity, but does not diminish the value of TbCLK1 as a potential therapeutic target.

In a continued effort to validate TbCLK1 as a therapeutic target and to study its role in cells, we hypothesized a chemical genetics (pharmacological) approach would be most appropriate. To generate selective inhibitors of TbCLK1, a second non-conserved active site cysteine, found in only two T. brucei kinases (TbCLK1/2), was exploited for the development of covalent inhibitors. Our most promising series of electrophilic compounds, based on AD57, are potent (IC₅₀ < 200 nM), allele-selective (> 50-fold) inhibitors of wild-type TbCLK1 in vitro with potent (EC₅₀ < 1 µM) anti-proliferative activity. These molecules, when used in combination with cells expressing compound-resistant TbCLK1, have the potential to be powerful tools to elucidate the cellular functions of TbCLK1 kinase activity.

Activity-based protein profiling was also used to identify targets of the cysteine protease inhibitor K777 and related analogs. These molecules were unexpectedly found to inhibit both cruzain and TcCYP51, a cysteine protease and a 14-α-demethylase enzyme, respectively, in Trypanosoma cruzi. Specifically, we designed and synthesized a chemical probe, propargyl-K777, with the aim of characterizing the cysteine protease related effects of K777 and the putative dual-targeted analogs. Attempts to label and identify parasite derived targets of propargyl-K777 proved challenging due to competitive reaction with an abundant host-cell protein. Chemoproteomics using propargyl-K777 identified this host cell off-target as cathepsin B. The probe was also used to show that some of our non-basic analogs do not significantly inhibit cathepsin B at anti-trypanosomal concentrations. Thus, this new probe can serve as a tool to assay future compounds for inhibition of cathepsin B in a cellular context in order to minimize this off-target activity.