Activity-Based Proteome Profiling of Potential Cellular Targets of Orlistat − An FDA-Approved Drug with Anti-Tumor Activities

Abstract

Orlistat, or tetrahydrolipstatin (THL), is an FDA-approved antiobesity drug with potential antitumor activities. Cellular off-targets and potential side effects of Orlistat in cancer therapies, however, have not been extensively explored thus far. In this study, we report the total of synthesis of THL-like protein-reactive probes, in which extremely conservative modifications (i.e., an alkyne handle) were introduced in the parental THL structure to maintain the native biological properties of Orlistat, while providing the necessary functionality for target identification via the bio-orthogonal click chemistry. With these natural productlike, cell-permeable probes, we were able to demonstrate, for the first time, this chemical proteomic approach is suitable for the identification of previously unknown cellular targets of Orlistat. In addition to the expected fatty acid synthase (FAS), we identified a total of eight new targets, some of which were further validated by experiments including Western blotting, recombinant protein expression, and site-directed mutagenesis. Our findings have important implications in the consideration of Orlistat as a potential anticancer drug at its early stages of development for cancer therapy. Our strategy should be broadly useful for off-target identification against quite a number of existing drugs and/or candidates, which are also covalent modifiers of their biological targets.

Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

“…THL-alkyne analogs, rhodamine-azide, biotin-azide and the trifunctional probe were synthesized as described previously (25). THL and its analogs were dissolved in DMSO for all experiments.…”

“…THL was previously shown to inhibit both active and latent forms of mycobacteria (11, 20 -22) but the bacterial target spectrum remains poorly characterized. Therefore, to (1) define the THL target spectrum in a mycobacterial species and (2) to obtain biochemical insights into regulation of lipases and esterases in different metabolic states, we employed a chemical-proteomics approach using activity-based protein profiling (ABPP) with a bait that has been described to bind to lipolytic enzymes (23)(24)(25). We identified several known lipases (as anticipated), putative lipase and esterases, and hypothetical proteins of unknown functions, thereby providing a comprehensive resource of experimentally determined THL targets in mycobacteria.…”

A Quantitative Chemical Proteomics Approach to Profile the Specific Cellular Targets of Andrographolide, a Promising Anticancer Agent That Suppresses Tumor Metastasis

“…To overcome this limitation, several groups have used activity-based protein profiling (ABPP) combined with bio-orthogonal click chemistry to identify drug targets both in vitro and in vivo (supplemental Fig. S1) (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). ABPP probes exert their functions via covalent reactions with the target proteins or photoaffinity-based labeling via the incorporation of photoreactive groups.…”

Click-based synthesis and proteomic profiling of lipstatin analogues

“…1). 4 The b-lactone pharmacophore and amino ester would thus be preserved (to maintain sufficient binding/activity to FAS) and a terminal alkyne handle introduced to the right aliphatic side chain (for identification of cellular targets via the downstream conjugation to reporter tags). 6,7 A left alkyne-terminated aliphatic side chain would then be diversified through a triazole ring (cf.…”