Proteomic Ligandability Maps of Phosphorus(V) Stereoprobes Identify Covalent TLCD1 Inhibitors

Activity-based protein profiling
(ABPP) of stereoisomerically
defined
sets of electrophilic compounds (‘stereoprobes’) offers
a versatile way to discover covalent ligands for proteins in native
biological systems. Here we report the synthesis and chemical proteomic
characterization of stereoprobes bearing a P(V)-oxathiaphospholane
(OTP) reactive group. ABPP experiments identified numerous proteins
in human cancer cells that showed stereoselective reactivity with
OTP stereoprobes, and we confirmed several of these liganding events
with recombinant proteins. OTP stereoprobes engaging the poorly characterized
transmembrane protein TLCD1 impaired the incorporation of monounsaturated
fatty acids into phosphatidylethanolamine lipids in cells, a lipidomic
phenotype that mirrored genetic disruption of this protein. Using
AlphaFold2, we found that TLCD1 structurally resembles the ceramide
synthase and fatty acid elongase families of coenzyme A-dependent
lipid processing enzymes. This structural similarity included conservation
of catalytic histidine residues, the mutation of which blocked the
OTP stereoprobe reactivity and lipid remodeling activity of recombinant
TLCD1. Taken together, these data indicate that TLCD1 acts as a lipid
acyltransferase in cells, and that OTP stereoprobes function as inhibitors
of this enzymatic activity. Our findings thus illuminate how the chemical
proteomic analysis of electrophilic compounds can facilitate the functional
annotation and chemical inhibition of a key lipid metabolic enzyme
in human cells.