Tool compounds are used to explore the role of a specific protein in a biological context and – it goes without saying – that to obtain meaningful results in a complex situation, the tool compound should have appropriate potency and selectivity. The family of phospholipase C (PLC) enzymes play important regulatory roles and a small molecule inhibitor, U73122, has been extensively used to provide evidence for the involvement of PLCs in many cellular pathways. Recent reports, however, have questioned the selectivity of U73122 and scientists at the University of North Carolina and GlaxoSmithKline have now discovered that, even in its interaction with PLCs, U73122 may not be quite what it seems. When the team explored the effects of U73122 on human PLCs in cell-free micellar systems, they found that the compound actually increased the enzymatic activity of a number of isoforms in a concentration- and time-dependent manner. At micromolar concentrations, U713122 increased the activity of PLCβ3 by up to eight-fold, that of PLCγ1 by more than ten-fold, and that of PLCβ2 by around two-fold; PLCδ1 was neither activated nor inhibited.
Activation of PLCβ3 was attenuated by competing nucleophiles, suggesting that activation involves covalent modification of the protein by the reactive maleimide group of U73122; the analogous succinimide, U73343, was not effective as an activator. Involvement of specific cysteine residues in the protein was demonstrated by LC/MS/MS experiments. Although N-ethyl maleimide (NEM) itself did not activate PLCβ3, excess NEM attenuated the U73122-mediated activation in a concentration-dependent manner. The authors propose an activation model in which U73122 irreversibly binds to multiple cysteine residues on PLCβ3 and acts as either a lipid anchor or interfacial recognition site for the enzyme, facilitating adsorption to the substrate interface (i.e. the micelle surface). The protein-linked U73122 increases the rate of lipase activity by keeping the enzyme in close proximity to substrate which is held in the membrane.
The study, which is published in the Journal of Biological Chemistry, provides strong evidence that U73122 activates PLC enzymes in cell-free systems, in contrast to its ‘established’ role as a specific inhibitor of this family. The authors suggest that U73122 may have opposing effects on cytosolic and membrane-bound enzymes and/or may modify other cellular nucleophiles and advise great caution when forming hypotheses based on the observed effects of U73122 in cellular systems.