Influence of side chains on phorbol ester binding to protein kinase C

The phorbol esters, natural products derived from plants of the family Euphorbiaceae, are potent tumor promoters and modulators of biological response. Their major site of action is protein kinase C. Three classes of derivatives were examined to better assess the influence of the ester side chains on phorbol ester binding affinity for protein kinase C. A series of phorbol diesters substituted at the 13‐OH with biotin separated from the phorbol nucleus by linker arms of variable length were prepared. Although all showed good potency by themselves (half‐maximally effective doses of 12–67 nM), addition of avidin greatly inhibited activity, suggesting significant steric constraints on the side chains for retention of activity. At the other extreme, very high concentrations of the unsubstituted parent alcohol phorbol were examined to determine whether phorbol was actually inactive or whether it might simply possess weak binding activity. Inhibition was in fact observed with an apparent K i of approximately 10 mM. The inhibition was time dependent and not stereospecific, however, suggesting that the inhibition was not due to competitive binding. The third class of compounds examined were phorbol 12‐ and 13‐monoesters. Although approximately 20‐fold less potent than the 13‐monoesters, the 12‐monoesters showed substantial binding activity, in contrast to their reported lack of activity in vivo .