The development of activating and inhibiting camelid VHH domains against human protein kinase C epsilon

The 10 isozymes of the protein kinase C (PKC) family can have different roles on the same biological process, making isozyme specific analysis of function crucial. Currently, only few pharmacological compounds with moderate isozyme specific effects exist thus hampering research into individual PKC isozymes. The antigen binding regions of camelid single chain antibodies (VHHs) could provide a solution for obtaining PKC isozyme specific modulators. In the present study, we have successfully selected and characterized PKC epsilon specific VHH antibodies from two immune VHH libraries using phage display. The VHHs were shown to exclusively bind to PKC epsilon in ELISA and immunoprecipitation studies. Strikingly, five of the VHHs had an effect on PKC epsilon kinase activity in vitro. VHHs A10, C1 and D1 increased PKC epsilon kinase activity in a concentration-dependent manner (EC50 values: 212-310 nM), whereas E6 and G8 inhibited PKC epsilon activity (IC50 values: 103-233 nM). None of these VHHs had an effect on the activity of the other novel PKC isozymes PKC delta and PKC theta. To our knowledge, these antibodies are the first described VHH activators and inhibitors for a protein kinase. Furthermore, the development of PKC epsilon specific modulators is an important contribution to PKC research. (C) 2011 Elsevier B.V. All rights reserved.