UC San Diego

Activation of protein kinase G (PKG) Iα in nociceptive neurons induces long-term hyperexcitability that causes chronic pain. Recently, a derivative of the fungal metabolite balanol, N46, has been reported to inhibit PKG Iα with high potency and selectivity and attenuate thermal hyperalgesia and osteoarthritic pain. Here we determined co-crystal structures of the PKG Iα C-domain and cAMP-dependent protein kinase (PKA) Cα, each bound with N46, at 1.98 Å and 2.65 Å, respectively. N46 binds the active site with its external phenyl ring, specifically interacting with the glycine-rich loop and the αC helix. Phe-371 at the PKG Iα glycine-rich loop is oriented parallel to the phenyl ring of N46, forming a strong π-stacking interaction, whereas the analogous Phe-54 in PKA Cα rotates 30° and forms a weaker interaction. Structural comparison revealed that steric hindrance between the preceding Ser-53 and the propoxy group of the phenyl ring may explain the weaker interaction with PKA Cα. The analogous Gly-370 in PKG Iα, however, causes little steric hindrance with Phe-371. Moreover, Ile-406 on the αC helix forms a hydrophobic interaction with N46 whereas its counterpart in PKA, Thr-88, does not. Substituting these residues in PKG Iα with those in PKA Cα increases the IC₅₀ values for N46, whereas replacing these residues in PKA Cα with those in PKG Iα reduces the IC₅₀, consistent with our structural findings. In conclusion, our results explain the structural basis for N46-mediated selective inhibition of human PKG Iα and provide a starting point for structure-guided design of selective PKG Iα inhibitors.