Structural basis for selective inhibition of human PKG Iα by the balanol-like compound N46

Liying Qin,Banumathi Sankaran,Sahar Aminzai,Darren E Casteel,Choel Kim

doi:10.1074/jbc.ra118.002427

Abstract

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.

Highlights

Activation of protein kinase G (PKG) I␣ in nociceptive neurons induces long-term hyperexcitability that causes chronic pain
We determined co-crystal structures of the PKG I␣ C-domain and cAMP-dependent protein kinase (PKA) C␣, each bound with N46, at 1.98 Å and 2.65 Å, respectively
Several crystal structures have been solved for mammalian PKG I, but these are of various fragments of the R-domains (36 –39)

Summary

Results and discussion

Several crystal structures have been solved for mammalian PKG I, but these are of various fragments of the R-domains (36 –39). The A-ring binds the adenine subsite, and the interactions in this region are essentially the same as in PKG I␣ These include hydrogen bonds between the A-ring and the backbone atoms of Glu-121 and Val-123 at the hinge and VDW contacts with a hydrophobic pocket consisting of Leu-49, Val-57, Ala-70, Val104, Met-120, Leu-173, and Phe-327 (Fig. 3B). The B-ring docks onto the ribose subsite, its amine group interacts only with the hinge residue Glu-127 through a hydrogen bond, not with the activation loop residue Asp-184 (Fig. 3B). The d-ring is oriented parallel to the side chain of Phe-54, allowing a parallel ␲-stacking interaction between them as well as a lone pair–␲ interaction between the carbonyl group and Phe-54 None of these interactions are preserved in the PKA–N46 complex, a new hydrogen bond forms between the propoxy group and the backbone amide of Ser-53. Our structural and biochemical data in part explain N46’s selectivity for PKG I␣ and provide a starting point for structure-guided design of selective PKG I␣ inhibitors

Experimental procedures

Crystallization and structure determination

In vitro kinase assays