Conformationally constrained analogs of protein kinase inhibitor(6–22)amide: Effect of turn structures in the center of the peptide on inhibition of cAMP‐dependent protein kinase

Abstract

The high-affinity interaction between protein kinase inhibitor (PKI)(6-22)amide (Thr6-Tyr-Ala-Asp-Phe-Ile-AlaSer-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-IleZ2-NH2) and the catalytic subunit of CAMP-dependent protein kinase requires both the N-terminal Thr6 to Ile" sequence of the inhibitor peptide and its C-terminal pseudosubstrate site comprised of Arg" to Ilez2. Small angle X-ray scattering data indicate that PKI(6-22)amide has a compact, rather than extended, structure in solution (Reed J et al., 1989, Biochem 5264371-380). CD spectroscopic analysis of the PKI peptide led to the suggestion that a p-turn structure might be located in the -Ala'2-Ser-Gly-Arg15-connecting sequence in the middle of the molecule (Reed J, Kinzel V, Cheng HC, Walsh DA, 1987, Biochemistry267641-7647). To investigate this possibility further, conformationally constrained and flexible analogs of PKI(6-22)amide were synthesized and used to study the structure-function relationships of this central portion of the inhibitor. (Desl2-14)PKI(6-22) amide exhibited over a 200-fold loss in inhibitory activity. Replacement of the omitted -Ala12-Ser-Gly14-sequence with aminocaprylicacid yielded an analog that regained more than 90% of the lost binding energy. The alanine'^ PKI analog was as potent as the parent peptide, whereas the /3-alanine14 and the sarcosine14 analogs were only 10-fold less active. Several peptides that promoted a &turn structure at residues 12-15 showed about 200-fold decreases in inhibitory activity. Two constrained analogs that could not assume a @-turn conformation were only 30-fold less potent than PKI(6-22)amide. Thus, the structure of the central connecting portion of the PKI peptide, encompassing residues 12-15, greatly influences its ability to effectively bind to and inhibit the catalytic subunit. We conclude, however, that a formal &turn at this position is not required and is actually detrimental for a high-affinity interaction of PKI(6-22)amide with the enzyme. These resdts are interpreted in light of the Fourier-transform infrared spectra of the peptide analogs and the crystal structure of the peptide bound at the active site of the protein kinase (Knighton DR et al., 1991b, Science253:414-420 its inhibitory activity have been delineated using analog peptides Aib, 2-aminoisobutyric acid; DAh, D-alanine; Acy, aminocaprylic acid that correspond to the amino acid sequence of its active portion (8-aminooctanoic acid

Protein Kinase Inhibitor Peptide as a Tool to Specifically Inhibit Protein Kinase A

“…As discussed above, PKA mediates the signals from the extracellular side to the intracellular side and various kinds of life processes demand to termination and/or reverse phosphorylation of proteins catalyzed by PKA (Akabane et al, 2016;Bachmann et al, 2016;Tao et al, 2016;Yamauchi et al, 2016). So far, several kinds of methods have been developed for the inhibition of PKA in research, including the development of small molecules such as H89, and the construction of synthetic peptide analogs of PKI as well as the creation of PKA specific inhibition animal models taking advantage of PKI (Glass et al, 1995;Lochner and Moolman, 2006;Inoue et al, 2013;Zhang et al, 2013a;Zynda et al, 2014). Although in this review, we mainly aim to discuss the related knowledge of PKI, we also introduce widely used small molecular compounds, Rp-cAMP, KT-5720 and H89, to facilitate the subsequent discussion of PKI.…”