Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit^,

Abstract

The protein kinase family is a prime target for therapeutic agents, since unregulated protein kinase activities are linked to myriad diseases. Balanol, a fungal metabolite consisting of four rings, potently inhibits Ser/Thr protein kinases and can be modified to yield potent inhibitors that are selectives characteristics of a desirable pharmaceutical compound. Here, we characterize three balanol analogues that inhibit cyclic 3′,5′-adenosine monophosphate-dependent protein kinase (PKA) more specifically and potently than calcium-and phospholipid-dependent protein kinase (PKC). Correlation of thermostability and inhibition potency suggests that better inhibitors confer enhanced protection against thermal denaturation. Crystal structures of the PKA catalytic (C) subunit complexed to each analogue show the Gly-rich loop stabilized in an "intermediate" conformation, disengaged from important phosphoryl transfer residues. An analogue that perturbs the PKA C-terminal tail has slightly weaker inhibition potency. The malleability of the PKA C subunit is illustrated by active site residues that adopt alternate rotamers depending on the ligand bound. On the basis of sequence homology to PKA, a preliminary model of the PKC active site is described. The balanol analogues serve to test the model and to highlight differences in the active site local environment of PKA and PKC. The PKA C subunit appears to tolerate balanol analogues with D-ring modifications; PKC does not. We attribute this difference in preference to the variable B helix and C-terminal tail. By understanding the details of ligand binding, more specific and potent inhibitors may be designed that differentiate among closely related AGC protein kinase family members.As cellular processes are better understood at the molecular level, especially in the context of recent genomic information, there has been an increased effort to target specific proteins that are linked to disease. Protein kinases are a diverse family of enzymes that have various regulatory roles yet function similarly by catalyzing the phosphoryl transfer of the γ-phosphate of ATP 1 to an enzyme-specific protein substrate. Since many diseases, including cancer, autoimmune disorders, cardiac disease, and diabetes, are associated with defects in protein phosphorylation, and there are an estimated ∼500 protein kinases in the human genome (1), this family is a major target in the design of pharmaceutical agents and inhibitors. A major challenge for the development of therapeutics is the identification of compounds that have high selectivity. To better understand binding diversity and how this correlates with specificity for protein kinases, three analogues of balanol were studied that specifically inhibit † Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the Office of Basic Energy Sciences, U.S.

Orthogonal Multipolar Interactions in Structural Chemistry and Biology

“…X-ray crystal structure analysis shows that balanol analogues, which are inhibitors of the cyclic 3',5'-adenosine monophosphate dependent kinase (PKA), establish short orthogonal contacts of the ester carbonyl group to the protein backbone C = O functionality of Gly 50. . [60] A second corresponding combination of interacting C = O units is found when examining the X-ray crystal structure of ketobis(5-amidino-2-benzimidazolyl)methane bound to the serine protease trypsin (Figure 10 b, PDB-code: 1XUI). [61] In this complex, the side chain carbonyl group of Gln 192 is ideally positioned to interact with the inhibitor keto group forming a short dipolar orthogonal contact (d(O···C) = 3.00 , angle (O protein -C-O) = 878, angle (C protein -O protein -C) = 1668).…”

“…a) Short intermolecular, orthogonal C=O(ligand)···C=O(protein) contact observed in the X-ray crystal structure (PDB code: 1REJ) of a balanol analogue inhibitor complexed with PKA. [60] b) Ketobis(5-amidino-2-benzimidazolyl)methane establishes a short, orthogonal C = O(protein)···C=O(ligand) contact when binding to the serine protease trypsin, as observed in the X-ray crystal structure (PDB-code: 1XUI) of the complex. [61] Color code: inhibitor skeletons: green; C: gray; O: red; N: blue; S: yellow.…”

Recent progress in the discovery of Akt inhibitors as anticancer agents

“…Its binding to the ATP pocket of PKA has been well characterized. The 4-hydroxybenzamide binds to the hinge via the hydroxyl group and the C-6 hydroxyl group on the benzophenone hydrogen binds to Lys72 [103,104] . Akt inhibitors that were made by replacing the hinge binding phenol of balanol with 4-pyridinyl or other heteroaryl groups were reported by scientists at Hoffmann-La Roche [307] .…”

Total Synthesis of (−)‐Balanol, All Stereoisomers, Their N‐Tosyl Analogues, and Fully Protected Ophiocordin: An Easy Route to Hexahydroazepine Cores from Garner Aldehydes

“…Ophiocordin (3), [14,15] a regioisomer of (À)-balanol (1 a), was isolated from Cordyceps ophioglossoides in 1977 and serves as an antibiotic with antifungal activity. While several efforts to achieve selectivity among PKC isoforms have been undertaken, [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] there is still a constant need for more selective and potent PKC inhibitors.…”