Molecular design and biological activity of potent and selective protein kinase inhibitors related to balanol

Koide, Kazunori; Bunnage, Mark E.; Paloma, Luigi Gomez; Kanter, Joan R.; Taylor, Susan S.; Brunton, Laurence L.; Nicolaou, K. C.

doi:10.1016/1074-5521(95)90124-8

Cited by 65 publications

(85 citation statements)

References 32 publications

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“…In the previously solved crystal structure of PKA C subunit complexed with balanol (C:Bal), balanol occupied the ATPbinding site (11), as predicted by kinetic studies (7,8) ( Figure 1B). Briefly, the p-hydroxybenzamide group (A-ring) occupied the adenine ring subsite of ATP, and the hexahydroazepine ring (B-ring) occupied the ribose subsite.…”

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confidence: 58%

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

et al. 2003

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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.

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confidence: 58%

“…This is surprising since neither the A-and B-rings together nor the C-and D-rings together could inhibit PKA and PKC (7). Since only three rings are necessary for creating a potent PKA inhibitor, the synthesis of balanol-based inhibitors can be simplified.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2003

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“…CMPD103A and CMPD101 are markedly more selective than balanol, another potent inhibitor of GRK2 . Balanol has a K i of 4 nM against PKA (Koide et al, 1995) and ϳ2 nM against bovine GRK2, whereas CMPD101 has IC 50 values of Ͼ2000 and 35 nM for PKA and GRK2, respectively (Ikeda et al, 2007). A comparison of the PKA-balanol structure (Narayana et al, 1999) with that of GRK2-CMPD101-G␤␥ suggests that the D ring of CMPD101 would collide with Phe187 of PKA in its hydrophobic subsite, thereby precluding binding.…”

Section: Discussionmentioning

confidence: 99%

“…CMPD103A and CMPD101 are potential cardiotonic drugs discovered by Takeda Pharmaceutical Company, Ltd. (Ikeda et al, 2007). Balanol is a less selective AGC kinase inhibitor isolated from the fungus Verticillium balanoides (Koide et al, 1995). The ring structures are labeled as follows for CMPD103A/CMPD101: A, pyrimidine/pyridine; B, substituted 1,2,4-triazole; C, aminobenzamide; D, substituted benzene; and for balanol: A, p-hydroxybenzamide; B, azepane; C and D, substituted and esterified benzophenones.…”

Section: Introductionmentioning

confidence: 99%

Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

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Yeow

Schoenau³

et al. 2011

Mol Pharmacol

105

162

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G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-G␤␥ complex in the presence of two of these inhibitors. Comparison with the apoGRK2-G␤␥ structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.

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“…1A) have been synthesized chemically (Lampe et al, 1994, Nicolaou et al, 1994. We have found that balanol is a potent inhibitor of cyclic AMP (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC) but not of two tyrosine protein kinases, pp60 src and the epidermal growth factor receptor kinase (Koide et al, 1995;Setyawan et al, 1999). In in vitro assays with purified components, balanol inhibits protein kinase activity by competing with ATP for binding at the enzyme's catalytic site (Koide et al, 1995).…”

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Differential and Selective Inhibition of Protein Kinase A and Protein Kinase C in Intact Cells by Balanol Congeners

Gustafsson

Brunton

1999

Mol Pharmacol

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The fungal metabolite balanol is a potent inhibitor of protein kinase A (PKA) and protein kinase C (PKC) in vitro that acts by competing with ATP for binding (K i ϳ 4 nM); congeners of balanol show specificity for PKA over PKC. We have characterized the effects of balanol and 10Љ-deoxybalanol in intact cells to determine whether these compounds cross the cell membrane and whether the potency and specificity noted in vitro are preserved in vivo. In neonatal rat myocytes and cultured A431 cells transiently transfected with a cyclic AMP response element-luciferase reporter construct, balanol inhibits the induction of luciferase activity by isoproterenol, indicating inhibition of PKA. Western analysis shows that both balanol and 10Љ-deoxybalanol reduce phosphorylation of cAMP response element-binding protein in isoproterenol-stimulated A431 cells; inhibition is concentration dependent with an IC 50 value of ϳ3 M. Balanol, but not 10Љ-deoxybalanol, inhibits phosphorylation of the myristoylated alanine-rich C kinase substrate protein, a PKC substrate, in phorbol ester-stimulated A431 cells (IC 50 ϳ 7 M). Our data demonstrate that balanol is a potent inhibitor of PKA and PKC in several whole-cell systems and causes no obvious toxicity. In addition, balanol congeners inhibit PKA and PKC with the specificity and potency predicted by in vitro experiments.Balanol is a fungal metabolite produced by Verticillium balanoides (Kulanthaivel et al., 1993). The parent structure and a number of congeners (Fig. 1A) have been synthesized chemically (Lampe et al., 1994, Nicolaou et al., 1994. We have found that balanol is a potent inhibitor of cyclic AMP (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC) but not of two tyrosine protein kinases, pp60 src and the epidermal growth factor receptor kinase (Koide et al., 1995;Setyawan et al., 1999). In in vitro assays with purified components, balanol inhibits protein kinase activity by competing with ATP for binding at the enzyme's catalytic site (Koide et al., 1995). In fact, balanol interacts with PKA and PKC with an affinity (K i ϭ 4 nM) that is more than three orders of magnitude greater than that for ATP (Koide et al., 1995). We have recently taken advantage of the high affinity of the balanol-kinase interaction to isolate and analyze the structure of a crystal of the balanol-PKA complex, confirming that balanol binds in the ATP cleft of the catalytic core of PKA (Narayana et al., 1999).Minor modification of the balanol structure produces congeners that exhibit substantial specificity toward PKA over PKC. For instance, 10Љ-deoxybalanol (Fig. 1A) inhibits PKA with a K i value of 4 nM and PKC with a K i value of 640 nM in studies with purified enzymes in vitro (Koide et al., 1995;Setyawan et al., 1999). These data suggest that balanol might be a useful template on which a family of specific protein kinase inhibitors can be developed. However, biologically useful inhibitors need to enter cells and have desired effects at modest concentrations that do not injure the ...

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Molecular design and biological activity of potent and selective protein kinase inhibitors related to balanol

Cited by 65 publications

References 32 publications

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

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

Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

Differential and Selective Inhibition of Protein Kinase A and Protein Kinase C in Intact Cells by Balanol Congeners

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Molecular design and biological activity of potent and selective protein kinase inhibitors related to balanol

Cited by 65 publications

References 32 publications

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

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

Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

Differential and Selective Inhibition of Protein Kinase A and Protein Kinase C in Intact Cells by Balanol Congeners

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

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