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Disadvantages and Potential Pitfalls of Protein Kinase A Inhibitors Blocking the Catalytic Subunit of Protein Kinase A ( e.g. H 89, KT 5720 a.o. )

and comparison with cyclic nucleotide-based inhibitors for the regulatory subunits of PKA


Due to the tetrameric composition of protein kinase A (PKA) of both, regulatory and catalytic subunits, inhibition of PKA can be achieved either by inhibitory analogues of cyclic AMP or by structures that block the ATP binding site (isoquinolines, staurosporine analogues). While inhibition of the regulatory subunits prevents the PKA holoenzyme from dissociation and liberation of free active regulatory subunits, a blockade of the catalytic ATP sites stops the phosphorylation process directly. Especially, membrane-permeant ATP site inhibitors, such as H 89, H 8, KT 5720 have become very popular in signal transduction studies, however, there are some serious limitations and potential pitfalls which should be known, in order to avoid drawbacks and disappointments:

Different Working Principle

Due to their different sites of action, ATP-site inhibitors and blockers of the regulatory subunits can differ in their effects on a given signaling pathway1. While H 89 and KT 5720 will interfere solely with the phosphorylation process of PKA, Rp-cAMPS and its analogues prevent the PKA holoenzyme from dissociation and thus act one step earlier. This can be an important difference, if the released regulatory subunits have special tasks on their own. Differences between e.g. H 89 and Rp-cAMPS and analogues were already reported:

  • Rp-8-Br-cAMPS inhibits PKA effects, while H 89 fails (neuroblastoma 1,  hepatocytes 2)
  • Rp-8-CPT-cAMPS inhibits PKA effect, while H 89 fails (pinealocytes 3 )
  • Rp-8-CPT-cAMPS has no influence, while H 89 has effects although cAMP is not involved 4
  • H 8 shows no effect on smooth muscle relaxation 5, while cyclic nucleotides work 6

Questionable Selectivity

Although the selectivity of ATP-site inhibitors of PKA, such as H 89 or KT 5720, seems to be rather good with respect to protein kinases C and G, this does obviously not hold true for other protein kinases. In addition, the usefulness of such compounds is at least questionable under the impression of numerous different types of ATP receptors in a cell, which are of course potential competitive binding sites as well. Thus it is not really astonishing, that more and more disturbing side effects are discovered, and that an increasing number of papers have already reported on that topic:

  • H 89 has unwanted side effects, e.g. contracts smooth muscle or influences ion currents 7
  • H 89 can obviously also activate protein kinase G pathway 8
  • H 89 effect is not persistant in comparison to Rp-8-CPT- cAMPS 9
  • H 89 has unspecific effects on sarcoplasmatic reticulum Ca2+ ATPase 10, 14
  • Isochinoline derivatives such as H 89 interact with serotonin transport 11
  • Isochinoline derivatives block cyclic GMP-gated ion channels 12
  • Isochinoline derivative H 8 is not specific for protein kinase A 13
  • Isochinoline derivatives promote dephosphorylation of RNA polymerase subunit 15
  • H 89 and other isochinoline derivatives stimulate amiloride-sensitive Na+ transport16
  • H 89 antagonizes beta adrenergic receptor ligand binding 17
  • H 89 and KT 5720 lack specificity for PKA but inhibit other kinases with similar potency 20
  • H 89 blocks shaker-type K+-channels 22
  • H 89 inhibits Rho kinase23
  • H 89 has effects different from PKA inhibition24

In contrast to ATP, the number of cyclic AMP receptors is much more limited (PKA, PKG, PDE, CAP, CNG, EPAC/GEF), so a correspondingly selective inhibitor based on a cyclic nucleotide, such as Rp-cAMPS and its analogues would be much more focused on PKA, and thus have a clear advantage here.

Unclear Metabolic Fate

ATP-site directed inhibitors such as H 89 or KT 5720 are relatively complex organic compositions, whereas cyclic nucleotide analogues vary only slightly from natural cyclic AMP structure.
Thus, the metabolic fate of H 89 or KT 5720 and the side effects of their metabolites in different tissues is more or less unknown, while cyclic nucleotide-based PKA inhibitors (Rp-cAMPS) are resistant against mammalian phosphodiesterases, the compulsory first step in metabolic degradation of these structures.


ATP-site directed inhibitors of PKA can be a useful additional control in concert with other tools, but should be used with caution and not be the single source of evidence for the involvement of PKA.


H 89: {N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide}

KT 5720: (8R, 9S, 11S)- (-)- 9- hydroxy- 9- hexoxycarbonyl- 8- methyl- 2, 3, 9, 10- tetrahydro- 8, 11- epoxy- 1H, 8H, 11H- 2, 7b, 11a- triazadibenzo [a,g] cycloocta[cde]trinden- 1- one

Rp-cAMPS: Adenosine- 3', 5'- cyclic monophosphorothioate, Rp-isomer (BIOLOG Cat. No. A 002)

Rp-8-Br-cAMPS: 8-Bromoadenosine- 3', 5'- cyclic monophosphorothioate, Rp-isomer (BIOLOG Cat. No. B 001)

Rp-8-CPT-cAMPS: 8-(4-Chlorophenylthio)adenosine- 3', 5'- cyclic monophosphorothioate, Rp-isomer (BIOLOG Cat. No. C 011)

Selected references


Petersen, R.K.; Madsen, L.; Pedersen, L.M.; Hallenborg, P.; Hagland, H.; Viste, K.; Døskeland, S.O.; Kristiansen, K., Mol. Cell. Biol., 28, 3804 - 3816 (2008): "Cyclic AMP (cAMP)-mediated Stimulation of Adipocyte Differentiation Requires the Synergistic Action of Epac- and cAMP-dependent Protein Kinase-dependent Processes"


Lee, T.H.; Linstedt, A.D., Mol. Biol. Cell, 11, 2577 - 2590 (2000): "Potential Role for Protein Kinases in Regulation of Bidirectional Endoplasmic Reticulum-to Golgi Transport Revealed by Protein Kinase Inhibitor H89"


Leemhuis, J.; Boutillier, S.; Schmidt, G.; Meyer, D.K., J. Pharmacol. Exp. Therap., 300, 1000 - 1007, (2002): "The Protein Kinase A Inhibitor H89 Acts on Cell Morphology by Inhibiting Rho Kinase"


Choi, J.-S.; Choi, B.H.; Hahn, S.J.; Yoon, S.H.; Min, D.S.; Jo, Y.-H.; Kim, M.-S., Biochem. Pharmacol., 61, 1029 - 1032 (2001): "Inhibition of Kv1.3 Channels by H-89 (N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) Independent of Protein Kinase A"


Oki, N.; Takahashi, S.-I.; Hidaka, H.; Conti, M., J. Biol. Chem., 275, 10831 - 10837 (2000): "Short Term Feedback Regulation of cAMP in FRTL-5 Thyroid Cells"


Davies, S.P.; Reddy, H.; Caivano, M.; Cohen, P., Biochem. J., 351, 95 - 105 (2000): "Specificity and Mechanisms of Action of Some Commonly Used Protein Kinase Inhibitors"


Osinski, M.; Weber, A.A.; Schrör, K, Eur. J. Pharmacol., 395, 173 - 176 (2000): "Complex Actions of Protein Kinase A Inhibitors on Mitogenesis of Bovine Coronary Artery Smooth Muscle Cells"


Bode, H. P.; Moormann, B.; Dabew, R.; Göke, B., Endocrinology, 140, 3919 - 3927 (1999): "Glucagon-like Peptide 1 Elevates Cytosolic Calcium in Pancreatic Beta-Cells Independently of Protein Kinase A"


Penn, R.B.; Parent, J.-L.; Pronin, A.N.; Panettieri, R.A.; Benovic, J.L, J. Pharmacol. Exper. Ther., 288, 428 - 437 (1999): "Pharmacological Inhibition of Protein Kinases in Intact Cells: Antagonism of Beta Adrenergic Receptor Ligand Binding by H-89 Reveals Limitations of Usefulness"


Niisato, N.; Ito, Y.; Marunaka, Y., Life Sci., 65, PL 109 - 114 (1999): "Effect of PKA Inhibitors, H-compounds, on Epithelial Na+ Channels Via PKA-independent Mechanisms"


Dubois, M.-F.; Nguyen, V.T.; Bellier, S.; Bensaude, O., J. Biol. Chem., 269, 13331 - 13336 (1994): "Inhibitors of Transcription Such as 5,6-Dichloro-1-ß-D-ribofuranosylbenzimidazole and Isoquinoline Sulfonamide Derivatives (H-8 and H-7) Promote Dephosphorylation of the Carboxyl-terminal Domain of RNA Polymerase II Largest Subunit"


Lahouratate, P.; Guibert, J.; Camelin, J.-C.; Bertrand, I., Biochem. Pharmacol., 54, 991 - 998 (1997): "Specific Inhibition of Cardiac and Skeletal Muscle Sarcoplasmic Reticulum Ca2+ Pumps by H-89"


Hughes, S.J., Hollingsworth, M.; Elliott, K.R.F., J. Reproduction & Fertility 109, 289 - 296 (1997): "The role of a cAMP-dependent Pathway in the Uterine Relaxant Action of Relaxin in Rats"


Wei, J.-Y.; Cohen, E.D.; Barnstable, C.J., Neurosci. Lett., 233, 37 - 40 (1997): "Direct Blockade of both cloned rat rod photoreceptor cyclic nucleotide-gated non-selective cation (CNG) channel alpha-subunit and native CNG channels from Xenopus rod outer segments by H-8, a non-specific cyclic nucleotide-dependent protein kinase inhibitor"


Helmeste, D.M.; Tang, S.W., Eur. J. Pharmacol., 267, 239 - 242 (1994): "Kinase inhibitors compete with imipramine for binding and inhibition of serotonin transport"


Hussain M., Drago, G.A.; Bhogal, M.; Colyer, J.; Orchard, C.H., Pflügers Arch. 437, 529 - 537 (1999): "Effects of the protein kinase inhibitor H-89 on Ca2+ regulation in isolated ferret ventricular myocytes"


Schubert, R., University of Rostock, 1993, personal communication


Satake, N., Fujimoto, S.; Shibata, S., Gen. Pharmacol., 27, 701 - 705 (1996): "The Potentation of Nitroglycerin-induced Relaxation by PKG Inhibition in Aortic Rings"


Yuan, W.L.; Bers, D.M., Amer. J. Physiol., 37, C651 - C659 (1995): "Protein kinase inhibitor H-89 reverses forskolin stimulation of cardiac L-type calcium current"


There are numerous papers dealing with cyclic-nucleotide-based inhibitors and smooth muscle relaxation. Please inquire for a printout from our data base.


Daugirdas, J.T.; Zhou, H.L.; Tamulaitis, V.V.; Nutting, C.W.; Fiscus, R.R., Blood Vessels, 28, 366 - 371 (1991): "Effect of H-8, an isoquinolinesulfonamide inhibitor of cyclic nucleotide-dependent protein kinase, on cAMP- and cGMP-mediated vasorelaxation"


Warskulat et al., Biol. Chem. Hoppe-Seyler, 377, 57 - 65 (1996): "Anisoosmotic Regulation of Hepatic Gene Expression"


Maronde, E., University of Frankfurt, 1998, personal communication


Holen, I.; Gordon, P.B.; Strømhaug, P.E.; Seglen, P.O., Eur. J. Biochem., 236, 163 - 170 (1996): "Role of cAMP in the Regulation of Hepatocytic Autophagy"


Boundy, V.A.; Chen, J.S.; Nestler, E.J., J. Pharm. Exp. Ther., 286, 1058 - 1065 (1998): "Regulation of cAMP-dependent Protein Kinase Subunit Expression in CATH.a and SH-SY5Y cells"