Selective Activation of Protein Kinase A Isozymes I or II Taking Advantage of Synergistic Pairs of cAMP Analogs with Opposite Site Selectivity
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Introduction:
Eucaryotic cAMP-dependent protein kinase (cAK) exists as two different isozymes type I and II. In its deactivated status both types form a tetrameric complex consisting of two regulatory and two catalytic subunits. Each regulatory subunit offers two cooperative binding sites A and B for the allosteric activator cyclic AMP (cAMP). After binding of cAMP to the regulatory subunits the holoenzyme complex dissociates, releasing free catalytic subunits which then are able to phosphorylate suitable substrates.
Chemically modified analogues of cAMP were shown to bind with different affinity to the A and B sites of the regulatory subunits (site selectivity) and in contrast to cAMP itself thus can discriminate between them.
If a pair of differently modified cAMP analogues with opposite site selectivity is used simultaneously for kinase activation, a synergism is observed which exceeds the normally additive effect of both compounds. If correspondingly selected, these pairs can be rather specific and powerful activators of their target cAK isozymes, often providing stability towards phosphodiesterases (PDE) and membrane permeability as well. Thus the following advantages can be taken:
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By combining appropriate couples of analogues only one of both cAK isozymes I or II can be activated preferably; giving hints which of both types is mainly responsible for the biological effect observed. It has been shown recently that cAK isozymes can act funtionally different and are not necessarily redundant.
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The fact of synergism gives strong evidence for a cAK-mediated biological effect, since for other cAMP receptors known so far (e.g. ion channels), this behaviour is not possible.
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Decrease of the applicable concentration of analogues in cell culture experiments which in the case of expensive compounds is an economic benefit as well.
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As soon as the responsible cAK isozyme is identified, modulation of the corresponding signal pathway can be performed by more optimal agonists and antagonists designed to give maximal stimulation and inhibition, respectively, instead of high site selectivity.
Pairs of Analogs Suitable for Preferential Activation of cAK I / PKA I (Selection):
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Analogue Pairs |
BIOLOG |
Relative Affinity for cAK I |
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Relative Affinity for cAK II |
Selectivity e |
References |
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A site |
B site |
A site |
B site |
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6-Bnz-8-PIP-cAMP |
1.7 |
0.018 |
|
0.0011 |
0.065 |
105 : 1 |
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8-AHA-2-Cl-cAMP |
0.0052 |
3.9 |
0.0012 |
0.5 |
||||
|
|
||||||||
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8-PIP-cAMP |
2.3 |
0.065 |
|
0.046 |
3.2 |
10 : 1 |
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2-Cl-8-MA-cAMP |
0.04 |
6.7 |
0.0095 |
0.72 |
||||
|
|
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8-PIP-cAMP |
2.3 |
0.065 |
|
0.046 |
3.2 |
5 : 1 |
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8-HA-cAMP |
0.033 |
1.8 |
0.086 |
0.87 |
||||
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6-MB-cAMP b |
3.6 |
0.093 |
|
0.74 |
0.041 |
5 : 1 |
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8-AHA-cAMP |
0.11 |
1.6 |
0.021 |
0.29 |
||||
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8-PIP-cAMP |
2.3 |
0.065 |
|
0.046 |
3.2 |
5 : 1 |
||
|
8-AHA-cAMP |
0.11 |
1.6 |
0.021 |
0.29 |
||||
|
6-MB-cAMP b |
3.6 |
0.093 |
|
0.74 |
0.041 |
2 : 1 |
||
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8-HA-cAMP |
0.033 |
1.8 |
0.086 |
0.87 |
||||
The following pair of analogues results in potent stimulation of cAK I but is lacking isozyme selectivity. It can be used if only cAK I is present, if cAK II activation does not disturb or, of course, if both isozymes shall be activated:
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6-Phe-cAMP |
18.0 |
0.48 |
|
40.0 |
0.44 |
1 : 4.3 |
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5,6-DCl-cBIMPc |
0.11 |
5.1 |
0.38 |
42.0 |
Pairs of Analogues Suitable for Preferential Activation of cAK II /PKA II:
The pairs shown were either already used successfully or are suggested due to their site selectivity data. Of course, other combinations can be formed, however, selectivity, metabolic stability as well as membrane permeability should be considered. Data are from references 4, 5, 8, 12 and are relative to cAMP (1.0 for all sites).
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|
BIOLOG |
Relative Affinity for cAK I |
|
Relative Affinity for cAK II |
Selectivity e cAK I / cAK II |
References |
||
|
A site |
B site |
A site |
B site |
|||||
|
6-MBC-cAMP |
0.48 |
0.068 |
|
16.0 |
0.13 |
1 : 60 |
||
|
Sp-5,6-DCl-cBIMPS |
0.022 |
0.13 |
0.034 |
14.0 |
||||
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6-MBC-cAMP |
0.48 |
0.068 |
|
16.0 |
0.13 |
1 : 33 |
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|
Sp-8-Br-cAMPS |
0.19 |
0.054 |
0.0033 |
2.2 |
||||
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6-MBC-cAMP |
0.48 |
0.068 |
|
16.0 |
0.13 |
1 : 18 |
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8-PIP-cAMP |
2.3 |
0.065 |
0.046 |
3.2 |
||||
|
6-MBC-cAMP |
0.48 |
0.068 |
|
16.0 |
0.13 |
1 : 13 |
||
|
Sp-8-PIP-cAMPS |
0.098 |
0.0009 |
0.000005 |
0.33 |
||||
|
6-MBC-cAMP |
0.48 |
0.068 |
|
16.0 |
0.13 |
1 : 9 |
||
|
8-Br-cAMP |
1.3 |
1.0 |
0.11 |
6.8 |
||||
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6-Phe-cAMP |
18.0 |
0.48 |
|
40.0 |
0.44 |
1 : 8 |
||
|
Sp-5,6-DCl-cBIMPS |
0.022 |
0.13 |
0.034 |
14.0 |
||||
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6-Bnz-cAMP |
3.5 |
0.18 |
|
4.1 |
0.034 |
1 : 6 |
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Sp-8-CPT-cAMPS |
0.92 |
0.09 |
0.006 |
5.2 |
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The following pairs of analogues are combined to achieve maximal stimulation of cAK II, but are lacking isozyme selectivity. They should only be used if solely cAK II is present, if cAK I activation does not disturb or, of course, if both isozymes want to be activated:
|
6-Phe-cAMP |
18.0 |
0.48 |
|
40.0 |
0.44 |
1 : 5 |
||
|
8-CPT-cAMP d |
3.9 |
1.7 |
0.045 |
19.0 |
||||
|
6-Phe-cAMP |
18.0 |
0.48 |
|
40.0 |
0.44 |
1 : 4.3 |
||
|
5,6-DCl-cBIMP c |
0.11 |
5.1 |
0.38 |
42.0 |
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a) This analogue has only low membrane permeability.
b) N6-Monobutyryl- cAMP ( 6-MB-cAMP) is formed by metabolic degradation of dibutyryl-cAMP (Db-cAMP) releasing butyrate.
c) This analogue is not very resistant towards PDE hydrolysis. Addition of an unspecific PDE inhibitor such as IBMX is advisable here.
d) Caution: 8-CPT-cAMP is unselective and stimulates cGMP-dependent protein kinase as well.
e) Calculated by (A I x B I)½ : (A II x B II)½ . Actual selectivity should be higher due to synergism.
References:
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Øgreid, D.; Døskeland, S.O.; Miller, J.P., J. Biol. Chem., 258, 1041 - 1049 (1983) |
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Robinson-Steiner, A. M.; Corbin, J. D., J. Biol. Chem., 258, 1032 - 1040 (1983) |
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Øgreid, D.; Ekanger, R.; Suva, R.H.; Miller, J.P.; Sturm, P.A.; Corbin, J.D.; Døskeland, S.O., Eur. J. Biochem.,150, 219 - 227 (1985) |
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Øgreid, D.; Ekanger, R.; Suva, R.H.; Miller, J.P.; Døskeland, S.O., Eur. J. Biochem., 181,19 - 31 (1989) |
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Dostmann, W.R.G.; Taylor, S.S.; Genieser, H.-G.; Jastorff, B.; Døskeland, S.O.; Øgreid, D., J. Biol. Chem., 265, 10484 - 10491(1990) |
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Steinberg, R.A.; Gorman, K.B.; Øgreid, D.; Døskeland, S.O.; Weber, I.T., J. Biol. Chem., 266, 3547 - 3553 (1991) |
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Otten, A.D.; Parenteau, L.A.; Døskeland, S.; McKnight, G.S., J. Biol. Chem., 266, 23074 - 23082 (1991) |
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Genieser, H.-G.; Winkler, E.; Butt, E.; Zorn, M.; Schulz, S.; Iwitzki, F.; Störmann, R.; Jastorff, B.; Døskeland, S.O.; Øgreid, D.; Ruchaud, S.; Lanotte, M., Carbohydr. Res., 234, 217 - 235 (1992) |
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Mira-y-Lopez, R.; Jaramillo, S.; Waxmann, S., J. Biol. Chem., 267, 23063 - 23068 (1992) |
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Skalhegg, B.S.; Landmark, B.F.; Døskeland, S.O.; Hansson, V.; Lea, T.; Jahnsen, T., J. Biol. Chem., 267,15707 - 15714 (1992) |
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Døskeland, S.O.; Maronde, E.; Gjertsen, B.T., Biochim. Biophys. Acta 1178, 249 - 258 (1993) |
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Øgreid, D.; Dostmann, W.; Genieser, H.-G.; Niemann, P.; Døskeland, S.O.; Jastorff, B., Eur. J. Biochem., 221, 1089 - 1094 (1994) |
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Maronde, E.; Middendorff, R.; Telgmann, R.; Müller, D.; Hemmings, B.; Tasken, K.; Olcese, J., J. Neurochem., 68, 770 - 777 (1997) |
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Maronde, E.; Wicht, H.; Tasken, K.; Genieser, H.G.; Olcese, J.; Korf, H.W., J. Pineal Res., 27, 170 - 182 (1999) |
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Schwede, F.; Christensen, A.; Liauw, S.; Hippe, T.; Kopperud, R.; Jastorff, B.; Døskeland, S. O., Biochemistry, 39, 8803 - 8812 (2000) |
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Kopperud, R.; Krakstad, C.; Selheim, F.; Døskeland, S.O., FEBS Lett., 546, 121 - 126 (2003) |
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Huseby et al., Cell Death Dis., e-pub, Dec 8; 2: e237. doi 10.1038/cddis.2011.124 |
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