Comparative Structure-Affinity Relations by MTD for Binding of Cycloadenosine Monophosphate Derivatives to Protein Kinase Receptors (original) (raw)
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European Journal of Biochemistry, 1989
cAMP analogs, all 96 of which were modified in the adenine moiety, were examined quantitatively for their ability to inhibit the binding of [3H]cAMP to each of the two classes (A and B) of cAMP-binding sites of type I (rabbit skeletal muscle) and type II (bovine heart) cAMP-dependent protein kinase. The study showed that analogs can be constructed that have a higher affinity than cAMP for a binding site. N6-phenyl-cAMP had 18-fold increased affinity for site A of RI (AI) and 40-fold increased affinity for site AII. 2-chloro-8-methylamino-cAMP had a 7-fold increased affinity for BI, and 8-(4-chlorophenylthio)-cAMP had 17-fold increased affinity for BII. Analogs could discriminate between the two classes of binding sites by more than two orders of magnitude in binding affinity: 2-chloro-8-methylamino-cAMP had 170-fold higher affinity for BI than for AI, and 2-n-butyl-8-thiobenzyl-cAMP had 700-fold higher affinity for BII than for AII. Analogs could also discriminate between the homologous binding sites of the isozymes: 2-n-butyl-8-bromo-cAMP had 260-fold higher affinity for AI than for AII (22-fold higher for BII than BI), and 8-piperidino-cAMP had 50-fold higher affinity for BII than for BI (and 50-fold higher for AI than for AII). The data suggest the following conclusions. (a) Stacking interactions are important for the binding of cAMP to all the binding sites. (b) Subtle differences exist between the sites as to the optimal electron distribution in the adenine ring since modifications that withdraw electrons at C2 and donate at C8 favour binding to BI, and disfavour binding to AI and AII. (c) There are no hydrogen bonds between the adenine ring of cAMP and any of the binding sites. (d) All sites bind cAMP in the syn conformation. (e) The subsites adjacent to the N6 and C8 positions may have nonpolar neighbouring regions since hydrophobic substituents at N6 could increase the affinity for AI and AII and similar substituents at C8 could increase the affinity for BII. Finally, (f) the sites differed in their ability to accomodate bulky substituents at C2 and C8. For all compounds tested, their potency as activators of protein kinases I and II was found to correlate, in a predictable fashion, to their mean affinity for the two classes of binding sites, rather than to the affinity for only one of the sites.
European Journal of Biochemistry, 1994
Received January 19Eebruary 21, 1994) -EJB 94 005913 8-Piperidino-CAMP has been shown to bind with high affinity to site A of the regulatory subunit of CAMP-dependent protein kinase type I (AI) whereas it is partially excluded from the homologous site (AII) of isozyme I1 [Ggreid, D., Ekanger, R., Suva, R. H., Miller, J. P., and Dmkeland, S. 0. (1989), Eul: J. Biochem. 181,[28][29][30][31]. To further increase this selectivity, the (I?,)and (S,)diastereoisomers of 8-piperidino-CAMP[ S] were synthesized and analyzed for their potency to inhibit binding of 13H]cAMP to site A and site B from type I (rabbit skeletal muscle) and type I1 (bovine myocardium) CAMP-dependent protein kinases.
QSAR of Adenosine Receptor Antagonists II
QSAR & Combinatorial Science, 2003
Considering potential of selective adenosine receptor subtype ligands in the development of prospective drug candidates, A 1 and A 3 receptor binding affinity data of 2arylpyrazolo[3,4-c]quinoline derivatives have been subjected to QSAR analyses to explore the physicochemical requirements for selective binding. The study has been carried out with Wang-Ford charges of the common atoms of the molecules calculated from their energy minimized conformations using AM1 technique. Apart from the charge parameters, physicochemical variables like partition coefficient and molar refractivity of the whole molecules have been used along with suitable indicator variables. The study shows that substituents on the appended 2-phenyl ring and 4-amino or 4-keto substitution on the pyrazolo[3,4-c]quinoline nucleus modulate the selectivity pattern. Further, negative charge on the quinoline nitrogen and volume and lipophilicity of the whole molecules are important contributors to the selectivity.
A Model for Agonism and Antagonism in an Ancient and Ubiquitous cAMP-binding Domain
Journal of Biological Chemistry, 2006
The cAMP-binding domain (CBD) is an ancient and conserved regulatory motif that allosterically modulates the function of a group of diverse proteins, thereby translating the cAMP signal into a controlled biological response. The main receptor for cAMP in mammals is the ubiquitous regulatory (R) subunit of protein kinase A. Despite the recognized significant potential for pharmacological applications of CBDs, currently only one group of competitive inhibitor antagonists is known: the (R p)-cAMPS family of phosphorothioate cAMP analogs, in which the equatorial exocyclic oxygen of cAMP is replaced by sulfur. It is also known that the diastereoisomer (S p)-cAMPS with opposite phosphorous chirality is a cAMP agonist, but the molecular mechanism of action of these analogs is currently not fully understood. Previous crystallographic and unfolding investigations point to the enhanced CBD dynamics as a key determinant of antagonism. Here, we investigate the (R p)-and (S p)-cAMPS-bound states of R(CBD-A) using a comparative NMR approach that reveals a clear chemical shift and dynamic NMR signature, differentiating the (S p)-cAMPS agonist from the (R p)-cAMPS antagonist. Based on these data, we have proposed a model for the (R p /S p)-cAMPS antagonism and agonism in terms of steric and electronic effects on two main allosteric relay sites, Ile 163 and Asp 170 , respectively, affecting the stability of a ternary inhibitory complex formed by the effector ligand, the regulatory and the catalytic subunits of protein kinase A. The proposed model not only rationalizes the existing data on the phosphorothioate analogs, but it will also facilitate the design of novel cAMP antagonists and agonists.
Biochemistry, 2004
Cyclic adenosine 5′-monophosphate (cAMP) is an ancient signaling molecule, and in vertebrates, a primary target for cAMP is cAMP-dependent protein kinase (PKA). (R p )-adenosine 3′,5′-cyclic monophosphothioate ((R p )-cAMPS) and its analogues are the only known competitive inhibitors and antagonists for cAMP activation of PKA, while (S p )-adenosine 3′,5′-cyclic monophosphothioate ((S p )-cAMPS) functions as an agonist. The crystal structures of a ∆(1-91) deletion mutant of the RIR regulatory subunit of PKA bound to (R p )-cAMPS and (S p )-cAMPS were determined at 2.4 and 2.3 Å resolution, respectively. While the structures are similar to each other and to the crystal structure of RIR bound to cAMP, differences in the dynamical properties of the protein when (R p )-cAMPS is bound are apparent. The structures highlight the critical importance of the exocyclic oxygen's interaction with the invariant arginine in the phosphate binding cassette (PBC) and the importance of this interaction for the dynamical properties of the interactions that radiate out from the PBC. The conformations of the phosphate binding cassettes containing two invariant arginine residues (Arg209 on domain A, and Arg333 on domain B) are somewhat different due to the sulfur interacting with this arginine. Furthermore, the B-site ligand together with the entire domain B show significant differences in their overall dynamic properties in the crystal structure of ∆(1-91) RIR complexed with (R p )-cAMPS phosphothioate analogue ((R p )-RIR) compared to the cAMP-and (S p )-cAMPS-bound type I and II regulatory subunits, based on the temperature factors. In all structures, two structural solvent molecules exist within the A-site ligand binding pocket; both mediate water-bridged interactions between the ligand and the protein. No structured waters are in the B-site pocket. Owing to the higher resolution data, the N-terminal segment (109-117) of the RIR subunit can also be traced. This strand forms an intermolecular antiparallel -sheet with the same strand in an adjacent molecule and implies that the RIR subunit can form a weak homodimer even in the absence of its dimerization domain.
The Journal of Peptide Research, 2009
Peptides derived from the inhibitor of CAMP-dependent protein kinase, PKI, have been studied by 2D 'H NMR techniques. These include the inhibitor PKI(6-22), the substrate [Ala2"-Ser"]PKI(5-24), and a phosphorylated form of the latter [Ala'o-Ser"P]PKI(5-24). A homologous fold was found in the three peptides which consisted of an h'-terminal segment in helical conformation to residue 13 and a C-terminal segment poorly defined conformationally. A parallel study was carried out by molecular dynamics (MD) for the inhibitor peptide PKI(5-24). The N-terminal helix, as observed in the crystal structure of the catalytic subunit-PKI(5-24) complex. was conserved in the M D simulations with the enzyme-free inhibitor. Similarly the Gly'J-Glyl' turn was apparent in all M D structures, whereas the C-terminal region, residues 18-24, was directed towards the N-terminal helix in contrast to the extended conformation of this segment pointing away from the A'-terminal helix in the crystal structure. This is primarily due to ionic interaction between Asp' and Arg". Indeed. a detailed analysis of the NOE contacts by NOESY at low temperature (2-C) shows the occurrence of pH-dependent contacts with Phe'". We conclude that the binding of short inhibitors, such as PKI(5-24), to the enzyme involves a conformational rearrangement of the C-terminal region. The substrate [Ala'o-Ser"]PKI(5-24) and the product [Alazo-SerZ1P]PKI(5-24), give very similar structures with local rearrangements involving some of the side chains. 0 Munksgaard 1997. K r j itorris: CAMP-dependent protein kinase: P K I x NMR: molecular dlnamics The heat-and acid-stable protein kinase inhibitor (PKI) was initially purified from rabbit skeletal muscle (l) , and the entire 75-amino acid sequence of this isoform, PKIr, has been determined (2). PKI specifically inhibits the catalytic subunit of the CAMPdependent protein kinase (3). The inhibitory region of PKI was localized, using proteolytic cleavage. to be in the first third of the molecule (4). Inhibition by the peptide PKI(5-24), as well as by full length PKI, is competitive with respect to protein substrate (5). The C-terminal segment of PKI(5-24) contains the pseudo-substrate site required for inhibition of the catalytic subunit of CAMP-dependent protein kinase (5, 6). The sequence Arg' 5-Thr-Gly-Arg-Arg-Asn-Ala-Ile2' of PKI is similar to the typical-Arg-X-X-Arg-Arg-X-Ser-hyd-phosphorylation consensus sequence found for substrates of the CAMP-depend-en{ protein kinase (7). The N-terminal domiin of Abbreviations: PKI, the heat-stable inhibitor of CAMP-dependent p~1 (5~2 4) has been shown to confer tight binding to the CAMP-dependent protein kinase (5, 6).