| 1. |
- Dominguez, Jose L., et al.
(author)
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Experimental and 'in silico' analysis of the effect of pH on HIV-1 protease inhibitor affinity : Implications for the charge state of the protein ionogenic groups
- 2012
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In: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 20:15, s. 4838-4847
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Journal article (peer-reviewed)abstract
- The pH dependence of the HIV-1 protease inhibitor affinity was studied by determining the interaction kinetics of a series of inhibitors at three pH values by surface plasmon resonance (SPR) biosensor analysis. The results were rationalized by molecular mechanics based protocols that have as a starting point the structures of the HIV-1 protease inhibitor complexes differing in the protonation states as predicted by our calculations. The SPR experiments indicate a variety of binding affinity pH dependencies which are rather well reproduced by our simulations. Moreover, our calculations are able to pinpoint the possible changes in the charged state of the protein binding site and of the inhibitor that underlie the observed effects of the pH on binding affinity. The combination of SPR and molecular mechanics calculations has afforded novel insights into the pH dependence of inhibitor interactions with their target. This work raises the possibility of designing inhibitors with different pH binding affinity profiles to the ones described here.
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| 2. |
- Gorny, Xenia, et al.
(author)
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AKAP79/150 interacts with the neuronal calcium-binding protein caldendrin
- 2012
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In: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 122:4, s. 714-726
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Journal article (peer-reviewed)abstract
- The A kinase-anchoring protein AKAP79/150 is a postsynaptic scaffold molecule and a key regulator of signaling events. At the postsynapse it coordinates phosphorylation and dephosphorylation of receptors via anchoring kinases and phosphatases near their substrates. Interactions between AKAP79 and two Ca2+ -binding proteins caldendrin and calmodulin have been investigated here. Calmodulin is a known interaction partner of AKAP79/150 that has been shown to regulate activity of the kinase PKC in a Ca2+-dependent manner. Pull-down experiments and surface plasmon resonance biosensor analyses have been used here to demonstrate that AKAP79 can also interact with caldendrin, a neuronal calcium-binding protein implicated in regulation of Ca2+ -influx and release. We demonstrate that calmodulin and caldendrin compete for a partially overlapping binding site on AKAP79 and that their binding is differentially dependent on calcium. Therefore, this competition is regulated by calcium levels. Moreover, both proteins have different binding characteristics suggesting that the two proteins might play complementary roles. The postsynaptic enrichment, the complex binding mechanism, and the competition with calmodulin, makes caldendrin an interesting novel player in the signaling toolkit of the AKAP interactome.
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| 3. |
- Gossas, Thomas, et al.
(author)
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Aliskiren displays long-lasting interactions with human renin
- 2012
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In: Naunyn-Schmiedeberg's Archives of Pharmacology. - : Springer Science and Business Media LLC. - 0028-1298 .- 1432-1912. ; 385:2, s. 219-224
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Journal article (peer-reviewed)abstract
- Aliskiren is a selective renin inhibitor recently approved for use in hypertension. Efficacy duration appears longer than what would be expected based on its circulating half-life. The aim was therefore to characterize the kinetics of the interaction between aliskiren and renin. The interaction was evaluated in three assays and compared with two other renin inhibitors including remikiren. First, the inhibition of recombinant human renin was assessed by monitoring the cleavage of fluorescent substrate. Second, human plasma renin activity (PRA) was monitored by measuring generated angiotensin I over 1 h in the presence or absence of inhibitor. Finally, the affinity, association and dissociation rate constants were determined by using a surface plasmon resonance (SPR) biosensor assay. Aliskiren and remikiren were found to be equipotent inhibitors of recombinant renin activity (K (i) ≤ 0.04 nM) while compound 1 displayed a K (i) value of 1 nM. PRA was efficiently inhibited by both aliskiren and remikiren with IC(50) values of 0.2-0.3 nM. Remikiren and aliskiren also displayed long-lasting interactions with immobilized renin having k (off) values of 0.18 and 0.11 × 10(-3) s(-1) respectively. These dissociation rate constants corresponded to residence times of 1.5 and 2.5 h, respectively, while compound 1 had a residence time lasting only 3 min. It is therefore concluded that the long-lasting interaction between aliskiren and human renin may contribute to the 24 h anti-hypertensive effect seen in clinical trials and possibly also to target-mediated drug disposition.
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| 4. |
- Seeger, Christian, et al.
(author)
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Histaminergic pharmacology of homo-oligomeric beta 3 gamma-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology
- 2012
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In: Biochemical Pharmacology. - : Elsevier BV. - 0006-2952 .- 1356-1839. ; 84:3, s. 341-351
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Journal article (peer-reviewed)abstract
- A surface plasmon resonance biosensor assay was established for studying the interactions of 51 histaminergic and 15 GABAergic ligands with homo-oligomeric beta 3 GABA(A) receptors. Detergent solubilized receptors were successfully immobilized via affinity-capture on biosensor surfaces. The interaction kinetics of both histaminergic and GABAergic ligands were very rapid but affinities could be determined by steady-state analysis. Binding of several GABAergic ligands was observed, in agreement with previous data. Histamine and 16 histaminergic ligands were detected to directly bind to beta 3 GABA(A) receptors with micromolar affinity (K-D <300 mu M), thus extending previous evidence that beta 3 GABA(A) receptors can interact with histaminergic ligands. Histamine exhibited an affinity for these receptors comparable to that for human histamine type 1 (H1) or type 2 (H2) receptors. Furthermore, 13 of these histaminergic ligands appeared to compete with histamine. The discovery that H2, H3 and H4 receptor ligands interact with beta 3 receptors indicates a unique histaminergic pharmacology of these receptors. Due to their low affinity for the homo-pentameric beta 3 receptors these histaminergic drugs are not expected to modulate these receptors at clinically relevant concentrations. The results support the use of the new biosensor assay for the identification of drugs interacting with full length receptors and for fragment-based drug discovery of high affinity ligands for beta 3 receptors. Drugs with high affinity and selectivity for these receptors can be used to clarify the question whether beta 3 receptors do exist in the brain, and provide new avenues for the development of therapeutically active compounds targeting this novel histamine binding site.
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| 5. |
- Seeger, Christian, et al.
(author)
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Kinetic and mechanistic differences in the interactions between caldendrin and calmodulin with AKAP79 suggest different roles in synaptic function
- 2012
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In: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 25:10, s. 495-503
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Journal article (peer-reviewed)abstract
- The kinetic and mechanistic details of the interaction between caldendrin, calmodulin and the B-domain of AKAP79 were determined using a biosensor-based approach. Caldendrin was found to compete with calmodulin for binding at AKAP79, indicating overlapping binding sites. Although the AKAP79 affinities were similar for caldendrin (KD?=?20?n m) and calmodulin (KD?=?30?n m), their interaction characteristics were different. The calmodulin interaction was well described by a reversible one-step model, but was only detected in the presence of Ca2+. Caldendrin interacted with a higher level of complexity, deduced to be an induced fit mechanism with a slow relaxation back to the initial encounter complex. It interacted with AKAP79 also in the absence of Ca2+, but with different kinetic rate constants. The data are consistent with a similar initial Ca2+-dependent binding step for the two proteins. For caldendrin, a second Ca2+-independent rearrangement step follows, resulting in a stable complex. The study shows the importance of establishing the mechanism and kinetics of proteinprotein interactions and that minor differences in the interaction of two homologous proteins can have major implications in their functional characteristics. These results are important for the further elucidation of the roles of caldendrin and calmodulin in synaptic function.
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