Molecular Mechanism for Inhibition of G Protein-Coupled Receptor Kinase 2 by a Selective RNA Aptamer

Tesmer, Valerie M.; Lennarz, Sabine; Mayer, Günter; Tesmer, John J.G.

doi:10.1016/j.str.2012.05.002

Cited by 51 publications

(58 citation statements)

References 50 publications

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“…The residues that paroxetine interact with in the active site are highly conserved among GRKs, and thus the mechanism underlying its selectivity for GRK2 could not be divined from this structure alone. However, because paroxetine stabilizes the kinase domain of GRK2 in a conformation that has not been observed in other reported GRK2·inhibitor complexes (Thal et al, 2011;Tesmer et al, 2012), this structure could serve as a unique platform for the development of GRK2 selective inhibitors distinct from other classes of currently known GRK2 inhibitors as well as from other protein kinase A, G, and C (AGC kinase) family members.…”

Section: Introductionmentioning

confidence: 87%

Structural and Functional Analysis of G Protein–Coupled Receptor Kinase Inhibition by Paroxetine and a Rationally Designed Analog

Homan¹,

Wu²,

Wilson³

et al. 2013

Mol Pharmacol

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Recently we identified the serotonin reuptake inhibitor paroxetine as an inhibitor of G protein-coupled receptor kinase 2 (GRK2) that improves cardiac performance in live animals. Paroxetine exhibits up to 50-fold selectivity for GRK2 versus other GRKs. A better understanding of the molecular basis of this selectivity is important for the development of even more selective and potent small molecule therapeutics and chemical genetic probes. We first sought to understand the molecular mechanisms underlying paroxetine selectivity among GRKs. We directly measured the K D for paroxetine and assessed its mechanism of inhibition for each of the GRK subfamilies and then determined the atomic structure of its complex with GRK1, the most weakly inhibited GRK tested. Our results suggest that the selectivity of paroxetine for GRK2 largely reflects its lower affinity for adenine nucleotides. Thus, stabilization of off-pathway conformational states unique to GRK2 will likely be key for the development of even more selective inhibitors. Next, we designed a benzolactam derivative of paroxetine that has optimized interactions with the hinge of the GRK2 kinase domain. The crystal structure of this compound in complex with GRK2 confirmed the predicted interactions. Although the benzolactam derivative did not significantly alter potency of inhibition among GRKs, it exhibited 20-fold lower inhibition of serotonin reuptake. However, there was an associated increase in the potency for inhibition of other AGC kinases, suggesting that the unconventional hydrogen bond formed by the benzodioxole ring of paroxetine is better accommodated by GRKs.

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Section: Introductionmentioning

confidence: 87%

Structural and Functional Analysis of G Protein–Coupled Receptor Kinase Inhibition by Paroxetine and a Rationally Designed Analog

Homan¹,

Wu²,

Wilson³

et al. 2013

Mol Pharmacol

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“…Such direct inhibition has been inferred from aptamer-protein structures such as NFκB or GRK2 (Reiter et al 2008;Tesmer et al 2012). However, Lys1.2minE (and Lys1.2minF) inhibits lysozyme cleavage of large substrates by an unexpected mechanism.…”

Section: Discussionmentioning

confidence: 99%

“…The other complexes that have been deposited in the Protein Data Bank (PDB) to date are nucleolin (Allain et al 2000;Bouvet et al 2001), NF-κB (Huang et al 2003), bacteriophage MS2 (Convery et al 1998;Horn et al 2004), thrombin (Long et al 2008), human IgG (Nomura et al 2010), and the G protein-coupled receptor kinase GRK2 (Tesmer et al 2012). A feature common to these specific aptamer-protein interactions is the positive potential of protein surfaces to which each aptamer binds (Fig.…”

Section: Discussionmentioning

confidence: 99%

An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates

Padlan

Malashkevich

Almo

et al. 2014

RNA

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RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid-protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Our 2.0-Å crystal structure of the aptamer-protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein-nucleic acid interface; (1) only 410 Å 2 of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (∼18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na + stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE-lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform.

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“…Some remarkable advantages are their low immunogenicity, a well implemented synthesis, relative small size, flexibility, excellent biocompatibility and the possibility of chemical modification very useful for functionalization purposes. 59 Moreover aptamers, oppositely to proteins, are not denaturalized under thermal shocks because they are able to refold to their active three-dimensional configuration once heating arrests, 60 (Table 1). 57 In general, aptamers have been widely employed for drug delivery strategies 57 in many different platforms such as aptamer-drug conjugates, 64,65 polymeric-based nanoparticles, 66 liposomes, 67 hard inorganic nanoparticles 68,69 or even chimeras.…”

Section: Aptamer Functionalized Mesoporous Silica Nanoparticlesmentioning

confidence: 99%

Recent applications of the combination of mesoporous silica nanoparticles with nucleic acids: development of bioresponsive devices, carriers and sensors

Castillo¹,

Baeza²

2017

Biomater. Sci.

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The discovery and control of the biological roles mediated by nucleic acids have turned them into a powerful tool for the development of advanced biotechnological materials. Much is the importance of those gene-keeping biomacromolecules that even nanomaterials have succumbed to the claimed benefits of DNA and RNA. Currently, there could be found in the literature a practically intractable number of examples which report the use in combination of nanoparticles with nucleic acids, which demands boundedness. Following this premise, this revision will only cover the most recent and powerful strategies developed to exploit the possibilities of nucleic acids as biotechnological materials when in combination with mesoporous silica nanoparticles. The extensive research done on nucleic acids has significantly incremented the technological possibilities for those biomacromolecules, which could be employed in many different applications; where substrate or sequence recognition or modulation of biological pathways due to its coding role in living cells are the most promising. In the present revision, the chosen counterpart, mesoporous silica nanoparticles, also with unique properties, became a reference material for drug delivery and biomedical applications due to their high biocompatibility and porous structure suitable for hosting and delivering small molecules. Although most of revisions deal with significant advances in the use of nucleic acid and mesoporous silica nanoparticles in biotechnological applications, a rationale classification of those new generation hybrid materials is still uncovered. Along this review there will be covered promising strategies for living cell and biological sensors, DNA-based molecular gates with targeting, transfection or silencing properties which could provide a significant advance of current nanomedicine.

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Molecular Mechanism for Inhibition of G Protein-Coupled Receptor Kinase 2 by a Selective RNA Aptamer

Cited by 51 publications

References 50 publications

Structural and Functional Analysis of G Protein–Coupled Receptor Kinase Inhibition by Paroxetine and a Rationally Designed Analog

Structural and Functional Analysis of G Protein–Coupled Receptor Kinase Inhibition by Paroxetine and a Rationally Designed Analog

An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates

Recent applications of the combination of mesoporous silica nanoparticles with nucleic acids: development of bioresponsive devices, carriers and sensors

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