| 1. |
- T. Tegler, Lotta, et al.
(author)
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Efficient protein binders for the C-reactive protein from a designed chemically modified peptide library
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Other publication (other academic/artistic)abstract
- A polypeptide conjugate synthesized by coupling a small organic molecule to the side chain of an amino acid residue in a designed 42-residue polypeptide binds the C-reactive protein (CRP) essentially irreversibly. The specificity in human serum is equal to that of an avian antibody although the size is only 1/30 and the structure unordered. The polypeptide conjugate binds CRP several orders of magnitude more tightly than the small molecule due to the fact that one amino acid has been modified to include a more strongly interacting side chain. The polypeptide was selected from a 16-membered set of sequences with no prior relationship to the target protein and designed to fold into a helix-loop-helix motif. The results suggest that synthetic amino acid alphabets with more strongly interacting side chains can be used to form polypeptides with improved binding properties in comparison to those engineered by biological methods.
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| 2. |
- Tegler, Lotta T., et al.
(author)
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Powerful protein binders from designed polypeptides and small organic molecules : a general concept for protein recognition
- 2011
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In: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 50:8, s. 1823-1827
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Journal article (peer-reviewed)abstract
- High-affinity binders for the C-reactive protein (CRP), with dissociation constants in the pM to nM range and selectivities in human serum comparable to those of antibodies, were obtained by conjugation of 16 designed polypeptides to phosphocholine, a small molecule that binds CRP with a KDvalue of 5I . The polypeptides were not designed specifically to recognize CRP and bind by an adapted fit mechanism.
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| 3. |
- Christopeit, Tony, et al.
(author)
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A surface plasmon resonance-based biosensor with full-length BACE1 in a reconstituted membrane
- 2011
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In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 414:1, s. 14-22
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Journal article (peer-reviewed)abstract
- A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (β-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer's disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer's disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution.
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| 4. |
- Christopeit, Tony, et al.
(author)
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Characterization of Ca2+ and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor
- 2009
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In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 391:1, s. 39-44
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Journal article (peer-reviewed)abstract
- The interactions between Ca2+ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca2+ or phosphocholine were injected. Binding of Ca2+ induced a 10-fold higher signal than expected from the molecular weight of Ca2+. It was interpreted to result from the conformational change that occurs on binding of Ca2+. Two sites with different characteristics were distinguished: a high-affinity site with K-D = 0.03 mM and a low-affinity site with K-D = 5.45 mM. The pH dependencies of the two Ca2+ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K-D = 5 mu M at 10 mM Ca2+. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and Suggest that Ca2+ is an efficient regulator under physiological conditions.
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| 5. |
- Domínguez, José L, et al.
(author)
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Effect of the Protonation State of the Titratable Residues on the Inhibitor Affinity to BACE-1
- 2010
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 49:34, s. 7255-7263
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Journal article (peer-reviewed)abstract
- BACE-1 is one of the aspartic proteases involved in the cleavage of beta amyloid peptide, an initial step in the formation of amyloid plaques whose toxicity induces neuron death in Alzheimer's disease patients. One of the central issues in the search of novel BACE-1 inhibitors is the optimum pH for the binding of inhibitors to the enzyme. It is known that the enzyme has optimal catalytic activity at acidic pH, while cell active inhibitors may bind optimally at higher pH. In this work we determine the effect of the pH on the affinities of a set of inhibitors, with a variety of chemical motifs, for the ectodomain region of BACE-1 by a surface plasmon resonance (SPR) biosensor based assay. In order to understand the molecular interactions that underlie the diverse optimum pH for the binding of the various inhibitors as observed experimentally, we have calculated the titration curves for a set of BACE-1 ligand complexes. The results indicate that the pK(a) values of the titratable residues of the protein depend on the nature of the ligand involved, in disagreement with previous work. The enzyme-inhibitor structures with the resulting protonation states at pH values 4.5 and 7.4 served as the starting point for the prediction of the pH-dependent binding ranking. Our calculations reproduced the entire affinity ranking observed upon pH increase and most of the binding trends among inhibitors, especially at low pH. Finally, our cell-based assays indicate a possible correlation between high inhibitor affinity at both acidic and neutral pH values, with optimal cell response, a result that may open new venues for the search of potent BACE-1 inhibitors that are active at the cellular level.
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| 6. |
- 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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| 7. |
- Elinder, Malin, et al.
(author)
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Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology
- 2011
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In: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 16:1, s. 15-25
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Journal article (peer-reviewed)abstract
- A new fragment library for lead discovery has been designed and experimentally validated for use in surface plasmon resonance (SPR) biosensor-based screening. The 930 compounds in the library were selected from 4.6 million commercially available compounds using a series of physicochemical and medicinal chemistry filters. They were screened against 3 prototypical drug targets: HIV-1 protease, thrombin and carbonic anhydrase, and a nontarget: human serum albumin. compound solubility was not a problem under the conditions used for screening. The high sensitivity of the sensor surfaces allowed the detection of interactions for 35% to 97% of the fragments, depending on the target protein. None of the fragments was promiscuous (i.e., interacted with a stoichiometry ≥5:1 with all 4 proteins), and only 2 compounds dissociated slowly from all 4 proteins. The use of several targets proved valuable since several compounds would have been disqualified from the library on the grounds of promiscuity if fewer target proteins had been used. The experimental procedure allowed an efficient evaluation and exploration of the new fragment library and confirmed that the new library is suitable for SPR biosensor-based screening.
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| 8. |
- 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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| 9. |
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| 10. |
- Gossas, Thomas, et al.
(author)
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Characterization of Ca2+ interactions with matrix metallopeptidase-12 : implications for matrix metallopeptidase regulation
- 2006
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In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 398:3, s. 393-398
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Journal article (peer-reviewed)abstract
- Matrix metallopeptidase-12 (MMP-12) binds three calcium ions and a zinc ion, in addition to the catalytic zinc ion. These ions are thought to have a structural role, stabilizing the active conformation of the enzyme. To characterize the importance of Ca2+ binding for MMP-12 activity and the properties of the different Ca2+ sites, the activity as a function of [Ca2+] and the effect of pH was investigated. The enzymatic activity was directly correlated to calcium binding and a Langmuir isotherm for three binding sites described the activity as a function of [Ca2+]. The affinities for two of the binding sites were quantified at several pH values. At pH 7.5, the K-D was 0.1 mM for the high-affinity binding site, 5 mM for the intermediate-affinity binding site and > 100 mM for the low-affinity binding site. For all three sites, the affinity for calcium decreased with reduced pH, in accordance with the loss of interactions upon protonation of the calcium-co-ordinating aspartate and glutamate carboxylates at acidic pH. The pK(a) values of the calcium binding sites with the highest and intermediate affinities were determined to be 4.3 and 6.5 respectively. Optimal pH for catalysis was above 7.5. The low-, intermediate-and high-affinity binding sites were assigned on the basis of analysis of three-dimensional-structures of MMP-12. The strong correlation between MMP-12 activity and calcium binding for the physiologically relevant [Ca2+] and pH ranges studied suggest that Ca2+ may be involved in controlling the activity of MMP-12.
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| 11. |
- Gossas, Thomas, 1976-
(author)
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Protease Activity, Inhibition and Ligand Interaction Analysis : Developments and Applications for Drug Discovery
- 2007
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Doctoral thesis (other academic/artistic)abstract
- The present study has focused on characterising protease-ligand interactions in the context of drug discovery. The proteases that have been studied are human matrix metallopeptidase 12 (MMP-12), HIV-protease and Hepatitis C virus (HCV) NS3/NS4A protease. These studies have involved kinetic characterisation of protease-inhibitor interactions using biosensor technology, as well as determination of inhibition and activity regulation by using activity assays.The regulation of MMP-12 activity by calcium was proposed, based on the study of the calcium dependence of MMP-12 activity. Furthermore, it was shown that the high affinity of hydroxamate-based inhibitors of MMP-12 were due to slow dissociation of the enzyme-inhibitor complex by using a new biosensor assay for the study of interactions between MMP-12 and ligands.A study of the pH-dependency of protease-inhibitor interactions revealed that the interaction kinetics of HIV-protease inhibitors differed with pH in a way that could be related to the inhibitor structures. This suggested that the forces of interaction are different in the association and dissociation phases of an interaction. Furthermore, it demonstrated the usefulness of pH as a variable in characterising protein-ligand interactions.Results applicable in the discovery of drugs against Hepatitis C were obtained, with the analysis of structure-activity relationships of novel inhibitors. Furthermore, the mode of binding imposed by key functional groups of the inhibitors was explored by investigating the effect of pH on the interactions with NS3.The results show the importance of using appropriate model systems for drug discovery by selecting relevant targets and assay conditions. Furthermore, the usefulness of kinetic rate information in drug discovery is demonstrated. Thus, by contributing to the knowledge of protease-ligand interactions, applicable to both protease inhibitor interactions and protease activity regulation, this thesis is expected to have an impact on the field of protease inhibitor development and drug discovery in general.
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| 12. |
- Gossas, Thomas, et al.
(author)
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The advantage of biosensor analysis over enzyme inhibition studies for slow dissociating inhibitors : characterization of hydroxamate-based matrix metalloproteinase-12 inhibitors
- 2013
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In: MedChemComm. - : Royal Society of Chemistry (RSC). - 2040-2503 .- 2040-2511. ; 4:2, s. 432-442
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Journal article (peer-reviewed)abstract
- The kinetic characteristics of hydroxamate-based inhibitors of matrix metalloproteinase (MMP)-12 were explored using an SPR biosensor-based assay and enzyme inhibition analysis. These high-affinity inhibitors were shown to dissociate very slowly from the enzyme-inhibitor complex while a carboxylate analogue had a much faster dissociation rate, verifying the importance of the hydroxamate group for the slow dissociation. Progress curve enzyme inhibition analysis confirmed that the hydroxamate compounds but not the carboxylate compound acted as time-dependent inhibitors. The slow dissociation excluded steady-state estimation of IC50-values and K-i values but also made K-i values from progress curve analysis unreliable. Although a full characterization of the inhibitors using biosensor analysis was limited by slow dissociation, it provided kinetic and mechanistic information of relevance for MMP drug discovery and avoided some pitfalls of conventional enzyme inhibition assays.
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| 13. |
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| 14. |
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| 15. |
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| 16. |
- Nordström, Helena, et al.
(author)
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Identification of MMP-12 Inhibitors by Using Biosensor-Based Screening of a Fragment Library
- 2008
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In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 51:12, s. 3449-3459
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Journal article (peer-reviewed)abstract
- Small inhibitors of matrix metalloproteinase 12 (MMP-12) have been identified with a biosensor-based screening strategy and a specifically designed fragment library. The interaction between fragments and three variants of the target and a reference protein with an active-site zinc ion was measured continuously by surface plasmon resonance. The developed experimental design overcame the inherent instability of MMP-12 and allowed the identification of fragments that interacted specifically with the active-site of MMP-12 but not with the reference protein. The interaction with MMP-12 for selected compounds were analyzed for concentration dependence and saturability. Compounds interacting distinctly with the target were further evaluated by an activity-based assay, verifying MMP-12 inhibition. Two effective inhibitors were identified, and the compound with highest affinity was confirmed to be a competitive inhibitor with an IC50 of 290 nM and a ligand efficiency of 0.7 kcal/mol heavy atom. This procedure integrates selectivity and binding site identification into the screening procedure and does not require structure determination.
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| 17. |
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| 18. |
- Rönn, Robert, et al.
(author)
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Evaluation of a diverse set of potential P1 carboxylic acid bioisosteres in hepatitis C virus NS3 protease inhibitors
- 2007
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In: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 15:12, s. 4057-4068
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Journal article (peer-reviewed)abstract
- There is an urgent need for more efficient therapies for people infected with hepatitis C virus (HCV). HCV NS3 protease inhibitors have shown proof-of-concept in clinical trials, which make the virally encoded NS3 protease an attractive drug target. Product-based NS3 protease inhibitors comprising a P1 C-terminal carboxylic acid have shown to be effective and we were interested in finding alternatives to this crucial carboxylic acid group. Thus, a series of diverse P1 functional groups with different acidity and with possibilities to form a similar, or an even more powerful, hydrogen bond network as compared to the carboxylic acid were synthesized and incorporated into potential inhibitors of the NS3 protease. Biochemical evaluation of the inhibitors was performed in both enzyme and cell-based assays. Several non-acidic C-terminal groups, such as amides and hydrazides, were evaluated but failed to produce inhibitors more potent than the corresponding carboxylic acid inhibitor. The tetrazole moiety, although of similar acidity to a carboxylic acid, provided an inhibitor with mediocre potencies in both assays. However, the acyl cyanamide and the acyl sulfinamide groups rendered compounds with low nanomolar inhibitory potencies and were more potent than the corresponding carboxylic acid inhibitor in the enzymatic assay. Additionally, results from a pH-study suggest that the P1 C-terminal of the inhibitors comprising a carboxylic acid, an acyl sulfonamide or an acyl cyanamide group binds in a similar mode in the active site of the NS3 protease.
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| 19. |
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| 20. |
- Rönn, Robert, et al.
(author)
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Hepatitis C Virus NS3 Protease Inhibitors Comprising a Novel Aromatic P1 Moiety
- 2008
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In: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 16:6, s. 2955-2967
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Journal article (peer-reviewed)abstract
- Inhibition of the hepatitis C virus (HCV) NS3 protease has emerged as an attractive approach to defeat the global hepatitis C epidemic. In this work, we present the synthesis and biochemical evaluation of HCV NS3 protease inhibitors comprising a non-natural aromatic P-1 moiety. A series of inhibitors with aminobenzoyl sulfonamides displaying submicromolar potencies in the full-length NS3 protease assay was prepared through a microwave-irradiated, palladium-catalyzed, amidocarbonylation protocol.
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| 21. |
- Spurny, Radovan, et al.
(author)
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Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the alpha 7 nicotinic acetylcholine receptor
- 2015
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 112:19, s. E2543-E2552
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Journal article (peer-reviewed)abstract
- The alpha 7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native alpha 7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed alpha 7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal alpha-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the alpha-helix as the fragment wedges between the alpha-helix and a loop homologous to the main immunogenic region of the muscle alpha 1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human a7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the alpha 7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential.
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| 22. |
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| 23. |
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| 24. |
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| 25. |
- Örtqvist, Pernilla, et al.
(author)
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Phenylglycine as a Novel P2 Scaffold in Hepatitis C Virus NS3 Protease Inhibitors
- 2007
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In: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 15:3, s. 1448-1474
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Journal article (peer-reviewed)abstract
- Molecular modeling and inhibitory potencies of tetrapeptide protease inhibitors of HCV NS3 proposed phenylglycine as a new promising P2 residue. The results suggest that phenylglycine might be capable of interacting with the NS3 (protease-helicase/NTPase) in ways not possible for the common P2 proline-based inhibitors. Thus, a series of tripeptides, both linear and macrocyclic, based on p-hydroxy-phenylglycine in the P2 position were prepared and their inhibitory effect determined. When the p-hydroxy group was replaced by methoxy, isoquinolin-, or quinolinyloxy functions, inhibitors with improved potencies were obtained. The P2 phenylglycine-based inhibitors were further optimized by C-terminal extension to acyl sulfonamides and by P1–P3 cyclization, which gave products with inhibition constants in the nanomolar range (75 nM).
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