| 1. |
- T. Tegler, Lotta, et al.
(författare)
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Efficient protein binders for the C-reactive protein from a designed chemically modified peptide library
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Annan publikation (övrigt vetenskapligt/konstnärligt)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.
(författare)
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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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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 50:8, s. 1823-1827
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Tidskriftsartikel (refereegranskat)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.
(författare)
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A surface plasmon resonance-based biosensor with full-length BACE1 in a reconstituted membrane
- 2011
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 414:1, s. 14-22
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Tidskriftsartikel (refereegranskat)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.
(författare)
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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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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 391:1, s. 39-44
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Tidskriftsartikel (refereegranskat)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.
(författare)
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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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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 49:34, s. 7255-7263
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Tidskriftsartikel (refereegranskat)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.
(författare)
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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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Ingår i: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 20:15, s. 4838-4847
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology
- 2011
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Ingår i: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 16:1, s. 15-25
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Aliskiren displays long-lasting interactions with human renin
- 2012
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Ingår i: 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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Tidskriftsartikel (refereegranskat)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.
(författare)
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Characterization of Ca2+ interactions with matrix metallopeptidase-12 : implications for matrix metallopeptidase regulation
- 2006
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 398:3, s. 393-398
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Tidskriftsartikel (refereegranskat)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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