Beactica

Aliskiren displays long-lasting interactions with human renin.

maleli — Fri, 10 Feb 2012 11:15:07 +0000

Gossas T, Vrang L, Henderson I, Sedig S, Sahlberg C, Lindström E, Danielson U. H. (2011) N-S Arch. Pharmaco. 385(2);219-24

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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A surface plasmon resonance based biosensor with full-length BACE1 in a reconstituted membrane.

maleli — Fri, 10 Feb 2012 11:14:39 +0000

Christopeit, T., Stenberg, G., Gossas, T., Nyström., S., Baraznenok, V., Lindström, E., Danielson, U.H. (2011) Analytical Biochem. 414; 14-22.

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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Powerful protein binders from designed polypeptides and small organic molecules – a general concept for protein recognition.

maleli — Fri, 10 Feb 2012 11:13:13 +0000

Tegler, L.T. Nonglaton, G., Büttner, F., Caldwell, K., Christopeit, T., Danielson, U.H., Fromell, K., Gossas, T., Larsson, A., Longati, P., Norberg, T., Ramapanicker, R., Rydberg, J. and Baltzer. (2011) Angew. Chem. 50; 1823-1827.

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 K_D value of 5 μM (see picture). The polypeptides were not designed specifically to recognize CRP and bind by an adapted fit mechanism.

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Deconstruction of Non-Nucleoside Reverse Transcriptase Inhibitors of Human Immunodeficiency Virus Type 1 for Exploration of the Optimization Landscape of Fragments

maleli — Fri, 10 Feb 2012 11:12:44 +0000

Brandt, P., Geitmann, M. and Danielson, U.H. (2011) J. Med. Chem. 54(3); 709-718.

Abstract

This study has taken a closer look at the theoretical basis for protein−fragment interactions. The approach involved the deconstruction of 3 non-nucleoside inhibitors of HIV-1 reverse transcriptase and investigation of the interaction between 21 substructures and the enzyme. It focused on the concept of ligand efficiency and showed that ligand independent free energy fees (ΔG_ind) are crucial for the understanding of the binding affinities of fragments. A value of 7.0 kcal mol⁻¹ for the ΔG_ind term is shown to be a lower limit for the NNRTI binding pocket of HIV-1 RT. The addition of the ΔG_ind term to the dissociation free energy in the calculation of a corrected ligand efficiency, in combination with the lack of an efficient ligand binding hot spot in the NNIBP, fully explains the existence of nonbinding NNRTI substructures. By applying the concept to a larger set of ligands, we could define a binding site profile that indicates the absence of an efficient fragment binding hot spot but an efficient binding of full-sized NNRTIs. The analysis explains some of the challenges in identifying fragments against flexible targets involving conformational changes and how fragments may be prioritized.

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Identification of a novel scaffold for allosteric inhibition of wild type and drug resistant HIV-1 Reverse transcriptase by fragment library screening.

maleli — Fri, 10 Feb 2012 11:11:16 +0000

Geitmann, M., Elinder, M., Seeger, C., Brandt, P., de Esch, I. and Danielson, U.H. (2011) J. Med. Chem. 54(3); 699-708.

Abstract

A novel scaffold inhibiting wild type and drug resistant variants of human immunodeficiency virus type 1 reverse transcriptase (HIV-1RT) has been identified in a library consisting of 1040 fragments. The fragments were significantly different from already known non-nucleoside reverse transcriptase inhibitors (NNRTIs), as indicated by a Tversky similarity analysis. A screening strategy involving SPR biosensor-based interaction analysis and enzyme inhibition was used. Primary biosensor-based screening, using short concentration series, was followed by analysis of nevirapine competition and enzyme inhibition, thus identifying inhibitory fragments binding to the non-nucleoside reverse transcriptase inhibitor (NNRTI) binding site. Ten hits were discovered, and their affinities and resistance profiles were evaluated with wild type and three drug resistant enzyme variants (K103N, Y181C, and L100I). One fragment exhibited submillimolar K_D and IC₅₀ values against all four tested enzyme variants. A substructure comparison between the fragment and 826 structurally diverse published NNRTIs confirmed that the scaffold was novel. The fragment is a bromoindanone with a ligand efficiency of 0.42 kcal/mol⁻¹.

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Quantification of interactions between drug leads and serum proteins by use of “binding efficiency”.

maleli — Fri, 10 Feb 2012 11:10:09 +0000

Svahn, S., Vrang, L., Terelius, Y., Danielson, U.H. (2011) Anal. Biochem. 409; 163–175.

Abstract

To develop efficient and reliable methods for prediction of serum protein binding of drug leads, the kinetic characteristics for the interactions between selected compounds and human serum albumin and α₁-acid glycoprotein have been explored using a surface plasmon resonance biosensor. Conventional methods for quantification of interactions (i.e., using rate constants or affinities determined on the basis of a reasonable mechanistic model) were applicable for only a few of the compounds. The affinity of a primary interaction and the contribution of lower affinity secondary interactions could be estimated for some compounds, but the affinity of many compounds could not be quantified by either of these methods. To have a quantification method that could be used for all compounds, independent of affinity and complexity of interaction mechanisms, the concept of “binding efficiency,” analogous to “catalytic efficiency” used for enzymes, was developed. It allowed the quantification of the binding of compounds interacting with weak affinity and for which saturation is not reached within a concentration range where the compound is soluble or when the influence of interactions with secondary sites makes interpretations difficult. In addition, compounds with large fractional binding can be identified by this strategy and simply quantified relative to reference compounds. This approach will enable ranking and identification of structure-activity relationships of compounds with respect to their serum protein binding profile.

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Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors.

maleli — Fri, 10 Feb 2012 11:09:26 +0000

Geitmann, M., Dahl, G., Danielson, U.H.(2011) J. Mol. Recog. 24(1):60-70.

Abstract

The mechanism and kinetics of the interactions between ligands and immobilized full-length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3-NS4A interaction consisted of a high-affinity (K(D) = 50 nM) and a low-affinity (K(D) = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism-based inhibitor VX 950 exhibited a time-dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN-191 showed no signs of time-dependent interactions, but ITMN-191 had the highest affinity of the tested compounds, with both the slowest dissociation (k(off)) and fastest association rate, closely followed by BILN 2061. The k(off) for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti-HCV lead discovery and optimization.

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Experimental Validation of a Fragment Library for Lead Discovery using SPR Biosensor Technology.

maleli — Fri, 10 Feb 2012 11:08:52 +0000

Elinder, M., Geitmann, M., Gossas, T., Källblad, P., Winquist, J., Nordström, H., Hämäläinen, M. and Danielson, U.H.(2011) J. Biomol.Screen. 16(1); 15-25.

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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Surface Plasmon Resonance Biosensor Based Fragment Screening Using Acetylcholine Binding Protein Identifies Ligand Efficiency Hot Spots (LE Hot Spots) by Deconstruction of Nicotinic Acetylcholine Receptor α7 Ligands.

maleli — Fri, 10 Feb 2012 11:07:23 +0000

De Kloe, G., Retra, K., Geitmann, M., Källblad, P., Nahar, T., van Elk, R., Smit, A.B., van Muijlwijk-Koezen, J.E., Leurs, R., Irth, H., Danielson, U.H. and de Esch, I.J.P. (2010) Journal of Medicinal Chemistry 53; 7192–7201.

Abstract

The soluble acetylcholine binding protein (AChBP) is a homologue of the ligand-binding domain of the nicotinic acetylcholine receptors (nAChR). To guide future fragment-screening using surface plasmon resonance (SPR) biosensor technology as a label-free, direct binding, biophysical screening assay, a focused fragment library was generated based on deconstruction of a set of α7 nAChR selective quinuclidine containing ligands with nanomolar affinities. The interaction characteristics of the fragments and the parent compounds with AChBP were evaluated using an SPR biosensor assay. The data obtained from this direct binding assay correlated well with data from the reference radioligand displacement assay. Ligand efficiencies for different (structural) groups of fragments in the library were correlated to binding with distinct regions of the binding pocket, thereby identifying ligand efficiency hot spots (LE hot spots). These hot spots can be used to identity the most promising hit fragments in a large scale fragment library screen.

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Interaction kinetic and structural dynamic analysis of ligand binding to acetylcholine-binding protein.

maleli — Fri, 10 Feb 2012 11:06:12 +0000

Geitmann, M., Retra, K., de Kloe, G., Homan, E., Källblad, P., de Esch, I., and Danielson, U.H. (2010) Biochemistry 49; 8143–8154.

Abstract

The mechanism of agonist interactions with Cys-loop ligand-gated ion channels has been studied using the acetylcholine-binding protein (AChBP) from Lymnaea stagnalis as a model protein and acetylcholine, nicotine, epibatidine, and a series of substituted quinuclidines as ligands. A biosensor-based assay for direct interaction studies of immobilized AChBP and small molecule ligands was developed. It allowed the characterization of the interaction kinetics of the ligands and the structural dynamics of the protein. The interactions with AChBP were very sensitive to variations in the experimental conditions and showed several types of complexities. These could be resolved into two types of ligand-induced secondary effects with different kinetics, representing fast and slow conformational changes. The data could be rationalized in a mechanistic model, and a structural interpretation of the interaction was obtained by molecular modeling involving induced fit and loop flexibility simulations. The data suggest that AChBP exhibits ligand-induced structural dynamics, as expected for the ligand gating mechanism of Cys-loop receptors. It shows that the formation of the initial encounter complex between AChBP and ligands is very rapid, in accordance with the functional characteristics required of neurotransmission. These developed procedures will enable further exploration of the mechanism of Cys-loop receptor function and the identification of specific ligands suitable for pharmacological use.

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