uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
Effect of the Protonation State of the Titratable Residues on the Inhibitor Affinity to BACE-1
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Show others and affiliations
2010 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 34, 7255-7263 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2010. Vol. 49, no 34, 7255-7263 p.
National Category
Chemical Sciences
URN: urn:nbn:se:uu:diva-129426DOI: 10.1021/bi100637nISI: 000281052600003PubMedID: 20687525OAI: oai:DiVA.org:uu-129426DiVA: diva2:343704
Available from: 2010-08-15 Created: 2010-08-15 Last updated: 2013-02-11Bibliographically approved
In thesis
1. Protein Interaction Studies with Low Molecular Weight Ligands: Applications for Drug Discovery, Basic Research and Diagnostic Tool Design
Open this publication in new window or tab >>Protein Interaction Studies with Low Molecular Weight Ligands: Applications for Drug Discovery, Basic Research and Diagnostic Tool Design
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, the interactions between different proteins and small ligands were characterized by surface plasmon resonance spectroscopy (SPR) and fluorescence resonance energy transfer (FRET) based assays.   

For the C-reactive protein (CRP), a new type of artificial binder was identified which allows designing diagnostic assays superior to commonly used standard assays. Furthermore, an interaction study with the endogenous ligand phosphocholine revealed the importance of the avidity of pentameric CRP for the distinction of different types of lipid membranes. The interaction study with calcium showed how SPR based assays can be used to study ion-protein interactions despite the low atomic weight of ions.   

The transmembrane protease BACE1, an important drug target for Alzheimer’s disease, was immobilized to an SPR biosensor surface and embedded into a lipid membrane. An interaction study with a set of known BACE1 inhibitors showed that the transmembrane region has only minor effects on the interactions. Furthermore the pH-dependencies of the interactions were investigated and revealed new important conclusions for inhibitor design. Computer aided modelling showed that the protonation state of the aspartic dyad is dependent on the interacting inhibitor which offers new perspectives for in silico screenings.

The SPR assay developed for BACE1 was adapted to a more complex membrane protein, the pentameric β3 GABAA receptor. The assay allowed the pharmacological characterisation for histaminergic and GABAergic ligands and gave further evidence for cross-talk between the two signal transduction pathways. This study shows that the immobilisation method used for BACE1 and the ß3 GABAA receptor has the potential to become a standard method for handling membrane proteins.  

The identification of new drug leads from natural sources is a common strategy for drug discovery. A combination of SPR and FRET based activity assays were explored to increase the efficiency of this process. For HIV-1 protease, secreted aspartic protease (SAP) 1, 2 and 3 extracts from a marine vertebrate were identified containing potent inhibitors which interacted with the active site of the enzymes.

The studies in this thesis show that the investigation of protein interactions is crucial for understanding protein functions and can help to develop novel drugs for the treatment of different diseases.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 34 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1007
National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-188328 (URN)978-91-554-8566-5 (ISBN)
Public defence
2013-02-14, B21, BMC, Husargatan 3, Uppsala, 13:15 (English)
Available from: 2013-01-24 Created: 2012-12-14 Last updated: 2013-04-02Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Christopeit, TonyDanielson, U Helena
By organisation
Department of Biochemistry and Organic Chemistry
In the same journal
Chemical Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 499 hits
ReferencesLink to record
Permanent link

Direct link