Screening a library of potential prion therapeutics against cellular prion proteins and insights into their mode of biological activities by surface plasmon resonance

doi:10.1016/j.jpba.2005.08.011

Journal of Pharmaceutical and Biomedical Analysis

Volume 40, Issue 4, 3 March 2006, Pages 822-832

https://doi.org/10.1016/j.jpba.2005.08.011 Get rights and content

Abstract

The conversion of cellular prion protein (PrP^C) to the protease resistant isoform (PrP^Sc) is considered essential for the progression of transmissible spongiform encephalopathies (TSEs). A potential therapeutic strategy for preventing the accumulation of PrP^Sc is to stabilize PrP^C through the direct binding of a small molecule to make conversion less energetically favourable. Using surface plasmon resonance (SPR)-based technology we have developed a procedure, based on direct binding, for the screening of small molecules against PrP^C immobilized on a sensor chip. In this paper we report some problems associated with the immobilization of PrP^C onto the sensor surface for conducting drug screening and how these problems were overcome. We demonstrated that the conformational change of PrP^C on the chip surface leads to increased exposure of the C-terminal which was observed by the increase in quinacrine binding over time, and loss of heparin binding to the N-terminal. In addition, we also report the results of the successful screening of a library of 47 compounds of known activity in cell line or cell free conversion studies for direct binding to three forms of PrP^C (huPrP^C, t-huPrP^C and moPrP^C). These results show the usefulness of this technique for the identification of PrP^C binding ligands and to gain some insight as to their potential mode of action.

Introduction

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases affecting animals and humans [1]. The human form of TSE is known as Creuzfeld–Jakob disease (CJD). Formation of amyloidal deposits in affected brain is a hallmark of the disease similar to many other neurodegenerative conditions such as Alzheimer's disease. These deposits are constituted mainly of aggregated prion proteins in a misfolded state. The cause for the misfolding/aggregation of the host proteins at a molecular level is unknown and occurs spontaneously (sporadic type) or can be attributed to genetic reasons (familial type). Strikingly different from other neurodegenerative diseases, TSEs are also infective (iatrogenic type) and can be transmitted via transplants, contaminated biological products from cadavers, blood transfusion, contaminated surgical instruments and ingestion of infected materials. The latter has been observed in humans after consumption of cattle affected by bovine spongiform encephalopathy (BSE) and has been termed variant CJD (vCJD). No effective treatments for CJDs are currently available, nor has a bio-molecular component been fully validated as a drug target. However, it is believed that the formation of protease resistant insoluble prion protein (PrP^Sc), which is the main component of amyloidal deposits, from the cellular prion protein (PrP^C), is essential for the progression of the disease. Therefore, both PrP^Sc and PrP^C are currently being used as potential drug targets [2].

PrP^C is a membrane protein of unknown physiological function [3], [4], [5]. It consists of an unstructured, flexible N-terminal domain (AA23–110) which contains five octarepeats (AA51–91), a globular C-terminal domain (AA111–230) which contains two glycosylation sites (Asn181, Asn197) [6] and a glycosylphosphatidyl-inositol (GPI) anchor (Ser230) [7]. Several compounds are known to interact directly with PrP^C including the natural binding partners heparin and glycosaminoglycans (GAGs) [8], [9], [10], [11], copper [12], [13], [14], [15], [16], nucleic acids (RNA and DNA) [17], [18], [19], [20], [21], plasminogens [22], [23], laminin receptors [24], PrP fragments and PrP^C itself [23]. Other binding partners have been reported including various antibodies [25], [26], Congo Red and quinacrine [27]. However, only one compound, quinacrine, can be considered as a small drug-like molecule.

Several studies have reported binding events between PrP^C and antibodies, heparin and plasminogen using surface plasmon resonance (SPR) [8], [23], [25], [28], [29]. In all of these studies the quantity of PrP^C immobilized on the SPR sensor chip was low (500–1000 RU). Baseline instability was not reported in any of these studies although changes in binding behaviour of PrP^C to some antibodies raised against the N-terminal epitopes over time was observed [26].

As part of an ongoing medicinal chemistry program towards the automated screening of potential therapeutic compounds against CJD, a direct binding assay for small ligands with recombinant human prion protein (huPrP^C) was explored. SPR was selected as an assay platform due to the ease of assay development, medium sample throughput and automated sample handling, as well as the reported success of the technique as a binding assay for small drug-like molecules against other drug targets [30], [31], [32]. However, it is well known that prion protein binds strongly to metal surfaces [33], [34], [35]. It may also bind to the carboxymethylated dextran (CM-dextran) on the gold surface because, structurally, CM-dextran resembles heparin, which is known to bind to prion proteins [8], [9], [10], [11]. For the convenience and optimal throughput in drug screening for prion lead discovery, immobilizing PrP^C on the surface and injecting a compound of interest to study their interactions would be ideal. But drug like compounds normally have a low molecular weight between 300 and 800 Da. To observe binding between a prion protein and those compounds it is essential to be able to immobilize prion proteins at a level between 3000 and 10 000 RU and keep the baseline as stable as possible. A number of problems were encountered and overcome during the development of an optimal protocol for the use of SPR for screening and mechanistic studies.

In this paper we describe the problems associated with the high huPrP^C immobilization levels required for drug screening, possible causes and changes to the immobilization procedure that overcame these problems. We demonstrate that it is a repositioning of PrP^C on the sensor surface that leads to the increased exposure of the C-terminal, and an increase in quinacrine binding. Interactions between PrP^C and CM-dextran also resulted in the loss of heparin binding to the N-terminal. We also report the results of the successful screening of a library of 47 compounds of known activity in cell line or cell free conversion studies for direct binding to three forms of PrP^C (huPrP^C, t-huPrP^C and moPrP^C).

Section snippets

Materials

N-hydroxysuccinimide (NHS), N-ethyl-N′-(3–diethylaminopropyl) carbodiimide hydrochloride (EDC), 1 M ethanolamine, HBS-EP buffer, surfactant P20, regeneration solution (10 mM glycine–HCl, pH 3.0) and CM-dextran (MW ≈ 13 000 Da) were purchased from BIAcore. Sodium phosphate, ethylenediaminetetraacetic acid (EDTA), sodium chloride, sodium hydroxide and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich. Bovine serum albumin (BSA) was purchased from New England BioLabs. Recombinant full length

Preparation of sensor surface

The standard immobilization conditions, as recommended by the manufacturer of the BIAcore 3000, were followed using huPrP^C to establish a general protocol. Various protein concentrations lead to immobilization levels from 500 to 10 000 RU. In order to accurately measure the interaction of small compounds with huPrP^C, an immobilization level of approximately 4000 RU was selected. A protein concentration of 2 μg/mL was used for the immobilization. A set of compounds including quinacrine, Congo Red

Conclusions

An SPR system using Biacore 3000 has been identified as a useful tool for the screening of compounds binding to PrP^C. An optimal immobilization procedure was developed, which significantly reduced the observed baseline increase. The intensive studies of interactions between PrP^C and CM-dextran revealed that the interactions between immobilized PrP^C and the CM-dextran matrix are fast and irreversible. It results in the heparin binding site on the N-terminal of PrP^C becoming unavailable therefore

Acknowledgements

We would like to thank A. Gill for the generous gift of the recombinant huPrP^C, t-huPrP^C and moPrP^C. Funding was provided by the Department of Health (Contract No. DH007/0102).

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Screening a library of potential prion therapeutics against cellular prion proteins and insights into their mode of biological activities by surface plasmon resonance

Abstract

Introduction

Section snippets

Materials

Preparation of sensor surface

Conclusions

Acknowledgements

Lancet

FEBS Lett.

Biochimie

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Mol. Biol.

J. Mol. Biol.

C.R. Biologies

J. Mol. Biol.

J. Pharmacol. Sci.

J. Biol. Chem.

Eur. J. Med. Chem.

Cell

Int. J. Antimicrob. Agents

Proc. Natl. Acad. Sci. U.S.A.

Clin. Microbiol. Rev.

Cell Biochem. Biophys.

Biochem. J.

Biochem. J.

J. Virol.

Biochemistry

Biochemistry

Prog. Biochem. Biophys.

Proc. Natl. Acad. Sci. U.S.A.

Nature