Biochem. J. (2007) 408
(131–138) (Printed in Great Britain)
Comparative in vitro and ex vivo activities of selected inhibitors of transthyretin aggregation: relevance in drug design
Isabel CARDOSO*, Maria Rosário ALMEIDA*†, Nelson FERREIRA*†, Gemma ARSEQUELL‡, Gregorio VALENCIA‡ and Maria João SARAIVA*†1
*Molecular Neurobiology Unit, IBMC, University of Porto, 4150-180 Porto, Portugal, †ICBAS, University of Porto, Porto, Portugal, and ‡Instituto de Investigaciones Químicas y Ambientales de Barcelona, Consejo Superior de Investigaciones Científicas (IIQAB-CSIC), 08034 Barcelona, Spain
Destabilization of the tetrameric fold of TTR (transthyretin) is important for aggregation of the protein which culminates in amyloid fibril formation. Many TTR mutations interfere with tetramer stability, increasing the amyloidogenic potential of the protein. The vast majority of proposed TTR fibrillogenesis inhibitors are based on in vitro assays with isolated protein, limiting their future use in clinical assays. In the present study we investigated TTR fibrillogenesis inhibitors using a cellular system that produces TTR intermediates/aggregates in the medium. Plasmids carrying wild-type TTR, V30M or L55P cDNA were transfected into a rat Schwannoma cell line and TTR aggregates were investigated in the medium using a dot-blot filter assay followed by immunodetection. Results showed that, in 24 h, TTR L55P forms aggregates in the medium, whereas, up to 72 h, wild-type TTR and V30M do not. A series of 12 different compounds, described in the literature as in vitro TTR fibrillogenesis inhibitors, were tested for their ability to inhibit L55P aggregate formation; in this system, 2-[(3,5-dichlorophenyl) amino] benzoic acid, benzoxazole, 4-(3,5-difluorophenyl) benzoic acid and tri-iodophenol were the most effective inhibitors, as compared with the reference iododiflunisal, previously shown by ex vivo and in vitro procedures to stabilize TTR and inhibit fibrillogenesis. Among these drugs, 2-[(3,5-dichlorophenyl) amino] benzoic acid and tri-iodophenol stabilized TTR from heterozygotic carriers of V30M in the same ex vivo conditions as those used previously for iododiflunisal. The novel cellular-based test herein proposed for TTR fibrillogenesis inhibitor screens avoids not only lengthy and cumbersome large-scale protein isolation steps but also artefacts associated with most current in vitro first-line screening methods, such as those associated with acidic conditions and the absence of serum proteins.
Key words: aggregation, amyloid, anti-amyloidogenic drug, iododiflunisal, transthyretin.
Abbreviations used: AA, amyloid A associated amyloidosis; DCPA, 2-(3,5-dichlorophenyl) amino benzoic acid; DES, diethylstilbestrol; DFPB, 4-(3, 5-difluorophenyl) benzoic acid; DNP, dinitrophenol; FAP, familial amyloidotic polyneuropathy; FCS, foetal calf serum; IEF, isoelectric focusing; PS2, presenilin 2; TEM, transmission electron microscopy; TIP, tri-iodophenol; TTR, transthyretin; WT, wild-type.
1To whom correspondence should be addressed (email mjsaraiv@ibmc.up.pt).
Received 23 May 2007/3 August 2007; accepted 8 August 2007
Published as BJ Immediate Publication 8 August 2007, doi:10.1042/BJ20070689
© The Authors Journal compilation © 2007 Biochemical Society
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