The X-ray crystal structure of the sulfonated azo dye Congo Red, a non-peptidic inhibitor of HIV-1 protease which also binds to reverse transcriptase and amyloid proteins

Abstract

Congo Red is a sulfonated azo dye and widely used biological stain that has recently been the focus of intense interest because it has been shown to bind to proteins involved in viral recognition and replication. Congo Red also finds wide use as a histological stain for amyloid proteins of the type found in neurodegenerative conditions such as Alzheimer's disease, transmissible spongiform encephalopathies in cattle and mink, and scrapie in sheep. Congo Red has been demonstrated to protect normal prion protein from being converted to the protease-resistant form, an important step in the pathology of the so-called 'slow viral' diseases. The range of biological molecules to which Congo Red binds makes it an important lead compound in drug development, for example in the development of new anti-HIV and anti-Alzheimer's therapeutic agents. In this report we present the first high-resolution structure of Congo Red: the low-temperature (173 K) X-ray crystal structure determination of its calcium salt. Two conformations of the molecule are found in the same crystal structure, one in which the central biphenyl group assumes a twisted (25°) conformation, and one in which the biphenyl group is planar and is located on a crystallographic inversion centre. In both conformations the sulfonate groups are oriented and with respect to the long molecular axis and assume eclipsed conformations with respect to the naphthalene rings. A comparison is made with a published structure [Turnell, W.G., and Finch, J.T. (1992) J Mol Biol 227: 1205-1223] in which Congo Red is bound to porcine insulin, this complex serving as a model for amyloid binding. The results illustrate the conformational flexibility possessed by the biphenyl spacer, which allows the hydrophobic portion of the molecule to assume an optimum fit in the hydrophobic binding pockets of target proteins. A model is presented for the binding of Congo Red to the HIV protease in which the sulfonate groups interact with the side-chains of arginine residues. This proposed binding mode is consistent with the observed binding for other sulfonated aromatic inhibitors such as Evans Blue.