Modeling, synthesis and biological evaluation of potential retinoid X receptor-selective agonists: novel halogenated analogues of 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (bexarotene).

The synthesis of halogenated analogues of 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (1), known commonly as bexarotene, and their evaluation for retinoid X receptor (RXR)-specific agonist performance is described. Compound 1 is FDA approved to treat cutaneous T-cell lymphoma (CTCL); however, bexarotene treatment can induce hypothyroidism and elevated triglyceride levels, presumably by disrupting RXR heterodimer pathways for other nuclear receptors. The novel halogenated analogues in this study were modeled and assessed for their ability to bind to RXR and stimulate RXR homodimerization in an RXRE-mediated transcriptional assay as well as an RXR mammalian-2-hybrid assay. In an array of eight novel compounds, four analogues were discovered to promote RXR-mediated transcription with EC(50) values similar to that of 1 and are selective RXR agonists. Our approach also uncovered a periodic trend of increased binding and homodimerization of RXR when substituting a halogen atom for a proton ortho to the carboxylic acid on 1.

Self-interaction nanoparticle spectroscopy: a nanoparticle-based protein interaction assay.

Solution conditions conducive to protein crystallization are identified mainly in an empirical manner using screening methods. Measurements of a dilute solution thermodynamic parameter, the osmotic second virial coefficient, have been shown to be useful in guiding this search, yet the measurement of this parameter remains difficult. In this work, a nanoparticle-based assay, self-interaction nanoparticle spectroscopy, is presented as an efficient alternative. The method involves adsorbing proteins on the surface of gold nanoparticles and adding the protein/gold conjugates to solutions of interest for crystallization. The optical properties of gold colloid, including macroscopic ones such as color, are sensitive to the interparticle separation distance, and they are demonstrated to correlate with the value of the second virial coefficient for BSA and ovalbumin. Serendipitously, the conditions that correspond to second virial coefficient values within the thermodynamic region ideal for protein crystallization lead to the maximum change in color of the gold suspensions. Given the remarkable efficiency of this method, it holds significant potential to aid in the crystallization of proteins that have not been crystallized previously. Moreover, this method may find utility in the analysis of weak homo- and heterotypic interactions involved in other biological applications, including preventing protein aggregation and formulating therapeutic proteins.

Predictive crystallization of ribonuclease A via rapid screening of osmotic second virial coefficients.

Important progress has been made in recent years toward developing a molecular-level understanding of protein phase behavior in terms of the osmotic second virial coefficient, a thermodynamic parameter that characterizes pairwise protein interactions. Yet there has been little practical application of this knowledge to the field of protein crystallization, largely because of the difficult and time-consuming nature of traditional techniques for characterizing protein interactions. Self-interaction chromatography has recently been proposed as a highly efficient method for measuring the osmotic second virial coefficient. The utility of the technique is examined in this work by characterizing virial coefficients for ribonuclease A under 59 solution conditions using several crystallization additives, including PEG, sodium chloride, ammonium sulfate, and propanol. The virial coefficient measurements show some counterintuitive trends and shed light on the previous difficulties in crystallizing ribonuclease A. Crystallization experiments at the corresponding solution conditions were conducted by using ultracentrifugal crystallization. Using this methodology, ribonuclease A crystals were obtained under conditions for which the virial coefficients fell within the "crystallization slot." Crystallographic characterization showed that the crystals diffract to high resolution. Metastable crystals were also obtained for conditions outside, but near, the "crystallization slot," and they could also be frozen and used to collect structural information.