Abstract
Vitamin A derived retinoid compounds have multiple, powerful roles in the cellular growth and development cycle and, as a result, have attracted significant attention from both academic and pharmaceutical research in developing and characterizing synthetic retinoid analogues. Simplifying the hit development workflow for retinoid signaling will improve options available for tackling related pathologies, including tumor growth and neurodegeneration. Here, we present a novel assay that employs an intrinsically fluorescent synthetic retinoid, DC271, which allows direct measurement of the binding of nonlabeled compounds to relevant proteins. The method allows for straightforward initial measurement of binding using existing compound libraries and is followed by calculation of binding constants using a dilution series of plausible hits. The ease of use, high throughput format, and measurement of both qualitative and quantitative binding offer a new direction for retinoid-related pharmacological development.
Highlights
Retinoids are a class of molecules derived from vitamin A, which play a wide and active role within the growth and development cycle of mammalian cells.[1]
This control over cell differentiation, proliferation, and apoptosis is mediated by the interaction of all-trans retinoic acid (ATRA, Figure 1) with the retinoic acid receptor (RAR) proteins, which form DNA
Cellular retinoic acid binding proteins, CRABP-I and CRABP-II, are small, intracellular shuttling proteins associated with retinoic acid trafficking in the cytosol.[9]
Summary
DC271 is a further development of EC23, based on a donor−acceptor diphenylacetylene structure, which exhibits solvatochromic fluorescence when excited at wavelengths suitable for microscopy and as a fluorescent probe (absorbing at 340−410 nm).[17,18] The compound’s emission is highly dependent on the local environment; intense and blue-shifted in nonpolar solvents, weak and red-shifted in polar solvents. Each plate was read using a Biotek Synergy H4 plate reader, in fluorescence intensity mode, exciting at 355 nm and reading emission at 460 nm These wavelengths were deemed the most suitable by examination of the absorption/emission spectra and demonstrate the useful near-visible fluorescence of the compound.[19] A Z′ value of >0.60 was routinely achieved, and the assay shows several compounds with binding affinity including ATRA and 13-cis retinoic acid. Subsequent fitting of the curve using DynaFit[21] gives an estimated binding constant (Kd) of 160 nM, consistent with the reported activity of EC23 and within the same nanomolar range previously reported for ATRA binding to both CRABPII and CRABPII.[16,23,24] This quantitative method is becoming increasingly relevant and offers more insight into a compound’s binding efficiency than a single concentration library screen alone.[25] Analogous to a full competition titration, but using less material and equipment time, the rapid evaluation of potential compounds for binding efficiency will prove invaluable for hit-to-lead progression, as well as in the development of isoform specific binding compounds.
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