Photoactivated cell-killing involving a low molecular weight, donor-acceptor diphenylacetylene.

Photoactivation of photosensitisers can be utilised to elicit the production of ROS, for potential therapeutic applications, including the destruction of diseased tissues and tumours. A novel class of photosensitiser, exemplified by DC324, has been designed possessing a modular, low molecular weight and 'drug-like' structure which is bioavailable and can be photoactivated by UV-A/405 nm or corresponding two-photon absorption of near-IR (800 nm) light, resulting in powerful cytotoxic activity, ostensibly through the production of ROS in a cellular environment. A variety of in vitro cellular assays confirmed ROS formation and in vivo cytotoxic activity was exemplified via irradiation and subsequent targeted destruction of specific areas of a zebrafish embryo.

Fluorescent Retinoic Acid Analogues as Probes for Biochemical and Intracellular Characterization of Retinoid Signaling Pathways.

Retinoids, such as all- trans-retinoic acid (ATRA), are endogenous signaling molecules derived from vitamin A that influence a variety of cellular processes through mediation of transcription events in the cell nucleus. Because of these wide-ranging and powerful biological activities, retinoids have emerged as therapeutic candidates of enormous potential. However, their use has been limited, to date, due to a lack of understanding of the complex and intricate signaling pathways that they control. We have designed and synthesized a family of synthetic retinoids that exhibit strong, intrinsic, solvatochromatic fluorescence as multifunctional tools to interrogate these important biological activities. We utilized the unique photophysical characteristics of these fluorescent retinoids to develop a novel in vitro fluorometric binding assay to characterize and quantify their binding to their cellular targets, including cellular retinoid binding protein II (CRABPII). The dihydroquinoline retinoid, DC360, exhibited particularly strong binding ( Kd = 34.0 ± 2.5 nM), and we further used X-ray crystallography to determine the structure of the DC360-CRABPII complex to 1.8 Å, which showed that DC360 occupies the known hydrophobic retinoid binding pocket. Finally, we used confocal fluorescence microscopy to image the cellular behavior of the compounds in cultured human epithelial cells, highlighting a fascinating nuclear localization, and used RNA sequencing to confirm that the compounds regulate cellular processes similar to those of ATRA. We anticipate that the unique properties of these fluorescent retinoids can now be used to cast new light on the vital and highly complex retinoid signaling pathway.

Probing biological activity through structural modelling of ligand-receptor interactions of 2,4-disubstituted thiazole retinoids.

Retinoids, such as all-trans-retinoic acid (ATRA), regulate cellular differentiation and signalling pathways in chordates by binding to nuclear retinoic acid receptors (RARα/ß/γ). Polar interactions between receptor and ligand are important for binding and facilitating the non-polar interactions and conformational changes necessary for RAR-mediated transcriptional regulation. The constraints on activity and RAR-type specificity with respect to the structural link between the polar and non-polar functions of synthetic retinoids are poorly understood. To address this, predictions from in silico ligand-RAR docking calculations and molecular dynamics simulations for a small library of stable, synthetic retinoids (designated GZ series) containing a central thiazole linker structure and different hydrophobic region substituents, were tested using a ligand binding assay and a range of cellular biological assays. The docking analysis showed that these thiazole-containing retinoids were well suited to the binding pocket of RARα, particularly via a favorable hydrogen bonding interaction between the thiazole and Ser232 of RARα. A bulky hydrophobic region (i.e., present in compounds GZ23 and GZ25) was important for interaction with the RAR binding pockets. Ligand binding assays generally reflected the findings from in silico docking, and showed that GZ25 was a particularly strongly binding ligand for RARα/ß. GZ25 also exhibited higher activity as an inducer of neuronal differentiation than ATRA and other GZ derivatives. These data demonstrate that GZ25 is a stable synthetic retinoid with improved activity which efficiently regulates neuronal differentiation and help to define the key structural requirements for retinoid activity enabling the design and development of the next generation of more active, selective synthetic retinoids as potential therapeutic regulators of neurogenesis.

Novel Fluorescence Competition Assay for Retinoic Acid Binding Proteins.

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.

The molecular basis of the interactions between synthetic retinoic acid analogues and the retinoic acid receptors.

All-trans-retinoic acid (ATRA) and its synthetic analogues EC23 and EC19 direct cellular differentiation by interacting as ligands for the retinoic acid receptor (RARα, ß and γ) family of nuclear receptor proteins. To date, a number of crystal structures of natural and synthetic ligands complexed to their target proteins have been solved, providing molecular level snap-shots of ligand binding. However, a deeper understanding of receptor and ligand flexibility and conformational freedom is required to develop stable and effective ATRA analogues for clinical use. Therefore, we have used molecular modelling techniques to define RAR interactions with ATRA and two synthetic analogues, EC19 and EC23, and compared their predicted biochemical activities to experimental measurements of relative ligand affinity and recruitment of coactivator proteins. A comprehensive molecular docking approach that explored the conformational space of the ligands indicated that ATRA is able to bind the three RAR proteins in a number of conformations with one extended structure being favoured. In contrast the biologically-distinct isomer, 9-cis-retinoic acid (; 9CRA), showed significantly less conformational flexibility in the RAR binding pockets. These findings were used to inform docking studies of the synthetic retinoids EC23 and EC19, and their respective methyl esters. EC23 was found to be an excellent mimic for ATRA, and occupied similar binding modes to ATRA in all three target RAR proteins. In comparison, EC19 exhibited an alternative binding mode which reduces the strength of key polar interactions in RARα/γ but is well-suited to the larger RARß binding pocket. In contrast, docking of the corresponding esters revealed the loss of key polar interactions which may explain the much reduced biological activity. Our computational results were complemented using an in vitro binding assay based on FRET measurements, which showed that EC23 was a strongly binding, pan-agonist of the RARs, while EC19 exhibited specificity for RARß, as predicted by the docking studies. These findings can account for the distinct behaviour of EC23 and EC19 in cellular differentiation assays, and additionally, the methods described herein can be further applied to the understanding of the molecular basis for the selectivity of different retinoids to RARα, ß and γ.

Practical synthetic strategies towards lipophilic 6-iodotetrahydroquinolines and -dihydroquinolines.

The synthesis of novel tetrahydroquinolines (THQ) and dihydroquinolines (DHQ) are reported using three practical, scalable synthetic approaches to access highly lipophilic analogues bearing a 6-iodo substituent, each with a different means of cyclisation. A versatile and stable quinolin-2-one intermediate was identified, which could be reduced to the corresponding THQ with borane reagents, or to the DHQ with diisobutylaluminium hydride via a novel elimination that is more favourable at higher temperatures. Coupling these strongly electron-donating scaffolds to electron-accepting moieties caused the resulting structures to exhibit strong fluorescence.

Conjugate Addition of 3-Buytn-2-one to Anilines in Ethanol: Alkene Geometric Insights through In Situ FTIR Monitoring.

A convenient, mild and effective conjugate addition of 3-butyn-2-one to a variety of anilines in ethanol is reported. The reaction was monitored and characterized through in situ FTIR, and the dynamics of the facile E/Z alkene geometry interconversion of the resultant aniline-derived enaminones was explored through NMR, FTIR and X-ray crystallography. A straightforward purification protocol that employs direct Kugelrohr distillation was identified, and the method was further extended to other amines and ynones, allowing rapid access to these interesting compounds.

Synthesis and applications of 2,4-disubstituted thiazole derivatives as small molecule modulators of cellular development.

Understanding how the structure of molecules relates to their function and biological activity is essential in the development of new analogues with targeted activity. This is especially relevant in mediating developmental processes in mammalian cells and the regulation of stem cell differentiation. In this study, thiazole-containing small molecules were synthesised and investigated for their ability to induce the differentiation of human pluripotent stem cells and their derivatives. Analyses of cell morphology, cell viability, expression of cell surface markers and ability to induce cell differentiation and regulate neurite formation identified the analogue with the longest and most bulky hydrophobic side chain as possessing comparable or enhanced activity to all-trans-retinoic acid (ATRA). Interestingly, a shorter, less bulky, known thiazole compound reported to be isoform selective for the retinoic acid receptor ß2 (RARß2) agonist did not mediate differentiation under the conditions tested; however, activity could be restored by adjusting the structure to a longer, more bulky molecule. These data provide further insight into the complexity of compound design in terms of developing small molecules with specific biological activities to control the development and differentiation of mammalian cells.