Novel N-Arylmethyl-aniline/chalcone hybrids as potential VEGFR inhibitors: synthesis, biological evaluations, and molecular dynamic simulations.

Significant advancements have been made in the domain of targeted anticancer therapy for the management of malignancies in recent times. VEGFR-2 is characterised by its pivotal involvement in angiogenesis and subsequent mechanisms that promote tumour cells survival. Herein, novel N-arylmethyl-aniline/chalcone hybrids 5a-5n were designed and synthesised as potential anticancer and VEGFR-2 inhibitors. The anticancer activity was evaluated at the NCI-USA, resulting in the identification of 10 remarkably potent molecules 5a-5j that were further subjected to the five-dose assays. Thereafter, they were explored for their VEGFR-2 inhibitory activity where 5e and 5h emerged as the most potent inhibitors. 5e and 5h induced apoptosis with cell cycle arrest at the SubG0-G1 phase within HCT-116 cells. Moreover, their impact on some key apoptotic genes was assessed, suggesting caspase-dependent apoptosis. Furthermore, molecular docking and molecular dynamics simulations were conducted to explore the binding modes and stability of the protein-ligand complexes.

New phenothiazine conjugates as apoptosis inducing agents: Design, synthesis, In-vitro anti-cancer screening and 131I-radiolabeling for in-vivo evaluation.

Phenothiazines (PTZs) are a group of compounds characterized by the presence of the 10H-dibenzo-[b,e]-1,4-thiazine system. PTZs used in clinics as antipsychotic drugs with other diverse biological activities. The current aim of the study is to investigate and understand the effect of potent PTZs compounds using a group of In-vitro and In-vivo assays. A total of seventeen novel phenothiazine derivatives have been designed, synthesized, and evaluated primarily in-vitro for their ability to inhibit proliferation activity against NCI-60 cancer cell lines, including several multi-drug resistant (MDR) tumor cell lines. Almost all compounds were active and displayed promising cellular activities with GI50 values in the sub-micromolar range. Four of the most promising derivatives (4b, 4h, 4g and 6e) have been further tested against two selected sensitive cancer cell lines (colon cancer; HCT-116 and breast cancer; MDA-MB231). The apoptosis assay showed that all the selected compounds were able to induce early apoptosis and compound 6e was able to induce additional cellular necrosis. Cell cycle assay showed all selected compounds were able to induce cell cycle arrest at sub-molecular phase of G0-G1 with compound 6e induced cell cycle arrest at G2M in HCT-116 cells. Accordingly, the apoptotic effect of the selected compounds was extensively investigated on genetic level and Casp-3, Casp-9 and Bax gene were up-regulated with down-regulation of Bcl-2 gene suggesting the activation of both intrinsic and extrinsic pathways. In-vivo evaluation of the antitumor activity of compound 4b in solid tumor bearing mice showed promising therapeutic effect with manifestation of dose and time dependent toxic effects at higher doses. For better evaluation of the degree of localization of 4b, its 131I-congener (131I-4b) was injected intravenously in Ehrlich solid tumor bearing mice that showed good localization at tumor site with rapid distribution and clearance from the blood. In-silico study suggested NADPH oxidases (NOXs) as potential molecular target. The compounds introduced in the current study work provided a cutting-edge phenothiazine hybrid scaffold with promising anti-proliferation action that may suggest their anti-cancer activity.

Augmented Therapeutic Potential of EC-Synthetic Retinoids in Caco-2 Cancer Cells Using an In Vitro Approach.

Colorectal cancer therapies have produced promising clinical responses, but tumor cells rapidly develop resistance to these drugs. It has been previously shown that EC19 and EC23, two EC-synthetic retinoids, have single-agent preclinical anticancer activity in colorectal carcinoma. Here, isobologram analysis revealed that they have synergistic cytotoxicity with retinoic acid receptor (RAR) isoform-selective agonistic retinoids such as AC261066 (RARß2-selective agonist) and CD437 (RARγ-selective agonist) in Caco-2 cells. This synergism was confirmed by calculating the combination index (lower than 1) and the dose reduction index (higher than 1). Flow cytometry of combinatorial IC50 (the concentration causing 50% cell death) confirmed the cell cycle arrest at the SubG0-G1 phase with potentiated apoptotic and necrotic effects. The reported synergistic anticancer activity can be attributed to their ability to reduce the expression of ATP-binding cassette (ABC) transporters including P-glycoprotein (P-gp1), breast cancer resistance protein (BCRP) and multi-drug resistance-associated protein-1 (MRP1) and Heat Shock Protein 70 (Hsp70). This adds up to the apoptosis-promoting activity of EC19 and EC23, as shown by the increased Caspase-3/7 activities and DNA fragmentation leading to DNA double-strand breaks. This study sheds the light on the possible use of EC-synthetic retinoids in the rescue of multi-drug resistance in colorectal cancer using Caco-2 as a model and suggests new promising combinations between different synthetic retinoids. The current in vitro results pave the way for future studies on these compounds as possible cures for colorectal carcinoma.

Role of single nucleotide polymorphisms of the HSD3B1 gene (rs6203 and rs33937873) in the prediction of prostate cancer risk.

3­ß­hydroxysteroid dehydrogenase 1 (HSD3B1) is shown to affect dihydrotestosterone level in prostatic tissue which is a risk factor for prostate cancer (PC). The present study aimed to determine whether rs33937873 (G313A) and rs6203 (C338T) single nucleotide polymorphisms (SNP) in HSD3B1 gene was a potential risk factor for PC susceptibility and can predict the recurrence of PC in Egyptian patients. A total of 186 Egyptian patients were selected with incident primary PC and compared with 180 age healthy controls. The frequencies and the main effect of rs33937873 and rs6203 in HSD3B1 were compared and investigated between the patients and control using genotyping technique and statistical analysis. The mutant GA genotype of G313A in rs33937873 SNP was considered as an independent risk for PC in the multivariate regression analysis [odds ratio (OR)=2.7, 95% confidence intervals (CI): 1.2­5.5, P=0.01] together with positive history of hypertension (HTN) (OR=6.2, 95% CI: 3.2­12.1, P=0.0001) and begin prostatic hyperplasia (BPH; OR=8.9, 95% CI: 4.5­17.5, P=0.0001). Conversely, in rs6203 (C338T), C allele is considered as major risk allele in the development of PC (OR=1.8, 95% CI: 1.3­2.4, P=0.0003). The univariate logistic regression analyses indicated that CC genotype of rs6203 was a PC risk factor (OR=1.9, 95% CI: 1.3­2.9, P=0.002). In addition, the frequency of the A­C haplotype established by rs33937873­rs6203 was also significantly higher for PC (P=0.013). The predication of PC recurrence was associated only with positive family history (OR=7.7, 95% CI: 2.3­25.9, P=0.001) and not for The G313A and C338T SNPs. These results suggested that the two HSD3B1 polymorphisms rs33937873 and rs6203 may modify the risk of PC, particularly among patients with HTN and history of BPH, suggesting them as prominent future markers for prediction of PC risk.

The potential roles of retinoids in combating drug resistance in cancer: implications of ATP-binding cassette (ABC) transporters.

Multidrug resistance (MDR) means that tumour cells become unresponsive during or after the course of treatment to one or more of chemotherapeutic drugs. Chemotherapeutic resistance critically limits the treatment outcomes and remains a key challenge for clinicians. The alternation in intracellular drug concentration through the modulation of its transport across the plasma membrane is the major cause for MDR and is adopted by various mediators, including ATP-requiring enzymes (ATPases). Among these ATPases, ABC transporters have been extensively studied, and found to be highly implicated in tumorigenesis and MDR. The present review sheds light on the documented effects of retinoids on ABC enzymes to understand their mechanism in combating cancer cell resistance. This would open the gate to test the mechanism and applicability of different new synthetic retinoids in literature and market as modulators of ATP-dependent efflux pumping activity, and promote their applicability in diminishing anti-cancer drug resistance.

Growth Inhibition and Apoptotic Effect of Pine Extract and Abietic Acid on MCF-7 Breast Cancer Cells via Alteration of Multiple Gene Expressions Using In Vitro Approach.

In vitro anti-proliferative activity of Pinus palustris extract and its purified abietic acid was assessed against different human cancer cell lines (HepG-2, MCF-7 and HCT-116) compared to normal WI-38 cell line. Abietic acid showed more promising IC50 values against MCF-7 cells than pine extract (0.06 µg/mL and 0.11 µM, respectively), with insignificant cytotoxicity toward normal fibroblast WI-38 cells. Abietic acid triggered both G2/M cell arrest and subG0-G1 subpopulation in MCF-7, compared to SubG0-G1 subpopulation arrest only for the extract. It also induced overexpression of key apoptotic genes (Fas, FasL, Casp3, Casp8, Cyt-C and Bax) and downregulation of both proliferation (VEGF, IGFR1, TGF-ß) and oncogenic (C-myc and NF-κB) genes. Additionally, abietic acid induced overexpression of cytochrome-C protein. Furthermore, it increased levels of total antioxidants to diminish carcinogenesis and chemotherapy resistance. P. palustris is a valuable source of active abietic acid, an antiproliferative agent to MCF-7 cells through induction of apoptosis with promising future anticancer agency in breast cancer therapy.

Elevated Expression of MiR-17 in Microglia of Alzheimer's Disease Patients Abrogates Autophagy-Mediated Amyloid-ß Degradation.

Autophagy is a proposed route of amyloid-ß (Aß) clearance by microglia that is halted in Alzheimer's Disease (AD), though mechanisms underlying this dysfunction remain elusive. Here, primary microglia from adult AD (5xFAD) mice were utilized to demonstrate that 5xFAD microglia fail to degrade Aß and express low levels of autophagy cargo receptor NBR1. In 5xFAD mouse brains, we show for the first time that AD microglia express elevated levels of microRNA cluster Mirc1/Mir17-92a, which is known to downregulate autophagy proteins. By in situ hybridization in post-mortem AD human tissue sections, we observed that the Mirc1/Mir17-92a cluster member miR-17 is also elevated in human AD microglia, specifically in the vicinity of Aß deposits, compared to non-disease controls. We show that NBR1 expression is negatively correlated with expression of miR-17 in human AD microglia via immunohistopathologic staining in human AD brain tissue sections. We demonstrate in healthy microglia that autophagy cargo receptor NBR1 is required for Aß degradation. Inhibiting elevated miR-17 in 5xFAD mouse microglia improves Aß degradation, autophagy, and NBR1 puncta formation in vitro and improves NBR1 expression in vivo. These findings offer a mechanism behind dysfunctional autophagy in AD microglia which may be useful for therapeutic interventions aiming to improve autophagy function in AD.

Novel thienopyrimidine analogues as potential metabotropic glutamate receptors inhibitors and anticancer activity: Synthesis, In-vitro, In-silico, and SAR approaches.

There is a continuous need in drug development approach for synthetic anticancer analogues with new therapeutic targets to diminish chemotherapeutic resistance of cancer cells. This study presents new group of synthetic thienopyrimidine analogues (1-9) aims as mGluR-1 inhibitors with anticancer activity. In-vitro antiproliferative assessment was carried out using viability assay against cancer cell lines (MCF-7, A-549 and PC-3) compared to WI-38 normal cell line. Analogues showed variable anticancer activity with IC50 ranging from 6.60 to 121 µg/mL with compound 7b is the most potent analogue against the three cancer cell lines (MCF-7; 6.57 ± 0.200, A-549; 6.31 ± 0.400, PC-3;7.39 ± 0.500 µg/mL) compared to Doxorubicin, 5-Flurouracil and Riluzole controls. Selected compounds were tested as mGluR-1 inhibitors in MCF-7 cell line and results revealed compound 7b induced significant reduction in extracellular glutamate release (IC50; 4.96 ± 0.700 µM) compared to other analogues and next to Riluzole (IC50; 2.80 ± 0.500 µM) of the same suggested mode of action. Furthermore, both cell cycle and apoptosis assays confirmed the potency of compound 7b for early apoptosis of MCF-7 at G2/M phase and apoptotic positive cell shift to (91.4%) compared to untreated control (19.6%) and Raptinal positive control (51.4%). On gene expression level, compound 7b induced over-expression of extrinsic (FasL, TNF-α and Casp-8), intrinsic (Cyt-C, Casp-3, Bax) apoptotic genes with down-regulation of anti-apoptotic Bcl-2 gene with boosted Bax/Bcl-2 ratio to 2.6-fold increase. Molecular docking and dynamic studies confirmed the biological potency through strong binding and stability modes of 7b where it was faster in reaching the equilibrium point and achieving the stability than Riluzole over 20 ns MD. These results suggest compound 7b as a promising mGluR inhibitory scaffold with anticancer activity that deserves further optimization and in-depth In-vivo and clinical investigations.

Gasdermin D restricts Burkholderia cenocepacia infection in vitro and in vivo.

Burkholderia cenocepacia (B. cenocepacia) is an opportunistic bacterium; causing severe life threatening systemic infections in immunocompromised individuals including cystic fibrosis patients. The lack of gasdermin D (GSDMD) protects mice against endotoxin lipopolysaccharide (LPS) shock. On the other hand, GSDMD promotes mice survival in response to certain bacterial infections. However, the role of GSDMD during B. cenocepacia infection is not yet determined. Our in vitro study shows that GSDMD restricts B. cenocepacia replication within macrophages independent of its role in cell death through promoting mitochondrial reactive oxygen species (mROS) production. mROS is known to stimulate autophagy, hence, the inhibition of mROS or the absence of GSDMD during B. cenocepacia infections reduces autophagy which plays a critical role in the restriction of the pathogen. GSDMD promotes inflammation in response to B. cenocepacia through mediating the release of inflammasome dependent cytokine (IL-1ß) and an independent one (CXCL1) (KC). Additionally, different B. cenocepacia secretory systems (T3SS, T4SS, and T6SS) contribute to inflammasome activation together with bacterial survival within macrophages. In vivo study confirmed the in vitro findings and showed that GSDMD restricts B. cenocepacia infection and dissemination and stimulates autophagy in response to B. cenocepacia. Nevertheless, GSDMD promotes lung inflammation and necrosis in response to B. cenocepacia without altering mice survival. This study describes the double-edged functions of GSDMD in response to B. cenocepacia infection and shows the importance of GSDMD-mediated mROS in restriction of B. cenocepacia.

Revealing the Potential Application of EC-Synthetic Retinoid Analogues in Anticancer Therapy.

(1) Background and Aim: All-trans retinoic acid (ATRA) induces differentiation and inhibits growth of many cancer cells. However, resistance develops rapidly prompting the urgent need for new synthetic and potent derivatives. EC19 and EC23 are two synthetic retinoids with potent stem cell neuro-differentiation activity. Here, these compounds were screened for their in vitro antiproliferative and cytotoxic activity using an array of different cancer cell lines. (2) Methods: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, AV/PI (annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)), cell cycle analysis, immunocytochemistry, gene expression analysis, Western blotting, measurement of glutamate and total antioxidant concentrations were recruited. (3) Results: HepG2, Caco-2, and MCF-7 were the most sensitive cell lines; HepG2 (ATRA; 36.2, EC19; 42.2 and EC23; 0.74 µM), Caco-2 (ATRA; 58.0, EC19; 10.8 and EC23; 14.7 µM) and MCF-7 (ATRA; 99.0, EC19; 9.4 and EC23; 5.56 µM). Caco-2 cells were selected for further biochemical investigations. Isobologram analysis revealed the combined synergistic effects with 5-fluorouracil with substantial reduction in IC50. All retinoids induced apoptosis but EC19 had higher potency, with significant cell cycle arrest at subG0-G1, -S and G2/M phases, than ATRA and EC23. Moreover, EC19 reduced cellular metastasis in a transwell invasion assay due to overexpression of E-cadherin, retinoic acid-induced 2 (RAI2) and Werner (WRN) genes. (4) Conclusion: The present study suggests that EC-synthetic retinoids, particularly EC19, can be effective, alone or in combinations, for potential anticancer activity to colorectal cancer. Further in vivo studies are recommended to pave the way for clinical applications.

Raptinal silver nanoparticles: new therapeutic advances in hepatocellular carcinoma mouse model.

Raptinal is a novel antineoplastic agent that induces an expeditious intrinsic apoptotic pathway, in addition to the shutdown of mitochondrial function for cancerous cells, because of silver nanoparticles (AgNPs) that have been shown to provide a worthy approach to overcome tumors. In this study, Both Raptinal and Raptinal-loaded silver nanoparticles (AgNPs) were tested as the first time in hepatocellular carcinoma-induced mice to evaluate its efficacy and targeting to HCC. Seventy-two albino male mice of comparable age were classified into six groups; early stage of HCC was induced using diethyl nitrosamine (DEN)/carbon tetrachloride (CCL4). Liver function was assessed in all groups using ALT, AST, total bilirubin, and alpha-fetoprotein (AFP) as well as histopathological examination. Quantitative gene expression of key apoptotic gene markers p53, cytochrome c, and caspase 3 was assessed in all liver homogenates. The results showed that Raptinal-loaded AgNPs group had significant increase in both apoptotic genes of cytochrome c and Caspase 3 at P = 0.0001 compared with Raptinal-free drug group. AFP levels were significantly decreased in Raptinal-loaded AgNPs group compared with both Raptinal-free drug and HCC groups at P = 0.0001. Degenerative changes in the hepatocytes with focal necrosis and inflammatory cell infiltration in histopathology confirm the biochemical analysis. Our study is considered one of the first studies using Raptinal in vivo. Moreover, it showed that Raptinal and/or the combination between Raptinal and AgNPs showed a promising therapeutic agent in treating early HCC.

Biological Screening and Radiolabeling of Raptinal as a Potential Anticancer Novel Drug in Hepatocellular Carcinoma Model.

New synthetic compound Raptinal (RAP) was investigated on different biological levels for its potential anticancer activity. RAP showed higher antiproliferative activity on HepG2 cell line with IC50 0.62µM compared to MCF-7 and HCT-116 (4.03 and 92.3 µM) respectively. Moreover, RAP induces early stage of apoptosis in the most sensitive HepG2 treated cells after 24 hr with cell cycle arrest in both subG0-G1 and G0-G1 phases and minimal cell count in G2/M mitotic phase with apoptotic index 9.25-fold higher than to control. RAP induces over-expression of key apoptotic genes such as Fas receptor, Caspase-8, Caspase-9, Bax and P53. Western blotting confirm the observation on protein level via over-expression of Caspase-9, Cytochrome-C and higher ration of Bax/Bcl-2. In addition, RAP was radiolabeled using one of the most important diagnostic radioactive isotopes, technetium-99m (99mTc), with a radiochemical yield of 92.7 ± 0.41 %. Quality control and biological distribution of 99mTc-RAP in both healthy and HCC rat model were investigated. Biodistribution profile revealed the localization of RAP in liver tissues (20.5±2.6 %) of HCC models at half an hour post intravenous injection. Histopathological examination confirmed the biodistribution of RAP into liver tissue with induction of karyomegaly in the nuclei of hepatocytes as well as others that proceeded into apoptosis. Molecular docking suggested RAP binds in binding pocket of p53 cancer mutant Y220C making reactivation of the mutant form which is a promising strategy for further investigation on molecular level as a novel anticancer therapeutics. All the results support the use of RAP as a potential anticancer drug in HCC and its 99mTc complex as an imaging probe.

Synthesis, biological evaluation and molecular docking studies of novel thiopyrimidine analogue as apoptotic agent with potential anticancer activity.

This study synthesizes novel 6-amino-5-cyano-4-aryl-2-mercapto pyrimidines and condensed pyrimidines analogues in order to investigate their potential activity as anticancer agents. The compounds were synthesized via one-pot condensation of p-nitrobenzaldehyde or p-anisaldehyde with malononitrile and thiourea to prepare 6-amino-5-cyano-4-aryl-2-mercaptopyrimidines series (1-9a,b). The pyrimidine analogues were biologically screened In-vitro in HepG2 and MCF-7 compared to normal WI-38. Compound 8a showed higher antiproliferative activity to MCF-7 cells with sensitivity and minimal cytotoxic effect (IC50 53.3 µM- HepG2, 12.9 µM- MCF-7 and >100 µM- WI-38). Compound 8a was able to induce 40% of total antioxidants and 60% following treatment with 50 µM of H2O2 for 3hrs as external source of oxidative stress in MCF-7. 8a was able to significantly induce early stage apoptosis of 74.37% MCF-7 and cell cycle arrest with cells accumulation in subG0-G1 phase to 69.42% and reduction of cells in G2M phase to 3.6% and high apoptotic index. Compound 8a induced over-expression of Fas receptor and Cyto C genes. Molecular docking studies suggested that 8a can bind to both phosphodiesterase 4B and 4D binding pockets and inhibit their action through network of hydrophobic interactions in Q-P pockets with preferential selectivity to PDE4B through invariant Glu443. The chemical profile and the biological results suggest that 8a can be a promising anticancer agent.

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.

Neurogenesis in Response to Synthetic Retinoids at Different Temporal Scales.

All-trans retinoic acid (ATRA) plays key roles in neurogenesis mediated by retinoic acid receptors (RARs). RARs are important targets for the therapeutic regulation of neurogenesis but effective drug development depends on modelling-based strategies to design high-specificity ligands in combination with good biological assays to discriminate between target-specificity and off-target effects. Using neuronal differentiation as a model, the aim of this study was to test the hypothesis that responses across different temporal scales and assay platforms can be used as comparable measures of retinoid activity. In biological assays based on cell phenotype or behaviour, two structurally similar synthetic retinoids, differing in RAR affinity and specificity, retained their relative activities across different temporal scales. In contrast, assays based on the transcriptional activation of specific genes in their normal genomic context were less concordant with biological assays. Gene-induction assays for retinoid activity as modulators of neurogenesis require careful interpretation in the light of variation in ligand-receptor affinity, receptor expression and gene function. A better characterization of neuronal phenotypes and their regulation by retinoids is badly needed as a framework for understanding how to regulate neuronal 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.