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ABSTRACT Modeling, Synthesis and Biological Evaluation of Potential Retinoid-X-Receptor (RXR) Selective Agonist for the Treatment of Cutaneous T-cell Lymphoma.

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Presentation on theme: "ABSTRACT Modeling, Synthesis and Biological Evaluation of Potential Retinoid-X-Receptor (RXR) Selective Agonist for the Treatment of Cutaneous T-cell Lymphoma."— Presentation transcript:

1 ABSTRACT Modeling, Synthesis and Biological Evaluation of Potential Retinoid-X-Receptor (RXR) Selective Agonist for the Treatment of Cutaneous T-cell Lymphoma JK Furmick 1, PW Jurutka 1, PA Marshall 1, A van der Vaart 2, SL Badshah 3, CE Wagner 1 1 Arizona State University at the West Campus, Glendale, AZ, 2 University of South Florida, Tampa, FL, 3 Arizona State University, Tempe, AZ MATERIALS AND METHODS RESULTS Retinoids, a class of compounds derived from vitamin A, can be used clinically to treat a variety of skin disorders and certain cancers. An isoform of vitamin A, 9-cis retinoic acid (9-cis RA), binds to the nuclear retinoid X receptor (RXR) and induces RXR homodimer formation and RXR-mediated transcription. Bexarotene (Bex) is a synthetic ligand (rexinoid) modeled after 9-cis RA that is indicated for the treatment of cutaneous T-cell lymphoma (CTCL). Bex can stimulate RXR homodimer formation and modulate the activity of 9-cis RA target genes. However, Bex can also dysregulate other RXR-requiring pathways since other nuclear receptors (e.g., retinoic acid and vitamin D receptors) form heterodimers with RXR. Therefore, we sought to model (via docking studies) and synthesize novel analogs of Bex, including nitro- and fluoro-substituted compounds, that bind RXR and mediate regulation of anti-tumor genes, without disrupting other RXR pathways. Employing both a mammalian two-hybrid system (M2H), and an RXRE-mediated transcriptional assay, we tested 19 analogs of Bex and discovered three compounds that best induce homodimerization and RXR-mediated transcriptional activity (20-120% of Bex). These three analogs also stimulate significant apoptosis in CTCL cells, and have similar (analog 2 and 19) or better (analog 18) Ki and EC50 values when compared to the Bex parent compound. These observations are consistent with modeling studies that predicted analogs 2 and 19 would possess the best binding affinities. We also evaluated Bex and the three analogs for their "residual" retinoic acid receptor (RAR) agonist activity employing expression of human RAR and a retinoic acid responsive element (RARE)-luciferase reporter system and found that these three analogs are selective RXR agonists (especially analog 19). Taken together, these results suggest that modification of Bex with a halogen atom on the aromatic ring that bears the carboxylic acid may reduce the activation of RAR (analog 19), or increase its ability to activate RXR (analog 18). Based on these novel results, we have designed three new halogenated compounds, and other compounds functionalized with hydrogen bonding groups, that are being evaluated to test this hypothesis. In conclusion, our experimental approach suggests that rational drug design can be employed to develop rexinoids with improved biological properties. BACKGROUND FUTURE DIRECTIONS CONCLUSION Molecular Modeling Figure 8. Evaluation of Bexarotene and selected analogs for apoptotic activity utilizing a Caspase 3/7 assay in CTCL cells. Human T-cell lymphoma cells (CTCL) were plated and immediately dosed with the indicated treatments. Cells were allowed to incubate for 24 hours and then lysis was initiated by the addition of a Caspase-Glo lysis/substrate mix. Upon programmed cell death, caspases released by the lysed cells cleave the added substrate and generate a measurable luminescent signal. Analog-induced apoptosis was compared to the parent compound, Bexarotene. Sodium butyrate (Na Bu), a known inducer of apoptosis, was used as a positive control. Antagonist Experiment Figure 3. Identification of potential RXR agonists via a mammalian two-hybrid assay in an intact cell system. Caco-2 human colon cancer cells were cotransfected using both a pCMV-hRXR binding domain vector (BD) and a hRXR-activation domain (AD) plasmid along with a pFR-Luc reporter gene containing BD-binding sites, and renilla control plasmid. Cells were transfected for six hours utilizing a liposome-mediated transfection protocol, and then treated with ethanol vehicle, M Bexarotene, or the indicated analog. After a 24 hour incubation, cells were lysed and a luciferase assay was completed. Analog-mediated RXR binding and homodimerization, as measured by luciferase output, was compared to the parent compound Bexarotene (value set to 1.0). The analog that displays the highest activity (analog 18), demonstrates nearly 120% of Bexarotene's binding ability in this assay. Transcriptional Assay- RXRE Apoptosis Assay Figure 2. Structure of the RXR ligand binding pocket docked with Bexarotene and select analogs. The docked structures of analogs 18 (A) and 2 (B) are shown; in both cases the lowest energy structures are overlayed with the docked structure of Bexarotene. As the figure illustrates, the orientation and binding mode of the ligands is markedly similar. The binding pocket is almost entirely hydrophobic, except for the Arg-316 residue which hydrogen bonds to the carboxylate groups of the ligands. Our preliminary docking studies suggest a high binding potential of our proposed compounds. Figure 4. Identification of potential RXR agonists via an RXRE-luciferase reporter based assay in human colon cancer cells. Caco-2 cells were transfected with hRXRα, an RXRE-luciferase reporter gene, renilla control plasmid, and carrier DNA (pTZ18U). Cells were transfected for six hours utilizing a liposome-mediated transfection protocol, and then treated with ethanol vehicle, M Bexarotene, or the indicated analog. After a 24 hour incubation, cells were lysed and a luciferase assay was completed. Analog-stimulated, RXR-mediated transcription, as measured by luciferase output, was compared to the parent compound Bexarotene (value set to 1.0). The analog that displays the highest activity (analog 18), demonstrates 100% of Bexarotene's transcriptional activity in this assay. Mammalian Two-Hybrid Assay Generation Two Analogs- M2HGenechip- Bexarotene Genechip- Analog 18 Figure 1. Methods employed in our in vitro biological assays. A transcriptional assay (A) measures how effectively the ligand induces receptor activation and transcriptional activity. The mammalian two-hybrid assay (B) detects the affinity of the ligand for the receptor by assessing induction of homodimerization. In a mammalian two-hybrid assay, the RXR receptor is fused separately to a DNA binding domain (BD) and an activator domain (AD). If an analog can bind to RXR as an agonist, then this will result in RXR homodimerization, binding of RXR-BD to its binding site, recruitment of RXR-AD and coactivators, and activation of the luciferase gene. Thus, the mammalian two- hybrid system allows us only to evaluate the analogs for their ability to bind to the receptor and induce dimerization. In a transcriptional assay, we are measuring the ability of the analogs to recognize and bind directly to the RXR. Next, the receptor must form the appropriate dimer, bind to a specific DNA responsive element, and recruit necessary transcription factors. This system allows us to identify possible RXR agonists capable of mediating transcription of anti-cancer target genes. A transcriptional assay was also used to evaluate Bex, A2, 18, and 19 for their "residual" retinoic acid receptor (RAR) agonist activity. Human RAR and a retinoic acid responsive element (RARE)-luciferase reporter system was used (as described in the legend to Figure 6). A mammalian two-hybrid system was used to determine the EC50 values of the most promising first generation analogs, as well as to test for any possible antagonist activity of those analogs that do not regulate RXR-mediated transcription. In both experiments we were solely interested in the binding affinity of the ligand for the receptor therefore a mammalian two-hybrid assay was the most appropriate experimental design. Employing mammalian two-hybrid (M2H) technology and RXRE-based transcription assays, we have identified Bexarotene analogs that can bind RXR, induce homodimerization, and modulate transcription. Six compounds were identified whose activity ranges from 20% - 120% when compared to Bexarotene. Those compounds that did not demonstrate the ability to activate RXR were then tested for any residual antagonist activity via mammalian two-hybrid assays. None of the analogs appeared to effectively compete with Bexarotene for binding as antagonists to RXR. The most promising first generation analogs were tested for their ability to induce programmed cell death, or apoptosis. Based on previous studies, it is known that Bexarotene induces significant apoptosis in malignant tissue, therefore we hypothesized that any analog capable of activating RXR-mediated transcription might also stimulate apoptosis when tested in a CTCL cell model system. This hypothesis was supported by our results in that our analogs displayed apoptotic activity ranging from % when compared to Bex. Bexarotene, A2, A18, and A19 were tested for any retinoic acid receptor (RAR) agonist activity, a suspected side effect of Targretin®. Interestingly, analog A19 displayed less RAR agonism than Bexarotene, while the other analogs were comparable to Bex. Base on molecular docking studies and the biological evaluation of A1 through A19, we designed eight novel, or "generation two" analogs, three of which are halogenated. Employing a mammalian two- hybrid assay we observed that 3 of the 8 analogs, all of which are halogenated, bind to RXR and induce homodimerization. Human gene microarray technology was used to compare gene regulation mediated by Bexarotene and A18. Three genes were down regulated by both rexinoids, including MGC2408, REEP2, and RAC GAP1, a GTPase activating protein which is required for malignant cell movement. In Bexarotene-treated CTCL cells, we observed the up regulation of protein kinase 3, a kinase activated in apoptosis as well as the induction of urocortin, a protein that inhibits tumorigenesis. With A18 treatment, we found the suppression of EIF3S6IP (eukaryotic translation factor 3) a gene that is known to be over-expressed in patients with gastric cancer, as well as the activation of macrophage erythroblast attacher, which is responsible for producing mature erythrocytes. Bexarotene, commercially known as Targretin®, is a chemotherapeutic indicated for the treatment of cutaneous T-cell lymphoma (CTCL). It is most often prescribed to patients who have been resistant to at least one prior systemic therapy. Bexarotene works by binding with high affinity to the nuclear retinoid X receptor and inducing homodimerization. However, since Bexarotene binds specifically to RXR, and RXR is required as a heterodimeric partner for other nuclear receptors (i.e., RAR, VDR, TR), one contraindication for Targretin® is its potential to dysregulate these other RXR requiring pathways. More specifically, when Bexarotene enters the nucleus of the target cell it recognizes and binds to all retinoid X receptors, including those active in heterodimerization. Thus, when Bexarotene binds to RXR as part of a heterodimer, it can cause the heterodimer to dissociate resulting in the loss of function of the "primary" receptor which can lead to detrimental side effects in patients treated with this medication. We have synthesized the Bexarotene analogs above whose structural features were chosen based on data from a combination of preliminary molecular modeling studies as well as the biological evaluation of the “generation one” analogs. Our preliminary modeling of novel analogs 1-3, 18 and 19 indicated that the presence of the carboxylic acid group at its locus in Bexarotene is essential for a Bexarotene analog to bind effectively to RXR. Molecular modeling suggested that compounds 18 and 19, which possess fluorine atoms proximal to the carboxylic acid group, would bind to RXR, but that 16 and 17 would not due to the addition of a ketone. These compounds allowed us to systematically gauge the variation of halogen atoms proximal to the carboxylic acid and thus also generate analogs A Transcriptional Assay- RARE Relative Binding Affinities Figure 6. Identification of potential RAR agonists via an RARE-luciferase reporter based assay in human colon cancer cells. Caco-2 cells were transfected with hRAR, an RARE-luciferase reporter gene, renilla control plasmid, and carrier DNA (pTZ18U). Cells were transfected for six hours utilizing a liposome-mediated transfection protocol, and then treated with either ethanol vehicle, M All trans- RA, Bexarotene, or the indicated analog. After a 24 hour incubation, cells were lysed and a luciferase assay was completed. Analog-stimulated, RAR-mediated transcription, as measured by luciferase output, was compared to the natural RAR ligand, all-trans-retinoic acid (RA) (value set to 1.0). The A19 analog displayed significantly lower RAR agonist activity, while the other analogs were similar to Bexarotene in activating RAR. Figure 5. Identification of potential RXR antagonists via a mammalian two-hybrid assay in an intact cell system. Caco-2 human colon cancer cells were cotransfected using both a pCMV-hRXR binding domain vector (BD) and a hRXR-activation domain (AD) plasmid along with a pFR-Luc reporter gene containing BD-binding sites, and renilla control plasmid. Cells were transfected for six hours utilizing a liposome-mediated transfection protocol, and then treated with either the ethanol vehicle or a combination of 5 x M Bexarotene and indicated analog. After a 24 hour incubation, cells were lysed and a luciferase assay was completed. The ability of each analog to compete with Bexarotene in binding to RXR and inducing homodimerization, as measured by luciferase output, was compared to Bexarotene alone (value set to 1.0). None of the analogs appear to significantly compete with Bexarotene in RXR binding (as would be indicated by a decrease in luciferase activity). Figure 9. Identification of potential “second generation” RXR agonists via a mammalian two-hybrid assay in an intact cell system. Caco-2 human colon cancer cells were cotransfected using both a pCMV-hRXR binding domain vector (BD) and a hRXR-activation domain (AD) plasmid along with a pFR-Luc reporter gene containing BD-binding sites, and renilla control plasmid. Cells were transfected for six hours utilizing a liposome-mediated transfection protocol, and then treated with ethanol vehicle, M Bexarotene, or the indicated analog. After a 24 hour incubation, cells were lysed and a luciferase assay was completed. Analog-mediated RXR binding and homodimerization, as measured by luciferase output, was compared to the parent compound Bexarotene (value set to 1.0). The analog that displays the highest activity (analog 20), demonstrates nearly 60% of Bexarotene's binding ability in this assay. Figure 9. Gene microarray analysis of human Caco-2 cells treated with M Bexarotene. Total RNA was extracted from human Caco-2 cells treated with (red) and without (green) M of Bexarotene for 4 hours. The RNA was used to synthesize cDNA using a Genisphere 350 kit. The human microarrays were hybridized using the Genisphere 350 high sensitivity protocol at 42°C. The chip was scanned with a GenePix Personal 4100A Microarray Scanner. Target genes regulated by Bexarotene include RAC GAP1, protein kinase 3, and urocortin. Figure 10. Gene microarray analysis of human Caco-2 cells treated with M analog 18. Total RNA was extracted from human Caco-2 cells treated with (red) and without (green) 1 X M of analog 18 for 4 hours. The RNA was used to synthesize cDNA using a Genisphere 350 kit. The human microarrays were hybridized using the Genisphere 350 high sensitivity protocol at 42°C. The chip was scanned with a GenePix Personal 4100A Microarray Scanner. Target genes regulated by A18 include RAC GAP1, EIF3S6IP, and MAEA. The aim of our study was to develop more efficient synthetic analogs of the current chemotherapeutic Bexarotene that increase transcription of anti-cancer target genes without significantly disrupting the activity of other receptors/pathways that require RXR heterodimerization. Our research not only concerns the development of novel drug analogs, but it may also yield further insights into the mechanism of ligand binding and activation of RXR. We hope to conduct successful M2H assays and transcriptional assays in a CTCL cell model system for all promising analogs. We will continue to employ preliminary molecular modeling studies, as well as results from our biological assays, to help drive the design and synthesis of additional Bexarotene analogs. Finally, gene microarray technology will be used to compare target gene activation and suppression mediated by Bexarotene and the most promising analogs. This has been done with analog 18 and needs to be completed for additional potential RXR agonists. Evaluation of expression profiles in CTCL cells will help us to evaluate the mechanism(s) by which Bexarotene and promising analogs mediate both anti-proliferation and pro-apoptosis to suppress the growth of lymphoma cells. This research was supported in part by funds from the Howard Hughes Medical Institute through the Undergraduate Science Education Program and from the ASU School of Life Sciences. A Special thanks goes out to Anthony Witten for his help in conducting the Genechip portion of this study. Also the human microarrays were obtained through Genome Consortium for Active Teaching and without their support this part of the project would not have been possible. Figure 7. Quantification of the binding affinity and EC50 values of potential RXR agonists. Binding affinities (Ki values) were determined by competition of 10 nM [3H]-9-cis-retinoic acid (RA) with unlabeled test rexinoids as described in Experimental Section. EC50 values were determined from full dose-response curves ranging from to M in transfected Caco-2 cells using an RXR mammalian two-hybrid system. Analog 18 has the lowest Ki and EC50 values suggesting the highest affinity for the nuclear RXR receptor.


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