Synthesis and mechanism of action of novel classes of retinoids and rexinoids with antineoplastic activities

RA isomers induce a strong growth inhibitory effect on Epstein-Barr virus (EBV)-immortalized lymphoblastoid B cell lines (LCLs), a model of EBV+ lymphoproliferations of immuno-suppressed patients. This effect is mainly mediated by RARa and occurs through the up-regulation of the CDK inhibitor p27kip-1. RA down-regulates the expression of the p45Skp2 and Cks1 proteins, two essential components of the SCFSkp2 ubiquitin ligase complex that target p27Kip1 for degradation, resulting in a decreased ubiquitination and proteasome-dependent degradation of p27Kip1. The analysis of the effects of RA on IL-6-induced signalling in LCLs showed that RA induced a down-regulation of IL-6R a-chain (gp80) whereas the expression of the ß-chain (gp130) was unaffected. RA induced a down-regulation of sgp80 (IL-6 agonist) and a slight increase in the levels of sgp130 (IL-6 antagonist). Analysis of post-receptor events demonstrated that IL-6 stimulation of LCLs triggers JAK1 (but not TYK2) phosphorylation, which results in increased STAT3 phosphorylation and activation. The finding that RA severely impairs IL-6-dependent signalling in LCLs and inhibits their growth despite the presence of constitutively active JAK/STAT and MAPK cascades provide additional support for a role of RA in the prevention and treatment of EBV-related lymphoproliferative disorders of immuno-suppressed patients.

Study of retinoid and rexinoid receptors role in growth arrest differentiation and apoptosis in acute myeloid leukemias. Investigation on the role of the specific receptors in these biological functions and indications for combination treatments. Molecular investigation on the role of RAR and RXR in the regulation of cell death alone or in combination with other signal transduction pathways. Novel paradigms for therapy.

HDAC inhibitors (HDACi's) induce expression of the death ligand TRAIL and this molecular event is responsible for apoptosis in acute myeloid leukaemia systems after epigenetic treatment. This has been the first time in which TRAIL is specifically linked to HDACi's and explains the previously noted tumour-selective action of some HDACi's. We: - showed that TRAIL-induction causes HDACi-induced apoptosis, - described the corresponding mechanisms, - separated the effects of HDACi action on inhibition of cell cycle progression, differentiation and apoptosis, and - demonstrated TRAIL induction in AML patient's blasts.

It was shown that retinoids have anti-HHV-8 activity and, in particular, new retinoid compounds with stronger antiviral activity on HHV-8 were identified. The compounds specifically inhibited viral promoters, both during the early and the late phases of infection in all the cell systems tested, and resulted in significant reductions of viral replication and release of progeny virus. The inhibition of viral replication displayed by retinoids in endothelial cells, primary target of in vivo HHV-8-associated neoplastic transformation in the course of Kaposi's Sarcoma (KS), was reflected by fact that endothelial cells did not develop the characteristic changes associated to HHV-8 infection and neoplastic activity, i.e. spindle morphology and tube formation in Matrigel. Therefore, specific retinoids might be good candidates for the development of antiviral drugs and for antineoplastic therapy of HHV-8 associated lesions.

A novel system for producing HHV-8 cell free virus inoculum was established. This was obtained from activated BC3 cells and subsequent purification and concentration by high-speed centrifugation. The second step was in identifying a continuous cell line supporting viral production. The experiments showed that Vero cells are permissive to HHV-8 replication, and efficiency of virus production is similar (even higher) than that of primary endothelial cells, a well-recognized cell target of HHV-8 infection. We are trying to enhance the system, by production of Vero cell clones stably transformed with ORF50, a key promoter of HHV-8 replication.

Known and potent, as well as novel RAR and RXR modulators (agonists, antagonists, inverse agonists&) for use in the research carried out by the Consortium members were prepared. These benchmark retinoids are important biological tools for research. Among the compounds prepared through ligand design and synthesis, two are particularly noteworthy: - UVI3003, a powerful and selective RXR antagonist. - UVI2034, a selective RAR-antagonist Compound UVI3003 has been used in the context of RXR signalling pathways, as well as in the context of structural-activity determination. In addition, compound UVI2003 has served as a derivative of TTNPB to show the role of the halogen at the benzoic acid end in the RARß ligand discrimination.

Role of TRAIL in apoptosis induced by retinoids and rexinoids. TRAIL as effector of different signalling pathways during the induction of cell death. Role of TRAIL inducibility in cancer cells. Tumour specific cell death. Investigation of alternative rexinoid-based pathways of apoptosis involving TRAIL induction. Molecular investigation of retinoid resistance in leukemias.

A series of 9-cis-retinoic acid analogs modified at the hydrophobic ring with a cyclohexenyl moiety derived from natural terpenes has been stereoselectively prepared using a Suzuki cross-coupling as key step to connect alkenyl fragments functionalized as iodide or boronic acid. For convenience, due to the instability of the iodotetraenyl esters, the sequence is best carried out without isolation of the intermediates. In turn, the boronic acids were obtained by trapping with a borate the alkenyl organolithium intermediate obtained by Shapiro reaction of the hydrazones derived from either naturally-occurring ketones or derivatives thereof.

Ligand recognition to retinoic receptors (RXRa and RAR) has been studied using a molecular mechanics-based docking method. The protocol is able to rank the affinity of the ligands for a single retinoid receptor, the highest values corresponding to those that adapt better to the shape of the binding site and generate the optimal set of electrostatic and van der Waals interactions with the receptor. Moreover, our studies shed light into some of the energetic contributions to retinoid receptor ligand selectivity. In this regard we show that there is a difference in polarity between the binding site regions that anchor the carboxylate in RAR and RXR, which translates itself in large differences in interaction energy of both receptors for the same ligand. We observe that the latter energy change is cancelled off by the solvation energy penalty upon binding. This energy compensation is borne out as well by experiments that address the effect of site directed mutagenesis in ligand binding to RAR. The hypothesis that the difference in binding site polarity might be exploited to build RXR selective ligands is tested with some compounds having a thiazolidinedione anchoring group.

Proliferation of MCL cells is not significantly enhanced by physiologic concentrations of dexamethasone or hydrocortisone. Moreover, these steroid hormones are unable to antagonize the antiproliferative effects exerted by RA in MCL cells. These results seem to rule out that endogenous GC could promote the growth of MCL cells, as instead observed for EBV-immortalized B lymphocytes. Moreover, 9-cis-RA sensitizes MCL cells to TRAIL-dependent apoptosis by a mechanism not involving TRAIL receptors. RA isomers, and particularly 9-cis-RA, are able to inhibit the growth promoting effect induced in primary MCL cells by CD40 activation alone or in combination with IL-4. Immunohistochemical analysis showed that significant numbers of CD40L-expressing lymphoid cells are present in lymph node biopsies of MCL patients. These results, therefore, further strengthen the possibility that triggering of CD40 by infiltrating CD40L+ cells may continuously promote the growth of MCL cells in vivo. On these grounds, our findings that RA inhibits basal MCL proliferation as well as MCL growth-promoting effects exerted by microenvironmental factors make these compounds highly attractive in terms of potential clinical efficacy in this setting.

MCL is an aggressive B-cell non-Hodgkin's lymphoma with poor response to therapy and unfavourable prognosis. RA isomers significantly inhibit the proliferation of both primary MCL cultures and established cell lines. RA induces cell accumulation in G0/G1 together with a marked up-regulation of p27Kip-1 by inhibiting ubiquitination and proteasome-dependent degradation of the protein. Most of RA-induced p27Kip-1 binds to cyclin D1/CDK4 complexes, thus contributing to the inhibit Cdk4 kinase activity and pRb hypophosphorylation. Experiments with receptor-selective ligands indicate that RARa co-operates with RXRs in mediating RA-dependent MCL cell growth inhibition. MCL is characterized by a constitutively active PI3-K signalling whose pharmacologic inhibition severely impairs the proliferation of these cells. Interestingly, RA treatment decreases the levels of phosphorylated, active Akt in these cells, suggesting the possibility that the antiproliferative effects exerted by RA in MCLs are at least in part mediated by RA-induced inhibition of the PI3-K/Akt signalling. IFN-a enhances the antiproliferative activity exerted by RA in MCL cells and, more importantly, the 9-cis-RA/IFN-a combination also induces caspase-dependent apoptosis. These findings provide the rationale for the evaluation of the possible efficacy of 9-cis-RA in the treatment of MCL patients. The drawing up of a specific clinical trial is in progress.

It was determined that several cell lines are susceptible and permissive to exogenous HHV-8 infection. The cell types suitable to conduct HHV-8 in vitro infection include: human endothelial cells from umbilical cord (HUVEC, a primary cell culture representing one of the natural targets of in vivo HHV-8 infection), Vero (a continuous cell line derived from monkey kidney and permissive for several human viruses), 293 (a human kidney epithelial derived continuous cell line), ECV-304 (a human cell line presenting markers specific of endothalial cells), U937 (a monocytic cell line) and primary human macrophages from healthy blood donors. HHV-8 did not induce any lytic cytopathic effect, but in HUVEC cells induced a typical spindle shape phenotype. All cell lines supported lytic replication, as shown by analysis of viral transcription, and by release on infectious virus in culture supernatant.

Sequential Pd-catalyzed cross-coupling reactions of precursors dihalogenated heterocycles (thiazoles, thiophenes), using position-selective Stille or Suzuki coupling reactions, provided the scaffold for the synthesis of nuclear receptor modulators. In addition to several families of retinoids, compound GW501516, the most potent PPARd agonist yet described was also synthesized.

Leistungen

Diese Seite teilen

Herunterladen