Potencial epigenético do Pterocarpano em combinação com Vorinostat: da modelagem computacional à biologia celular em câncer hematológico e de próstata

Abstract

Introduction: Epigenetic alterations allow gene expression to be modulated without changing the DNA sequence. Epigenetics has advanced as a promising area in cancer treatment. Epigenetic drugs, or simply epidrugs, act on key enzymes, reactivating epigenetically silenced genes involved in tumor suppression and DNArepair. Inthe context ofcancertherapy, naturally occurring compounds such as flavonoids, isoflavonoids, and pterocarpan stand out for their pro apoptotic, antiproliferative, and regulatory properties of epigenetic targets, such as DNMTs, HDACs and HATs. Given the need to develop new approaches that overcome the limitations of monotherapy, the combination of pterocarpane (+)-PTC with the HDAC inhibitor Vorinostat (SAHA) emerges as an alternative strategy for cancer treatment. The main objective of this thesis was to investigate the epigenetic potential of (+)-PTC pterocarpane and its combination with the HDAC inhibitor Vorinostat (SAHA) in the treatment of hematological and prostate tumors. Methodology: In silico and in vitro approaches were applied in a complementary manner. ADME profiling and molecular docking tools allowed us to predict interactions between compounds and epigenetic targets, guiding in vitro assays with greater precision and economy. This thesis is structured in two chapters: the first includes a literature review of flavonoids, isoflavonoids, and pterocarpans with epigenetic activity against different types of cancer and a prediction of their pharmacokinetic properties; the second presents molecular docking interaction assays, evaluation of the synergistic potential of the combination ((+) PTC+SAHA), and cytotoxicity of (+)-PTC and SAHA on cell viability and cell cycle progression in cancer cell lines. Results: The first chapter highlights the potential of flavonoids, isoflavonoids, and pterocarpans in epigenetic modulation and other molecular pathways, such as p19Arf-p53-p21Cip1, PI3K/AKT/mTOR, and TRPM3/miR-204, inhibiting cell proliferation, restoring homeostasis, and reprogramming gene expression, which makes these compounds promising candidates in oncological research. The SwissADME BOILED-Egg model highlighted the (+)-PTC molecule with good gastrointestinal (GI) absorption and blood brain barrier (BBB) penetration capacity, in addition to indicating it as a likely substrate of Pgp glycoprotein, all valuable characteristics for a drug candidate for oncohematological treatments. The second chapter highlights the therapeutic potential of the ((+)-PTC+SAHA) combination, given its enhanced antitumor response and indication of synergism between the compounds. The molecular docking results indicated that the chosen ligands showed attraction to the site of interest in most of the selected poses, with emphasis on HDACs 1 and 2, and HSP90, whose results were above 90% attraction for both ligands (control and (+)-PTC). It should be noted that (+)-PTC tends to bind spontaneously as much as the control ligands, as observed by the very close difference between ΔG values (-0.549 kcal/mol for HDAC8 and 1.874 kcal/mol for HDAC3- site I0P). However, the stability of the binding of most control ligands is more robust than that of (+)-PTC. Even though the reactions between HDACs and (+)-PTC are favorable, the latter tends to form more stable complexes with the HSP90 protein. Thesimulation of potential synergistic effects in CompuSyn suggested concentrations that were unfeasible for practical effect for 24h and 48h (respectively, Fa = 0.5 (+)-PTC 8.466 µM and SAHA 66.659 µM; Fa = 0.5 (+)-PTC 5.226 µM and SAHA 78.556 µM). Morphological changes were observed in the MOLM-13 and KG-1 cell lines caused by exposure to (+)-PTC and SAHA compounds, either separately or in combination. For cells of the MOLM-13 line treated with the compound (+)-PTC, morphological changes such as cytoplasmic bleb formation, plasma membrane rupture, pyknosis, and karyolysis were observed, while in the groups treated with the compound Vorinostat, membrane rupture and the presence of vacuoles were identified in a more noticeable way. For KG-1 cells treated with the combination ((+) PTC+SAHA), more marked morphological changes for apoptosis were observed, such as vacuolization, pyknosis, and karyolysis. Conclusion: Despite methodological limitations, the combination ((+)-PTC+SAHA) showed promising effects against leukemia cell lines. These results predict a reduction in adverse effects compared to monotherapies and suggest new avenues for epigenetic therapeutic strategies. Although further studies covering gene and protein expression analyses, systemic toxicity assessment, and in vivo experiments are still needed, this study demonstrated that the integration of in silico and in vitro approaches is an essential element for research progress and the discovery of new ways to fight cancer.