RESUMO
Phosphatidylinositide-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) have recently been identified as potential cancer targets. In our work, a new family of quinoline analogues was designed, developed, and evaluated as dual inhibitors of PI3Kδ/mTOR. The preliminary biological activity analysis led to the discovery of the lead compounds 5h and 5e. Compounds 5h and 5e exhibited excellent anti-tumor potency with IC50 of 0.26 µM and 0.34 µM against Ramos cells, respectively. Importantly, based on the enzymatic activity assay results, compounds 5h and 5e were identified as dual inhibitors of PI3Kδ and mTOR, with IC50 values of 0.042 µM and 0.056 µM for PI3Kδ and 0.059 µM and 0.073 µM for mTOR, respectively. Furthermore, these compounds showed superior selectivity for blocking PI3Kδ compared to other PI3K isoforms (α, ß, and γ), supporting the concept of developing inhibitors that specifically target PI3Kδ/mTOR. The most effective compound 5h was chosen for additional biological testing. At a low dose of 0.5 µM, a western blot investigation confirmed the anticancer effects by inhibiting the PAM cascade, which in turn reduced downstream biomarkers pAkt (Ser473), pAkt (Thr308), and pRPS6 (Ser235/236). Furthermore, it increased apoptosis at the early (10.03 times) and late (17.95 times) stages in the Annexin-V assay as compared to the standard. In addition, the expression of p53, caspase-3, caspase-9, and the Bax/BCl-2 ratio were all significantly increased by compound 5h in the ELISA assay. Based on these results, it appears that 5h may activate the intrinsic apoptosis pathway, which in turn triggers cell death. Furthermore, the anticancer effects could be attributed to the inhibition of PI3Kδ/mTOR, as shown by docking interactions. Lastly, it demonstrated improved in vitro metabolic stability and passed the in silico ADMET/drug-likeness test. This profile recommends 5h for future in vivo PK-PD and efficacy investigations in animal cancer models.
RESUMO
Pyrazole, as a small molecule, was discovered for higher cytotoxicity and affinity towards Aurora-A kinase. Based on these facts, a novel pyrazole substituted at the 4th position was designed, synthesized, and evaluated against MCF-7, MDA-MB-23, and Vero (non-cancerous kidney cell) cell lines. Compounds5hand5eexhibited greater cytotoxicity in the series against MCF-7 and MDA-MB-231, with GI50 values of 0.12 µM and 0.63 µM, respectively, as compared to Imatinib (GI50 values of 16.08 µM and 10.36 µM). All of the compounds displayed selective cytotoxicity against cancer cells but not on normal Vero cells, supporting the design strategy to be a selective anticancer agent. Furthermore, compounds 5h and 5e inhibited Aurora-A kinase with IC50 values of 0.78 µM (4.70-fold) and 1.12 µM (2.84-fold), respectively, as compared to alisertib (IC50 = 3.36 µM). In addition, compound 5h significantly arrested the cell cycle at G2/M (34.89 %, 5.56-fold) and the apoptotic phase (25.04 %, 11.81-fold) compared to the control. It also triggered an increase in early (7.43 %) and late (14.89 %) phase apoptosis compared to vehicle (0.235 and 0.36 %, respectively), causing 37.89-fold higher total apoptosis in the annexin-V assay. These data imply that Aurora-A kinase inhibition may be linked to apoptosis induction and cell cycle arrest. Furthermore, their higher docking score in the study confirmed evidence of Aurora-kinase suppression. It was observed that fluorine and imidazole increased the H-bond and lipophilic interactions with the binding residue. Also, the substitution of electron-rich and lipophilic groups increased hydrophobic interactions. Moreover, the three-atom linkage (CH2NHCH2) expanded compound 5h to fill the cavity. Based on current findings, it is concluded that compounds 5h and 5e with strong Aurora-A kinase suppression may be promising anticancer agents.
