Thienopyrimidine–Chalcone Hybrid Molecules Inhibit Fas-Activated Serine/Threonine Kinase: An Approach To Ameliorate Antiproliferation in Human Breast Cancer Cells

Abstract

Apoptotic evasion by cancerous cells being one of the striking hallmarks of cancer has turned into a new arena of drug discovery. A large number of pathways reported that govern the apoptotic evasion have been reported. Fas-activated serine/threonine kinase (FASTK) is a member of Ser/Thr kinase family, and it has been implicated in the apoptotic evasion and, hence, the development of cancer. Keeping this in view, a series of novel thienopyrimidine-based chalcones have been synthesized and evaluated to modulate the FASTK mediated apoptotic evasion. Initial screening was done by enzyme inhibition assay and binding studies, which showed that out of 15 synthesized compounds, 3 thienopyrimidine-based chalcone derivatives possess considerably high binding affinity and enzyme inhibitory potential (nM range) for FASTK. Cell proliferation assessment of selected compounds was performed on HEK-293 and MCF-7 cells. For MCF-7 cells, compounds 2, 10, and 12 show IC values of 20.22 ± 1.50, 6.52 ± 0.82, and 8.20 ± 0.61 μM, respectively. Annexin-V and PI staining suggested that these molecules induce apoptosis in MCF-7 cells, arrest the cell cycle in the G0/G1 phase, and subsequently inhibit cell migration presumably by inhibiting FASTK and reactive oxygen species production. In conclusion, we have successfully designed, synthesized, and characterized thienopyrimidine-based chalcones that inhibit FASTK and induce apoptosis. These compounds may be exploited as potential anticancer agents.

Design and Development of Small-Molecule Arylaldoxime/5-Nitroimidazole Hybrids as Potent Inhibitors of MARK4: A Promising Approach for Target-Based Cancer Therapy

“…Additionally, as our laboratory is working on different human kinases named calcium–calmodulin-dependent protein kinase IV (CAMKIV), Fas-activated serine/threonine kinase (FASTK), PDK3, and integrin-linked kinase (ILK), so we have also studied the selectivity/inhibition potential of compound 4h by malachite green assay, as per our previously published protocol. 64 …”

“…Binding affinities of the synthesized arylaldoxime hybrid 4h with MARK4 were determined using the fluorescence intensity change in the emission spectrum of MARK4, as per our published protocol. 64 The titration of protein was done in triplicates and, for analysis, their average was taken. A significant decrease in the fluorescence intensity of protein with increasing concentration of the selected arylaldoxime hybrid was used to calculate the binding constant ( K a ) and the number of binding sites ( n ) present on the protein molecule by using the modified Stern–Volmer equation.…”

Probing the Inhibition of Microtubule Affinity Regulating Kinase 4 by N-Substituted Acridones

“…Enzyme activity of MARK4 was evaluated with the help of standard Malachite Green (Biomol Green reagent, Enzo Life Sciences) microtitre-plate assay as described earlier 41 . Estimated amount of MARK4 (200 ng) and 20 µM ATP as a substrate were incubated for 10–15 min at 25 °C with increasing concentrations (0–20 µM) of synthesized acridone derivatives, in 20 mM Tris buffer, pH 8.0.…”

“…ITC experiments were performed on a VP-ITC microcalorimeter (MicroCal, Inc. GE, MicroCal, USA) by following our previously published protocols 41,47 . For sample preparation, recombinant MARK4 was extensively dialyzed in 20 mM Tris buffer, pH 8.5 and the working solutions of acridone derivatives were prepared in last dialyzing buffer.…”

“…DCF fluorescence helps to measures innumerable ROS such as H 2 O 2 and hydroxyl radicals 49 . That why DCFDA staining was used to quantify the reactive oxygen species (ROS) level inside the cell as described earlier 17,41 . Briefly, the MCF-7 cells (70–80% confluent) were incubated with IC 50 concentration of each compound and positive control H 2 O 2, respectively in a 24-well culture plates.…”