RAF (Ras activating factor) kinases are important and attractive targets for cancer therapy. With the aim of discovering RAF inhibitors that bind to DFG-out inactive conformation created by the movement of Asp-Phe-Gly (DFG), we conducted structure-based drug design using the X-ray cocrystal structures of BRAF (v-raf murine sarcoma viral oncogene homolog B1), starting from bisarylurea derivative based on 1H-pyrazolo[3,4-d]pyrimidine scaffold 1a. Most of the synthesized compounds showed good to excellent inhibitory activities against BRAF^V600E kinase, possessed moderate to potent anti-proliferative activities against four tumor cell lines (A375, HT-29, PC-3 and A549) and good selectivity towards cancer cells rather normal cells (Madin-Darby canine kidney, MDCK). The most promising compound, 1v, exhibited potent inhibitory activity against not only BRAF^V600E (half maximal inhibitory concentration, IC₅₀ = 23.6 nM) but also wild-type BRAF (IC₅₀ = 51.5 nM) and C-RAF (IC₅₀ = 8.5 nM), and effective cellular anti-proliferative activities against A375, HT-29, PC-3 and A549 cell lines as well as a very good selectivity profile. Moreover, compound 1v mainly arrested the A375 cell line in the G0/G1 stage, and showed significant suppression of MEK (mitogen-activated protein kinase kinase) phosphorylation in A375 and HT-29 cell lines. Taken together, the optimal compound 1v showed excellent in vitro potency as a pan-RAF inhibitor. In addition, the promise of compound 1v was further confirmed by molecular dynamics simulation and binding free energy calculations.

Enhancing the horticultural and commercial value of Ananas comosus through polyploidy has great potential, as does meeting market demands. This study used different colchicine treatments, durations, and concentrations to induce polyploidy in pineapple somatic embryo callus. Consequently, a maximum polyploid induction rate of 40.00% was achieved in somatic embryogenic callus exposed to 0.1% colchicine for 20 days. Flow cytometry revealed 202 tetraploids among the regenerated plants analyzed. Polyploid plants, in contrast to diploid ones, are morphologically characterized by having thicker, heavier, wider, and shorter leaves, a larger leaf area, a darker color, a shorter overall height, thicker stems, and fewer, shorter leaf spines that are more irregularly distributed and differently shaped. In polyploids, the stomata were both larger and less numerous than in diploid plants, and the stomatal guard cells and leaf epidermal cells were larger too. The study revealed that polyploid plants exhibited much higher levels of chlorophyll and PEPC enzyme activity than the diploid control plants. Compared to diploid control plants, polyploid plants displayed enhanced resistance to salt and heat following treatment. Overall, this research outlined an efficient strategy for inducing polyploidy in pineapples, paving the way for future advancements in pineapple germplasm resources.