Affinity Crosslinking of Y1036 to Nerve Growth Factor Identifies Pharmacological Targeting Domain for Small Molecule Neurotrophin Antagonists

Classically, small molecule antagonists have targeted membrane bound receptors and intracellular enzyme targets. While this drug discovery strategy is extremely successful, the number of new chemical entities in the pharmaceutical pipeline is diminishing and complementary strategies are in need. A particularly attractive therapeutic strategy is to neutralize soluble signalling proteins using small molecules. Small molecule-based technologies have the potential to sufficiently alter the molecular topology of a given ligand and inhibit ligand/receptor interactions—effectively neutralizing the ligand’s signalling capacity. Recent technical advances in the field of structural biology have enabled the elucidation of ligand/receptor complexes at atomic resolution enabling a detailed appreciation of the molecular interactions governing ligand-mediated receptor activation. Exploiting molecular modeling techniques to study these signalling complexes allows for a paradigm shift from “receptorcentric” to “ligandcentric” screening strategies. Nerve growth factor (NGF) is a prototypical protein signalling ligand, which binds two receptors, TrkA and p75NTR. We first explore the molecular landscape governing the ligand/receptor interactions of NGF/TrkA and NGF/p75 structures. Next, we use the recently reported NGF neutralizing small-molecule, Y1036, as an affinity probe to determine residues in proximity to the pharmacological targeting domain of NGF and perform theoretical docking experiments to predict the residues which comprise distinct pharmacological targeting domains on the surface of NGF. Exploiting such strategies may facilitate “ligandcentric” drug discovery and could further the development of a trophic-factor-selective compound such as a BDNF-selective antagonist.

Effect of quercetin on secretion and gene expression of leptin in breast cancer

OBJECTIVE: To investigate the possible inhibitory action of pure quercetin on secretion and gene expression of leptin in the T47 D breast cancer cell line.METHODS: In this experimental study, T47 D cells were cultured in monolayers in RPMI 1640. IC50 was determined by MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay after 24 h treatment at different concentrations of quercetin.The levels of leptin gene expression were measured by reverse-transcription real-time polymerase chain reaction(PCR). Secreted leptin was measured in the supernatant of cells by an enzyme-linked immuno sorbent assay.RESULTS: Analysis of MTT assay data showed that quercetin has a cytotoxic effect on T47 D breast cancer cells with 40 μM IC50 after 24 h exposure. Dataanalysis of real-time PCR showed that with increases in quercetin concentration, a decreasing trend was seen in m RNA levels of leptin of treated cells compared with the control cells(P < 0.05). Also,measurement of secreted leptin in the culture media showed a similar decreasing trend in the amount of leptin protein in the treated cells compared with the control cells(P < 0.05).CONCLUSION: Quercetin significantly inhibits the growth of T47 D cells through inhibition of leptin secretion and gene expression in T47 D breast cancer cells. Therefore, it might be an alternative approach to breast cancer therapy through leptin targeting.