PCI-34051 is an effective and selective HDAC8 inhibitor (IC50: 10 nM).

HDAC8:10 nM

PCI-34051 has a good potency for HDAC8 (Ki: 10 nM). PCI-34051 has high specificity (about 5-fold) for HDAC8 relative to the other class I HDACs including HDAC1. PCI-34051 has higher 200-fold selectivity than HDAC1/6 and higher 1000-fold selectivity than HDAC2/3//10. PCI-34051 inhibits ovarian tumor line OVCAR-3 (GI50: 6 μM) and 15% cell death. Neither significant tubulin nor histone acetylation is observed in the sensitive cell lines treated with PCI-34051 (<25 μM) at 24 hours nor at earlier time points. PCI-34051 induces caspase-dependent apoptosis. When caspase-3 activity is measured at various times after treatment with 5 μM PCI-34051, increasing levels of activity are observed from 12 to 24 to 48 hours. PCI-34051 does not stimulate Bid cleavage. While J.RT3-T.5 and P116 are sensitive to PCI-34051, the PLCγ1-deficient J.gamma1 line shows a marked decrease in the extent of PCI-34051-induced apoptosis. Furthermore, steady-state calcium levels strongly influence the apoptosis induced by PCI-34051. PCI-34051 induces cytochrome c release from mitochondria.

Administration of PCI-34051 and Dexamethasone reduces eosinophilic inflammation and airway hyperresponsiveness in asthma, thereby mitigating airway remodeling [2].

Histone deacetylase activity:For PCI-34051 characterization, measurements are performed in a reaction volume of 100 μL using 96-well assay plates in a fluorescence plate reader. For each isozyme. The HDAC protein in reaction buffer (50 mM HEPES, 100 mM KCl, 0.001% Tween-20, 5% dimethyl sulfoxide, pH7.4, supplemented with bovine serum albumin at concentrations of 0-0.05%) is mixed with PCI-34051 at various concentrations and allowed to incubate for 15 min. Trypsin is added to a final concentration of 50 nM, and acetyl-Gly-Ala-(N-acetyl-Lys)-amino-4-methyl coumarin is added to a final concentration of 25-100 μM to initiate the reaction. After a 30 min lag time, the fluorescence is measured over a 30 min time frame using an excitation wavelength of 335 nm and a detection wavelength of 460 nm. The increase in fluorescence with time is used as the measure of the reaction rate.

Cell lines: A549 cell line,Ovcar-3 cell line. Concentrations: 5 μM. Incubation Time: 24 hours. Method: Tumor cell lines and human umbilical vein endothelial cells are cultured for at least two doubling times,and growth is monitored at the end of PCI-34051 exposure using an Alamar Blue fluorometric cell proliferation assay as recommended by the manufacturer.PCI-34051 is assayed in triplicate wells in 96-well plates.The concentration required to inhibit cell growth by 50% (GI50) and 95% confidence intervals are estimated from the nonlinear regression using a four-parameter logistic equation.