A selective and rapid cell-permeable inhibitor of human caspase-3

Abstract

The individual roles and overlapping functionalities the twelve human caspases have during apoptosis and other cellular processes remain poorly resolved primarily due to a lack of chemical tools. Here we present a new selective caspase-3 inhibitor, termed Ac-ATS010-KE, with rapid and irreversible binding kinetics. Relative to previously designed caspase-3-selective molecules that have tremendously abated inhibitory rates and thus limited use in biological settings, the improved kinetics of Ac-ATS010-KE permit its use in a cell-based capacity. We demonstrate that Ac-ATS010-KE prevents apoptosis with comparable efficacy to the general caspase inhibitor Ac-DEVD-KE and surprisingly does so without side-chain methylation. This observation is in contrast to the well-established peptide modification strategy typically employed for improving cellular permeability. Ac-ATS010-KE protects against extrinsic apoptosis, which demonstrates the utility of a thiophene carboxylate leaving group in biological settings, challenges the requisite neutralization of free carboxylic acids to improve cell permeability, and provides a tool-like compound to interrogate the role of caspase-3 in a variety of cellular processes.Cell Culture. Jurkat A3 cells were cultured as described by ATCC using 10% FBS and pen/strep/glutamine at 37 °C with 5% CO2. For all experiments, Jurkat cells were grown to near confluence, harvested and resuspended in fresh media to the indicated density. Apoptosis was induced using 10 ng/mL MegaFasL (AdipoGen® Life Sciences).Apoptotic protection assays. Cells were plated into sterile Nunc Edge 96-well tissue culture-treated plates at a density of 10,000 cells/well and preincubated (or chased) with 20 µM of the indicated inhibitor or DMSO vehicle for the indicated time before (or after) apoptotic induction. 3 h after induction, cellular viability was measured using CellTiter-Glo® on a PerkinElmer EnVision plate reader.

Technologies for Sustainable Biomass Supply—Overview of Market Offering

“…Some tools focus on analytical predictions of production output, development of pests, and market evolution in order to help make better decisions in advance, and thus maximize the profitability and sustainability of the production. For instance, tools such as Olivia [26], Solania [27], and Berria [28] rely on machine learning algorithm to make predictions.…”