February 9, 2018
... ozone plate as described previously. An aliquot of 5 Οl of tazobactam, sulbactam, and clavulanic acid solutions (at starting concentrations of 100 ΟM) were added to each microzone using a Tecan Freedom Evo 200 liquid-handling robot (Tecan, Switzerland). The inhibitor was incubated with the printed β-lac for 20 min, after which the assay was initiated with the addition of 5 Οl of nitroc ... of 200 ΟM. β-lac activity was then assessed via UV-vis spectrophotometry by tracking the hydrolysis of nitrocefin by monitoring solution absorbance at 492 nm (Infinite M1000 spectrophotometer, Tecan). Printed hydrogel-based β-lac assay POH ink solution was prepared with a final concentration of 50 nM β-lac and used to print hydrogel spots on a 96-well paper microzone plate as described
A significant problem in high-throughput drug screening is the disproportionate number of false hits associated with drug candidates that form colloidal aggregates. Such molecules, referred to as promiscuous inhibitors, nonspecifically inhibit multiple enzymes and are thus not useful as potential drugs. Here, we report a printable hydrogel-based drug-screening platform capable of non-ambiguously differentiating true enzyme inhibitors from promiscuous aggregating inhibitors, critical for accelerating the drug discovery process. The printed hydrogels can both immobilize as well as support the activity of entrapped enzymes against drying or treatment with a protease or chemical denaturant. Furthermore, the printed hydrogel can be applied in a high-throughput microarray-based screening platform (consistent with current practice) to rapidly ( <25 min) and inexpensively identify only clinically promising lead compounds with true inhibitory potential as well as to accurately quantify the dose-response relationships of those inhibitors, all while using 95% less sample than required for a solution assay.
Mateen, R; Ali, MM; Hoare, T;
Journal: Nat Commun Pages: 602