(736b) A Printable Paper-Based Hydrogel Microarray for Drug Screening Enabling Discrimination between True and Promiscuous Enzyme Inhibitors
AIChE Annual Meeting
2019 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Omics and High-Throughput Technologies
Thursday, November 14, 2019 - 3:48pm to 4:06pm
Methodology: A drop-on-demand syringe solenoid printer was used to sequentially print hydrazide (POH) and aldehyde (POA) functionalized poly(oligoethylene glycol methacrylate) (PO) precursor polymers, previously shown to rapidly gel upon mixing via hydrazone bond formation18, on a nitrocellulose substrate using glycerol as a humectant/printing aid. Enzymes were added into the hydrazide polymer solution as desired to enable their physical entrapment within the hydrazone crosslinked hydrogel formed. The thin film hydrogel was characterized via X-ray photoelectron spectroscopy (XPS) and fluorescence imaging to confirm the expected gelation/entrapment mechanism. The activity of the loaded enzymes under different conditions (e.g. long-term storage, drying, proteolytic degradation) was assessed by adding the appropriate colorimetric enzyme substrate (e.g. nitrocefin for beta-lactamase, the target enzyme to inhibit in this work based on the prevalence of beta-lactam rings in antibiotic structures whose cleavage by bacterial enzymes is a key contributor to antibiotic resistance19) and comparing to controls of directly printed enzymes and fresh enzymes in solution. The potential to discriminate between true and promiscuous inhibitors was assessed by adding known true (tazobactum, sulbactam, clavulanic acid) and promiscuous (rottlerin, TIPT, BIS) inhibitors of beta-lactamase and repeating the enzyme activity tests.
Results: Well-defined and uniform thin film hydrogels could be reproducibly printed on a nitrocellulose support, with XPS providing evidence of hydrazone crosslinking to facilitate gelation following printing. Efficient (>85%) encapsulation of a variety of enzymes (e.g. alkaline phosphatase, urease, beta-galactosidase, and beta-lactamase) was demonstrated by simply mixing the enzyme in the hydrazide precursor polymer solution. The hydrogel enabled long term stabilization of the enzyme in both wet and dry conditions, with >90% enzyme notably maintained upon dry storage of the printed enzyme-hydrogel constructs on the benchtop. The presence of the hydrogel also protects the enzyme against degradation via proteinase K, with the large size of proteinase K facilitating its steric exclusion from the encapsulated enzyme sites. True inhibitors of beta-lactamase remained as soluble small molecules and as such freely diffused into the hydrogel, enabling us to directly reproduce the IC50 curves achieved when the same assay is done in solution (albeit using only 5% of the total number of reagents as would be required for the equivalent 96-well multiwell plate solution assay). However, when known promiscuous inhibitors are added, the colloidal aggregates cannot diffuse into the encapsulated enzyme active sites such that the printed hydrogel reports no significant change in enzyme activity while the solution assay shows substantial enzyme inhibition.
Significance: The printed hydrogel microarray described herein can accurately reproduce the enzyme inhibition curves currently assessed using solution-based assays but can also successfully discriminate between true and promiscuous inhibitors of a target enzyme during drug screening. Given that >95% of the âhitsâ identified during early stage drug screening are in reality false hits due to promiscuous inhibition20, our approach offers a significant advantage in terms of accurately characterizing these false hits early and thus saving the time and money now invested in follow-up screening of these ultimately sure-to-fail compounds. In addition, the design of our screening system results in significantly preserved enzyme stability upon storage as well as significantly lower reagent demands due to the use of an interfacial rather than solution-based approach, reducing the cost of the assay while still preserving its potential to be easily implemented in existing high-throughput drug screening workflows.
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