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Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair

preprint
submitted on 05.06.2018 and posted on 06.06.2018 by Todd D. Gruber, Chithra Krishnamurthy, Jonathan B. Grimm, Michael R. Tadross, Laura M. Wysocki, Zev J. Gartner, Luke D. Lavis

The utility of small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. ‘Masking’ the activity of small molecules using a general chemical modification and ‘unmasking’ it only within target cells could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of E. coli nitroreductase (NTR) and a 2‑nitro-N-methylimidazolyl (NM) masking group. To discover and optimize this NTR–NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.

Funding

Howard Hughes Medical Institute. Z.J.G.: CZ Biohub Investigator, National Science Foundation (MCB-1330864 and DBI-1548297) and Department of Defense (W81XWH-13-1-0221).

History

Email Address of Submitting Author

todd.gruber@cnu.edu

Email Address(es) for Other Author(s)

lavisl@janelia.hhmi.org

Institution

Janelia Research Campus, Howard Hughes Medical Institute

Country

United States of America

ORCID For Submitting Author

0000-0003-1664-1497

Declaration of Conflict of Interest

No conflicts of interest to declare.

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