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  • Simultaneous Control of Endogenous and User-Defined Genetic Pathways Using Unique ecDHFR Pharmacological Chaperones.

Simultaneous Control of Endogenous and User-Defined Genetic Pathways Using Unique ecDHFR Pharmacological Chaperones.

Cell chemical biology (2020-04-25)
Prerana Ramadurgum, DaNae R Woodard, Steffi Daniel, Hui Peng, Prema L Mallipeddi, Hanspeter Niederstrasser, Melina Mihelakis, Viet Q Chau, Peter M Douglas, Bruce A Posner, John D Hulleman
摘要

Destabilizing domains (DDs), such as a mutated form of Escherichia coli dihydrofolate reductase (ecDHFR), confer instability and promote protein degradation. However, when combined with small-molecule stabilizers (e.g., the antibiotic trimethoprim), DDs allow positive regulation of fusion protein abundance. Using a combinatorial screening approach, we identified and validated 17 unique 2,4-diaminopyrimidine/triazine-based ecDHFR DD stabilizers, at least 15 of which were ineffective antibiotics against E. coli and S. aureus. Identified stabilizers functioned in vivo to control an ecDHFR DD-firefly luciferase in the mouse eye and/or the liver. Next, stabilizers were leveraged to perform synergistic dual functions in vitro (HeLa cell death sensitization) and in vivo (repression of ocular inflammation) by stabilizing a user-defined ecDHFR DD while also controlling endogenous signaling pathways. Thus, these newly identified pharmacological chaperones allow for simultaneous control of compound-specific endogenous and user-defined genetic pathways, the combination of which may provide synergistic effects in complex biological scenarios.

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Sigma-Aldrich
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