Abstract
Destabilizing domains (DDs), such as a mutated formof 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 proteinabundance. Using a combinatorial screening approach, we identified and validated 17unique 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 invivo to control an ecDHFR DD-firefly luciferase in the mouse eye and/or the liver. Next, stabilizers were leveraged to perform synergistic dual functions invitro (HeLa cell death sensitization) and invivo (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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