| Literature DB >> 32330442 |
Prerana Ramadurgum1, DaNae R Woodard1, Steffi Daniel1, Hui Peng1, Prema L Mallipeddi2, Hanspeter Niederstrasser2, Melina Mihelakis3, Viet Q Chau1, Peter M Douglas3, Bruce A Posner2, John D Hulleman4.
Abstract
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.Entities:
Keywords: E. coli dihydrofolate reductase; chemical biology; conditional regulation; destabilizing domain; dual use; gene therapy; high-throughput screening; ocular; pharmacological chaperone
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Year: 2020 PMID： 32330442 PMCID： PMC7245562 DOI： 10.1016/j.chembiol.2020.03.006
Source DB: PubMed Journal: Cell Chem Biol ISSN： 2451-9448 Impact factor: 8.116