Language

English

Publication Date

2-1-2026

Journal

Bioorganic & Medicinal Chemistry Letters

DOI

10.1016/j.bmcl.2025.130463

PMID

41197881

PMCID

PMC12619934

PubMedCentral® Posted Date

11-17-2025

PubMedCentral® Full Text Version

Author MSS

Abstract

Chemically-controlled genetic tools are useful for studying biological systems due to their ability to dose-dependently and temporally modulate intracellular function. Chemically-disrupted proximity (CDP) systems, which involve the pre-localization of two interacting protein components that can be disrupted with a small molecule, are complementary to more commonly used chemically-inducible dimerization (CID) systems. However, fewer CDP systems have been developed, and the genetically-encoded protein components have not been as optimized for intracellular applications. Here, we describe a transcriptional activation reporter assay for screening the intracellular interaction between the 21-amino acid peptide ANR and HCVp NS3a, which are the genetically-encoded components of a CDP system that utilizes clinically-approved antiviral drugs. We used this assay to screen a library of single amino acid substitution ANR variants and identified several that increase the intracellular interaction between ANR and NS3a. By combining affinity-optimized single substitutions, we achieved improved transcriptional activation and engineered an autoinhibited signaling switch with low basal activity. Together, our study describes a functional assay for screening genetically-encoded CDP components and a more optimized version of ANR for intracellular applications.

Keywords

Viral Nonstructural Proteins, Peptides, Humans, Hepacivirus, Molecular Structure, Antiviral Agents, Viral Proteases, Serine Endopeptidases, Nucleoside-Triphosphatase, DEAD-box RNA Helicases

Published Open-Access

yes

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