Biology subjects

Mercer, J. A. M.

Publications and source records attributed to Mercer, J. A. M..

3 recordsLinked to original sources

bioRxiv2025

Ultrasmall chemogenetic tags with group-transfer ligands

Chemogenetic tags are valuable tools for studying functions of a given protein-of-interest (POI) lacking small-molecule ligands, but most tags are too large for several POIs. Here, we report two ultrasmall chemogenetic tags (mgTag and cTag) of 36 and 50 amino acids (aa) that, to the best of our knowledge, are the smallest reported. These tags exhibit transferase-type reactivity with their ligands to append any moiety of interest to the tag. cTag utilizes an engineered C1 domain-bearing cysteine that undergoes group-transfer reaction with its ligand. Likewise, mgTag utilizes an engineered zinc-finger domain-bearing cysteine that undergoes group-transfer reaction with its molecular-glue ligand in the presence of cereblon (CRBN). We applied these tags in the context of cell signaling and proximity induction. While the fusion of HaloTag (297 aa) to the KRASG12D (188 aa) disrupted its ability to activate the growth-signaling pathway, fusion of mgTag or cTag did not. Group-transfer of BRD4 binder to tags appended to Abelson kinase (ABL) induced proximity between ABL and BRD4, resulting in the latters phosphorylation. Deletion of the transferase-type reactivity reduced phosphorylation levels, suggesting that proximity-inducing chimeras with group-transfer design may be more efficacious in certain scenarios. We envision these ultrasmall tags to have wide-ranging applications, including in basic science, biotechnology, and medicine. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/653252v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1f99888org.highwire.dtl.DTLVardef@18d3e0corg.highwire.dtl.DTLVardef@1e4a044org.highwire.dtl.DTLVardef@d610ee_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Small-molecule binding and sensing with a designed protein family

Despite transformative advances in protein design with deep learning, the design of small-molecule-binding proteins and sensors for arbitrary ligands remains a grand challenge. Here we combine deep learning and physics-based methods to generate a family of proteins with diverse and designable pocket geometries, which we employ to computationally design binders for six chemically and structurally distinct small-molecule targets. Biophysical characterization of the designed binders revealed nanomolar to low micromolar binding affinities and atomic-level design accuracy. The bound ligands are exposed at one edge of the binding pocket, enabling the de novo design of chemically induced dimerization (CID) systems; we take advantage of this to create a biosensor with nanomolar sensitivity for cortisol. Our approach provides a general method to design proteins that bind and sense small molecules for a wide range of analytical, environmental, and biomedical applications.

biochemistry↗

bioRxiv2021

Proteolysis Targeting Chimeras With Reduced Off-targets

Proteolysis Targeting Chimeras (PROTACs), a class of heterobifunctional molecules that recruit target proteins to E3 ligases, have gained traction for targeted protein degradation. However, pomalidomide, a widely used E3 ligase recruiter in PROTACs, can independently degrade other targets, such as zinc-finger (ZF) proteins, that hold key functions in normal development and disease progression. This off-target degradation of pomalidomide-based PROTACs raises concerns about their therapeutic applicability and long-term side effects. Therefore, there is a crucial need to develop rules for PROTAC design that minimize off-target degradation. In this study, we developed a high-throughput platform that interrogates the off-target degradation of ZF domains and discovered, using this platform, that PROTACs with the current design paradigm induce degradation of several ZF proteins. To identify new rules for PROTAC design, we generated a library of pomalidomide analogs that allowed systematic exploration of the impact of positional isomerism (e.g., C4 and C5 positions of the phthalimide ring), hydrogen bonding, steric and hydrophobic effects on propensities for ZF protein degradation. We found that modifications of appropriate size on the C5 position reduced off-target ZF degradation. We validated these results using immunoblotting, target engagement, and global mass spectrometric studies. We applied our newfound design principles on a previously developed ALK oncoprotein-targeting PROTAC and generated PROTACs with enhanced potency and minimal off-target degradation. We envision the reported off-target profiling platform and pomalidomide analogs will find utility in design of specific PROTACs.

pharmacology and toxicology↗