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@article{pmid42538903,

title = {A  designed enzyme for photo-proximity labeling of E3 ligase neighborhoods in live cells},

author = {Nam Hyeong Kim and Zhi Lin and Sangwon Nam and Danielle L Swaney and Nevan Krogan and Yong Ho Kim and Michael J Therien and William F DeGrado and James A Wells},

doi = {10.64898/2026.07.22.739993},

issn = {2692-8205},

year  = {2026},

date = {2026-07-01},

urldate = {2026-07-01},

journal = {bioRxiv},

abstract = {Photocatalytic proximity labeling proteomics (photo-PLP) has emerged as a powerful technology for rapid capture of protein interactomes in situ. Typically, photo-PLP relies on chemical conjugation of the photocatalyst to the target of interest which creates practical challenges for derivatized photocatalyst synthesis and bioconjugation specificity. Integrating the precision of genetically encodable enzymes with the versatility of chemically defined photocatalysts provides a modular approach to further expand the scope of neighborhood mapping. Here, we present EYClamp, a  designed proximity labeling enzyme harnessing the off-the-shelf photocatalyst Eosin Y (EY) as a cofactor. Using a domain-swapped dimer architecture, we designed a scaffold that binds EY with high affinity (   = 10 nM) and lengthens its triplet excited-state lifetime by 29-fold. EYClamp enables efficient, multi-scale photocatalytic proximity labeling in live cells with aryl-diazirine-, aryl-azide- and phenol-biotin. We genetically fused EYClamp to a panel of six important E3 ligases. Using EYClamp, we identified over 1,500 candidate neighbors for KEAP1, MDM2, ASB7 and STUB1, providing a broad and unbiased view of these important neighborhoods. Critical functional networks were revealed including ASB7 engagement with HP1a/CUL5 complex for heterochromatin remodeling. Our EYClamp provides a genetically encodable "plug-and-play" solution for photo-PLP interactome discovery of the large family of E3 ligases and establishes domain-swapping as a promising strategy for  photoenzyme design.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid42476294,

title = {Epitope-guided detection of a molecular glue-induced ternary complex using engineered synthetic antibody fragments},

author = {Kelly M O'Leary and Tomasz Slezak and Duc Anh Le and Anthony A Kossiakoff},

doi = {10.1016/j.jmb.2026.169953},

issn = {1089-8638},

year  = {2026},

date = {2026-07-01},

urldate = {2026-07-01},

journal = {J Mol Biol},

pages = {169953},

abstract = {Molecular glues represent a class of small molecules that enable the modulation of proteins lacking traditional ligand-binding pockets. A defining feature of molecular glues is their ability to bind cooperatively at weak or neomorphic protein-protein interfaces. Despite growing interest in these compounds, tools for selectively isolating native molecular glue-induced protein assemblies from heterogeneous populations in cells remain limited. Here, synthetic antibody fragment (Fab) reporters were generated by phage display to enable selective detection of the rapamycin-induced FKBP12-mTOR ternary complex as a model molecular glue-induced protein assembly. Crystallographic studies revealed the structural basis for three distinctive epitope recognition mechanisms to monitor the molecular glue activity of rapamycin using synthetic binders. Fab-1A and Fab-2C reported ternary complex formation with low to moderate precision by exploiting rapamycin-induced allosteric and interaction-gated epitopes, respectively. Fab-4R exhibited superior accuracy as a molecular glue reporter by sensing a rapamycin-gated junctional epitope that bridges the FKBP12-rapamycin-mTOR architecture. Structure-guided mutagenesis showed that the paratope of Fab-4R confers specificity for the ternary complex through hot spot interactions positioned directly across the conditional interface formed between FKBP12 and mTOR. Cell-based immunoprecipitation assays demonstrated that both Fab-4R and scFv-4R function as versatile molecular probes to report FKBP12-dependent mTOR inhibition by rapamycin with high fidelity and picomolar sensitivity. Taken together, these findings delineate the recognition properties for multiple antibody-based molecular glue reporters and highlight the versatility of synthetic binders for sensing conditionally formed epitopes within the proteome.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid42178617,

title = {Dual targeting of inhibitory EGFR epitopes with synthetic antibodies in therapeutic-resistant cancers},

author = {Jarrett J Adams and Evan Mallette and Max London and Ryan J Liang and Dewald van Dyk and Zvezdan Pavlovic and Isabelle Pot and C Ronald Geyer and Heather A Bruce and Levi L Blazer and Craig A Hokanson and Michael D L Suits and Alexander U Singer and Sachdev S Sidhu},

doi = {10.1002/pro.70645},

issn = {1469-896X},

year  = {2026},

date = {2026-06-01},

urldate = {2026-06-01},

journal = {Protein Sci},

volume = {35},

number = {6},

pages = {e70645},

abstract = {Therapeutic antibodies that inhibit the epidermal growth factor receptor (EGFR) are limited to a subset of EGFR-driven cancers. This is in part due to resistance mechanisms that attenuate efficacy. All approved therapeutic antibodies target the closed form of EGFR and compete with the ligand. However, tumors can be desensitized to these antibodies by upregulation of EGFR ligands or through EGFR mutations that uncouple kinase activity from ligand binding. We sought to overcome these resistance mechanisms by developing antibodies targeting alternative epitopes of EGFR. Using phage-displayed libraries, we developed two distinct antibodies, one that competed with EGF and another that did not. Crystal structures revealed that the competitive antibody bound to a site that overlapped the EGF-binding site, whereas the other antibody bound to the arm that induces receptor dimerization. Because the libraries used a common light chain, we were able to assemble a biparatopic antibody that incorporated both antigen-binding sites and thus targeted both epitopes on EGFR. We showed that the antibody that targeted the dimerization arm inhibited the growth of cancer cell lines that were resistant to the antibody that targeted the EGF-binding site. Moreover, the biparatopic antibody was more potent than the monoparatopic antibodies. Our results suggest that antibodies that target the dimerization arm of EGFR may be effective across a broader range of cancers than antibodies that target the EGF-binding site, and that a biparatopic antibody targeting both epitopes may be the most effective therapeutic for inhibiting aberrant EGFR signaling in cancer.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}