Biology subjects

Ryan, J. K.

Publications and source records attributed to Ryan, J. K..

2 recordsLinked to original sources

bioRxiv2026

An epigenetic bifunctional that toggles between transactivation and repression

The targeted modulation of gene expression with bifunctional small molecules enables the precise control of cellular and biological processes. To screen for ligands that could be used to induce gene expression, we conjugated the high affinity FKBP(F36V) binder, AP1867, to known high-affinity binders of activating epigenetic machinery. We tested these bifunctionals in a FKBP(F36V)-tagged transcription factor reporter system and found bifunctional induced transactivation is relatively common, being observed for bifunctionals with BET ligand JQ1, p300/CBP ligand GNE-781, CDK9 ligand SNS-032, and BRD9 ligand iBRD9. aTAG-2 (mAP1867-C8-GNE781) was identified as the strongest and most potent transactivator, possessing single-digit nanomolar activity. When tested in models where oncogenic RNA binding protein-transcription factor fusion proteins have been FKBP(F36V)-tagged, we unexpectedly observed rapid collapse of the fusion transcriptional program. In a tagged Ewing sarcoma model, aTAG-2 exhibits at least three distinct mechanisms of action: i) RIPTAC mediated p300/CBP inhibition, ii) ubiquitination- and ternary complex-dependent EWS/FLI degradation, and iii) replacement of p300 with CBP at EWS/FLI bound chromatin loci. Together, these data establish bifunctionals targeting p300/CBP that toggle between a program of ultra-potent transactivation and repression depending on cellular context. Overall demonstrating that induced proximity with a given ligand does not encode a fixed functional outcome.

biochemistry↗

bioRxiv2025

Development of Degraders and 2-pyridinecarboxyaldehyde (2-PCA) as a recruitment Ligand for FBXO22

Targeted protein degradation (TPD) is a promising therapeutic strategy that requires the discovery of small molecules that induce proximity between E3 ubiquitin ligases and proteins of interest. FBXO22 is an E3 ligase that is overexpressed in many cancers and implicated in tumorigenesis. While FBXO22 was previously identified as capable of recognizing ligands bearing a primary amine degron, further investigation and development of recruitment ligands is required to enable its broader utility for TPD. Here, we describe the discovery of chemical probes that can either selectively degrade FBXO22 or recruit this ligase for TPD applications. First, we describe AHPC(Me)-C6-NH2 as a potent and selective FBXO22 degrader (DC50 = 77 nM, Dmax = 99%) that is suitable for interrogating the effects of FBXO22 loss of function. Further, we discovered that the simple hexane-1,6-diamine acts as a minimal FBXO22 self-degrader, whereas shorter C4 (putrescine) to C5 (cadaverine) analogs, found in mammalian cells, do not induce degradation. Finally, we found that 2-pyridinecarboxaldehyde (2-PCA) functions as a novel electrophilic degron capable of forming a reversible thioketal with cysteine 326 for recruiting FBXO22. Conjugating 2-PCA to various ligands successfully induced FBXO22-dependent degradation of BRD4 and CDK12. Collectively, these chemical probes will facilitate the study of FBXO22 biology and broaden its applicability in TPD.

biochemistry↗