We developed molecular tension probes (TPs) that report traction forces of adherent cells with high spatial resolution, can be linked to virtually any surface, and obviate monitoring deformations of elastic substrates. TPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific forces. We applied TPs to reveal that cellular traction forces are heterogeneous within focal adhesions and localized at their distal edges.

ngaging the mostly undruggable proteome to uncover new disease therapies not only requires technological innovations that facilitate rapid discovery of ligandable hotspots across the proteome but also demands new therapeutic modalities that alter protein function through novel mechanisms 1,2 . Targeted protein degradation (TPD) tackles the undruggable proteome by targeting specific proteins for ubiquitination and proteasomal degradation. One major class of small-molecule effectors of TPD, proteolysis-targeting chimeras (PROTACs), are heterobifunctional molecules that consist of an E3 ligase recruiter linked to a protein-targeting ligand to induce the formation of ternary complexes that bring together an E3 ubiquitin ligase and the target protein as a neo-substrate [3][4][5] . PROTACs have enabled the targeted and specific degradation of numerous disease-causing proteins in cells 3,6 . New approaches for TPD have also arisen that exploit endosomal and lysosomal degradation pathways with lysosome-targeting chimeras or autophagy with autophagy-targeting chimeras 7,8 . New approaches for chemically induced proximity beyond degradation have also been developed in recent years, including targeted phosphorylation with phosphorylation-inducing chimeric small molecules and targeted dephosphorylation, but no small-molecule-based induced proximity approaches exist for targeted deubiquitination and subsequent stabilization of proteins 9,10 .Active ubiquitination and degradation of proteins is the root cause of several classes of diseases, including many tumor suppressors in cancer (for example, TP53, CDKN1A, CDN1C and BAX), and mutated and misfolded proteins, such as ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis or glucokinase in pancreatic cells in maturity-onset diabetes of the young type 2. In these cases, a TPS therapeutic strategy, rather than degradation, would be beneficial [11][12][13][14] . Analogous to TPD, we hypothesized that TPS could be enabled by the discovery of a small-molecule recruiter of a deubiquitinase (DUB) that could be linked to a protein-targeting ligand to form a chimeric molecule, which would induce the deubiquitination and stabilization of proteins of interest. We call this heterobifunctional stabilizer a DUBTAC (Fig. 1a). In this study, we report the discovery of a covalent recruiter for the K48-ubiquitin chain-specific DUB OTUB1, which when linked to a protein-targeting ligand stabilizes an actively degraded target protein to demonstrate proof of concept for the DUBTAC platform. ResultsIdentifying allosteric ligandable sites within DUBs. To enable the DUBTAC platform, our first goal was to identify a small-molecule recruiter that targeted an allosteric site on a DUB without inhibiting DUB function, as the recruitment of a functional DUB would be required to deubiquitinate and stabilize the target protein. While many DUBs possess well-defined active sites bearing a catalytic and highly nucleophilic cysteine, there have not yet been systematic evaluations of ...

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Table of Contents I. Peptide Synthesis and Purification SI-2 i. Table SI-1. Peptide Sequences SI-2 ii. Table SI-2. Peptide calculated and observed masses SI-3 II. Peptide Characterization SI-4 A. Circular Dichroism SI-4 i. Figure SI-1. Thermostability of aPP and variants SI-5 ii. Figure SI-2. Thermostability of YY2 and variants SI-6 iii. Table SI-3. Peptide Melting Temperatures SI-7 B. Flow Cytometry SI-7 C. Confocal microscopy SI-8 D. Cell Viability SI-8 i. Figure SI-3. Cell Viability SI-9 III. References Cited SI-9 Supplemental Information SI-2 I. Peptide Synthesis and PurificationTable SI-1. Sequences of peptides and miniature proteins described in this work. aPP GPSQPTYPGDDAPVEDLIRFYNDLQQYLNVVTRHRY 1 aPP 4R1 GPSQPTYPGDDAPVRDLIRFYRDLQRYLNVVTRHRY aPP 5R1 GPSQPTYPGDDAPVRDLIRFYRDLRRYLNVVTRHRY aPP 6R1 GPSQPTYPGDDAPVRDLRRFYRDLRRYLNVVTRHRY YY2 APPLPPRNRGEDASPEELSRYYASLRHYLNLVTRQRY 2 YY2 3R1 APPLPPRNRGEDASPEELSRYYRSLRHYLNLVTRQRY YY2 4R1 APPLPPRNRGEDASPRELSRYYRSLRHYLNLVTRQRY YY2 5R1 APPLPPRNRGEDASPRELRRYYRSLRHYLNLVTRQRY R 8 Y RRRRRRRRY Tat (48-60) GRKKRRQRRRPPQY 3 Penetratin RQIKIWFQNRRMKWKK 4 Transportan GWTLNSAGYLLKINLKALAALAKKIL 5 KLA KLALKLALKALKAALKLA 6Synthesis. All peptides were synthesized using standard solid-phase Fmoc chemistry on a 25 µmol scale with a Symphony ® multi-channel solid phase synthesizer (Protein Technologies, Inc., Tuscon, AZ). All α-amino acids and resins were purchased from Novabiochem (San Diego, CA) and solvents were purchased from American Bioanalytical (Natick, MA). All peptides were synthesized to carry free amines at their N-termini and carboxamides at their C-termini. Peptides were labeled on their N-termini on resin for at least one hour with fluorescein-5-EX, succinimidyl ester (Invitrogen, Carlsbad, CA, Cat. # F-6130), which was dissolved in 1 mL DMF and added to the reaction vessel with 20 µL N,N-diisopropylethylamine (EDIPA).Purification. Peptides were purified by reverse-phase HPLC using Grace Vydac C8 preparative or semi-preparative scale columns (300 silica, 10 µm particle size, 22 mm x 250 mm) and water/acetonitrile gradients containing 0.1% TFA. Peptide identity was confirmed by mass spectrometry on an Applied Biosystems Voyager-DE Pro MALDI-TOF mass spectrometer