Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only a few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome-and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.

The interaction of conjugated polyelectrolyte, PPE-SO(3)(-), with platinum(II) complexes, [Pt(tpy)(C≡CC(6)H(4)-CH(2)NMe(3)-4)](OTf)(2) (1) and [Pt(tpy)(C≡C-CH(2)NMe(3))](OTf)(2) (2), has been studied by UV-vis, and steady-state and time-resolved emission spectroscopy. A unique FRET from PPE-SO(3)(-) to the aggregated complex 1 on the polymer chain with Pt···Pt interaction has been demonstrated, resulting in the growth of triplet metal-metal-to-ligand charge transfer ((3)MMLCT) emission in the near-infrared (NIR) region. This two-component ensemble has been employed in a "proof-of-principle" concept for the sensitive and selective label-free detection of human serum albumin (HSA) by the emission spectral changes in the visible and in the NIR region. The spectral changes have been ascribed to the disassembly of the polymer-metal complex aggregates upon the binding of PPE-SO(3)(-) to HSA, which is rich in arginine residues and hydrophobic patches, leading to the decrease in FRET from PPE-SO(3)(-) to the aggregated platinum(II) complex. The ensemble is found to have high selectivity toward HSA over a number of polyelectrolytes, proteins and small amino acids. This has been suggested to be a result of the extra stabilization gained from the Pt···Pt and π-π interactions in addition to the electrostatic and hydrophobic interactions found in the polymer-metal complex aggregates.

Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest–activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.

Pd(II) complexes with long-lived emissive excited states found applications in photo-catalysis and PSF-OLEDs.

Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification, and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling.

Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with one of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome-and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications. Main textTargeted protein degradation is a groundbreaking drug discovery approach for tackling the undruggable proteome by exploiting the cellular protein degradation machinery to selectively eliminate target proteins 1,2 .This technology most often involves the utilization of heterobifunctional degrader molecules consisting of a substrate-targeting ligand linked to an E3 ligase recruiter. These degraders are capable of recruiting E3 ligases to specific protein targets to ubiquitinate and mark targets for degradation in a proteasome-dependent manner.As functional inhibition of the target is not necessary for degrader efficacy, this strategy has the potential to target and degrade any protein in the proteome for which there exists a ligand. However, a major challenge in the application of this technology is relatively small number of E3 ligase recruiters. While there are ~600 different E3 ligases, there are only a few E3 ligases that have been successfully exploited in suhc a strategy , including small-molecule recruiters for cereblon, VHL, MDM2, and cIAP 2,3 . Identifying facile strategies for discovering ligands that bind to E3 ligases remains crucial for expanding the set of E3 ligase recruiters that can be utilized for targeted protein degradation applications.Activity-based protein profiling (ABPP) has arisen as a powerful platform for ligand discovery against targets of interest, including proteins commonly considered as undruggable 4-10 . ABPP utilizes reactivity-based chemical probes to m...

Excited state properties, photo-catalysis and cellular imaging of photo-stable bis-NHC Ir(iii) complexes are described.

Water-soluble alkynylplatinum(II) terpyridine complexes, [Pt{tpy(C 6 H 4 CH 2 NMe 3 -4)-have been synthesized and characterized.The photophysical and electrochemical properties of the complexes have been studied. Complex 1 has been found to undergo aggregation at low pHs, leading to metal-metal and/or p-p interactions and the emergence of a triplet metal-metal-to-ligand charge transfer ( 3 MMLCT) emission in the nearinfrared (NIR) region, the intensity of which has been enhanced 1350-fold over that at physiological pH.Such 'switchable' NIR emission of complex 1 was employed in cell-imaging experiments. The pH response of the 3 MMLCT emission of complex 1 in cellular compartments has been studied using experiments with fixed Madin-Darby canine kidney (MDCK) cells, while live cell-imaging experiments revealed that complex 1 could function as a NIR luminescent probe for the tracking of the location of acidic organelles such as lysosomes.