Development of sulfonyl fluoride chemical probes to advance the discovery of cereblon modulators.

Sulfonyl fluoride EM12-SF was developed previously to covalently engage a histidine residue in the sensor loop of cereblon (CRBN) in the E3 ubiquitin ligase complex CRL4CRBN. Here, we further develop the structure-activity relationships of additional sulfonyl fluoride containing ligands that possess a range of cereblon binding potencies in cells. Isoindoline EM364-SF, which lacks a key hydrogen bond acceptor present in CRBN molecular glues, was identified as a potent binder of CRBN. This led to the development of the reversible molecular glue CPD-2743, that retained cell-based binding affinity for CRBN and degraded the neosubstrate IKZF1 to the same extent as EM12, but unlike isoindolinones, lacked SALL4 degradation activity (a target linked to teratogenicity). CPD-2743 had high permeability and lacked efflux in Caco-2 cells, in contrast to the isoindolinone iberdomide. Our methodology expands the repertoire of sulfonyl exchange chemical biology via the advancement of medicinal chemistry design strategies.

Development of a covalent cereblon-based PROTAC employing a fluorosulfate warhead.

Many cereblon (CRBN) ligands have been used to develop proteolysis targeting chimeras (PROTACs), but all are reversible binders of the E3 ubiquitin ligase. We recently described the use of sulfonyl exchange chemistry to design binders that covalently engage histidine 353 in CRBN for the first time. Here we show that covalent CRBN ligands can be used to develop efficient PROTAC degraders. We demonstrate that the fluorosulfate PROTAC FS-ARV-825 covalently labels CRBN in vitro, and in cells the BRD4 degrader is insensitive to wash-out and competition by potent reversible CRBN ligands, reflecting enhanced pharmacodynamics. We anticipate that covalent CRBN-based PROTACs will enhance degradation efficiencies, thus expanding the scope of addressable targets using the heterobifunctional degrader modality.

Catalytic Degraders Effectively Address Kinase Site Mutations in EML4-ALK Oncogenic Fusions.

Heterobifunctional degraders, known as proteolysis targeting chimeras (PROTACs), theoretically possess a catalytic mode-of-action, yet few studies have either confirmed or exploited this potential advantage of event-driven pharmacology. Degraders of oncogenic EML4-ALK fusions were developed by conjugating ALK inhibitors to cereblon ligands. Simultaneous optimization of pharmacology and compound properties using ternary complex modeling and physicochemical considerations yielded multiple catalytic degraders that were more resilient to clinically relevant ATP-binding site mutations than kinase inhibitor drugs. Our strategy culminated in the design of the orally bioavailable derivative CPD-1224 that avoided hemolysis (a feature of detergent-like PROTACs), degraded the otherwise recalcitrant mutant L1196M/G1202R in vivo, and commensurately slowed tumor growth, while the third generation ALK inhibitor drug lorlatinib had no effect. These results validate our original therapeutic hypothesis by exemplifying opportunities for catalytic degraders to proactively address binding site resistant mutations in cancer.

HiBiT-SpyTag: A Minimal Tag for Covalent Protein Capture and Degrader Development.

The ability to rapidly and selectively modulate cellular protein levels using small molecules is essential for studying complex biological systems. Degradation tags, such as dTAG, allow for selective protein removal with a specific degrader molecule, but their utility is limited by the large tag size (>12 kDa) and the low efficiency of fusion product gene knock-in. Here, we describe the development of a short 24 amino acid peptide tag that enables cell-based quantification and covalent functionalization of proteins to which it is fused. The minimalistic peptide, termed HiBiT-SpyTag, incorporates the HiBiT peptide for protein level quantification and SpyTag, which forms a spontaneous isopeptide bond in the presence of the SpyCatcher protein. Transient expression of dTAG-SpyCatcher efficiently labels HiBiT-SpyTag-modified BRD4 or IRE1α in cells, and subsequent treatment with the dTAG13 degrader results in efficient protein removal without the need for full dTAG knock-in. We also demonstrate the utility of HiBiT-SpyTag for validating the degradation of the endoplasmic reticulum (ER) stress sensor IRE1α, which led to the development of the first PROTAC degrader of the protein. Our modular HiBiT-SpyTag system represents a valuable tool for the efficient development of degraders and for studying other proximity-induced pharmacology.

Advancing targeted protein degrader discovery by measuring cereblon engagement in cells.

Measurement of target engagement in cells is critical to understand the molecular pharmacology of drugs and chemical probes. Many targeted protein degraders engage the E3 ligase CRL4CRBN and induce proximity with target neosubstrates resulting in their polyubiquitination and subsequent proteasomal degradation. Here we describe the development of a sensitive and robust cellular NanoBRET-based assay that measures occupancy of the CRBN ligand binding site. The assay is based on a bioluminescence resonance energy transfer (BRET) between NanoLuc luciferase tagged CRBN and a BODIPY-lenalidomide tracer which can be competed out by CRBN ligands, including PROTACs and molecular glues. The assay is compatible with a 384-well plate setup, does not require transfections and can be performed in a single day with only 3-4h of laboratory time. The protocols can be used to design other NanoLuc fusion engagement assays based on BODIPY tracers.

Cereblon covalent modulation through structure-based design of histidine targeting chemical probes.

Electrophilic biocompatible warheads, particularly cysteine-reactive acrylamides, have enabled the development of covalent inhibitor drugs and chemical biology probes, but cysteine is rarely present in protein binding sites. Therefore, expansion of the list of targetable amino acid residues is required to augment the synthetic bology toolkit of site-selective protein modifications. This work describes the first rational targeting of a specific histidine residue in a protein binding site using sulfonyl exchange chemistry. Structure-based drug design was used to incorporate sulfonyl fluoride and triazole reactive groups into the isoindolinone thalidomide congener EM12 to yield potent covalent inhibitors of the cereblon E3 ubiquitin ligase complex through engagement of His353. Conversely, the fluorosulfate derivative EM12-FS labels His353, but degrades a novel neosubstrate, the protein N-terminal glutamine amidohydrolase NTAQ1, which is involved in the N-end rule pathway and DNA damage response. Targeted protein degradation using cereblon ligands has become an important new drug discovery modality and the chemical probes and covalent labeling strategy described here will broadly impact this exciting area of therapeutic research.

Tuning site-specific dynamics to drive allosteric activation in a pneumococcal zinc uptake regulator.

MarR (multiple antibiotic resistance repressor) family proteins are bacterial repressors that regulate transcription in response to a wide range of chemical signals. Although specific features of MarR family function have been described, the role of atomic motions in MarRs remains unexplored thus limiting insights into the evolution of allostery in this ubiquitous family of repressors. Here, we provide the first experimental evidence that internal dynamics play a crucial functional role in MarR proteins. Streptococcus pneumoniae AdcR (adhesin-competence repressor) regulates ZnII homeostasis and ZnII functions as an allosteric activator of DNA binding. ZnII coordination triggers a transition from somewhat independent domains to a more compact structure. We identify residues that impact allosteric activation on the basis of ZnII-induced perturbations of atomic motions over a wide range of timescales. These findings appear to reconcile the distinct allosteric mechanisms proposed for other MarRs and highlight the importance of conformational dynamics in biological regulation.

Conserved residues of the Pro103-Arg115 loop are involved in triggering the allosteric response of the Escherichia coli ADP-glucose pyrophosphorylase.

The synthesis of glycogen in bacteria and starch in plants is allosterically controlled by the production of ADP-glucose by ADP-glucose pyrophosphorylase. Using computational studies, site-directed mutagenesis, and kinetic characterization, we found a critical region for transmitting the allosteric signal in the Escherichia coli ADP-glucose pyrophosphorylase. Molecular dynamics simulations and structural comparisons with other ADP-glucose pyrophosphorylases provided information to hypothesize that a Pro103-Arg115 loop is part of an activation path. It had strongly correlated movements with regions of the enzyme associated with regulation and ATP binding, and a network analysis showed that the optimal network pathways linking ATP and the activator binding Lys39 mainly involved residues of this loop. This hypothesis was biochemically tested by mutagenesis. We found that several alanine mutants of the Pro103-Arg115 loop had altered activation profiles for fructose-1,6-bisphosphate. Mutants P103A, Q106A, R107A, W113A, Y114A, and R115A had the most altered kinetic profiles, primarily characterized by a lack of response to fructose-1,6-bisphosphate. This loop is a distinct insertional element present only in allosterically regulated sugar nucleotide pyrophosphorylases that could have been acquired to build a triggering mechanism to link proto-allosteric and catalytic sites.