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Target-anchored monovalent degraders are more drug-like than their bivalent counterparts, Proteolysis Targeting Chimeras (PROTACs), while offering greater target specificity control than E3 ligase-anchored monovalent degraders, also known as molecular glues. However, their discovery has typically been serendipitous, and the rules governing their identification remain unclear. This study focuses on the intentional discovery of SMARCA2/A4 monovalent degraders using a library based on SMARCA2/A4 bromodomain-binding ligands. Compound G-6599 emerged as a lead candidate, showing exceptional degradation potency and specificity for SMARCA2/A4. Mechanistic studies reveal that G-6599 operates through the ubiquitin-proteasome pathway and the E3 ligase FBXO22. G-6599 promotes ternary complex formation between SMARCA2 and FBXO22 involving covalent conjugation to a cysteine residue on the latter. Unlike other recently identified FBXO22-dependent degraders, it does not require biotransformation. The selective degradation ability of G-6599 , along with its unique mechanism, highlights the therapeutic potential of target-anchored monovalent degraders. Degraders of SMARCA2/4 have so far relied on bivalent designs. Here, a targeted discovery campaign identified the first monovalent degraders, revealing a highly potent, selective compound that recruits FBXO22 through a covalent mechanism.

TEAD (transcriptional enhanced associate domain) transcription factors (TEAD1-4) serve as the primary effectors of the Hippo signaling pathway in various cancers. Targeted therapy leads to the emergence of resistance and the underlying mechanism of resistance to TEAD inhibition in cancers is less characterized. We uncover that upregulation of the AP-1 (activator protein-1) transcription factors, along with restored YAP (yes-associated protein) and TEAD activity, drives resistance to GNE-7883, a pan-TEAD inhibitor. Acute GNE-7883 treatment abrogates YAP-TEAD binding and attenuates FOSL1 (FOS like 1) activity. TEAD inhibitor resistant cells restore YAP and TEAD chromatin occupancy, acquire additional FOSL1 binding and exhibit increased MAPK (mitogen-activated protein kinase) pathway activity. FOSL1 is required for the chromatin binding of YAP and TEAD. This study describes a clinically relevant interplay between the Hippo and MAPK pathway and highlights the key role of MAPK pathway inhibitors in mitigating resistance to TEAD inhibition in Hippo pathway dependent cancers. The underlying mechanism of acquired resistance to targeted therapy in cancer remains to be explored. Here, the authors show that upregulation of the FOSL1 transcription factor restores YAP/TEAD occupancy on chromatin to drive resistance to GNE-7883, an allosteric TEAD inhibitor.

We describe a process for rapid antibody affinity optimization by repertoire mining to identify clones across B cell clonal lineages based on convergent immune responses where antigen-specific clones with the same heavy (V H ) and light chain germline segment pairs, or parallel lineages, bind a single epitope on the antigen. We use this convergence framework to mine unique and distinct V H lineages from rat anti-triggering receptor on myeloid cells 2 (TREM2) antibody repertoire datasets with high diversity in the third complementarity-determining loop region (CDR H3) to further affinity-optimize a high-affinity agonistic anti-TREM2 antibody while retaining critical functional properties. Structural analyses confirm a nearly identical binding mode of anti-TREM2 variants with subtle but significant structural differences in the binding interface. Parallel lineage repertoire mining is uniquely tailored to rationally explore the large CDR H3 sequence space in antibody repertoires and can be easily and generally applied to antibodies discovered in vivo. Identification of specificity from antibody sequence is challenging especially when different clonotype lineages recognise antigens similarly in a process of convergence. Here using TREM2 reactive antibodies in rats as an example the authors characterise convergent antibodies and identify similarities in recognition between different lineages.

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.

Many G protein-coupled receptors (GPCRs) are organized as dynamic macromolecular complexes in human cells. Unraveling the structural determinants of unique GPCR complexes may identify unique protein:protein interfaces to be exploited for drug development. We previously reported α 1D -adrenergic receptors (α 1D -ARs) – key regulators of cardiovascular and central nervous system function – form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping α 1D -AR complex architecture, biolayer interferometry (BLI) revealed the α 1D -AR C-terminal PDZ ligand selectively binds the PDZ protein scribble (SCRIB) with >8x higher affinity than known interactors syntrophin, CASK and DLG1. Complementary in situ and in vitro assays revealed SCRIB PDZ domains 1 and 4 to be high affinity α 1D -AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α 1D -AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110 PDZ4 as a unique, critical residue dictating SCRIB:α 1D -AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α 1D -AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α 1D -AR interaction sites, and potential drug targets to treat diseases associated with aberrant α 1D -AR signaling.

Circadian rhythms are generated by a transcription-based feedback loop where CLOCK:BMAL1 drive transcription of their repressors (PER1/2, CRY1/2), which bind to CLOCK:BMAL1 to close the feedback loop with ~24-hour periodicity. Here we identify a key biochemical and structural difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. While both cryptochromes bind the BMAL1 transactivation domain with similar affinity to sequester it from coactivators, CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We identify a dynamic loop in the secondary pocket that regulates differential binding of cryptochromes to the PAS domain core. Notably, PER2 binding remodels this loop in CRY2 to enhance its affinity for CLOCK:BMAL1, explaining why CRY2 forms an obligate heterodimer with PER2, while CRY1 is capable of repressing CLOCK:BMAL1 both with and without PER2.

The COMPASS complex represents the prototype of the SET1/MLL family of methyltransferases that controls gene transcription by H3K4 methylation (H3K4me). Although H2B monoubiquitination (H2Bub) is well-known as a prerequisite histone mark for COMPASS activity, how the H2Bub-H3K4me crosstalk is catalyzed by COMPASS remains unclear. Here, we report the cryo-EM structures of an extended COMPASS catalytic module (CM) bound to the H2Bub and free nucleosome. The COMPASS CM clamps onto the nucleosome disk-face via an extensive interface to capture the flexible H3 N-terminal tail. The interface also sandwiches a critical Set1 arginine-rich motif (ARM) that auto-inhibits COMPASS. Unexpectedly, without enhancing COMPASS-nucleosome interaction, H2Bub activates the enzymatic assembly by packing against Swd1 and alleviating the inhibitory effect of the Set1 ARM upon fastening it to the acidic patch. By unmasking the spatial configuration of the COMPASS-H2Bub-nucleosome assembly, our studies establish the structural framework for understanding the long-studied H2Bub-H3K4me histone modification crosstalk.

Many G protein-coupled receptors (GPCRs) are organized as dynamic macromolecular complexes in human cells. Unraveling the structural determinants of unique GPCR complexes may identify unique protein:protein interfaces to be exploited for drug development. We previously reported α -adrenergic receptors (α -ARs) - key regulators of cardiovascular and central nervous system function - form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping α -AR complex architecture, biolayer interferometry (BLI) revealed the α -AR C-terminal PDZ ligand selectively binds the PDZ protein scribble (SCRIB) with >8x higher affinity than known interactors syntrophin, CASK and DLG1. Complementary in situ and in vitro assays revealed SCRIB PDZ domains 1 and 4 to be high affinity α -AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α -AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110 as a unique, critical residue dictating SCRIB:α -AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α -AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α -AR interaction sites, and potential drug targets to treat diseases associated with aberrant α -AR signaling.

Post-translational modification of histone H4 by the small ubiquitin-like modifier (SUMO) protein was associated with gene repression. However, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using H4 chemically modified at Lys12 by SUMO-3 (H4K12su) that was incorporated into mononucleosomes and chromatinized plasmids. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, and transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed H4K12su inhibits RNA polymerase II-mediated transcription and H4 tail acetylation. Thus, we have uncovered negative biochemical crosstalk with acetylation/methylation and the direct inhibition of RNAPII-mediated transcription by H4K12su.