Spiteri VA, Segal D, Correa-Sáez A
… +12 more, Iso K, Casement R, Muñoz I Ordoño M, Nakasone MA, Sathe G, Schätz C, Peters HE, Doward M, Kainacher L, Cowan AD, Ciulli A, Winter GE
Proteolysis-targeting chimeras (PROTACs) and molecular glue degraders (MGDs) target proteins for degradation by co-opting an E3 ligase. While heterotrivalent PROTACs that can recruit multiple E3 ligases have been describ...Proteolysis-targeting chimeras (PROTACs) and molecular glue degraders (MGDs) target proteins for degradation by co-opting an E3 ligase. While heterotrivalent PROTACs that can recruit multiple E3 ligases have been described, all MGDs reported to date depend on a single E3. Using orthogonal genetic screening, biophysical and structural analyses, we show that a monovalent MGD can recruit CUL4 and CRL1 in parallel to degrade SMARCA2/4. Deep mutational scanning identifies C173 in DCAF16 as essential for degrader activity and intact protein mass spectrometry confirms covalent modification at this site. Elucidating the ternary complex structure reveals a unique binding mode and a distinct interface of neointeractions that underlie degrader specificity. We demonstrate that ligase dependency is chemically and genetically tunable. Minimal compound modifications shift preference from DCAF16 to FBXO22, while a single substitution boosts degrader dependency on DCAF16. These results establish a framework for designing tunable dual E3 ligase degraders to mitigate potential resistance mechanisms.
Stoddard EG, Perera BGK, Fang L
… +6 more, Brush DS, Zhong Y, Potter ZE, Simon JJ, Golkowski M, Maly DJ
Nat Chem Biol
· 2026 May · PMID 42120500
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RAF kinases are key effectors in the RAS-RAF-MEK-ERK signaling pathway, making them important targets for the development of cancer therapeutics. Here we investigate the variable potency of DFG-out-stabilizing RAF inhibi...RAF kinases are key effectors in the RAS-RAF-MEK-ERK signaling pathway, making them important targets for the development of cancer therapeutics. Here we investigate the variable potency of DFG-out-stabilizing RAF inhibitors in mutant KRAS-expressing cell lines. We demonstrate that inhibitor potency correlates with basal RAF activity, with more active RAF being more sensitive to inhibition. We further show that DFG-out-stabilizing inhibitors disrupt high-affinity RAF-MEK interactions, promoting the formation of inhibited RAF dimers. Furthermore, we identify cobimetinib as an MEK inhibitor that uniquely sensitizes RAF kinases to DFG-out-stabilizing inhibitors by disrupting autoinhibited RAF-MEK complexes. Building on this insight, we developed cobimetinib analogs with enhanced sensitization properties. Together, our findings provide a mechanistic framework for understanding the cellular determinants of DFG-out-stabilizing inhibitor sensitivity and offer strategies for optimizing synergistic RAF-MEK inhibitor combinations.
Algov I, Van Heest A, Hopton M
… +4 more, Liang F, Holmes A, Hao L, Zhou X
Nat Chem Biol
· 2026 May · PMID 42115695
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Dysregulated extracellular proteolytic activity is a prominent hallmark of cancer and can thus be exploited for tumor detection and therapeutic development. However, the discovery of tumor-responsive probes has been hind...Dysregulated extracellular proteolytic activity is a prominent hallmark of cancer and can thus be exploited for tumor detection and therapeutic development. However, the discovery of tumor-responsive probes has been hindered by the lack of methods to directly screen proteolytic events in specific tissue samples. Here we report PSurf, a platform that enables the identification of tissue-specific protease sensors with tissue specimens. Through differential selection of tumor-specific sequences over healthy tissue, PSurf identifies context-specific tumor-activated probes that precisely distinguish metastatic lesions in lung tissue slices. Using these substrates, we engineered nanobody-targeted biosensors that release urinary reporters upon tumor-specific cleavage in vivo, enabling precise non-invasive tumor detection in a mouse lung metastasis model. PSurf provides a foundation for developing conditionally activated agents through tissue-specific activity mapping and probe discovery.
Dopaminylation, the covalent attachment of dopamine to the side chain of glutamine in proteins, represents a newly characterized class of posttranslational modifications. Because of the limited identification of substrat...Dopaminylation, the covalent attachment of dopamine to the side chain of glutamine in proteins, represents a newly characterized class of posttranslational modifications. Because of the limited identification of substrates, the functions and molecular mechanisms associated with dopaminylation remain largely uncharacterized. Using an alkyne-functionalized dopamine probe, we developed a method for selectively enriching dopaminylated proteins in whole-cell systems. This approach provided a comprehensive resource of 4,133 dopamine-enriched protein candidates and peptide-level analysis with acid-cleavable tags identified 1,181 putative dopaminylated proteins, including histone H4 dopaminylation at Q27 (H4Q27dop), which we further validated. Functionally, H4Q27dop acts as a transcriptional repressor in a neuroblastoma model, where it blocks CEBPD binding at the CCND1 promoter, leading to transcriptional downregulation of CCND1 and subsequent suppression of cell proliferation. Our findings provide both a valuable resource of dopaminylated substrate proteins and a distinct mechanistic insight into how dopamine regulates neuroblastoma cell growth.
Posttranslational modifications (PTMs) vastly expand the diversity of the human proteome, dynamically reshaping protein activity, interactions and localization in response to environmental, pharmacologic and disease-asso...Posttranslational modifications (PTMs) vastly expand the diversity of the human proteome, dynamically reshaping protein activity, interactions and localization in response to environmental, pharmacologic and disease-associated cues. However, their proteome-wide impact on small-molecule recognition-and, thus, druggability-remains largely unexplored. Here we present a chemical proteomic strategy to delineate how PTM states remodel protein ligandability in human cells. Using broad-spectrum photoaffinity probes, we identified more than 400 functionally diverse proteins whose ability to engage small molecules is impacted by phosphorylation or N-linked glycosylation status. Integrating binding site mapping with structural analyses reveals a diverse array of PTM-dependent pockets. Among these, we discovered that the phosphorylation status of common oncogenic KRAS mutants impacts the action of small molecules, including clinically approved inhibitors. These findings illuminate a previously underappreciated layer of proteome plasticity governed by PTMs and highlight opportunities to develop chemical probes that selectively target proteins in defined modification states.
Chemical modifications on RNA represent an additional regulatory layer of gene expression analogous to epigenetic marks on DNA and histones. Over the past decade, the fundamental mechanisms of N-methyladenosine and other...Chemical modifications on RNA represent an additional regulatory layer of gene expression analogous to epigenetic marks on DNA and histones. Over the past decade, the fundamental mechanisms of N-methyladenosine and other mRNA modifications have been extensively characterized, establishing their roles in nearly all aspects of RNA metabolism with broad implications for physiological and pathological processes. These advances lay the groundwork for future therapeutic approaches targeting mRNA modification pathways. By contrast, emerging evidence indicates that RNA methylation on chromatin-associated RNAs (caRNAs) intersects with chromatin regulators to modulate chromatin state and transcription, adding a new dimension to epigenetic regulation with biological significance. Here we summarize established principles of post-transcriptional RNA modifications and their therapeutic potential, highlight the rapidly developing chromatin-related functions of RNA modifications as regulatory elements on caRNAs and discuss future directions that emphasize the need to investigate additional regulatory elements on these RNAs in shaping gene expression programs.
Decarboxylative condensation drives chain elongation and translocation of polyketides and fatty acids. However, the mechanism by which nonelongating modules in trans-acyltransferase polyketide synthases (trans-AT PKSs) e...Decarboxylative condensation drives chain elongation and translocation of polyketides and fatty acids. However, the mechanism by which nonelongating modules in trans-acyltransferase polyketide synthases (trans-AT PKSs) enable intramodular polyketide chain translocation without decarboxylation remains poorly understood. Here we elucidate a condensation-independent intramodular translocation mechanism in which KS within the nonelongating module operates as a transacylase, directly translocating the polyketide chain to its downstream cognate acyl carrier protein (ACP). Notably, the inherent demalonylation activity of trans-AT facilitates efficient ACP recycling ensuring intramodular translocation. Structural modeling and site-directed mutagenesis studies uncover a conserved KS-ACP binding mode that underpins intramodular translocation across diverse nonelongating modules. Additionally, the strict discrimination of polyketide intermediate by the nonelongating module highlights its critical role in maintaining biosynthetic precision and efficiency. These findings provide mechanistic insights into evolutionary adaptation and sophisticated crosstalk between catalytic domains within trans-AT PKS, illuminating how metabolic flux and fidelity are maintained and opening avenues for polyketide engineering.