In neurons, treatment with E64d lead to about 50-fold PLD3 accumulation and dysregulation of its proteolytic cleavage, while there was only a minor overall change on the whole proteome level, which suggests that their activation might be responsible for decreased PLD3 levels in neurons of patients with Alzheimer’s diseases.
Abstract
Catalytic activity of 5′-3′ exonuclease Phospholipase D3 (PLD3) is associated with immune signaling and neurodegeneration including Alzheimer’s disease. PLD3 undergoes multiple post-translational modifications and proteolytic cleavage to establish its catalytically active form. However, the proteases catalyzing the cleavage of PLD3 have remained unidentified. To study the proteolytic cleavage of PLD3, we have evaluated the small molecule covalent inhibitor E64d that blocks proteolysis catalyzed by cysteine cathepsins. To validate the selectivity of E64d, we have designed and synthetized an E64d propargyl analogue and carried out a detailed activity-based protein profiling to reveal a broad engagement of the compound with other protein targets including bleomycin hydrolase (BLMH), Kelch-like ECH-associated protein 1 (KEAP1), transcription elongation factor SPT5 (SUPT5H) and asparagine synthetase (ASNS). The specificity of the E64d-protein interactions was confirmed by biochemical assays and mass spectrometry-based site identifications. In neurons, treatment with E64d lead to about 50-fold PLD3 accumulation and dysregulation of its proteolytic cleavage, while there was only a minor overall change on the whole proteome level. Taken together, this study provides insights into previously unknown E64d selectivity and renders cysteine cathepsins responsible for PLD3 degradation in neurons. It highlights the importance of cysteine cathepsins activity in neuronal lysosomes for proper PLD3 processing and hence it suggests that their activation might be responsible for decreased PLD3 levels in neurons of patients with Alzheimer’s diseases. These findings are key for further elucidation of PLD3 function in neurodegenerative diseases.
UBA5 is the E1 enzyme that initiates UFMylation, a ubiquitin-like modification implicated in proteostasis, neurodegeneration, and cancer. Here, we report a chemical biology approach to interrogate UBA5 function through small-molecule inhibition. We developed a robust high-throughput screening assay based on AMP-GloTM and screened a library of blood-brain barrier-permeable compounds, identifying five inhibitors spanning three distinct scaffolds with low-micromolar potency. Orthogonal biochemical and gel-based assays confirmed that these compounds directly inhibit UBA5-mediated UFM1 activation and conjugation, with selectivity over other E1 enzymes, including UBA1. In cells, these compounds suppressed endogenous UFMylation without significantly affecting global polyubiquitination, supporting pathway and target engagement. Together, these studies establish UBA5 as a tractable enzymatic target and provide first-in-class chemical tools to probe UFMylation. Given the emerging role of UFMylation in disease, these inhibitors offer a foundation for developing therapeutics targeting proteostasis pathways.
Vigyasa Singh, Yuansong Wan, Zhisong Gao et al.· ACS Chemical Biology· 0 citations
Expanding the repertoire of usable E3 ubiquitin ligases remains a critical challenge in the field of targeted protein degradation. For the emerging E3 ligase Kelch domain-containing protein 2 (KLHDC2), currently reported ligands have a carboxylate moiety in common that mimics the natural degron but compromises cellular permeability. Here we report the discovery of carboxylate-free ligands for KLHDC2 through high-throughput screening and cellular functional evaluation. A fluorescence polarization-based screening assay identified NL1 as a micromolar KLHDC2 binder, which was confirmed by orthogonal biophysical methods. Although the structure-activity study did not improve affinity, a degron reporter assay demonstrated measurable cellular target engagement. Encouraged by these results, an expanded screen further identified NL2 as a neutral ligand with improved cellular activity. Computational analysis suggests that NL2 adopts a distinct binding mode, in which the conformational flexibility of two loops of KLHDC2 enables accommodation of its aromatic scaffold, while hydrophobic and π-driven interactions compensate for the canonical ionic contacts of other carboxylate-containing ligands. This work demonstrates that KLHDC2 can accommodate non-carboxylate scaffolds and expands the chemical space for developing KLHDC2-based targeted protein degradation strategies.
Yao Chen, Rui-Chen Gao, Jia-Jia Li et al.· Protein Science· 0 citations
Molecular editing, rooted in synthetic chemistry, has emerged as a powerful tool for structure optimization in medicinal chemistry but remains underutilized in targeted protein degradation. Here, we demonstrate the utility of molecular editing by repurposing the NSD2 degrader LLC0424 with GSPT1 neosubstrate engagement to generate two structurally analogous molecules with divergent modes of action: 424-ND, a PROTAC selective for NSD2, and 424-GD, a selective GSPT1 molecular glue. 424-ND drove NSD2 degradation selectively in a CRBN- and proteasome-dependent manner and functionally suppressed androgen receptor (AR) signaling in prostate cancer cells. 424-GD induced selective degradation of GSPT1 and robustly upregulated the integrated stress response markers ATF4 and ATF3. Biolayer interferometry revealed distinct ternary complex cooperativity profiles in the presence of NSD2 or GSPT1, which directly correlate with the observed biased degradation activity. Further molecular dynamics and metadynamics simulations showed that the compounds adopt distinct low-energy conformational ensembles with different spatial orientations, likely underlying their divergent cooperativity and selectivity. These findings not only deliver highly selective chemical probes for NSD2 and GSPT1 but also demonstrate that molecular editing enables precision control over target selectivity, providing a generalizable strategy to tune selectivity in targeted protein degradation. Table of Contents Graphic
Wei-Zhong Shen, Yi-Han Liu, Lian-Chao Liu et al.· bioRxiv· 0 citations
Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that induce selective degradation of target proteins by hijacking the ubiquitin-proteasome system (UPS). Despite their transformative potential in eliminating disease-associated proteins, comprehensively identifying off-target degradation events remains technically challenging. Here, we employed an integrated proteomic and ubiquitinomic strategy to systematically profile the degradation landscape of the PROTAC molecule dBET1 in Jurkat T cells. By capturing the upstream ubiquitination events─which serve as earlier and more sensitive indicators than total protein abundance─our approach enabled the identification of previously overlooked off-target candidates. While dBET1 efficiently degraded its canonical BET family targets, our data also revealed the mitochondrial outer membrane protein VDAC1 as a putative off-target, evidenced by its depletion and increased multisite ubiquitination. Notably, our analysis framework enabled site-specific resolution of degradation events within BRD3, revealing preferential ubiquitination at functionally essential bromodomains, suggesting that degron-enriched regions may underlie domain-selective degradation. Additionally, dBET1 treatment was associated with mitochondrial depolarization and calcium homeostasis disruption, defects that we hypothesize may be functionally linked to the observed VDAC1 depletion. Together, this study demonstrates that integrating ubiquitomics provides a superior sensitivity layer for PROTAC safety assessment, capable of uncovering mechanism-based liabilities that escape conventional global proteomic screening.
Hongtao Wen, Hui Liu, Jinfang Liu et al.· Analytical Chemistry· 0 citations
N-terminal acetyltransferase A (NatA) catalyzes 40-57% of eukaryotic Nα-acetylation, a co-translational modification critical for protein stability, localization, and function, yet no validated small-molecule probe of NatA has been reported. Here, we describe the discovery and characterization of tideglusib (YD3139), a clinical-stage GSK-3β inhibitor, as the first small-molecule probe for NatA through a unprecedented catalysis-dependent mechanism. This dual-event mechanism was established through intact protein MS, rapid dilution assays, and substrate-independent IC50 profiling. Tideglusib binds allosterically to Saccharomyces cerevisiae NatA (yNatA), whereupon the CoA released during catalysis chemically modifies tideglusib to form a reversible covalent adduct, a new paradigm for acetyltransferase inhibition. Tideglusib was identified by screening of an in-house library (∼700 compounds) and confirmed by structural optimization of the thiadiazolidinedione scaffold, yielding IC50 values of 0.68-1.8 μM against yeast and human NatA with >20-fold selectivity over other NAT family members. Quantitative proteomics confirmed on-target suppression of NatA-mediated Nα-acetylation in yeast. These findings establish tideglusib as a mechanistic chemical probe for NatA, reveal NatA as a previously unrecognized molecular target for a promiscuous clinical-stage investigational compound, and introduce a conceptual framework for exploiting catalysis-dependent adduct formation as a new strategy for acetyltransferase inhibitor design.
Youchao Deng, Zhuojun Luo, Sarah M. Gardner et al.· ACS Chemical Biology· 0 citations
It is established that metabolically activated compounds can redirect E3 ligase function, thereby expanding the scope of targeted protein degradation and chemically induced proximity.
Hojong Yoon, F. Wachter, Katharine A Barrett et al.· Nature· 1 citation
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