The SARS-CoV-2 main protease (Mpro) is a key antiviral drug target due to its essential role in the pathogen's replication cycle. The development of potent next-generation Mpro inhibitors is of great importance to ensure the availability of safe and effective COVID-19 therapeutics. Associated research necessitates robust and highly sensitive biochemical assays for the early kinetic characterization of Mpro inhibitor candidates. These assays often reach their limits when characterizing highly potent inhibitors, with a common bottleneck being the insufficient catalytic efficiency and competitive capacity of employed Mpro substrates. To optimize assays for highly active Mpro inhibitors by providing Mpro substrates with improved kinetic features, we created a library of fluorogenic substrates (2-8). These reporters were structurally derived from the well-established Mpro substrate Boc-Abu-Tle-Leu-Gln-AMC (1) and the clinically approved covalent-reversible Mpro inhibitor nirmatrelvir. Kinetic evaluation of 1-8 identified hit compound 6 with about ten-fold improved catalytic efficiency (kcat/Km = 21,400 M-1 s-1) compared to parent substrate 1 (kcat/Km = 2410 M-1 s-1). Substrate 6 was successfully applied for the kinetic characterization of three highly potent Mpro inhibitors. Comparative X-ray crystallographic analyses revealed high similarity in the molecular interactions of the C145A mutant Mpro with nirmatrelvir and with a peptide whose sequence was derived from a natural Mpro substrate. The tetrapeptidic AMC derivative 6 will serve as a valuable biochemical tool contributing to the optimization of SARS-CoV-2 Mpro-directed drug research.
Fe-S clusters are emerging as important cofactors in viral replication but are frequently misassigned as Zn due to their O2-sensitivity and overlapping cysteine-based coordination chemistry. The Hepatitis B virus regulatory protein HBx, which is essential for viral replication and hepatocarcinogenesis, has long remained mechanistically intractable because of uncertainty surrounding its physiologically relevant metallocofactor. Although HBx can bind either an Fe-S cluster or Zn, its intrinsic disorder and extensive mutational tolerance have hindered precise characterization of its metal-binding environment. Here, we combine chemoproteomics with HYSCORE spectroscopy to define the metal-coordinating ligands in HBx and overcome limitations associated with conventional mutational analysis of disordered proteins. We exclude histidine coordination and identify C61, C69, C143, and C148 as the primary cysteine ligands for Fe-S cluster binding, with C137 functioning as a conditional auxiliary ligand. These residues also support Zn binding and overlap with regions implicated in HBx transactivation and clinically relevant variants. In addition, HBx engages the host cytosolic Fe-S cluster assembly machinery and displays sensitivity to Fe-S-targeting reagents, behavior consistent with Fe-S cluster acquisition and cofactor lability. Together, these findings propose HBx as an Fe-S cluster-associated viral protein and expand the growing class of viral Fe-S proteins that are critical for infection.
Human mucin-1 (MUC1) is an attractive tumor antigen due to its high expression levels on tumor cells and its important roles in tumor development. Due to the aberrant glycosylation of MUC1 on tumor cells, tumor-associated MUC1 (tMUC1) has a shortened O-glycan attached on the peptide backbone, distinguishing it from its counterpart on normal cells. Preclinical evaluation of tMUC1-based vaccine candidates typically involved the immunization of mice that are tumor-free. However, tumor development is often associated with immune suppression. To better mimic clinical conditions encountered in cancer treatment, a breast cancer mouse model was built by crossing mouse mammary tumor virus (MMTV)-polyoma middle T (PyMT) mice with human MUC1 transgenic (MUC1.Tg) mice. The resulting female double-transgenic mice (MUC1/MMTV) spontaneously develop palpable breast cancer from 6 weeks of age. A promising vaccine candidate composed of a short 9-amino-acid Thomsen-nouveau (Tn) antigen containing tMUC1 glycopeptide conjugated with bacteriophage Qβ was evaluated in MUC1/MMTV mice. The vaccine was found to be immunogenic and capable of inducing high levels of anti-tMUC1 IgG antibody responses. The antibodies were selective toward both the glycan and the peptide sequence of tMUC1, as deciphered from a glycopeptide microarray study. Furthermore, the induced antibodies bound tumor cells in a MUC1-dependent manner and could kill a range of tumor cells via complement-dependent cytotoxicity. Vaccination with a Qβ-tMUC1 conjugate significantly prolonged the survival of MUC1/MMTV mice, with 83% of treated MUC1/MMTV mice outliving the control group. The Qβ-tMUC1 vaccine exhibited promising results in an aggressive and rapidly growing spontaneous tumor model, laying the groundwork for its clinical translation to human patients.
Fluorescent indicators are indispensable imaging tools for visualizing the spatiotemporal dynamics of biological processes. Red fluorescent indicators are in particularly high demand because they offer compatibility with existing green fluorescent indicators or optogenetic tools, and longer wavelength fluorescence has inherent advantages for biological applications. We previously described a chemigenetic indicator design that combines a green fluorescent protein and a synthetic chelator in an effort to combine the advantages of conventional protein-based biosensors and synthetic chemosensors. We now demonstrate that this chemigenetic design can be extended to red fluorescent proteins. Through screening of variants with a range of chromophore-chelator orientations, followed by directed evolution, we developed a red fluorescent calcium ion (Ca2+) indicator with 5.4-fold fluorescence intensity change when going from 0 to 39 μM Ca2+ with purified proteins. Although the functionality of the current version is lost when expressed in mammalian cells and the selectivity is low, these results establish this chemigenetic design as a strategy that can be extended to other fluorescent protein color variants.
DNA replication fidelity depends on the integrity of the replication fork to prevent DNA damage and preserve genome stability. Disruptions to this process can trigger replication stress, leading to the accumulation of single-strand DNA (ssDNA) and double-strand breaks (DSBs), which drive mutagenesis and ultimately contribute to disease. While the roles of core histones and their post-translational modifications in this context have been more well-studied, far less is known about how linker histones regulate the replication stress response. Here, we demonstrate that the S-phase-phosphorylated form of the linker histone H1 (pH1) plays a key role in DNA damage repair at collapsed replication forks, both in vitro and in cells. Using phosphomimic and phosphonull H1 mutants, we show that phosphorylation enhances H1 assembly with ssDNA. Utilizing intein chemistry for the site-specific incorporation of a photocrosslinker to the C-terminus of H1, we map the direct interactors of H1. We identify phosphorylated H1 at replication forks, where it engages replication machinery and DNA damage response factors, including Histone PARylation Factor 1 (HPF1). We further demonstrate that ssDNA induces pH1-HPF1 interactions that promote liquid-like assemblies, correlating with reduced DNA damage and histone PARylation. Consistent with this role, reduction of total H1 increases cellular sensitivity to DNA damage, a phenotype that is partially rescued by reintroduction of H1.4. Together, these findings establish pH1 as a new regulator of DNA damage repair at collapsed replication forks through the controlled sequestration of repair factors.
Dermatan sulfate epimerase 1 (DS-epi1, DSE) and dermatan sulfate 4-O-sulfotransferase (DS 4-OST, CHST14) are critical enzymes involved in the biosynthesis of dermatan sulfate. In this study, structurally homogeneous chondroitin oligosaccharide substrates were used to investigate the substrate specificity of each individual enzyme, as well as their combined actions. The structures of the enzyme-modified oligosaccharides were confirmed by high-resolution mass spectrometry (MS) and NMR or determined by LC-MS-based sequencing analysis. Our results demonstrate that DS-epi1 catalyzes the reversible conversion between glucuronic acid (GlcA) and iduronic acid (IdoA), with GlcA being the predominant product once the reaction reaches equilibrium. DS 4-OST selectively sulfates the 4-hydroxyl group of N-acetylgalactosamine (GalNAc) residues that are linked to the reducing end of an IdoA unit (-IdoA-GalNAc-). Furthermore, DS-epi1 and DS 4-OST act in a concerted and ordered manner to generate clustered -IdoA-GalNAc4S- domains. During the reaction, the enzyme mixture initiates modifications at the non-reducing end of the saccharide chain and proceeds toward the reducing end consecutively. These findings provide new insights into the regulatory mechanisms governing dermatan sulfate biosynthesis and demonstrate the feasibility of synthesizing structurally defined dermatan sulfate oligosaccharides.
Maintenance of lipid homeostasis requires cells to coordinate lipid synthesis precisely. Sterol regulatory element-binding proteins (SREBPs) represent one of the central mechanisms by which cells sense lipid availability and adjust the biosynthesis of fatty acids, cholesterol, and phospholipids in response to metabolic and other changes. Dysregulation of SREBP signaling has been implicated in a broad spectrum of diseases, where altered lipid metabolism reshapes membrane organization, cellular signaling, and stress adaptation. In this review, we summarize the molecular mechanisms governing SREBP activation and systems-level regulation. Particular emphasis is placed on the approaches that have enabled interrogation of SREBP signaling. We discuss imaging-based assays, perturbation strategies, pharmacological inhibitors, lipidomics, and other approaches that collectively allow SREBP activity to be interrogated across spatial, biochemical, and metabolic scales. Finally, we highlight how integration of systems-level approaches can refine our understanding of membrane homeostasis and reveal new opportunities for therapeutic intervention in diseases driven by altered SREBP metabolism.
N 6-methyladenosine (m6A) is a widespread RNA modification that regulates RNA metabolism and gene expression, yet most sequencing methods rely on isolated RNA and therefore lose its spatial context. This In Focus article surveys methodological advances in transcriptome-wide m6A detection and highlights m6A-ARTR-DBiT, a spatial m6A profiling assay that leverages reverse-transcription-based detection and deterministic barcoding in tissue to map transcriptome-wide m6A distribution while preserving native tissue context. This platform enables researchers to investigate how m6A patterns vary across distinct tissue regions, developmental structures, and local gene regulatory networks, bridging the gap between spatially patterned epitranscriptomic deposition and gene regulation.
CCAAT/enhancer-binding protein alpha (C/EBPα) is a critical lineage-defining transcription factor and tumor suppressor. Despite its clinical significance in conditions like acute myeloid leukemia and hepatocellular carcinoma (HCC), C/EBPα has remained undruggable due to its intrinsically disordered regions and lack of small-molecule binding pockets. In this study, we describe the development of a novel Oligonucleotide-based PROteolysis-TArgeting Chimera (Oligo-PROTAC or O'PROTAC) strategy designed to achieve targeted degradation of endogenous C/EBPα. We designed and synthesized a 10-nucleotide double-stranded DNA decoy derived from a C/EBPα-binding motif and conjugated it via variable linkers to the E3 ligase ligands pomalidomide or VH032. A total of six candidates were synthesized and evaluated in 3T3-J2 and Huh7 cell lines. We demonstrate that these O'PROTACs successfully localize to the nucleus and achieve dose-dependent degradation of C/EBPα, with the most potent candidates exhibiting sub-micromolar DC50 via lipofection delivery. Notably, this degradation occurred without inducing significant cytotoxicity. We observe a transcriptional shift consistent with loss of C/EBPα, including downregulation of cooperative factors involved in liver lineage specification and their downstream target genes. This work establishes O'PROTACs as a viable modality for targeting C/EBPα, offering a novel strategy for mechanistic investigation and future therapeutic targeting.
Inositols are a family of cyclic sugar alcohols comprising nine stereoisomers. myo-Inositol is the most abundant isomer found in humans and has been studied most extensively. It plays an important role in osmoregulation and is incorporated into membrane-anchored phosphatidylinositols. scyllo-Inositol is the second most abundant inositol isomer in the human brain, and aberrant concentrations are associated with various diseases; however, its biological functions remain unclear. Here, the development and application of [13C6]scyllo-inositol as an isotopic tracer to study its metabolism are reported. A concise and robust synthetic route was established to obtain [13C6]scyllo-inositol from [13C6]myo-inositol in good yield. The uptake of [13C6]scyllo-inositol at concentrations used in clinical trials and the responses of endogenous inositol isomers were measured in multiple immortalized mammalian cell lines by hydrophilic liquid interaction (HILIC)-MS/MS. [13C6]scyllo-Inositol proved to be a versatile isotopic tracer when coupled with mass spectrometry (MS)-based lipidomics and 2D nuclear magnetic resonance (NMR) experiments. These experiments provide evidence that scyllo-inositol is incorporated into phosphatidylinositols in different immortalized mammalian cell lines and suggest a previously underappreciated role of scyllo-inositol. Future research utilizing [13C6]scyllo-inositol can elucidate scyllo-inositol metabolism and its role in physiological and diseased states.
Ewing sarcoma is an aggressive pediatric cancer for which no targeted therapies have been approved. This disease is driven by EWSR1::ETS family fusions that, like most fusion transcription factors, have proven difficult to target directly. Here, we focus on identifying actionable dependencies within the high-risk STAG2-mutant (STAG2-mut) disease subtype of Ewing sarcoma. In STAG2-mut Ewing sarcoma, STAG1 knockout is synthetically lethal but is not readily druggable. Using the Cancer Dependency Map (DepMap), we identify histone deacetylase 8 (HDAC8) as an enriched vulnerability in this molecular context. CRISPR validation and a newly developed HDAC8 degrader, XY-09-36, confirm this selective dependency and suggest increased reliance on HDAC8-mediated cohesin regulation when STAG2 is lost. These findings establish HDAC8 as a context-dependent, pharmacologically accessible vulnerability and outline a framework for discovering rational targets in tumor subtypes where primary drivers are intrinsically hard to drug.
Irreversible covalent inhibitors have garnered significant attention in recent years. Despite encouraging progress, the vast majority contain electrophiles that target the least abundant amino acid, cysteine, substantially limiting target inhibitor design for therapeutic intervention. Here, we generalize 2-ethynylbenzaldehyde as a proximity-induced electrophile for generating irreversible covalent peptide and protein inhibitors that specifically target native lysine residues. Leveraging this warhead, we designed a covalent de novo peptide that potently engages MCL1 to block its interaction with Bak. We show it is faster, more site-selective, and increases potency by 61-fold for MCL1 relative to a sulfonyl fluoride warhead. Additionally, with the guide of a computational script to predict "reactive hotspots" at the protein level, we developed a minibinder that labels PD-L1 in vitro and in live cells, displays a slower off-rate, and potently blocks the native PD-1 and PD-L1. These results establish isoquinolinium capture as a promising strategy to inhibit protein-protein interactions and for the development of novel covalent peptide and protein therapeutics.
Activity-based ubiquitin probes have emerged as powerful tools for dissecting protein ubiquitination. In this work, we discovered that an α-amidomethyl-substituted acrylate electrophile exhibits markedly enhanced reactivity. Based on this finding, we designed and synthesized a new activity-based probe, ubiquitin-α-amidomethyl acrylate (Ub-AMA). In vitro biochemical assays demonstrated that Ub-AMA effectively crosslinked with deubiquitinases, E1 and E3s. In addition, compared to the classic Ub-VME and Ub-PA probes, Ub-AMA achieves improved labeling efficiency for E2 enzymes. Finally, proteomic profiling in cell lysates confirmed that Ub-AMA can capture E1, E2, and E3 enzymes as well as DUBs in a complex biological environment. In summary, the Ub-AMA probe developed here provides a new tool for in-depth analysis of the dynamic regulatory mechanisms of ubiquitination. Moreover, this work offers valuable insights for the design of other activity-based probes based on α,β-unsaturated carbonyl scaffolds.
Mitochondria serve as central hubs of cellular bioenergetics and signaling, yet the dynamic role of their lipid composition in cellular adaptation remains underappreciated. Unlike most organelles, mitochondria possess a unique dual-bilayer membrane architecture shaped by lipid transport and de novo synthesis. The mitochondrial lipidome, dominated by phosphatidylcholine, phosphatidylethanolamine, and the signature phospholipid cardiolipin, influences cristae organization, oxidative phosphorylation capacity, and metabolite transport, collectively determining whether mitochondria undergo stabilization, remodeling, or degradation. In this review, we explore how mitochondrial lipid dynamics sustain organelle-wide homeostasis while coordinating cellular adaptation across multiple temporal scales and how failure of lipid homeostasis drives rare monogenic disorders and complex pathologies. We propose that environmental shifts transiently disrupt the balance between phospholipid biosynthesis and utilization, generating changes in mitochondrial lipid homeostasis that promote cellular adaptation through complementary biophysical and biochemical signaling mechanisms. Specifically, membrane lipid remodeling rapidly alters membrane biophysical properties to regulate membrane protein activity, whereas bioactive phospholipid intermediates and side-products support long-term adaptive reprogramming. Mitochondrial lipids therefore function not merely as passive structural components but as active regulatory nodes that drive cellular plasticity, positioning lipid dynamics at the nexus of metabolic adaptation and human disease.
Assigning causal function to post-translational modifications (PTMs) remains a central challenge in molecular biology, as most modification events cannot be readily interrogated in their native cellular context. Here, we present a generalizable chemical biology strategy for investigating the functional consequences of lysine acetylation through programmable induced proximity. By combining modular effector recruitment with chemically controlled proximity, this approach enables systematic elucidation of how enzyme identity shapes acetylation outcomes on target proteins in living cells. Across multiple substrates, including histone H3 and p53, we find that distinct acetyltransferases generate reproducible and target-dependent site-selective acetylation patterns, indicating that effector identity encodes predictable features of modification outcomes. These observations establish a framework for linking enzyme recruitment to site-specific PTM deposition and provide a route to identify candidate functional modification events. Rather than providing a single mechanistic insight, this work introduces a broadly applicable strategy for interrogating causal relationships between proximity-driven enzyme recruitment and protein modification, as demonstrated by the impact of p53 acetylation on downstream transcripts. This platform is readily extensible to additional effectors and targets and enables systematic discovery of functional PTMs in cellular systems.
Strategies for site-specific incorporation of noncanonical amino acids have advanced substantially, enabling access to proteins containing numerous analogues. That noted, many interesting analogues are not α-L-amino acids, and the incorporation of such compounds often requires the development and use of engineered ribosomes. Recently, elongation factor P (EF-P), a translation factor reported to alleviate ribosomal stalling during the incorporation of contiguous L-proline residues, has been shown to facilitate the incorporation of a diverse range of noncanonical amino acids in the presence of native bacterial ribosomes. However, the specific experimental parameters that enable EF-P-mediated enhancement of extensively modified amino acids remain poorly understood. Presently, we examined EF-P-mediated incorporation of four conformationally constrained cyclic dipeptides into position 24 of human RRM1, a nucleic acid recognition motif, using an in vitro transcription-translation system. We compared the effects of ring substitution and conformational isomerism on protein incorporation. Each was incorporated using mono- and bisaminoacylated tRNACUAPhe in comparison to L-phenylalanine. Supplemental EF-P enhanced the incorporation of all analogues significantly, but varied in magnitude substantially between congeners. Bisaminoacylated tRNAs exhibited distinct profiles relative to their monoaminoacylated counterparts. In the absence of exogenous EF-P, bisaminoacylated tRNAs activated with cyclic dipeptides produced similar or lower protein yields relative to their monoaminoacylated counterparts. In contrast, in the absence of added EF-P bisphenylalanyl-tRNA maintained higher incorporation yields than monophenylalanyl-tRNA, as we have observed previously for many aminoacyl-tRNAs containing α-L-amino acids. In the presence of exogenous EF-P, tRNAs activated with two cyclic dipeptides exhibited disproportionately greater yield enhancement, surpassing the corresponding monoaminoacylated analogues. These findings demonstrate that exogenous EF-P-mediated enhancement is influenced by changes within the local structure of the analogues. Additionally, bisaminoacylated tRNAs have the potential of affording significantly greater protein yields, especially in the presence of supplementary EF-P.
β-Ketoacyl-ACP synthase (KAS) III enzymes play central roles in fatty acid biosynthesis and the production of various natural products. While their canonical function involves catalyzing C-C bond formation between acetyl-CoA and malonyl acyl-carrier-proteins (ACPs), an increasing number of non-canonical activities have been reported among KAS III homologues, including C-O and C-N bond formation. Here, we describe a KAS III family enzyme, CalO4, that catalyzes C-S bond formation during the biosynthesis of the potent antitumor agent calicheamicin. We successfully reconstituted its transacylation activities, demonstrated C-S bond formation, and assessed its substrate selectivity. Furthermore, X-ray crystallography combined with molecular docking and mutational analysis identified key residues likely involved in substrate selection and catalysis. This work not only expands the functional diversity of the KAS III family but also provides a potential biocatalyst for hindered C-S bond formation and a target for engineering novel calicheamicin-like or other bioactive compounds.
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.
The DNA/RNA helicase Schlafen11 (SLFN11) is considered a sensitivity marker of various tumor cells for cytotoxic treatment with DNA damaging agents (DDA), such as cisplatin and PARP inhibitors (PARPi). Cells can epigenetically silence SLFN11 expression to gain chemoresistance, which can be targeted by HDAC inhibitors (HDACi). Here, we investigate the predictive value of SLFN11 expression for the response to three different PARPi in ovarian cancer cells. Furthermore, we target its epigenetic downregulation via HDACi for sensitization to cisplatin and PARPi. We show that HDAC3 plays a key role in SLFN11 silencing in W1 and W1CR ovarian cancer cells, confirmed by an HDAC3 knockdown approach. Therefore, addressing HDAC3 by novel class I HDACi DS-103 and FL-007 at non-toxic concentrations restored SLFN11 expression with various consequences for DDA treatment in W1, W1CR, and SKOV-3 cells. While DS-103 and FL-007 significantly sensitized cisplatin-resistant W1CR cells to cisplatin to the level of W1 cells, the response to various PARPi remains divergent. Whereas both HDACi induced significant sensitization in W1 and W1CR cells, and DS-103 in SKOV-3 cells for niraparib cytotoxicity, cells remain unaffected in response to olaparib and rucaparib. Both compounds were also shown to be less dependent on, or independent of, SLFN11 in different ovarian cancer cells. In sum, the first time reported functional link of HDAC3 and SLFN11 appears an attractive co-treatment opportunity of DDA with HDACi. This is particularly promising for niraparib as it has a broader approval independent of BRCA status compared to other PARPi.
