RSC Chemical Biology

The biosynthetic machineries of ribosomally synthesized and post-translationally modified peptides (RiPPs) are often substrate tolerant. A remarkable example is the class II lanthipeptide synthetase ProcM, which naturally functions as a generalist enzyme that has not evolved to use a specific substrate during its evolutionary history. Although ProcM has been studied extensively, the sequence features associated with productive modification remain underexplored. In this study, we use the ultrahigh-throughput mRNA display technique to map the sequence compatibility of ProcM across a focused library. This approach expands the landscape of ProcM reactivity beyond native substrates and individually characterized variants. Machine learning (ML) is used as a tool to demonstrate that the selected dataset contains learnable signatures and classification architectures revealed a balanced accuracy of 0.73. This performance contrasts sharply with the near-perfect accuracy of specialized enzyme models as the sequence-fitness landscape of the generalist enzymes are characterized by class imbalance and limited by intrinsic dataset features. Our results provide a high-throughput view of ProcM reactivity and highlight differences with previous high-throughput studies on substrate selectivity of RiPP modification enzymes. Future studies will need to assess whether these differences are common when comparing generalist with specialist enzymes.

Chemically induced proximity has not been systematically applied to control RNA fate. Here, a programmable platform was developed to identify RNA-binding proteins (RBPs) that can be recruited by small molecules to destabilize RNA. Using microtubule-associated protein Tau (MAPT) pre-mRNA as a model target, a heterobifunctional molecule was designed to bind both a ligandable structure in MAPT pre-mRNA and FKBP12F36V-tagged RBPs. Screening of a library of tagged RBPs identified several proteins that reduced MAPT RNA levels, including zinc finger protein 36 (ZFP36) and nanos C2HC-type zinc finger 3 (NANOS3). The approach was then extended from engineered proteins to an endogenous RBP. Using small molecule ligandability maps, a cysteine-reactive ligand for ZFP36 was identified. When this ligand was linked to the MAPT-binding small molecule, endogenous ZFP36 was recruited to MAPT mRNA, reducing its abundance in cells. Genetic and chemical controls demonstrated that activity was dependent on both RNA binding and ZFP36 recruitment, supporting an induced-proximity mechanism. These studies establish a general strategy for identifying new recruitable RBP effectors and should advance ribonuclease-targeting chimera (RiboTAC) technology by expanding the repertoire of effector proteins that can be harnessed for RNA degradation. More broadly, new effectors can be discovered through model reporter-based screens and translated to endogenous systems by mining known protein binders and ligandability maps, providing a systematic path to develop small molecules that control RNA stability, including RNAs targeted through structured regions of nuclear pre-mRNAs.

Selenocysteine (Sec), the 21st amino acid, is a rare non-canonical amino acid that represents an attractive target for protein engineering due to its desirable chemical properties such as high affinity for metals, strong nucleophilicity, and reversible covalent bond formation. To bypass the natural constraints on Sec placement within proteins, several strategies have been developed to rewire the native translational machinery to enable site-specific incorporation. However, these usually abolish the quality control mechanism that excludes the serine-charged selenocysteinyl-tRNA (Ser-tRNASec), the immediate biosynthetic precursor, from translation resulting in heterogenous protein species. This challenge is confounded by a lack of genetic tools to accurately report the selenylation state of the tRNA pool as most are blind to competing process of Ser incorporation, which can only be observed using analytical methods. To resolve this issue, we have developed a new fluorescent reporter, Selenocysteine Adjusted Ratiometric Chromophore (SeARCh), which exhibits two distinct spectral outputs dependent on the incorporation of either Ser (red) or Sec (green). Using SeARCh, we define several factors which influence the observed Sec:Ser ratio and construct a new hybrid biosynthetic pathway with improved performance, achieving 90% Sec incorporation. Furthermore, SeARCh displays unusually complex mass spectra due to the isotope distribution of selenium and heterogenous nature of the protein in solution and we report specific methods to account for this behaviour and precisely quantify the rare Ser-containing species found at high Sec incorporation efficiencies. Our findings suggest that the equilibrium between selenoprotein and tRNASec expression levels is a key driver of incorporation efficiency and implies a process that is broadly biosynthetically constrained. Collectively these tools represent a significant advance in the metrology of selenocysteine biosynthesis and incorporation and can be used to inform and standardize future engineering efforts.

The initial attachment of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) to host cell sialosides is critical for infection, yet its precise receptor specificity remains poorly understood. Here, we describe a chemoenzymatic methodology to synthesize a comprehensive panel of 6-sulfo sialyl Lewisx (6-sulfo-SLex) containing glycans. Our approach entails the enzymatic assembly of an oligo-lactosamine chain modified at specific positions with N-trifluoroacetyl-glucosamine (GlcNTFA) moieties. Mild base treatment removes the TFA group to yield glucosamine, which effectively blocks enzymatic fucosylation. By leveraging this approach alongside the unique substrate selectivity of GlcNAc-6-O-sulfotransferases 2 (CHST-2), we achieved the selective preparation of fucosylated 6-sulfo-SLex glycans. Microarray screening of these printed glycans revealed that a 6-sulfo-SLex derivative presented on an extended LacNAc chain is the preferred host receptor for MERS-CoV. Conjugation of this lead compound to a polyglycerol-based dendrimer generated a multivalent inhibitor that potently blocks hemagglutination of human red blood cells by the MERS-CoV spike protein N-terminal domain (NTD). Furthermore, computational modeling demonstrated that the fucose moiety does not directly contact the viral spike protein. Instead, it pre-organizes the ligand into a favorable conformation, enabling a critical salt bridge between the glycans sulfate group and the guanidinium side chain of viral residue Arg307.

Contemporary RNA-binding ligand collections are biased toward aromatic scaffolds, although it remains unclear whether this over-representation reflects an intrinsic requirement for productive RNA recognition or historical discovery bias. Here, using a modular "host-guest" ligand design strategy targeting the env8 cobalamin (Cbl) riboswitch, we established a common molecular framework to directly evaluate whether aromaticity is fundamentally required for RNA binding. We synthesized a focused series of cyclic aliphatic {beta}-axial Cbl derivatives, expanding the ligand library and enabling matched-pair comparisons to isolate the contribution of aromaticity to molecular recognition. Aliphatic ligands supported high-affinity RNA binding and regulatory activity comparable to aromatic analogues, with several derivatives exhibiting equal or greater affinity than their matched aromatic counterparts. Structural analyses revealed that aromatic and aliphatic ligands engage the same cryptic RNA binding site through distinct modes of molecular recognition, including nucleobase {pi}-stacking and alternative van der Waals packing arrangements. Machine learning analyses further demonstrated that the physicochemical features associated with affinity extend beyond aromaticity itself and instead reflect a broader combination of shape, surface, heteroatom, and electronic properties. Together, these findings demonstrate that high-affinity RNA binding can arise from multiple structural and physicochemical solutions, suggesting that aromaticity is not uniquely privileged as a strategy for RNA-targeted ligand design and supporting broader exploration of underrepresented RNA-binding chemotypes.

Site-specific protein modification allows diverse functionalities to be introduced while minimizing perturbations to the protein structure and activity. Considerable efforts have been made to achieve site-specific modification of native proteins to overcome the heterogeneity resulting from conventional stochastic Lys or Cys modification. We have previously achieved the selective modification of Lys65 in a native immunoglobulin G1 (IgG1) antibody (trastuzumab) using EzMTG-pG(Fab), which is an engineered zymogen of microbial transglutaminase (EzMTG) fused to a Fab-binding protein G [pG(Fab)]. However, this approach cannot be widely applied to different types of IgG antibodies. Here, we designed pG(Fab)-EzMTG by fusing pG(Fab) to the N-terminus of EzMTG. Notably, switching the fusion partners dramatically altered the IgG modification site from Lys65 to Lys225, which is located in the hinge site of native IgG1 antibodies. This Lys225-selective labeling was applicable to different IgG1 antibodies. As a functional application, the cytotoxic drug monomethyl auristatin E (MMAE) was conjugated to Lys225 of trastuzumab, and the resulting antibody-drug conjugate exhibited antigen-specific cytotoxicity. These findings demonstrate that fusion-protein architecture determines site selectivity in proximity-directed enzymatic modification, providing a strategy for the site-specific functionalization of native antibodies.

Achieving predictable, tunable, and temporal control over mRNA function would grant direct regulation of gene expression, facilitating the development of new therapeutics and biotechnologies. Although several approaches for stimuli-responsive control of nucleic acids have been explored, most are limited to short oligonucleotides, lack a timed-release mechanism, or both. We envisioned a complementary method using glyoxal as a caging reagent. Glyoxal readily reacts with amidine groups found on the faces of nucleobases to give stable bis-hemiaminal adducts, directly disrupting hydrogen bonding. Fortuitously, this reaction is readily reversible, enabling spontaneous time-release decaging that varies with temperature. However, when applied previously to full-length mRNAs, the sequence length and excessive adduct formation resulted in no reactivation under relevant physiological conditions. To address this challenge, we developed chemical lithography in which portions of longer RNAs are "masked" through hybridization to complementary DNAs, permitting selective caging on only non-masked regions and preventing excessive adduct formation. We present an optimized glyoxalation protocol applied to EGFP as a model mRNA sequence and evaluate masking effectiveness through qualitative and quantitative studies. Using EGFP fluorescence, we monitored and assessed the ability of selective glyoxalation to control gene expression over time in vitro. We demonstrate the direct dependence of both the initial inhibited expression and the respective activity recovery based on the amount and location of glyoxalation. We also highlight distinct caging patterns exhibiting total inhibition upon initial treatment and complete reactivation following decaging. We anticipate that this approach will improve the mechanistic study of mRNA and gene expression and also facilitate new investigations and methods within chemical biology and biomedicine.

Cereblon (CRBN) is widely used in targeted protein degradation, but its ligand space has remained dominated by a narrow set of cyclic imide chemotypes. Here, we show that the accessible CRBN ligand space extends substantially beyond this canonical space. A high-throughput screen of > 40,000 compounds, followed by orthogonal biophysical validation, X-ray crystallography and SAR analyses, identified several chemically distinct ligand classes. These include linear acetyl-based motifs, a phthalide-derived scaffold, steroidal compounds, and a range of bicyclic ligands. They engage CRBN through distinct recognition modes, several of which deviate from the canonical hydrogen-bonding pattern. Steroidal scaffolds were particularly notable: cortisone binds the human CRBN thalidomide-binding domain with an affinity comparable to thalidomide, with its A-ring occupying the tri-tryptophan pocket in a glutarimide-like orientation despite lacking the canonical imide NH donor. SAR within this series showed substantial tolerance for chemical modification and scaffold simplification, raising the possibility that endogenous steroidal metabolites may contribute to the physiological ligand landscape of CRBN. Bicyclic lactams additionally provided synthetically accessible scaffolds with tunable affinity and promising sites for linker attachment. Across the identified ligand classes, none of the tested representatives induced detectable degradation of canonical CRBN neosubstrates, and several showed largely clean proteomic profiles. Together, these findings broaden the chemical, mechanistic and potential physiological landscape of CRBN recognition and provide diverse starting points for alternative, potentially neosubstrate-sparing CRBN recruiters.

Small-molecule drug discovery relies on identifying compounds that modulate specific protein targets, a process often hindered by cellular complexity. Through phenotypic screening of a kinase-focused diazaquinazoline library, we serendipitously identified CEM198 as the first high-affinity ligand of tubulin-tyrosine ligase (TTL). Functional assays combining live-cell TTL inhibition, microtubule polymerization, cell cycle analysis, and proteomics revealed that CEM198 acts through a dual mechanism: directly binding to TTL and altering /{beta}-tubulin conformation. This interaction restricts -tubulin tyrosination and disrupts tubulin polymerization, leading to microtubule destabilization. The differential effects observed between SH-SY5Y and HEK293T cells indicate that effective TTL inhibition depends on both direct binding and structural modulation of the tubulin heterodimer. These findings introduce CEM198 as a chemical probe for investigating the tubulin tyrosination-detyrosination and demonstrate the potential of chemoproteomics to uncover novel modulators of microtubule dynamics.

We present a robust and versatile in vitro transcription (IVT) assay based on an optimized Broccoli RNA aptamer sequence. When paired with the fluorophore DFHBI-1T, this system enables real-time monitoring of multi-round transcription over several hours. To facilitate streamlined promoter analysis, we developed the pIVT3 plasmid backbone. The system was validated using both the single-subunit T7 RNA polymerase and the multi-subunit Escherichia coli RNA polymerase; notably, the activity of the E. coli enzyme remained strictly dependent on the presence of a {sigma} factor and a cognate promoter. To optimize the signal-to-noise ratio, we incorporated two rrnBT1 terminators upstream of the promoter of interest. This modification effectively eliminated background transcription for weak promoters (PlivJ) and prevented interference from read-through transcription in strong synthetic promoters (Ptrc*). Furthermore, we demonstrated the assays utility for drug discovery by characterizing the time- and dose-dependent inhibitory kinetics of rifampicin. Collectively, these results establish the Broccoli-based IVT system as a highly adaptable platform for quantifying promoter strength and screening small-molecule inhibitors of bacterial transcription. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/744185v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1e0c991org.highwire.dtl.DTLVardef@d154aeorg.highwire.dtl.DTLVardef@10e95fcorg.highwire.dtl.DTLVardef@98ea80_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bacterial sortases are widely used in sortase-mediated ligation (SML) experiments for various protein engineering applications. The power of these enzymes to bind and cleave a specific recognition motif, followed by ligation to another substrate using a ping-pong reaction mechanism has numerous applications in vaccine and antibody/nanobody drug conjugate development, as a diagnostic and therapeutic tool, in creating novel insulin derivatives, etc. The most widely used sortase for SML is the class A sortase (SrtA) from Staphylococcus aureus (saSrtA), and its engineered derivatives. Despite its utility, saSrtA and other endogenous sortases are relatively inefficient enzymes and use can be limited by the need for specific recognition of the Cell Wall Sorting Signal (CWSS), sequence Leu-Pro-X-Thr-Gly, where X=any amino acid. Therefore, there is a need to continue to identify new tools for SML and to develop screening assays towards these endeavors. Here, we present optimization procedures for a FRET-based assay utilizing the GFP derivatives mTurquoise2 and SYFP2 to directly monitor formation of ligation products generated via SML. Similar to related assays, our recombinant substrates can be easily manipulated to screen either the substrate recognition motif, second substrate nucleophile, and/or sortase variants themselves. We believe continued optimization of this assay for a variety of high throughput uses in sortase screening strategies is possible, providing a proof-of-concept approach for continued SML reagent development.

Oxalyl-coenzyme A (oxalyl-CoA) is a key intermediate in oxalate metabolism in plants, fungi and oxalate-degrading bacteria, but its limited availability has restricted biochemical investigations of oxalyl-CoA-dependent enzymes. Here, we describe a practical semisynthetic procedure for the preparation of oxalyl-CoA based on rapid oxalyl transfer from S-oxalyl p-thiocresol to coenzyme A. The reaction was monitored directly by 1H NMR spectroscopy, allowing optimisation of pD and reaction conditions. Following removal of thiocresol and purification by reversed-phase HPLC, oxalyl-CoA was obtained in 39% yield as determined by quantitative 1H NMR. The product was characterised by high-resolution electrospray mass spectrometry and comprehensive 1H, 13C and 31P NMR spectroscopy, confirming its structure unequivocally. During the study, the limited stability of oxalyl-CoA in aqueous solution was documented, leading to recommendations for its purification and storage. The semisynthetic protocol provides a convenient source of analytically pure oxalyl-CoA suitable for biochemical assays and supplies reference spectroscopic data for its unambiguous identification. The biological utility of the semisynthetic oxalyl-CoA was demonstrated by its application as an acyl donor substrate in assays of PnBAHD15, enabling quantitative kinetic characterisation of the enzyme and illustrating its suitability for biochemical studies of oxalyl-CoA-dependent enzymes.

Membrane protein stability is strongly influenced by the surrounding lipid environment, yet how individual lipid species shape membrane proteome stability remains poorly understood. Here, we systematically examined the impact of sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesteryl hemisuccinate (CHS) on membrane proteomes using membrane mimetic platforms combined with membrane mimetic thermal proteome profiling (MM-TPP). CHS shifted the proteome composition away from soluble proteins and toward integral membrane proteins, and induced concentration-dependent thermal stabilization of the mouse liver membrane proteome. Organellar membrane proteins, which displayed greater intrinsic lability than plasma membrane proteins, showed preferential stabilization by CHS. CHS supplementation of E. coli membranes similarly produced broad stabilization, indicating that this effect occurs even in cholesterol-naive systems. CHS responses were reproducible across Peptidisc and DDM and independent of CRAC/CARC motif density, supporting a broad, sequence-independent mechanism rather than selective lipid binding, although stabilization was greater among proteins with more transmembrane helices. Accordingly, individual purified proteins reconstituted with CHS exhibited only modest stabilization, consistent with a broad effect that is more apparent at the proteome scale than for any single protein examined in isolation. Together, these findings redefine CHS as a general sterol scaffold that broadly stabilizes membrane proteins and establish MM-TPP as a versatile platform for investigating lipid-dependent effects on membrane proteome stability. Subject areaIntegral Membrane Proteins, Thermal Proteome Profiling, Membrane Mimetics, Cholesterol, Lipid-Protein Interactions, Mass Spectrometry HighlightsO_LICHS broadly stabilizes membrane proteins across diverse membrane mimetics. C_LIO_LIOrganellar membrane proteins exhibit the strongest CHS-mediated stabilization. C_LIO_LICHS stabilization is conserved in cholesterol-naive E. coli. C_LIO_LIMM-TPP enables proteome-wide analysis of lipid-dependent protein stability. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/745344v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1a3e017org.highwire.dtl.DTLVardef@1cd4464org.highwire.dtl.DTLVardef@1453da7org.highwire.dtl.DTLVardef@d2dc1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Actinomycin D (Act D) is a global transcriptional inhibitor widely used in research and clinical practice; however, its effects on RNA-binding protein (RBP) dynamics remain poorly understood. Analysis of an RNA-seq dataset from Act D-treated HeLa cells revealed a compensatory stress response enriched in RNA metabolism, processing, and translation. Here, we investigated the effects of Act D on the subcellular localization of RBPs using HuR as a model mRNA stabilizing RBP. Short-term Act D treatment markedly increased cytoplasmic HuR localization in HCT116 and HeLa cells where the protein is known to be active. Analysis of known pathways regulating HuR nucleocytoplasmic translocation did not fully explain this redistribution, suggesting alternative mechanisms. To identify proteins proximal to HuR following Act D treatment, we performed TurboID labeling followed by LC-MS/MS in HCT116 cells. Several proteins involved in RNA regulation were identified. Probabilistic modeling highlighted FUS, an RBP with established roles in phase-separated granule dynamics, as a candidate proximal protein. The Act D-dependent interaction between HuR and FUS was interrogated using molecular dynamics simulations and validated with proximity ligation assays. Furthermore, increased cytoplasmic localization of RBPs following Act D treatment was accompanied by formation of granular structures that were relatively fluid and could be disrupted by hypotonic shock. Collectively, our findings demonstrate that Act D induces cytoplasmic redistribution of multiple RBPs and their sequestration into dynamic granular structures, revealing a previously unrecognized cellular response to transcriptional inhibition. Graphical AbstractAct D induced cytoplasmic re-localization of HuR along with FUS and other RBPs in dynamic, hypotonic shock-sensitive granular structures. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/745449v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@515322org.highwire.dtl.DTLVardef@128f0d5org.highwire.dtl.DTLVardef@db4302org.highwire.dtl.DTLVardef@10c4a2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Rational design of covalent protein-labeling reagents in complex biological environments requires a molecular-level understanding of how the protein microenvironment governs chemical reactivity; yet, such mechanistic details remain inaccessible to experimental methods alone. In living neurons, Ligand-Directed Acyl Imidazole (LDAI) chemistry has been used to label AMPA receptors as a traceless, affinity-based protein labeling method. Although LDAI labeling reagents have been optimized in the lab, the atomic details of their interactions with the protein and the underlying mechanism remain elusive. In this work, we combined Quantum Mechanical (QM) calculations and molecular dynamics (MD) simulations to propose a detailed reaction mechanism for AMPAR labeling by LDAI reagents and to clarify how the protein microenvironment governs reactivity. Although Lys residues are usually protonated at physiological pH and therefore less nucleophilic in water, our QM results show that Lys labeling is energetically more favorable than competing reactions with Ser or water. MD simulations reveal that PFQX ---the LDAI reagent precursor--- binds dynamically to the GluA2 AMPAR as an antagonist, inducing conformational changes that reshape the local environment of the acyl imidazole (AI) warhead, underscoring that ligand identity strongly affects labeling outcomes. We also identified intra and intermolecular hydrogen bond networks that may contribute to further immobilize and pre-organize the LDAI reagent. Moreover, the probe's chemical nature shapes its interactions with the Ligand Binding Domain (LBD), offering a plausible rationale for the previously experimentally observed ligand-dependent fluorescent response. Taken together, our results establish design principles for exploiting the reagent geometry and binding pocket hydrogen-bonding networks for the rational design of LDAI reagents.

Glioblastoma (GBM) is the most common primary malignant brain tumor and is typically fatal. GBM therapies are hindered by the impermeability of the blood brain barrier (BBB), the diffuse and infiltrative nature of the tumor, and the high heterogeneity of intratumoral GBM cells. Aptamers are short, synthetic, folded single strands of RNA or DNA or analogs that bind targets with high affinity and specificity. Aptamers are developed via the principles of natural selection, permitting an unbiased approach to therapeutic development. Thus, rather than using rational design to select a target and develop a targeting moiety, cycles of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) are employed in cell culture or in vivo to identify aptamers against unknown targets. Antibody drug conjugates (ADCs) have shown some efficacy for GBM but are limited by their large size and thus depend on leakiness of the BBB. We have recently applied in vivo SELEX to develop anti-GBM aptamers (six-fold smaller in mass than IgG antibodies) and to select aptamer-drug conjugates. Here we report attempts to focus aptamer selection toward internalizing drug-delivery targets and resulting challenges involving loss of tumor specificity in vivo.

Catalytic activity of 5'-3' exonuclease Phospholipase D3 (PLD3) is associated with immune signaling and neurodegeneration including Alzheimers 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 Alzheimers diseases. These findings are key for further elucidation of PLD3 function in neurodegenerative diseases.

Transcription factors remain among the most challenging therapeutic targets in part because they lack well-defined ligandable binding pockets. We recently showed that aberrantly reactive cysteines in transcription factors can be directly targeted with electrophilic small molecules to induce selective transcription factor destabilization and degradation. Here, we extend this strategy to the lineage-defining oncogenic transcription factor PAX8, a critical driver of ovarian cancer. Screening of a chemically diverse library of more than 3,000 cysteine-reactive compounds against an endogenously HiBiT-tagged PAX8 reporter identified a sulfinyl aziridine chemotype that selectively reduced PAX8 abundance. Structure-activity and stereochemical analyses revealed highly enantio- and diastereoselective activity, identifying KL6-159A as the lead compound. Quantitative proteomics demonstrated selective loss of PAX8, while cellular thermal shift analysis and chemoproteomic profiling established direct covalent engagement of PAX8 at cysteine C57. Mutation of C57 completely abolished KL6-159A-induced PAX8 depletion, demonstrating that this residue is essential for compound activity. Transcriptomic profiling revealed broad suppression of the PAX8 transcriptional program, with FOXM1 emerging as the most significantly downregulated regulatory network together with numerous established PAX8 target genes. Collectively, these studies establish direct covalent engagement, transcriptional inhibition, and destabilization of PAX8 and further demonstrate the generality of covalent chemoproteomic approaches for drugging previously intractable transcription factors.

Small RNAs (sRNAs) regulate the expression of many genes including those involved in antibiotic resistance and bacterial virulence, making them potential therapeutic targets. A molecule that binds an sRNA could interfere with its ability to bind its target mRNA and disrupt the regulation mechanism. Randomization and screening of natural arginine rich motif (ARM) peptides led to peptides capable of interfering with the sRNA MicF's ability to regulate ompF in Escherichia coli. Molecular dynamics simulations suggested that this effect was not a result of a direct disruption of the MicF-ompF interaction. Instead, the peptides interfere with binding of the chaperone Hfq, which is required for MicF-mediated regulation. Subsequent testing demonstrated peptide specificity for MicF over two other Hfq scaffolds and the ability to disrupt regulation of two additional MicF targets. Together, these findings support the use of synthetic ARMs as a potential tool for modulating sRNA function in bacteria.

Proteins are dynamic molecules which sensitively adapt according to their environment. Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS) provides unique insights into protein conformational processes. However, existing methodology cannot selectively enrich proteins post-labeling because D-to-H back exchange must be minimized by rapid processing at pH 2.3-3.0 and 0 {degrees}C, where affinity purification fails. Here, we create LemonCatcher, a protein superglue that spontaneously forms an amide bond to the LemonTag peptide under these harsh acidic and cold quench conditions, even at -20 {degrees}C. Engineering of a bead-coupled LemonCatcher purification system introduces fast and selective quench-capture HDX-MS (SelQueX) on LemonTagged fusion proteins. We demonstrate targeted measurement of protein dynamics in living bacterial cells, revealing ligand-induced conformational changes in maltose-binding protein. Moreover, probing a stalled membrane protein nascent-chain supports a role for the ribosome in maintaining partially unfolded folding intermediates. Thus, SelQueX makes possible selective characterization of protein structural dynamics within the complex cellular milieu.