Antisense oligonucleotides (ASOs) are widely used as therapeutics. One class of ASOs enhances protein expression by sequestering the mature microRNA (miRNA) in a double-stranded structure within the RNA-induced silencing complex (RISC). An alternative approach for the targeted control of gene expression is to use ASOs that bind to precursor miRNAs (pre-miRNAs) and modulate their enzymatic processing. Here, we demonstrate that ASOs that disrupt the junction of pre-miR-31, a region we previously identified as a critical regulatory element, are potent inhibitors of Dicer/TRBP processing in vitro but do not inhibit Dicer/TRBP binding. Furthermore, we extend and validate this strategy to pre-miR-144, which has a similar junction-dependent structure-function relationship. Through analysis of pre-miRNA secondary structures, we find that nearly 20% of human pre-miRNAs are predicted to contain junctions, and we validate our ASO approach on several members of this group. Importantly, we also verify the application of junction-targeting ASOs for the specific inhibition of pre-miRNA processing in cellulo. Our study reemphasizes the important role of RNA structure in regulating Dicer/TRBP processing of pre-miRNAs and provides the framework to develop structure-informed ASOs that serve to inhibit miRNA production.
A fundamental question in cell signaling is how a single kinase, with a single primary substrate, can activate multiple distinct cellular programs. Kinases can be characterized by speed, duration, and amplitude. They should be efficient-but not necessarily maximally-as signaling functions often require slow kinetics. How the physical behavior of individual kinases, especially the top-tier kinase, coordinates the collective cascade for functional output-essential for molecular engineering and pharmacology-remains a challenge. Here we decipher the structural "why" behind the specific kinase order in cascades as the key design principle of cellular signaling, which resolves these questions. We elucidate the conformational mechanisms and catalytic actions of the sequentially ordered component kinases in seven related and distinct cascades, including MAPKs. We propose that the order of kinases in cascades is dictated by their conformational character, a mechanism amplified in cascade-enriched biomolecular condensates-establishing design principles for pathway-selective, cascade-directed therapeutics.
We report the discovery and engineering of a new (R)-selective transaminase (RTA-223, UniProt W9Z089), identified from Capronia coronata. The wild-type enzyme was found to display broad activity on a range of bulky aryl and alkyl ketone substrates, with high enantioselectivity using both d-alanine and isopropyl amine as amine donors. We then investigated activity towards pro-sitagliptin ketone, a well-known challenging pharmaceutical target of industrial significance. Although no forward amination of pro-sitagliptin was initially detected by wild-type RTA-223, low-level activity in the reverse deamination reaction enabled engineering without the need for truncated sitagliptin analogues. Adopting this reverse screening strategy, two active-site mutations unlocked forward amination activity, and subsequent rounds of directed evolution delivered a quadruple mutant (H53L/V60G/F113A/V148A) capable of converting pro-sitagliptin to (R)-sitagliptin with high levels of stereoselectivity (e.r. 93 : 7). We further performed a kinetic analysis of the candidates from across the evolution process, using a previously reported coupled assay system linked to d-alanine oxidation. This work validates reverse screening as an effective approach for evolving transaminases toward sterically hindered substrates and highlights RTA-223 as a promising candidate for further biocatalyst development.
Selective disassembly of multifunctional molecules through the use of cleavable linkers is central to many aspects of chemical biology, biochemistry, and pharmaceutical science. Ideally, cleavable linkers are structurally small and chemically stable on their own but fragment rapidly in the presence of a specific trigger without damaging the rest of the molecule. Here, we report the discovery of the facile oxidative cleavage of 2-(1-amidovinyl)azole-containing peptides (ΔAlaAz; Az: thiazole or oxazole) in the presence of aromatic thiols and air oxygen under ambient conditions. The reaction leads to quantitative peptide cleavage in minutes (<5 min for some substrates) in aqueous buffers at room temperature. We demonstrate that the ΔAlaAz cleavable linkers can be utilized to (i) linearize complex cyclic peptides, (ii) aid the structure determination of ΔAlaAz-containing natural products, and (iii) facilitate the isolation of peptides from cell lysates. Altogether, our work adds an efficient method to the toolbox of cleavable linkers in chemical biology.
Filamentous fungi are major contributors to diverse secondary metabolites with broad applications to medicine, agriculture, and biotechnology. Advances in genome sequencing and bioinformatic tools have revealed that fungal genomes encode far more biosynthetic gene clusters (BGCs) than are expressed under normal laboratory conditions, leaving much biosynthetic potential transcriptionally silent. Overcoming this gap between predicted and observed secondary metabolism has become a major challenge in fungal natural product discovery. In this review, we summarize current strategies for activating silent or weakly expressed fungal BGCs through regulatory engineering, with an emphasis on approaches validated in Aspergillus, Penicillium, Monascus, and related filamentous fungi. We focus on genetic and chemical manipulations that enable coordinated activation of multiple biosynthetic pathways through chromatin-level modifiers, global transcriptional regulators, and developmental regulators. By framing these regulators as practical tools rather than solely biological components, we demonstrate their strengths, limitations, and applications in Aspergillus and related filamentous fungi. We further discuss emerging combinatorial and integrative approaches that use regulatory engineering alongside omics technologies and predictive tools, outlining alternatives and future directions for improving the interpretability of silent pathway activation.
Polysaccharide monooxygenases (PMOs) degrade carbohydrate polymers via an oxidative mechanism involving activation of O2 or H2O2 at a mononuclear copper active site. The reaction with O2 requires the timely delivery of two electrons to the copper during each catalytic cycle. Large polymeric substrates might occlude the active site from reductant access, which raises questions of whether the substrate stays associated with the PMO during catalysis and whether there is an alternate path for electrons to reach the active site. Previous work provided evidence for electron transfer through residues Y168 (the axial tyrosine) and Y62 in an AA9 PMO, MtPMO9E. Results reported here further investigate these redox-active residues by comparing the Y62F/Y168F double variant (2F) to the wild-type (WT) enzyme. Oxidase activity and oxygenase activity were compared using three different reductants: cysteine, ascorbate, and cellobiose dehydrogenase. Unlike cysteine and ascorbate, cellobiose dehydrogenase is comparatively large and thought to be the native electron donor to fungal PMOs. The oxidase and oxygenase activities of 2F are significantly decreased compared to WT, but the peroxygenase activity is unaffected. The slow oxygenase activity of 2F suggests the PMO is preferentially reduced via the redox-active residues, although some reduction may occur directly at the active site when the substrate is present. Engineering tyrosine residues into an AA9 PMO that natively lacks the redox-active residues increases oxidase activity. The oxygenase reaction is first-order in the reductant, suggesting the rate-limiting step involves electron transfer between the reductant and the redox-active residues. These observations underscore the importance of redox-active residues in O2 utilization by PMOs.
Norovirus is a leading cause of global acute gastroenteritis, yet the mechanisms underlying its replication remain incompletely understood. As a positive-sense single-stranded RNA (+ ssRNA) virus, norovirus utilizes a nucleotidylated viral protein (VPg) as a primer for synthesis of its genomic and subgenomic mRNAs. In this study, we report the efficient solid-phase synthesis of a guanylylated (also known as GMPylated) VPg-derived peptide fragment using a novel pre-guanylylated tyrosine building block equipped with acid-sensitive protecting groups. This approach enables a streamlined, one-step deprotection and cleavage process. The resulting synthetic peptide was characterised via LC-MS/MS, establishing its gas-phase fragmentation behaviour and confirming prior observations of a diagnostic guanine nucleobase peak at 152.0572 m/z. We subsequently applied these findings to develop a parallel reaction monitoring (PRM) assay to investigate endogenous VPg guanylylation in murine microglial cells infected with murine norovirus (MNV). Our results demonstrate that while unmodified VPg is detectable from 4 hours post-infection, the guanylylated form appears predominantly during later stages (8-12 hpi). The timing of VPg guanylylation is consistent with a model where guanylylation may serve as a regulator in the switch between anti-sense and sense RNA synthesis, though at this time this hypothesis is speculative and remains to be proven. These synthetic molecular tools and mass spectrometry assays provide a robust framework for further exploring the role of nucleotidylation in viral pathogenesis and in broader cell and pathogen biology.
Small interfering RNAs (siRNAs) are programmable nucleic acids that play key roles in chemical biology and can selectively silence disease-associated genes through RNA interference (RNAi). These programmable nucleic acids have emerged as a powerful class of medicines and chemical biology tools that can rewire tumor-immune signaling, target immunosuppressive genes, stimulate immune responses, and boost the immune system against immune-mediated diseases. Recent success in the rapid synthesis and applications of siRNA highlights the potential of this technology to address previously "undruggable" targets across a range of genetic, metabolic, and oncologic diseases. Despite the potential of these siRNA-based therapies, including those used in cancer immunotherapy, challenges such as off-target effects during delivery, chemical degradation of siRNA in the body, and immunogenicity limit their efficacy. This review provides a comprehensive overview of the chemical biology and chemical modifications inherent to the design of robust siRNA therapies; the nucleic acid structure-function relationships that dictate the cellular mechanisms underlying siRNA-mediated gene silencing and efficacy; and the current clinical landscape and safety of approved siRNA therapeutics for immunotherapy. We examine the growing role of computationally guided design strategies and emerging machine-learning-based methods in optimizing siRNA chemical design, and outline how recent advances in siRNA chemical modification are expected to improve targeted gene modulation in the clinic. Additionally, we examine the role of delivery systems in enhancing siRNA potency, with an emphasis on tumor-targeted and tissue-specific approaches, as well as emerging combination therapies integrating siRNA with chemotherapy, immune checkpoint blockade, siRNA and mRNA co-delivery, and prodrug activation.
Thioneins are cysteine-rich apoproteins that regulate divalent metal homeostasis by virtue of their metal-chelation properties resulting in the ligand-bound metallothionein state. Previous studies show transient upregulation of the metallothionein (MT) gene cluster as part of a complex transcriptional response to a class of histone H3K27me3 demethylase tool compounds targeting human Fe2+ dependent ketoglutarate oxygenases KDM6A (UTX) and KDM6B (JmjD3). The prototypic bioactive KDM6 inhibitor GSK-J4 induces apoptotic cell death in multiple myeloma cells and corresponding transcriptomic profiles are dominated by metal and integrated stress response (ISR) signatures, also observed in primary human myeloma cells. Here we investigate the hypothesis that metal-chelation by GSK-J4 provides the means for transport and intracellular release of Zn2+ leading to a metallothionein transcriptomic response signature. Live cell imaging of myeloma cells shows transient increases in intracellular free Zn2+ concentrations when exposed to GSK-J4, consistent with a model of inhibitor-mediated metal transport, further supported by direct metal-inhibitor complex formation as determined by MALDI-TOF mass spectrometry and 1H NMR. Comparisons of GSK-J4 and ZnSO4 treatments in the presence or absence of metal chelators show that both treatment conditions induce different transcription factor repertoires with an overlapping MTF1 transcriptional regulation responsible for metallothionein and metal ion transport regulation. The data provide a possible explanation for the observed metal response upon GSK-J4 inhibition however the relationship with the pro-apoptotic ISR mechanism in myeloma cells requires further investigation.
Fragment-based drug discovery (FBDD) is a powerful workflow for the development of drug candidates and probe molecules that begins with the discovery of one or more low affinity, low molecular weight ligands for a protein of interest (POI). Recently, we developed a simple fragment discovery platform in which TentaGel beads displaying many copies of the fragment are incubated with a fluorescently labeled multimeric protein. Association of two or more bead-displayed ligands with the multimeric target stabilizes the complex sufficiently to allow its detection even after rigorous washing. However, this simple “pull-down” assay cannot be used for monomeric protein targets since these complexes are too kinetically labile to survive being taken out of equilibrium. Here we explore various strategies to multimerize monomeric targets, thus allowing them to be used as targets in this fragment discovery platform. We find that a particularly effective and convenient strategy is to express a fusion of the POI with FOLDON, a 30-residue, homo-trimeric peptide.
Tetrazine-trans-cyclooctene (Tz-TCO) click chemistry has been widely adopted for various applications due to its faster reaction kinetics and higher biocompatibility compared to conventional copper-catalyzed click chemistry. However, most click chemistry groups do not appear to be fully bioorthogonal, as several of them—including tetrazine—have been found to directly modify proteins. Here, we report the unexpected discovery of non-specific protein modifications by TCO during the development of a tetrazine-based proximity labeling method. TCO probes readily conjugate to cysteine residues on proteins, and pre-treatment with iodoacetamide significantly reduces TCO-mediated background labeling. Chemoproteomic analyses reveal that proteins involved in translation are heavily modified by TCO, pointing to potential off-target effects when TCO probes are used in live cells. Our findings underscore the need for further improving the biocompatibility of click chemistry to enable more precise biological applications.
The development of efficient bioconjugation methods is essential for enhancing the therapeutic potential of oligonucleotides, especially antisense oligonucleotides (ASOs). Two main strategies are used for modification during oligonucleotide solid-phase synthesis: the early incorporation of functionalized monomers, or the post-synthetic modification of precursors bearing small reactive groups-the latter offering greater versatility and yield. Extensive efforts have been dedicated to incorporating bioorthogonal groups into phosphoramidite building blocks to enable the controlled chemical synthesis of reactive oligonucleotides suitable for post-synthetic modifications. Among these, alkynes and cyclooctynes are the most widely used, enabling Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC), respectively. We recently demonstrated the compatibility of the sydnone group with automated solid-phase chemistry. This chemical motif shows significant promise in oligonucleotide chemistry, as it allows for strain-promoted sydnone-alkyne cycloaddition (SPSAC), a reaction that has recently emerged as an efficient alternative to SPAAC for cellular studies. Herein, we present the synthesis of three sydnone-functionalized phosphoramidite monomers, their incorporation into ASOs, and an evaluation of their chemical and biological properties. These developments aim to expand the bioconjugation toolbox for ASO tracking, targeting, and imaging, thereby improving their therapeutic application and the understanding of intracellular mechanisms.
The dysregulation of protein-protein interactions (PPIs) in disease states is well established, yet they are challenging to target, owing to the large surface area and featureless nature of protein binding interfaces. For targeting helix-mediated interactions, α-helix mimetics present a promising strategy. These are versatile small molecule scaffolds, capable of mimicking the hotspot residues on an α-helix. A wide range of such scaffolds have been reported, yet their target protein selectivity in the context of a whole proteome requires further exploration. Here, we report the affinity-based protein profiling of three structurally distinct classes of α-helix mimetics, N-substituted oligobenzamides, pyrrolopyrimidines, and oxopiperazines. This represents the first direct cross-comparison of different helix mimetic scaffolds, revealing significant differences in proteome-wide selectivity.
Orthoesters are unique functional groups consisting of three alkoxy substituents attached to a central carbon atom, which endow orthoester-bearing compounds with structural complexity and therapeutically valuable bioactivity. As orthoester natural products are rarely encountered in nature, their biosynthesis is poorly understood, particularly regarding the generation of their orthoester functionalities. Over the past decade, the isolation of putative biosynthetic precursors of orthoester natural products in plants and animals as well as the discovery of orthoester-forming enzymes in four species of microorganisms including bacteria and fungi have shed light on the cryptic biosynthetic machinery behind orthoester biogenesis. Protein structural analyses and biochemical enzymatic investigations have since revealed a variety of unique catalytic strategies for activating substrate scaffolds and stabilizing reactive intermediates to enable the formation of highly constrained orthoester moieties. Accordingly, this review summarizes recent advances made in the biosynthesis of orthoester natural products, highlighting the structural bases of unusual enzymatic mechanisms involved in orthoester formation. Additionally, established and putative biosynthetic pathways of prominent orthoester compounds are examined to inform future studies for discovering new orthoester natural products as well as for uncovering novel biocatalysts for the generation of orthoester compounds.
DCAF11 is a substrate receptor of the Cullin-RING ligase 4 (CRL4) ubiquitin ligase complex and an emerging effector supporting targeted protein degradation. DCAF11 exists as two major isoforms, but their functional differences remain incompletely understood. Here, we show that DCAF11 isoforms 1 and 2 assemble into CRL4 complexes with similar efficiency and exhibit largely overlapping endogenous substrate profiles, including proteins implicated in electrophile detoxification. In contrast, they differ in their compatibility with small-molecule degraders: covalent PROTACs engage both isoforms, whereas a non-covalent molecular glue selectively utilizes isoform 1. These findings reveal isoform-dependent control of protein degradation and highlight opportunities for isoform-selective targeting.
The therapeutic potential of peptides is severely limited by rapid metabolism mediated by proteases. Traditional stabilization strategies often compromise pharmacological profiles by disrupting native conformation or physicochemical properties. Here we show that backbone amidine substitution offers a minimal, site-specific modification that enhances metabolic stability. Using the pentapeptide Leu-enkephalin as a model, amidine replacement of metabolically labile amides attenuated or blocked proteolysis in a position-dependent manner, with one analog preserving and even enhancing G-protein signaling at the μ-opioid receptor while reducing β-arrestin2 recruitment. Amidines thus provide a modular strategy to rescue promising peptide leads limited by metabolic instability, with the potential of conferring protease resistance without the need for wholesale scaffold redesign.
Photocatalytic proximity labeling has transformed the mapping of local molecular environments in living cells, but many existing platforms depend on flavin, heme, or metal cofactors, limiting their broader deployment. Here we repurpose HyperNova, a monomeric photosensitizer with an autocatalytically formed chromophore, as a genetically encoded photocatalyst for photocatalytic proximity labeling. Using biotin-aniline as the substrate, we show that HyperNova enables 560 nm light-activated proximity labeling across multiple subcellular compartments in living cells. We further engineer a blue-light-activated variant, HyperNova Green, and demonstrate that HyperNova and HyperNova Green can be selectively activated by 560 nm and 460 nm light, respectively, enabling dual-wavelength orthogonal labeling within the same sample and in distinct compartments of the same cell. In addition, fusion of HyperNova to Antares2 enables bioluminescence resonance energy transfer (BRET)-mediated activation, offering an external-illumination-free mode of proximity labeling. Together, these results establish NovaID as a compact and versatile proximity labeling platform with broad potential for spatiotemporally-resolved mapping of local subcellular environments.
Circadian rhythms are innate biological processes that cycle over periods of approximately 24 hours and are crucial in regulating many physiological and metabolic functions. At the molecular level in mammals, these cell-autonomous oscillations are controlled by a network of self-regulating feedback loops comprised of transcriptional and translational regulators. Reporter platforms for following promoter activity and/or proteins via detectable fluorescent and bioluminescent entities have facilitated the tracking of circadian elements in cells and organisms in real-time, which is especially important for studying such a dynamic biological system. As a result, reporters have been pivotal in uncovering fundamental aspects of the molecular core clock, changes in individual components in healthy versus various disease models, and screening for synthetic clock modulators. While the initially established tools of in vivo reporters (i.e., Per2::Luc mice) continue to be widely utilized, newer ones have capabilities for evaluating other components and performing simultaneous assessments of multiple circadian components across different levels of expression. Concurrently, in vitro reporter models have been developed to track alterations in rhythms and protein presence in various cellular models, including those of disease. In this review, we discuss various reporter systems, including historical context, and their applications for monitoring rhythms at transcriptional and translational levels of expression, in vitro and in/ex vivo, with a focus on mammalian systems. Furthermore, we assess the advantages, disadvantages, and limitations of employing bioluminescent and fluorescent reporters in monitoring biological rhythms and components. Our goals are to provide an account of available reporter systems and pose considerations for their use and the development of technologies that can be used to expand our tracking and understanding of the molecular clock.
