The two technologies at the heart of the study capture fundamentally different views of drug activity. The first, thermal proteome profiling, or TPP, grew out of the cellular thermal shift assay introduced in 2013. The principle is elegant: when a small molecule binds a protein, it often shifts the protein’s melting temperature, either stabilizing or destabilizing it. Researchers heat intact cells to a range of temperatures before lysis, then use mass spectrometry to measure how much of each protein remains soluble at each temperature. Coupling this to whole-proteome analysis, first demonstrated by Savitski and colleagues in 2014, allows an unbiased survey of thousands of drug-protein interactions in a single experiment. A typical whole-cell TPP experiment yields tens to hundreds of proteins with perturbed thermal stability, but these include not only direct drug targets but also proteins in complexes with, or downstream of, the true target, making it difficult to separate cause from consequence.

The second technology, Cell Painting, works from the opposite direction. Instead of measuring protein behavior directly, it captures the morphological fingerprint a drug imposes on a cell. Cells are treated with compounds and then stained with a fixed panel of fluorescent dyes labeling the nucleus, endoplasmic reticulum, nucleoli and cytoplasmic RNA, Golgi apparatus and actin cytoskeleton, and mitochondria. Software such as CellProfiler then extracts thousands of features per cell, producing a rich quantitative description of each treatment’s morphological perturbation. Because the assay is inexpensive and highly multiplexable, it can be applied to thousands of compounds at once. Compounds with similar mechanisms of action tend to induce similar morphological changes, so machine learning can infer the mechanism of an unknown compound by comparing it to well-annotated neighbors. The weakness is that small molecules often have many targets, and the accuracy of predictions depends heavily on the quality of available annotations.

The Uppsala team’s insight was that these two data types are complementary in a very specific way. TPP detects target engagement directly but misses proteins, sometimes because a peptide fails to ionize in the mass spectrometer, sometimes because a protein is more thermally stable than the tested temperature range, and sometimes because membrane-associated proteins sediment during centrifugation. Cell Painting, meanwhile, can suggest targets through compounds that produce similar shapes but cannot confirm physical binding. The researchers therefore built a pipeline that starts with the list of thermally stabilized or destabilized proteins from a TPP experiment and constructs a protein-protein interaction network using the STRING database, which aggregates experimentally scored interactions, curated pathway databases, and text mining. In parallel, the compound of interest is located within a Cell Painting dataset of 5259 compounds from the SPECS drug repurposing repository, profiled in U2OS bone cancer cells, and the known targets of its closest morphological neighbors are used to fill gaps in the network.

A key technical challenge was clustering the Cell Painting data reliably. Popular algorithms such as k-means and HDBSCAN are stochastic and can produce very different cluster structures on repeated runs, particularly on high-dimensional, poorly separated data like morphological profiles. The team turned to SC3s, a consensus clustering method originally developed for single-cell RNA sequencing, which combines principal component analysis with thousands of repeated k-means runs and summarizes the results into a consensus matrix. Running the algorithm 2000 times across five different cluster numbers, between 100 and 180, produced highly reproducible clusters for most test compounds. The cluster-derived target candidates were then merged with the TPP network, and betweenness centrality scores, a graph-theoretic measure of how often a node lies on shortest paths between others, were used to filter the merged network. Because drug targets have been shown to occupy central positions in interaction networks, the researchers reasoned that true targets would rank among the highest-scoring nodes.

To validate the approach, the team used publicly available TPP datasets for five well-characterized drugs: the BET bromodomain inhibitors (+)-JQ1 and I-BET151, the BRAF inhibitor vemurafenib, the ALK and MET inhibitor crizotinib, and the histone deacetylase inhibitor panobinostat. For (+)-JQ1, 67 proteins showed dose-dependent thermal shifts, and the resulting network correctly placed the known targets BRD4, BRD3, and BRD2, along with the documented off-target HADHA, among the top-ranked nodes. For I-BET151, 40 perturbed proteins yielded a network containing BRD4, BRD3, and BRD2. Intriguingly, although the two compounds share targets, only 11 perturbed proteins overlapped between them, suggesting they regulate different pathways downstream of target binding. Gene Ontology and Reactome enrichment analysis of the network communities recovered mechanisms consistent with the known biology of (+)-JQ, including lysine-acetylated histone binding and activation of cell death.

The validation extended further. For panobinostat, TPP alone detected only three of eleven known targets, but Cell Painting cluster analysis contributed eight more, and the combined network placed six targets among the top-ranked proteins. For vemurafenib, the primary target BRAF was entirely absent from the TPP data, while the off-target FECH was missing from the Cell Painting cluster, so only the integrated network captured both. The method correctly identified known targets and mechanisms for four of the five compounds. The exception, crizotinib, proved instructive rather than disappointing: its true targets ALK, ROS1, and MET are transmembrane proteins that classical TPP protocols rarely detect, and neither U2OS nor K-562 cells expressed ALK or ROS1. Instead, the analysis highlighted the Bcr-Abl pathway through the off-targets ABL1 and BCR, a biologically plausible finding given the high expression of these proteins in the K-562 leukemia cells used for TPP. The team then scaled the validation to a public Proteome Integral Solubility Alteration, or PISA, dataset covering 49 compounds, a high-throughput TPP variant in which all temperature treatments are pooled before mass spectrometry. The combined model achieved target prediction with a ROC AUC of at least 0.7 for 21 compounds, compared with 15 for PISA alone and 14 for Cell Painting alone, and outperformed the individual methods in the majority of cases.

As a proof of principle, the researchers applied the pipeline to sinomenine, a plant-derived alkaloid used in China to treat rheumatoid arthritis and pain, whose mechanism of action has remained largely obscure. They generated their own PISA data in U2OS cells treated with 10 and 30 micromolar sinomenine, finding 175 and 121 significant thermal shifts respectively, with 94 proteins affected at both doses. The integrated network revealed a strikingly multimodal profile. Among the highest centrality scores were the beta-2 adrenergic receptor ADRB2 and the NMDA receptor subunit GRIN2C, both endogenously expressed in U2OS cells, supporting an action on nervous system receptors consistent with the compound’s analgesic use and with previous mouse studies showing elevated GABA signaling. Enrichment analysis also flagged neurotransmitter receptor activity and chemical synaptic transmission. Equally intriguing was the appearance of cyclin-dependent kinase 2, CDK2, alongside pathways governing RNA binding, DNA replication, chromosome organization, and double-strand break repair, pointing to potential anti-tumor effects that corroborate earlier sinomenine studies. Additional communities implicated actin cytoskeleton organization, membrane trafficking, RHO GTPase signaling, and metabolic processes including the citric acid cycle, with the structural protein ACTB and the mitochondrial transporter SFXN1 scoring highly.

The authors are careful to note the method’s limits. It cannot detect a target unless that target shows thermal shifts or is annotated for morphologically similar compounds, and the workflow deliberately trusts TPP-derived nodes over Cell Painting-derived ones, which meant that in 7 of 15 cases a target seen only in imaging data failed to enter the final network. Cell line choice matters enormously, as the crizotinib case demonstrated, and the team recommends using the same cell line for both assays when investigating a compound with unknown targets. Clustering reproducibility also faltered for vemurafenib, the compound with the weakest morphological signal. Even so, the payoff is substantial: a typical mass spectrometry run covers only about half the proteome, roughly 10,000 of 20,000 canonical proteins, and TPP alone can leave researchers with 35 to 64 candidate targets to chase. The integrated workflow narrows that list to ten or fewer, promising to slash the time and cost of follow-up validation experiments. With the full pipeline written in Python and released on GitHub, the Uppsala team has handed chemical biologists a practical tool for turning two imperfect lenses on drug action into one considerably sharper picture, one that could accelerate both drug development and drug repurposing for compounds whose secrets have resisted decryption for decades.

Subject of Research: Integration of Cell Painting morphological profiling and thermal proteome profiling for drug target identification and mechanism of action inference

Article Title: Cell painting and thermal proteome profiling for inference of drug targets and mechanism of action

Article References: Johansson, C., Johansson, M., Kasi, P. B., Larsson, M., Jakobsson, P.-J., Göransson, U., Carreras Puigvert, J., Spjuth, O., & Jansson, E. T. (2026). Cell painting and thermal proteome profiling for inference of drug targets and mechanism of action. Molecular Systems Biology, 22(8), 1270-1291. https://doi.org/10.1038/s44320-026-00214-9

Image Credits: AI Generated

DOI: 10.1038/s44320-026-00214-9

Keywords: Cell Painting, thermal proteome profiling, drug discovery, mechanism of action, target deconvolution, protein-protein interaction networks, proteomics, sinomenine, PISA, mass spectrometry, chemical biology, Uppsala University

The nucleolus is organized into three subcompartments: the fibrillar centers, the dense fibrillar component and the granular component, each hosting a distinct stage of ribosomal RNA production and processing. Recent work has shown that these subcompartments are maintained as coexisting liquid phases, and that a pH gradient across them helps set up the biochemical conditions each phase requires. Yet while pH is recognized as a key variable controlling condensate dynamics and function, how cells actually control pH inside these compartments under physiological and pathological conditions has remained poorly understood. The Xiamen team set out to close that gap, focusing on a structure that appears when nucleolar transcription is disrupted.

When rRNA synthesis is interrogated by stress, whether from DNA damage or from drugs that block transcription, the nucleolus undergoes a characteristic reorganization. Some of its components segregate into cap-like structures at the nucleolar periphery, structures that were first described more than sixty years ago in cells treated with the chemotherapeutic agent actinomycin D. Among these are the so-called dark nucleolar caps, or DNCs, marked by proteins such as SFPQ and DDX18. The researchers discovered that under specific stressed conditions, SRSF1, a canonical splicing factor that normally resides in nuclear speckles, is recruited into these DNCs. Using super-resolution imaging and knock-in fluorescent tags in human cells, they tracked this redistribution in detail, finding that it occurs not only in one cell line but across multiple human cell types, including 293T, HCT 116 and Huh7 cells, after ultraviolet treatment followed by a recovery period.

Recruitment, however, is not random. Through immunoprecipitation combined with mass spectrometry, the team identified a molecular interaction between SRSF1 and DDX18, a DEAD-box RNA helicase that localizes to the granular component and the DNCs. When DDX18 was depleted with short hairpin RNA, SRSF1’s ability to accumulate in the dark nucleolar caps was impaired, indicating that this interaction is partially required for SRSF1’s stress-induced localization. The finding adds a new dimension to the biology of SR proteins, a family of splicing factors already known to redistribute to nuclear stress bodies and segregated nucleolar components in response to DNA damage, and it suggests that the nucleolar caps act as staging grounds where splicing machinery performs an unexpected, non-splicing job.

That job, the study shows, is pH management. Using live-cell imaging with the ratiometric pH-sensitive dye BCECF-AM and a genetically encoded pHluorin biosensor, the researchers measured the pH inside individual nucleolar subcompartments with remarkable precision. They found that the pH microenvironments of the granular component, the dense fibrillar component and the fibrillar centers remain largely unchanged upon ultraviolet exposure, but the dark nucleolar caps behave differently. When SRSF1 was knocked down, the pH homeostasis of the DNCs was disrupted, and this disturbance cascaded into a failure of the whole organelle to recover. Cells lacking SRSF1 could not properly restore the nucleolar multiphase architecture after stress, and functional readouts of nucleolar health deteriorated accordingly.

The functional consequences were striking. In control cells subjected to ultraviolet treatment or actinomycin D and then allowed to recover, the granular component protein NPM1 repartitioned back into the nucleolus, nascent RNA synthesis resumed as measured by 5-ethynyl uridine incorporation, and precursors of ribosomal RNA detected by probes against the 5-prime external transcribed spacer returned to normal levels. In SRSF1-depleted cells, all of these recovery markers were blunted. The team also observed elevated levels of gamma-H2AX, a marker of DNA damage, in the stressed cells lacking SRSF1, consistent with the idea that impaired nucleolar recovery leaves the genome more vulnerable. Rescue experiments in which shRNA-resistant SRSF1 was reintroduced restored DNC pH and nucleolar function, confirming the specificity of the effect.

The mechanistic heart of the paper lies in SRSF1’s arginine/serine-rich, or RS, domain. Because of its high positive charge, this domain is accountable for alkalizing the DNC microenvironment. When the researchers mutated the charged residues of the RS domain to uncharged alanines, the mutant SRSF1 failed to modulate the pH of the dark nucleolar caps even though it could still localize there. Importantly, the pH-modulating activity of SRSF1 was shown to operate independently of its canonical role in splicing regulation, separating this newly discovered function from the protein’s day job in RNA processing. The work resonates with a growing body of evidence that biomolecular condensates can sustain electrochemical gradients and modulate reactions at their interfaces, and with theoretical work suggesting that charge neutralization allows condensates to maintain pH gradients at equilibrium.

Perhaps the most translationally exciting result is the demonstration that the pH-restoring function can be mimicked by a synthetic molecule. The team designed arginine-rich dipeptide derivatives based on the SRSF1 RS domain, varying their length and net charge, and expressed them in stressed cells. These synthetic dipeptides localized to the nucleolus, enriched in the DNC region as measured against the DDX18 fluorescence mask, and safeguarded the nucleolus from pH and functional disturbance. In cells treated with actinomycin D and then allowed to recover, the arginine-arginine dipeptides preserved nucleolar size, maintained 5-prime ETS and rRNA levels, and protected the multiphase organization of the organelle. In other words, a minimal positively charged peptide was sufficient to stand in for the splicing factor’s pH-buffering role, offering a proof of principle that nucleolar pH can be pharmacologically tuned.

The implications extend well beyond basic cell biology. The nucleolus is increasingly viewed as a therapeutic target in human disease, and its disorganization is a hallmark of stress responses in cancer chemotherapy, since many chemotherapeutic drugs inhibit ribosome biogenesis at various levels. Nucleolar dysfunction has also been implicated in neurodegenerative conditions: dipeptide repeat proteins produced by the C9orf72 repeat expansion, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, are known to disturb biomolecular phase separation and disrupt nucleolar function. A pathway that actively restores nucleolar pH homeostasis after stress, and that can be enhanced with simple arginine-rich peptides, suggests new angles for intervening in diseases where condensate chemistry goes wrong. It also reframes SRSF1, long studied as a splicing factor and oncoprotein, as a multifunctional regulator whose charged domains carry out electrochemical work inside membrane-less organelles.

Technically, the study stands out for the rigor of its measurements. The team combined structured illumination microscopy with knock-in fluorescently tagged endogenous proteins, ratiometric live-cell pH imaging validated against standard buffers, fluorescence recovery after photobleaching to assess condensate dynamics, quantitative RNA FISH, immunostaining and transcriptome-wide analyses. Mass spectrometry datasets were deposited to the ProteomeXchange Consortium and RNA-seq data to the Gene Expression Omnibus, with source data provided for every figure. By connecting a single charged protein domain to the electrochemical state of a nucleolar subcompartment, and that state to the recovery of an entire organelle, the work delivers a mechanistic model in which stress-recruited SRSF1 alkalizes the dark nucleolar caps, stabilizes the pH gradient of the multiphase nucleolus and enables the cell’s ribosome factory to resume production. It is a vivid demonstration that the chemistry of condensates, not merely their composition, governs how cellular compartments endure and recover from adversity.

Subject of Research: pH regulation of nucleolar condensates by the splicing factor SRSF1 during stress recovery

Article Title: SRSF1 modulates the dark nucleolar cap pH to restore nucleolar integrity and function

Article References: Ma, S., Guo, J., Zhan, X., Wu, F., Yang, S., Huang, C., & Wang, B. (2026). SRSF1 modulates the dark nucleolar cap pH to restore nucleolar integrity and function. Nature Chemical Biology. https://doi.org/10.1038/s41589-026-02316-9

Image Credits: AI Generated

DOI: 10.1038/s41589-026-02316-9

Keywords: nucleolus, SRSF1, pH gradient, biomolecular condensates, dark nucleolar caps, DDX18, RS domain, ribosome biogenesis, cellular stress, phase separation, arginine-rich dipeptides, chemical biology

The core challenge that PRADA addresses is a long-standing bottleneck in proximity labeling. The most widely used tools, such as APEX2 and TurboID, genetically fuse an engineered enzyme to a protein of interest. The enzyme then converts a supplied small molecule into a reactive species that tags nearby biomolecules, which can later be fished out and identified by mass spectrometry or sequencing. The catch is the trigger. APEX2 requires a bolus of exogenous hydrogen peroxide, a reactive oxidant that damages cells and diffuses poorly through intact tissues. TurboID requires biotin, which works in vivo but generates its own background labeling. Neither approach has proven ideal for the delicate, three-dimensional environment of a living organism.

The Shanghai- and Singapore-based team, led by Shuo Han of the Chinese Academy of Sciences together with Yue Wan of the Genome Institute of Singapore, took a different route: instead of supplying hydrogen peroxide directly, they engineered the system to make it on the spot. PRADA pairs a genetically fused peroxidase with an engineered D-amino acid oxidase, or DAAO, derived from the yeast Rhodotorula gracilis. When researchers deliver a nonproteinogenic D-amino acid such as D-phenylalanine or D-alanine — molecules that animal cells largely ignore — the oxidase converts them into hydrogen peroxide right next to the peroxidase. That locally generated oxidant then activates the peroxidase, which converts phenol probes such as biotin-phenol into short-lived radicals that covalently tag whatever proteins or nucleic acids sit within roughly a few tens of nanometers.

The engineering itself was nontrivial. The team used structural modeling and rational mutagenesis to delete a flexible C-terminal loop of the R. gracilis oxidase, producing a monomeric variant that fuses cleanly with APEX2 without disrupting either enzyme’s activity. Extended data show the researchers systematically tested fusion orientations, split-enzyme designs and D-amino acid concentrations, using fluorescence assays to confirm that the cascade produces hydrogen peroxide efficiently and that the labeling signal colocalizes precisely with the tagged protein’s known subcellular address. When the two enzymes were expressed separately rather than fused, labeling dropped sharply, confirming that the reaction is genuinely confined to the immediate vicinity of the fusion protein.

That spatial confinement is what makes the method safe enough for living animals. Because the hydrogen peroxide is generated in situ at nanometer scales and consumed almost immediately by the peroxidase, it never accumulates to toxic levels. The researchers measured malondialdehyde levels, a marker of lipid peroxidation, and protein carbonylation after PRADA labeling and found both below detection limits or unchanged compared with controls. Mitochondrial superoxide, a sensitive indicator of oxidative stress, was actually lower in PRADA-labeled cells than in cells labeled conventionally with APEX2 and exogenous peroxide. Cell viability assays showed no measurable harm from the D-amino acid treatment itself. In effect, PRADA converts the most dangerous step of proximity labeling into a self-limiting, locally contained reaction.

The versatility of the platform is striking. In cultured cells, the team targeted PRADA to the nucleus, nucleolus, endoplasmic reticulum, plasma membrane and mitochondria, and in each case recovered proteomic profiles consistent with the known composition of those compartments. They also showed that PRADA can drive functional polymer assembly — the genetically targeted chemical assembly of conducting or insulating materials inside living cells — extending the technique beyond mapping into materials synthesis. And, crucially, they demonstrated that the peroxidase step can label RNA as well as protein, opening a door that most proximity-labeling systems leave closed.

That RNA reactivity became the foundation of the study’s most inventive application. When peroxidase radicals oxidize RNA, they leave behind chemical lesions that cause reverse transcriptase to misread bases during cDNA synthesis. The team realized these misincorporations could be read as a molecular fingerprint of RNA structure: nucleotides that are chemically accessible — that is, single-stranded and exposed — accumulate more mutations than nucleotides buried inside base-paired helices. Building on the logic of mutational profiling sequencing, they developed PRADA-MaPseq, a strategy that converts peroxidase labeling directly into a spatiotemporally resolved map of RNA secondary structure inside living cells.

They benchmarked PRADA-MaPseq against the well-established mitochondrial DMS-MaPseq datasets, showing that uracil and guanine reactivity scores accurately reproduce known structures of mitochondrial messenger RNAs, with area-under-the-curve values reaching 0.60 to 0.82 across transcripts and inter-experimental correlation coefficients as high as 0.96 for individual transcripts. The approach worked with both 4-thiouridine and 6-selenoguanosine metabolic labeling, and the team optimized reverse transcriptase choice to maximize mutational signal. In short, they turned a proximity-labeling enzyme into a structure-probing reagent that reports on RNA folding in a defined cellular compartment at a defined moment in time.

The payoff came in a mouse xenograft model. The researchers implanted HEK293T cells expressing mitochondria-targeted PRADA into immunodeficient mice, then administered D-amino acids and phenol probes systemically. Labeling proceeded efficiently inside the tumors in living animals. Proteomic analysis recovered the expected mitochondrial proteome, transcriptomic enrichment captured mitochondrial RNAs with high specificity, and PRADA-MaPseq yielded the first in vivo structurome of mitochondrial RNA within a living mammalian tumor. Comparing these in vivo structure maps with in vitro measurements of the same transcripts revealed systematic differences — evidence that RNA folding inside a living organism is actively shaped by its environment rather than being a fixed property of the sequence.

Those differences carried biological meaning. The team focused on LRPPRC, a mitochondrial RNA-binding protein known to organize the folding of the mitochondrial transcriptome. When they knocked down LRPPRC in the xenografts, changes in RNA structure propagated to changes in mitochondrial gene expression, demonstrating that RNA architecture functions as a regulatory layer controlling how mitochondrial genes are read. This finding elevates the structurome from a descriptive atlas to a mechanistic insight: the three-dimensional shapes of RNAs inside mitochondria help determine how much protein the organelle makes, and those shapes are tunable in vivo.

Beyond mitochondria, the breadth of validated applications suggests PRADA could become a workhorse across biology. The team demonstrated labeling in the cytoplasm and nuclei of body wall muscle cells in Caenorhabditis elegans, recovering nuclear-enriched transcripts and detecting retained introns as expected; in rat neurons and Drosophila; and in zebrafish. Because the trigger molecule — a simple D-amino acid — is cell-permeable, inexpensive and largely inert, the method should scale to tissues and organisms where peroxide bolus delivery is impractical, such as dense neural tissue, developing embryos or intact tumors. The authors have filed patent applications, and the analysis code for RNA structuromics is freely available on GitHub under an MIT license, with sequencing and proteomic data deposited in public repositories.

Limitations remain, as with any new technology. The labeling window is measured in tens of minutes rather than seconds, so fast molecular events may still escape capture. The peroxidase chemistry, while gentler than exogenous peroxide, still relies on radical intermediates whose diffusion radius sets the spatial resolution. And the D-amino acid oxidase must be carefully engineered for each fusion context to avoid perturbing the protein under study. Nevertheless, PRADA represents a genuine conceptual advance: it decouples proximity labeling from externally supplied oxidants, unifies protein, RNA and polymer chemistries under one enzymatic umbrella, and — for the first time — brings RNA structure mapping into living animals. If the platform generalizes as its developers hope, the spatial organization of biomolecules in health and disease may soon be readable not just in culture dishes, but in the bodies of living organisms, one D-amino acid at a time.

Subject of Research: An engineered D-amino-acid-activated peroxidase cascade for in vivo multiomic proximity labeling of proteins and RNA structures

Article Title: Multiomic proximity labeling in vivo by D-amino-acid-activated peroxidase reaction

Article References: Liu, J., Han, J., Wang, Y., Song, M., Zhu, J., Liang, Y., Chen, F., Liu, Z., Yan, X., Wang, Z., Peng, W., Tu, R., Zhang, Z., Zhong, B., Sun, H., Yin, J., Chang, J., Long, Y., Men, Y., … Han, S. (2026). Multiomic proximity labeling in vivo by D-amino-acid-activated peroxidase reaction. Nature Chemical Biology. https://doi.org/10.1038/s41589-026-02336-5

Image Credits: AI Generated

DOI: 10.1038/s41589-026-02336-5

Keywords: proximity labeling, PRADA, D-amino acid oxidase, APEX2, RNA structure, structurome, mitochondria, spatial proteomics, chemical biology, in vivo imaging, xenograft, LRPPRC

The center’s mission is rooted in a deceptively straightforward observation about how modern science is organized. Infectious disease biologists at KU have long been skilled at identifying the gene products and factors that contribute to disease, mapping the molecular machinery that pathogens deploy to infect, evade and persist. Chemists and medicinal chemists, meanwhile, excel at developing chemical tools, probes and approaches that can interrogate and manipulate biological systems. Too often, Hefty noted, that expertise is not shared between the two camps. The CBID center was created precisely to bridge that divide, connecting infectious disease biologists with colleagues in chemistry, medicinal chemistry, pharmaceutical chemistry and bioengineering so that the identification of a disease-relevant target can be followed rapidly by the design of molecular tools to study and defeat it. As Hefty put it, both sides are good at what they do individually, but together is where they really excel.

What makes the Kansas effort notable is its scale and its reach across institutional boundaries. CBID recruits, mentors and supports investigators not only at the University of Kansas in Lawrence but also at KU Medical Center, Kansas State University, Wichita State University and other regional partners. The center gives participating researchers access to four core research labs organized around infectious disease high-throughput screening, computational chemical biology and molecular modeling, synthetic chemical biology and flow cytometry. These cores function as shared scientific infrastructure, allowing a researcher with a promising hypothesis but limited equipment to test it using capabilities that would be prohibitively expensive to build in an individual laboratory. Investigators can apply for pilot project funds and vouchers for low-barrier use of the core facilities, lowering the entry cost for exploratory science that might otherwise never get off the ground.

The center also invests heavily in something less tangible but arguably more important: bringing people together. Monthly programs and annual symposia create regular occasions for researchers from different departments and institutions to share results and swap ideas. You just have to get scientists in a room, sharing science, talking science, and great things come out of that, Hefty said, adding that collaborations and events often happen organically. Anyone who has watched interdisciplinary research struggle against the gravitational pull of departmental silos will recognize what the center is attempting. The most productive ideas in chemical biology frequently emerge at the boundaries between disciplines, where a biologist’s unmet need meets a chemist’s unexpected capability, and those encounters require deliberate cultivation.

The track record from the first two phases of funding offers evidence that the model works. Over its first decade, the center has recruited seven faculty members, contributed to 15 startups, and helped generate roughly $25 million in NIH funding along with $90 million from broader research projects, pilot projects and cores. Researchers affiliated with the center have authored more than 600 publications, and the scientific network now includes 80 affiliates. Those numbers matter beyond vanity metrics. Faculty recruitment supported by COBRE funding builds permanent intellectual capacity at a university, and the startup activity suggests that basic research at the center is feeding into the kind of translational pipeline that can eventually carry laboratory discoveries toward real-world applications against infectious disease.

Beyond the publications and the grant dollars, Hefty emphasized the center’s role in training and workforce development, a contribution he argued cannot be understated. Undergraduates, graduate students and postdocs have spent a decade learning techniques, tools and approaches in the center’s labs, and will continue to do so for at least another five years under the new award. Those trainees will disperse into professions across Kansas and beyond, carrying chemical biology skills into academia, industry and the biotechnology sector. In a state working to build a high-tech regional workforce, the center functions as an engine of human capital as much as a generator of papers. Hefty also credited the KU Office of Research, the College of Liberal Arts & Sciences and the Kansas Board of Regents for their support, and pointed to the School of Pharmacy and researchers at KU Medical Center in microbiology and biochemistry as key partners in the enterprise.

Scientifically, the Phase 3 era will be guided by the theme of fighting antibiotic resistance, and the strategy Hefty describes represents a meaningful departure from the traditional pharmaceutical playbook. Rather than pursuing broad-spectrum antibiotics, the center’s researchers will develop targeted treatments designed to protect the body’s beneficial microbiome while defeating resistance mechanisms. The logic is compelling. Conventional broad-spectrum antibiotics cannot distinguish between a pathogenic invader and the trillions of beneficial microbes that inhabit the human body, and the collateral damage they inflict on the microbiome has been linked to a range of secondary health problems, including opportunistic infections such as Clostridioides difficile. Hefty framed the ambition in concrete terms: if a patient has an infection with staph, strep or enterococcus, can researchers develop chemical tools, probes and approaches that target just those organisms without disrupting the rest of the flora in our bodies? And can new tools be developed to address the antibiotic resistance mechanisms that currently exist?

That question sits at the frontier of chemical biology, a field that uses small molecules as precision instruments for understanding and manipulating biological systems. Pathogen-selective compounds require an intimate knowledge of the biochemical differences between a pathogen and its human host, and between the pathogen and the harmless commensal bacteria that share the same ecological niche. Computational chemical biology and molecular modeling, one of the center’s four core capabilities, allow researchers to identify selective targets and predict how candidate molecules will interact with them. High-throughput screening can then test hundreds of thousands of compounds against those targets, while synthetic chemical biology provides the capacity to design and build new molecules from scratch. Flow cytometry, meanwhile, enables researchers to sort and analyze individual cells at high speed, a capability that is essential for studying heterogeneous bacterial populations and the emergence of resistant subpopulations within an infection.

The collaborative spirit underpinning the award will be on public display on October 16 and 17, when researchers and stakeholders gather for the fifth annual Chemical Biology Symposium at KU. The event is expected to draw more than 100 attendees, with regional participation from Kansas State and Wichita State. According to organizers, the symposium seeks to provide a forum for Graduate Training in Chemical Biology trainees and Chemical Biology of Infectious Disease researchers to present their work and to cover special topics with invited speakers. For Hefty, this cross-pollination of ideas among institutions, departments and disciplines is the whole point of the enterprise. We’re breaking down a lot of those disciplinary walls and barriers so that interdisciplinary science can occur and happen in a very productive way, he said.

Looking a decade ahead, Hefty described what success would look like: an established, formal research chemical biology center with the scientists recruited and supported over the years still participating in its activities. Such a center could continue to focus on infectious disease or expand into other areas, whether cancer, Alzheimer’s or neurological disorders, but the essential outcome would be a durable research structure that continues to support investigators at KU, KU Medical Center and other institutions. In an era when federal research funding is increasingly competitive and antibiotic resistance continues to outpace the development of new drugs, the Kansas experiment offers a test of whether deliberate, sustained investment in collaborative infrastructure can produce science that no single laboratory, department or discipline could achieve alone. With $5.8 million in new support and a decade of accumulated momentum, CBID now has the resources and the runway to find out.

Subject of Research: NIH Phase 3 COBRE funding for a chemical biology center addressing infectious disease and antibiotic resistance

Article Title: KU receives $5.8 million NIH Phase 3 COBRE funding for Chemical Biology of Infectious Disease center

Article References: KU receives $5.8 million NIH Phase 3 COBRE funding for Chemical Biology of Infectious Disease center. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: University of Kansas, NIH, COBRE, chemical biology, infectious disease, antibiotic resistance, microbiome, drug discovery, high-throughput screening, molecular modeling, workforce development, interdisciplinary research

The original call for papers read like a catalogue of the field’s hottest frontiers: targeted protein degradation, molecular glues, covalent inhibitors, antibody-drug conjugates, macrocycles, nucleoside therapeutics, immuno-oncology agents, natural-product discovery, multicomponent reactions, advanced imaging and screening technologies, and artificial intelligence. That breadth is significant in itself. A decade ago, many of these approaches were considered speculative or confined to a handful of specialist laboratories. Today they constitute a mainstream toolkit, and the 58 accepted articles show researchers across medicinal chemistry, chemical biology, and pharmacology routinely combining them rather than working in silos. The result, according to the editorial framing the collection, is a discovery enterprise that is faster, more efficient, and increasingly sophisticated in how it approaches molecular design.

A central theme running through the issue is the development of efficient and diversity-oriented synthetic strategies for bioactive molecules. The published articles describe asymmetric and visible-light-promoted reactions, multicomponent and one-pot syntheses, iron-catalyzed functionalization, nanocatalytic and sonochemical methods, skeletal editing, total synthesis, and optimized synthetic routes. Each of these techniques addresses a persistent bottleneck in drug discovery: the speed and reliability with which chemists can actually make the molecules that computational and biological studies suggest might work. Visible-light photocatalysis, for example, allows bond formations under mild conditions that were previously difficult or impossible, while skeletal editing permits late-stage modifications of molecular frameworks that would once have required rebuilding a candidate from scratch. Multicomponent reactions compress multi-step sequences into single operations, dramatically shortening the path from idea to testable compound.

Crucially, the synthetic advances described in the collection are not presented as ends in themselves. The resulting compounds, which include diverse indoles, indolizines, heterocycles, molecular hybrids, peptides, and natural-product analogues with enhanced structural and stereochemical complexity, were investigated as anticancer, antimicrobial, antitubercular, antiviral, anti-inflammatory, antidiabetic, antiseizure, and neuroprotective agents. This direct linkage between methodology and biological application is what distinguishes the current wave of synthetic innovation from earlier eras in which method development and drug hunting often proceeded on separate tracks. When a new catalytic reaction can be evaluated within weeks against disease-relevant cell models, the feedback loop between chemistry and pharmacology tightens, and the odds that an interesting molecule becomes a therapeutic candidate improve accordingly.

Another striking pattern in the collection is the continuing shift beyond the traditional one drug-one target paradigm that dominated pharmaceutical research for much of the past half-century. Among the highlighted examples are dual inhibitors targeting BTK/FLT3, COX-2/5-LOX, and FAAH/sEH, enzyme pairs relevant to cancer and inflammation, alongside multifunctional agents designed for Alzheimer’s disease and reviews of xanthone hybrids and pyrazolopyrimidine-based dual inhibitors. Multi-target design acknowledges that complex diseases rarely hinge on a single protein, and that modulating several nodes of a pathological network simultaneously can be more effective than maximal blockade of one. The approach demands a different kind of medicinal chemistry, one in which selectivity is engineered across multiple binding sites rather than maximized against a single target, and the articles in the issue show that scaffold design and mechanistic understanding are being integrated to meet exactly that challenge.

The issue also surveys work on established and emerging molecular targets, including FAK, HDAC, ERα, PI3Kδ, p38 MAPK, EZH2, DprE1, histamine H1 and H2 receptors, SFRP1, PDE4B, and nitric oxide synthase. This list spans kinases, epigenetic enzymes, nuclear receptors, phosphodiesterases, and bacterial cell-wall biosynthesis machinery, reflecting the wide biological terrain on which modern medicinal chemistry now operates. A dedicated review on PROTAC technology underscores the prominence of targeted protein degradation, one of the most promising new therapeutic modalities of the past decade. Rather than inhibiting a protein’s activity, degraders recruit cellular disposal machinery to eliminate the disease-causing protein altogether, an approach that can succeed against targets long considered undruggable by conventional small molecules. Its inclusion alongside classical target families illustrates how new modalities are being folded into, rather than replacing, the existing discovery apparatus.

Natural products and biomolecule-inspired scaffolds remain a major focus of the collection, and the evidence assembled suggests that nature is far from exhausted as a source of molecular diversity. Studies on phorbazole D, menominin A, polyprenylated acylphloroglucinols, oleanolic and alepterolic acids, Eucommiae folium, µ-conotoxins, honey-bee antimicrobial peptides, and marine cyclopeptides demonstrate the remarkable chemical inventiveness of the natural world, from terrestrial plants to venomous cone snails and social insects. What has changed is the technology brought to bear on these molecules. Total synthesis, analogue generation, chemical ligation, mass spectrometry, network pharmacology, and cell-based screening are overcoming longstanding challenges in natural-product discovery and optimization, problems of supply, structural complexity, and limited optimization potential that historically kept many natural products out of the clinic despite compelling biological activity.

The synergy between computation and experimentation emerges as perhaps the defining feature of the modern discovery pipeline. Molecular docking, molecular dynamics simulations, pharmacophore modeling, network pharmacology, and integrated in silico-in vitro workflows now support compound prioritization and mechanistic studies across the collection. These are not decorative additions; they determine which of millions of conceivable molecules get synthesized and tested, effectively allocating scarce laboratory resources. AI-assisted analysis, label-free cell-based screening, high-resolution LC-Orbitrap mass spectrometry, and zebrafish disease models further illustrate advances in compound characterization, phenotypic screening, and translational validation. Phenotypic screening in whole organisms such as zebrafish is particularly notable, because it allows compounds to be evaluated for efficacy and toxicity in a living system before the costly transition to mammalian models, catching failures earlier and more cheaply than traditional pipelines allow.

The collective message of the 58 articles is that new technologies in drug discovery are not defined by any single platform or methodology, but by the integration of innovative chemistry, emerging therapeutic modalities, computational prediction, advanced screening technologies, and rigorous biological validation. This multidisciplinary convergence is expanding druggable chemical space, the universe of molecules that can realistically be made, characterized, and developed into medicines, while accelerating therapeutic discovery and enabling increasingly sophisticated approaches to drug design. For decades, the pharmaceutical industry has grappled with declining productivity per research dollar, and collections like this one suggest a credible path forward: rather than betting on any single breakthrough, the field is stacking multiple incremental advantages in synthesis, target biology, computation, and screening into compounding gains across the entire pipeline.

In closing the special issue, Shi thanks the authors, reviewers, and the editorial team of Molecular Diversity, and expresses the hope that the collection will stimulate new collaborations, inspire continued technological innovation, and contribute to making drug discovery faster, more efficient, and more successful while preserving the molecular and mechanistic diversity that underpins transformative medicines. That emphasis on diversity is more than rhetorical. History shows that transformative drugs often emerge from unexpected chemical territory, and the deliberate cultivation of varied scaffolds, modalities, and screening strategies is the best insurance against the field narrowing prematurely around fashionable targets. If the technologies surveyed here continue to mature and interconnect, the coming decade of drug discovery may look markedly different from the last, with molecules designed, synthesized, and validated at a pace and precision that earlier generations of researchers could scarcely have imagined.

Subject of Research: Emerging technologies and multidisciplinary approaches in drug discovery

Article Title: New technologies in drug discovery

Article References: Shi, T. (2026). New technologies in drug discovery. Molecular Diversity. https://doi.org/10.1007/s11030-026-11711-2

Image Credits: AI Generated

DOI: 10.1007/s11030-026-11711-2

Keywords: drug discovery, medicinal chemistry, targeted protein degradation, PROTAC, molecular glues, natural products, artificial intelligence, multicomponent reactions, phenotypic screening, dual inhibitors, chemical biology, Molecular Diversity

Proximity labeling rests on a deceptively simple idea. Instead of trying to capture fragile or transient interactions directly, researchers fuse an engineered enzyme to a molecule of interest and let that enzyme chemically tag everything nearby. The tagged neighbors can then be purified and identified by mass spectrometry or sequencing, producing a snapshot of the local molecular environment. Enzymes such as APEX2, which uses hydrogen peroxide to drive radical generation, and TurboID, an evolved derivative of biotin ligase, have transformed the study of protein complexes and organelle proteomes. Applying the same logic to RNA, however, has proven far more difficult, because RNA molecules are chemically distinct, often abundant, and embedded in ribonucleoprotein assemblies that are easily disrupted by harsh labeling conditions.

Lantern was developed to address precisely this gap. The enzyme is designed to label molecules in the immediate vicinity of a chosen RNA, generating a record of the proteins and other RNAs that associate with it in living cells. Early versions of the tool, like many first-generation proximity labeling systems, faced limitations in catalytic rate, background activity, and the conditions required to drive the labeling reaction. Slow enzymes require long labeling periods, during which the cell continues to change, blurring the temporal resolution of the resulting map. High background activity, meanwhile, can swamp genuine neighbors in a haze of nonspecific tags, obscuring the very interactions researchers hope to detect.

Directed evolution offers a systematic way out of this impasse. The approach mimics natural selection in the laboratory: researchers generate large libraries of enzyme variants carrying random mutations, screen or select the variants that perform best on a defined task, and then iterate the process, accumulating beneficial mutations over successive rounds. Applied to Lantern, this strategy allowed the team to interrogate enormous sequence space and identify combinations of mutations that jointly improved catalytic turnover, reduced background, and preserved the enzyme’s ability to function inside the complex chemical environment of a mammalian cell. The result is an evolved Lantern variant that labels proximal RNA and protein molecules far more rapidly than its predecessors.

The significance of speed in proximity labeling is difficult to overstate. Cellular states are not static; signaling events, stress responses, and cell-cycle transitions can remodel the interactome of an RNA within minutes. A labeling reaction that requires hours effectively averages over all of these changes, producing a composite picture that may not correspond to any real biological moment. A fast enzyme, by contrast, can capture a molecular neighborhood on a timescale that approaches the dynamics of the underlying biology. This temporal precision matters enormously for studying processes such as RNA granule assembly, stress granule formation, and the rapid redistribution of RNAs during cellular responses to external stimuli.

Dual labeling of both RNA and protein by the same enzyme is another defining feature of the evolved Lantern system. Most existing tools are specialized: some tag proteins efficiently but leave RNA untouched, while RNA-targeting approaches often rely on separate chemistries that are difficult to reconcile in a single experiment. A unified catalyst that marks both classes of molecules in the vicinity of a target simplifies experimental design and enables genuinely integrated maps of ribonucleoprotein architecture. Because RNA-binding proteins and their RNA partners form tightly interwoven networks, the ability to profile both sides of the interface from a single labeling event provides a more complete and internally consistent picture than combining results from separate, independently optimized systems.

The technical challenges that directed evolution had to overcome are worth appreciating in detail. An ideal proximity labeling enzyme must remain inactive until deliberately deployed, tolerate fusion to diverse RNA-targeting modules such as Cas proteins or RNA-binding domains, operate at physiological temperature and pH, and generate reactive intermediates that diffuse only over a short range before reacting with nearby molecules. Balancing these competing demands is not intuitive; mutations that boost catalytic activity often increase background or alter substrate specificity in undesirable ways. Screening strategies that evaluate variants directly in cellular contexts, rather than in simplified biochemical assays, are therefore essential for identifying enzymes that perform well where it matters, inside living cells rather than in a test tube.

Beyond its immediate technical achievements, the evolved Lantern system points toward broader applications across biology and medicine. Mapping the protein companions of disease-associated noncoding RNAs could reveal how long noncoding RNAs execute their regulatory functions and how mutations disrupt these interactions in conditions ranging from cancer to neurodegeneration. Viral RNAs, which recruit host factors into specialized replication and packaging complexes, could be profiled with unprecedented temporal resolution, illuminating points of vulnerability for antiviral therapeutics. In developmental biology, tracking the changing molecular neighborhoods of specific transcripts as cells differentiate could clarify how post-transcriptional regulation shapes cell fate decisions. The combination of speed, dual specificity, and genetic encodability makes the tool adaptable to virtually any RNA that can be targeted with a suitable binding module.

The study also contributes to a growing appreciation of directed evolution as an engine of innovation in chemical biology. Time and again, natural enzymes have proven to be starting points rather than finished solutions, and laboratory evolution has repeatedly delivered variants with properties that no rational design effort could have predicted. The Lantern work exemplifies this pattern: by letting mutation and selection explore sequence space under experimentally defined pressures, researchers obtained a catalyst whose performance characteristics reflect the specific demands of proximity labeling in living cells. As screening technologies improve and libraries grow larger and more diverse, the pace at which such optimized tools emerge is likely to accelerate, equipping the community with an ever-richer toolkit for interrogating molecular proximity.

For the field of RNA biology in particular, the arrival of a rapid, dual-function proximity labeling enzyme marks a meaningful step forward. The interactomes of RNAs have long been studied through laborious biochemical purification methods that require large quantities of material and inevitably perturb the very assemblies under investigation. A genetically encodable, fast-acting labeling system brings the study of RNA neighborhoods into the same experimental regime that has already revolutionized protein interaction mapping, with all the advantages of sensitivity, scalability, and compatibility with living systems. As researchers begin to apply evolved Lantern to their own questions, the coming years are likely to see a substantial expansion in our understanding of the molecular ecosystems that surround RNA, and of the roles those ecosystems play in health and disease.

Subject of Research: Directed evolution of the Lantern enzyme for rapid proximity labeling of RNA and proteins in living cells

Article Title: Directed evolution of Lantern enables rapid RNA and protein proximity labeling

Article References: Fang, Y., Ren, Z., Zheng, F., Wang, R., Zhao, S., Zhang, Y., Wang, W., Li, C., Liu-Yang, L., Lin, C., Liu, J., & Zou, P. (2026). Directed evolution of Lantern enables rapid RNA and protein proximity labeling. Nature Chemical Biology. https://doi.org/10.1038/s41589-026-02313-y

Image Credits: AI Generated

DOI: 10.1038/s41589-026-02313-y

Keywords: directed evolution, Lantern, proximity labeling, RNA biology, protein labeling, RNA-protein interactions, chemical biology, ribonucleoprotein complexes, enzyme engineering, molecular interactome, living cells, Nature Chemical Biology

The conceptual foundation of the study rests on a tension that immunologists have wrestled with for decades. The classical view of phagocytosis describes a one-way road: a target particle is recognized by receptors on the macrophage membrane, enveloped by actin-driven membrane extension, sealed inside an intracellular vesicle called a phagosome, and then progressively acidified and enzymatically degraded as the phagosome matures through fusion with lysosomes. Under this model, anything the macrophage eats is destined for the degradative pathway. Peptides derived from digested proteins are loaded onto major histocompatibility complex molecules and presented to lymphocytes, closing the loop between innate scavenging and adaptive surveillance. The new work suggests that this pathway is not the only fate available to captured material, and that functional protein transfer to the plasma membrane competes with degradation during uptake.

Technically, the distinction between internalization and surface transfer is not trivial to demonstrate, because material that remains attached to the outside of a cell can masquerade as internalized cargo in conventional flow cytometry and bulk fluorescence assays. Experiments of this kind therefore depend on approaches that spatially resolve the membrane. The study’s conclusions hinge on the ability to distinguish proteins that have genuinely been routed to the macrophage surface from those merely riding on incompletely internalized particles or trapped in membrane ruffles. Proteins delivered to the surface in a functional state must retain at least some of their biochemical activity, a criterion that separates this phenomenon from passive adsorption of denatured fragments. The authors’ characterization of functionally active proteins appearing on the macrophage membrane after uptake thus implies a controlled trafficking event rather than an artifact of sample handling.

One implication of the finding concerns the growing appreciation of trogocytosis, the process by which cells exchange fragments of their plasma membrane and surface molecules through contact. Trogocytosis has been documented most extensively among lymphocytes and antigen-presenting cells, where a cell can literally strip membrane-associated ligands from a partner and wear them on its own surface. The macrophage behavior described in the new study can be understood as a related but distinct phenomenon: rather than acquiring proteins from another cell through direct intermembrane contact during a competitive interaction, the macrophage appears to reroute a portion of the cargo it engulfs back to its own membrane during the uptake process itself. The phrase live-cell uptake in the study’s title is significant, because it indicates that this transfer occurs when the macrophage consumes material from living cells, situations in which the membrane chemistry of the target and the dynamics of receptor engagement differ substantially from uptake of dead cells or inert particles.

The biochemical questions raised by the work are considerable. For a protein to appear on the external face of the macrophage plasma membrane in a functional form, it must traverse or bypass several membrane barriers. Cargo internalized by phagocytosis is enclosed within a vesicle whose lumen is topologically extracellular, which means that, in principle, a protein could reach the cell surface by fusion of recycling vesicles with the plasma membrane without ever entering the cytosol. This recycling route is well established for receptors that are internalized and returned to the surface, and the new study suggests that at least some captured functional proteins can piggyback on analogous recycling traffic. Alternatively, transfer could involve direct membrane continuity between the forming phagosome and the plasma membrane, or regurgitation of incompletely sealed uptake structures. Distinguishing among these routes is a central challenge for follow-up work.

Functional display of captured proteins could have far-reaching consequences for immune regulation. A macrophage that presents an active, intact protein on its surface is not merely advertising peptides for T cell inspection; it is offering other cells the opportunity to bind that protein, respond to its enzymatic activity, engage it as a ligand, or be inhibited by it. If the transferred proteins include, for example, receptors, adhesion molecules, complement regulators, or signaling ligands derived from the cells the macrophage has consumed, the macrophage could effectively adopt surface properties of its prey. Such molecular mimicry at the single-cell level would provide a mechanism by which tissue-resident macrophages continually update their surface identity to reflect the local environment they patrol, blurring the boundary between self-display and scavenged display.

The finding also speaks to long-standing puzzles in the biology of macrophage interactions with living cells. Macrophages routinely sample healthy cells through brief contacts and transient uptake events without triggering inflammation, a process that depends on the balance of activating and inhibitory signals received through receptors such as those in the signal regulatory protein and integrin families. If live-cell uptake can leave functional proteins on the macrophage surface, then even a fleeting phagocytic event could durably alter the macrophage’s signaling landscape. Proteins acquired from a healthy cell might include inhibitory ligands that reinforce tolerance, whereas proteins acquired from a stressed or transformed cell might advertise danger. In this way, surface protein transfer could convert every meal a macrophage takes into a change in its own phenotype, coupling immune surveillance at the level of tissues to reprogramming at the level of the single cell.

From the perspective of chemical biology, the study exemplifies a broader trend of interrogating immune phenomena with tools that track molecules rather than populations. Understanding that captured proteins can remain functional after transfer requires assays that measure activity, localization, and trafficking simultaneously, integrating live-cell imaging, biochemical fractionation of membrane compartments, and perturbation of vesicular transport pathways. The paper’s home in Nature Chemical Biology underscores this methodological character: the question is not only what the macrophage does, but how molecular movement between intracellular compartments and the plasma membrane can be resolved, quantified, and manipulated. Insights of this kind are likely to inform the design of drug delivery systems, because nanoparticles and antibody conjugates engineered for macrophage uptake may likewise find themselves displayed, intact and active, on the macrophage surface rather than sequestered internally.

Therapeutically, the implications span several domains. In cancer immunotherapy, macrophages infiltrating tumors are known to engulf tumor cells and tumor-derived material, and their subsequent behavior profoundly shapes the antitumor response. If live-cell uptake leaves functional tumor proteins on the macrophage surface, this could either help prime adaptive immunity by displaying intact targets for antibody binding, or subvert it by presenting tolerogenic ligands. In infectious disease, pathogens that manipulate phagocytosis might exploit the transfer pathway to decorate macrophages with their own surface molecules, a strategy that could aid immune evasion. In transplantation and autoimmunity, acquired display of donor- or self-derived functional proteins could tilt local immune signaling toward acceptance or attack. Each of these scenarios remains speculative pending direct evidence about which proteins are transferred and under what physiological conditions, but they illustrate why a shift in the fate map of phagocytosed material matters well beyond cell biology.

The study ultimately reframes the macrophage surface as an interface in constant negotiation with the cell’s dietary history. Rather than a fixed identity defined by genome-encoded receptor expression, the macrophage membrane emerges as a composite structure, continuously edited by the functional proteins the cell captures from its surroundings during live-cell uptake. Future work will need to identify the molecular machinery that directs captured proteins to the surface, determine the breadth of cargo that follows this route, establish how long acquired proteins persist and signal, and test whether the phenomenon operates in vivo across tissues and disease states. What the current finding establishes is that the degradative pipeline of phagocytosis has a branch point that earlier models did not anticipate, and that branch point places captured, functional proteins directly in the traffic of the immune system’s most voracious and influential scavenger cells.

Subject of Research: Protein transfer to the macrophage surface during live-cell phagocytic uptake

Article Title: Macrophages transfer functional proteins to their surface during live-cell uptake

Article References: Volk, R. F., Fan, A. C., Casebeer, S. W., Tejus, V. R., Condon, A. C., Zirak, B., Manon, N. A., Irkliyenko, I., Torralba, D. M., Tao, S., Pollini, T., Ramani, V., Maker, A. V., Krummel, M. F., Goodarzi, H., & Zaro, B. W. (2026). Macrophages transfer functional proteins to their surface during live-cell uptake. Nature Chemical Biology. https://doi.org/10.1038/s41589-026-02292-0

Image Credits: AI Generated

DOI: 10.1038/s41589-026-02292-0

Keywords: macrophages, phagocytosis, live-cell uptake, protein transfer, cell surface display, trogocytosis, innate immunity, membrane trafficking, Nature Chemical Biology, immune regulation, phagosome recycling, chemical biology

The heart of the device is MspA, a porin from the soil bacterium Mycobacterium smegmatis that has already earned a storied reputation in DNA sequencing. MspA forms a stable, conical pore roughly one nanometre wide at its constriction, a scale at which a single small molecule can obstruct the flow of ions and produce a characteristic electrical signature. When a voltage is applied across a membrane containing the pore, analytes that wander into the aperture transiently block or modulate the ionic current, and the resulting blips carry fingerprints of the molecule’s size, shape, charge and chemistry.

The problem has always been versatility. Nanopores are exquisitely selective, which is a virtue for detecting one target molecule but a liability when the target is unknown. Different chemical classes interact with the pore environment in different ways: saccharides are neutral and hard to trap, amino acids span a dramatic range of charge and hydrophobicity, and nucleotides carry dense negative charges that make them rush through too quickly to be read. Earlier engineered pores succeeded with individual analyte classes—discriminating all twenty proteinogenic amino acids in one design, or distinguishing monosaccharides in another—but no single pore had tackled all of them at once.

The new work solves this by grafting a chemical adapter into the pore. The team modified MspA with a maleimido-C2-FPBA moiety, a benzaboronic acid derivative attached through a short linker to a defined site inside the pore lumen. Boronic acids are famous in chemical biology for forming reversible covalent complexes with cis-diols, the pairing of hydroxyl groups found abundantly on sugars. But the adapter does more than catch carbohydrates. Inspired by iminoboronate chemistry, the reversible interaction between boronic acids and nitrogen-containing functional groups allows the adapter to transiently capture amines as well, slowing the passage of amino acids and peptides so that their signals can be recorded.

The result is a pore that no longer lets any of these small molecules simply tumble through. Instead, each analyte is repeatedly captured, held and released at the adapter site, producing long trains of current fluctuations rather than a single fleeting blip. Amino acids produce residence events whose depths and durations reflect their side chains; nucleoside monophosphates generate distinct blockade patterns shaped by their base and phosphate; saccharides, bound through their diols, yield slow, stuttering signals; and peptides produce composite signatures reflecting both their terminal amines and their residue composition. Crucially, all four classes can be present in the same solution and still be told apart, because their event statistics cluster in separate regions of feature space.

Distinguishing those clusters is where machine learning enters. The researchers extracted features from thousands of individual events—mean and variance of the blockade current, dwell times, recurrence rates and higher-order statistics of the fluctuation patterns—and trained classifiers on labelled mixtures. Using a few-shot learning strategy, which requires only a small number of labelled examples per analyte, the system assigned unseen events to the correct analyte with 98.7% accuracy across the four chemical classes. The approach is notable for its data efficiency: instead of demanding enormous training sets, the engineered pore’s physically distinct binding chemistry produces such characteristic signals that a handful of reference measurements per molecule suffices.

The most striking demonstration involves glycopeptides, molecules that marry a peptide backbone to one or more covalently attached glycans. Glycopeptides are central to biology—most secreted and membrane proteins carry them—and their analysis is a major bottleneck in proteomics, typically requiring enzymatic release of the sugars and elaborate liquid chromatography tandem mass spectrometry workflows. Because the FPBA adapter engages the cis-diols of the glycan while the pore constriction senses the peptide, the sensor reads both parts of the molecule in one event. The team showed that the device could determine the compositional makeup of native, unlabelled glycopeptides, distinguishing variants that differ in their sugar content without any prior chemical derivatization.

Experts in bioanalytical chemistry will recognize how much engineering subtlety underlies this apparent simplicity. The site-specific attachment of the adapter required a genetically introduced handle in the MspA protein, and the linker length and chemistry had to be tuned so that analytes of wildly different sizes—from a single amino acid of around one hundred daltons to glycopeptides several times larger—could all access and interact with the boronic acid. The ionic current readout itself is straightforward, but converting raw fluctuation trains into reliable chemical identities demands rigorous control of pH, salt concentration and voltage, conditions the authors establish and characterize in the study.

The broader significance lies in what a practical four-class nanopore sensor could enable. A benchtop or even portable device that identifies metabolites, nucleotides, sugars and peptide fragments from a crude mixture would be transformative for point-of-care diagnostics, metabolic flux studies, quality control in biopharmaceutical manufacturing and the emerging field of glycomics. It also complements the rapid progress in nanopore peptide sequencing, where related engineered pores have recently been used to read stepwise-shortened peptides, and with nanopore single-molecule chemistry more generally, which now extends far beyond its nucleic-acid origins. A universal small-molecule reader, in other words, no longer seems fanciful.

Challenges remain before the technology migrates from research lab to routine use. Real biological samples contain hundreds of analytes, some present at vanishingly low concentrations, and the classifier’s performance on such dense, unbalanced mixtures will need to be demonstrated. Sensor lifetime, throughput and the standardization of pore fabrication will also matter for adoption. But the conceptual hurdle has been cleared: a single chemically versatile protein pore, read by a modest machine-learning model, can simultaneously name molecules from four different chemical worlds. As nanopore sensing matures, the humble ionic-current trace is quietly becoming one of the most information-dense signals in all of analytical science.

Subject of Research: An engineered MspA nanopore sensor that simultaneously identifies amino acids, nucleoside monophosphates, saccharides and peptides, including native glycopeptides, using machine-learning classification.

Article Title: A versatile nanopore identifies four classes of analytes simultaneously

Article References: A versatile nanopore identifies four classes of analytes simultaneously. (2026). Nature Biotechnology. https://doi.org/10.1038/s41587-026-03322-x

Image Credits: AI Generated

DOI: 10.1038/s41587-026-03322-x

Keywords: nanopore sensing, MspA, biosensor, machine learning, amino acids, saccharides, peptides, nucleoside monophosphates, glycopeptides, single-molecule detection, bioanalytical chemistry, chemical biology

The new work, published in Nature Chemical Biology, tackles a problem that has frustrated the apoptosis field since BAX was first implicated in ischemic injury: the protein is a moving target. In healthy cells, BAX lounges in the cytosol as an inactive monomer, its lethal membrane-penetrating helices tucked away inside its own structure. Only when death signals accumulate does BAX undergo a dramatic conformational transformation, exposing its N-terminus, unfurling its ninth alpha helix, and migrating to the mitochondrial outer membrane, where it oligomerizes into pores. Small molecules that bind the resting state have been described before, but they tend to be weak, poorly characterized, or reactive with many unrelated proteins, making them unreliable tools and even less reliable medicines.

The team behind the new study took a different approach: rather than hunting for a generic binder, they engineered a covalent warhead aimed at a specific cysteine residue on the surface of inactive BAX. Covalent inhibitors have enjoyed a renaissance in recent years, most famously in the form of acrylamide-based drugs that target a non-catalytic cysteine in EGFR-mutant lung cancer. The strategy offers the allure of prolonged target engagement at low drug concentrations, but it carries a well-known risk: off-target reactivity with the many cysteine-rich proteins floating in any cell. The challenge, therefore, was to design a ligand whose reactivity is only unleashed in the precise geometric context of the BAX binding pocket.

That is where the concept of enantioselectivity becomes central. Small drug-like molecules typically exist as two enantiomers, mirror-image forms that are chemically identical in an achiral test tube but profoundly different in the chiral environment of a living cell. Enzymes, receptors, and protein binding pockets distinguish between these mirror images with exquisite sensitivity, often binding one form tightly while ignoring the other. The researchers exploited this principle twice over: first by synthesizing both enantiomers of their candidate inhibitor and then by demonstrating that only one of them engages BAX efficiently, while the opposite enantiomer is essentially inert. This one-handed specificity is a hallmark of a well-behaved chemical probe and stands in sharp contrast to earlier BAX inhibitors whose activity could not be cleanly separated from nonspecific protein damage.

The design process began with structural analysis of the inactive BAX monomer, using prior nuclear magnetic resonance structures and molecular docking to identify a pocket adjacent to a reactive cysteine. The team then iterated through a series of analogues, tuning the electrophilic warhead and the surrounding scaffold until they achieved a compound that reacts with BAX rapidly and selectively in competition assays against a broad panel of cysteine-containing proteins. Chemoproteomic experiments in cell lysates confirmed the selectivity on a proteome-wide scale, showing that the compound’s covalent footprint is dominated by BAX rather than by the hundreds of other cysteine residues available for reaction. This kind of global reactivity profiling has become the gold standard for validating covalent chemistry, and its successful application here lends substantial credibility to the probe.

With a selective inhibitor in hand, the researchers turned to functional testing. In cell culture, the compound protected cells from apoptotic death provoked by a variety of stresses, and the protection was abolished when BAX was removed or when a non-reactive analogue was substituted, establishing that the cytoprotective effect runs through the intended target. Biochemical assays showed that the covalently modified BAX can no longer expose its membrane-inserting helix or translocate to mitochondria in response to activating signals, effectively locking the protein in its harmless resting conformation. The modification also prevented BAX oligomerization, the downstream step that converts individual protein molecules into the pore-forming assemblies that rupture the mitochondrial membrane and release cytochrome c.

The most consequential experiments, however, were performed in living animals. In a mouse model of ischemia-reperfusion injury, a scenario that mirrors the cellular damage that follows a heart attack or stroke, administration of the active enantiomer significantly reduced tissue damage compared with vehicle controls. Critically, the mirror-image enantiomer, which lacks BAX reactivity in vitro, provided no protection, a rigorous in vivo control that ties the therapeutic benefit directly to the covalent engagement of BAX. Pharmacokinetic measurements confirmed that the compound reaches relevant tissues at concentrations sufficient to modify the target, and the treated animals tolerated the drug without overt toxicity, an encouraging early signal for a strategy that modifies a protein involved in fundamental cellular quality control.

Experts in the apoptosis field have long debated whether inhibiting BAX systemically is safe or even desirable, given the protein’s role in eliminating damaged or potentially cancerous cells. The new study does not resolve that debate, but it sharpens the terms of the discussion. Because the inhibitor is covalent and long-acting, dosing regimens could in principle be tailored to acute, short-term scenarios, such as the hours surrounding reperfusion therapy after a myocardial infarction, where transient BAX inhibition might salvage tissue without the long-term cancer risks that chronic suppression might entail. The authors’ demonstration that a single enantiomer drives the entire pharmacological effect also suggests that medicinal chemistry optimization can proceed with confidence, since the inactive mirror image provides a built-in negative control for every future experiment.

The work also carries broader lessons for chemical biology. Covalent inhibitors were once viewed as liabilities to be engineered out of drug candidates; today they are a deliberate design choice, provided that selectivity is demonstrated rigorously. The BAX program illustrates the full pipeline: structural insight to identify a ligandable site, warhead tuning to balance reactivity and selectivity, enantiomer pairing to isolate specific from nonspecific effects, chemoproteomics to survey the proteome, and animal models to test whether the molecular mechanism translates into tissue-level protection. Each step reinforces the others, and the resulting probe is far more than a tool; it is a proof of concept that a notoriously difficult, conformationally dynamic protein can be drugged with precision.

Looking ahead, the researchers and their colleagues face the familiar gauntlet of translation: optimizing potency and pharmacokinetics, assessing safety across longer time horizons, and identifying the clinical settings where BAX inhibition offers the greatest benefit at the least risk. Beyond ischemic injury, candidates include neurodegenerative conditions in which mitochondrial apoptosis contributes to neuronal loss, and organ transplantation, where protecting donor tissue from programmed death could extend viability and improve outcomes. Whatever the ultimate therapeutic destination, the demonstration that an enantioselective covalent inhibitor of BAX can confer cytoprotection in vivo marks a milestone in the long campaign to control the machinery of cell death, and it hands the field a chemical instrument of unprecedented specificity for dissecting BAX biology in health and disease.

Subject of Research: Development of an enantioselective covalent small-molecule inhibitor of the pro-apoptotic protein BAX that prevents mitochondrial apoptosis and provides cytoprotection in vivo.

Article Title: An enantioselective covalent inhibitor of BAX confers cytoprotection in vivo

Article References: An enantioselective covalent inhibitor of BAX confers cytoprotection in vivo. (n.d.). https://doi.org/10.1038/s41589-026-02297-9

Image Credits: AI Generated

DOI: 10.1038/s41589-026-02297-9

Keywords: BAX, apoptosis, covalent inhibitor, enantioselectivity, mitochondria, cytoprotection, ischemia-reperfusion injury, chemical biology, drug design, chemoproteomics, Nature Chemical Biology, cell death

The problem the researchers set out to solve has long frustrated chemical biologists. Most strategies for controlling amino acid function in living cells rely on caging groups built around heteroatoms — oxygen, nitrogen, boron, or iodine-bearing linkages that can be cleaved by light, enzymes, or reactive chemicals. These approaches work beautifully for residues like serine, lysine, cysteine, tyrosine, and histidine, all of which carry reactive heteroatoms in their side chains. Phenylalanine, by contrast, is a purely hydrocarbon residue: a nonpolar benzene ring dangling from the protein backbone with no convenient chemical handle. Traditional caging chemistry simply has nothing to grab onto. As a result, although phenylalanine is one of the twenty canonical amino acids and is functionally critical in contexts ranging from amyloid formation to immune recognition, its activity could not previously be masked and restored at will in a living system.

The Peking University group, led by Peng R. Chen and Xinyuan Fan, with Yuchao Zhu, Shibo Liu, and Shan Qin as co-first authors, approached the challenge from an unconventional angle. Rather than trying to attach a bulky protecting group to an impossible target, they systematically evaluated caging strategies based on exogenous heteroatoms — oxygen, nitrogen, boron, and iodine — under physiological conditions. The winning design turned out to be remarkably simple: iodine atoms installed directly onto phenylalanine’s aromatic ring. This haloatom-assisted caging strategy exploits the fact that aryl iodides can undergo clean, traceless reduction, stripping the iodine away and regenerating the native phenylalanine residue without leaving any molecular scar behind.

The trigger for this restoration is palladium, a transition metal that has become a workhorse of bioorthogonal chemistry over the past decade. Palladium catalysts can mediate deprotection and bond-cleavage reactions inside living cells because they operate through mechanisms that native biochemistry simply does not use — no enzyme, no metabolite, and no cellular component competes for the reaction. When the researchers delivered palladium alongside a mild reducing system to iodinated phenylalanine residues, the aromatic cage was lifted and the amino acid sprang back to life in its native form. Crucially, the team demonstrated that the reaction works not only in solution but also in cell lysates and inside living cells, a benchmark that few bioorthogonal decaging reactions have reached.

To show that the chemistry could control real biology, the researchers first turned their attention to small molecules and peptides. They demonstrated that iodination could silence the fluorescence of a fluorophore and that palladium-triggered decaging could restore it, providing a convenient optical readout for the reaction. They then applied the strategy to peptide self-assembly, showing that iodinated phenylalanine residues could direct the disassembly of peptide structures — a finding with implications for the growing field of peptide-based nanomedicine, where the phenylalanine-phenylalanine motif is a celebrated driver of supramolecular assembly. By removing the iodine on demand, the researchers could toggle assembly states at will, effectively writing and erasing structural information in a peptide system.

The most striking demonstrations, however, came at the level of proteins and cells. Using genetic code expansion — the technique of engineering cells to site-specifically incorporate unnatural amino acids into proteins at chosen positions — the team installed iodinated phenylalanine into engineered HER2-targeting affibodies, small binding proteins directed against the HER2 receptor that is overexpressed in many breast cancers. lodinating a phenylalanine at the binding interface crippled the affibody’s ability to engage its receptor. When palladium was added, the cage was lifted, the native phenylalanine was restored, and ligand-receptor binding on the cell surface surged back to full strength. This dynamic control of a protein-protein interaction on a living cell membrane represents exactly the kind of precise, externally triggered molecular switch that the bioorthogonal chemistry community has pursued for years.

The team then extended the strategy into immunology, an area where the stakes are particularly high. Antigenic peptides presented on the surface of tumor cells by major histocompatibility complex class I molecules are the signals that tell cytotoxic T cells to attack. The researchers showed that iodinating phenylalanine residues within antigenic peptides could reshape their immunogenicity, dampening the presentation landscape until palladium-triggered decaging flipped it back on. In practical terms, this means tumor cells could be chemically tuned in their engagement with T cells — a concept that suggests future therapeutic strategies in which the immune visibility of a tumor is masked or unmasked on demand. The experiments demonstrated temporally controlled reshaping of the immunopeptidome, rewiring the tumor-T cell interface from the outside in, with chemistry rather than genetics as the controlling hand.

What makes this work especially significant is its scope. Phenylalanine is not a niche residue; it is ubiquitous, appearing in roughly four percent of protein sequences on average and clustering disproportionately at binding interfaces, active sites, and recognition motifs. Previous decaging efforts from the same laboratory and others had conquered tryptophan, tyrosine, lysine, and other functionalized residues, but the hydrophobic aromatic core of phenylalanine had remained out of reach. By establishing that a halogen atom can serve as both a functional disruptor and a removable cage — and that palladium chemistry can reverse the modification under fully physiological conditions — the study unlocks an entire class of nonpolar groups for chemical manipulation in living systems. The authors note that the strategy complements photocaged amino acids and other genetically encoded approaches, adding a small-molecule trigger that can penetrate cells and act without light.

The technical achievements underlying the paper are considerable. The researchers systematically compared oxygen-, nitrogen-, boron-, and iodine-based caging groups, finding that monoidinated phenylalanine offered the best balance of stability under physiological conditions and clean, traceless decaging. Computational modeling helped them understand how iodination perturbs binding energetics at protein interfaces, and LC-MS analysis confirmed complete consumption of caged substrates with well-defined products. In living cells, the palladium-mediated decaging restored roughly half of the fluorescence of a caged pyrene reporter, a substantial efficiency for intracellular bioorthogonal catalysis. The work also builds on a decade of progress in palladium-mediated intracellular chemistry, from early demonstrations of palladium-mediated deprotection on cell surfaces to nanopalladium catalysts operating inside living animals, and it extends that legacy into a domain — nonpolar aromatic residues — that was previously considered chemically inaccessible.

Looking forward, the implications ripple outward across chemical biology, drug development, and immunotherapy. Prodrug strategies could exploit palladium-triggered phenylalanine decaging to activate therapeutics at disease sites where catalysts are delivered. Synthetic biologists could build protein circuits whose interactions are gated by a small-molecule cue rather than by transcription or light. Cancer immunologists now have a chemical tool to modulate how tumor cells present themselves to the immune system, potentially improving the precision of adoptive cell therapies and vaccine design. And because the reaction is traceless, the decaged product is indistinguishable from the native biomolecule, sidestepping concerns about residual chemical artifacts. What the Peking University team has delivered is not merely a new reaction but a new degree of freedom: the ability to silence and restore, on command, one of biology’s most essential and least manipulable building blocks. In a field where the grand ambition is to exert the precision of synthetic chemistry inside the messiness of living systems, that is a milestone worth pausing over.

Subject of Research: Palladium-triggered bioorthogonal decaging of iodine-caged phenylalanine for controlling protein and cell functions in living systems

Article Title: Palladium-triggered bioorthogonal phenylalanine decaging

Article References: Zhu, Y., Liu, S., Qin, S., Wang, X., Liu, Y., Zhang, X., Shan, Y., Fan, X., & Chen, P. R. (2026). Palladium-triggered bioorthogonal phenylalanine decaging. Nature Chemistry. https://doi.org/10.1038/s41557-026-02226-2

Image Credits: AI Generated

DOI: 10.1038/s41557-026-02226-2

Keywords: bioorthogonal chemistry, palladium catalysis, phenylalanine decaging, genetic code expansion, protein-protein interactions, iodination, chemical biology, tumor immunology, peptide self-assembly, HER2 affibody, amino acid caging, Nature Chemistry