Rachita Dash

Chembiochem. 2026 May 14;27(9):e70329. doi: 10.1002/cbic.70329.

ABSTRACT

We investigated the passive permeability and cytotoxicity of cyclic heptapeptides based on the mortiamide family of natural products. Of all possible stereoisomeric backbones, the natural product's scaffold was among the two most lipophilic as measured by hydrocarbon-water partition coefficients. Using one-bead-one-compound synthesis, we generated a ∼66,000-member library based on the mortiamide scaffold and identified numerous compounds that showed low micromolar cytotoxicity against synovial sarcoma and breast cancer cell lines. Physicochemical characterization revealed that these compounds, including the known mortiamides, have very low aqueous solubilities and form amyloid-like fibril aggregates. Multiple lines of evidence-including detergent-reversible enzyme inhibition, thioflavin T fluorescence, transmission electron microscopy, and equipotent enantiomeric pairs-demonstrate that the observed bioactivity arises from colloidal aggregation rather than specific target engagement. Our findings highlight the critical importance of early physicochemical evaluation in natural product-inspired drug discovery and underscore how aggregation-prone scaffolds can generate misleading structure-activity relationships.

PMID:42131907 | PMC:PMC13173399 | DOI:10.1002/cbic.70329

J Med Chem. 2026 May 28;69(10):12669-12677. doi: 10.1021/acs.jmedchem.6c00871. Epub 2026 May 15.

ABSTRACT

Membrane permeability is critical to the function of proteolysis targeting chimeras (PROTACs), yet common methods such as the parallel artificial membrane permeability assay (PAMPA) and Caco-2 transwell assays are limited by their experimental configuration and fail to directly measure a compound's ability to access the cytosolic space of a target cell. Here, we present a method to measure the membrane permeability of unmodified PROTACs and other E3 ligase ligands directly into the cytosol of living cells. By utilizing NanoBRET live-cell target engagement in real time, we quantify permeability rates that match those from transwell systems and reveal rates that transwell setups fail to measure. Several previously undetectable BET-targeting PROTACs, with subtle structural changes, display differing permeabilities that rationalize discrepancies in live-cell degradation efficiency and cytotoxicity. Ultimately, this approach enables quantitative permeability profiling of PROTACs previously considered unmeasurable in transwell assays, providing a powerful tool to guide rational design and lead optimization.

PMID:42139371 | DOI:10.1021/acs.jmedchem.6c00871

ACS Infect Dis. 2026 May 22. doi: 10.1021/acsinfecdis.6c00053. Online ahead of print.

ABSTRACT

Bacterial cells are surrounded by a dynamic cell wall which is made up of a mesh-like peptidoglycan (PG) layer that provides the cell with structural integrity and resilience. In Gram-positive bacteria, this layer is thick and robust, whereas in Gram-negative bacteria, it is thinner and flexible as the cell is supported by an additional outer membrane. PG undergoes continuous turnover, with degradation products being recycled to maintain cell wall homeostasis. Some Gram-negative species can bypass de novo PG biosynthesis, relying instead on PG recycling to sustain growth and division. Legionella pneumophila (hereafter Legionella), the causative agent of Legionnaires' disease, encodes such recycling machinery within its genome. This study investigates the biochemical, genetic, and pathogenic roles of PG recycling in Legionella. Here, two PG recycling gene homologues in the Legionella genome lpg0296 (amgK) and lpg0295 (murU) were identified; chemical biology strategies were used to illuminate incorporation of "click"-PG-probes into whole PG. Copper-free click chemistry with ultrafast tetrazine-NAM probes enabled live-cell PG labeling further supported the use of recycling programs in Legionella. Deletion of amgK abolished PG labeling, while genetic complementation restored labeling. The data suggest that under conditions where de novo peptidoglycan synthesis is blocked, amgK plays a critical role in maintaining cell wall integrity, as its deletion led to increased antibiotic susceptibility and impaired survival in host alveolar macrophages. An intracellular survival assay demonstrated that while PG recycling is not essential for internalization, survival of Legionella within MH-S murine alveolar macrophages requires functional amgK. These findings underscore the essential role of AmgK in Legionella's intracellular survival, emphasizing the importance of PG recycling in pathogenicity, and establishing a foundation for developing novel Legionella-specific antibiotic strategies.

PMID:42172232 | DOI:10.1021/acsinfecdis.6c00053

Proc Natl Acad Sci U S A. 2026 May 12;123(19):e2523631123. doi: 10.1073/pnas.2523631123. Epub 2026 May 4.

ABSTRACT

Protein kinases regulate almost every major signaling pathway. Visualizing spatiotemporal dynamics of kinase activity is thus essential to understand cell signaling. Here, we report a de novo-designed activity reporter of kinase, dubbed NOVARK, which contains a single polypeptide chain with multiple modular motifs that act as specific kinase substrates and reporters. NOVARK undergoes phosphorylation-induced higher-order assembly, which are detectable as ultrabright green fluorescent protein (GFP) droplets with a large dynamic range. We designed versions of NOVARK that rapidly and reversibly report intracellular activity of protein kinase A, C, and extracellular signal-regulated kinase (ERK) following stimulation/inhibition by upstream G protein-coupled receptor (GPCR) agonists. Our work provides a generalizable platform that enables the design of ultrabright biosensors for illuminating dynamic architecture of kinase signaling.

PMID:42081727 | PMC:PMC13167765 | DOI:10.1073/pnas.2523631123

Nat Chem. 2026 May 4. doi: 10.1038/s41557-026-02125-6. Online ahead of print.

ABSTRACT

Binding and catalysis play central roles in living systems. While natural proteins have finely tuned affinities for their primary ligands, they also bind weakly and promiscuously to other molecules, which serve as starting points for the incremental evolution of different specificities. Thus, modern proteins have emerged from the joint exploration of sequence and structural space. Interactions between natural proteins and small molecules can be systematically profiled by crystallographic fragment screening in defined geometries, yet this approach has not been applied to highly designable de novo proteins. Here we apply this method to explore the binding specificity of a de novo small-molecule-binding protein, apixaban-binding helical bundle. As in nature, we found that it formed weak complexes, which were excellent starting points for the design of entirely distinct functions, including a turn-on fluorophore binder and a highly efficient Kemp eliminase with a catalytic efficiency of 3,200,000 M^-1 s^-1, approaching the diffusion limit. This work illustrates how simultaneous consideration of sequence and chemical structure diversity can guide the emergence of different functions in designed proteins.

PMID:42082787 | DOI:10.1038/s41557-026-02125-6

Nat Protoc. 2026 Apr 29. doi: 10.1038/s41596-026-01348-8. Online ahead of print.

ABSTRACT

The ability to precisely modify proteins and peptides is fundamental to studying their function and creating new variants or topologies with improved properties. Recent studies have transformed the scope of transpeptidases as versatile tools for site-specific modification of proteins and peptides. The engineered asparaginyl ligase OaAEP1 is an ultrafast transpeptidase that stands out owing to its ability to efficiently catalyze a diverse range of modifications that extend well beyond its natural function to generate backbone cyclic peptides in plants. In this Protocol Extension, we describe a framework for the design and application of noncanonical reactions catalyzed by OaAEP1 that provide access to engineered products with customized terminal or side-chain modifications. The reactions proceed cleanly under mild, nondenaturing conditions and can be applied to a broad array of substrates produced by chemical synthesis or recombinant expression, including folded proteins and peptides. After preparing the required substrates and reagents (~5 d) and expressing the recombinant enzyme in Escherichia coli (~3 d), OaAEP1-catalyzed reactions can be carried out in a matter of minutes to hours. We describe methods for installing non-native C-terminal modifications, including by conjugating commercially available nonpeptidic amines (reactive handles, carbohydrates and so on) or ligating a reversed (retro) substrate mimetic that enables production of genetically inaccessible C-to-C fusions. We also describe procedures for OaAEP1-catalyzed side-chain modification of proteins and peptides, which can be applied to generate side-chain-to-tail macrocyclic products, to label a specific side-chain amine with a dye or other reporter tag, or to produce defined protein-cyclic peptide fusions.

PMID:42056561 | DOI:10.1038/s41596-026-01348-8

Nature. 2026 Apr;652(8112):1139-1152. doi: 10.1038/s41586-026-10328-7. Epub 2026 Apr 29.

ABSTRACT

With deep-learning-powered advances in protein design methods, there is an ongoing paradigm shift in protein engineering from random selection to intentional computational design methods. Here we describe the current state of de novo protein design. While there is still room for improvement in success rates and activities, the long-standing challenges of designing new protein structures, assemblies and protein binders are close to being solved. The key current questions in these areas are not how to design, but what to design, and open-source design methodology such as RFdiffusion and ProteinMPNN together with protein structure prediction tools enable biochemists and molecular biologists to broadly explore possible applications. There has also been considerable progress in the de novo design of small-molecule target binders, enzymes and multistate protein systems. Current challenges for methods development include design of catalysts for reactions with high energy barriers and, more generally, design of switches and nanomachines that integrate binding, conformational change and catalysis. Over the next five to ten years, we anticipate the design of sophisticated protein nanomachines and materials with functionality ranging far beyond that generated during natural evolution for a wide range of applications in medicine, technology and sustainability.

PMID:42056544 | DOI:10.1038/s41586-026-10328-7

J Med Chem. 2026 May 14;69(9):11620-11637. doi: 10.1021/acs.jmedchem.6c00889. Epub 2026 Apr 27.

ABSTRACT

Protein N-terminal cysteines (NCys) are valuable for selective modification and posttranslational regulation, but their proteome-wide landscape remains unexplored. We constructed a library of electrophilic probes, including cyanobenzothiazole (CBT), to systematically map ligandable native NCys residues. Modifiable NCys sites were identified in proteins such as GFPT1 and CSTB, revealing accessible NCys in the human proteome. Integrating CBT into a BRD4-targeting ligand generated a reversible covalent degrader (DC₅₀ = 16.7 nM, D_max = 98%). Mechanistic studies showed that CBT modifies DCAF16 at Cys58, promoting ternary complex formation and enabling efficient ubiquitination and degradation. The platform achieved potent and selective degradation of challenging targets like EGFR^{L858R/T790M/C797S} and HER2 without hook effects, outperforming clinical inhibitors. This work provides the first proteome-wide map of ligandable NCys residues and establishes CBT as a versatile platform for covalent targeted protein degradation (TPD), opening new avenues for precision therapeutics.

PMID:42045146 | DOI:10.1021/acs.jmedchem.6c00889

Nat Chem Biol. 2026 Apr 17. doi: 10.1038/s41589-026-02199-w. Online ahead of print.

ABSTRACT

Nanobodies are emerging as attractive biopharmaceuticals due to their small size, stability and target specificity. However, their therapeutic use has largely been restricted to extracellular targets because of a lack of efficient delivery methods. This limitation is particularly relevant for diseases caused by dysfunctional intracellular proteins, such as cystic fibrosis. Here we show that cell-permeable nanobodies can modulate an intracellular disease-relevant target: the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel carrying the common F508del mutation. By combining a CFTR-binding nanobody with cell-penetrating peptides, we achieved intracellular delivery in cystic fibrosis bronchial epithelial cells. The delivered nanobody stabilizes misfolded F508del-CFTR, promotes its maturation and trafficking to the apical membrane and restores chloride channel activity. Moreover, the cell-permeable nanobody enhances the efficacy of approved CFTR modulator drug combination in primary airway epithelial cultures from patients with cystic fibrosis. These findings establish cell-permeable nanobodies as promising biopharmaceuticals for intracellular protein targeting and therapeutic modulation.

PMID:41998105 | DOI:10.1038/s41589-026-02199-w

Angew Chem Int Ed Engl. 2026 Jun 1;65(23):e24419. doi: 10.1002/anie.202524419. Epub 2026 Apr 14.

ABSTRACT

Recent advances in cell-penetrating peptide (CPP)-mediated intracellular protein delivery emphasized the critical role of sustained membrane association in enhancing delivery efficiency. Here, we report cell-surface-reactive, polyfluoroalkyl-tagged polyarginine peptides with varying fluorine content as CPP-additives that significantly enhance protein delivery in living cells. At low micromolar concentrations (2.5 µM), CPP-additives containing 11-13 fluorine atoms enhanced intracellular protein delivery over 2-fold relative to a tagless control without observable cytotoxicity. Live-cell time-lapse fluorescence imaging revealed that a CPP-additive with 13 fluorine atoms showed prolonged membrane association (>5 min) relative to a tagless control and facilitated rapid protein internalization within 10 min. Remarkably, surface-enhanced infrared absorption spectroscopy (SEIRAS) with POPC membranes showed that fluorous CPP-additives initially interacted with the lipid bilayer predominantly as aggregates but subsequently inserted into the membrane interior as monomers without fluorous tag-tag association. Complementary molecular dynamics simulations of the initial membrane-association step provided atomistic insight, showing partial lipid insertion of a monomeric CPP-additive with 13 fluorine atoms while no insertion was observed for a tagless control within the same time scale. Collectively, our findings establish polyfluoroalkyl-tagged CPP-additives as potent, non-cytotoxic vectors for intracellular protein delivery and provide mechanistic detail regarding the molecular basis of their lipid bilayer interactions.

PMID:41981976 | DOI:10.1002/anie.202524419

Chembiochem. 2026 Apr 14;27(7):e202500972. doi: 10.1002/cbic.202500972.

ABSTRACT

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a well-established target for lowering cholesterol and is abundantly present in the extracellular space. Inhibitors of PCSK9 have achieved marked success in the clinic, but an alternative strategy for therapeutic modulation is emerging through the degradation of PCSK9. This novel strategy has been enabled by the identification of cell surface receptors such as the asialoglycoprotein receptor (ASGPR), which mediates the lysosomal degradation of extracellular ligands. Given the importance of this therapeutic mechanism, we investigated the synthesis of bifunctional molecules comprising Tri-GalNAc (an ASGPR binder) with a peptide inhibitor we previously reported. In addition to chemical synthesis, we report a novel method for the production of Tri-GalNAc-conjugated peptides, involving the use of enzymatically mediated ligation postsynthesis. We demonstrate that both the synthetic constructs and chemoenzymatic constructs have the intended structures and in vitro activities. While these molecules did not show cellular activities, the chemical and biochemical methods reported here could be broadly applied to the construction of LYTACs in general. One significant challenge that this work overcomes is the C-terminal attachment of Tri-GalNAc, which remains hitherto a difficult experimental task for not only peptides but also larger biologics in particular.

PMID:41948978 | PMC:PMC13059054 | DOI:10.1002/cbic.202500972

J Am Chem Soc. 2026 Apr 22;148(15):15919-15929. doi: 10.1021/jacs.5c23019. Epub 2026 Apr 7.

ABSTRACT

Incorporation of α-hydrazino acid (α-Hza) into peptide chain can stabilize the secondary structures, such as helices and turns, and give rise to rigidification of peptide conformation. Consequently, such peptides potentially acquire enhanced binding affinity to target proteins. Moreover, the hydrazidic bond in α-Hza-containing peptides contributes to proteolytic resistance. Despite such favorable characteristics for therapeutic peptides, there is no example of success in screening de novo α-Hza-containing peptides against target proteins of interest. Here we report the construction of diverse, mRNA-encoded α-Hza-containing macrocyclic peptide libraries and their application to the Random nonstandard Peptides Integrated Discovery (RaPID) selection against two enzyme targets, Janus kinase 2 (JAK2) and human factor XIIa (FXIIa). The affinity-based enrichment of ligands from these libraries yielded potent binders with low-to-sub-nM dissociation constants, also exhibiting potent inhibitory activity, target specificity, proteolytic stability, and membrane permeability. Mutational studies of active macrocycles underscored critical roles of α-Hza residues in functional potencies. This study establishes a platform for de novo discovery of bioactive α-Hza-containing macrocyclic peptides against proteins of choice, thereby expanding the accessible chemical space for the RaPID system.

PMID:41944810 | DOI:10.1021/jacs.5c23019

Angew Chem Int Ed Engl. 2026 May 18;65(21):e7837067. doi: 10.1002/anie.7837067. Epub 2026 Apr 6.

ABSTRACT

Backbone-cyclic peptides (BMPs) are an attractive class of molecules appeared in diverse natural bioactive products. However, mRNA display technology coupled with ribosomal synthesis is intrinsically inapplicable to such peptide phenotypes due to loss of the C-terminal peptide region linking to the mRNA genotypes. To overcome this issue, we have devised a new strategy to link the sidechain-to-S-mainchain bond via an S-to-N acyl-shift to connect BMPs to the C-terminal fragment of the peptide. Here, we report the application of this strategy to construct a library of BMPs fused to cognate mRNAs. The library was applied for the selection of BMP ligands targeting Akt2, which is involved in the signal pathway to cancer pathogenesis. Consequently, BMP ligands against Akt2 were successfully uncovered from the library. The most potent Akt2 inhibitor, BMPakti-3, showed 1.3 nM of dissociation constant and 34 nM of half-maximal inhibitory concentration (IC₅₀). This system offers a unique platform for the de novo discovery of bioactive BMP ligands against various protein targets of interest.

PMID:41940682 | PMC:PMC13182207 | DOI:10.1002/anie.7837067

bioRxiv [Preprint]. 2026 Mar 24:2026.03.23.713626. doi: 10.64898/2026.03.23.713626.

ABSTRACT

De novo biosynthesis of cell wall peptidoglycan is essential for bacterial viability under many growth conditions and is a well-validated antibiotic target. Although generally not essential for bacterial fitness under standard laboratory growth conditions, peptidoglycan recycling can aid bacterial survival under host or antibiotic stress. Peptidoglycan consists of alternating sugars N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) cross-linked by peptides. Recycling of these sugars can proceed via GlcNAc and glucosamine intermediates (Escherichia coli-type) or, in the case of MurNAc, bypass these intermediates altogether (Pseudomonas-type). We serendipitously discovered that the pathogen Mycobacterium tuberculosis and model organism M. smegmatis assimilate 2-modified MurNAc probes into their peptidoglycan despite lacking the Pseudomonas-type machinery that is normally required for incorporation of these molecules. Our data suggest that unmodified and 2-modified MurNAc incorporate into M. smegmatis peptidoglycan via multiple pathways, the former preferentially via an E. coli-type route and the latter preferentially via a non-E. coli, non-Pseudomonas-type route with GlcNAc but not glucosamine intermediates. These findings reveal metabolic flexibility in mycobacterial cell wall recycling that encompasses a previously undescribed pathway.

PMID:41929186 | PMC:PMC13041923 | DOI:10.64898/2026.03.23.713626

bioRxiv [Preprint]. 2026 Mar 26:2026.03.09.710535. doi: 10.64898/2026.03.09.710535.

ABSTRACT

The LC3/GABARAP protein family is a promising target for selective inhibition of autophagy. Further, LC3/GABARAP ligands have been used as targeted degraders of soluble proteins, protein aggregates, mitochondria, lipid droplets, and RNA. However, the small molecules used for such applications have poor binding affinity and known off-target effects. LC3/GABARAP proteins are challenging targets for small-molecule drug development due to their long, shallow binding grooves. In this work, we evaluate multiple approaches to stabilizing the extended structure of the native binding motif, producing N -methylated peptides and stapled peptides with low nanomolar affinity. A crystal structure and molecular dynamics simulations support a model where the N -methylation pre-organizes the motif into an extended, strand-like structure. N -methylation allowed minimization of the binding motif to a tetrapeptide that retained sub-micromolar affinity while minimizing charge and overall molecular weight. The truncated, N -methylated tetrapeptide showed passive permeability in artificial membrane and cell-based transwell assays. These results highlight new drug-like space for LC3/GABARAP ligands with high affinity and subfamily selectivity.

TABLE OF CONTENTS GRAPHIC: Drawn and developed by Mollie McGibbon and Joshua Kritzer.

PMID:41928939 | PMC:PMC13041900 | DOI:10.64898/2026.03.09.710535

bioRxiv [Preprint]. 2026 Apr 12:2026.03.22.713512. doi: 10.64898/2026.03.22.713512.

ABSTRACT

The blood-brain barrier (BBB) poses a major obstacle to the delivery of therapeutics into the central nervous system (CNS) due to its highly restrictive permeability. Here, we introduce glycan-targeted delivery vehicles, or GlycoShuttles, that traverse the BBB by harnessing the cerebrovascular glycocalyx, a carbohydrate-rich layer lining the BBB lumen. We discover that mucin-domain glycoproteins within this structure serve as novel entry portals for brain delivery and engineer mucin-binding protein shuttles that enable efficient transport of diverse molecular cargo across the BBB into multiple key brain cell types. This modular platform facilitates enhanced brain delivery of a variety of payloads, including antibodies and lysosomal proteins, and demonstrates therapeutic efficacy in mouse models of dementia. Our findings establish mucin-targeted GlycoShuttles as a versatile platform for noninvasive brain delivery of therapeutics, opening new avenues for the treatment of CNS diseases.

PMID:41929072 | PMC:PMC13041825 | DOI:10.64898/2026.03.22.713512

Nat Commun. 2026 Mar 28. doi: 10.1038/s41467-026-70953-8. Online ahead of print.

ABSTRACT

The de novo design of small-molecule-binding proteins holds great promise as a potential tool to develop sensors on-demand for arbitrary small molecules. Here we combine deep learning and physics-based methods to generate a family of proteins with diverse and designable pocket geometries, which we employ to computationally design binders for six small-molecule targets. Biophysical characterization of the designed binders reveals nanomolar to low micromolar binding affinities and atomic-level design accuracy. Additionally, we use a cortisol binder to design a chemically induced dimerization (CID) system that enables the construction of a biosensor for cortisol detection. The approach described here demonstrates the potential of the NTF2 fold and deep learning-based protein design in sensor development, paving the way for future platforms to design binders and sensors for small molecules across analytical, environmental, and biomedical applications.

PMID:41904144 | DOI:10.1038/s41467-026-70953-8

bioRxiv [Preprint]. 2026 Mar 16:2026.03.16.712108. doi: 10.64898/2026.03.16.712108.

ABSTRACT

Mycobacterium tuberculosis withstands acidic conditions to survive and replicate within macrophages. To define the genetic determinants of this adaptation, we performed a transposon screen in a lipid-rich, acidic medium that mimics the host environment and supports robust M. tuberculosis growth. This screen identified ldtB, encoding an L,D-transpeptidase, as essential for growth and survival under acid stress. Loss of LdtB decreased 3-3 peptidoglycan cross-linking, disrupted cell wall architecture, and impaired intrabacterial pH homeostasis, resulting in increased susceptibility to cell wall-active antibiotics. Notably, M. tuberculosis lacking LdtB displayed heightened sensitivity to meropenem within macrophages, suggesting that targeting this enzyme could potentiate β-lactam efficacy during infection. These findings establish LdtB as a key mediator linking peptidoglycan homeostasis to acid stress resistance and underscores the importance of in vitro culture models that recapitulate the host microenvironment for uncovering new in vivo active therapeutic targets.

PMID:41889872 | PMC:PMC13015542 | DOI:10.64898/2026.03.16.712108

J Med Chem. 2026 Apr 9;69(7):7689-7708. doi: 10.1021/acs.jmedchem.5c02876. Epub 2026 Mar 24.

ABSTRACT

Macrocyclic peptides (MPs) are valuable molecular formats for drug development, bridging small molecules and larger biologics due to their favorable pharmacological properties. Here, we describe the discovery of low-nanomolar inhibitors of human angiotensin-converting enzyme 2 (hACE2) by quantitatively screening millions of structurally diverse disulfide-cyclized peptide ligands using yeast display technology. The most potent yeast-encoded "one-ring" and "two-ring" MP inhibit hACE2 with K_i values of 1.9 and 1.5 nM, respectively. These inhibitory potencies are comparable to those of other cyclic peptides discovered using well-established in vitro display technologies. Crystal structures of the two MPs in complex with hACE2 reveal the adoption of either a rigid β-hairpin or a cysteine-stabilized α-helix/α-helix motif. Both MPs exhibit binding modes distinct from those of previously reported inhibitors. Thus, yeast display is a valid technology to rapidly generate MPs with desired binding properties for the development of potential therapeutics.

PMID:41875055 | PMC:PMC13071878 | DOI:10.1021/acs.jmedchem.5c02876