Rachita Dash

FEMS Yeast Res. 2025 Jan 30;25:foaf069. doi: 10.1093/femsyr/foaf069.

ABSTRACT

Cyclic disulfide-rich peptides have become increasingly popular in drug development because their structures enhance molecular stability and allow for mutagenesis to introduce non-native functions. This review focuses on yeast-based platform technologies and their utility in advancing cyclic disulfide-rich peptides as drug modalities and for large-scale biomanufacturing. These technologies include yeast surface display which facilitates the screening of large libraries to develop peptide binders with strong affinity and selectivity for protein targets, while maintaining the innate high stability of the peptide scaffold via protease-based selection pressure. We also describe a recently developed platform that leverages yeast's ability to secrete correctly folded disulfide-rich peptides while simultaneously displaying peptide or protein tags on their surfaces. In combination with microfluidics technology, the platform creates single-cell yeast-in-droplets reactors, enabling the screening of large libraries based on functional output rather than solely on binding affinity. After identifying cyclic peptide candidates through library-based discovery, these candidates can be produced using a versatile yeast-based bioproduction platform. Traditionally, cyclic disulfide-rich peptides are produced through solid-phase synthesis, a method that generates significant amounts of toxic waste. In contrast, yeast-based bioproduction offers an environmentally sustainable alternative. It has the capability to produce structurally distinct peptides with minimal adjustments and is easily scalable using microbial fermenters, making it an ideal choice for large-scale production.

PMID:41410484 | PMC:PMC12715861 | DOI:10.1093/femsyr/foaf069

Chemistry. 2026 Jan 2;32(1):e02315. doi: 10.1002/chem.202502315. Epub 2025 Nov 29.

ABSTRACT

Bioorthogonal chemistry has emerged as a transformative strategy for detecting and quantifying biomolecules in complex biological systems. This review highlights recent advances in catalyst-free bioorthogonal reactions specifically applied to semi-quantitative and quantitative biomolecular analysis. We exclude reactions that require toxic or complex catalysts and focus on four reactions: Staudinger ligation, strain-promoted azide-alkyne cycloaddition, inverse electron-demand Diels-Alder reaction, and 2-cyanobenzothiazole-cysteine condensation. For each, we discuss reaction kinetics and strategies for representative applications in biomolecular quantification. The scope of target biomolecules varies by reaction, including proteins, nucleic acids, glycans, and small molecules. Quantification techniques such as fluorescence spectroscopy, luminescence spectroscopy, and mass spectrometry are examined, with reported limits of detection typically ranging from nanomolar to micromolar, and a few advanced techniques reaching femtomolar or attomolar sensitivity. Each reaction is discussed in terms of kinetics, molecular compatibility, and analytical sensitivity. Finally, we outline key challenges and future opportunities, emphasizing the need for faster reaction kinetics, improved probe design, enhanced integration with advanced analytical platforms, and standardized methods to improve reproducibility and cross-study comparability in biomolecular quantification.

PMID:41318924 | PMC:PMC12759176 | DOI:10.1002/chem.202502315

ACS Infect Dis. 2025 Dec 12;11(12):3391-3393. doi: 10.1021/acsinfecdis.5c00956. Epub 2025 Nov 25.

ABSTRACT

Bacterial glycans are complex and often presented on the surface of the cell as a level of protection. These glycans offer an opportunity to screen for new antibiotic targets and immunological markers. Here recent developments in the field of glycan arrays are presented as opportunities to advance therapies for human health.

PMID:41287554 | PMC:PMC13078616 | DOI:10.1021/acsinfecdis.5c00956

bioRxiv [Preprint]. 2025 Oct 16:2025.10.16.682811. doi: 10.1101/2025.10.16.682811.

ABSTRACT

Glycosylation is instrumental in governing essential biological processes that dictate health outcomes. Herein we describe a mini-tetrazine N-acetylglucosamine (HTz-GlcNAc) that is incorporated into N-linked glycans and through rapid live-cell labeling with trans-cyclooctene (TCO) reagents enables real-time visualization and evaluation of glycoconjugate trafficking in living cells. We utilize cell-permeable and impermeable TCO-fluorophores to perform time-resolved, multicolor live labeling of cell-surface glycoconjugates, as well as intracellular, and internalized glycoconjugates. We observe intracellular glycoconjugate trafficking throughout the secretory pathway of live cells in real-time, as well as cell surface glycan turnover and extracellular vesicle secretion across three days. In the toolkit of probes for metabolic oligosaccharide engineering, HTz-GlcNAc brings the capability of precise interrogation of intracellular and extracellular glycoconjugate trafficking, and selective tagging of intra-and-extracellular vesicles, while providing a handle for the purification and enrichment of glycans for immunoblot and proteomic analyses.

PMID:41279695 | PMC:PMC12633079 | DOI:10.1101/2025.10.16.682811

Structure. 2026 Feb 5;34(2):284-295.e5. doi: 10.1016/j.str.2025.10.018. Epub 2025 Nov 24.

ABSTRACT

Muscle-specific receptor tyrosine kinase (MuSK) is a single-pass transmembrane protein expressed on skeletal muscle. MuSK is activated by binding of nerve-derived agrin with the help of muscle coreceptor LRP4, leading to the clustering of acetylcholine receptors (AChR), which is required for the formation and maintenance of functional neuromuscular junctions. The structural mechanism of MuSK activation by physiological and artificial agonistic agents has remained elusive. In this study, we isolated a 27-residue linear peptide (L1) that binds human MuSK with high affinity. Genetic fusion of L1 to either the N or C termini of the human IgG Fc resulted in two different versions of MuSK dimerizers, denoted as L1-Fc and Fc-L1. Only Fc-L1 activated MuSK on myotubes and induced AChR clustering. Crystallographic analysis of MuSK-L1 interactions revealed that MuSK activation requires a particular dimeric conformation, pointing toward the importance of the lateral size of the receptor complex at the muscle cell surface.

PMID:41290004 | DOI:10.1016/j.str.2025.10.018

bioRxiv [Preprint]. 2025 Oct 16:2025.10.16.682811. doi: 10.1101/2025.10.16.682811.

ABSTRACT

Glycosylation is instrumental in governing essential biological processes that dictate health outcomes. Herein we describe a mini-tetrazine N-acetylglucosamine (HTz-GlcNAc) that is incorporated into N-linked glycans and through rapid live-cell labeling with trans-cyclooctene (TCO) reagents enables real-time visualization and evaluation of glycoconjugate trafficking in living cells. We utilize cell-permeable and impermeable TCO-fluorophores to perform time-resolved, multicolor live labeling of cell-surface glycoconjugates, as well as intracellular, and internalized glycoconjugates. We observe intracellular glycoconjugate trafficking throughout the secretory pathway of live cells in real-time, as well as cell surface glycan turnover and extracellular vesicle secretion across three days. In the toolkit of probes for metabolic oligosaccharide engineering, HTz-GlcNAc brings the capability of precise interrogation of intracellular and extracellular glycoconjugate trafficking, and selective tagging of intra-and-extracellular vesicles, while providing a handle for the purification and enrichment of glycans for immunoblot and proteomic analyses.

PMID:41279695 | PMC:PMC12633079 | DOI:10.1101/2025.10.16.682811

J Am Chem Soc. 2025 Dec 10;147(49):45495-45505. doi: 10.1021/jacs.5c16108. Epub 2025 Nov 24.

ABSTRACT

The widespread expression of therapeutic targets in both diseased and healthy tissues poses a major challenge for protein-based therapeutics, often leading to dose-limiting side effects. One promising strategy to enhance selectivity is reversible inactivation via affinity masks tethered through cleavable linkers responsive to disease-specific cues. Here, we introduce a workflow for the de novo design of peptide masks that reversibly inactivate miniprotein binders. By extending the C-terminus of the binder with a protease-cleavable linker and a masking helix, we generated minimal constructs that sterically block the receptor-binding interface. We applied this strategy to four therapeutically relevant targets, EGFR domains I and III, FGFR2, and IL7Rα, demonstrating broad applicability. Nearly half of the 20 designs achieved >100-fold affinity reduction, with the most effective mask decreasing EGFR binding by over 3 orders of magnitude. Upon cleavage by tumor-associated proteases, binding was restored in 19 out of 20 cases, confirming reversibility. We further show that micromolar or weaker affinity between the binder and the isolated mask is sufficient for robust inactivation and rapid activation. Additionally, by chemically conjugating a photocleavable linker, we created a light-responsive version of the masked binder, enabling external control with comparable efficiency to protease-sensitive designs. This work establishes a generalizable, rapid, and efficient platform for designing cleavable peptide masks from scratch, paving the way for conditionally active protein therapeutics responsive to endogenous or exogenous stimuli.

PMID:41284265 | PMC:PMC12703739 | DOI:10.1021/jacs.5c16108

Angew Chem Int Ed Engl. 2026 Jan 9;65(2):e17689. doi: 10.1002/anie.202517689. Epub 2025 Nov 23.

ABSTRACT

Macrocyclic peptides are promising scaffolds for drug discovery due to their structural rigidity and high target specificity. Here, we report a strategy for in vitro ribosomal translation of thioisoindole-bridged bicyclic peptides. Central to this approach is a newly developed flexizyme substrate, Ac-Ala(NtBA)^Sc-CME, which features a semicarbazone-masked 2-nicotinoyl benzaldehyde sidechain. We show that this amino acid can be efficiently charged onto tRNA with flexizyme and incorporated into ribosomal peptides using a customized flexible in vitro translation (FIT) system. The semicarbazone group can be post-translationally removed under mild conditions, triggering spontaneous intramolecular cyclization to cysteine and lysine sidechains in the same substrate to yield thioisoindole-bridged bicyclic (TiB) peptides. This strategy was leveraged to synthesize structurally diverse bicyclic peptides with varying sequences and ring sizes. The method maintains the integrity of mRNA and is therefore compatible with mRNA display, which opens the possibility of constructing topologically defined bicyclic peptide libraries for therapeutic peptide discovery.

PMID:41277055 | PMC:PMC12790353 | DOI:10.1002/anie.202517689

Methods Enzymol. 2025;723:195-229. doi: 10.1016/bs.mie.2025.08.021. Epub 2025 Sep 23.

ABSTRACT

Cyclic peptides are a class of ligands readily discovered from nucleic acid-encoded libraries by affinity-based screening methodologies developed in recent years. Advancing these cyclic peptide ligands into drug candidates has been limited, often due to the peptide's poor pharmaco-dynamic and -kinetic properties such as cell permeability and metabolic stability. However, their broad protein surface complementarity and straightforward discovery methods make cyclic peptides ideal assay ligand probes to enable high throughput screening (HTS) assays against protein targets that were traditionally difficult or prohibitive to screen. In this chapter, we will discuss the discovery and characterization of macrocyclic peptide ligands and their development and utilization as assay probes in a variety of ligand-displacement assays. We will cover two examples where prior assay designs were either inefficient or intractable for HTS, co-factor independent phosphoglycerate mutases (iPGM) and the secreted Mycobacterium tuberculosis chorismate mutase (*MtbCM). In the context of these enzymes, we will describe eight different ligand-displacement assay formats, including direct 'one-component' assays such as fluorescence binding and fluorescence polarization assays as well as more complex 'two-component' assays such as laser scanning cytometry, homogeneous time-resolved fluorescence (HTRF), and NanoLuc luciferase bioluminescence resonance energy transfer (NanoBRET).

PMID:41266032 | DOI:10.1016/bs.mie.2025.08.021

ACS Nano. 2025 Dec 2;19(47):40293-40303. doi: 10.1021/acsnano.5c11721. Epub 2025 Nov 20.

ABSTRACT

Endosomal escape remains a critical bottleneck for the intracellular delivery of nucleic acids by lipid nanoparticles (LNPs), largely due to its low efficiency and poorly understood mechanism. While various models, including proton sponge effect/osmotic lysis and membrane destabilization/fusion, have been proposed, none are fully validated or sufficient for guiding rational LNP design. Herein, I reevaluate existing data, presenting strong evidence that LNPs escape the endosomal compartment through the recently discovered vesicle budding-and-collapse (VBC) mechanism. A critical subsequent finding is that endosomal escape triggers the formation of an insoluble lipid/nucleic acid aggregate within the cytoplasm. The slow dissolution of this aggregate emerges as an additional, potentially rate-limiting, bottleneck to functional nucleic acid delivery. By reconciling previously puzzling experimental observations, the VBC mechanism provides a powerful theoretical framework for the rational design of LNPs with enhanced endosomal escape and overall functional delivery efficiencies.

PMID:41263288 | PMC:PMC12848894 | DOI:10.1021/acsnano.5c11721

bioRxiv [Preprint]. 2025 Sep 30:2025.09.29.678898. doi: 10.1101/2025.09.29.678898.

ABSTRACT

Phosphorylation on tyrosine is a key step in many signaling pathways. Despite recent progress in de novo design of protein binders, there are no current methods for designing binders that recognize phosphorylated proteins and peptides; this is a challenging problem as phosphate groups are highly charged, and phosphorylation often occurs within unstructured regions. Here we introduce RoseTTAFold Diffusion 2 for Molecular Interfaces (RFD2-MI), a deep generative framework for the design of binders for protein, ligand, and covalently modified protein targets. We demonstrate the power and versatility of this method by designing binders for four critical phosphotyrosine sites on three clinically relevant targets: Cluster of Differentiation 3 (CD3ε), Epidermal Growth Factor Receptor (EGFR) and Insulin Receptor (INSR). Experimental characterization shows that the designs bind their phospho-tyrosine containing targets with affinities comparable to native binding sites and have negligible binding to non-phosphorylated targets or phosphopeptides with different sequences. X-ray crystal structures of generated binders to CD3ε and EGFR are very close to the design models, demonstrating the accuracy of the design approach. RFD2-MI provides a generalizable all-atom diffusion framework for probing and modulating phosphorylation-dependent signaling, and more generally, for developing research tools and targeted therapeutics against post-translationally modified proteins.

PMID:41256494 | PMC:PMC12621789 | DOI:10.1101/2025.09.29.678898

Nutrients. 2025 Nov 6;17(21):3496. doi: 10.3390/nu17213496.

ABSTRACT

Non-proteinogenic amino acids, such as L-citrulline and L-theanine, have garnered attention for their potential health benefits, including enhanced immunity, antioxidant activity, and cardiovascular support. The application of natural amino acids in disease treatment and health supplementation is and will remain a research hotspot in pharmaceutics. Plant-derived L-citrulline and L-theanine have demonstrated multifaceted benefits, primarily through mechanisms involving nitric oxide (NO) bioavailability (for L-citrulline) and mitochondrial regulation or immune modulation (for both). Critical gaps are identified: (1) the role of D-amino acids (e.g., D-citrulline and D-theanine) in health and metabolism remains underexplored, particularly regarding chiral-specific bioactivity; (2) derivatives and co-administration strategies of L-forms warrant systematic evaluation for drug. However, while these compounds show promise, evidence is predominantly from animal and cell studies, with limited long-term human data on efficacy and safety. Potential side effects, dosing limitations, and sourcing challenges are discussed. This review emphasizes the need for cautious interpretation of their benefits, acknowledging that while promising, some effects, such as those on muscle protein synthesis, require further validation compared to established nutrients like branched-chain amino acids. By bridging mechanistic insights with translational challenges, this work aims to guide future research toward sustainable nutraceutical production.

PMID:41228568 | PMC:PMC12610250 | DOI:10.3390/nu17213496

J Am Chem Soc. 2025 Nov 26;147(47):43330-43341. doi: 10.1021/jacs.5c08850. Epub 2025 Nov 13.

ABSTRACT

Mutanobactin D is an interkingdom communicator derived from the human oral microbiome. The lipopeptide prevents yeast-to-hyphae morphogenesis in Candida albicans, notably without fungicidal or fungistatic activity. The mode of action and structure-activity relationship of mutanobactin D are unknown and prompt an interdisciplinary program of study. Stereoselective synthesis of designed mutanobactin D analogs reveals that the C26 configuration is crucial for bioactivity associated with inhibition of pathogenesis, or yeast-to-hyphae transition, in C. albicans. To shed light on this finding, we employ molecular dynamics (MD) simulations of mutanobactin D and selected analogs in increasingly complex environments: Monophasic (water or CHCl₃), interfacial (water/CHCl₃), and explicit lipid membrane (phosphatidylcholine) models. Monophasic MD simulations do not distinguish between bioactive and inactive compounds. In contrast, at a polar/apolar interphase, a dominant, stable conformation emerges for mutanobactin D and bioactive analogs. Explicit lipid membrane simulations reinforce these results and further reveal the formation of a continuous, structured water cushion, which is not found for inactive analogs. Our studies collectively reveal how the stereodefined attachment of the lipid in the C26-C28 motif governs activity against C. albicans and provide a framework for understanding the membrane behavior of mutanobactin D, which may be coupled to its role in the human oral microbiome. The approach described herein, consisting of synthesis and evaluation of designed analogs complemented by MD simulations, provides a blueprint for the study of bioactive natural products in various contexts, including the human microbiome.

PMID:41231122 | DOI:10.1021/jacs.5c08850

Anal Chem. 2025 Nov 25;97(46):25928-25934. doi: 10.1021/acs.analchem.5c06327. Epub 2025 Nov 12.

ABSTRACT

The invasion and intracellular survival of Staphylococcus aureus (S. aureus) within phagocytic cells is a major cause of serious antibiotic resistance and recurrent infections in clinical settings, making the detection of intracellular S. aureus highly desirable prior to initiating effective treatment. In this study, we proposed a dual-targeting and cascaded aggregation-induced emission luminogen (AIEgen) probe for washing-free and enhanced fluorescence imaging of intracellular S. aureus. The AIEgen probes consist of an AIE molecule (TPE), cleavable peptide (YVADC(StBu)GKK), targeting peptide (RP)₃, and the disulfide Cys(StBu) and 2-cyanobenzothiazole (CBT) units, YVADC(StBu)GK(TPE)K(RP)₃G-CBT (YTR-CBT). Upon encountering elevated levels of intracellular biomarkers (GSH and Caspase-1) associated with bacterial infection, the YTR-CBT probe undergoes a CBT-Cys click reaction, triggering its self-assembly into AIEgen nanoparticles (TR-CBT-NPs) and consequently activating the AIEgen fluorescence. These nanoparticles then undergo cascaded aggregation on the surface of S. aureusvia the (RP)₃ targeting unit, leading to a significantly enhanced fluorescence signal around the intracellular bacteria compared to that of control groups lacking either the targeting unit or bacterial infection. Moreover, the AIEgen probe specifically imaged intracellular bacteria without interference from extracellular bacteria, enabling a washing-free imaging strategy. This capability allowed for rapid monitoring of therapeutic efficacy in a mouse model of septic arthritis involving intracellular bacteria, demonstrating its considerable potential for clinical application.

PMID:41222298 | DOI:10.1021/acs.analchem.5c06327

J Am Chem Soc. 2025 Nov 19;147(46):42524-42531. doi: 10.1021/jacs.5c13391. Epub 2025 Nov 4.

ABSTRACT

Affinity-driven reactions have allowed chemists to perform site-selective modifications of native proteins. By combining the high cysteine chemoselectivity of hypervalent iodine-based ethynylbenziodoxolones (EBXs) with the site selectivity of peptide ligands known to inhibit protein-protein interactions, we achieved site-selective labeling of Cys434 in the KELCH domain of Kelch-like epichlorohydrin-associated protein 1 (KEAP1), a key protein in the regulation of oxidative stress. EBXs could be used either as traceless reagents with release of the peptide ligand to introduce reactive handles such as azides or alkynes or as covalent reagents leading to the formation of peptide-protein adducts, which could be cleaved in a separated step.

PMID:41187164 | PMC:PMC12643532 | DOI:10.1021/jacs.5c13391