Rachita Dash

Biochemistry. 2026 Apr 7;65(7):985-993. doi: 10.1021/acs.biochem.6c00003. Epub 2026 Mar 11.

ABSTRACT

Cell-penetrating peptides (CPPs) are powerful tools for delivering membrane-impermeable biomolecules into eukaryotic cells, with broad applications ranging from therapeutics to biopesticides. However, conventional linear CPPs typically require a high density of positive charges (at least +6) to function, often resulting in dose-limiting toxicity and off-target effects. Reducing this charge without sacrificing delivery efficiency remains a significant challenge. In this study, we performed a structure-activity relationship (SAR) analysis and medicinal chemistry optimization of the bismuth-mediated bicyclic CPP, BCP16. This campaign led to the discovery of BCP16e, a potent analog that carries only a +2 charge at physiological pH. Compared to its parent molecule, BCP16e exhibits significantly higher cytosolic entry efficiency, similar proteolytic stability, and a superior safety profile. Our findings demonstrate that high cationic charge is not a prerequisite for efficient translocation, providing a framework for the design of minimally charged, high-efficiency vehicles for intracellular delivery.

PMID:41810515 | PMC:PMC13043234 | DOI:10.1021/acs.biochem.6c00003

J Am Chem Soc. 2026 Mar 25;148(11):12154-12165. doi: 10.1021/jacs.6c00367. Epub 2026 Mar 13.

ABSTRACT

Streptothricin (ST) antibiotics are promising agents against multidrug-resistant pathogens and are structurally classified into two groups, containing either a β-lysyl or a glycyl pendant moiety attached via an amide bond to an amino sugar core. These pendant moieties are essential determinants of the biological activity and selective toxicity of ST antibiotics. We previously demonstrated that during ST biosynthesis, the β-lysyl pendant moiety is installed by nonribosomal peptide synthetases, whereas the glycyl pendant moiety is generated by a Gly-tRNA^Gly-dependent amide-forming enzyme. Here, we present a chemoenzymatic approach to transform aminoacyl pendant moieties using the promiscuous tRNA-dependent amide-forming enzyme Sba18. Remarkably, Sba18 generates two new ST derivatives, alanylthricin and serylthricin, by utilizing Ala-tRNA^Ala and Ser-tRNA^Ser, respectively. Moreover, Sba18 accepts aminoacyl-tRNA mimics prepared by flexizyme-mediated charging of chemically synthesized aminoacyl groups and produces additional 11 ST derivatives. Alanylthricin and serylthricin retain antibiotic activity, demonstrating that this tRNA-dependent chemoenzymatic approach provides a viable strategy for expanding the structural diversity of streptothricin antibiotics. Furthermore, structural comparison of Sba18 with its Gly-tRNA^Gly-specific orthologue Sbb17 elucidates the catalytic and substrate-recognition mechanisms underlying the broad specificity of Sba18. These structural insights provide a foundation for expanding the structural diversity not only of ST antibiotics but also of other peptide natural products biosynthesized using aminoacyl-tRNAs.

PMID:41823201 | DOI:10.1021/jacs.6c00367

Proc Natl Acad Sci U S A. 2026 Mar 24;123(12):e2520462123. doi: 10.1073/pnas.2520462123. Epub 2026 Mar 16.

ABSTRACT

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a Cytosine-Adenosine-Guanine (CAG) repeat expansion in the Huntingtin (HTT) gene, with no disease-modifying therapies currently available. The precise molecular function of the HTT protein is unclear, and the lack of selective chemical tools has limited functional studies. We have identified and characterized macrocyclic peptide binders targeting HTT. These binders exhibit low-nanomolar affinity in vitro and engage distinct HTT and HTT-HAP40 interfaces, as revealed by hydrogen-deuterium exchange mass spectrometry and cryoelectron microscopy. Chemoproteomics confirmed selective binding in cell extracts from wildtype but not HTT-null cell lines. HAP40 consistently and stoichiometrically copurified with HTT across cell lines, including with HTT variants containing different CAG repeat lengths, highlighting the broad presence of the HTT-HAP40 complex.

PMID:41838909 | PMC:PMC13012115 | DOI:10.1073/pnas.2520462123

J Am Chem Soc. 2026 Apr 1;148(12):13174-13185. doi: 10.1021/jacs.5c23173. Epub 2026 Mar 17.

ABSTRACT

Although numerous strategies for the identification of biologically active peptides are available, the methodologies to discover functional peptide pairs remain relatively scarce. The pairing of two combinatorial peptide libraries can furnish very large chemical spaces, which can be leveraged in ligand discovery to identify potent binders for proteins of interest. Here, we report the development of a library-vs-library in vitro selection platform for the discovery of heterodimeric macrocyclic peptide ligands. The platform is built upon the Random nonstandard Peptides Integrated Discovery (RaPID) system and utilizes protein-templated ligation of peptides from two mRNA display libraries to select for functional heterodimers. We report the method development and demonstrate the utility of the resulting protocols by identifying an ultrapotent heterodimeric ligand of 14-3-3ζ protein (h1.2, K_D = 120 pM), which forms via a proximity-enabled inverse electron demand Diels-Alder reaction of two cyclic peptide monomers upon binding to the protein. The formation of h1.2 from a1 and b2 monomers was accelerated 560-fold in the presence of 14-3-3ζ, and the heterodimer had a superior affinity compared to the constituent monomers [K_D(a1) = 430 pM and K_D(b2) = 700 pM]. Our strategy may be useful in drug discovery to develop high-affinity ligands against oligomeric proteins, targets without prominent binding pockets, or for disrupting protein-protein interactions characterized by large interaction areas.

PMID:41841560 | PMC:PMC13048255 | DOI:10.1021/jacs.5c23173

J Med Chem. 2026 Mar 3. doi: 10.1021/acs.jmedchem.6c00507. Online ahead of print.

ABSTRACT

AI-based multiparameter optimization for membrane permeability of cyclic peptides is hampered by the limited availability of ground truth biological data. This Viewpoint highlights the Featured Article reporting an AI model, integrating a high-throughput assay classifying billions of cyclic peptides for permeability-related characteristics. Combining this with a generative AI, the first de novo designed permeable cyclic peptides containing polar groups were reported.

PMID:41774122 | DOI:10.1021/acs.jmedchem.6c00507

Nature. 2026 Apr;652(8111):1049-1059. doi: 10.1038/s41586-026-10323-y. Epub 2026 Mar 3.

ABSTRACT

Cells constantly change their molecular state in response to internal and external cues¹. Mapping cellular activity in tissues with spatiotemporal precision is essential for understanding organ physiology, pathology and regenerative processes. Current cell-sensing modalities primarily rely on either end point analysis that takes static snapshots² or real-time sensing that monitors a small subset of cells^3,4. Here we introduce granularly expanding memory for intracellular narrative integration (GEMINI), an in cellulo recording platform that leverages a computationally designed protein assembly as an intracellular memory device to record the history of individual cells. GEMINI grows predictably within live cells, capturing cellular events as tree-ring-like fluorescent patterns for imaging-based retrospective readout. Absolute chronological information of activity histories is attainable with hour-level accuracy. GEMINI effectively maps differential NF-κB-mediated transcriptional changes, resolving fast dynamics of 15 min and providing quantifiable signal amplitudes. In a xenograft model, GEMINI records inflammation-induced signalling dynamics across tissue, revealing spatial heterogeneity linked to vascular density. When expressed in the mouse brain, GEMINI minimally impacts neuronal functions and can resolve both transcriptional changes and activity patterns of neurons. Together, GEMINI provides a robust and generalizable means for spatiotemporal mapping of cell dynamics underlying physiological and pathological processes in both culture and intact tissues.

PMID:41775935 | PMC:PMC13102709 | DOI:10.1038/s41586-026-10323-y

PNAS Nexus. 2026 Feb 13;5(2):pgag031. doi: 10.1093/pnasnexus/pgag031. eCollection 2026 Feb.

ABSTRACT

Herein, we report a bead-surface protein display method based on a peptidyl transferase reaction, termed peptide ligase-mediated display (PL display). This technique enables the covalent linkage of genotypic DNA and phenotypic protein variants on beads via a minimal nine-amino acid linker in a fully cell-free system. Using this method, hemagglutinin (HA)-tag sequences introduced at a 0.01% frequency were completely isolated in a single round of selection via fluorescence-activated cell sorting (FACS) against an anti-HA-tag antibody. Furthermore, consensus sequences that bind to the anti-HA-tag antibody were enriched from a random peptide library with a sequence diversity of 1.7 × 10⁶ in two rounds of selection using FACS. This quantitative affinity selection platform using PL display is applicable under diverse conditions, as it is not constrained by cellular physiological properties, fluctuations in gene expression, or the structural and functional limitations of linker proteins involved in genotype-phenotype linkage. These advantages arise from the use of a fully cell-free system and covalent linkage with a minimal linker.

PMID:41756107 | PMC:PMC12934352 | DOI:10.1093/pnasnexus/pgag031

bioRxiv [Preprint]. 2026 Feb 16:2026.02.13.705776. doi: 10.64898/2026.02.13.705776.

ABSTRACT

Antibodies and other protein therapeutics have revolutionized medicine, but their application is largely limited to extracellular targets. The lack of efficient intracellular delivery methods remains a major bottleneck. Here, we engineered a family of small (~90 amino acids), metabolically stable membrane translocation domains (MTDs) by modifying the loop sequences of a human fibronectin type III (FN3) domain. The most potent variant, MTD4, is highly cell-permeable and can be recombinantly fused to the N- or C-terminus of any peptide or protein, serving as a versatile "plug-and-play" vehicle. We demonstrate that MTD4 fusions efficiently deliver a wide variety of functional peptides and proteins into the cytosol and nucleus of eukaryotic cells, both in vitro and in vivo. Following systemic administration, MTD4 fusion proteins exhibit broad biodistribution and homogenous tissue penetration in mice. Importantly, MTD4 is effective at low nanomolar (nM) concentrations, making it a promising platform for addressing a vast range of intracellular and previously "undruggable" targets.

PMID:41756926 | PMC:PMC12934630 | DOI:10.64898/2026.02.13.705776

bioRxiv [Preprint]. 2026 Feb 22:2026.02.21.706835. doi: 10.64898/2026.02.21.706835.

ABSTRACT

Gene editing has been used to enhance CAR T-cell function by disrupting negative regulators but has limitations. Here we show that de novo-designed generated targeted degraders (bioPROTACs) provide an alternative approach. Expression of bioPROTACs in CAR T-cells targeting DNMT3A, a key regulator of T-cell exhaustion, phenocopied gene knockout. Our reversible, non-gene editing approach provides a tunable strategy to reprogram T-cell fate which should be broadly applicable for next-generation cell therapies.

PMID:41757106 | PMC:PMC12934639 | DOI:10.64898/2026.02.21.706835

Nat Commun. 2026 Feb 24;17(1):1963. doi: 10.1038/s41467-026-69891-2.

ABSTRACT

Recent work has demonstrated that some bacterial antibiotics that inhibit protein synthesis by binding the peptidyl transferase center (PTC) of the ribosome act in a context-dependent manner, inhibiting translation elongation only at specific amino acids. However, this phenomenon has yet to be documented for compounds that inhibit the PTC of the human ribosome. Here, we use structure-based design to guide the synthesis of such PTC-binding, context-dependent inhibitors of the human ribosome, termed interdictors. In the PTC, these compounds preferentially interact with nascent protein residues that exhibit complementary physiochemical properties to the moieties of the small molecule, causing structural rearrangements in both the nascent polypeptide chain and ribosomal RNA. Further, the compounds differentially impact ribosome surveillance pathways, including the ribotoxic stress response. Finally, we confirm their anti-tumor activity after oral dosing in a mouse xenograft model of triple-negative breast cancer. Together, our data establish targeting oncogenic dependency factors through context-dependent inhibition of translation as a potential small molecule therapeutic modality for historically difficult to address cancers.

PMID:41735331 | PMC:PMC12932737 | DOI:10.1038/s41467-026-69891-2

Biochemistry. 2026 Mar 3;65(5):571-578. doi: 10.1021/acs.biochem.5c00808. Epub 2026 Feb 18.

ABSTRACT

Asparaginyl ligases are powerful tools for peptide and protein engineering due to their ability to efficiently catalyze a variety of site-specific transpeptidation reactions. Although engineering efforts have enhanced the transpeptidation efficiency of several enzymes, attempts to modify their substrate specificity have been more limited. In a recent study, we produced the first asparaginyl ligase with engineered P2' substrate specificity by mutating Tyr188 to Ala in OaAEP1. Here, we report the engineering of two additional asparaginyl ligases from different plant families, VyPAL2 and butelase 1. We show that mutating the corresponding Tyr residue located in the S2' pocket of these enzymes also expands their substrate scope, enabling the mutant enzymes to process substrates for peptide cyclization, protein-protein ligation, and N-terminal protein labeling that their parent enzymes process poorly. These findings further establish the role of the conserved S2' Tyr residue as a general determinant of substrate specificity for asparaginyl ligases and provide a path toward more extensive engineering efforts.

PMID:41705341 | DOI:10.1021/acs.biochem.5c00808

J Am Chem Soc. 2026 Feb 25;148(7):7772-7781. doi: 10.1021/jacs.5c22349. Epub 2026 Feb 10.

ABSTRACT

Covalent inhibitors and chemical probes targeting ligandable cysteine residues have emerged as powerful tools for drug discovery and proteomics. In this study, we introduce vinyl phosphonamidates (VPAs) as a novel class of latent cysteine electrophiles and assess their reactivity, selectivity, and potential for developing covalent inhibitors. Compared to well-established chloroacetamide and acrylamide electrophiles, VPAs exhibit a significantly lower intrinsic reactivity toward the model thiol glutathione. Moreover, VPA-derived covalent fragments displayed only very limited nonspecific reactivity in human cell lysate. Encouraged by these results, we developed VPA-functionalized derivatives of the FDA-approved covalent inhibitors Afatinib and Ibrutinib and evaluated their ability to engage the target protein by gel-based and mass spectrometry-based activity-based protein profiling (ABPP). Compared to commonly employed Michael acceptor-based electrophilic groups, VPA-functionalized drug ligands displayed significantly less off-targets while maintaining inhibitor efficiency. Furthermore, we leveraged the modular nature and accessibility of VPAs to develop a bifunctional proteolysis targeting chimera (PROTAC) for targeted protein degradation. The demonstrated selectivity and modularity, as exemplified by the incorporation of various ligands on the phosphorus O-substituent, of the vinyl phosphonamidate group as a cysteine-directed electrophile highlight its ability to expand the chemical space in the development of covalent inhibitors with a favorable proteome-wide reactivity profile.

PMID:41667386 | PMC:PMC12951430 | DOI:10.1021/jacs.5c22349

J Phys Chem B. 2026 Feb 19;130(7):2031-2043. doi: 10.1021/acs.jpcb.5c06368. Epub 2026 Feb 5.

ABSTRACT

Cyclic peptides have gained interest as potential therapeutics due to their ability to target specific protein-protein interactions and be membrane-permeable. Understanding the sequence-structure relationship of cyclic peptides would greatly benefit their rational design. However, cyclic peptides tend to adopt multiple conformations in solution, and it remains challenging to use experimental techniques such as solution NMR to delineate their structural ensembles: i.e., the different structures a cyclic peptide adopts and the associated populations. Alternatively, molecular dynamics (MD) simulations can be used to provide such information. However, MD simulations are computationally expensive and not applicable for large-scale screening. Our group has developed the StrEAMM (Structural Ensembles Achieved by Molecular Dynamics and Machine Learning) computational platform and applied it to predict structural ensembles of head-to-tail cyclized pentapeptides and hexapeptides. However, head-to-tail cyclized peptides can be challenging to synthesize due to low yield and complicated reaction workup and product isolation. Furthermore, head-to-tail cyclized peptides are not compatible with screening techniques like mRNA display. Here, we expand the StrEAMM method to thioether-linked cyclic peptides, a popular scaffold in mRNA display. The trained graph neural network models are able to provide fast and simulation-quality structural ensembles for thioether-linked cyclic peptides. Using these models, we identify four thioether-linked cyclic pentapeptides that are predicted to be the best-structured and subsequently experimentally synthesize and characterize them by solution NMR. We observe general agreement between the predicted structures and the NMR results. Ultimately, we envision that StrEAMM-thioether models can work synergistically with the current mRNA platform to streamline the resource-intensive process of drug discovery and design of cyclic peptides.

PMID:41645422 | DOI:10.1021/acs.jpcb.5c06368

bioRxiv [Preprint]. 2026 Jan 17:2026.01.16.699845. doi: 10.64898/2026.01.16.699845.

ABSTRACT

Asparagine-linked protein glycosylation is among the most frequent modifications of proteins trafficking through the secretory pathway. These glycans are manufactured in an assembly line process to a common precursor that is then subject to individual modifications with different levels of complexity. An important biosynthetic modulator is the incorporation of N -acetylglucosamine (GlcNAc) at distinct positions in N-linked glycan biosynthesis, commencing with the activity of the glycosyltransferase MGAT1. While mapping of N-glycans to their corresponding protein attachment sites is generally possible, not much is known about the glycoprotein substrate choice for MGAT1 and related transferases. Analogs of GlcNAc with small bioorthogonal tags can be incorporated into N-glycans. However, due to the promiscuity of some GlcNAc transferases, incorporation is of little specificity towards individual positions. Here, we report an iterative bump-and-hole approach in the design of a bioorthogonal precision tool for the activity of MGAT1 in mammalian cells. Structure-informed protein engineering abrogated the activity of MGAT1 towards the nucleotide-sugar UDP-GlcNAc while retaining activity towards bumped, azide-modified analogs. Kinetic and computational analyses using a neural network approach informed the synthesis of a tailored UDP-GlcNAc analog with preferential acceptance by the engineered enzyme. Following substrate biosynthesis, the strategy allowed selective incorporation of a chemical tag on MGAT1 substrate proteins in living mammalian cells with little background incorporation by other GlcNAc transferases. Our work expands the toolbox for glycan-based reporter compounds.

PMID:41648446 | PMC:PMC12871145 | DOI:10.64898/2026.01.16.699845

Angew Chem Int Ed Engl. 2026 Mar 2;65(10):e14067. doi: 10.1002/anie.202514067. Epub 2026 Jan 28.

ABSTRACT

Rhomboid proteases, a class of intramembrane proteases characterized by a Ser-His catalytic dyad, have recently emerged as promising therapeutic targets. While inhibitors for soluble serine proteases have been extensively studied, the spectrum of potent rhomboid protease inhibitor chemotypes is limited to active-site targeted nucleophiles. To address this limitation, we conducted a high-throughput screen of over 68,000 compounds targeting the E. coli rhomboid protease GlpG, using a fluorescent liposome-based assay. A selection of 326 inhibitory compounds was evaluated in a subsequent IC₅₀ screen against two variants of GlpG (core domain and full length), a soluble serine protease (chymotrypsin), as well as the human mitochondrial rhomboid PARL. Of these, the selective inhibitory effects of 2 compounds and their analogues on GlpG were confirmed through further biochemical and biophysical characterisation, molecular docking, and solid-state NMR spectroscopy. This study paves the way for developing small-molecule tool compounds and drug-like molecules targeting rhomboid proteases.

PMID:41607018 | PMC:PMC12955534 | DOI:10.1002/anie.202514067

J Am Chem Soc. 2026 Feb 11;148(5):4777-4790. doi: 10.1021/jacs.5c10593. Epub 2026 Jan 28.

ABSTRACT

Lasso peptides have emerged as a promising class of ribosomally synthesized and post-translationally modified peptide natural products, with entangled slipknot-like structures produced through multienzyme biosynthetic pathways. Their threaded structure provides exceptional thermal and proteolytic stability, along with diverse biological activities, especially against infectious diseases and cancer. Over the past two decades, making the native lasso fold through chemical synthesis has been considered a paramount challenge that is not yet solved. Nevertheless, in recent years, there has been significant progress in overcoming technical barriers associated with the discovery and development of lasso peptides that unlock their broader potential. In this perspective, we outline recent advances in the production of lasso peptides via biosynthesis and chemical synthesis approaches, as well as late-stage modification strategies to introduce chemical diversity onto recombinantly produced lasso peptides. We conclude by discussing remaining challenges and opportunities for further advances in this field.

PMID:41604624 | DOI:10.1021/jacs.5c10593

Nat Commun. 2026 Jan 24;17(1):2008. doi: 10.1038/s41467-026-68717-5.

ABSTRACT

Gram-negative bacterial pathogens pose a significant challenge in drug development because their outer membranes hinder the permeation of small molecules. The lack of widely adoptable methods for measuring the cytosolic accumulation of compounds in bacterial cells further hinders drug discovery efforts. To address this challenge, we report the development of the Chloroalkane Azide Membrane Permeability (CHAMP) assay, which we designed specifically to assess molecule accumulation in the cytosol of Gram-negative bacteria. The CHAMP analysis utilizes bioorthogonal epitopes anchored within HaloTag-expressing bacteria and measures the cytosolic arrival of azide-bearing test molecules through strain-promoted azide-alkyne cycloaddition. This workflow enables robust and rapid accumulation measurements of thousands of azide-tagged small molecules. Our approach consistently produces comprehensive accumulation profiles that surpass the scale of previous measurements in Escherichia coli (E. coli). We validated the CHAMP assay across various chemical and biological contexts, including hyperporinated cells, membrane-permeabilized cells, and E. coli strains with impaired TolC function, a key component of the efflux pump. The CHAMP platform provides a simple, high-throughput, and accessible method that enables the analysis of over 1000 molecules within hours. This technique addresses a critical gap in antimicrobial research and has the potential to accelerate the development of effective agents against Gram-negative pathogens.

PMID:41580407 | PMC:PMC12936067 | DOI:10.1038/s41467-026-68717-5