Rachita Dash

ACS Phys Chem Au. 2025 Mar 10;5(2):134-138. doi: 10.1021/acsphyschemau.4c00103. eCollection 2025 Mar 26.

ABSTRACT

Differing in pseudoatom, three macrocycles with isosteric substitutions (geminal dimethyl, cyclopropyl, cyclobutyl) can be described as choreoisosteres. Under ambient conditions, they share a dynamic hinge-like motion that can be described as fully revolute in solution. The barriers to hinging, ΔG ^‡, are identical within experimental error: ΔG ^‡ = 14.2-15.2 kcal/mol as judged by variable-temperature ¹³C NMR spectroscopy. Consistent with conserved dynamic behavior and isosterism, other physical properties including hydrophobicity and solution/membrane diffusion constants are amenable to prediction.

PMID:40160939 | PMC:PMC11950847 | DOI:10.1021/acsphyschemau.4c00103

Proc Natl Acad Sci U S A. 2025 Apr;122(13):e2415845122. doi: 10.1073/pnas.2415845122. Epub 2025 Mar 28.

ABSTRACT

The translocation of bacteria from intestinal tracts into blood vessels and distal organs plays pivotal roles in the pathogenesis of numerous severe diseases. Intravital monitoring of bacterial translocation, however, is not yet feasible, which greatly hinders us from comprehending this spatially and temporally dynamic process. Here we report an in vivo fluorogenic labeling method, which enables in situ imaging of mouse gut microbiota and real-time tracking of the translocated bacteria. By mimicking the peptidoglycan stem peptide in bacteria, a tetrapeptide probe composed of alternating D- and L-amino acids and separately equipped with a fluorophore and a quencher on the N- and C-terminal amino acid, is designed. Because of its resistance to host proteases, it can be directly used in gavage and achieves fluorogenic labeling of the microbiota in the gut via the functioning of the L,D-transpeptidases of the labeled bacteria. Using intravital two-photon microscopy, we then successfully visualize the translocation of gut bacteria into the bloodstream and liver in obesity mouse models. This technique can help further exploration into the spatiotemporal activities of gut microbiota in vivo, and be valuable in investigating the less understood pathogenicity of bacterial translocation in many severe diseases.

PMID:40153461 | PMC:PMC12002288 | DOI:10.1073/pnas.2415845122

JACS Au. 2025 Feb 16;5(3):1299-1307. doi: 10.1021/jacsau.4c01148. eCollection 2025 Mar 24.

ABSTRACT

Histone deacetylase (HDAC) enzymes are epigenetic regulators that affect diverse protein function by removing acyl groups from lysine side chains throughout the proteome. The most recently discovered human isozyme, HDAC11, differs from other HDACs in substrate preference and tissue expression profile. Elucidation of the biological function of this enzyme has been scarce and only a few chemical probes to help advance this insight have been developed thus far. Here we discovered macrocyclic inhibitors that exhibit selectivity for HDAC11 and penetrate the cytoplasmic membrane in cultured cells as determined by the chloroalkane penetration assay. Our work establishes the combination of de novo macrocycle synthesis with incorporation of N-alkylated hydroxamic acid moieties as a viable strategy for targeting HDAC11. Further, this study demonstrates the potential of applying macrocyclic peptide-based library synthesis to directly furnish high-affinity, cell-permeating ligands. The discovered inhibitors comprise tool compounds for the investigation of the biological function of HDAC11.

PMID:40151233 | PMC:PMC11938020 | DOI:10.1021/jacsau.4c01148

Nat Biotechnol. 2025 May;43(5):702-711. doi: 10.1038/s41587-025-02592-1. Epub 2025 Mar 26.

ABSTRACT

Molecular proximity is a governing principle of biology that is essential to normal and disease-related biochemical pathways. At the cell surface, protein-protein proximity regulates receptor activation, inhibition and protein recycling and degradation. Induced proximity is a molecular engineering principle in which bifunctional molecules are designed to bring two protein targets into close contact, inducing a desired biological outcome. Researchers use this engineering principle for therapeutic purposes and to interrogate fundamental biological mechanisms. This Review focuses on the use of induced proximity at the cell surface for diverse applications, such as targeted protein degradation, receptor inhibition and activating intracellular signaling cascades. We see a rich future for proximity-based modulation of cell surface protein activity both in basic and translational science.

PMID:40140559 | PMC:PMC13041533 | DOI:10.1038/s41587-025-02592-1

Proc Natl Acad Sci U S A. 2025 Apr;122(13):e2426006122. doi: 10.1073/pnas.2426006122. Epub 2025 Mar 25.

ABSTRACT

Macrocyclic peptides have emerged as promising drug candidates, filling the gap between small molecules and large biomolecules in drug discovery. The antiapoptotic protein myeloid cell leukemia 1 (MCL1) is crucial for numerous cancers, yet it presents challenges for selective targeting by traditional inhibitors. In this study, we identified a macrocyclic peptide, 5L1, that strongly binds to MCL1, with a dissociation constant (K_D) of 7.1 nM. This peptide shows the potential to specifically inhibit the function of MCL1, and demonstrates effective antitumor activity against several blood tumor cell lines with the half maximal inhibitory concentration (IC₅₀) values for cell-penetrating peptide-conjugated 5L1 in the range of 0.6 to 3 μM. Structural analysis revealed that it functions similarly to molecular glue, capable of binding to two MCL1 molecules simultaneously and inducing their homodimerization. This unique mechanism of action distinguishes it from traditional small-molecule MCL1 inhibitors, underscoring the potential of macrocyclic peptides functioning as molecular glues. Moreover, it inspires the development of highly selective inhibitors targeting MCL1 and other related targets with this glue-like mechanism.

PMID:40131955 | DOI:10.1073/pnas.2426006122

Proc Natl Acad Sci U S A. 2025 Apr;122(13):e2426745122. doi: 10.1073/pnas.2426745122. Epub 2025 Mar 24.

ABSTRACT

There is considerable interest in the targeted degradation of proteins implicated in human disease. The use of sequence-specific proteases for this purpose is severely limited by the difficulty in engineering the numerous enzyme-substrate interactions required to yield highly selective proteases while maintaining catalytic activity. Herein, we report a strategy to evolve a protease for the programmed degradation of α-Synuclein, a presynaptic protein closely linked to Parkinson's disease. Our structure-guided evolution campaign uses the protease from botulinum neurotoxin and showcases the stepwise change of specificity from its native substrate SNAP25 to the selective degradation of α-Synuclein. The protease's selectivity is further demonstrated in human cells where near complete degradation of overexpressed human α-Synuclein is observed with no significant effects on cell proliferation. This stepwise strategy may serve as a general approach to evolve highly selective proteases targeting dysregulated proteins.

PMID:40127273 | PMC:PMC12002255 | DOI:10.1073/pnas.2426745122

J Control Release. 2025 May 10;381:113651. doi: 10.1016/j.jconrel.2025.113651. Epub 2025 Mar 20.

ABSTRACT

The majority of cellular functions are regulated by intracellular proteins, and regulating their interactions can unlock fundamental insights in biology and open new avenues for drug discovery. Because the vast majority of intracellular targets remain undruggable, there is significant current interest in developing protein-based agents especially monoclonal antibodies due to their specificity, availability, and established screening/engineering methods. However, efficient delivery of proteins into the cytoplasm has been a major challenge in biological engineering and drug discovery. We previously reported a platform technology based on a Coomassie blue-cholesterol conjugate (CB-tag) capable of delivering small proteins directly into the cytoplasm. Here, we report a new generation of CB-tag that can bring proteins with a wide size range into the cytoplasm, bypassing endosomal sequestration. Remarkably, intracellular targets with distinct structures were visualized. Overall, the new CB-tag demonstrated a robust ability in protein delivery with broad applications ranging from live-cell immunofluorescence to protein-based therapeutic development.

PMID:40120690 | DOI:10.1016/j.jconrel.2025.113651

Biochemistry. 2025 Apr 1;64(7):1437-1449. doi: 10.1021/acs.biochem.4c00845. Epub 2025 Mar 13.

ABSTRACT

Peptides that have a head-to-tail cyclic backbone tend to be more stable than linear peptides, as do peptides that contain one or more cross-linking disulfide bond. Some of these cyclic and/or disulfide rich peptides have been reported to penetrate cells. These include peptides from a wide range of natural sources, including plants, spiders, crabs, and humans. In this review we describe the structures and biophysical properties of a selected set of such peptides that have been studied in our laboratories. We further describe how they can be engineered to enhance their stability and cellular uptake, and to fine-tune selective cell entry and activity toward intracellular therapeutic targets. Examples of targets described include intracellular protein-protein interactions implicated in cancer, intracellular malarial parasites and intracellular bacterial targets. In addition to the important advances being made with these nature-inspired peptides, the rapid strides in machine learning and artificial intelligence seen over recent years promise to accelerate the use of de novo design methods to produce peptides that are able to pass through biological membranes. We describe examples where such approaches have been used to design macrocyclic peptides and peptide-drug conjugates that can penetrate cell membranes and even have significant oral bioavailability in some cases.

PMID:40082248 | DOI:10.1021/acs.biochem.4c00845

Angew Chem Int Ed Engl. 2025 Jul;64(27):e202500493. doi: 10.1002/anie.202500493. Epub 2025 Jun 1.

ABSTRACT

Cyclic peptides are attractive for drug discovery due to their excellent binding properties and the potential to cross cell membranes. However, by far, not all cyclic peptides are cell permeable, and measuring or predicting their membrane permeability is not trivial. In this work, we assessed the membrane permeability of thioether-cyclized peptides, a widely used format in drug discovery. We developed a strategy for synthesizing hundreds of cyclic peptides carrying a short chloroalkane tag for the bulk quantification of membrane permeability in live cells using the chloroalkane penetration assay. Permeability data for random cyclic peptides established design rules, indicating the probability of peptides entering cells is strongly increasing if the molecular weight is below 800 Da, the polar surface is smaller than 250 Ų, or if there are less than six hydrogen bond donors. From this, machine learning could predict the membrane permeability of random peptides with good confidence, facilitating the future development of membrane-permeable cyclic peptide drugs.

PMID:40052878 | DOI:10.1002/anie.202500493

bioRxiv [Preprint]. 2025 Feb 17:2025.02.12.637979. doi: 10.1101/2025.02.12.637979.

ABSTRACT

The cell surface is a dynamic interface that controls cell-cell communication and signal transduction relevant to organ development, homeostasis and repair, immune reactivity, and pathologies driven by aberrant cell surface phenotypes. The spatial organization of cell surface proteins is central to these processes. High-resolution fluorescence microscopy and proximity labeling have advanced studies of surface protein associations, but the spatial organization of the complete surface proteome remains uncharted. In this study, we systematically mapped the surface proteome of human T-lymphocytes and B-lymphoblasts using proximity labeling of 85 antigens, identified from over 100 antibodies tested for binding to surface-exposed proteins. These experiments were coupled with an optimized data-independent acquisition mass spectrometry workflow to generate a robust dataset. Unsupervised clustering of the resulting interactome revealed functional modules, including well-characterized complexes such as the T-cell receptor and HLA class I/II, alongside novel clusters. Notably, we identified mitochondrial proteins localized to the surface, including the transcription factor TFAM, suggesting previously unappreciated roles for mitochondrial proteins at the plasma membrane. A high-accuracy machine learning classifier predicted over 6,000 surface protein associations, highlighting functional associations such as IL10RB's role as a negative regulator of type I interferon signaling. Spatial modeling of the surface proteome provided insights into protein dispersion patterns, distinguishing widely distributed proteins, such as CD45, from localized antigens, such as CD226 pointing to active mechanisms of regulating surface organization. This work provides a comprehensive map of the human surfaceome and a resource for exploring the spatial and functional dynamics of the cell membrane proteome.

PMID:40027624 | PMC:PMC11870420 | DOI:10.1101/2025.02.12.637979