Rachita Dash

bioRxiv [Preprint]. 2024 Aug 27:2024.08.27.609898. doi: 10.1101/2024.08.27.609898.

ABSTRACT

Pathogenic fungi rely on the cell wall component, chitin, for critical structural and immunological functions. Here a chitin labeling method to visualize the hyphal pathogenic response was developed. The data show that filamentous fungi, Candida albicans , transport N -acetylglucosamine (NAG) bio-orthogonal probes and incorporate them into the cell wall, indicating the probes utility for in vivo study of the morphological, pathogenic switch. As yeast reside in complex microenvironments, The data show that the opportunistic microbe C. albicans , has developed processes to utilize surrounding bacterial cell wall fragments to initiate the morphogenic switch. The probes are utilized for visualization of growth patterns of pathogenic fungi, providing insights into novel mechanisms for the development of antifungals. Remodeling chitin in fungi using NAG derivatives will advance yeast pathogenic studies.

PMID:39253419 | PMC:PMC11383299 | DOI:10.1101/2024.08.27.609898

ACS Chem Biol. 2024 Oct 18;19(10):2131-2140. doi: 10.1021/acschembio.4c00250. Epub 2024 Sep 24.

ABSTRACT

Among bacteria used as anticancer vaccines, attenuated Listeria monocytogenes (Lm^at) stands out, because it spreads from one infected cancer cell to the next, induces a strong adaptive immune response, and is suitable for repeated injection cycles. Here, we use click chemistry to functionalize the Lm^at cell wall and turn the bacterium into an "intelligent carrier" of the chemotherapeutic drug doxorubicin. Doxorubicin-loaded Lm^at retains most of its biological properties and, compared to the control fluorophore-functionalized bacteria, shows enhanced cytotoxicity against melanoma cells both in vitro and in a xenograft model in zebrafish. Our results show that drugs can be covalently loaded on the Lm^at cell wall and pave the way to the development of new two-in-one therapeutic approaches combining immunotherapy with chemotherapy.

PMID:39317359 | PMC:PMC11494506 | DOI:10.1021/acschembio.4c00250

Nature. 2024 Sep 25. doi: 10.1038/s41586-024-07948-2. Online ahead of print.

ABSTRACT

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by endogenous ligands. Therapeutic approaches such as lysosome-targeting chimaeras^1,2 (LYTACs) and cytokine receptor-targeting chimeras³ (KineTACs) have used this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. Although powerful, these approaches can be limited by competition with native ligands and requirements for chemical modification that limit genetic encodability and can complicate manufacturing, and, more generally, there may be no native ligands that stimulate endocytosis through a given receptor. Here we describe computational design approaches for endocytosis-triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for insulin-like growth factor 2 receptor (IGF2R) and asialoglycoprotein receptor (ASGPR), sortilin and transferrin receptors, and show that fusing these tags to soluble or transmembrane target protein binders leads to lysosomal trafficking and target degradation. As these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. EndoTag fusion to a PD-L1 antibody considerably increases efficacy in a mouse tumour model compared to antibody alone. The modularity and genetic encodability of EndoTags enables AND gate control for higher-specificity targeted degradation, and the localized secretion of degraders from engineered cells. By promoting endocytosis, EndoTag fusion increases signalling through an engineered ligand-receptor system by nearly 100-fold. EndoTags have considerable therapeutic potential as targeted degradation inducers, signalling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody-drug and antibody-RNA conjugates.

PMID:39322662 | DOI:10.1038/s41586-024-07948-2

Commun Biol. 2024 Sep 18;7(1):1173. doi: 10.1038/s42003-024-06785-3.

ABSTRACT

The essential L,D-transpeptidase of Mycobacterium tuberculosis (Ldt_Mt2) catalyses the formation of 3 → 3 cross-links in cell wall peptidoglycan and is a target for development of antituberculosis therapeutics. Efforts to inhibit Ldt_Mt2 have been hampered by lack of knowledge of how it binds its substrate. To address this gap, we optimised the isolation of natural disaccharide tetrapeptide monomers from the Corynebacterium jeikeium bacterial cell wall through overproduction of the peptidoglycan sacculus. The tetrapeptides were used in binding / turnover assays and biophysical studies on Ldt_Mt2. We determined a crystal structure of wild-type Ldt_Mt2 reacted with its natural substrate, the tetrapeptide monomer of the peptidoglycan layer. This structure shows formation of a thioester linking the catalytic cysteine and the donor substrate, reflecting an intermediate in the transpeptidase reaction; it informs on the mode of entrance of the donor substrate into the Ldt_Mt2 active site. The results will be useful in design of Ldt_Mt2 inhibitors, including those based on substrate binding interactions, a strategy successfully employed for other nucleophilic cysteine enzymes.

PMID:39294212 | PMC:PMC11410929 | DOI:10.1038/s42003-024-06785-3

J Am Chem Soc. 2024 Sep 25;146(38):26320-26330. doi: 10.1021/jacs.4c08656. Epub 2024 Sep 14.

ABSTRACT

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that binds thousands of different proteins, including substrates that it glycosylates and nonsubstrate interactors that regulate its biology. OGT also has one proteolytic substrate, the transcriptional coregulator host cell factor 1 (HCF-1), which it cleaves in a process initiated by glutamate side chain glycosylation at a series of central repeats. Although HCF-1 is OGT's most prominent binding partner, its affinity for the enzyme has not been quantified. Here, we report a time-resolved Förster resonance energy transfer assay to measure ligand binding to OGT and show that an HCF-1-derived polypeptide (HCF3R) binds with picomolar affinity to the enzyme (K_D ≤ 85 pM). This high affinity is driven in large part by conserved asparagines in OGT's tetratricopeptide repeat domain, which form bidentate contacts to the HCF-1 peptide backbone; replacing any one of these asparagines with alanine reduces binding by more than 5 orders of magnitude. Because the HCF-1 polypeptide binds so tightly to OGT, we tested its ability to inhibit enzymatic function. We found that HCF3R potently inhibits OGT both in vitro and in cells and used this finding to develop a genetically encoded, inducible OGT inhibitor that can be degraded with a small molecule, allowing for reversible and tunable inhibition of OGT.

PMID:39276112 | PMC:PMC11440498 | DOI:10.1021/jacs.4c08656

J Am Chem Soc. 2024 Sep 18;146(37):25397-25402. doi: 10.1021/jacs.4c03361. Epub 2024 Sep 9.

ABSTRACT

Phototriggered release of various cargos, including soluble protein factors and small molecules, has the potential to correct aberrant biological events by offering spatiotemporal control over local therapeutic levels. However, the poor penetration depth of light historically limits implementation to subdermal regions, necessitating alternative methods of light delivery to achieve the full potential of photodynamic therapeutic release. Here, we introduce a strategy exploiting bioluminescence resonance energy transfer (BRET)-an energy transfer process between light-emitting Nanoluciferase (NLuc) and a photosensitive acceptor molecule-to drive biomolecule release from hydrogel biomaterials. Through a facile, one-pot, and high-yielding synthesis (60-70%), we synthesized a heterobifunctional ruthenium cross-linker bearing an aldehyde and an azide (CHO-Ru-N₃), a compound that we demonstrate undergoes predictable exchange of the azide-bearing ligand under blue-green light irradiation (>550 nm). Following site-specific conjugation to NLuc via sortase-tag enhanced protein ligation (STEPL), the modified protein was covalently attached to a poly(ethylene glycol) (PEG)-based hydrogel via strain-promoted azide-alkyne cycloaddition (SPAAC). Leveraging the high photosensitivity of Ru compounds, we demonstrate rapid and equivalent release of epidermal growth factor (EGF) via either direct illumination or via BRET-based bioluminolysis. As NLuc-originated luminescence can be controlled equivalently throughout the body, we anticipate that this unique protein release strategy will find use for locally triggered drug delivery following systemic administration of a small molecule.

PMID:39250821 | DOI:10.1021/jacs.4c03361

Chem Sci. 2024 Aug 13;15(37):15463-73. doi: 10.1039/d4sc04238g. Online ahead of print.

ABSTRACT

Dialkyldiazirines have emerged as a photo-reactive group of choice for interactome mapping in live cell experiments. Upon irradiation, 'linear' dialkyldiazirines produce dialkylcarbenes which are susceptible to both intramolecular reactions and unimolecular elimination processes, as well as diazoalkanes, which also participate in intermolecular labeling. Cyclobutylidene has a nonclassical bonding structure and is stable enough to be captured in bimolecular reactions. Cyclobutanediazirines have more recently been studied as photoaffinity probes based on cyclobutylidene, but the mechanism, especially with respect to the role of putative diazo intermediates, was not fully understood. Here, we show that photolysis (365 nm) of cyclobutanediazirines can produce cyclobutylidene intermediates as evidenced by formation of their expected bimolecular and unimolecular products, including methylenecyclopropane derivatives. Unlike linear diazirines, cyclobutanediazirine photolysis in the presence of tetramethylethylene produces a [2 + 1] cycloaddition adduct. By contrast, linear diazirines produce diazo compounds upon low temperature photolysis in THF, whereas diazo compounds are not detected in similar photolyses of cyclobutanediazirines. Diazocyclobutane, prepared by independent synthesis, is labile, reactive toward water and capable of protein alkylation. The rate of diazocyclobutane decomposition is not affected by 365 nm light, suggesting that the photochemical conversion of diazocyclobutane to cyclobutylidene is not an important pathway. Finally, chemical proteomic studies revealed that a likely consequence of this primary conversion to a highly reactive carbene is a marked decrease in labeling by cyclobutanediazirine-based probes relative to linear diazirine counterparts both at the individual protein and proteome-wide levels. Collectively, these observations are consistent with a mechanistic picture for cyclobutanediazirine photolysis that involves carbene chemistry with minimal formation of diazo intermediates, and contrasts with the photolyses of linear diazirines where alkylation by diazo intermediates plays a more significant role.

PMID:39246352 | PMC:PMC11372447 | DOI:10.1039/d4sc04238g

J Am Chem Soc. 2024 Sep 11;146(36):25371-25382. doi: 10.1021/jacs.4c10533. Epub 2024 Sep 2.

ABSTRACT

Cell-penetrating peptides (CPPs) enter the cell by two different mechanisms-endocytosis followed by endosomal escape and direct translocation at the plasma membrane. The mechanism of direct translocation remains unresolved. In this work, the direct translocation of nonaarginine (R9) and two cyclic CPPs (CPP12 and CPP17) into Jurkat cells was monitored by time-lapse confocal microscopy. Our results provide direct evidence that all three CPPs translocate across the plasma membrane by a recently discovered vesicle budding-and-collapse (VBC) mechanism. Membrane translocation is preceded by the formation of nucleation zones. Up to four different types of nucleation zones and three variations of the VBC mechanism were observed. The VBC mechanism reconciles the enigmatic and conflicting observations in the literature.

PMID:39221867 | PMC:PMC11610503 | DOI:10.1021/jacs.4c10533

Mol Pharm. 2024 Oct 7;21(10):5255-5260. doi: 10.1021/acs.molpharmaceut.4c00688. Epub 2024 Sep 2.

ABSTRACT

Intracellular delivery of biological cargos, which would yield new research tools and novel therapeutics, remains an active area of research. A convenient and potentially general approach involves the conjugation of a cell-penetrating peptide to a cargo of interest. However, linear CPPs lack sufficient cytosolic entry efficiency and metabolic stability, while previous backbone cyclized CPPs have several drawbacks including the necessity for chemical synthesis and posttranslational conjugation to peptide/protein cargos and epimerization during cyclization. We report here a new class of bismuth cyclized CPPs with excellent cytosolic entry efficiencies, proteolytic stability, and potential compatibility with genetic encoding and recombinant production.

PMID:39223839 | PMC:PMC11610496 | DOI:10.1021/acs.molpharmaceut.4c00688

Brief Bioinform. 2024 Jul 25;25(5):bbae417. doi: 10.1093/bib/bbae417.

ABSTRACT

Cyclic peptides are versatile therapeutic agents that boast high binding affinity, minimal toxicity, and the potential to engage challenging protein targets. However, the pharmaceutical utility of cyclic peptides is limited by their low membrane permeability-an essential indicator of oral bioavailability and intracellular targeting. Current machine learning-based models of cyclic peptide permeability show variable performance owing to the limitations of experimental data. Furthermore, these methods use features derived from the whole molecule that have traditionally been used to predict small molecules and ignore the unique structural properties of cyclic peptides. This study presents CycPeptMP: an accurate and efficient method to predict cyclic peptide membrane permeability. We designed features for cyclic peptides at the atom-, monomer-, and peptide-levels and seamlessly integrated these into a fusion model using deep learning technology. Additionally, we applied various data augmentation techniques to enhance model training efficiency using the latest data. The fusion model exhibited excellent prediction performance for the logarithm of permeability, with a mean absolute error of $0.355$ and correlation coefficient of $0.883$. Ablation studies demonstrated that all feature levels contributed and were relatively essential to predicting membrane permeability, confirming the effectiveness of augmentation to improve prediction accuracy. A comparison with a molecular dynamics-based method showed that CycPeptMP accurately predicted peptide permeability, which is otherwise difficult to predict using simulations.

PMID:39210505 | PMC:PMC11361855 | DOI:10.1093/bib/bbae417

Angew Chem Int Ed Engl. 2025 Feb 17;64(8):e202414256. doi: 10.1002/anie.202414256. Epub 2024 Nov 5.

ABSTRACT

Matrix metallopeptidase 7 (MMP7) plays a crucial role in cancer metastasis and progression, making it an attractive target for therapeutic development. However, the development of selective MMP7 inhibitors is challenging due to the conservation of active sites across various matrix metalloproteinases (MMPs). Here, we have developed mirror-image random nonstandard peptides integrated discovery (MI-RaPID) technology to discover innate protease-resistant macrocyclic peptides that specifically bind to and inhibit human MMP7. One identified macrocyclic peptide against D-MMP7, termed D20, was synthesized in its mirror-image form, D'20, consisting of 12 D-amino acids, one cyclic β-amino acid, and a thioether bond. Notably, it potently inhibited MMP7 with an IC₅₀ value of 90 nM, and showed excellent selectivity over other MMPs with similar substrate specificity. Moreover, D'20 inhibited the migration of pancreatic cell line CFPAC-1, but had no effect on the cell proliferation and viability. D'20 exhibited excellent stability in human serum, as well as in simulated gastric and intestinal fluids. This study highlights that MI-RaPID technology can serve as a powerful tool to develop in vivo stable macrocyclic peptides for therapeutic applications.

PMID:39215490 | PMC:PMC11833282 | DOI:10.1002/anie.202414256