Rachita Dash

mBio. 2025 Feb 24:e0395024. doi: 10.1128/mbio.03950-24. Online ahead of print.

ABSTRACT

The cell envelope of gram-negative bacteria consists of two membranes sandwiching the peptidoglycan (PG) cell wall. The outer membrane (OM) contains integrated beta-barrel proteins and has an outer leaflet composed of lipopolysaccharide (LPS). LPS is transported from the inner membrane where it is made to the OM surface by the Lpt system. In the polarly elongating alpha-proteobacterium Brucella abortus, LPS transport has been localized to the polar growth zone and division site. However, LPS transport has not been tracked in live proteobacteria like Escherichia coli that elongate by dispersed incorporation of envelope material along their cell body. Here, we report an investigation into the binding target of fluorescently labeled wheat germ agglutinin (FL-WGA) on E. coli cells that led to the development of a method for visualizing LPS transport. We show that instead of PG or enterobacterial common antigen for which FL-WGA labeling has been used to detect in the past, this probe recognizes LPS modified with a terminal N-acetylglucosamine formed by the defective O-antigen synthesis pathway of laboratory strains of E. coli. This finding enabled the construction of mutants inducible for LPS modification that were used together with FL-WGA labeling to track LPS transport to the cell surface. We show that new LPS is inserted throughout the cell cylinder and at the division site, but not at the cell poles. A similar pattern was observed previously for PG synthesis and OM protein insertion in E. coli, suggesting that LPS transport to the OM is coordinated with these processes.IMPORTANCEGram-negative bacteria like Escherichia coli are surrounded by a multilayered cell envelope that includes an outer membrane (OM) responsible for their high intrinsic resistance to antibiotics. The outer leaflet of this membrane is composed of a glycolipid called lipopolysaccharide (LPS). Here, we report the development of an imaging method to track the transport of LPS to the E. coli outer membrane. The results indicate that transport occurs throughout the cell cylinder and at the division site, but not at the cell poles. A similar pattern was observed previously when cell wall synthesis and the insertion of proteins into the OM were tracked. Our results therefore suggest that LPS transport to the OM is coordinated with other essential processes that underly gram-negative cell envelope biogenesis.

PMID:39992125 | DOI:10.1128/mbio.03950-24

RSC Chem Biol. 2025 Jan 31;6(4):563-570. doi: 10.1039/d4cb00218k. eCollection 2025 Apr 2.

ABSTRACT

The stabilisation of recombinant glycosidases by exogenous ligands, known as pharmacological chaperones or enzyme stabilisers, has recently garnered great clinical interest. This strategy can prevent enzyme degradation in the blood, reducing required dosages of recombinant enzyme and extending IV injection intervals, thereby reducing side effects, improving patient lifestyles and treatment costs. While this therapeutic approach has been successfully implemented for treating Pompe and Fabry diseases, clinical studies for Gaucher disease using chaperones alone or in combination with enzyme replacement therapy (ERT) have been limited, and no small molecule chaperones have yet been approved for this condition. Developing such therapies requires selective and effective reversible GBA1 ligands. Here, we describe the development of a new class of selective macrocyclic peptide GBA1 ligands using random nonstandard peptides integrated discovery (RaPID) technology, and demonstrate their ability to bind and stabilise rhGBA1 in plasma at nanomolar concentrations. These cyclic peptides do not inhibit endogenous GBA1 in cells due to poor cell permeability but can stabilise extracellular rhGBA1 in plasma, presenting significant potential as a combinatorial ERT-pharmacological chaperone therapy for Gaucher disease.

PMID:39936129 | PMC:PMC11808397 | DOI:10.1039/d4cb00218k

Angew Chem Int Ed Engl. 2025 May 26;64(22):e202423057. doi: 10.1002/anie.202423057. Epub 2025 Feb 19.

ABSTRACT

Precise modulation of dynamic and complex tumor microenvironment (TME) to disrupt tumorigenesis and reshape intratumoral immune infiltration has emerged as promising approaches for enhanced cancer therapy. Among recent innovations, proteolysis-targeting chimeras (PROTACs) represent a burgeoning chemical knockdown technology capable of degrading oncogenic protein homeostasis and inducing dynamic alternations within carcinoma settings, offering potential for antitumor manipulation. However, achieving selectivity in PROTACs that respond to disease environmental stimulation and precisely perturb on-target proteins remains challenging. The multi-step synthesis and limited permeability, attributed to high-molecular-weight and heterobifunctional structures, further hinder their in vivo efficacy. Herein, we present a unique TME-responsive enzyme-activated clickable PROTACs, which features a short peptide-tagged pomalidomide derivative to undergo tumor-specific cleavage by cathepsin protease to induce orthogonal crosslinking of the exposed cysteine with 2-cyanobenzothiazole-labeled epigenetic protein-ligand JQ1, facilitating in situ degrader formation within tumor regions only. Systematic protein profiling and proteomic analysis revealed that such TME-specific clickable-PROTACs not only selectively eliminate epigenetic proteins without tedious pre-synthesis to bridge disparate small-molecule bi-warhead fragments, but also demonstrated superior tumor penetration compared to conventional high-molecular-weight PROTACs. Importantly, these clickable-PROTACs efficiently downregulated immune checkpoint programmed death-ligand 1 (PD-L1) both in vitro and in vivo, remodeling TME for enhanced therapeutics, especially in anti-tumoral immunomodulation.

PMID:39932237 | DOI:10.1002/anie.202423057

Nat Commun. 2025 Feb 10;16(1):1484. doi: 10.1038/s41467-025-56317-8.

ABSTRACT

Targeting host proteins that are crucial for viral replication offers a promising antiviral strategy. We have designed and characterised antiviral PROteolysis TArgeting Chimeras (PROTACs) targeting the human protein cyclophilin A (CypA), a host cofactor for unrelated viruses including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The PROTAC warheads are based on fully synthetic macrocycles derived from sanglifehrin A, which are structurally different from the classical Cyp inhibitor, cyclosporine A. Our Cyp-PROTACs decrease CypA levels in cell lines and primary human cells and have high specificity for CypA confirmed by proteomics experiments. Critically, CypA degradation facilitates improved antiviral activity against HIV-1 in primary human CD4+ T cells compared to the non-PROTAC parental inhibitor, at limiting inhibitor concentrations. Similarly, we observe antiviral activity against HCV replicon in a hepatoma cell line. We propose that CypA-targeting PROTACs inhibit viral replication potently and anticipate reduced evolution of viral resistance and broad efficacy against unrelated viruses. Furthermore, they provide powerful tools for probing cyclophilin biology.

PMID:39929804 | PMC:PMC11811207 | DOI:10.1038/s41467-025-56317-8

J Am Chem Soc. 2025 Feb 19;147(7):5632-5641. doi: 10.1021/jacs.4c11554. Epub 2025 Feb 9.

ABSTRACT

The fungal cell wall is essential for the integrity of the cell, providing strength and shape, as well as protection against environmental stimuli. For pathogenic fungi, the cell wall is also the initial point of contact with the host. Specific cell wall features such as hypha tails and smaller glycan components modulate a wide range of fungal interactions with the immune defenses. Here, a bio-orthogonal labeling method utilizing N-acetyl-glucosamine (NAG) probes is developed to fluorescently label native, pathogenic yeast via the chitin scavenging pathway. A panel of NAG probes was assembled, synthesized, and characterized for the ability to label the chitin in pathogenic yeast. Enzymatic data show that the native scavenging biosynthetic enzyme, Hxk1, is promiscuous, permitting the labeling of the native chitin biopolymer. This chitin labeling method was validated via the development of mass spectrometry protocols. When compared to the current available labeling systems for chitin, the probes do not affect the integrity of the cell wall and do not interrupt cell growth. Furthermore, the NAG probes enabled multiple "click" platforms across pathogenic Candida species including Candida albicans and Candida tropicalis. Budding and filamentous hyphal states were observed. The results indicate the probes' utility for in vivo study of the morphological, pathogenic switch, and visualization of growth patterns. Thus, the use of these probes in pathogenic Candida strains is ideal for a variety of future applications including strain specific antifungals, diagnostic tools, and immunomodulators.

PMID:39925016 | PMC:PMC11849683 | DOI:10.1021/jacs.4c11554

ACS Synth Biol. 2025 Feb 5. doi: 10.1021/acssynbio.4c00671. Online ahead of print.

ABSTRACT

To overcome the difficulty of building large nonribosomal peptide synthetase (NRPS) gene cluster libraries, an efficient one-pot method using Bacillus subtilis was developed. This new method, named Seamed Express Assembly Method (SEAM)-combi-Ordered Gene Assembly in Bacillus subtilis (OGAB), combines the SEAM-OGAB approach for NRPS gene cluster construction with the combi-OGAB method for combinatorial DNA library construction to randomly swap DNA fragments for NRPS modules. In this study, NRPS gene clusters of plipastatin and gramicidin S were used as the starting material. The full length of each gene cluster was prepared as plasmid DNA by introducing restriction enzyme SfiI sites into the module border according to SEAM-OGAB. These two plasmids were mixed, digested with SfiI, ligated in a tandem repeat form, and used to transform B. subtilis according to the combi-OGAB method. While 64 of all the possible combinations were used in the calculation, 32 types of plasmid DNA were obtained from 50 randomly selected transformants. These transformants produced at least 30 types of peptides, including cyclic and linear variations with lengths ranging from 5 to 10 amino acids. Thus, this method enabled an efficient construction of NRPS gene cluster libraries with more than five module members, making it advantageous for applications in peptide libraries.

PMID:39907600 | DOI:10.1021/acssynbio.4c00671

ACS Infect Dis. 2025 Mar 14;11(3):703-714. doi: 10.1021/acsinfecdis.4c00798. Epub 2025 Feb 4.

ABSTRACT

The secreted Chorismate mutase enzyme of Mycobacterium tuberculosis (*MtbCM) is an underexplored potential target for the development of new antitubercular agents that are increasingly needed as antibiotic resistance rises in prevalence. As an enzyme suspected to be involved in virulence and host-pathogen interactions, disruption of its function could circumvent the difficulty of treating tuberculosis-infected granulomas. Drug development, however, is limited by novel ligand discovery. Currently, *MtbCM activity is measured by using a low throughput acid/base-mediated product derivatization absorbance assay. Here, we utilized an RNA-display affinity selection approach enabled by the Random Peptides Integrated Discovery (RaPID) system to screen a vast library of macrocyclic peptides (MCP) for novel *MtbCM ligands. Peptides identified from the RaPID selection, and analogs thereof identified by analyzing the selection population dynamics, produced a new class of *MtbCM inhibiting MCPs. Among these were two noteworthy "chorismides", whose binding modes were elucidated by X-ray crystallography. Both were potent inhibitors of the CM enzyme activity. One was identified as an allosteric binding peptide revealing a novel inhibition approach, while the other is an active-site binding peptide that when conjugated to a fluorescent probe allowed for the development of a series of alternative fluorescence-based ligand-displacement assays that can be utilized for the assessment of potential *MtbCM inhibitors.

PMID:39903128 | DOI:10.1021/acsinfecdis.4c00798

bioRxiv [Preprint]. 2025 Jan 22:2025.01.17.633674. doi: 10.1101/2025.01.17.633674.

ABSTRACT

Lasso peptides are a unique class of natural products with distinctively threaded structures, conferring exceptional stability against thermal and proteolytic degradation. Despite their promising biotechnological and pharmaceutical applications, reported attempts to prepare them by chemical synthesis result in forming the nonthreaded branched-cyclic isomer, rather than the desired lassoed structure. This is likely due to the entropic challenge of folding a short, threaded motif prior to chemically mediated cyclization. Accordingly, this study aims to better understand and enhance the relative stability of pre-lasso conformations-the essential precursor to lasso peptide formation-through sequence optimization, chemical modification, and disulfide incorporation. Using Rosetta fixed backbone design, optimal sequences for several class II lasso peptides are identified. Enhanced sampling with well-tempered metadynamics confirmed that designed sequences derived from the lasso structures of rubrivinodin and microcin J25 exhibit a notable improvement in pre-lasso stability relative to the competing nonthreaded conformations. Chemical modifications to the isopeptide bond-forming residues of microcin J25 further increase the probability of pre-lasso formation, highlighting the beneficial role of non-canonical amino acid residues. Counterintuitively, the introduction of a disulfide cross-link decreased pre-lasso stability. Although cross-linking inherently constrains the peptide structure, decreasing the entropic dominance of unfolded phase space, it hinders the requisite wrapping of the N-terminal end around the tail to adopt the pre-lasso conformation. However, combining chemical modifications with the disulfide cross-link results in further pre-lasso stabilization, indicating that the ring modifications counteract the constraints and provide a cooperative benefit with cross-linking. These findings lay the groundwork for further design efforts to enable synthetic access to the lasso peptide scaffold.

SIGNIFICANCE: Lasso peptides are a unique class of ribosomally synthesized and post-translationally modified natural products with diverse biological activities and potential for therapeutic applications. Although direct synthesis would facilitate therapeutic design, it has not yet been possible to fold these short sequences to their threaded architecture without the help of biosynthetic enzyme stabilization. Our work explores strategies to enhance the stability of the pre-lasso structure, the essential precursor to de novo lasso peptide formation. We find that sequence design, incorporating non-canonical amino acid residues, and design-guided cross-linking can augment stability to increase the likelihood of lasso motif accessibility. This work presents several strategies for the continued design of foldable lasso peptides.

PMID:39896618 | PMC:PMC11785075 | DOI:10.1101/2025.01.17.633674

Proc Natl Acad Sci U S A. 2025 Feb 4;122(5):e2411316122. doi: 10.1073/pnas.2411316122. Epub 2025 Jan 30.

ABSTRACT

The design of organic-peptide hybrids has the potential to combine our vast knowledge of protein design with small molecule engineering to create hybrid structures with complex functions. Here, we describe the computational design of a photoswitchable Ca²⁺-binding organic-peptide hybrid. The designed molecule, designated Ca²⁺-binding switch (CaBS), combines an EF-hand motif from classical Ca²⁺-binding proteins such as calmodulin with a photoswitchable group that can be reversibly isomerized between a spiropyran (SP) and merocyanine (MC) state in response to different wavelengths of light. The MC/SP group acts both as a photoswitch as well as an optical sensor of Ca²⁺ binding. Photoconversion of the SP to the corresponding MC unmasks an acidic phenol, which CaBS uses as an integral part of both its Ca²⁺-binding site as well as its tertiary and quaternary structure. By design, the SP state of CaBS is monomeric, while the Ca²⁺-bound form of the MC state is an obligate dimer, with two Ca²⁺-binding sites formed at the interface of a domain-swapped dimer. Thus, light and Ca²⁺ were expected to serve as an "AND gate" that powers a change in backbone structure/dynamics, oligomerization state, and fluorescence properties of the designed molecule. CaBS was designed using Rosetta and molecular dynamics simulations, and experimentally characterized by nuclear magnetic resonance, isothermal titration calorimetry, and optical titrations. These data illustrate the potential of combining small molecule engineering with de novo protein design to develop sensors whose conformation, association state, and optical properties respond to multiple environmental cues.

PMID:39883844 | PMC:PMC11804555 | DOI:10.1073/pnas.2411316122

bioRxiv [Preprint]. 2025 Mar 12:2025.01.17.633618. doi: 10.1101/2025.01.17.633618.

ABSTRACT

Understanding the factors that influence the accumulation of molecules beyond the mycomembrane of Mycobacterium tuberculosis (Mtb) - the main barrier to accumulation - is essential for developing effective antimycobacterial agents. In this study, we investigated two design principles commonly observed in natural products and mammalian cell-permeable peptides: backbone N-alkylation and macrocyclization. To assess how these structural edits impact molecule accumulation beyond the mycomembrane, we utilized our recently developed Peptidoglycan Accessibility Click-Mediated Assessment (PAC-MAN) assay for live-cell analysis. Our findings provide the first empirical evidence that peptide macrocyclization generally enhances accumulation in mycobacteria, while N-alkylation influences accumulation in a context-dependent manner. We examined these design principles in the context of two peptide antibiotics, tridecaptin A1 and griselimycin, which revealed the roles of N-alkylation and macrocyclization in improving both accumulation and antimicrobial activity against mycobacteria in specific contexts. Together, we present a working model for strategic structural modifications aimed at enhancing the accumulation of molecules past the mycomembrane. More broadly, our results also challenge the prevailing belief in the field that large and hydrophilic molecules, such as peptides, cannot readily traverse the mycomembrane.

PMID:39868157 | PMC:PMC11760776 | DOI:10.1101/2025.01.17.633618

Curr Org Synth. 2025 Jan 14. doi: 10.2174/0115701794334314241212114056. Online ahead of print.

ABSTRACT

INTRODUCTION: An efficient procedure was reported for the synthesis of novel hybrid dithiazoles 7 and thiazoles 15, in good yields, by applying hydrazonyl chlorides 4 with thiocarbohydrazone derivatives 3 and 12.

METHODS: The thiazole derivatives were evaluated for their antimicrobial and antioxidant activities.

RESULTS: According to the results, thiazoles revealed marked potency as antimicrobial and antioxidant agents. Thus, 7a's DPPH radical scavenging activity was excellent (38.19±0.33 and 14.37±0.4) at concentrations of 2.0 and 1.0 mg/mL, respectively. In addition, compound 3 exhibited activity against all bacterial strains tested, as evidenced by inhibition zones measuring that ranged from 8.5±0.43 mm for E. faecalis to 16.5±0.43 mm for S. mutans.

CONCLUSION: The MIC results showed that compound 3 was effective against E. coli, S. aureus, E. faecalis, P. aeruginosa, and S. mutans at concentrations of 1.0, 1.0, 2.0, 1.0, and 1.0 mg/mL, respectively. Furthermore, molecular docking has shown lower binding energy with different types of interactions at the active sites of Dihydropteroate synthase, Sortase A, LasR, and penicillin-binding protein pockets, indicating that these compounds could inhibit the enzyme and cause promising antimicrobial effects.

PMID:39844414 | DOI:10.2174/0115701794334314241212114056

Chem Biol Drug Des. 2025 Jan;105(1):e70051. doi: 10.1111/cbdd.70051.

ABSTRACT

Drug targeting strategies, such as peptide-drug conjugates (PDCs), have arisen to combat the issue of off-target toxicity that is commonly associated with chemotherapeutic small molecule drugs. Here we investigated the ability of PDCs comprising a human protein-derived cell-penetrating peptide-platelet factor 4-derived internalization peptide (PDIP)-as a targeting strategy to improve the selectivity of camptothecin (CPT), a topoisomerase I inhibitor that suffers from off-target toxicity. The intranuclear target of CPT allowed exploration of PDC design features required for optimal potency. A suite of PDCs with various structural characteristics, including alternative conjugation strategies (such as azide-alkyne cycloaddition and disulfide conjugation) and linker types (non-cleavable or cleavable), were synthesized and investigated for their anticancer activity. Membrane permeability and cytotoxicity studies revealed that intact PDIP-CPT PDCs can cross membranes, and that PDCs with disulfide- and protease-cleavable linkers liberated free CPT and killed melanoma cells with nanomolar potency. However, selectivity of the PDIP carrier peptide for melanoma compared to noncancerous epidermal cells was not maintained for the PDCs. This study emphasizes the distinct role of the peptide, linker, and drug for optimal PDC activity and highlights the need to carefully match components when assembling PDCs as targeted therapies.

PMID:39834140 | PMC:PMC11747586 | DOI:10.1111/cbdd.70051