Ananya Naick

Cell Rep. 2025 Oct 28;44(10):116389. doi: 10.1016/j.celrep.2025.116389. Epub 2025 Oct 7.

ABSTRACT

Peptidoglycan, the major constituent of bacterial cell walls, is a giant macromolecule made of glycan strands cross-linked by short peptides, which provides resistance to the turgor pressure of the cytoplasm. We explored the mechanism of insertion of newly synthesized subunits into the existing peptidoglycan mesh by high-resolution mass spectrometry analysis of peptidoglycan labeled with heavy carbon and nitrogen isotopes in Escherichia coli mutants conditionally producing essential peptidoglycan biosynthesis enzymes. We show that side-wall expansion at a constant diameter is mediated by endopeptidases that cleave existing peptidoglycan cross-links in order to enable the one-at-a-time insertion of newly synthesized glycan strands into the stress-bearing peptidoglycan layer by transpeptidases. These results provide direct experimental evidence for the essential participation of endopeptidases in the biosynthesis of the lateral wall. They also suggest that transpeptidases and endopeptidases might act in a coordinated, rather than sequential, manner.

PMID:41060808 | DOI:10.1016/j.celrep.2025.116389

J Med Chem. 2025 Sep 25;68(18):18908-18929. doi: 10.1021/acs.jmedchem.5c00544. Epub 2025 Sep 8.

ABSTRACT

Proteasome inhibitors are effective in treating hematologic cancers but have limited utility in brain tumors due to poor blood-brain barrier (BBB) penetration and metabolic instability. In this study, we developed novel macrocyclic peptide epoxyketone inhibitors with improved drug-like properties. Compounds were screened for cytotoxicity against brain cancer cell lines, permeability (PAMPA-BBB and Caco-2), and metabolic stability. Lead compound 10 demonstrated potent in vitro activity (IC₅₀ < 100 nM), low P-gp efflux, and favorable microsomal and plasma stability. In vivo pharmacokinetic studies in mice showed that compound 10 maintained therapeutic plasma levels and achieved measurable brain concentrations without toxicity. Co-administration of a P-gp inhibitor significantly enhanced brain exposure of compound 35, confirming efflux as a key parameter. The incorporation of fluorinated phenyl and α,α-dimethylglycine moieties contributed to improved BBB permeability and metabolic stability. These findings support further development of macrocyclic epoxyketone inhibitors as promising candidates for brain cancer therapy.

PMID:40922058 | DOI:10.1021/acs.jmedchem.5c00544

J Med Chem. 2025 Sep 25;68(18):19377-19395. doi: 10.1021/acs.jmedchem.5c01540. Epub 2025 Sep 12.

ABSTRACT

Melanoma-associated antigen A4 (MAGE-A4) is a cancer/testis antigen (CTA) that interacts with the E3 ubiquitin ligase RAD18 to enhance DNA damage tolerance in tumor cells. Here, we report the structure-guided optimization of a previously reported potent but cell-impermeable cyclic peptide, called MTP-1. Building off our previous peptide inhibitor efforts, we aimed to develop next-generation peptide inhibitors with significantly improved cell permeability. Through systematic structure-activity relationship (SAR) studies employing an mRNA display site-saturation mutagenesis library (SSML) and strategic scaffold optimization with modified cyclization strategy, we developed JWP24, the first cell-permeable peptide inhibitor of MAGE-A4. Evaluation across multiple assays demonstrates intracellular target engagement, maintained binding potency, and exhibits no cytotoxicity at effective concentrations. This study provides a valuable framework for transforming potent but larger, macrocyclic peptide inhibitors into cell-permeable probes. The work presented here demonstrates progress toward further establishing MAGE-A4 as a chemically tractable oncology target.

PMID:40937506 | DOI:10.1021/acs.jmedchem.5c01540

Acc Chem Res. 2025 Sep 16;58(18):2852-2861. doi: 10.1021/acs.accounts.5c00451. Epub 2025 Aug 29.

ABSTRACT

ConspectusAlkynes are one of the most fundamental functional groups in organic synthesis due to the versatile chemistry of the triple bond, their unique rigid structure, and their use in bioconjugation. The introduction of alkynes onto organic molecules traditionally relies on nucleophilic activation, often requiring strong bases or metal catalysts. These conditions, however, restrict applications involving biomolecules such as peptides and proteins due to functional group incompatibility. To address this limitation, our group developed an "umpolung" approach, utilizing hypervalent iodine compounds to create electrophilic alkyne transfer reagents such as benziodoxol(on)es (Bx(X)s) and benziodazolones (BZs). The high reactivity of EBx/X/Z reagents enables efficient alkyne transfer to various nucleophilic residues in peptides and proteins under different reaction conditions, providing a versatile tool for biomolecule modification.In this Account, we highlight the residue-selective alkynylation and alkenylation of peptides enabled by the development of novel EBx/X/Z reagents with a focus on progress since 2021. This includes the following: (1) Selective residue modification: We have made significant progress in the residue-selective alkynylation and alkenylation of peptides and proteins. Building on our initial work with Cys-selective alkynylation, we enhanced reactivity and solubility by introducing a sulfonate group on the benziodoxolone arene core, facilitating lipophilic alkynylation in an aqueous environment. Furthermore, we developed perfluoroaryl-modified BZ reagents to achieve sequential Cys-Cys cross-linking and used them for antibody cross-linking with superior reactivity compared to that of conventional methods. Additionally, we expanded the reactivity beyond Cys to achieve Tyr-selective conjugation. All of these achievements underscored the tunability of EBx/X/Z reagents through strategic substituent modification on the iodine core. (2) Peptide stapling and macrocyclization: We designed EBx(X) reagents featuring an additional reactive site on the alkyne moiety, enabling Cys-Cys and Cys-Lys stapling in peptides. This approach enhanced their α-helicity and potential as PPI inhibitors with improved binding affinity to the MDM2 protein. For sequences lacking Cys, we incorporated the whole EBx(X) core onto Lys residues via an activated ester on the alkyne, forming peptide-EBx(X) conjugates. These conjugates facilitated the formation of rigid, functional peptide macrocycles using C-terminal or Trp-selective alkynylation. The utility of these macrocyclizations was demonstrated by achieving improved binding affinity to the KEAP1 protein and by generating fluorescent cyclic peptides suitable for live-cell imaging without additional fluorophores. (3) Broadening applicability with EBx-containing amino acids: We prepared EBx amino acids compatible with both solid-phase peptide synthesis (SPPS) and solution-phase synthesis (SPS), allowing us to apply our cyclization strategies to construct a diverse library of cyclic peptides.

PMID:40879758 | PMC:PMC12445010 | DOI:10.1021/acs.accounts.5c00451

Bioconjug Chem. 2025 Jun 18;36(6):1157-1168. doi: 10.1021/acs.bioconjchem.4c00575. Epub 2025 May 21.

ABSTRACT

The strain-promoted alkyne-azide cycloaddition (SPAAC) reaction can be used to modify the surface of bacteria for a variety of applications including drug delivery, biosensing, and imaging. This is usually accomplished by first installing a small azide group within the peptidoglycan and then delivering exogenous cargo (e.g., a protein or nanoparticle) modified with a cyclooctyne group, such as dibenzocyclooctyne (DBCO), for in situ conjugation. However, DBCO is comparatively bulky and hydrophobic, increasing the propensity of some payloads to aggregate. In this study, we sought to invert this paradigm by exploring two novel strategies for incorporating DBCO into the peptidoglycan of Staphylococcus aureus and compared them to an established approach using DBCO-vancomycin. We demonstrate that DBCO-modified small molecules belonging to all three classes─a sortase peptide substrate (LPETG), two d-alanine derivatives, and vancomycin─can selectively label the S. aureus surface to varying degrees. In contrast to DBCO-vancomycin, the DBCO-d-alanine variants do not adversely affect the growth of S. aureus or lead to off-target labeling or toxicity in HEK293T or RAW 264.7 cells. Finally, we show that, unlike IgG3-Fc labeled with DBCO groups, IgG3-Fc labeled with azide groups is stable (i.e., remains water-soluble) under normal storage conditions, retains its ability to bind the immune receptor CD64, and can be successfully attached to the surface of DBCO-modified S. aureus. We believe that the labeling strategies explored herein will expand the paradigm of specific, nontoxic SPAAC-mediated labeling of the surface of S. aureus and other Gram-positive bacteria, opening the door for new applications using azide-modified cargo.

PMID:40398634 | DOI:10.1021/acs.bioconjchem.4c00575

Chem Sci. 2025 May 31;16(27):12455-12466. doi: 10.1039/d5sc01586c. eCollection 2025 Jul 10.

ABSTRACT

Cell wall-deficient bacteria (CWDB) are key contributors to antimicrobial resistance (AMR), enabling persistent infections by evading antibiotics through their transition to L-form states. Therefore, molecular tools for detecting L-form conversion and AMR mechanisms are crucial for developing novel strategies against bacterial infections. Herein, we present the development of small-sized, peptidoglycan-specific fluorogenic probes employing a two-step bioorthogonal strategy that enables real-time visualization of CWDB formation. Tz-FL-S rapidly reacts with the novel d-alanine derivative TCO-d-Ala at a rate of (2.61 ± 0.07) × 10³ M^-1 s^-1, resulting in a 4.9-fold increase in fluorescence intensity. This platform exhibited excellent labeling of peptidoglycan in both Gram-positive and Gram-negative bacteria (signal-to-noise ratio: 15 to 305), effectively capturing the transition from N-form to L-form. Furthermore, we investigated the impact of 14 kinds of antibiotics on L-form conversion and found 13 of them induced CWDB. Besides, we explored the relationship between L-form conversion and AMR. This research enhances our understanding of bacterial adaptations and resistance mechanisms, paving the way for innovative strategies to combat drug-resistant infections.

PMID:40502822 | PMC:PMC12150066 | DOI:10.1039/d5sc01586c

Biophys Rep. 2025 Jun 30;11(3):172-179. doi: 10.52601/bpr.2024.240044.

ABSTRACT

The profound influence of gut microbiota on human health has been well-recognized; however, substantial gaps remain in our understanding of the highly diverse and dynamic processes of microbial growth and activities in the gut. Conventional methods, which primarily rely on DNA sequencing, provide limited insights into these aspects. This paper presents a protocol that integrates fluorescent D-amino acid (FDAA) metabolic labeling with fluorescence in situ hybridization (FISH) for imaging and quantitatively analyzing the in vivo growth of gut microbiota. By administering two FDAAs sequentially through mouse gavage, we label the peptidoglycan of gut bacteria in their native environment, allowing the labeling signals on bacterial cell walls to serve as markers of cellular proliferation and division. We have also demonstrated that the intensity of FDAA labeling directly correlates with the metabolic activity of gut bacteria. Additionally, FISH is employed to distinguish specific bacterial taxa of interest via fluorescence microscopy or flow cytometry. This integrative method greatly enhances our capacity to visualize and measure the in vivo growth and metabolic states of various gut bacteria, thereby illuminating the previously obscured "dark matter" in the gut ecosystem.

PMID:40612236 | PMC:PMC12213662 | DOI:10.52601/bpr.2024.240044

ACS Infect Dis. 2025 Sep 12;11(9):2422-2433. doi: 10.1021/acsinfecdis.5c00233. Epub 2025 Aug 18.

ABSTRACT

Infections with Mycobacterium tuberculosis (Mtb) cause tuberculosis (TB), which requires at least 6 months of treatment with multiple antibiotics. There is emergent interest in using β-lactam antibiotics to improve treatment outcomes for patients. These drugs target cell wall biosynthesis, but a comprehensive list of enzymes inhibited by β-lactams in Mtb is lacking. In the current study, we sought to identify and characterize Mtb enzymes inhibited by β-lactam antibiotics using physiological conditions representative of both acute and chronic TB disease. We used new activity-based probes based on the β-lactam antibiotic meropenem due to its approval by the World Health Organization for TB treatment. Activity-based probes label enzymes based on both substrate specificity and catalytic mechanism, enabling precise identification of drug targets. We identified previously undiscovered targets of meropenem in addition to known cell wall biosynthetic enzymes. We validated β-lactam binding and hydrolysis for six newly identified targets: Rv1723, Rv2257c, Rv0309, DapE (Rv1202), MurI (Rv1338), and LipD (Rv1923). Our results demonstrate that there are at least 30 enzymes in Mtb vulnerable to inhibition by meropenem. This is many more β-lactam targets than historically described, suggesting that efficacy in Mtb is a direct result of polypharmacology.

PMID:40824748 | DOI:10.1021/acsinfecdis.5c00233

JACS Au. 2025 Jun 27;5(7):3399-3407. doi: 10.1021/jacsau.5c00473. eCollection 2025 Jul 28.

ABSTRACT

Discovery of cyclic peptide hits using DNA-encoded libraries (DELs) has recently been extensively researched, with significant efforts directed toward developing DEL-compatible macrocyclization methods. To investigate how different cyclic linkers influence DEL selection outcomes, we constructed eight distinct sublibraries and screened them against two protein targets, MDM2 and GIT1, resulting in two representative yet contrasting scenarios. Validation studies for MDM2 revealed that structural similarity patterns observed across multiple sublibraries could enhance confidence in the authenticity of identified hits. Notably, cmp-10 demonstrated potent inhibitory activity, exhibiting an inhibition constant of 11 nM. In contrast, selections against GIT1 produced discrete enrichment patterns. From these outcomes, two exemplary compounds were selected and validated through both the on-DNA and off-DNA modes. cmp-17 was confirmed to bind specifically to the desired binding pocket, displaying a dissociation constant of 1.22 μM. Furthermore, ITC experiments using mutant GIT1 proteins (GIT1^L271A/L279A and GIT1^L271k/L279k) provided additional insights into the mechanism by which cmp-17 disrupts the interaction between GIT1 and β-PIX.

PMID:40747057 | PMC:PMC12308377 | DOI:10.1021/jacsau.5c00473

J Am Chem Soc. 2025 Aug 20;147(33):29930-29938. doi: 10.1021/jacs.5c06944. Epub 2025 Aug 8.

ABSTRACT

Side chain stapling of cysteine (Cys) residues offers convenient entry into constrained peptides with enhanced bioactivity and bioavailability. Despite its widespread application in the constraint of α-helical, PPII, and loop conformations, the stabilization of β-sheet folds via intrastrand side chain Cys stapling remains largely unexplored. Here, we demonstrate that i→i+2 stapling with E-butenyl, butynyl, and m-xylyl linkers significantly enhances the folded population of two distinct β-hairpin model peptides. High-resolution NMR structures reveal that these staples support canonical β-sheet backbone torsions and stabilize cross-strand interactions. Leveraging the maintenance of intact backbone hydrogen-bonding edges, we employed i→i+2 side chain macrocyclization in the design of constrained β-arch peptides derived from the tau protein. We show that intrastrand stapling of a nonaggregation-prone segment promotes self-assembly into β-sheet-like filaments. The resulting filaments also seed the aggregation of endogenous tau in a cell-based assay in a macrocycle- and sequence-dependent manner. These findings establish di-Cys i→i+2 stapling as a versatile and synthetically accessible method to stabilize β-sheet structure and modulate the self-assembly of seed-competent amyloidogenic peptides.

PMID:40778481 | DOI:10.1021/jacs.5c06944

Chemistry. 2025 Jul 24:e02044. doi: 10.1002/chem.202502044. Online ahead of print.

ABSTRACT

Lanthanide luminescent bioprobes incorporating a reactive group for the functionalization of biomolecules via click chemistry are of high interest for the straightforward preparation of smart luminescent tools for biological imaging and sensing applications. This article describes the synthesis and the photophysical properties of two lanthanide complexes based on the pyclen macrocycle bearing a terminal alkyne group on its pyridine moiety and a 4-(4-alkylthiophenyl)picolinate charge transfer chromophore to sensitize the lanthanide luminescence. The Eu³⁺ and Sm³⁺ complexes show excellent two-photon absorption and emission properties. These complexes were conjugated to a cell penetrating peptide bearing an azide via copper-catalyzed azide-alkyne cycloaddition. The resulting conjugates exhibit luminescence properties comparable to those of the parent alkyne-complex. Two-photon microscopy imaging of live HeLa cells was performed with both the Eu³⁺ and the Sm³⁺ conjugates. These bioprobes are efficiently delivered to the cytosol of the living cells. The Eu³⁺ luminescence was detected in cells even at very low incubation concentration (50 nM) and the Sm³⁺ complex gave excellent quality images despite a low emission quantum yield (< 1 %).

PMID:40702857 | DOI:10.1002/chem.202502044

J Am Chem Soc. 2025 Jul 30;147(30):26307-26318. doi: 10.1021/jacs.5c04424. Epub 2025 Jul 18.

ABSTRACT

Traditional methods for identifying selective protease substrates have primarily relied on synthetic libraries of linear peptides, which offer limited sequence and structural diversity. Here, we present an approach that leverages phage display technology to screen large libraries of chemically modified cyclic peptides, enabling the identification of highly selective substrates for a protease of interest. Our method uses a reactive chemical linker to cyclize peptides on the phage surface, while simultaneously incorporating an affinity tag and a fluorescent reporter. The affinity tag enables capture of the phage library and subsequent release of phages expressing optimal substrates upon incubation with a protease of interest. The addition of a turn-on fluorescent reporter allows direct quantification of cleavage efficiency throughout each selection round. The resulting identified substrates can then be chemically synthesized, optimized and validated using recombinant enzymes and cells. We demonstrate the utility of this approach using Fibroblast Activation Protein α (FAPα) and the related proline-specific protease, dipeptidyl peptidase-4 (DPP4), as targets. Phage selection and subsequent optimization identified substrates with selectivity for each target that have the potential to serve as valuable tools for applications in basic biology and fluorescence image-guided surgery (FIGS). Overall, our strategy provides a rapid and unbiased platform for effectively discovering highly selective, non-natural protease substrates, overcoming key limitations of existing methods.

PMID:40679920 | PMC:PMC12314892 | DOI:10.1021/jacs.5c04424

J Am Chem Soc. 2025 Jul 30;147(30):27095-27104. doi: 10.1021/jacs.5c10625. Epub 2025 Jul 19.

ABSTRACT

Macrocyclic peptides are emerging as a promising molecular framework in covalent drug development due to their high specificity, affinity, and low toxicity, addressing challenges such as off-target effects and nonspecific binding associated with traditional covalent binders. Although mRNA display technology has advanced the discovery of covalent peptide binders, it has primarily focused on cysteine residues, thereby limiting the diversity of targetable proteins. In this study, we ribosomally incorporated 4-fluorosulfonyloxy-l-phenylalanine (FSY) into macrocyclic peptides, enabling the construction of a diverse covalent macrocyclic peptide library for de novo screening against human α-thrombin and fibroblast activation protein. This led to the identification of novel peptides with covalent binding capabilities and a distinct dissociation profile, which in turn enabled potent inhibitory activities at concentrations within the low nanomolar range. Notably, FSY-bearing macrocyclic peptides conjugated with a radionuclide chelator demonstrated significantly improved tumor-selective uptake and prolonged retention, outperforming their noncovalent counterparts while underscoring their remarkable potential in targeted radionuclide therapy. This study provided a robust and efficient platform for the de novo discovery of FSY-bearing macrocyclic peptides, broadening the scope of covalent drug development for diverse biomedical applications.

PMID:40682595 | DOI:10.1021/jacs.5c10625

J Med Chem. 2025 Aug 14;68(15):16578-16592. doi: 10.1021/acs.jmedchem.5c01399. Epub 2025 Jul 24.

ABSTRACT

Unimolecular multireceptor coagonists have emerged as a promising approach in the development of next-generation GLP-1 therapeutics. Herein, we describe the development of a long-acting and stapled GLP-1R/GIPR/GCGR triple agonist that exhibits balanced bioactivities comparable with those of their native ligands along with improved pharmacokinetic parameters. A robust and straightforward solid-phase Ugi macrocyclization strategy enables the facile synthesis of targeted peptides with a side-chain protractor attached on the exocyclic lactam bridge. In obese mice, the lead candidate UTG-4 demonstrates enhanced efficacy in promoting weight loss, suppressing food intake, and improving glucose tolerance and liver health compared to the clinically approved GLP-1R monoagonist semaglutide and GLP-1R/GIPR dual agonist tirzepatide. UTG-4 also exhibits remarkable antiatherosclerotic effects in the Apoe knockout mice. Studies using human aortic endothelial cells reveal that UTG-4 effectively alleviates the endothelial-to-mesenchymal transition, a key process implicated in atherosclerosis progression. These results highlight the therapeutic potential of UTG-4 for combating metabolic disorders and reducing cardiovascular risks.

PMID:40707865 | DOI:10.1021/acs.jmedchem.5c01399

Front Microbiol. 2025 Jun 26;16:1613241. doi: 10.3389/fmicb.2025.1613241. eCollection 2025.

ABSTRACT

INTRODUCTION: Mycobacteria have a unique hydrophobic membrane with several lipid-enriched layers that are low in permeability, setting them apart from other bacteria. This complex structure, consisting of three distinct layers is crucial for cell growth, virulence, and providing a barrier to antibiotics. Previously, we identified a plectasin variant, NZX, which showed activity against Mycobacterium tuberculosis in several murine tuberculosis (TB) infection studies. In this study, we investigated another plectasin variant, NZ2114, known for its effectiveness against Gram-positive bacteria, as a potential antimycobacterial peptide both in vitro and in vivo.

METHODS: The resazurin microtiter assay (REMA) was used to determine MIC; a time-kill assay was performed to evaluate long-term effects; scanning electron microscopy (SEM) was employed to visualize peptide impact; a checkerboard assay assessed drug compatibility; MTT and WST-8 assays were used to estimate peptide toxicity; intracellular killing was evaluated using primary macrophages; peptide stability was assessed in human serum; and a murine tuberculosis (TB) infection model was used to verify the peptide's efficacy.

RESULTS: NZ2114 effectively killed mycobacteria at a minimal inhibitory concentration (MIC₉₉) of 6.1 µM, was non-toxic to primary human cells, and remained resistant to serum degradation while preserving its antimycobacterial capacity. In a checkerboard assay, NZ2114 demonstrated synergy with the first-line TB drugs isoniazid and ethambutol. The antimicrobial effect was also observed against several clinical isolates of Gram-positive bacteria, including Enterococcus faecalis, Enterococcus faecium, and Methicillin-Resistant Staphylococcus aureus (MRSA). In our murine TB infection model, compared to untreated controls, NZ2114 eliminated M. tuberculosis with a log reduction of 0.72 (81.14%) after three doses.

DISCUSSION: These studies suggest NZ2114 as a potential TB therapy, aiding in the control of this significant infectious disease.

PMID:40641884 | PMC:PMC12241124 | DOI:10.3389/fmicb.2025.1613241