Marcos Pires
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[ASAP] Synthesis of Membrane-Permeable Macrocyclic Peptides via Imidazopyridinium Grafting
A Bioorthogonal Dual Fluorogenic Probe for the Live‐Cell Monitoring of Nutrient Uptake by Mammalian Cells
Cells rely heavily on the uptake of exogenous nutrients for survival, growth, and differentiation. Yet quantifying the uptake of small molecule nutrients at the single cell level is difficult. Here we present a new approach to studying the nutrient uptake in live single cells using Inverse Electron-Demand Diels Alder (IEDDA) chemistry. We have modified carboxyfluorescein-diacetate-succinimidyl esters (CFSE) – a quenched fluorophore that can covalently react with proteins and is only turned on in the cytosol of a cell following esterase activity – with a tetrazine. This tetrazine serves as a second quencher for the pendant fluorophore. Upon reaction with nutrients modified with an electron-rich or strained dienophile in an IEDDA reaction, this quenching group is destroyed, thereby enabling the probe to fluoresce. This has allowed us to monitor the uptake of a variety of dienophile-containing nutrients in live primary immune cell populations using flow cytometry and live-cell microscopy.
An HLA-E-targeted TCR bispecific molecule redirects T cell immunity against Mycobacterium tuberculosis
[ASAP] Extending the Potency and Lifespan of Antibiotics: Inhibitors of Gram-Negative Bacterial Efflux Pumps
Targeted acidosis mediated delivery of antigenic MHC-binding peptides
Front Immunol. 2024 Apr 11;15:1337973. doi: 10.3389/fimmu.2024.1337973. eCollection 2024.
ABSTRACT
Cytotoxic T lymphocytes are the primary effector immune cells responsible for protection against cancer, as they target peptide neoantigens presented through the major histocompatibility complex (MHC) on cancer cells, leading to cell death. Targeting peptide-MHC (pMHC) complex offers a promising strategy for immunotherapy due to their specificity and effectiveness against cancer. In this work, we exploit the acidic tumor micro-environment to selectively deliver antigenic peptides to cancer using pH(low) insertion peptides (pHLIP). We demonstrated the delivery of MHC binding peptides directly to the cytoplasm of melanoma cells resulted in the presentation of antigenic peptides on MHC, and activation of T cells. This work highlights the potential of pHLIP as a vehicle for the targeted delivery of antigenic peptides and its presentation via MHC-bound complexes on cancer cell surface for activation of T cells with implications for enhancing anti-cancer immunotherapy.
PMID:38665920 | PMC:PMC11043575 | DOI:10.3389/fimmu.2024.1337973
Bacterial peptidoglycan acts as a digestive signal mediating host adaptation to diverse food resources in C. elegans
Nat Commun. 2024 Apr 16;15(1):3286. doi: 10.1038/s41467-024-47530-y.
ABSTRACT
Food availability and usage is a major adaptive force for the successful survival of animals in nature, yet little is known about the specific signals that activate the host digestive system to allow for the consumption of varied foods. Here, by using a food digestion system in C. elegans, we discover that bacterial peptidoglycan (PGN) is a unique food signal that activates animals to digest inedible food. We identified that a glycosylated protein, Bacterial Colonization Factor-1 (BCF-1), in the gut interacts with bacterial PGN, leading to the inhibition of the mitochondrial unfolded protein response (UPRmt) by regulating the release of Neuropeptide-Like Protein (NLP-3). Interestingly, activating UPRmt was found to hinder food digestion, which depends on the innate immune p38 MAPK/PMK-1 pathway. Conversely, inhibiting PMK-1 was able to alleviate digestion defects in bcf-1 mutants. Furthermore, we demonstrate that animals with digestion defects experience reduced natural adaptation capabilities. This study reveals that PGN-BCF-1 interaction acts as "good-food signal" to promote food digestion and animal growth, which facilitates adaptation of the host animals by increasing ability to consume a wide range of foods in their natural environment.
PMID:38627398 | PMC:PMC11021419 | DOI:10.1038/s41467-024-47530-y
Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation
Macrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol-containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol-containing elements and used them for the combinatorial synthesis of a 2,688-member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X-ray structure analysis of macrocycle-target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.
[ASAP] Bioluminescence-Based Determination of Cytosolic Accumulation of Antibiotics in Escherichia coli
Pyrrole-based inhibitors of RND-type efflux pumps reverse antibiotic resistance and display anti-virulence potential
by Nisha Mahey, Rushikesh Tambat, Ritu Kalia, Rajnita Ingavale, Akriti Kodesia, Nishtha Chandal, Srajan Kapoor, Dipesh Kumar Verma, Krishan Gopal Thakur, Sanjay Jachak, Hemraj Nandanwar
Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.[ASAP] Noninvasive Analysis of Peptidoglycan from Living Animals
[ASAP] Maturation and Conformational Switching of a De Novo Designed Phase-Separating Polypeptide
[ASAP] Chimeric NOD2 Mincle Agonists as Vaccine Adjuvants
[ASAP] Bioorthogonal Radiolabeling of Azide-Modified Bacteria Using [18F]FB-sulfo-DBCO
[ASAP] GlcNAc-1,6-anhydro-MurNAc Moiety Affords Unusual Glycosyl Acceptor that Terminates Peptidoglycan Elongation
[ASAP] Membrane Permeability in a Large Macrocyclic Peptide Driven by a Saddle-Shaped Conformation
[ASAP] Cu-Catalyzed Azide–Alkyne–Thiol Reaction Forms Ubiquitous Background in Chemical Proteomic Studies
A new type of antibiotic targets a drug-resistant bacterium
Nature, Published online: 03 January 2024; doi:10.1038/d41586-023-03988-2
Infections caused by drug-resistant strains of the bacterium Acinetobacter baumannii have been hard to treat in the clinic. A new class of antibiotics has been identified with the potential to tackle these microbes.A novel antibiotic class targeting the lipopolysaccharide transporter
Nature, Published online: 03 January 2024; doi:10.1038/s41586-023-06873-0
A tethered macrocyclic peptide antibiotic class described here—which shows potent antibacterial activity against carbapenem-resistant Acinetobacter baumannii—blocks the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane through inhibition of the LptB2FGC complex.A new antibiotic traps lipopolysaccharide in its intermembrane transporter
Nature, Published online: 03 January 2024; doi:10.1038/s41586-023-06799-7
A mechanism of lipid transport inhibition has been identified for a class of peptide antibiotics effective against resistant Acinetobacter strains, which may have applications in the inhibition of other Gram-negative pathogens.De novo development of small cyclic peptides that are orally bioavailable
Nature Chemical Biology, Published online: 28 December 2023; doi:10.1038/s41589-023-01496-y
Cyclic peptides show promise for modulating difficult disease targets; however, they often cannot be administered orally. The authors developed a method to synthesize and screen large libraries of small cyclic peptides while enabling the simultaneous interrogation of activity and permeability. This approach was applied to the disease target thrombin to discover peptides with high affinity, stability and oral bioavailability of up to 18% in rats.[ASAP] Quantification of Binding of Small Molecules to Native Proteins Overexpressed in Living Cells
Biocatalytic cyclization of small macrolactams by a penicillin-binding protein-type thioesterase
Nature Chemical Biology, Published online: 07 December 2023; doi:10.1038/s41589-023-01495-z
Macrocyclic peptides are promising scaffolds for chemical tools and potential therapeutics, but their synthesis is currently difficult. Here, the authors report the characterization of Ulm16, a peptide cyclase of the penicillin-binding protein (PBP)-type class of thioesterases, that catalyzes head-to-tail macrolactamization of nonribosmal peptides of 4–6 amino acids in length.Measurement of Accumulation of Antibiotics to Staphylococcus aureus in Phagosomes of Live Macrophages
Staphylococcus aureus can persist within host immune cells, potentially evading immune responses and antibiotics. Investigating antibiotic permeability into phagocytic vacuoles, we developed a permeability assay. By analyzing antibiotic arrival in phagosomes of infected macrophages, we identified permeability differences, offering insights into antibiotic contribution to intracellular pathogens.
Abstract
Staphylococcus aureus (S. aureus) has evolved the ability to persist after uptake into host immune cells. This intracellular niche enables S. aureus to potentially escape host immune responses and survive the lethal actions of antibiotics. While the elevated tolerance of S. aureus to small-molecule antibiotics is likely to be multifactorial, we pose that there may be contributions related to permeation of antibiotics into phagocytic vacuoles, which would require translocation across two mammalian bilayers. To empirically test this, we adapted our recently developed permeability assay to determine the accumulation of FDA-approved antibiotics into phagocytic vacuoles of live macrophages. Bioorthogonal reactive handles were metabolically anchored within the surface of S. aureus, and complementary tags were chemically added to antibiotics. Following phagocytosis of tagged S. aureus cells, we were able to specifically analyze the arrival of antibiotics within the phagosomes of infected macrophages. Our findings enabled the determination of permeability differences between extra- and intracellular S. aureus, thus providing a roadmap to dissect the contribution of antibiotic permeability to intracellular pathogens.
Porin-independent accumulation in Pseudomonas enables antibiotic discovery
Nature, Published online: 22 November 2023; doi:10.1038/s41586-023-06760-8
We use a whole-cell accumulation assay to assess the ability of non-antibiotic, structurally diverse small molecules to accumulate in Pseudomonas aeruginosa, with potential application in developing drugs to target this pathogen.[ASAP] Lysine-Reactive N-Acyl-N-aryl Sulfonamide Warheads: Improved Reaction Properties and Application in the Covalent Inhibition of an Ibrutinib-Resistant BTK Mutant
[ASAP] Cell-Surface-Retained Peptide Additives for the Cytosolic Delivery of Functional Proteins
Targeting PGLYRP1 promotes antitumor immunity while inhibiting autoimmune neuroinflammation
Nature Immunology, Published online: 12 October 2023; doi:10.1038/s41590-023-01645-4
Here, the authors show that deletion of Pglyrp1 promotes antitumor immunity owing to its inhibitory function in CD8+ T cells and that targeting it can inhibit development of autoimmune neuroinflammation. These findings indicate that PGLYRP1 might be a target for immunotherapy.