Rachita Dash

ACS Chem Biol. 2024 Mar 15;19(3):707-717. doi: 10.1021/acschembio.3c00724. Epub 2024 Mar 5.

ABSTRACT

Surface lipids on pathogenic mycobacteria modulate infection outcomes by regulating host immune responses. Phenolic glycolipid (PGL) is a host-modulating surface lipid that varies among clinical Mycobacterium tuberculosis strains. PGL is also found in Mycobacterium marinum, where it promotes infection of zebrafish through effects on the innate immune system. Given the important role this lipid plays in the host-pathogen relationship, tools for profiling its abundance, spatial distribution, and dynamics are needed. Here, we report a strategy for imaging PGL in live mycobacteria using bioorthogonal metabolic labeling. We functionalized the PGL precursor p-hydroxybenzoic acid (pHB) with an azide group (3-azido pHB). When fed to mycobacteria, 3-azido pHB was incorporated into the cell surface, which could then be visualized via the bioorthogonal conjugation of a fluorescent probe. We confirmed that 3-azido pHB incorporates into PGL using mass spectrometry methods and demonstrated selectivity for PGL-producing M. marinum and M. tuberculosis strains. Finally, we applied this metabolic labeling strategy to study the dynamics of PGL within the mycobacterial membrane. This new tool enables visualization of PGL that may facilitate studies of mycobacterial pathogenesis.

PMID:38442242 | PMC:PMC10949201 | DOI:10.1021/acschembio.3c00724

J Biol Chem. 2024 Apr;300(4):107125. doi: 10.1016/j.jbc.2024.107125. Epub 2024 Mar 1.

ABSTRACT

Cyclotides are plant-derived peptides characterized by a head-to-tail cyclic backbone and a cystine knot motif comprised of three disulfide bonds. Formation of this motif via in vitro oxidative folding can be challenging and can result in misfolded isomers with nonnative disulfide connectivities. Here, we investigated the effect of β-turn nucleation on cyclotide oxidative folding. Two types of β-turn mimics were grafted into kalata B1, individually replacing each of the four β-turns in the folded cyclotide. Insertion of d-Pro-Gly into loop 5 was beneficial to the folding of both cyclic kB1 and a linear form of the peptide. The linear grafted analog folded four-times faster in aqueous conditions than cyclic kB1 in optimized conditions. Additionally, the cyclic analogue folded without the need for redox agents by transitioning through a native-like intermediate that was on-pathway to product formation. Kalata B1 is from the Möbius subfamily of cyclotides. Grafting d-Pro-Gly into loop 5 of cyclotides from two other subfamilies also had a beneficial effect on folding. Our findings demonstrate the importance of a β-turn nucleation site for cyclotide oxidative folding, which could be adopted as a chemical strategy to improve the in vitro folding of diverse cystine-rich peptides.

PMID:38432638 | PMC:PMC10999817 | DOI:10.1016/j.jbc.2024.107125

J Am Chem Soc. 2024 Mar 13;146(10):6817-6829. doi: 10.1021/jacs.3c13644. Epub 2024 Mar 1.

ABSTRACT

N-Acetyl muramic acid (NAM) probes containing alkyne or azide groups are commonly used to investigate aspects of cell wall synthesis because of their small size and ability to incorporate into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions are not compatible with live cells, and strain-promoted alkyne-azide cycloaddition (SPAAC) reaction rates are modest and, therefore, not as desirable for tracking the temporal alterations of bacterial cell growth, remodeling, and division. Alternatively, the tetrazine-trans-cyclooctene ligation (Tz-TCO), which is the fastest known bioorthogonal reaction and not cytotoxic, allows for rapid live-cell labeling of PG at biologically relevant time scales and concentrations. Previous work to increase reaction kinetics on the PG surface by using tetrazine probes was limited because of low incorporation of the probe. Described here are new approaches to construct a minimalist tetrazine (Tz)-NAM probe utilizing recent advancements in asymmetric tetrazine synthesis. This minimalist Tz-NAM probe was successfully incorporated into pathogenic and commensal bacterial PG where fixed and rapid live-cell, no-wash labeling was successful in both free bacterial cultures and in coculture with human macrophages. Overall, this probe allows for expeditious labeling of bacterial PG, thereby making it an exceptional tool for monitoring PG biosynthesis for the development of new antibiotic screens. The versatility and selectivity of this probe will allow for real-time interrogation of the interactions of bacterial pathogens in a human host and will serve a broader utility for studying glycans in multiple complex biological systems.

PMID:38427023 | PMC:PMC10941766 | DOI:10.1021/jacs.3c13644

ACS Omega. 2024 Feb 5;9(7):8179-8187. doi: 10.1021/acsomega.3c08701. eCollection 2024 Feb 20.

ABSTRACT

Cyclic peptides that inhibit protein-protein interactions have significant advantages over linear peptides and small molecules for modulating cellular signaling networks in cancer and other diseases. However, the permeability barrier of the plasma membrane remains a formidable obstacle to the development of cyclic peptides into applicable drugs. Here, we test the ability of a family of synthetically evolved spontaneous membrane translocating peptides (SMTPs) to deliver phalloidin, a representative bioactive cyclic peptide, to the cytosol of human cells in culture. Phalloidin does not enter cells spontaneously, but if delivered to the cytosol, it inhibits actin depolymerization. We thus use a wound-healing cell mobility assay to assess the biological activity of phalloidin conjugated to three SMTPs that we previously discovered. All three SMTPs can deliver phalloidin to the cell cytosol, and one does so at concentrations as low as 3 μM. Delivery occurs despite the fact that the SMTPs were originally selected based on membrane translocation with no cargo other than a small fluorescent dye. These results show that SMTPs are viable delivery vehicles for cyclic peptides, although their efficiency is moderate. Further, these results suggest that one additional generation of synthetic molecular evolution could be used to optimize SMTPs for the efficient delivery of any bioactive cyclic peptide into cells.

PMID:38405535 | PMC:PMC10882622 | DOI:10.1021/acsomega.3c08701

Adv Healthc Mater. 2024 Feb 22:e2303654. doi: 10.1002/adhm.202303654. Online ahead of print.

ABSTRACT

Oral delivery of peptide therapeutics faces multiple challenges due to their instability in the gastrointestinal tract and low permeation capability. In this study, we aimed to develop a liposomal nanocarrier formulation to enable the oral delivery of the vancomycin-peptide derivative FU002. FU002 is a promising, resistance-breaking, antibiotic which exhibits poor oral bioavailability limiting its potential therapeutic use. To increase its oral bioavailability, we incorporated FU002 into tetraether lipid-stabilized liposomes modified with cyclic cell-penetrating peptides on the liposomal surface. This liposomal formulation showed strong binding to Caco-2 cells without exerting cytotoxic effects in vitro. Pharmacokinetics studies in vivo in rats revealed increased oral bioavailability of liposomal FU002 when compared to the free drug. In vitro and in vivo antimicrobial activity of FU002 was preserved in the liposomal formulation. As highlight, oral administration of liposomal FU002 resulted in significant therapeutic efficacy in a murine systemic infection model. Thus, the presented nanotechnological approach provides a promising strategy for enabling oral delivery of this highly active vancomycin derivative. This article is protected by copyright. All rights reserved.

PMID:38387090 | DOI:10.1002/adhm.202303654

J Nat Prod. 2024 Feb 22. doi: 10.1021/acs.jnatprod.3c00750. Online ahead of print.

ABSTRACT

The marine sponge-derived fungus Stachylidium bicolor 293 K04 is a prolific producer of specialized metabolites, including certain cyclic tetrapeptides called endolides, which are characterized by the presence of the unusual amino acid N-methyl-3-(3-furyl)-alanine. This rare feature can be used as bait to detect new endolide-like analogs through customized fragment pattern searches of tandem mass spectrometry data using the Mass Spec Query Language (MassQL). Here, we integrate endolide-specific MassQL queries with molecular networking to obtain substructural information guiding the targeted isolation and structure elucidation of the new proline-containing endolides E (1) and F (2). We showed that endolide F (but not E) is a moderate antagonist of the arginine vasopressin V_1A receptor, a member of the G protein-coupled receptor superfamily.

PMID:38385767 | DOI:10.1021/acs.jnatprod.3c00750

Pharmaceuticals (Basel). 2024 Feb 13;17(2):243. doi: 10.3390/ph17020243.

ABSTRACT

A total of nine TIDES (pepTIDES and oligonucleoTIDES) were approved by the FDA during 2023. The four approved oligonucleotides are indicated for various types of disorders, including amyotrophic lateral sclerosis, geographic atrophy, primary hyperoxaluria type 1, and polyneuropathy of hereditary transthyretin-mediated amyloidosis. All oligonucleotides show chemically modified structures to enhance their stability and therapeutic effectiveness as antisense or aptamer oligomers. Some of them demonstrate various types of conjugation to driving ligands. The approved peptides comprise various structures, including linear, cyclic, and lipopeptides, and have diverse applications. Interestingly, the FDA has granted its first orphan drug designation for a peptide-based drug as a highly selective chemokine antagonist. Furthermore, Rett syndrome has found its first-ever core symptoms treatment, which is also peptide-based. Here, we analyze the TIDES approved in 2023 on the basis of their chemical structure, medical target, mode of action, administration route, and common adverse effects.

PMID:38399458 | PMC:PMC10893093 | DOI:10.3390/ph17020243

Nat Commun. 2024 Feb 21;15(1):1611. doi: 10.1038/s41467-024-45766-2.

ABSTRACT

We introduce a computational approach for the design of target-specific peptides. Our method integrates a Gated Recurrent Unit-based Variational Autoencoder with Rosetta FlexPepDock for peptide sequence generation and binding affinity assessment. Subsequently, molecular dynamics simulations are employed to narrow down the selection of peptides for experimental assays. We apply this computational strategy to design peptide inhibitors that specifically target β-catenin and NF-κB essential modulator. Among the twelve β-catenin inhibitors, six exhibit improved binding affinity compared to the parent peptide. Notably, the best C-terminal peptide binds β-catenin with an IC₅₀ of 0.010 ± 0.06 μM, which is 15-fold better than the parent peptide. For NF-κB essential modulator, two of the four tested peptides display substantially enhanced binding compared to the parent peptide. Collectively, this study underscores the successful integration of deep learning and structure-based modeling and simulation for target specific peptide design.

PMID:38383543 | PMC:PMC10882002 | DOI:10.1038/s41467-024-45766-2

Future Med Chem. 2024 Mar;16(5):389-398. doi: 10.4155/fmc-2023-0017. Epub 2024 Feb 19.

ABSTRACT

Background: Traditional methods for chemical library generation in virtual screening often impose limitations on the accessible chemical space or produce synthetically irrelevant structures. Incorporating common chemical reactions into generative algorithms could offer significant benefits. Materials & methods: In this study, we developed NeuroClick, a graphical user interface software designed to perform in silico azide-alkyne cycloaddition, a widely utilized synthetic approach in modern medicinal chemistry. Results & conclusion: NeuroClick facilitates the generation and filtering of large combinatorial libraries at a remarkable rate of 10,000 molecules per minute. Moreover, the generated products can be filtered to identify subsets of pharmaceutically relevant compounds based on Lipinski's rule of five and blood-brain barrier permeability prediction. We demonstrate the utility of NeuroClick by generating and filtering several thousand molecules for dopamine D3 receptor ligand screening.

PMID:38372134 | DOI:10.4155/fmc-2023-0017

Eur J Med Chem. 2024 Jan 26;268:116175. doi: 10.1016/j.ejmech.2024.116175. Online ahead of print.

ABSTRACT

Microbial secondary metabolites are pivotal for the development of novel drugs. However, conventional culture techniques, have left a vast array of unexpressed biosynthetic gene clusters (BGCs) in microorganisms, hindering the discovery of metabolites with distinct structural features and diverse biological functions. To address this limitation, several innovative strategies have been emerged. The "One Strain Many Compounds" (OSMAC) strategy, which involves altering microbial culture conditions, has proven to be particularly effective in mining numerous novel secondary metabolites for the past few years. Among these, microbial cyclic peptides stand out. These peptides often comprise rare amino acids, unique chemical structures, and remarkable biological function. With the advancement of the OSMAC strategy, a plethora of new cyclic peptides have been identified from diverse microbial genera. This work reviews the progress in mining novel compounds using the OSMAC strategy and the applications of this strategy in discovering 284 microbial cyclic peptides from 63 endophytic strains, aiming to offer insights for the further explorations into novel active cyclic peptides.

PMID:38377824 | DOI:10.1016/j.ejmech.2024.116175

J Am Chem Soc. 2024 Feb 15. doi: 10.1021/jacs.3c11153. Online ahead of print.

ABSTRACT

We report piperazine-fused six-membered-cyclic disulfides as redox substrates that unlock best-in-class bioreduction probes for live cell biology, since their self-immolation after reduction is unprecedentedly rapid. We develop scalable, diastereomerically pure, six-step syntheses that access four key cis- and trans-piperazine-fused cyclic dichalcogenides without chromatography. Fluorogenic redox probes using the disulfide piperazines are activated >100-fold faster than the prior art monoamines, allowing us to deconvolute reduction and cyclization rates during activation. The cis- and trans-fused diastereomers have remarkably different reductant specificities, which we trace back to piperazine boat/chair conformation effects: the cis-fused disulfide C-DiThia is activated only by strong vicinal dithiol reductants, but the trans-disulfide T-DiThia is activated even by moderate concentrations of monothiols such as GSH. Thus, in cellular applications, cis-disulfide probes selectively report on the reductive activity of the powerful thioredoxin proteins, while trans-disulfides are rapidly but promiscuously reactive. Finally, we showcase late-stage diversifications of the piperazine-disulfides, promising their broad applicability as redox-cleavable cores for probes and prodrugs that interface powerfully with cellular thiol/disulfide redox biology, for solid phase synthesis and purification, and for stimulus-responsive linkers in bifunctional reagents and antibody-drug conjugates - in addition to their dithiols' potential as high-performance reducing agents.

PMID:38358897 | DOI:10.1021/jacs.3c11153

Curr Protoc. 2024 Feb;4(2):e957. doi: 10.1002/cpz1.957.

ABSTRACT

In neurodegenerative diseases like Alzheimer's disease (AD), endogenous proteins or peptides aggregate with themselves. These proteins may lose their function or aggregates and/or oligomers can obtain toxicity, causing injury or death to cells. Aggregation of two major proteins characterizes AD. Amyloid-β peptide (Aβ) is deposited in amyloid plaques within the extracellular space of the brain and Tau in so-called neurofibrillary tangles in neurons. Finding peptide ligands to halt protein aggregation is a promising therapeutical approach. Using mirror-image phage display with a commercially available, randomized 12-mer peptide library, we have selected D-amino acid peptides, which bind to the Tau protein and modulate its aggregation in vitro. Peptides can bind specifically and selectively to a target molecule, but natural L-amino acid peptides may have crucial disadvantages for in vivo applications, as they are sensitive to protease degradation and may elicit immune responses. One strategy to circumvent these disadvantages is the use of non-naturally occurring D-amino acid peptides as they exhibit increased protease resistance and generally do not activate the immune system. To perform mirror-image phage display, the target protein needs to be synthesized as D-amino acid version. If the target protein sequence is too long to be synthesized properly, smaller peptides derived from the full length protein can be used for the selection process. This also offers the possibility to influence the binding region of the selected D-peptides in the full-length target protein. Here we provide the protocols for mirror-image phage display selection on the PHF6* peptide of Tau, based on the commercially available Ph.D.™-12 Phage Display Peptide Library Kit, leading to D-peptides that also bind the full length Tau protein (Tau441), next to PHF6*. In addition, we provide protocols and data for the first characterization of those D-peptides that inhibit Tau aggregation in vitro. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mirror image phage display selection against D-PHF6* fibrils Support Protocol 1: Single phage ELISA Basic Protocol 2: Sequencing and D-peptide generation Basic Protocol 3: Thioflavin-T (ThT) test to control inhibition of Tau aggregation Support Protocol 2: Purification of full-length Tau protein Basic Protocol 4: ELISA to demonstrate the binding of the generated D-peptides to PHF6* and full-length Tau fibrils.

PMID:38372457 | DOI:10.1002/cpz1.957

Cell Rep Med. 2024 Feb 20;5(2):101418. doi: 10.1016/j.xcrm.2024.101418. Epub 2024 Feb 9.

ABSTRACT

The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) poses a major challenge to vaccines and antiviral therapeutics due to their extensive evasion of immunity. Aiming to develop potent and broad-spectrum anticoronavirus inhibitors, we generated A1-(GGGGS)7-HR2m (A1L35HR2m) by introducing an angiotensin-converting enzyme 2 (ACE2)-derived peptide A1 to the N terminus of the viral HR2-derived peptide HR2m through a long flexible linker, which showed significantly improved antiviral activity. Further cholesterol (Chol) modification at the C terminus of A1L35HR2m greatly enhanced the inhibitory activities against SARS-CoV-2, SARS-CoV-2 VOCs, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudoviruses, with IC₅₀ values ranging from 0.16 to 5.53 nM. A1L35HR2m-Chol also potently inhibits spike-protein-mediated cell-cell fusion and the replication of authentic Omicron BA.2.12.1, BA.5, and EG.5.1. Importantly, A1L35HR2m-Chol distributed widely in respiratory tract tissue and had a long half-life (>10 h) in vivo. Intranasal administration of A1L35HR2m-Chol to K18-hACE2 transgenic mice potently inhibited Omicron BA.5 and EG.5.1 infection both prophylactically and therapeutically.

PMID:38340726 | PMC:PMC10897629 | DOI:10.1016/j.xcrm.2024.101418

J Sci Food Agric. 2024 Feb 9. doi: 10.1002/jsfa.13370. Online ahead of print.

ABSTRACT

Dietary patterns play an important role in regards to the modulation and control of the gut microbiome composition and function. The interaction between diet and microbiota plays an important role in order to maintain intestinal homeostasis, which ultimately afftect the host's health. Diet directly impacts the microbes that inhabit the gastrointestinal tract (GIT), which then contributes to the production of secondary metabolites, such as short-chain fatty acids, neurotransmitters, and antimicrobial peptides. Dietary consumption with genetically modified probiotics can be the best vaccine delivery vector and protect cells from various illnesses. A holistic approach to disease prevention, treatment, and management take these intrinsically linked diet-microbes, microbe-microbe interactions, and microbe-host interactions into account. Dietary components, such as fibre can modulate beneficial gut microbiota, and they have resulting ameliorative effects against metabolic disorders. Medical interventions, such as antibiotic drugs can conversely have detrimental effects on gut microbiota by disputing the balance between Bacteroides and firmicute, which contribute to continuing disease states. We summarise the known effects of various dietary components, such as fibres, carbohydrates, fatty acids, vitamins, minerals, proteins, phenolic acids, and antibiotics on the composition of the gut microbiota in this paper in addition to the beneficial effect of genetically modified probiotics and consequentially its role in regards to shaping human health. This article is protected by copyright. All rights reserved.

PMID:38334314 | DOI:10.1002/jsfa.13370