Amphiphilic shuttle peptide delivers base editor ribonucleoprotein to correct the CFTR R553X mutation in well-differentiated airway epithelial cells.

Base editing could correct nonsense mutations that cause cystic fibrosis (CF), but clinical development is limited by the lack of delivery methods that efficiently breach the barriers presented by airway epithelia. Here, we present a novel amphiphilic shuttle peptide based on the previously reported S10 peptide that substantially improved base editor ribonucleoprotein (RNP) delivery. Studies of the S10 secondary structure revealed that the alpha-helix formed by the endosomal leakage domain (ELD), but not the cell penetrating peptide (CPP), was functionally important for delivery.

PEGylation of a Peptide-Based Amphiphilic Delivery Agent and Influence on Protein Delivery to Cells.

The cell membrane is a restrictive biological barrier, especially for large, charged molecules, such as proteins. The use of cell-penetrating peptides (CPPs) can facilitate the delivery of proteins, protein complexes, and peptides across the membrane by a variety of mechanisms that are all limited by endosomal sequestration. To improve CPP-mediated delivery, we previously reported the rapid and effective cytosolic delivery of proteins in vitro and in vivo by their coadministration with the peptide S10, which combines a CPP and an endosomal leakage domain.

Design and synthesis of dansyl-labeled inhibitors of steroid sulfatase for optical imaging.

Steroid sulfatase (STS) is an important enzyme regulating the conversion of sulfated steroids into their active hydroxylated forms. Notably, the inhibition of STS has been shown to decrease the levels of active estrogens and was translated into clinical trials for the treatment of breast cancer. Based on quantitative structure-activity relationship (QSAR) and molecular modeling studies, we herein report the design of fluorescent inhibitors of STS by adding a dansyl group on an estrane scaffold.

Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia.

The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides.

Lobaric acid and pseudodepsidones inhibit NF-κB signaling pathway by activation of PPAR-γ.

Herein we report the anti-inflammatory activity of lobaric acid and pseudodepsidones isolated from the nordic lichen Stereocaulon paschale. Lobaric acid (1) and three compounds (2, 7 and 9) were found to inhibit the NF-κB activation and the secretion of pro-inflammatory cytokines (IL-1β and TNF-α) in LPS-stimulated macrophages. Inhibition and docking simulation experiments provided evidence that lobaric acid and pseudodepsidones bind to PPAR-γ between helix H3 and the beta sheet, similarly to partial PPAR-γ agonists.

Chemical synthesis of C3-oxiranyl/oxiranylmethyl-estrane derivatives targeted by molecular modeling and tested as potential inhibitors of 17β-hydroxysteroid dehydrogenase type 1.

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is a promising therapeutic target known to play a pivotal role in the progression of estrogen-dependent diseases such as breast cancer, and endometriosis. This enzyme is responsible for the last step in the biosynthesis of the most potent estrogen, estradiol (E2) and its inhibition would prevent the growth of estrogen-sensitive tumors. Based on molecular modeling with docking experiments, we identified two promising C3-oxiranyl/oxiranylmethyl-estrane derivatives that would bind competitively and irreversibly in the catalytic site of 17β-HSD1.

CRISPR-Induced Deletion with SaCas9 Restores Dystrophin Expression in Dystrophic Models In Vitro and In Vivo.

Duchenne muscular dystrophy (DMD), a severe hereditary disease affecting 1 in 3,500 boys, mainly results from the deletion of exon(s), leading to a reading frameshift of the DMD gene that abrogates dystrophin protein synthesis. Pairs of sgRNAs for the Cas9 of Staphylococcus aureus were meticulously chosen to restore a normal reading frame and also produce a dystrophin protein with normally phased spectrin-like repeats (SLRs), which is not usually obtained by skipping or by deletion of complete exons. This can, however, be obtained in rare instances where the exon and intron borders of the beginning and the end of the complete deletion (patient deletion plus CRISPR-induced deletion) are at similar positions in the SLR.

Membrane permeabilizing amphiphilic peptide delivers recombinant transcription factor and CRISPR-Cas9/Cpf1 ribonucleoproteins in hard-to-modify cells.

Delivery of recombinant proteins to therapeutic cells is limited by a lack of efficient methods. This hinders the use of transcription factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleoproteins to develop cell therapies. Here, we report a soluble peptide designed for the direct delivery of proteins to mammalian cells including human stem cells, hard-to-modify primary natural killer (NK) cells, and cancer cell models.

Synthesis and biological evaluation of novel quinazoline-4-piperidinesulfamide derivatives as inhibitors of NPP1.

The ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) was recently shown to promote mineralization of the aortic valve, hence, its inhibition represents a significant target. A quinazoline-4-piperidine sulfamide compound (QPS1) has been described as a specific and non-competitive inhibitor of NPP1. We report herein the synthesis and in vitro inhibition studies of novel quinazoline-4-piperidine sulfamide analogues using QPS1 as the lead compound.

Extended Linkers Improve the Detection of Protein-protein Interactions (PPIs) by Dihydrofolate Reductase Protein-fragment Complementation Assay (DHFR PCA) in Living Cells.

Understanding the function of cellular systems requires describing how proteins assemble with each other into transient and stable complexes and to determine their spatial relationships. Among the tools available to perform these analyses on a large scale is Protein-fragment Complementation Assay based on the dihydrofolate reductase (DHFR PCA). Here we test how longer linkers between the fusion proteins and the reporter fragments affect the performance of this assay.

Insight into the mode of action and selectivity of PBRM, a covalent steroidal inhibitor of 17β-hydroxysteroid dehydrogenase type 1.

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is involved in the biosynthesis of estradiol, the major bioactive endogenous estrogen in mammals, and constitutes an interesting therapeutic target for estrogen-dependent diseases. A steroidal derivative, 3-{[(16β,17β)-3-(2-bromoethyl)-17-hydroxyestra-1,3,5(10)-trien-16-yl]methyl} benzamide (PBRM), has recently been described as a non-estrogenic, irreversible inhibitor of 17β-HSD1. However, the mode of action of this inhibitor and its selectivity profile have not yet been elucidated.

Characterization of the structure, dynamics and allosteric pathways of human NPP1 in its free form and substrate-bound complex from molecular modeling.

The ectonucleotide phosphodiesterase/pyrophosphatase-1 (NPP1) is a type II transmembrane glycoprotein that regulates extracellular inorganic purine nucleotide and inorganic diphosphate levels through the hydrolysis of ATP into AMP and diphosphate. NPP1 is a promising drug target as it plays a role in several disorders. In the present work, we report the 3D structure modeling and extensive molecular dynamics simulations of NPP1-h, both in its free and ATP-bound forms.

Revisiting the Juliá-Colonna enantioselective epoxidation: supramolecular catalysis in water.

We describe an efficient epoxidation process leading to chiral epoxyketones using the reusable homo-oligopeptide poly-l-leucine (PLL) in pure water, without any organic co-solvent. A range of substituted epoxyketones can be accessed with good conversions and high enantioselectivities. Based on the experimental results and computational studies, we propose a mechanism that demonstrates the importance of both the α-helical structure and the presence of a hydrophobic groove of the homo-oligopeptide catalyst for reactivity and selectivity.

The I427T neuraminidase (NA) substitution, located outside the NA active site of an influenza A(H1N1)pdm09 variant with reduced susceptibility to NA inhibitors, alters NA properties and impairs viral fitness.

Emergence of pan neuraminidase inhibitor (NAI)-resistant variants constitutes a serious clinical concern. An influenza A(H1N1)pdm09 variant containing the I427T/Q313R neuraminidase (NA) substitutions was previously identified in a surveillance study. Although these changes are not part of the NA active site, the variant showed reduced susceptibility to many NAIs.

Discovery of a sulfamate-based steroid sulfatase inhibitor with intrinsic selective estrogen receptor modulator properties.

Steroid sulfatase (STS), the enzyme which converts inactive sulfated steroid precursors into active hormones, is a promising therapeutic target for the treatment of estrogen-sensitive breast cancer. We report herein the synthesis and in vitro study of dual-action STS inhibitors with selective estrogen-receptor modulator (SERM) effects. A library of tetrahydroisoquinoline-N-substituted derivatives (phenolic compounds) was synthesized by solid-phase chemistry and tested on estrogen-sensitive breast cancer T-47D cells.

Antibiotic resistance due to an unusual ColE1-type replicon plasmid in Aeromonas salmonicida.

Aeromonas salmonicida subsp. salmonicida is a fish pathogen known to have a rich plasmidome. In the present study, we discovered an isolate of this bacterium bearing an additional unidentified small plasmid.

Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method.

The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift.

Impact of structural modifications at positions 13, 16 and 17 of 16β-(m-carbamoylbenzyl)-estradiol on 17β-hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity.

The chemical synthesis of four stereoisomers (compounds 5a-d) of 16β-(m-carbamoylbenzyl)-estradiol, a potent reversible inhibitor of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), and two intermediates (compounds 3a and b) was performed. Assignment of all nuclear magnetic resonance signals confirmed the stereochemistry at positions 13, 16 and 17. Nuclear overhauser effects showed clear correlations supporting a C-ring chair conformation for 5a and b and a C-ring boat conformation for 5c and d.

Quinazoline-4-piperidine sulfamides are specific inhibitors of human NPP1 and prevent pathological mineralization of valve interstitial cells.

Background And Purpose: Ectonucleotide pyrophosphatase/PDE1 (NPP1) is an ectoenzyme, which plays a role in several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack potency and specificity. Quinazoline-4-piperidine sulfamides (QPS) have been described as potent inhibitors of NPP1.

tRNAGlu increases the affinity of glutamyl-tRNA synthetase for its inhibitor glutamyl-sulfamoyl-adenosine, an analogue of the aminoacylation reaction intermediate glutamyl-AMP: mechanistic and evolutionary implications.

For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS).

Identification of fused 16β,17β-oxazinone-estradiol derivatives as a new family of non-estrogenic 17β-hydroxysteroid dehydrogenase type 1 inhibitors.

A new family of cyclic carbamate-estradiol derivatives has been designed to remove the intrinsic estrogenic activity of a parent acyclic compound reported as a potent inhibitor of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1). The synthesis of two series of fused 16β,17β-oxazinone-estradiol derivatives, saturated compounds 7a-d and unsaturated compounds 10a-d, led to the identification of 10b, a 17β-HSD1 inhibitor (IC50 = 1.4 μM) without estrogenic activity in estrogen-sensitive T-47D cells.

Peroxidase activity and involvement in the oxidative stress response of roseobacter denitrificans truncated hemoglobin.

Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd.

Discovery of a non-estrogenic irreversible inhibitor of 17β-hydroxysteroid dehydrogenase type 1 from 3-substituted-16β-(m-carbamoylbenzyl)-estradiol derivatives.

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is thought to play a pivotal role in the progression of estrogen-sensitive breast cancer by transforming estrone (E1) into estradiol (E2). We designed three successive series of E2-derivatives at position C3 of the potent inhibitor 16β-(m-carbamoylbenzyl)-E2 to remove its unwanted estrogenic activity. We report the chemical synthesis and characterization of 20 new E2-derivatives, their evaluation as 17β-HSD1 inhibitors, and their proliferative (estrogenic) activity on estrogen-sensitive cells.