Neutrophil proteinase 3 (PR3) is an important drug target for inflammatory lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Drug discovery efforts targeting PR3 require active enzyme for in vitro characterization, such as inhibitor screening, enzymatic assays, and structural studies. Recombinant expression of active PR3 overcomes the need for enzyme supplies from human blood and in addition allows studies on the influence of mutations on enzyme activity and ligand binding.
View Article and Find Full Text PDFThe role of aromatic amino acids in peripheral protein membrane binding has been reported to involve cation-π interactions with choline lipids. In this study, we have investigated the interactions of the model pentapeptide Ac-WL-X-LL-OH (where X = L, Y, F, or W) with the phospholipid membrane using solid-state NMR. The effect of guest residue X on the peptide-lipid interactome was complementary to the seminal report on the interfacial hydrophobicity scale by Wimley and White.
View Article and Find Full Text PDFUsing a combination of multisite λ-dynamics (MSλD) together with IC assays, we evaluated the polypharmacological potential of a scaffold currently in clinical trials for inhibition of human neutrophil elastase (HNE), targeting cardiopulmonary disease, for efficacious inhibition of Proteinase 3 (PR3), a related neutrophil serine proteinase. The affinities we observe suggest that the dihydropyrimidinone scaffold can serve as a suitable starting point for the establishment of polypharmacologically targeting both enzymes and enhancing the potential for treatments addressing diseases like chronic obstructive pulmonary disease.
View Article and Find Full Text PDFAcetylation of protein N-termini is one of the most common protein modifications in the eukaryotic cell and is catalyzed by the N-terminal acetyltransferase family of enzymes. The N-terminal acetyltransferase NAA80 is expressed in the animal kingdom and was recently found to specifically N-terminally acetylate actin, which is the main component of the microfilament system. This unique animal cell actin processing is essential for the maintenance of cell integrity and motility.
View Article and Find Full Text PDFACS Chem Biol
September 2022
Factor VII Activating protease (FSAP) has a protective effect in diverse disease conditions as inferred from studies in FSAP mice and humans deficient in FSAP activity due to single-nucleotide polymorphism. The zymogen form of FSAP in plasma is activated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear whether this activation mechanism is specific and amenable to manipulation.
View Article and Find Full Text PDFPurpose: Fluorescence imaging (FLI) using targeted near-infrared (NIR) conjugates aids the detection of tumour lesions pre- and intraoperatively. The optimisation of tumour visualisation and contrast is essential and can be achieved through high tumour-specificity and low background signal. However, the choice of fluorophore is recognised to alter biodistribution and clearance of conjugates and is therefore a determining factor in the specificity of target binding.
View Article and Find Full Text PDFFabF (3-oxoacyl-[acyl-carrier-protein] synthase 2), which catalyses the rate limiting condensation reaction in the fatty acid synthesis II pathway, is an attractive target for new antibiotics. Here, we focus on FabF from P. aeruginosa (PaFabF) as antibiotics against this pathogen are urgently needed.
View Article and Find Full Text PDFNitroreductases (NTR) are a family of bacterial enzymes used in gene directed enzyme prodrug therapy (GDEPT) that selectively activate prodrugs containing aromatic nitro groups to exert cytotoxic effects following gene transduction in tumours. The clinical development of NTR-based GDEPT has, in part, been hampered by the lack of translational imaging modalities to assess gene transduction and drug cytotoxicity, non-invasively. This study presents translational preclinical PET imaging to validate and report NTR activity using the clinically approved radiotracer, F-FMISO, as substrate for the NTR enzyme.
View Article and Find Full Text PDFPhorbazoles are polychlorinated heterocyclic secondary metabolites isolated from a marine sponge and several of these natural products have shown inhibitory activity against cancer cells. In this work, a synthesis of the trichlorinated phorbazole B using late stage electrophilic chlorination was developed. The synthesis relied on the use of an oxazole precursor, which was protected with an iodine in the reactive 4-position, followed by complete chlorination of all pyrrole positions.
View Article and Find Full Text PDFN-terminal (Nt) acetylation is a major protein modification catalyzed by N-terminal acetyltransferases (NATs). Methionine acidic N termini, including actin, are cotranslationally Nt acetylated by NatB in all eukaryotes, but animal actins containing acidic N termini, are additionally posttranslationally Nt acetylated by NAA80. Actin Nt acetylation was found to regulate cytoskeletal dynamics and motility, thus making NAA80 a potential target for cell migration regulation.
View Article and Find Full Text PDFLTX 315 is an oncolytic peptide with potent immunological properties. In the present study, we demonstrate that intratumoral treatment with LTX-315 resulted in a complete regression and systemic immune response in a rat fibrosarcoma model. The treatment was T-cell dependent, and also resulted in an abscopal effect as demonstrated by the regression of distal non-treated lesions.
View Article and Find Full Text PDFThe oncolytic peptide LTX-315, which has been de novo designed based on structure-activity relationship studies of host defense peptides, has the ability to kill human cancer cells and induce specific anticancer immune response when injected locally into tumors established in immunocompetent mice. The oncolytic effect of LTX-315 involves perturbation of plasma membrane and the mitochondria with subsequent release of danger-associated molecular pattern molecules, which highlights the ability of LTX-315 to induce complete regression and protective immune responses. Treatment with LTX-315 reprograms the tumor microenvironment by decreasing the local abundance of immunosuppressive cells and by increasing the frequency of effector T cells.
View Article and Find Full Text PDFHere we report a series of close analogues of our recently published scaffold-based tripeptidomimetic CXCR4 antagonists, containing positively charged guanidino groups in R and R, and an aromatic group in R. While contraction/elongation of the guanidine carrying side chains (R and R) resulted in loss of activity, introduction of bromine in position 1 on the naphth-2-ylmethyl moiety (R) resulted in an EC of 61μM (mixture of diastereoisomers) against wild-type CXCR4; thus, the antagonistic activity of these tripeptidomimetics seems to be amenable to optimization of the aromatic moiety. Moreover, for analogues carrying a naphth-2-ylmethyl substituent, we observed that a Pictet-Spengler like cyclization side reaction depended on the nature of the R substituent.
View Article and Find Full Text PDFN-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins.
View Article and Find Full Text PDFOncolytic immunotherapies represent a new promising strategy in the treatment of cancer. In our efforts to develop oncolytic peptides, we identified a series of chemically modified 9-mer cationic peptides that were highly effective against both drug-resistant and drug-sensitive cancer cells and with lower toxicity toward normal cells. Among these peptides, LTX-315 displayed superior anticancer activity and was selected as a lead candidate.
View Article and Find Full Text PDFPhenylalanine hydroxylase catalyzes the first step in the synthesis of pyomelanin, a pigment that aids in the acquisition of essential iron in certain bacteria. In this work, we present the development and application of a drug discovery protocol by targeting this enzyme in Legionella pneumophila, the major causative agent of Legionnaires' disease. We employ a combination of high-throughput screening to identify small-molecule binders, enzymatic activity measurements to identify inhibitors in vitro, and the verification of the inhibitory effect in vivo.
View Article and Find Full Text PDFWe here report an experimentally verified binding mode for the known tripeptidomimetic CXCR4 antagonist KRH-1636 (1). A limited SAR study based on the three functionalities of 1 was first conducted, followed by site-directed mutagenesis studies. The receptor mapping showed that both the potency and affinity of 1 were dependent on the transmembrane residues His(113), Asp(171), Asp(262), and His(281) and also suggested the involvement of Tyr(45) and Gln(200) (potency) and Tyr(116) and Glu(288) (affinity).
View Article and Find Full Text PDFOver the last 5 years, X-ray structures of CXCR4 in complex with three different ligands (the small-molecule antagonist IT1t, the polypeptide antagonist CVX15 and the viral chemokine antagonist vMIP-II) have been released. In addition to the inherent scientific value of these specific X-ray structures, they provide a reliable structural foundation for studies of the molecular interactions between CXCR4 and its key peptide ligands (CXCL12 and HIV-1 gp120), and serve as valuable templates for further development of small-molecule CXCR4 antagonists with therapeutic potential. We here review recent computational studies of the molecular interactions between CXCR4 and its peptide ligands - based on the X-ray structures of CXCR4 - and the current status of small-molecule peptide and peptidomimetic CXCR4 antagonists.
View Article and Find Full Text PDFThe first total synthesis of breitfussin A and B is described. The approach features two palladium-catalyzed cross-couplings installing the indole and pyrrole onto the oxazole core and selective lithiation/iodination of a common indole-oxazole fragment providing 2,4-diiodinated or 2-iodinated oxazoles as potential precursors for breitfussin A and B, respectively. An unexpected acid promoted deiodination was utilized in the synthesis of breitfussin B.
View Article and Find Full Text PDFNodularia spumigena is a filamentous cyanobacterium that forms toxic blooms in brackish waters around the world through the production of the pentapeptide toxin nodularin. This cyanobacterium also produces large amounts of protease inhibitors belonging to the aeruginosin and spumigin families. Here we report the discovery of previously unknown protease inhibitors, pseudoaeruginosins NS1 (1) and NS2 (2), from 33 strains of N.
View Article and Find Full Text PDFNeutrophil serine proteases, proteinase 3 (PR3) and human neutrophil elastase (HNE), are considered as targets for chronic inflammatory diseases. Despite sharing high sequence similarity, the two enzymes have different substrate specificities and functions. While a plethora of HNE inhibitors exist, PR3 specific inhibitors are still in their infancy.
View Article and Find Full Text PDFStructure-activity relationship studies of the cyclopentapeptide CXCR4 antagonists (cyclo(-l-/d-Arg(1)-Arg(2)-2-Nal(3)-Gly(4)-d-Tyr(5)-)) suggest that the l-/d-Arg(1)-Arg(2)-2-Nal(3) tripeptide sequence contained within these cyclopentapeptides serves as a recognition motif for peptidic CXCR4 antagonists. Starting by dissecting the cyclopentapeptide structure and reintroducing cyclic constraints in a stepwise manner, we here report a novel class of scaffold-based tripeptidomimetic CXCR4 antagonists based on the d-Arg-Arg-2-Nal motif. Biological testing of the prototype compounds showed that they represent new peptidomimetic hits; importantly, the modular nature of the scaffold provides an interesting starting point for future ligand optimization.
View Article and Find Full Text PDFNeutrophil serine proteases are specific regulators of the immune response, and proteinase 3 is a major target antigen in antineutrophil cytoplasmic antibody-associated vasculitis. FRET peptides containing 2-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) as fluorophore and quencher groups, respectively, have been widely used to probe proteases specificity. Using in silico design followed by enzymatic assays, we show that Abz and EDDnp significantly contribute to substrate hydrolysis by PR3.
View Article and Find Full Text PDFThe ability to visualize reporter gene expression in vivo has revolutionized all facets of biologic investigation and none more so than imaging applications in oncology. Near-infrared reporter gene imaging may facilitate more accurate evaluation of chemotherapeutic response in preclinical models of orthotopic and metastatic cancers. We report the development of a cell permeable, quenched squarine probe (CytoCy5S), which is reduced by Escherichia coli nitroreductase (NTR), resulting in a near-infrared fluorescent product.
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