1,357,131 research outputs found
Novel screening platforms for the identification of inhibitors of the human protease Furin
No full textreservedFurin is a cellular protease that cleaves proteins at polybasic motifs. The processing event modulates the activity of the substrates. So far, a large number of proteins are known to be cleaved by Furin, including some of viral origin. Recently, the processing of SARS-CoV-2 glycoprotein S by Furin has attracted much attention since the inhibition of this event was early shown to be an effective strategy to block infection. Despite Furin inhibitors are available, none is FDA approved thus prompting researchers towards the search of novel compounds against Furin catalytic activity in order to combat against the current pandemic. In this project, I characterized a novel in vitro assay for the identification of Furin inhibitors, using a SARS-CoV-2 S-derived fluorogenic substrate. This assay significantly outperformed the standard in vitro Furin tests and proved suitable for high throughput screenings. Based on this setting, in a proof-of-concept study, I analyzed the inhibitor potency of small compounds libraries, identifying the 3-((5-((5-bromothi- 38 ophen-2-yl) methylene)-4-oxo-4,5 dihydrothiazol-2-yl)(3-chloro-4-methylphenyl)amino)propanoic 39 acid molecule (namely, P3) as a potent new Furin inhibitor (IC50 = 35μM). Docking analysis further suggested that the mechanism of action of P3 may involve interaction with a Furin exosite rather than its catalytic pocket. In parallel, in vitro and in silico studies, were complemented by cell-based assays, using a new Furin sensor construct containing the cleavage site of the envelope glycoprotein S of SARS-CoV-2. This sensor is a chimeric protein designed to release luciferase upon cleavage, indicating Furin activity. In mammalian cells, the sensor was processed by endogenous Furin, an action that could be blocked by specific inhibitors. These preliminary studies indicate that this new system is promising for the development of a novel cell-based assays for the screening of Furin inhibitors. Overall, this thesis has contributed to the characterization of innovative platforms for identifying compounds that can modulate Furin enzymatic activity, specifically targeting a viral sequence. Of note, a promising new inhibitor (P3) was found as an original scaffold to tailor effective antivirals.Furin is a cellular protease that cleaves proteins at polybasic motifs. The processing event modulates the activity of the substrates. So far, a large number of proteins are known to be cleaved by Furin, including some of viral origin. Recently, the processing of SARS-CoV-2 glycoprotein S by Furin has attracted much attention since the inhibition of this event was early shown to be an effective strategy to block infection. Despite Furin inhibitors are available, none is FDA approved thus prompting researchers towards the search of novel compounds against Furin catalytic activity in order to combat against the current pandemic. In this project, I characterized a novel in vitro assay for the identification of Furin inhibitors, using a SARS-CoV-2 S-derived fluorogenic substrate. This assay significantly outperformed the standard in vitro Furin tests and proved suitable for high throughput screenings. Based on this setting, in a proof-of-concept study, I analyzed the inhibitor potency of small compounds libraries, identifying the 3-((5-((5-bromothi- 38 ophen-2-yl) methylene)-4-oxo-4,5 dihydrothiazol-2-yl)(3-chloro-4-methylphenyl)amino)propanoic 39 acid molecule (namely, P3) as a potent new Furin inhibitor (IC50 = 35μM). Docking analysis further suggested that the mechanism of action of P3 may involve interaction with a Furin exosite rather than its catalytic pocket. In parallel, in vitro and in silico studies, were complemented by cell-based assays, using a new Furin sensor construct containing the cleavage site of the envelope glycoprotein S of SARS-CoV-2. This sensor is a chimeric protein designed to release luciferase upon cleavage, indicating Furin activity. In mammalian cells, the sensor was processed by endogenous Furin, an action that could be blocked by specific inhibitors. These preliminary studies indicate that this new system is promising for the development of a novel cell-based assays for the screening of Furin inhibitors. Overall, this thesis has contributed to the characterization of innovative platforms for identifying compounds that can modulate Furin enzymatic activity, specifically targeting a viral sequence. Of note, a promising new inhibitor (P3) was found as an original scaffold to tailor effective antivirals
Characterization of Furin Protease Sensitive Site Processing and Its Effects on Sindbis Virus Assembly and Budding
No full textSindbis virus particles are composed of three structural proteins (C/E2/E1). The E1 glycoprotein is organized into a highly constrained, energy-rich conformation. Its hypothesized that this energy is utilized to drive events that deliver the viral genome to the cytoplasm of a host cell. The extraction of the E1 glycoprotein from virus membranes results in disulfide-bridge rearrangement and the collapse of the protein to a low-energy, non-native configuration. In a new approach to the production of membrane glycoproteins, furin protease recognition motifs were installed at various positions in the E1 glycoprotein ectodomain. Proteins containing the furin sensitive sites undergo normal folding and assembly in the endoplasmic reticulum and only experience the consequence of the mutation after transport to the cell surface. Processing by furin in the Golgi results in the release of the protein from the membrane from which they are assembled. This processing also impacts the envelopment of the nucleocapsid in the modified plasma membrane. E2 has been shown to be responsible for host receptor recognition and thus plays a critical role in the virus lifecycle. To expand on our previously characterize E1 furin mutant study, we installed furin protease recognition motifs at various positions in the ectodomain of E2. Mutants were analyzed for production of truncated proteins and the effect of the mutations on virus assembly and budding was also characterized. Processing of the E2 mutants by the enzyme furin results in the release of the truncated proteins from the membrane in a fashion that is similar to the processing observed in the E1 furin sensitive mutants. This processing was also observed to impact envelopment of the nucleocapsid with virus protein modified plasma membrane at a step consistent with an early event in the envelopment process. Overall, this technique provides a unique method for studying the mechanism of virus assembly and protein structure without altering crucial early events in protein assembly, folding and maturation
Furin-Catalyzed Enhanced Magnetic Resonance Imaging Probe for Differential Diagnosis of Malignant Breast Cancers
No full textMolecular magnetic resonance imaging (mMRI) of biomarkers
is essential
for accurate cancer detection in precision medicine. However, the
current clinically used contrast agents provide structural magnetic
resonance imaging (sMRI) information only and rarely provide mMRI
information. Here, a tumor-specific furin-catalyzed nanoprobe (NP)
was reported for differential diagnosis of malignant breast cancers
(BCs) in vivo. This NP with a compact structure of
Fe3O4@Gd-DOTA NPs (FFG NPs) contains an “always-on”
T2-weighted MR signal provided by the magnetic Fe3O4 core and a furin-catalyzed enhanced T1-weighted
MR signal provided by the Gd-DOTA moiety. The FFG NPs were found to
produce an activated T1 signal in the presence of furin
catalysis and an “always-on” T2 signal, providing
mMRI and sMRI information simultaneously. Ratiometric mMRI:sMRI intensity
can be used for differential diagnosis of malignant BCs MDA-MB-231
and MCF-7, where the furin levels relatively differ. The proposed
probe not only provides structural imaging but also enables real-time
molecular differential visualization of BC through enzymatic activities
of cancer tissues
Xenon Gas Inhibits the Enzyme Furin In Vitro
No full textAbstract: Aims: Certain cancers, pathogenic infections, and other diseases are facilitated by the host enzyme furin, a calcium-dependent serine protease that is the most prominent member of the family of proprotein convertases. Furin and the other proprotein convertases modify certain other proteins to change them from their inactive to active forms. Previous attempts to find an effective, non-toxic furin inhibitor to treat diseases facilitated by furin have had only limited success, due to toxicity or large molecular size that impedes absorption of the molecule. This has placed increased importance on the development of small-molecule furin inhibitors. The object of this study was to consider the effect of the noble gas xenon as a furin inhibitor. Methods: This study uses a fluorometric furin inhibition assay to compare the enzymatic activity of recombinant human furin after exposure to 99.999% xenon gas, compared to the enzymatic activity of untreated recombinant human furin. Results: Xenon exposure was found to decrease the in vitro enzymatic activity of recombinant human furin by 96-99%. Fluorescence measurements were taken for 24 hours and the enzyme inhibition persisted for the duration of the experiment. Further studies will be necessary to better characterize the exact duration of this inhibition. Conclusions: These findings appear to be the first to report xenon as a furin inhibitor. The observed inhibition continued throughout the duration of the experiment. The effectiveness of xenon as a furin inhibitor, its favorable side effect profile, and its long history of safe use as an anesthetic, when used under direct medical supervision, make it a promising treatment for diseases facilitated by furin or its substrates. Further studies in cell culture or clinical trials may expand its clinical role for such diseases.<br /
Influence of organic solvents on the furin activity
No full textFurin belongs to a family of calcium-dependent serine proprotein convertases, which transform the inactive protein precursors into mature polypeptides. In model experiments, we studied the effect of organic solvents such as acetone, dimethyl sulfoxide (DMSO), dioxane, isopropanol and ethanol on the furin activity. Furin was found to retain up to 88% of its initial activity in the presence of DMSO, whereas in the presence of acetone only 30%. Organic solvents formed the following decreasing sequence of their effects on furin: acetone> isopropanol> ethanol> dioxane> dimethyl sulfoxide. The relationship between the residual furin activity and solvent parameters such as relative polarity, dipole moment and log P were investigated. The effect of the organic solvent appeared not to correlate with any of the listed characteristics. Laidler-Sсatchard’s graphs, which according to a theory must be linear, demostrated non-linearity. These results indicate that not only electrostatic interactions play an important role in the studied enzymatic reaction but also other factors, e.g. hydrophobic contacts, hydrogen bonds can influence furin catalysis. This seems relevant for further research in this area
Furin-Guided Intracellular <sup>68</sup>Ga Nanoparticle Formation Enhancing Tumor MicroPET Imaging
No full textPositron-emission tomography (PET) is routinely used
in the clinic
for tumor imaging with ultrahigh sensitivity, but tumor-targeted PET
imaging probes are quite few. In this work, we rationally designed
a furin-responsive radiotracer Acetyl-Arg-Val-Arg-Arg-Cys(StBu)-Lys(DOTA-68Ga)-CBT (CBT-68Ga) and demonstrated that coinjection of the radiotracer with
its cold analogue CBT-Ga instructed the formation of 68Ga nanoparticles in furin-overexpressing MDA-MB-468 cancer
cells, which significantly enhanced microPET imaging of the tumor in vivo. In vitro results showed that CBT-Ga subjected to furin-initiated CBT-Cys condensation reaction
and self-assembly to form the nanoparticles CBT-Ga-NPs with an average diameter of 258.3 nm. In vivo microPET
imaging results indicate that the mice coinjected with CBT-68Ga and CBT-Ga, which warrants 68Ga nanoparticle formation in their
MDA-MB-468 tumors, had a tumor/liver ratio 9.1-fold of that of the
mice only injected with CBT-68Ga. We envisioned that, by replacing the RVRR substrate
of CBT-68Ga with
other enzyme-specific ones and using the strategy of intracellular
nanoparticle formation, a series of radioactive probes could be developed
for more sensitive and precise tumor microPET imaging in the near
future
Synthesis and investigation of the derivatives of amidinohydrazonelated aromatic compounds as furin inhibitors
No full textThe proprotein convertase furin plays a crucial role in a variety of pathogenic processes such as cancer, bacterial and viral diseases, neurodegenerative disorders and diabetes. Thus, furin inhibitors are promising therapeutics for the treatment of many diseases. In this study we synthesized some new non-peptide of furin inhibitors, with positively charged amidinohydrazone groups present in ortho-, meta– or para-positions in the benzene rings relative to the linker. From the results of biological testing it followed that the position of amidinohydrazone groups in aromatic rings was significant for the manifestation of antifurin activity. The replacement of linkers containing a propoxy group by a “bridge” with a benzene ring was found to cause an increase in the inhibitory effect of the compounds. The effect of synthesized bisamidinohydrazones on furin also depended on the substitution of the hydrogen atom in the amidinohydrazone group by the methyl group. These compounds were shown to block the enzyme activity mainly by the mechanism of mixed inhibition, and their Ki values were at the micromolar level
Heparin enhances the furin cleavage of HIV-1 gp160 peptides
No full textInfectious HIV-1 requires gp160 cleavage by furin at
the REKR511fl motif (site1) into the gp120/gp41 complex,
whereas the KAKR503 (site2) sequence remains uncleaved. We
synthesized 41mer and 51mer peptides, comprising site1 and
site2, to study their conformation and in vitro furin processing.
We found that, while the previously reported 19mer and 13mer
analogues represent excellent in vitro furin substrates, the present
extended sequences require heparin for optimal processing.
Our data support the hypothesis of a direct binding of heparin
with site1 and site2, allowing selective exposure/accessibility of
the REKR sequence, which is only then optimally cleaved by furin
Temporal constraints to major events of the Triassic of the Dolomites (Southern Alps, Italy)
No full textFive new U/Pb ages obtained from Triassic ash-fall beds are presented, and preliminary data on other five layers are discussed. Adopting an improved pre-treatment of the samples (Chemical abrasion; Mattinson, 2005) and a standard, highly-precise method to determine isotope ratios from single grains (Isotope Dilution Thermal Ionization Mass Spectrometry or ID-TIMS), the calculated ages approach the nowadays maximum possible accuracy. The volcanic horizons are interbedded with marine fossiliferous sedimentary rocks, which in turn were dated by ammonoid or conodont biostratigraphies at the zonal or subzonal level. A database of 18 recent CA-ID-TIMS ages from Triassic rocks have been compiled then to apply the Maximum Likelihood fitting of a Functional Relationship method of Agterberg (2004). The method allows estimating stages and substages boundaries’ ages. The new numerical time scale, integrated by the most updated biostratigraphies for the Western Tethys has been adopted to quantify subsidence rates and carbonate production rates in the Dolomites Area. The Dolomites are an exceptional area to study Triassic carbonate buildings for many reasons; one more reason is now the exceptional control over the time variable, given by extremely evolved biostratigraphies and by availability of direct, highly precise numerical ages. These new ages independently demonstrated the enormous duration of the Late Triassic and, on the other hand, the short time required building carbonatic edifices higher than 800 m at that time. Sedimentation rates recorded in the Dolomites are then some of the higher ever recorded, but still comparable with rates recorded during the last Holocene transgression. Some hypothesis about factors controlling sedimentation rates rise from previous points. They’ve been discussed to assess a hierarchy on their importance across the Triassic
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