Aminoethyl benzenesulfonyl fluoride and its hexapeptide (Ac-VFRSLK) conjugate are both in vitro inhibitors of subtilisin kexin isozyme-1

AbstractUsing a number of intramolecularly quenched fluorogenic (IQF) substrates encompassing the subtilisin kexin isozyme-1 (SKI-1)-mediated cleavage sites of various viral glycoproteins, it is revealed that 4-[2-Aminoethyl Benzene] SulfonylFluoride (AEBSF) can inhibit the proteolytic activity of SKI-1 mostly in a competitive manner. The measured IC50 values range from 200 to 800 nM depending on the nature of the substrate used. This is the first in vitro demonstration of a non-peptide inhibitor of SKI-1. In an effort to enhance the selectivity and potency of SKI-1 inhibition, a hexapeptidyl derivative containing SKI-1 consensus sequence, Ac-Val-Phe-Arg-Ser-Leu-Lys-AEBSF, was prepared. The peptide sequence was derived from the primary auto-activation site of prodomain of SKI-1 itself terminating at Leu-Lys138 and contains the crucial P4-basic and P2 alkyl side chain containing hydrophobic amino acids. Like AEBSF, the hexapeptidyl-AEBSF analog blocked SKI-1 cleavages of all IQF-substrates tested but with enhanced efficiency

A Novel Enediynyl Peptide Inhibitor of Furin That Blocks Processing of proPDGF-A, B and proVEGF-C

BACKGROUND: Furin represents a crucial member of secretory mammalian subtilase, the Proprotein Convertase (PC) or Proprotein Convertase Subtilisin/Kexin (PCSK) superfamily. It has been linked to cancer, tumorgenesis, viral and bacterial pathogenesis. As a result it is considered a major target for intervention of these diseases. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we report, for the first time, the synthesis and biological evaluation of a newly designed potent furin inhibitor that contains a highly reactive beta-turn inducing and radical generating "enediynyl amino acid" (Eda) moiety. "Eda" was inserted between P1 and P1' residues of hfurin(98-112) peptide, derived from the primary cleavage site of furin's own prodomain. The resulting hexadecapeptide derivative inhibited furin in vitro with IC(50) approximately 40 nM when measured against the fluorogenic substrate Boc-RVRR-MCA. It also inhibited furin-mediated cleavage of a fluorogenic peptide derived from hSARS-CoV spike protein with IC(50) approximately 193 nM. Additionally it also blocked furin-processing of growth factors proPDGF-A, B and VEGF-C that are linked to tumor genesis and cancer. Circular dichroism study showed that this inhibitor displayed a predominantly beta-turn structure while western blots confirmed its ability to protect furin protein from self degradation. CONCLUSION/SIGNIFICANCE: These findings imply its potential as a therapeutic agent for intervention of cancer and other furin-associated diseases

Rheology of stirred yogurt

Rheological behavior of two commercial brands of stirred yogurt were investigated using a Haake RV 20 rotational viscometer. For samples from both brands, the upward shear-rate flow behavior generally followed the Herschel-Bulkley model and the downward flow curves were linear. They demonstrated progressive structural degradation with repeated shearing. In the steady shear runs, all samples exhibited apparent thixotropic behavior and did not attain the equilibrium condition even after 60 min of continuous shearing. The time-dependent stress decay behavior of all samples were accurately described by Weltman's logarithmic time model. The rheological properties of both yogurt brands were qualitatively similar. Both Arrhenius and Turian models were found suitable to assess the temperature influence in the range 10-25$sp circ$C.The influence of pectin (0.0 to 0.5%) and fruit concentrates (raspberry and strawberry) (64$sp circ$B, 0 to 10%) on the rheology of stirred yogurt were evaluated. The influence of storage time at 2$sp circ$C up to a period of four weeks on the rheological properties of the two brands were evaluated

Purification and characterization of furin-Eda peptide.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone-0007700-g004" target="_blank">Figure 4A</a>. RP-HPLC chromatograms of furin-Eda-peptide (II). Upper panel: HPLC for crude material obtained directly from solid phase synthesis (using C₁₈-semi preparative column), lower panel: HPLC for purified material (using C₁₈ analytical column). mAUFS  =  milli absorbance units full scale. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone-0007700-g004" target="_blank">Figure 4B</a>. SELDI-tof mass spectrum of purified furin-Eda-peptide (II). It shows major peaks at m/z 2111 and 2118 for (M+H)⁺ and (M+H+oxygen)⁺ ions respectively.</p

Circular dichroism and fluorescence spectra of furin-Eda peptide (II).

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone-0007700-g011" target="_blank">Figure 11A</a>. Overlay of Circular Dichroism (CD) spectra of furin-Eda-peptide (II) (0.5 mg/ml) in water at various pHs as shown. An expanded portion of the spectra showing the effects of pH on the position of the peak minima was shown directly beneath the full spectra. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone-0007700-g011" target="_blank">Figure 11B</a>. Overlay of CD spectra of furin-Eda-peptide (II) (0.5 mg/ml) in various solvent systems as indicated. The gradual conversion of the spectra to more helix rich structure characterized by the presence of a maximum at ∼195 nm followed by two broad minima at ∼205 and ∼222 nm are noticeable. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone-0007700-g011" target="_blank">Figure 11C</a>. Emission spectra (λ_ex = 320 nm) of Eda-peptide (II) in tetrafluoro ethanol (TFE) at various CaCl₂ concentrations. The spectra were recorded in TFE solvent (100 µl, peptide concentration  = 0.1 mM)) in the absence. Addition of CaCl₂ to the medium quenches the fluorescence intensity in a dose dependent manner. 1 mM CaCl₂ can fully suppress the fluorescence as shown by the spectrum.</p

Western blots and SDS-gel electrophoresis with silver staining of fresh recombinant furin sample and samples after 24 h incubation in the absence and presence of various concentrations of furin-Eda-peptide (II).

<p>Left panel: Immunoblot analysis of various furin samples using furin-specific antibody. Right panel: Silver stains of same samples in SDS-gel electrophoresis for visualization of all protein bands. The 55 kDa band shown within a box represents the soluble form of recombinant furin protein. FF  =  Fresh furin sample; std  =  Standard.sample, Ab  =  Antifurin antibody.</p

Complete amino acid sequence of human preprofurin.

<p>Various domains are highlighted as indicated below: Underlined residues (<u>129–427</u>): Peptidase S8 (Subtilase) domain, (full catalytic domain of hfurin is considered as 108–438); Residues in italics (<u>1–24</u>): Signal peptide; Bold residues (<u>25–107</u>): Prosegment; Residues with underlined italics (<u>484–575</u>): P-domain; Residues with bold underlined (<u>719–741</u>): Transmembrane. The catalytic residues Asp¹⁵³, His¹⁹⁴, Ser³⁶⁸ are shown in bold larger fonts within circles. The peptide segment shown within the box was used for incorporation of “Eda” moiety.</p

Schematic diagrams showing processing of hproPDGF-A and hproVEGF-C labeled at the C-terminus with a FLAG leading to their mature forms.

<p>The two upper panel figures highlight the furin processing sites (shown by vertical arrow) of the two precursor proteins. The lower panels show the effects of various furin inhibitors including the furin-Eda peptide (II) on the processing of proPDGF-A (left) and proVEGF-C (right) in CHO cell lines using western blot analysis. Pep-cmk  =  Dec-RVRR-cmk (chloromethyl ketone), Furin-pro  =  Synthetically made 83-mer full length hfurin prodomain (hfurin^25–107) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone.0007700-Basak14" target="_blank">[49]</a>. Actin levels were measured by western blots and used as controls for quantitation purpose.</p