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

List of synthetic aromatic enediyne derivatives and their effects on protease activities of Proprotein Convertases Furin, PC5 and PC7.

<p>The table shows the IC₅₀ values for inhibition of recombinant proprotein convertases furin, PC5 and PC7 by various aromatic enediyne compounds. The enzyme assay was carried out by using the fluorogenic peptide substrate Boc-RVRR-MCA (50 µM final concentration) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone.0007700-Basak4" target="_blank">[27]</a>. ND  =  not determined.</p

Furin inhibition by furin-Eda peptide.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone-0007700-g005" target="_blank">Figure 5A</a>. Progress curves showing inhibition of furin activity by furin-Eda-peptide (II). The assay was conducted using Boc-RVRR-MCA (20 µM) as fluorogenic substrate. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone-0007700-g005" target="_blank">Figure 5B</a>. Dixon plots showing inhibition of furin activity by furin-Eda-peptide (II). The inhibition was studied using three different concentrations (as indicated) of Boc-RVRR-MCA fluorogenic substrate (<i>see </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#s4" target="_blank"><i>Materials and Methods</i></a><i>section for details</i>). RFU  =  Raw fluorescence unit. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone-0007700-g005" target="_blank">Figure 5C</a>. Inhibition of furin-mediated cleavage of hSARS-CoV fluorogenic peptide by furin-Eda-peptide (II). Furin-Eda-peptide blocks furin cleavage of intramolecularly quenched fluorogenic peptide hSARS-CoV spike^754–766 Abz-AEQDRNTR⁷⁶¹⇓ EVFAQ-Tyx-A (Abz  = 2-Amino benzoic acid, fluorescent group and Tyx  = 3-Nitro tyrosine, fluorescence quench group).</p

Design of enediyne-based furin inhibitor.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone-0007700-g002" target="_blank">Figure 2A</a>. Scheme for chemical synthesis of Fmoc protected enediynyl amino acid (Eda). The reagents used for coupling or chemical reaction in various steps are shown at the footnotes of the scheme. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone-0007700-g002" target="_blank">Figure 2B</a>. Design of β-turn mimetic furin inhibitor based on enediynyl amino acid (Eda) and profurin^98–112. The figure (left panel) shows the structures of furin-Eda peptide (II) and its cyclized form (IIa) following Bergman cyclo-aromatization reaction. The right panel shows the mechanism and electron transfer of Bergman cyclization reaction with the formation of a bis radical intermediate. R  =  Any group; the dotted line indicates hydrogen bonding.</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

Interaction of furin-Eda peptide (II/IIa) with furin enzyme.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone-0007700-g010" target="_blank">Figure 10A</a>. Docking of cyclized furin-Eda peptide (IIa) with furin P-domain (residue <u>487–560</u>). Cyclized furin-Eda-peptide (IIa) is shown in space filled structure in red while the furin P-domain (residue ⁴⁸⁷Ala-Gln-Ala-Arg---------Ala-Asn-Asn-Tyr⁵⁶⁰) is depicted in stick mode and CPK (Corey, Pauling, Koltun) colors. This is the best fit docking structure obtained between the cyclic furin-Eda peptide (IIa) and any part of furin protein. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007700#pone-0007700-g010" target="_blank">Figure 10B</a>. An expanded area of docking of furin P-domain with cyclized furin-Eda peptide (IIa) (shown in red). An enlarged segment of P-domain of furin characterized by the sequence ⁵⁴⁸VLEIENTSEA⁵⁵⁷ showing its strong interaction with cyclized furin-Eda-peptide (IIa) (shown in red). Several observed strong H-bondings between the two segments were shown by dotted lines.</p

Schematic diagram showing proteolytic processing of hproPDGF-B leading to its mature form.

<p>Upper panel highlights the two processing sites including one by furin that lead to the production of its functionally active mature form; Lower panel: The lower panel shows the effects of Eda peptide (II) (left) and α1-Pdx (right), a known furin inhibitor on the processing of proPDGF-B in CHO cells at various concentration levels (5–15 µM) as indicated. Actin levels were measured by western blots and used as controls for quantitation purpose.</p

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