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A novel enediynyl peptide inhibitor of furin that blocks processing of proPDGF-A, B and proVEGF-C.

ABSTRACT: 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.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.These findings imply its potential as a therapeutic agent for intervention of cancer and other furin-associated diseases.

SUBMITTER: Basak A

PROVIDER: S-EPMC2778948 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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A novel enediynyl peptide inhibitor of furin that blocks processing of proPDGF-A, B and proVEGF-C.

Basak Ajoy A Khatib Abdel-Majid AM Mohottalage Dayani D Basak Sarmistha S Kolajova Maria M Bag Subhendu Sekhar SS Basak Amit A

PloS one 20091126 11

<h4>Background</h4>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.<h4>Methodology/principal findings</h4>Herein, we report, for the first time, the synthesis and biological evaluation of a newly designed potent furin inhibitor th ...[more]

PMID: 19956642

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Project description:BackgroundThe myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKβ regulate actin-myosin contractility and have been implicated in cancer metastasis. Along with the related ROCK1 and ROCK2 kinases, the MRCK proteins initiate signalling events that lead to contractile force generation which powers cancer cell motility and invasion. A potential strategy for cancer therapy is to reduce metastasis by blocking MRCK activity, either alone or in combination with ROCK inhibition. However, to date no potent small molecule inhibitors have been developed with selectivity towards MRCK.ResultsScreening a kinase-focused small molecule chemical library resulted in the identification of compounds with inhibitory activity towards MRCK. Medicinal chemistry combined with in vitro enzyme profiling led to the discovery of 4-chloro-1-(4-piperidyl)-N-[5-(2-pyridyl)-1H-pyrazol-4-yl]pyrazole-3-carboxamide (BDP00005290; abbreviated as BDP5290) as a potent MRCK inhibitor. X-ray crystallography of the MRCKβ kinase domain in complex with BDP5290 revealed how this ligand interacts with the nucleotide binding pocket. BDP5290 demonstrated marked selectivity for MRCKβ over ROCK1 or ROCK2 for inhibition of myosin II light chain (MLC) phosphorylation in cells. While BDP5290 was able to block MLC phosphorylation at both cytoplasmic actin stress fibres and peripheral cortical actin bundles, the ROCK selective inhibitor Y27632 primarily reduced MLC phosphorylation on stress fibres. BDP5290 was also more effective at reducing MDA-MB-231 breast cancer cell invasion through Matrigel than Y27632. Finally, the ability of human SCC12 squamous cell carcinoma cells to invade a three-dimensional collagen matrix was strongly inhibited by 2 μM BDP5290 but not the identical concentration of Y27632, despite equivalent inhibition of MLC phosphorylation.ConclusionsBDP5290 is a potent MRCK inhibitor with activity in cells, resulting in reduced MLC phosphorylation, cell motility and tumour cell invasion. The discovery of this compound will enable further investigations into the biological activities of MRCK proteins and their contributions to cancer progression.

Project description:We have previously reported that rat prosomatostatin (rPSS) undergoes conversion at Arg decreases and Lys decreases monobasic sites to SS-28 and PSS-(1-10) respectively in COS-7 cells, and have proposed furin or a related enzyme of the constitutive secretory pathway as the endoproteinase responsible. Here we have tested directly the ability of furin to cleave rPSS at the two monobasic sites as well as at the RXRK dibasic site of SS-14 conversion (a furin motif, except for Lys substituting for Arg at P1). Recombinant vaccinia virus (VV) vectors were used to co-express rPSS with graded doses of furin in COS-7 cells and LoVo colon carcinoma cells deficient in furin. PSS and cleavage products in cell extracts and media were characterized by HPLC analysis and C-terminal [SS-14-like immunoreactivity (SS-14 LI)] and N-terminal [PSS-(1-10) LI] directed radioimmunoassays. There was a dose-dependent increase in SS-28 production from rPSS by furin in COS-7 cells from 29% (control) to 58% (high-dose furin) associated with a progressive decrease in unprocessed PSS from > 60% to approximately 20% of total SS-14 LI. Significant SS-14 production occurred only at high levels of furin infection. Control LoVo cells infected with VV:rPSS exhibited production of approximately 21% SS-28, approximately 15% PSS-(1-10) and 3.5% SS-14. Infection of LoVo cells with VV:hfurin (hfurin = human furin) enhanced SS-28 production to 30-34%. SS-14 synthesis also increased to 25-40%, probably by conversion from SS-28. Overexpression of furin in COS-7 or LoVo cells failed to increase PSS-(1-10) production. These results show that furin is a candidate SS-28 convertase. Arginine is the preferred residue at the P1 site of furin cleavage. Furin does not process rPSS to PSS-(1-10), suggesting the existence of another monobasic convertase with a preference for Lys rather than Arg at P1. Such an enzyme could also explain the presence of endogenous SS-28-, PSS-(1-10)- and SS-14-producing activities in LoVo cells.

Project description:HIV-associated neurocognitive disorders (HAND) is a syndrome defined by neurocognitive deficits that are driven by viral neurotoxins, cytokines, free radicals, and proteases expressed in the brain. This neurological disease has also been linked to activation of Protease-Activated Receptors 1 and 2 (PAR1,2). These receptors are highly expressed in the central nervous system and are upregulated in HAND. Secretory basic-amino-acid-specific Proprotein Convertases (PCs), which cleave precursor proteins at basic residues, are also induced in HAND. They are vital for many biological processes including HIV-1 entry into cells. The cytoprotective role of Furin, PC5, and PACE4 has been linked to the presence of a potential PC-cleavage site R41XXXXR46↓ in PAR1. Furthermore, Furin binds PAR1 and both are trapped in the trans-Golgi-network (TGN) as inactive proteins, likely due to the intermediary trafficking role of phospho-Furin acidic cluster sorting protein 1 (PACS1). Nothing is known about PAR2 and its possible recognition by PCs at its putative R31XXXXR36↓ processing site. The present study implicates PACS1 in the retrograde trafficking of PAR1 to the TGN and demonstrates that the cytosolic extreme C-terminal tail of PAR1 contains an acidic phosphorylatable PACS1-sensitive domain. We further show the requirement of Asn47 in PAR1 for its Furin-dependent TGN localization. Our data revealed that Furin is the only convertase that efficiently cleaves PAR2 at Arg36↓. N-glycosylation of PAR2 at Asn30 reduces the efficacy, but enhances selectivity of the Furin cleavage. Finally, in co-cultures comprised of human neuroblastoma SK-N-SH cells (stably expressing PAR1/2 and/or Furin) and HIV-1-infected primary macrophages, we demonstrate that the expression of Furin enhances neuronal cell viability in the context of PAR1- or PAR2-induced neuronal cytotoxicity. The present study provides insights into early stages of HIV-1 induced neuronal injury and the protective role of Furin in neurons co-expressing PAR1 and/or PAR2, as observed in HAND.

Dataset Information

A novel enediynyl peptide inhibitor of furin that blocks processing of proPDGF-A, B and proVEGF-C.

Publications

A novel enediynyl peptide inhibitor of furin that blocks processing of proPDGF-A, B and proVEGF-C.

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