Synthesis and characterization of novel fluorogenic substrates of coagulation factor XIII-A

Hardes, Kornelia; Hammamy, M. Zouhir; Steinmetzer, Torsten

doi:10.1016/j.ab.2013.07.043

Cited by 8 publications

(8 citation statements)

References 29 publications

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“…The peptides were synthesized by automated solid phase peptide synthesis on a Syro 2000 synthesizer (MultiSynTech GmbH, Witten, Germany) using approximately 100 mg Rink-amide-MBHA resin (loading 0.68 mmol/g) for each 2 ml reaction vessel and a standard Fmoc-protocol with double couplings (approximately 4-fold excess of Fmoc amino acid, HOBt and HBTU, respectively, and 8 equiv. DIPEA, 2 × 2 h coupling time) as described recently (Hardes et al, 2013). After final coupling of Boc-2-aminobenzoic acid, the resin was washed with 20 % piperidine in DMF (5 and 15 min) to remove an acylation on the 3-nitrotyrosine (Singh et al, 2002).…”

Section: Methodsmentioning

confidence: 99%

TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets

Bestle

Heindl

Limburg

et al. 2020

Preprint

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116

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In December 2019, a novel coronavirus named SARS-CoV-2 first reported in Wuhan, China, emerged and rapidly spread to numerous other countries globally, causing the current pandemic. SARS-CoV-2 causes acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. Currently, there is no approved antiviral drug for treating COVID-19 patients and there is an urgent need for specific antiviral therapies and vaccines.In order for SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study we investigated which host cell proteases activate the SARS-CoV-2 S protein in Calu-3 human airway epithelial cells. We show that S can be cleaved by both the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2’ site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 cells through antisense-mediated knockdown of TMPRSS2 expression. Further, we show that SARS-CoV-2 replication can be efficiently inhibited by two synthetic inhibitors of TMPRSS2 and also by the broad range serine protease inhibitor aprotinin. Additionally, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851. Combining various TMPRSS2 inhibitors with MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication.Our data demonstrate that both TMPRSS2 and furin are essential for SARS-CoV-2 activation in human airway cells and are promising drug targets for the treatment of COVID-19 either by targeting one of these proteases alone or by a combination of furin and TMPRSS2 inhibitors. Therefore, this approach has a high therapeutic potential for treatment of COVID-19.

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Section: Methodsmentioning

confidence: 99%

TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets

Bestle

Heindl

Limburg

et al. 2020

Preprint

Self Cite

116

View full text Add to dashboard Cite

show abstract

“…The peptides were synthesized by automated solid phase peptide synthesis on a Syro 2000 synthesizer (MultiSynTech GmbH) using 100 mg Rink-amide-MBHA resin (loading 0.68 mmol/g) for each 2 ml reaction vessel and a standard Fmoc-protocol with double couplings (approximately fourfold excess of Fmoc amino acid, HOBt and HBTU, respectively, and 8 equiv. DIPEA, 2 × 2 h coupling time) as described recently (64). After final coupling of Boc-2-aminobenzoic acid, the resin was washed with 20% piperidine in DMF (5 and 15 min) to remove an acylation on the 3-nitrotyrosine (65).…”

Section: Synthesis Of Fret Substratesmentioning

confidence: 99%

TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells

et al. 2020

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The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2′ site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.

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“…Enzyme activity was measured using a fluorogenic enzyme activity assay. 30 The measurements were carried out with the substrate H-Tyr(3-NO 2 )-Glu(NH-(CH 2 ) 4 -NH-Abz)-Val-Lys-Val-Ile-NH 2 as described previously. 10 IC 50 plots were fitted in Origin Pro 8G (Origin Lab Corporation, Northampton, MA, USA) by nonlinear regression using the equation y = v max / (1 + (x / x 0 ) s ) with y = response, v max = maximum velocity, x = inhibitor concentration, x 0 = relative IC 50 , and s = slope factor.…”

Section: Enzyme Activity Assaymentioning

confidence: 99%

“…Enzyme activity was measured using a fluorogenic enzyme activity assay. 30 Whole Blood and Plasma Preparation. Phlebotomy was performed on consenting healthy donors in accordance with the Declaration of Helsinki and the University of North Carolina Institutional Review Board.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Coagulation Factor XIIIa Inhibitor Tridegin: On the Role of Disulfide Bonds for Folding, Stability, and Function

et al. 2019

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Tridegin is a potent and specific 66mer peptide inhibitor of coagulation factor XIIIa with six cysteines involved in three disulfide bonds. Three of the fifteen possible 3-disulfide-bonded isomers have been identified, which share a bridge between cysteines 19 and 25. We synthesized the three possible 2-disulfide-bonded analogs using a targeted protecting-group strategy to investigate the impact of the C 19 -C 25 bond on tridegin's folding, stability, and function. The FXIIIa inhibitory activity of the analogs was retained, which was shown by in vitro fluorogenic activity and whole blood clotting assays. Molecular dynamics simulations of wild type tridegin and the analogs as well as molecular docking studies with FXIIIa were performed to elucidate the impact of the C 19 -C 25 bond on conformational stability and binding mode. The strategy of

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Synthesis and characterization of novel fluorogenic substrates of coagulation factor XIII-A

Cited by 8 publications

References 29 publications

TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets

TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets

TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells

Coagulation Factor XIIIa Inhibitor Tridegin: On the Role of Disulfide Bonds for Folding, Stability, and Function

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