Addition ofo-aminobenzoic acid during Fmoc solid phase synthesis of a fluorogenic substrate containing 3-nitrotyrosine

Singh, Satendra; Khaytin, Ilya; Botsko, Steve; Crossley, George H.; Plank, Denise K.; Lefievre, Yann; Giuffrida, Fred; Pennington, Michael W.

doi:10.1007/bf02538387

Cited by 3 publications

(4 citation statements)

References 12 publications

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“…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). The peptides were cleaved from the resin and deprotected by a mixture of TFA/triisopropylsilane/water (95/2.5/2.5, v/v/v) over 2 h at room temperature, followed by precipitation in cold diethyl ether.…”

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

117

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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

117

View full text Add to dashboard Cite

show abstract

“…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). The peptides were cleaved from the resin and deprotected by a mixture of TFA/triisopropylsilane/water (95/2.5/2.5, vol/vol/v) over 2 h at RT, followed by precipitation in cold diethyl ether.…”

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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“…108 Fmoc-3-Nitrotyrosine was incorporated using standard Fmoc solid-phase peptide synthesis protocols, coupling using 2-3 equivalents each of Fmoc-3-nitrotyrosine and HBTU. After amide coupling, the side chain phenol was protected as an acetate ester 105,106 using acetic anhydride (Scheme S1) or as a silyl ether using TBSCl (Scheme 1). 108 After in situ side chain protection, the remainder of the peptide was synthesized by standard solid-phase peptide synthesis.…”

Section: Resultsmentioning

confidence: 99%

“…To improve the synthesis of peptides containing nitrotyrosine, multiple protecting group strategies for the nitrotyrosine phenol were considered. Previously, nitrotyrosine has been protected using trityl, benzyl, or acetyl protecting groups. ,− However, the trityl group on nitrotyrosine has been observed to be labile under standard solid-phase peptide synthesis conditions . Alternatively, the installation of a benzyl protecting group involved multiple solution-phase synthesis and purification steps and only modestly improved yields of longer peptides containing nitrotyrosine …”

Section: Resultsmentioning

confidence: 99%

Design of a Protein Motif Responsive to Tyrosine Nitration and an Encoded Turn-Off Sensor of Tyrosine Nitration

Urmey

Zondlo

2019

Biochemistry

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Tyrosine nitration is a protein post-translational modification that is predominantly non-enzymatic and that is observed to be increased under conditions of nitrosative stress and in numerous disease states. A small protein motif (14-18 amino acids) responsive to tyrosine nitration has been developed. In this design, nitrotyrosine replaced the conserved Glu12 of an EF-Hand metalbinding motif. Thus, the non-nitrated peptide bound terbium weakly. In contrast, tyrosine nitration resulted in a 45-fold increase in terbium affinity. NMR spectroscopy indicated direct binding of nitrotyrosine to the metal and EF-Hand-like metal contacts in this designed peptide. Nitrotyrosine is an efficient quencher of fluorescence. In order to develop a sensor of tyrosine nitration, the initial design was modified to incorporate Glu residues at EF Hand positions 9 and 16 as additional metal-binding residues, to increase the terbium affinity of the peptide with unmodified tyrosine. This peptide with tyrosine at residue 12 bound terbium and effectively sensitized terbium luminescence. Tyrosine nitration resulted in a 180-fold increase in terbium affinity (K d = 1.6 µM) and quenching of terbium luminescence. This sequence was incorporated as an encoded protein tag and applied as a turn-off fluorescent protein sensor of tyrosine nitration. The sensor was responsive to nitration by peroxynitrite, with fluorescence quenched on nitration. The greater terbium affinity upon tyrosine nitration resulted in high dynamic range and sensitivity to substoichiometric nitration. An improved approach was also developed to the synthesis of peptides containing nitrotyrosine, via the in situ silyl protection of nitrotyrosine. This work represents the first designed, encodable protein motif that is responsive to tyrosine nitration.

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Addition ofo-aminobenzoic acid during Fmoc solid phase synthesis of a fluorogenic substrate containing 3-nitrotyrosine

Cited by 3 publications

References 12 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

Design of a Protein Motif Responsive to Tyrosine Nitration and an Encoded Turn-Off Sensor of Tyrosine Nitration

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