Treatment with N-tosyl-l-phenylalanine chloromethyl ketone after the onset of collagen-induced arthritis reduces joint erosion and NF-κB activation

Choi, Jin; Ha, Kyung Ho; Byun, Mi Sun; Min, Sung−Wook; Park, Min Jung; Park, Hyun Sil; Oh, Hye Joa; Kim, Ho Youn; Jue, Dae Myung

doi:10.1016/j.ejphar.2008.07.056

Cited by 9 publications

(4 citation statements)

References 35 publications

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“…Previous studies reported that Bay 11-7082 [24], Bay 11-7085 [25], N-p-tosyl-L-phenylalanine chloromethyl ketone [26], IRAK-1/4 inhibitor I [27], and parthenolide [28] had inhibitory effects on the NF-κB signaling pathway. PD 169316 [29] was reported to inhibit the p38 MAP kinase.…”

Section: Discussionmentioning

confidence: 99%

Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells

Leister¹,

Huang²,

Goodwin³

et al. 2011

Curr Chem Genomics

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Tumor Necrosis Factor-α (TNF-α), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders, and is a potential target for drug development. The traditional assays for detecting TNF-α, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay, are not suitable for the large size compound screens. Both assays suffer from a complicated protocol, multiple plate wash steps and/or excessive radioactive waste. A simple and quick measurement of TNF-α production in a cell based assay is needed for high throughput screening to identify the lead compounds from the compound library. We have developed and optimized two homogeneous TNF-α assays using the HTRF (homogeneous time resolved fluorescence) and AlphaLISA assay formats. We have validated the HTRF based TNF-α assay in a 1536-well plate format by screening a library of 1280 pharmacologically active compounds. The active compounds identified from the screen were confirmed in the AlphaLISA TNF-α assay using a bead-based technology. These compounds were also confirmed in a traditional ELISA assay. From this study, several beta adrenergic agonists have been identified as TNF-α inhibitors. We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316. The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening.

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Section: Discussionmentioning

confidence: 99%

Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells

Leister¹,

Huang²,

Goodwin³

et al. 2011

Curr Chem Genomics

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“…Although TPCK-induced blocking of these multiple proteins in NF-κB signaling may cause unwanted side effects, it also suggests that TPCK is more effective in inhibiting various canonical and noncanonical pathways of NF-κB activation than inhibitors more specific to IKK or NF-κB subunits. TPCK has been shown to have therapeutic effects in hypoxic-ischemic brain injury and collagen-induced arthritis in experimental animals, without any obvious adverse side effects , . Understanding the mechanism of compounds like TPCK, which blocks multiple steps in the NF-κB signaling pathway, may lead to the development of more effective therapeutics for ischemic and inflammatory diseases.…”

Section: Discussionmentioning

confidence: 99%

N-Tosyl-l-phenylalanine Chloromethyl Ketone Inhibits NF-κB Activation by Blocking Specific Cysteine Residues of IκB Kinase β and p65/RelA

Byun

Choi

et al. 2009

Biochemistry

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N-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), a serine/cysteine protease inhibitor, has been reported to inhibit expression of inflammatory mediators by blocking nuclear factor-kappaB (NF-kappaB) activation. We examined the effect of TPCK on the NF-kappaB activation pathway in HeLa cells by measuring the activity of IkappaB kinase (IKK) and p65/RelA-DNA binding. TPCK inhibited tumor necrosis factor-alpha-induced IKK activation and directly blocked IKK activity in vitro. TPCK-induced inhibition of NF-kappaB and IKK activation was abrogated by addition of the thiol-reducing agent dithiothreitol, suggesting that the effect of TPCK occurred through modification of a thiol group in IKK. Consistent with this, an IKKbeta mutant in which Cys-179 was substituted with alanine was not more susceptible to TPCK. Our result also showed that TPCK inhibits the DNA binding of transiently expressed p65/RelA in HeLa cells. Inhibition of p65/RelA-DNA binding was recovered in the presence of dithiothreitol, and substitution of Cys-38 with Ser in p65/RelA rendered the protein resistant to inhibition by TPCK. Mass spectrometry analysis of IKKbeta and p65/RelA isolated from cells treated with TPCK by UPLC-ESI-Q-TOF tandem MS revealed the labeling of Cys-179 of IKKbeta and Cys-38 of p65/RelA with a tosylphenylalanylmethyl group. These results suggest that TPCK inhibits NF-kappaB activation by directly modifying thiol groups on two different targets: Cys-179 of IKKbeta and Cys-38 of p65/RelA.

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“…In a hypoxic ischemic model study using neonatal rats treated intraperitoneally with TPCK, decreased ischemic lesions were observed [ 45 ], which was similar to that reported by Hara and coworkers in adult gerbils [ 46 ]. Furthermore, TPCK significantly attenuated inflammatory parameters in a rheumatoid arthritis model in vivo by reducing the production of proinflammatory cytokines [ 47 ].…”

Section: Discussionmentioning

confidence: 99%

Effects of a Serine Protease Inhibitor N-p-Tosyl-L-phenylalanine Chloromethyl Ketone (TPCK) on Leishmania amazonensis and Leishmania infantum

et al. 2022

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Studies have previously demonstrated the importance of serine proteases in Leishmania. A well-known serine protease inhibitor, TPCK, was used in the present study to evaluate its in vitro and in vivo antileishmanial effects and determine its mechanism of action. Despite slight toxicity against mammalian cells (CC50 = 138.8 µM), TPCK was selective for the parasite due to significant activity against L. amazonensis and L. infantum promastigote forms (IC50 = 14.6 and 31.7 µM for L. amazonensis PH8 and Josefa strains, respectively, and 11.3 µM for L. infantum) and intracellular amastigotes (IC50 values = 14.2 and 16.6 µM for PH8 and Josefa strains, respectively, and 21.7 µM for L. infantum). Leishmania parasites treated with TPCK presented mitochondrial alterations, oxidative stress, modifications in lipid content, flagellar alterations, and cytoplasmic vacuoles, all of which are factors that could be considered as contributing to the death of the parasites. Furthermore, BALB/c mice infected with L. amazonensis and treated with TPCK had a reduction in lesion size and parasite loads in the footpad and spleen. In BALB/c mice infected with L. infantum, TPCK also caused a reduction in the parasite loads in the liver and spleen. Therefore, we highlight the antileishmanial effect of the assessed serine protease inhibitor, proposing a potential therapeutic target in Leishmania as well as a possible new alternative treatment for leishmaniasis.

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Treatment with N-tosyl-l-phenylalanine chloromethyl ketone after the onset of collagen-induced arthritis reduces joint erosion and NF-κB activation

Cited by 9 publications

References 35 publications

Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells

Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells

N-Tosyl-l-phenylalanine Chloromethyl Ketone Inhibits NF-κB Activation by Blocking Specific Cysteine Residues of IκB Kinase β and p65/RelA

Effects of a Serine Protease Inhibitor N-p-Tosyl-L-phenylalanine Chloromethyl Ketone (TPCK) on Leishmania amazonensis and Leishmania infantum

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