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A novel bioluminescent protease assay using engineered firefly luciferase.

Wigdal SS, Anderson JL, Vidugiris GJ, Shultz J, Wood KV, Fan F - Curr Chem Genomics (2008)

Bottom Line: Understanding their biological functions underpins the efforts of drug discovery.Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold.The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased.

View Article: PubMed Central - PubMed

Affiliation: Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA.

ABSTRACT
Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening.

No MeSH data available.


Three compounds found in the LOPAC1280 library screen as TEV inhibitors were confirmed using both the CP234-Luc/ENLYFQC assay and the protein fusion cleavage assay. Their chemical structures are:  A. aurintricarboxylic acid (ATA),  B. 8,8'-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis(1,3,5-naphthalene-trisulfonic acid) hexasodium salt (NF-023), C. 4-[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxyacetic acid (L-165,041).
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Figure 7: Three compounds found in the LOPAC1280 library screen as TEV inhibitors were confirmed using both the CP234-Luc/ENLYFQC assay and the protein fusion cleavage assay. Their chemical structures are: A. aurintricarboxylic acid (ATA), B. 8,8'-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis(1,3,5-naphthalene-trisulfonic acid) hexasodium salt (NF-023), C. 4-[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxyacetic acid (L-165,041).

Mentions: The three confirmed TEV protease inhibitors were: aurintricarboxylic acid (ATA), 8,8'-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis(1,3,5-naphthalene-trisulfonic acid) hexasodium salt (NF-023), and 4-[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxyacetic acid (L-165,041). The chemical structures of the three compounds are shown in Fig. (7). ATA and NF-023 were strong TEV protease inhibitors while L-165,041 was a moderate inhibitor. Although ATA is listed as a DNA topoisomerase II inhibitor in the Sigma LOPAC library database, it has also been shown to inhibit cysteine proteases such as calpain [19] and caspases 3, 6, 7, and 9 [20]. Therefore, our results showing an inhibitory effect of ATA on the TEV cysteine protease are consistent with previous reports. NF-023, listed as a potent, selective P2X1 receptor antagonist in the Sigma LOPAC library database, is an analog of suramin, a hexasulfonated naphthylurea. Suramin has many, widely diverse uses such as to treat trypanosomiasis and onchocerciasis [21], as an anti-tumor drug [22-25], Malaria parasite Plamodium falciparum erythrocyte invasion inhibitor [26], HIV-1 reverse transcriptase inhibitor [27], and inhibitor of three neutrophil serine proteinases: neutrophil elastase, cathepsin G and proteinase 3 [28]. Interestingly, TEV protease is a serine-like cysteine protease. Thus it is possible that NF-023’s inhibitory mechanism of action on TEV protease may be the same as suramin’s on the neutrophil serine proteinases. Further supporting this idea is the fact that two (out of 12) additional hits found in the primary LOPAC screen were suramin and NF-449, another suramin analog. This suggests that a structure-activity relationship exists between suramin and related compounds and their TEV protease inhibitory properties. Finally, L-165,041 is a peroxisome proliferator-activated receptor beta agonist. A search of the literature failed to yield any previous reports which would help explain this compound’s TEV protease inhibitory effects.


A novel bioluminescent protease assay using engineered firefly luciferase.

Wigdal SS, Anderson JL, Vidugiris GJ, Shultz J, Wood KV, Fan F - Curr Chem Genomics (2008)

Three compounds found in the LOPAC1280 library screen as TEV inhibitors were confirmed using both the CP234-Luc/ENLYFQC assay and the protein fusion cleavage assay. Their chemical structures are:  A. aurintricarboxylic acid (ATA),  B. 8,8'-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis(1,3,5-naphthalene-trisulfonic acid) hexasodium salt (NF-023), C. 4-[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxyacetic acid (L-165,041).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 7: Three compounds found in the LOPAC1280 library screen as TEV inhibitors were confirmed using both the CP234-Luc/ENLYFQC assay and the protein fusion cleavage assay. Their chemical structures are: A. aurintricarboxylic acid (ATA), B. 8,8'-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis(1,3,5-naphthalene-trisulfonic acid) hexasodium salt (NF-023), C. 4-[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxyacetic acid (L-165,041).
Mentions: The three confirmed TEV protease inhibitors were: aurintricarboxylic acid (ATA), 8,8'-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis(1,3,5-naphthalene-trisulfonic acid) hexasodium salt (NF-023), and 4-[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxyacetic acid (L-165,041). The chemical structures of the three compounds are shown in Fig. (7). ATA and NF-023 were strong TEV protease inhibitors while L-165,041 was a moderate inhibitor. Although ATA is listed as a DNA topoisomerase II inhibitor in the Sigma LOPAC library database, it has also been shown to inhibit cysteine proteases such as calpain [19] and caspases 3, 6, 7, and 9 [20]. Therefore, our results showing an inhibitory effect of ATA on the TEV cysteine protease are consistent with previous reports. NF-023, listed as a potent, selective P2X1 receptor antagonist in the Sigma LOPAC library database, is an analog of suramin, a hexasulfonated naphthylurea. Suramin has many, widely diverse uses such as to treat trypanosomiasis and onchocerciasis [21], as an anti-tumor drug [22-25], Malaria parasite Plamodium falciparum erythrocyte invasion inhibitor [26], HIV-1 reverse transcriptase inhibitor [27], and inhibitor of three neutrophil serine proteinases: neutrophil elastase, cathepsin G and proteinase 3 [28]. Interestingly, TEV protease is a serine-like cysteine protease. Thus it is possible that NF-023’s inhibitory mechanism of action on TEV protease may be the same as suramin’s on the neutrophil serine proteinases. Further supporting this idea is the fact that two (out of 12) additional hits found in the primary LOPAC screen were suramin and NF-449, another suramin analog. This suggests that a structure-activity relationship exists between suramin and related compounds and their TEV protease inhibitory properties. Finally, L-165,041 is a peroxisome proliferator-activated receptor beta agonist. A search of the literature failed to yield any previous reports which would help explain this compound’s TEV protease inhibitory effects.

Bottom Line: Understanding their biological functions underpins the efforts of drug discovery.Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold.The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased.

View Article: PubMed Central - PubMed

Affiliation: Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA.

ABSTRACT
Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening.

No MeSH data available.