Firefly luciferase is a 61 kDa monomeric enzyme that catalyzes the oxidation of firefly luciferin in the presence of ATP and oxygen to emit yellow-green light. Upon binding of substrates, the structure of firefly luciferase undergoes conformational changes from open to closed forms. We created a circularly permuted luciferase by covalently joining the native N and C termini of firefly luciferase through the cloning in of a short polypeptide linker containing a protease recognition sequence. This results in restricting the movement between the two domains and locking the enzyme in the less active open form. Protease cleavage releases this constriction thereby restoring higher activity. A. To express this mutant luciferase, new N and C termini were inserted at amino acids 234 and 233, respectively. B. Insertion of the polypeptide linker greatly reduces luciferase activity. Proteolytic cleavage by the cognate protease (scissors) activates the mutant luciferase enzyme resulting in a luminescent signal in the presence of the luciferin substrate (yellow circle).

A. Cleavage dependent activation of CP-Luc/ENLYFQS fusion protein. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes ± 10 U TEV protease and luminescence was measured from 5 µL aliquots. Data are the mean values, N = 3. Error bars are the standard deviation of the mean. B. Five µL of the protease digest reaction was size-fractionated on NuPAGE^® gels and FluoroTect^TM labeled proteins were visualized on a fluoroimager. TEV protease digestion resulted in the expected sized fragments. Note, the digestions were not complete, and therefore, residual undigested 60 kDa protein remains in all of the “+” lanes. No expressed protein is visible in the no DNA lanes.

Cleavage dependent activation of CP₂₃₄-Luc/ENLYFQX fusion proteins. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes ± 10 U TEV protease and luminescence was measured from 5 µL aliquots. The X-axis is the amino acid at the P₁' position (X) of the CP₂₃₄-Luc/ENLYFQX fusion protein. The Y-axis is the fold activation of the CP₂₃₄-Luc/ENLYFQX fusion proteins calculated by dividing the mean TEV digested values by the mean undigested values (no TEV digest), N = 3. Error bars are the standard deviation of the mean.

TEV protease dose dependent activation of CP₂₃₄-Luc/ENLYFQS protein. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes with titrating amounts of TEV protease and luminescence was measured from 5 µL aliquots in 100 µL 1:1 diluted Bright Glo^TM Luciferase Assay reagent in dH₂O; N = 4. Results are plotted as signal to noise (S/N). The limit of detection was defined as the amount of TEV protease giving a S/N ratio = 3 (dotted line). The R² value was 0.99.

The LOPAC¹²⁸⁰ library was screened for TEV protease inhibitors using the CP₂₃₄-Luc/ENLYFQC assay. Shown here are the data from plate 1 (N = 320 compounds). There were three hits () based on greater than 60% TEV protease inhibition, blue line (= 345,311 RLU). The CP₂₃₄-Luc/42AA assay was also performed to detect potential non-specific affectors of the CP₂₃₄-Luc assay and the results, which are overlaid on the graph in gray, indicate that two compounds non-specifically affected the assay. () = CP₂₃₄-Luc/42AA activity in DMSO; () = CP₂₃₄-Luc/42AA activity with compound library; () = CP₂₃₄-Luc/ENLYFQC activity minus TEV enzyme in DMSO; () = CP₂₃₄-Luc/ENLYFQC activity plus TEV enzyme in DMSO, and () = CP₂₃₄-Luc/ENLYFQC activity plus TEV protease with compound library. Controls are plotted as the mean of N = 64 (CP₂₃₄-Luc/42AA in DMSO) or 32 (CP₂₃₄-Luc/ENLYFQC ± TEV protease in DMSO). Error bars are the standard deviation of the mean.

Five compounds were re-tested at titrating concentrations. The five compounds were: aurintricarboxylic acid (ATA), 8,8'-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis(1,3,5-naphthalene-trisulfonic acid) hexasodium salt (NF-023), 4-[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxyacetic acid (L-165,041), SCH-202676 hydrobromide or N-(2,3-diphenyl-1,2,4-thiadiazol-5-(2H)-ylidene)methanamine hydrobromide (SCH HBr), and 6-hydroxyl-DL-DOPA or 2,5-dihydroxy-DL-tyrosine (L-DOPA). A. Using the CP₂₃₄-Luc/ENLYFQC assay, NF-023 and ATA were tested from 0 – 0.2 mM and the other three compounds were tested from 0 – 1 mM. Data are the mean values, N = 3. Error bars are the standard deviation of the mean. B. Using the protein fusion cleavage assay, compounds were tested from 0 – 0.1 mM. Data are the average of N = 2.

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).