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Establishment of a selective evaluation method for DPP4 inhibitors based on recombinant human DPP8 and DPP9 proteins.

Liu J, Huan Y, Li C, Liu M, Shen Z - Acta Pharm Sin B (2014)

Bottom Line: Thus, the development of DPP4 inhibitors that do not have DPP8 and DPP9 inhibitory activity is critical for safe anti-diabetic therapy.The optimum concentrations of the recombinant DPP8 and DPP9 proteins were 30 ng/mL and 20 ng/mL, respectively, and the corresponding concentrations of their substrates were both 0.2 mmol/L.This method was highly reproducible and reliable for the evaluation of the DPP8 and DPP9 selectivity for DPP4 inhibitor candidates, which would provide valuable guidance in the development of safe DPP4 inhibitors.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Diabetes Research Center, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.

ABSTRACT
Dipeptidyl peptidase 4 (DPP4) is recognised as an attractive anti-diabetic drug target, and several DPP4 inhibitors are already on the market. As members of the same gene family, dipeptidyl peptidase 8 (DPP8) and dipeptidyl peptidase 9 (DPP9) share high sequence and structural homology as well as functional activity with DPP4. However, the inhibition of their activities was reported to cause severe toxicities. Thus, the development of DPP4 inhibitors that do not have DPP8 and DPP9 inhibitory activity is critical for safe anti-diabetic therapy. To achieve this goal, we established a selective evaluation method for DPP4 inhibitors based on recombinant human DPP8 and DPP9 proteins expressed by Rosetta cells. In this method, we used purified recombinant 120 kDa DPP8 or DPP9 protein from the Rosetta expression system. The optimum concentrations of the recombinant DPP8 and DPP9 proteins were 30 ng/mL and 20 ng/mL, respectively, and the corresponding concentrations of their substrates were both 0.2 mmol/L. This method was highly reproducible and reliable for the evaluation of the DPP8 and DPP9 selectivity for DPP4 inhibitor candidates, which would provide valuable guidance in the development of safe DPP4 inhibitors.

No MeSH data available.


Related in: MedlinePlus

Determination of the optimum concentrations of the purified recombinant DPP8/9 protein and substrate. The purified recombinant DPP8 (A) and DPP9 (B) proteins, with a range of concentrations from 0 to 60 ng/mL and 0.5 mmol/L substrate; The substrate with different concentrations, from 0.05 to 0.5 mmol/L at 30 ng/mL DPP8 (C) and 20 ng/mL DPP9 (D).
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f0015: Determination of the optimum concentrations of the purified recombinant DPP8/9 protein and substrate. The purified recombinant DPP8 (A) and DPP9 (B) proteins, with a range of concentrations from 0 to 60 ng/mL and 0.5 mmol/L substrate; The substrate with different concentrations, from 0.05 to 0.5 mmol/L at 30 ng/mL DPP8 (C) and 20 ng/mL DPP9 (D).

Mentions: The recombinant human DPP8 and DPP9 proteins were found to display specific dipeptidyl peptidase activities similar to DPP4, but the enzyme kinetic characteristics were unknown. By monitoring the increasing absorbance rate at 405 nm, concentration-activity experiments of recombinant DPP8/9 were performed. The experiment showed that the reactivity of recombinant DPP9 was higher than DPP8. To determine the reliable and practical reaction conditions that were suitable for the soluble DPP8 and DPP9 activity inhibition assay, the proven DPP4 inhibition assay was referenced, which indicated the changes of OD values were 0.1–0.2 in 60 min. The experiment indicated that the optimum concentrations of the purified recombinant DPP8 and DPP9 proteins were 30 ng/mL and 20 ng/mL for 60 min, respectively, with 0.5 mmol/L substrate (Fig. 3A and B), and the optimum substrate concentration for both DPP8 (30 ng/mL) and DPP9 (20 ng/mL) was 0.2 mmol/L (Fig. 3C and D).


Establishment of a selective evaluation method for DPP4 inhibitors based on recombinant human DPP8 and DPP9 proteins.

Liu J, Huan Y, Li C, Liu M, Shen Z - Acta Pharm Sin B (2014)

Determination of the optimum concentrations of the purified recombinant DPP8/9 protein and substrate. The purified recombinant DPP8 (A) and DPP9 (B) proteins, with a range of concentrations from 0 to 60 ng/mL and 0.5 mmol/L substrate; The substrate with different concentrations, from 0.05 to 0.5 mmol/L at 30 ng/mL DPP8 (C) and 20 ng/mL DPP9 (D).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4590293&req=5

f0015: Determination of the optimum concentrations of the purified recombinant DPP8/9 protein and substrate. The purified recombinant DPP8 (A) and DPP9 (B) proteins, with a range of concentrations from 0 to 60 ng/mL and 0.5 mmol/L substrate; The substrate with different concentrations, from 0.05 to 0.5 mmol/L at 30 ng/mL DPP8 (C) and 20 ng/mL DPP9 (D).
Mentions: The recombinant human DPP8 and DPP9 proteins were found to display specific dipeptidyl peptidase activities similar to DPP4, but the enzyme kinetic characteristics were unknown. By monitoring the increasing absorbance rate at 405 nm, concentration-activity experiments of recombinant DPP8/9 were performed. The experiment showed that the reactivity of recombinant DPP9 was higher than DPP8. To determine the reliable and practical reaction conditions that were suitable for the soluble DPP8 and DPP9 activity inhibition assay, the proven DPP4 inhibition assay was referenced, which indicated the changes of OD values were 0.1–0.2 in 60 min. The experiment indicated that the optimum concentrations of the purified recombinant DPP8 and DPP9 proteins were 30 ng/mL and 20 ng/mL for 60 min, respectively, with 0.5 mmol/L substrate (Fig. 3A and B), and the optimum substrate concentration for both DPP8 (30 ng/mL) and DPP9 (20 ng/mL) was 0.2 mmol/L (Fig. 3C and D).

Bottom Line: Thus, the development of DPP4 inhibitors that do not have DPP8 and DPP9 inhibitory activity is critical for safe anti-diabetic therapy.The optimum concentrations of the recombinant DPP8 and DPP9 proteins were 30 ng/mL and 20 ng/mL, respectively, and the corresponding concentrations of their substrates were both 0.2 mmol/L.This method was highly reproducible and reliable for the evaluation of the DPP8 and DPP9 selectivity for DPP4 inhibitor candidates, which would provide valuable guidance in the development of safe DPP4 inhibitors.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Diabetes Research Center, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.

ABSTRACT
Dipeptidyl peptidase 4 (DPP4) is recognised as an attractive anti-diabetic drug target, and several DPP4 inhibitors are already on the market. As members of the same gene family, dipeptidyl peptidase 8 (DPP8) and dipeptidyl peptidase 9 (DPP9) share high sequence and structural homology as well as functional activity with DPP4. However, the inhibition of their activities was reported to cause severe toxicities. Thus, the development of DPP4 inhibitors that do not have DPP8 and DPP9 inhibitory activity is critical for safe anti-diabetic therapy. To achieve this goal, we established a selective evaluation method for DPP4 inhibitors based on recombinant human DPP8 and DPP9 proteins expressed by Rosetta cells. In this method, we used purified recombinant 120 kDa DPP8 or DPP9 protein from the Rosetta expression system. The optimum concentrations of the recombinant DPP8 and DPP9 proteins were 30 ng/mL and 20 ng/mL, respectively, and the corresponding concentrations of their substrates were both 0.2 mmol/L. This method was highly reproducible and reliable for the evaluation of the DPP8 and DPP9 selectivity for DPP4 inhibitor candidates, which would provide valuable guidance in the development of safe DPP4 inhibitors.

No MeSH data available.


Related in: MedlinePlus