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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

Inhibition of recombinant DPP8/9 and DPP4 activities by UAMC00132 and Sitagliptin. Chemical structure of UAMC00132 (A) and sitagliptin (B). (C) The DPP8/9 and DPP4 inhibitory activities of sitagliptin and UAMC00132. In DPP8/9 inhibition assays, 30 ng/mL purified DPP8 protein or 20 ng/mL purified DPP9 protein and 0.2 mmol/L substrate were used, and in DPP4 inhibition assay, 1 mU/mL DPP4 protein and 0.1 mmol/L substrate were used. The result was presented as the percentage of enzyme activity inhibition.
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f0020: Inhibition of recombinant DPP8/9 and DPP4 activities by UAMC00132 and Sitagliptin. Chemical structure of UAMC00132 (A) and sitagliptin (B). (C) The DPP8/9 and DPP4 inhibitory activities of sitagliptin and UAMC00132. In DPP8/9 inhibition assays, 30 ng/mL purified DPP8 protein or 20 ng/mL purified DPP9 protein and 0.2 mmol/L substrate were used, and in DPP4 inhibition assay, 1 mU/mL DPP4 protein and 0.1 mmol/L substrate were used. The result was presented as the percentage of enzyme activity inhibition.

Mentions: Using the recombinant DPP8/9 activity assay with the above optimised reaction conditions and the DPP4 activity assay method, the effects of two reported DPP inhibitors on DPP8/9 and DPP4 activities were determined. DPP8/9 inhibitor UAMC00132 (10 μmol/L) could, while DPP4 inhibitor sitagliptin (10 μmol/L) could not, inhibit DPP8 and DPP9 activities. Meanwhile, sitagliptin strongly inhibited DPP4 activity strongly, while UAMC00132 only slightly inhibited DPP4 activity (Fig. 4), which indicated that the selective evaluation method was established to evaluate the DPP8 and DPP9 selectivity of DPP4 inhibitor candidates.


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)

Inhibition of recombinant DPP8/9 and DPP4 activities by UAMC00132 and Sitagliptin. Chemical structure of UAMC00132 (A) and sitagliptin (B). (C) The DPP8/9 and DPP4 inhibitory activities of sitagliptin and UAMC00132. In DPP8/9 inhibition assays, 30 ng/mL purified DPP8 protein or 20 ng/mL purified DPP9 protein and 0.2 mmol/L substrate were used, and in DPP4 inhibition assay, 1 mU/mL DPP4 protein and 0.1 mmol/L substrate were used. The result was presented as the percentage of enzyme activity inhibition.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0020: Inhibition of recombinant DPP8/9 and DPP4 activities by UAMC00132 and Sitagliptin. Chemical structure of UAMC00132 (A) and sitagliptin (B). (C) The DPP8/9 and DPP4 inhibitory activities of sitagliptin and UAMC00132. In DPP8/9 inhibition assays, 30 ng/mL purified DPP8 protein or 20 ng/mL purified DPP9 protein and 0.2 mmol/L substrate were used, and in DPP4 inhibition assay, 1 mU/mL DPP4 protein and 0.1 mmol/L substrate were used. The result was presented as the percentage of enzyme activity inhibition.
Mentions: Using the recombinant DPP8/9 activity assay with the above optimised reaction conditions and the DPP4 activity assay method, the effects of two reported DPP inhibitors on DPP8/9 and DPP4 activities were determined. DPP8/9 inhibitor UAMC00132 (10 μmol/L) could, while DPP4 inhibitor sitagliptin (10 μmol/L) could not, inhibit DPP8 and DPP9 activities. Meanwhile, sitagliptin strongly inhibited DPP4 activity strongly, while UAMC00132 only slightly inhibited DPP4 activity (Fig. 4), which indicated that the selective evaluation method was established to evaluate the DPP8 and DPP9 selectivity of DPP4 inhibitor candidates.

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