High-throughput assay for evaluating dipeptidyl peptidase I activity
A method of screening a compound which modulates dipeptidyl peptidase I (DPPI) activities comprises the steps of adding a peptide substrate of DPPI to a reaction mixture which comprises DPPI and a compound, wherein the peptide substrate of DPPI has at least 3 amino acids and binds to a binding site of DPPI in addition to the S 1 -S 2 site; and measuring the molecular weight of the substrate, wherein a change in the molecular weight of the substrate is indicative of the presence of DPPI activity.
1. A method of screening compounds to identify compounds which modulate dipeptidyl peptidase I (DPPI) activities, comprising the steps of:
a. adding a substrate peptide of DPPI to a reaction mixture which comprises DPPI and a compound to be tested, wherein said substrate of DPPI is selected from the group consisting of:
(i) an isolated peptide of SEQ ID NO: 1;
(ii) an isolated peptide of SEQ ID NO: 2;
(iii) an isolated peptide of SEQ ID NO: 3;
(iv) an isolated peptide of SEQ ID NO: 4;
(v) an isolated peptide of SEQ ID NO: 5;
(vi) an isolated peptide of SEQ ID NO: 6;
(vii) an isolated peptide of SEQ ID NO: 7; and
(viii) an isolated peptide of up to 100 amino acids comprising SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO: 7;
b. measuring the molecular weight of said substrate and/or mass ratio of said substrate peptide and corresponding product peptide;
c. comparing the results of step (b) to the results of a positive control containing no test compound;
d. determining whether the molecular weight and/or mass ratio differs from the control; and
e. selecting a compound for which the results differ from the control.
2. The method of claim 1 , wherein said substrate is label-free.
3. The method of claim 1 , wherein said substrate is up to about 50 amino acids long.
4. The method of claim 1 , wherein said substrate is an isolated peptide selected from the group consisting of SEQ ID NOs: 1-7.
5. The method of claim 1 , wherein said substrate is an isolated peptideof SEQ ID NO: 1.
6. The method of claim 1 , wherein said compound is 2-(piperidin-1-ylcarbonyl)phenol.
7. The method of claim 1 , wherein said compound is 2-(piperidin-1-ylcarbonyl)aniline.
8. A DPPI substrate selected from the group consisting of an isolated peptide of SEQ ID NOs: 1-7.
9. The DPPI substrate of claim 8 , wherein said substrate is the isolated peptide of SEQ ID NO: 1.
10. A method of screening compounds to identify compounds which modulate dipeptidyl peptidase I (DPPI) activities, comprising the steps of:
a. adding a substrate peptide of DPPI to a positive control mixture which comprises DPPI and lacks a compound to be tested, wherein said substrate of DPPI is selected from the group consisting of:
(i) an isolated peptide of SEQ ID NO: 1;
(ii) an isolated peptide of SEQ ID NO: 2;
(iii) an isolated peptide of SEQ ID NO: 3;
(iv) an isolated peptide of SEQ ID NO: 4;
(v) an isolated peptide of SEQ ID NO: 5;
(vi) an isolated peptide of SEQ ID NO: 6;
(vii) an isolated peptide of SEQ ID NO: 7; and
(viii) an isolated peptide of up to 100 amino acids comprising SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO: 7;
b. measuring the molecular weight of said substrate peptide and/or mass ratio of said substrate peptide and corresponding product peptide in the control mixture;
c. adding the substrate peptide of DPPI to a reaction mixture which comprises DPPI and the compound to be tested;
d. measuring the molecular weight of said substrate peptide and/or mass ratio of said substrate peptide and corresponding product peptide in the reaction mixture;
e. comparing the results of step b) and d) to determine whether the molecular weight and/or mass ratio differs, wherein a difference is indicative of a compound that modulates DPPI activities; and
f. selecting those compounds which modulate DPPI activity.
11. The method of claim 10 , wherein said substrate is label-free.
12. The method of claim 10 , wherein said substrate is up to about 50 amino acids long.
13. The method of claim 10 , wherein said substrate is an isolated peptide selected from the group consisting of SEQ ID NOs: 1-7.
14. The method of claim 10 , wherein said substrate is an isolated peptide of SEQ ID NO: 1.
15. The method of claim 1 , wherein the compounds selected are those that increase the activity of DPPI.
16. The method of claim 1 , wherein the compounds selected are those that decrease the activity of DPPI.
17. The method of claim 10 , wherein the compounds selected are those that increase the activity of DPPI.
18. The method of claim 10 , wherein the compounds selected are those that decrease the activity of DPPI.