Use of inhibtors of glutaminyl cyclases for treatment and prevention of disease
Novel physiological substrates of mammalian glutaminyl cyclase (QC, EC 2.3.2.5), new effectors of QC, methods for screening for such effectors, and the use of such effectors and pharmaceutical compositions comprising such effectors for the treatment of conditions that can be treated by modulation of QC-activity. Preferred compositions additionally comprise inhibitors of DP IV or DP IV-like enzymes for the treatment or alleviation of conditions that can be treated by modulation of QC- and DP IV-activity.
1. A screening method for the identification and selection of inhibitors of glutaminyl cyclase comprising the following steps:
a) combining at least one candidate compound, a human glutaminyl cyclase, and a substrate of glutaminyl cyclase to form a reaction mixture;
b) monitoring conversion of the substrate and/or measuring the residual glutaminyl cyclase activity; and
c) calculating changes in the substrate conversion and/or enzyme activity of glutaminyl cyclase to identify inhibition of glutaminyl cyclase;
wherein
said substrate of glutaminyl cyclase comprises an N-terminal glutamate residue, and
said substrate of glutaminyl cyclase comprises Aβ3-40/42.
2. The screening method according to claim 1 , wherein combining at least one candidate compound, glutaminyl cyclase, and a substrate of glutaminyl cyclase to form a reaction mixture comprises contacting the at least one candidate compound with glutaminyl cyclase under conditions which would permit binding there between.
3. The screening method according to claim 2 , wherein combining at least one candidate compound, glutaminyl cyclase, and a substrate of glutaminyl cyclase to form a reaction mixture comprises contacting the at least one candidate compound with glutaminyl cyclase under conditions which would permit binding there between; and adding a substrate of glutaminyl cyclase.
4. The screening method according to claim 1 , wherein calculating changes in the substrate conversion and/or enzyme activity of glutaminyl cyclase comprises calculating an inhibitor constant (Ki) for the at least one candidate compound.
5. The screening method according to claim 1 , further comprising selecting a candidate compound that decreases the conversion of the substrate and/or decreases the activity of glutaminyl cyclase.
6. The screening method according to claim 5 , further comprising selecting a candidate compound that inhibits the conversion of an N-terminal glutamic acid residue to a pyroglutamic acid residue of the Aβ3-40/42 substrate of glutaminyl cyclase.
7. The screening method according to claim 5 , wherein the selected candidate compound is a competitive inhibitor of glutaminyl cyclase.
8. The method according to claim 7 , wherein the selected candidate compound has an inhibitor constant (K i ) value of about 0.0071 mM or below.
9. The method according to claim 8 , wherein the selected candidate compound has a K i -value in the range of from about 0.00041 mM to about 0.0071 mM.
10. The screening method according to claim 5 , wherein the selected candidate compound binds to an active-site bound metal ion of glutaminyl cyclase.
11. The screening method according to claim 1 , wherein the at least one candidate compound is a peptide, imidazole derivative, or L-histamine derivative.
12. The screening method according to claim 11 , wherein the imidazole derivative is substituted at the N-1 nitrogen atom of the imidazole residue.
13. The screening method according to claim 1 , wherein the substrate of glutaminyl cyclase is Aβ3-40/42.
14. The method of claim 1 , wherein monitoring the conversion of the substrate comprises fluorometrically monitoring the conversion of the substrate.
15. The method of claim 1 , wherein measuring the residual glutaminyl cyclase activity comprises spectrophotmetrically determining glutaminyl cyclase activity.
16. A screening method for the identification and selection of inhibitors of glutaminyl cyclase comprising the following steps:
a) combining at least one candidate compound, a human glutaminyl cyclase, and a substrate of glutaminyl cyclase to form a reaction mixture;
b) monitoring conversion of the substrate and/or measuring the residual glutaminyl cyclase activity; and
c) calculating changes in the substrate conversion and/or enzyme activity of glutaminyl cyclase to identify inhibition of glutaminyl cyclase;
d) selecting a candidate compound that inhibits the conversion of an N-terminal glutamic acid residue to a pyroglutamic acid residue of the substrate of glutaminyl cyclase,
wherein
the substrate of glutaminyl cyclase is Aβ3-40/42, and
the selected candidate compound is a competitive inhibitor of glutaminyl cyclase with an inhibitor constant (Ki) value in the range of from about 0.00041 mM to about 0.0071 mM.