Identification of Human Glutaminyl Cyclase as a Metalloenzyme
Human glutaminyl cyclase (QC) was identified as a metalloenzyme as suggested by the time-dependent inhibition by the heterocyclic chelators 1,10-phenanthroline and dipicolinic acid. The effect of EDTA on QC catalysis was negligible. Inactivated enzyme could be fully restored by the addition of Zn2+ in the presence of equimolar concentrations of EDTA. Little reactivation was observed with Co2+ and Mn2+. Other metal ions such as K+, Ca2+, and Ni2+ were inactive under the same conditions. Additionally, imidazole and imidazole derivatives were identified as competitive inhibitors of QC. An initial structure activity-based inhibitor screening of imidazole-derived compounds revealed potent inhibition of QC by imidazole N-1 derivatives. Subsequent data base screening led to the identification of two highly potent inhibitors, 3-[3-(1H-imidazol-1-yl)propyl]-2-thioxoimidazolidin-4-one and 1,4-bis-(imidazol-1-yl)-methyl-2,5-dimethylbenzene, which exhibited respective Ki values of 818 ± 1 and 295 ± 5 nm. The binding properties of the imidazole derivatives were further analyzed by the pH dependence of QC inhibition. The kinetically obtained pKa values of 6.94 ± 0.02, 6.93 ± 0.03, and 5.60 ± 0.05 for and the values obtained by pKa the for for binding to QC. the pH dependence of the for the of to the of the The QC as a the metal a for with highly potent competitive Human glutaminyl cyclase (QC) was identified as a metalloenzyme as suggested by the time-dependent inhibition by the heterocyclic chelators 1,10-phenanthroline and dipicolinic acid. The effect of EDTA on QC catalysis was negligible. Inactivated enzyme could be fully restored by the addition of Zn2+ in the presence of equimolar concentrations of EDTA. Little reactivation was observed with Co2+ and Mn2+. Other metal ions such as K+, Ca2+, and Ni2+ were inactive under the same conditions. Additionally, imidazole and imidazole derivatives were identified as competitive inhibitors of QC. An initial structure activity-based inhibitor screening of imidazole-derived compounds revealed potent inhibition of QC by imidazole N-1 derivatives. Subsequent data base screening led to the identification of two highly potent inhibitors, 3-[3-(1H-imidazol-1-yl)propyl]-2-thioxoimidazolidin-4-one and 1,4-bis-(imidazol-1-yl)-methyl-2,5-dimethylbenzene, which exhibited respective Ki values of 818 ± 1 and 295 ± 5 nm. The binding properties of the imidazole derivatives were further analyzed by the pH dependence of QC inhibition. The kinetically obtained pKa values of 6.94 ± 0.02, 6.93 ± 0.03, and 5.60 ± 0.05 for and the values obtained by pKa the for for binding to QC. the pH dependence of the for the of to the of the The QC as a the metal a for with highly potent competitive (QC) acid. in and the of glutaminyl with the of and the of was to the were identified as of the in and and to be in the and and and the QC of to QC to to a enzyme in the of and QC the catalysis of of and the inhibition by were the of with the of a on the of and the of by the of the glutaminyl in a the of the revealed in by a of the inhibition of and to of QC inhibitor data for which by QC was to be by 1,10-phenanthroline and the of heterocyclic compounds imidazole and compounds were to be the competitive inhibitors of QC. The data the for further of the and of QC QC was in and as were as was and were and were and the imidazole derivatives were and QC was as were of was as the of concentrations of in 0.05 pH to 5 which to the enzyme Ki values of QC inhibition were by EDTA. the of the and acid. were by the addition of and was by the in nm. the for the of QC and as The were nm. were by the addition of QC. QC was a of under conditions. were the the for inhibitor the was the same as for the addition of the inhibitor to of the respective imidazole and a to the of the of the inhibition and of Ki the of the inhibitor on the was was on of the the of the QC inhibition. The inhibition were by the data of the obtained to the for competitive inhibition for the of the pH dependence of QC catalysis and the was were in a by and of and The a the pH Additionally, the of QC on to be in The pH dependence data were to for the inhibitors to for two in the of the of the and of the enzyme under and the was to the and of QC 1 was by a of 5 5 dipicolinic in 0.05 pH the was by of the 0.05 pH 1 EDTA. were for Ca2+, K+, and Co2+ ions concentrations of and in pH EDTA. QC were in 0.05 pH EDTA to a reactivation by the of metal ions in by were by imidazole in the the as as the could be The of the data competitive inhibition by imidazole in imidazole in the The Ki values of ± and ± obtained with the and QC as and a Ki of ± 5 the structure the binding of the under the by the of imidazole and derivatives of compounds were to for QC of in presence of concentrations of imidazole and 1 The a of the obtained of the the inhibitor The were 0.05 pH 5 EDTA and derivatives of the were and the of the was The to the imidazole of imidazole derivatives in the QC ± ± derivatives ± ± 1 ± ± ± ± ± ± 5 ± inhibition derivatives ± 1 ± ± ± ± ± ± derivatives ± 5 ± inhibition derivatives ± ± ± inhibition in a of the imidazole and imidazole N-1 and compounds with in the 5 of the imidazole in QC. to be a potent in and to on as by the inhibition of and imidazole and to binding of the compounds to the of the of the imidazole derivatives to the the imidazole could to The Ki values for and of the of the inhibitors on for of was observed of the of was to by was derivatives a binding to the of QC to a on binding by in the addition of a to the inhibition of the by of the Ki values for and the imidazole derivatives as inhibitors of compounds Ki values with imidazole the N-1 in the were for of be and as QC The data for QC binding of N-1 derivatives the for the of potent QC inhibitors by structure of N-1 imidazole of the obtained by N-1 of the imidazole to highly potent inhibitors of QC by data base of the potent inhibitors in the observed inhibition of as with in the initial led to the identification of compounds Ki values of the QC inhibition in the of QC by derivatives in a of and inhibition obtained for the imidazole derivatives for binding to the An of and were with to The effect of in and 5 was analyzed by the of and the two in the of and of of the of the led to a of by the to the to which further for the of the in binding of the imidazole derivatives to the of QC by and two derivatives in a pH of the of imidazole was further of the of QC inhibition. of a as as the of the enzyme was to a pH pH and imidazole a pKa was to be for the of and of the The inhibition of the a dependence on the pH the of imidazole a pH to in the pH Ki was the of QC inhibition on of the imidazole derivatives. was by of the data to a The the properties of imidazole was by a pKa in with the pKa of the of imidazole pH were obtained for QC inhibition by and the kinetically pKa values with the pKa values by The dependence of the and on the was analyzed the for of was in the pH and the a pH dependence with pH to in the and pH The pH dependence of revealed a of 1 in the pH the presence of a The kinetically pKa in the was to the pKa of the the pH data revealed a pKa of ± a of the QC of the inhibition of imidazole and QC of were to a and revealed pKa values of 6.94 ± 0.02, 6.93 ± and 5.60 ± 0.05 for and were in and to the respective pH by the addition of of the of inhibitors and a by the of and as as by ± ± ± ± ± ± ± ± ± in a by inhibition of QC by 1,10-phenanthroline the EDTA to effect on QC catalysis suggested inhibition by to metal in addition to by was in presence of dipicolinic inhibitor of chelators QC in a competitive and time-dependent initial was was to be further with the compounds EDTA inhibition of under of QC by dependence of inhibition by 1,10-phenanthroline and EDTA of QC in the presence of was the addition of QC with the respective for QC was 5 1,10-phenanthroline 5 dipicolinic acid. QC was to 1 reactivation was of QC by dipicolinic 1,10-phenanthroline was by the for with in presence of EDTA of QC was obtained Co2+ and ions for in the presence of a reactivation to of the was reactivation was observed Ca2+, of fully QC with ions effect on the enzyme of QC with and metal QC was by the addition of dipicolinic in pH the enzyme was to pH 1 EDTA. of the enzyme was by of the enzyme with metal ions a of in the presence of EDTA to reactivation by of metal ions in by enzyme were the EDTA as the enzyme and by enzyme were EDTA a QC to in with the for the a of a for glutaminyl by were observed in binding and of the glutaminyl QC by the the and by the suggested in the of the glutaminyl by Additionally, were to inhibition of QC by which QC in to the inhibition of QC by imidazole QC was by of as with QC by and dipicolinic to compounds a the with the metal of the reactivation by the addition of Zn2+ ions to and a of the structure of QC and the a of the of was the of QC with of two of the the binding of the two Zn2+ ions in of in QC. as in of two of the identified and which for metal binding to a of data further the and of the metal binding the in the catalysis of QC. the the to the of a the of a on the of the in binding the of the ions the of the and the to with to and the of the by of QC and of the was with The of the for QC the and the of the The in metal binding in and the of the to the The and were identified as for QC catalysis the catalysis by the of binding the metal could with the of the of the QC the of the in to the of the a metal in the of QC by the of the glutaminyl the by the of the on the of the of the and the metal by the observed inhibition of QC by the of with the of the and the of the of the by in the catalysis of the the metal of QC as a binding for the imidazole-derived inhibitors and the with in to the to the Zn2+ concentrations in QC and QC which was observed in to be QC two metal ions to the to the binding Zn2+ was a further Zn2+ was for glutaminyl The of such a binding concentrations of Zn2+ ions the of the be in could of QC. and potent inhibitors of in the of QC with the the of the of the in the of the structure the binding of and to QC by metal a for the the of the of the glutaminyl to be and a of the same of such the of the of the of inhibitors for a QC. for QC to was to the to be potent QC the identification of N-1 imidazole derivatives as highly potent competitive inhibitors, the revealed QC as a enzyme as by the the of inhibition by imidazole and imidazole the of QC by the 1,10-phenanthroline and dipicolinic the reactivation of the by metal and the of in the structure of QC. the observed of of the imidazole derivatives on as a for inhibitor (QC) acid. in and the of glutaminyl with the of and the of was to the were identified as of the in and and to be in the and and and the QC of to QC to to a enzyme in glutaminyl cyclase acid. glutaminyl cyclase acid. the of and QC the catalysis of of and the inhibition by were the of with the of a on the of and the of by the of the glutaminyl in a the of the revealed in by a of the inhibition of and to of QC inhibitor data for which by QC was to be by 1,10-phenanthroline and the of heterocyclic compounds imidazole and compounds were to be the competitive inhibitors of QC. The data the for further of the and of QC QC was in and as were as was and were and were and the imidazole derivatives were and QC was as were of was as the of concentrations of in 0.05 pH to 5 which to the enzyme Ki values of QC inhibition were by EDTA. the of the and acid. were by the addition of and was by the in nm. the for the of QC and as The were nm. were by the addition of QC. QC was a of under conditions. were the the for inhibitor the was the same as for the addition of the inhibitor to of the respective imidazole and a to the of the of the inhibition and of Ki the of the inhibitor on the was was on of the the of the QC inhibition. The inhibition were by the data of the obtained to the for competitive inhibition for the of the pH dependence of QC catalysis and the was were in a by and of and The a the pH Additionally, the of QC on to be in The pH dependence data were to for the inhibitors to for two in the of the of the and of the enzyme under and the was to the and of QC 1 was by a of 5 5 dipicolinic in 0.05 pH the was by of the 0.05 pH 1 EDTA. were for Ca2+, K+, and Co2+ ions concentrations of and in pH EDTA. QC were in 0.05 pH EDTA to a reactivation by the of metal ions in QC was in and as were as was and were and were and the imidazole derivatives were and QC was as were of QC was as the of concentrations of in 0.05 pH to 5 which to the enzyme Ki values of QC inhibition were by EDTA. the of the and acid. were by the addition of and was by the in nm. the for the of QC and as The were nm. were by the addition of QC. QC was a of under conditions. were the the for inhibitor the was the same as for the addition of the inhibitor to of the respective imidazole and a to the of the of the inhibition and of Ki the of the inhibitor on the was was on of the the of the QC inhibition. The inhibition were by the data of the obtained to the for competitive inhibition for pH the of the pH dependence of QC catalysis and the was were in a by and of and The a the pH Additionally, the of QC on to be in The pH dependence data were to for the inhibitors to for two in the of the of the and of the enzyme under and the was to the and of QC 1 was by a of 5 5 dipicolinic in 0.05 pH the was by of the 0.05 pH 1 EDTA. were for Ca2+, K+, and Co2+ ions concentrations of and in pH EDTA. QC were in 0.05 pH EDTA to a reactivation by the of metal ions in by were by imidazole in the the as as the could be The of the data competitive inhibition by imidazole in imidazole in the The Ki values of ± and ± obtained with the and QC as and a Ki of ± 5 the structure the binding of the under the by the of imidazole and derivatives of compounds were to and derivatives of the were and the of the was The to the imidazole of imidazole derivatives in the QC ± ± derivatives ± ± 1 ± ± ± ± ± ± 5 ± inhibition derivatives ± 1 ± ± ± ± ± ± derivatives ± 5 ± inhibition derivatives ± ± ± inhibition in a of the imidazole and imidazole N-1 and compounds with in the 5 of the imidazole in QC. to be a potent in and to on as by the inhibition of and imidazole and to binding of the compounds to the of the of the imidazole derivatives to the the imidazole could to The Ki values for and of the of the inhibitors on for of was observed of the of was to by was derivatives a binding to the of QC to a on binding by in the addition of a to the inhibition of the by of the Ki values for and the imidazole derivatives as inhibitors of compounds Ki values with imidazole the N-1 in the were for of be and as QC The data for QC binding of N-1 derivatives the for the of potent QC inhibitors by structure of N-1 imidazole of the obtained by N-1 of the imidazole to highly potent inhibitors of QC by data base of the potent inhibitors in the observed inhibition of as with in the initial led to the identification of compounds Ki values of the QC inhibition in the of QC by derivatives in a of and inhibition obtained for the imidazole derivatives for binding to the An of and were with to The effect of in and 5 was analyzed by the of and the two in the of and of of the of the led to a of by the to the to which further for the of the in binding of the imidazole derivatives to the of QC by and two derivatives in a pH of the of imidazole was further of the of QC inhibition. of a as as the of the enzyme was to a pH pH and imidazole a pKa was to be for the of and of the The inhibition of the a dependence on the pH the of imidazole a pH to in the pH Ki was the of QC inhibition on of the imidazole derivatives. was by of the data to a The the properties of imidazole was by a pKa in with the pKa of the of imidazole pH were obtained for QC inhibition by and the kinetically pKa values with the pKa values by The dependence of the and on the was analyzed the for of was in the pH and the a pH dependence with pH to in the and pH The pH dependence of revealed a of 1 in the pH the presence of a The kinetically pKa in the was to the pKa of the the pH data revealed a pKa of ± a of the QC of the inhibition of imidazole and QC of were to a and revealed pKa values of 6.94 ± 0.02, 6.93 ± and 5.60 ± 0.05 for and were in and to the respective pH by the addition of of the of inhibitors and a by the of and as as by ± ± ± ± ± ± ± ± ± in a by inhibition of QC by 1,10-phenanthroline the EDTA to effect on QC catalysis suggested inhibition by to metal in addition to by was in presence of dipicolinic inhibitor of chelators QC in a competitive and time-dependent initial was was to be further with the compounds EDTA inhibition of under of QC by dependence of inhibition by 1,10-phenanthroline and EDTA of QC in the presence of was the addition of QC with the respective for QC was 5 1,10-phenanthroline 5 dipicolinic acid. QC was to 1 reactivation was of QC by dipicolinic 1,10-phenanthroline was by the for with in presence of EDTA of QC was obtained Co2+ and ions for in the presence of a reactivation to of the was reactivation was observed Ca2+, of fully QC with ions effect on the enzyme of QC with and metal QC was by the addition of dipicolinic in pH the enzyme was to pH 1 EDTA. of the enzyme was by of the enzyme with metal ions a of in the presence of EDTA to reactivation by of metal ions in by enzyme were the EDTA as the enzyme and by enzyme were EDTA by were by imidazole in the the as as the could be The of the data competitive inhibition by imidazole in imidazole in the The Ki values of ± and ± obtained with the and QC as and a Ki of ± 5 the structure the binding of the under the by the of imidazole and derivatives of compounds were to and derivatives of the were and the of the was The to the imidazole and compounds with in the 5 of the imidazole in QC. to be a potent in and to on as by the inhibition of and imidazole and to binding of the compounds to the of the of the imidazole derivatives to the the imidazole could to The Ki values for and of the of the inhibitors on for of was observed of the of was to by was derivatives a binding to the of QC to a on binding by in the addition of a to the inhibition of the by of the Ki values for and N-1 the imidazole derivatives as inhibitors of compounds Ki values with imidazole the N-1 in the were for of be and as QC The data for QC binding of N-1 derivatives the for the of potent QC inhibitors by structure of N-1 imidazole of the obtained by N-1 of the imidazole to highly potent inhibitors of QC by data base of the potent inhibitors in the observed inhibition of as with in the initial led to the identification of compounds Ki values of the QC inhibition in the of and inhibition obtained for the imidazole derivatives for binding to the An of and were with to The effect of in and 5 was analyzed by the of and the two in the of and of of the of the led to a of by the to the to which further for the of the in binding of the imidazole derivatives to the pH of the of imidazole was further of the of QC inhibition. of a as as the of the enzyme was to a pH pH and imidazole a pKa was to be for the of and of the The inhibition of the a dependence on the pH the of imidazole a pH to in the pH Ki was the of QC inhibition on of the imidazole derivatives. was by of the data to a The the properties of imidazole was by a pKa in with the pKa of the of imidazole pH were obtained for QC inhibition by and the kinetically pKa values with the pKa values by The dependence of the and on the was analyzed the for of was in the pH and the a pH dependence with pH to in the and pH The pH dependence of revealed a of 1 in the pH the presence of a The kinetically pKa in the was to the pKa of the the pH data revealed a pKa of ± a of the QC by inhibition of QC by 1,10-phenanthroline the EDTA to effect on QC catalysis suggested inhibition by to metal in addition to by was in presence of dipicolinic inhibitor of chelators QC in a competitive and time-dependent initial was was to be further with the compounds EDTA inhibition of under conditions. Human QC was 5 1,10-phenanthroline 5 dipicolinic acid. QC was to 1 reactivation was of QC by dipicolinic 1,10-phenanthroline was by the for with in presence of EDTA of QC was obtained Co2+ and ions for in the presence of a reactivation to of the was reactivation was observed Ca2+, of fully QC with ions effect on the enzyme a QC to in with the for the a of a for glutaminyl by were observed in binding and of the glutaminyl QC by the the and by the suggested in the of the glutaminyl by Additionally, were to inhibition of QC by which QC in to the inhibition of QC by imidazole QC was by of as with QC by and dipicolinic to compounds a the with the metal of the reactivation by the addition of Zn2+ ions to and a of the structure of QC and the a of the of was the of QC with of two of the the binding of the two Zn2+ ions in of in QC. as in of two of the identified and which for metal binding to a of data further the and of the metal binding the in the catalysis of QC. the the to the of a the of a on the of the in binding the of the ions the of the and the to with to and the of the by the catalysis by the of binding the metal could with the of the of the QC the of the in to the of the a metal in the of QC by the of the glutaminyl the by the of the on the of the of the and the metal by the observed inhibition of QC by the of with the of the and the of the of the by in the catalysis of the the metal of QC as a binding for the imidazole-derived inhibitors and the with in to the to the Zn2+ concentrations in QC and QC which was observed in to be QC two metal ions to the to the binding Zn2+ was a further Zn2+ was for glutaminyl The of such a binding concentrations of Zn2+ ions the of the be in could of QC. and potent inhibitors of in the of QC with the the of the of the in the of the structure the binding of and to QC by metal a for the the of the of the glutaminyl to be and a of the same of such the of the of the of inhibitors for a QC. for QC to was to the to be potent QC the identification of N-1 imidazole derivatives as highly potent competitive inhibitors, the revealed QC as a enzyme as by the the of inhibition by imidazole and imidazole the of QC by the 1,10-phenanthroline and dipicolinic the reactivation of the by metal and the of in the structure of QC. the observed of of the imidazole derivatives on as a for inhibitor a QC to in with the for the a of a for glutaminyl by were observed in binding and of the glutaminyl QC by the the and by the suggested in the of the glutaminyl by Additionally, were to inhibition of QC by which QC in to the inhibition of QC by imidazole QC was by of as with QC by and dipicolinic to compounds a the with the metal of the reactivation by the addition of Zn2+ ions to and a of the structure of QC and the a of the of was the of QC with of two of the the binding of the two Zn2+ ions in of in QC. as in of two of the identified and which for metal binding to a of data further the and of the metal binding the in the catalysis of QC. the the to the of a the of a on the of the in binding the of the ions the of the and the to with to and the of the by the catalysis by the of binding the metal could with the of the of the QC the of the in to the of the a metal in the of QC by the of the glutaminyl the by the of the on the of the of the and the metal by the observed inhibition of QC by the of with the of the and the of the of the by in the catalysis of the the metal of QC as a binding for the imidazole-derived inhibitors and the with in to the to the Zn2+ concentrations in QC and QC which was observed in to be QC two metal ions to the to the binding Zn2+ was a further Zn2+ was for glutaminyl The of such a binding concentrations of Zn2+ ions the of the be in could of QC. and potent inhibitors of in the of QC with the the of the of the in the of the structure the binding of and to QC by metal a for the the of the of the glutaminyl to be and a of the same of such the of the of the of inhibitors for a QC. for QC to was to the to be potent QC the identification of N-1 imidazole derivatives as highly potent competitive inhibitors, the revealed QC as a enzyme as by the the of inhibition by imidazole and imidazole the of QC by the 1,10-phenanthroline and dipicolinic the reactivation of the by metal and the of in the structure of QC. the observed of of the imidazole derivatives on as a for inhibitor The of and to and for on the