Improved substrates for human cytomegalovirus protease

Details Improved substrates for human cytomegalovirus protease Improved substrates for human cytomegalovirus protease

C - Chemistry, Metallurgy

12

Q

C12Q 1/37 (2006.01) C07K 5/103 (2006.01) C07K 7/06 (2006.01)

Patent

CA 2276107

Human cytomegalovirus (HCMV) protease is a slow-processing enzyme in vitro. Disclosed herein are improved fluorogenic and radiometric peptide substrates for CMV protease. The improved substrates have a typically Tbg-Tbg-Asn(NMe2) sequence replacing the Val-Val-Asn sequence, corresponding to the P4-P2 residue of the maturation site of the enzyme. The incorporation of this new sequence in a variety of substrates has afforded substrates with improved kinetic parameters compared to homologues containing the natural sequence only. For example, the substrate 2-aminobenzoyl-Tbg-Tbg-Asn(NMe2)- Ala.dwnarw.Ser-Ser-Arg-Leu-Tyr(3-NO2)ARG-OH displayed a k out/Km value of 15940 M-1, s-1 i.e. over 60-fold greater than that of the equivalent, non- optimized substrate in identical conditions. The new substrates find use in assays which can evaluate and characterize HCMV protease inhibitors having low nanomolar potency.

La protéase du cytomégalovirus humain (NCMV) est une enzyme de lente maturation in vitro. L'invention a trait aux substrats peptides fluorogènes et radiométriques améliorés pour la protéase du CMV. Les substrats améliorés ont une séquence Tbg-Tbg-Asn(NMe2) remplaçant la séquence Val-Val-Asn, correspondant aux résidus P4-P2 du site de maturation de l'enzyme. L'incorporation de cette nouvelle séquence dans une variété de substrats a fourni des substrats avec des paramètres cinétiques améliorés par rapport à leurs homologues contenant exclusivement la séquence naturelle. Par exemple, le substrat 2-aminobenzoyle-Tbg-Tbg-Asn(NMe2)-Ala &darr& Ser-Ser-Arg-Leu-Tyr(3-NO2)ARG-OH affiche une valeur kcat/Km de 15940 M<-1>s<-1>, à savoir plus de 60 fois supérieure par rapport au substrat non amélioré équivalent dans les mêmes conditions. Les nouveaux substrats sont utilisés dans des tests qui évaluent et caractérisent les inhibiteurs de protéase HCMV à faible puissance nanomolaire.

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