Foot and mouth disease leader protease (Lbpro): Investigation of prime side specificity allows the synthesis of a potent inhibitor

Abstract

Foot and mouth disease virus expresses its genetic information as a single polyprotein that is translated from the single-stranded RNA genome. Proteinases contained within the polyprotein then generate the mature viral proteins. The leader protease (Lbpro) performs the initial cleavage by freeing itself from the growing polypeptide chain; subsequently, Lbpro cleaves the two homologues of the host cell protein eukaryotic initiation factor 4G (eIF4G). We showed that Lbpro possesses specific binding sites at the non prime side from S1 down to S7 [Santos et al. (2009) Biochemistry, 48, 7948–7958]. Here, we demonstrate that Lbpro has high prime side specificity at least down to the S′5 site. Lbpro is thus not only one of the smallest papain-like cysteine peptidases but also one of the most specific. It can still however cleave between both K↓G and G↓R pairs. We further determined the two-step irreversible inhibition (E + I ↔ EI→ E − I) kinetic parameters of two known irreversible epoxide-based inhibitors of cysteine proteinases, E64 and CA074 on Lbpro that show for the reversible step (E + I ↔ EI) Ki = 3.4 μM and 11.6 μM, and for the irreversible step (EI→E−I) k4 = 0.16 and 0.06 min−1, respectively. Knowledge of the Lbpro specificity led us to extend E64 by addition of the dipeptide R–P. This compound, termed E64-R-P-NH2, irreversibly inhibited Lbpro with a Ki = 30 nM and k4 = 0.01 min−1 and can serve as the basis for design of specific inhibitors of FMDV replication.