title: 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 (Lb(pro)) performs the initial cleavage by freeing itself from the growing polypeptide chain; subsequently, Lb(pro) cleaves the two homologues of the host cell protein eukaryotic initiation factor 4G (eIF4G). We showed that Lb(pro) possesses specific binding sites at the non prime side from S(1) down to S(7) [Santos et al. (2009) Biochemistry, 48, 7948-7958]. Here, we demonstrate that Lb(pro) has high prime side specificity at least down to the S'(5) site. Lb(pro) 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 Lb(pro) that show for the reversible step (E + I ↔ EI) K(i) = 3.4 μM and 11.6 μM, and for the irreversible step (EI→E-I) k(4) = 0.16 and 0.06 min(-1), respectively. Knowledge of the Lb(pro) specificity led us to extend E64 by addition of the dipeptide R-P. This compound, termed E64-R-P-NH(2), irreversibly inhibited Lb(pro) with a K(i) = 30 nM and k(4) = 0.01 min(-1) and can serve as the basis for design of specific inhibitors of FMDV replication.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-20 São Paulo, Brazil.
authors.forename: Jorge Alexandre
authors.initials: JA
authors.lastname: Nogueira Santos
authors.forename: Diego M
authors.initials: DM
authors.lastname: Assis
authors.forename: Iuri Estrada
authors.initials: IE
authors.lastname: Gouvea
authors.forename: Wagner A S
authors.initials: WA
authors.lastname: Júdice
authors.forename: Mario Augusto
authors.initials: MA
authors.lastname: Izidoro
authors.forename: Maria Aparecida
authors.initials: MA
authors.lastname: Juliano
authors.forename: Tim
authors.initials: T
authors.lastname: Skern
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
mesh_terms: D000818:Animals; D002403:Cathepsins / Q000378:metabolism; D057056:Cysteine Proteases / Q000737:chemistry / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism; D005536:Foot-and-Mouth Disease / Q000201:enzymology*; D011480:Protease Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D039603:Eukaryotic Initiation Factor-4G; D011480:Protease Inhibitors; D002403:Cathepsins; D057056:Cysteine Proteases