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

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 it...

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Bibliographic Details
Published in:Biochimie 2012-03, Vol.94 (3), p.711-718
Main Authors: Nogueira Santos, Jorge Alexandre, Assis, Diego M., Gouvea, Iuri Estrada, Júdice, Wagner A.S., Izidoro, Mario Augusto, Juliano, Maria Aparecida, Skern, Tim, Juliano, Luiz
Format: Article
Language:English
Subjects:
Animals
Cathepsin
Cathepsins - metabolism
Cysteine protease
Cysteine Proteases - chemistry
Cysteine Proteases - metabolism
Eukaryotic Initiation Factor-4G - metabolism
Fluorescent peptides
Foot-and-Mouth Disease - enzymology
Picornavirus
Protease inhibitors
Protease Inhibitors - chemical synthesis
Protease Inhibitors - chemistry
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Summary: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. ► Lbpro is a small papain-like cysteine peptidase but has prime side specificity at least down to the S5′ site. ► S1 and S1′ specificities depend on their occupancy allowing cleavages at K↓G, R↓G or G↓R bonds but not at K–R. ► Like other cysteine peptidases proline was very well accepted by S2′ site of Lbpro. ► Lbpro potent inhibitor derived E64 was synthesized, E64-R-P-NH2.
ISSN:0300-9084
1638-6183
DOI:10.1016/j.biochi.2011.10.016