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. 2021:50:301-333.
doi: 10.1016/bs.enz.2021.09.004. Epub 2021 Nov 17.

Viral proteases: Structure, mechanism and inhibition

Jacqueto Zephyr  1 , Nese Kurt Yilmaz  1 , Celia A Schiffer  2
Affiliations

Viral proteases: Structure, mechanism and inhibition

Jacqueto Zephyr et al. Enzymes. 2021.

Abstract

Viral proteases are diverse in structure, oligomeric state, catalytic mechanism, and substrate specificity. This chapter focuses on proteases from viruses that are relevant to human health: human immunodeficiency virus subtype 1 (HIV-1), hepatitis C (HCV), human T-cell leukemia virus type 1 (HTLV-1), flaviviruses, enteroviruses, and coronaviruses. The proteases of HIV-1 and HCV have been successfully targeted for therapeutics, with picomolar FDA-approved drugs currently used in the clinic. The proteases of HTLV-1 and the other virus families remain emerging therapeutic targets at different stages of the drug development process. This chapter provides an overview of the current knowledge on viral protease structure, mechanism, substrate recognition, and inhibition. Particular focus is placed on recent advances in understanding the molecular basis of diverse substrate recognition and resistance, which is essential toward designing novel protease inhibitors as antivirals.

Keywords: Coronavirus; Drug resistance; Enterovirus; Flavivirus; HCV; HIV-1; HTLV-1; Protease inhibitors; Substrate envelope; Viral protease.

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Figures

Fig. 1
Fig. 1
HIV-1 Protease. (A) Schematic representation of HIV-1 polyprotein (Gag-Pro-Pol), with viral protease (PR) cleavage sites between structural proteins (MA: matrix; CA: capsid; NC: nucleocapsid), enzymes (RT: reverse transcriptase; RH: RNase H; IN: integrase) and peptides (TF: trans-frame; spacer peptides P1, P2) indicated by arrows. (B) Amino acid sequence of polyprotein cleavage sites recognized by HIV-1 protease. The protease cleaves between P1-P1’. (C) The crystal structure of HIV-1 protease (PDB ID: 1T3R) with the two monomers colored cyan and magenta and the catalytic residue (D25) depicted in yellow sticks and labeled, and (D) a close-up view of the active site with S2-S2’ pockets annotated. (E) The chemical structure of the latest FDA-approved HIV-1 protease inhibitor, darunavir. (F) Cocrystal structure of darunavir (green sticks) bound to HIV-1 protease, with location of primary (red) and secondary (blue) resistance mutations indicated by spheres.
Fig. 2
Fig. 2
HCV NS3/4A Protease. (A) Schematic representation of HCV polyprotein, with viral protease cleavage sites between non-structural proteins (NS) indicated by arrows. (B) Amino acid sequence of polyprotein cleavage sites recognized by HCV NS3/4A protease. The protease cleaves between P1-P1’. (C) The crystal structure of HCV NS3/4A protease (PDB ID: 3M5O) with the catalytic residues depicted as yellow sticks and labeled, and (D) a close-up view of the active site with S4-S1’ pockets annotated. (E) The chemical structure of HCV NS3/4A protease inhibitors (top, left) simeprevir, (top, right) grazoprevir, (bottom, left) voxilaprevir, and (bottom, right) glecaprevir. (F) Cocrystal structure of grazoprevir (green sticks) bound to HCV NS3/4A protease, with primary (red) and secondary (blue) resistance-associated substitutions labeled.
Fig. 3
Fig. 3
HTLV-1 Protease. (A) Schematic representation of HTLV-1 polyprotein, with viral protease cleavage sites indicated by arrows. (B) Amino acid sequence of polyprotein cleavage sites. The protease cleaves between P1-P1’. (C) The crystal structure of HTLV-1 protease (PDB ID: 6W6Q) with the catalytic residues depicted as yellow sticks and labeled, and (D) a close-up view of the active site with S2-S2’ pockets annotated. (E) (left) Indinavir and (right) PU6 (e), with their respective cocrystal structures bound to the HTLV-1 protease (PDB ID: 3WSJ and 6W6S) (F and G).
Fig. 4
Fig. 4
Flavivirus Proteases. (A) Schematic representation of flavivirus polyprotein, with viral protease cleavage sites indicated by arrows (B) Amino acid sequence of polyprotein cleavage sites. The protease cleaves between P1-P1’. (C) The chemical structure of flaviviral protease inhibitor, compound 1. (D) The crystal structure of Zika virus (ZIKV) NS2B/3 protease in the (left) closed and (right) open conformation (PDB ID: 5LC0 and 5GXJ), with the catalytic residues labeled. (E) The active site topology of the closed (E) and open (F) conformation with S3-S1’ pockets annotated. (G) The cocrystal structure of compound 1 bound to the ZIKV NS2B/3 protease in the closed conformation.
Fig. 5
Fig. 5
Enterovirus 3C Protease (3Cpro). (A) Schematic representation of the enterovirus polyprotein, with 3Cpro cleavage sites indicated by arrows. (B) Amino acid sequence of polyprotein cleavage sites. The protease cleaves between P1-P1’. (C) The crystal structure of the EV-A71 3Cpro (PDB ID: 7DNC) with the catalytic residues labeled, and (D) a close-up view of the active site with S4-S1’ pockets annotated. (E) Chemical structures of rupintrivir and AG7404. (F) Cocrystal structure of rupintrivir bound to EV-A71 3Cpro (PDB ID: 3SJO).
Fig. 6
Fig. 6
Coronavirus Main Protease (Mpro). (A) Schematic representation of the coronovirus polyprotein, with Mpro cleavage sites indicated by arrows. (B) Amino acid sequence of polyprotein cleavage sites. The protease cleaves between P1-P1’. (C) The crystal structure of SARS-CoV-2 Mpro homodimer (PDB ID: 7KHP) with the catalytic residues labeled and the N-finger, domains I, II, and III colored accordingly on one of the monomers, and (D) a close-up focused view of the active site with S4-S2’ pockets annotated. (E) Chemical structures of Mpro inhibitors PF-07304814 and PF-07321332. (F) Cocrystal structure of PF-00835231 bound to SARS-CoV-2 Mpro (PDB ID: 6XHM).
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