Polymorphic Structure Determination of the Macrocyclic Drug Paritaprevir by MicroED

Abstract

Paritaprevir is an orally bioavailable, macrocyclic drug used for treating chronic Hepatitis C virus infection. Its structures had been elusive to the public until recently when one of the crystal forms was solved by MicroED. In this work, we report the MicroED structures of two distinct polymorphic crystal forms of paritaprevir from the same experiment. The different polymorphs show conformational changes in the macrocyclic core, as well as the cyclopropylsulfonamide and methylpyrazinamide substituents. Molecular docking shows that one of the conformations fits well into the active site pocket of the NS3/4A serine protease target, and can interact with the pocket and catalytic triad via hydrophobic interactions and hydrogen bonds. These results can provide further insight for optimization of the binding of acylsulfonamide inhibitors to the NS3/4A serine protease. In addition, this also demonstrate the opportunity of deriving different polymorphs and distinct macrocycle conformations from the same experiments using MicroED.

Keywords: CryoEM; HCV Protease; Macrocycles; MicroED; Molecular Chameleons; Polymorphism.

Figure 1.

a: Chemical structure of paritaprevir with the macrocyclic core highlighted in green. b: Representative crystal image of needle-like paritaprevir form α and MicroED pattern. c: Representative crystal image of rod-like paritaprevir form β and MicroED pattern. Scale bars corresponds to 2 μm.

Figure 2.

a: Unit cell packing of paritaprevir form α viewed along the crystallographic b axis with the unit cell box shown in black. Atom colors: C, light blue; N, blue; O, red; S, yellow. The inter- and intramolecular hydrogen bonds are shown in magenta and cyan dashed lines, respectively. The one-dimensional solvent channels are shown in gray contoured surface extending along the crystallographic a axis. All hydrogens are omitted for clarity. b: Unit cell packing of paritaprevir form β viewed along the crystallographic b axis with the unit cell box shown in black. Atom colors: C, orange; N, blue; O, red; S, yellow. The inter- and intramolecular hydrogen bonds are shown in magenta and blue dashed lines, respectively. The void space are shown in gray contoured surface. All hydrogens are omitted for clarity. c: Overlay of all heavy atoms on the aromatic rings, 5-membered ring and macrocyclic core amides between paritaprevir form α (light blue) and form β (orange), showing conformational changes in the macrocyclic core, methylpyrazinamide moiety and cyclopropylsulfonamide moiety, as indicated by the pink arrows. The intramolecular hydrogen bonds are shown in dashed lines. Non-polar hydrogens are omitted for clarity.

Figure 3.

a: Docking of paritaprevir conformer β into the simeprevir-bound crystal structure of HCV NS3/4A protease (PDB ID: 3KEE). Atom colors: C, pink; N, blue; O, red; S, yellow. All hydrogen atoms are omitted for clarity b: Potential interactions between paritaprevir and the target observed from the molecular docking. The intra- and intermolecular hydrogen bonds on paritaprevir are shown as dashed lines in cyan and magenta, respectively.