Crystal Structure of Feline Infectious Peritonitis Virus Main Protease in Complex with Synergetic Dual Inhibitors

Abstract

Coronaviruses (CoVs) can cause highly prevalent diseases in humans and animals. Feline infectious peritonitis virus (FIPV) belongs to the genus Alphacoronavirus, resulting in a lethal systemic granulomatous disease called feline infectious peritonitis (FIP), which is one of the most important fatal infectious diseases of cats worldwide. No specific vaccines or drugs have been approved to treat FIP. CoV main proteases (M pro s) play a pivotal role in viral transcription and replication, making them an ideal target for drug development. Here, we report the crystal structure of FIPV M pro in complex with dual inhibitors, a zinc ion and a Michael acceptor. The complex structure elaborates a unique mechanism of two distinct inhibitors synergizing to inactivate the protease, providing a structural basis to design novel antivirals and suggesting the potential to take advantage of zinc as an adjunct therapy against CoV-associated diseases. C oronaviruses (CoVs) infect humans and animals, causing various highly prevalent and severe diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) (1, 2). Feline infectious peritonitis virus (FIPV) belongs to the genus Alphacoronavirus in the subfamily Coronavirinae. It is a highly virulent mutant of the feline enteric coronavirus (FECV), which is closely related to transmissible gastroenteritis coronavirus (TGEV) of pigs and canine coronavirus (CCV) (3). In contrast with FECV, which causes asymptomatic or mild infection in cats and other felines, FIPV is an etiologic agent resulting in a lethal systemic granulomatous disease called feline infectious peritonitis (FIP), one of the most important fatal infectious diseases of cats worldwide (4). There are no effective drugs specific for FIP. The development of vaccines toward FIPV has also failed due to the antibody-dependent enhancement, where infection of the monocyte/macrophage lineage by FIPV is enhanced in the presence of antibodies (5). Thus, discovery of effective antivirals against FIPV is desired for the treatment of FIP.Similar to other alphacoronaviruses, FIPV contains a single positive-stranded RNA genome that is composed of two overlapping open reading frames (ORFs), ORF1a and ORF1b at the 5= end, encoding two large polyproteins, pp1a and pp1ab (6). These two polyproteins are subsequently cleaved into 16 nonstructural proteins (nsp1 to nsp16), which assemble into a membrane-anchored replication machinery for transcription/replication. Cleavage is regulated by two proteases: the main protease (M pro , also called nsp5 or 3C-like protease), and the papain-like protease (PL pro ). PL pro processes the N-terminal end of pp1a/pp1ab into nsp1, nsp2, and nsp3, while M pro cleaves the polyproteins at 11 sites to release nsp4 to nsp16 (6). The essential roles M pro plays in the viral life cycle and the lack of a cellular homologue in the human genome make it an attractive target for drug design.To date, several crystal structures of CoV M pro and the complex of M pro -inhibitor...

In silico and in vitro analysis of small molecules and natural compounds targeting the 3CL protease of feline infectious peritonitis virus

“…FIPV 3CL pro contains a Cys residue as the nucleophile of the catalytic dyad (St. John et al, 2015;Wang et al, 2015). Usually, the inhibitors of 3CL pro are designed to facilitate the nucleophilic attack by protease at the carbonyl residue for the formation of a covalent bond with the inhibitor and trap the enzymatic nucleophile, terminating its catalytic activity.…”

“…1016/j.antiviral.2019.104697 Received 28 September 2019; Received in revised form 15 December 2019; Accepted 16 December 2019 (Pedersen, 2014a;St. John et al, 2015;Wang et al, 2015). Similar to other coronaviruses (CoV), FCoV pp1ab contains two cysteine proteases, the papain-like protease (PL pro ) and the 3-chymotrypsin-like protease (3CL pro ).…”

“…The 3CL pro is also known as the main protease (M pro ) (Pedersen, 2014a;St. John et al, 2015;Wang et al, 2015;Berry et al, 2015) that plays a pivotal role in the viral life cycle, including the processing of polyprotein and viral replication. The polyprotein pp1a and pp1ab are subsequently enzymatically cleaved into 16 nonstructural functional proteins (nsp1 to nsp16) that are mainly involved in proteolytic processing and viral RNA synthesis, including the replication of the genome and synthesis of the sub-genomic mRNA.…”

Discovery of unsymmetrical aromatic disulfides as novel inhibitors of SARS-CoV main protease: Chemical synthesis, biological evaluation, molecular docking and 3D-QSAR study

“…The lines displayed in reciprocal plots intersect at a same point, indicating that both inhibitors serve as a non-competitive inhibitor with a < 1 (Part 3 of the supplementary data) [27]. On this basis, the kinetic parameters (aK i ¼ 0.20 mM, K i ¼ 0.24 mM) of 3-31 were determined [28] (Fig. 3), which clearly proved that, the noncompetitive inhibitor 3-31 is characterized by smaller equilibrium-binding constant compared to some known inhibitors such as N3 (K i ¼ 9.0 mM) and N9 (K i ¼ 6.7 mM) [3].…”

“…The further characterization of the inhibitor as a noncompetitive inhibitor employs the methods described in earlier work [28]. Basically, the enzymatic velocity of SARS-CoV M pro versus substrate concentrations with presence of inhibitors is depicted by equation (2) [27], where K i is the dissociation constant for the SARS-CoV M pro complexed with inhibitor 3-31; factor a reflects the effect of inhibitor 3-31 on the affinity of the enzyme for its substrate; V max and K m represent the maximum velocity and Michaelis-Menten constant, respectively.…”