In December 2019, a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the etiological agent of viral pneumonia cases that occurred in Wuhan, Hubei Province, China. As of July 1st 2020, the pandemic has resulted in more than 10 million infections, and 500,000 deaths worldwide according to the World Health Organization.
There is currently no standardized treatment protocol, and there is no antiviral treatment of proven efficacy recommended for COVID-19. Clinical management of patients is mainly supportive, including supplementary oxygen and mechanical ventilation if needed. However, given the overwhelming burden of the pandemic on national healthcare systems and the global economy, experimental therapies have been attempted, which are predominantly based on the repurposing of FDA approved antivirals, antimalarials, arthritis drugs, and blood plasma derivatives [1].
The HIV protease inhibitors (PIs) lopinavir and ritonavir have gained particular interest, having shown documented in vitro activity against SARS-CoV and the Middle East respiratory syndrome coronavirus (MERS), however, these studies did not identify a molecular target for the drugs, since their efficacy was solely determined based on the inhibition of cytopathic effects or viral replication, respectively [2 3]. Given the 94.4% identity in amino acid sequence between SARS-CoV and SARS-CoV-2 [4], studying the efficacy of HIV protease inhibitors against SARS-CoV-2 would be of major relevance.
The genome of SARS-CoV-2 encodes for two viral cysteine proteases; nsp3 (papain-like protease) and nsp5 (main protease) [5]. The main protease (Mpro) of SARS-CoV-2; also named chymotrypsin-like protease (3CLpr), plays a crucial role in the viral life cycle, cleaving the initial polyproteins translated from the viral RNA at at least 11 of its 14 cleavage sites. Mpro of SARS-CoV-2 shares 96% sequence identity to that of SARS-CoV. The enzyme consists of three domains; two domains (I and II) which consist of antiparallel β-barrels, and an α-helical domain (I), which is responsible for dimerization and enzymatic activity [6 7]. Recent structure determination confirmed the similarities between the two enzymes [8].
One potential target for the HIV PIs is the Mpro. In silico screening identified nelfinavir as its potential inhibitor [9], while lopinavir and ritonavir were found to be potential inhibitors of the viral enzyme by molecular dynamics simulation [10].
It is important to note that the HIV protease is a C2-symmetric homodimeric aspartyl protease, composed of two identical subunits that are 99 amino acids each. The active site is located at the interface between the two monomers, and contains the catalytic Asp-Thr-Gly residues [11]. Mpro on the other hand, is a cysteine protease that can potentially be targeted by peptide mimetics. Given the structural difference between the two proteases, the efficacy of HIV protease inhibitors against SARS-CoV and SARS-CoV-2 is questionable.
Previous studies reported that a combination of lopinavir/ritonavir and ribavirin was effective against SARS-associated coronavirus, with concentrations of 4 µg/ml and 50 µg/ml, respectively [12]. However, a recent clinical trial of 99 patients with laboratory-confirmed SARS-CoV-2 infection who were treated with lopinavir–ritonavir concluded that no significant benefit was observed in the treated group compared to those who received standard care [13]. Recently, a short communication reported that lopinavir inhibited SARS-CoV-2 replication in Vero E6 cells with IC50 of 26.63 µM, ritonavir, however, showed no inhibition of viral replication [14].
Early in vitro reports from China showed that darunavir inhibited SARS-CoV-2 replication, although at a very high concentration (300 µM) [15]. Clinical trials are currently ongoing [16].
Our aim was to test the efficacy of a panel of HIV PIs against SARS-CoV-2 Mpro, using a cell culture-based model. In this study, we determined the IC50 of the PIs with the aid of a dark-to-bright GFP substrate system that had been developed and applied previously for the investigation of caspases [17]. Moreover, in vitro enzymatic inhibitory assays were also carried out using purified Mpro and an oligopeptide substrate representing the AVLQ*SGFR cleavage site of SARS-CoV-2 polyprotein 1 ab (PP1ab).