A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells (preprint)

K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 M inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of [≤] 100 M. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2 , differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.

Ivermectin repurposing for COVID-19 therapy: Safety and pharmacokinetic assessment of a novel nasal spray formulation in a pig model (preprint)

High ivermectin (IVM) concentrations suppress in vitro SARS-CoV-2 replication. Nasal IVM spray (NIVM spray) administration may contribute to attaining high drug concentrations in nasopharyngeal (NP) tissue, a primary site of virus entrance/replication. The safety and pharmacokinetic performance of a new NIVM spray formulation in a piglet model were assessed. Crossbred piglets (10/12 kg) were treated with either one or two (12 h apart) doses of N IVM spray (2 mg, 1 puff/nostril) or orally (0.2 mg/kg). The overall safety of NIVM-spray was assessed (clinical, haematological, serum biochemical determinations), and histopathology evaluation of the application site tissues performed. The IVM concentration profiles measured in plasma and respiratory tract tissues (nasopharynx and lungs) after the nasal spray treatment (one and two applications) were compared with those achieved after the oral administration. Animals tolerated well the novel NIVM spray formulation. No local/systemic adverse events were observed. After nasal administration, the highest IVM concentrations were measured in NP and lung tissues. Significant increases in IVM concentration profiles in both NPtissue and lungs were observed after the 2 dose nasal administrations. The nasal/oral IVM concentration ratios in NP and lung tissues (at 6 h postdose) markedely increased by repeating the spray application. The fast attainment of high and persistent IVM concentrations in NP tissue is the main advantage of the nasal over the oral route. These original results are encouraging to support the undertaking of further clinical trials to evaluate the safety/efficacy of the nasal IVM spray application in the treatment and/or prevention of COVID-19.

Evidence for ZAP-independent CpG reduction in SARS-CoV-2 genome, and pangolin coronavirus origin of 5'UTR (preprint)

SARS-CoV-2, the causative agent of COVID-19, has an RNA genome, which is, overall, closely related to the bat coronavirus sequence RaTG13. However, the ACE2-binding domain of this virus is more similar to a coronavirus isolated from pangolin. In addition to this unique feature, the genome of SARS-CoV-2 (and its closely related coronaviruses) has a low CpG content. This has been postulated to be signature of an evolutionary pressure exerted by the host antiviral protein ZAP. Here, we analyzed the sequences of a wide range of viruses using both alignment-based and alignment free approaches to investigate the origin of SARS-CoV-2 genome. Our analyses revealed a high level of similarity between the 5UTR of SARS-CoV-2 and that of a Guangdong pangolin coronavirus. These data suggest that not only ACE2, but also the 5UTR of SARS-CoV-2 likely has a pangolin coronavirus origin. Additionally, we performed a detailed analysis of viral genome compositions as well as expression and RNA binding data of ZAP to show that the low CpG abundance in SARS-CoV-2 is not related to an evolutionary pressure from ZAP.

SARS-CoV-2 RBD mutations, ACE2 genetic polymorphism, and stability of the virus-receptor complex: The COVID-19 host-pathogen nexus. (preprint)

We herein report a computational study on the implications of SARS-CoV-2 RBD mutations and the Angiotensin Converting Enzyme 2 (ACE2) receptor genetic variations on the stability of the virus-host association. In silico analysis of the complex between the virus mutated forms and ACE2 isoform 1 showed that out of 351 RBD point mutations, 83% destabilizes the complex, while 17% have mild stabilizing effect. Study of the complex SARS-CoV-2 Wuhan strain RBD region /ACE2 isoform 1, 6LZG PDB 3D model revealed 18 contact residues. Interestingly, mutations occurring in 15 out of these residues show variations in the patterns of polar and hydrophobic interactions as compared to the original complex. Similarly, comparison of the effect on the complex stability of different ACE2 variants showed that the pattern of molecular interactions and the virus-receptor complex stability varies also according to ACE2 polymorphism. This could explain the large inter-individual variation of disease susceptibility and/or severity. The observation of a high variability in the interactions patterns highlights the complexity of the molecular interplay between SARS-CoV-2 and the ACE2 receptor. We infer that it is important to consider both ACE2 genetic variants and SARS-CoV-2 RBD mutations to assess the stability of the virus-receptor association and evaluate the infectivity of circulating SARS-CoV-2. These findings point toward the importance of individuals genetic typing of the circulating viral form as well as the ACE2 receptor. This will offer a good molecular ground to adjust the mitigation efforts for a better control of the virus spreading.

Machine Learning Models Identify Inhibitors of SARS-CoV-2 (preprint)

With the ongoing SARS-CoV-2 pandemic there is an urgent need for the discovery of a treatment for the coronavirus disease (COVID-19). Drug repurposing is one of the most rapid strategies for addressing this need and numerous compounds have been selected for in vitro testing by several groups already. These have led to a growing database of molecules with in vitro activity against the virus. Machine learning models can assist drug discovery through prediction of the best compounds based on previously published data. Herein we have implemented several machine learning methods to develop predictive models from recent SARS-CoV-2 in vitro inhibition data and used them to prioritize additional FDA approved compounds for in vitro testing selected from our in-house compound library. From the compounds predicted with a Bayesian machine learning model, CPI1062 and CPI1155 showed antiviral activity in HeLa-ACE2 cell-based assays and represent potential repurposing opportunities for COVID-19. This approach can be greatly expanded to exhaustively virtually screen available molecules with predicted activity against this virus as well as a prioritization tool for SARS-CoV-2 antiviral drug discovery programs. The very latest model for SARS-CoV-2 is available at www.assaycentral.org.Competing Interest StatementSE is CEO and owner of Collaborations Pharmaceuticals, Inc. DHF, KMZ, TRL, AP are employees of Collaborations Pharmaceuticals, Inc.View Full Text