Targeting the Fusion Process of SARS-CoV-2 Infection by Small Molecule Inhibitors

SARS-CoV-2 is an enveloped virus that requires membrane fusion for entry into host cells. Since the fusion process is relatively conserved among enveloped viruses, we tested our HCV fusion inhibitors, dichlorcyclizine and fluoxazolevir, against SARS-CoV-2.

Discovery of Highly Potent Fusion Inhibitors with Potential Pan-Coronavirus Activity That Effectively Inhibit Major COVID-19 Variants of Concern (VOCs) in Pseudovirus-Based Assays

We report the discovery of several highly potent small molecules with low-nM potency against severe acute respiratory syndrome coronavirus (SARS-CoV; lowest half-maximal inhibitory concentration (IC50: 13 nM), SARS-CoV-2 (IC50: 23 nM), and Middle East respiratory syndrome coronavirus (MERS-CoV; IC50: 76 nM) in pseudovirus-based assays with excellent selectivity index (SI) values (>5000), demonstrating potential pan-coronavirus inhibitory activities. Some compounds showed 100% inhibition against the cytopathic effects (CPE; IC100) of an authentic SARS-CoV-2 (US_WA-1/2020) variant at 1.25 µM. The most active inhibitors also potently inhibited variants of concern (VOCs), including the UK (B.1.1.7) and South African (B.1.351) variants and the Delta variant (B.1.617.2) originally identified in India in pseudovirus-based assay. Surface plasmon resonance (SPR) analysis with one potent inhibitor confirmed that it binds to the prefusion SARS-CoV-2 spike protein trimer. These small-molecule inhibitors prevented virus-mediated cell–cell fusion. The absorption, distribution, metabolism, and excretion (ADME) data for one of the most active inhibitors, NBCoV1, demonstrated drug-like properties. An in vivo pharmacokinetics (PK) study of NBCoV1 in rats demonstrated an excellent half-life (t1/2) of 11.3 h, a mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect these lead inhibitors to facilitate the further development of preclinical and clinical candidates.

Click chemistry and molecular modeling methods in computer-aided design and identification of potential HIV-1 inhibitors

An integrated approach including the click chemistry methodology, molecular docking, quantum mechanics, and molecular dynamics was used to perform the computer-aided design of potential HIV-1 inhibitors able to block the membrane- proximal external region (MPER) of HIV-1 gp41 that plays an important role in the fusion of the viral and host cell membranes. Evaluation of the binding efficiency of the designed compounds to the HIV-1 MPER peptide was performed using the methods of molecular modeling, resulting in nine chemical compounds that exhibit the high-affinity binding to this functionally important site of the trimeric “spike” of the viral envelope. The data obtained indicate that the identified compounds are promising for the development of novel antiviral drugs, HIV fusion inhibitors blocking the early stages of HIV infection.

Design and Identification of Potential HIV-1 Entry Inhibitors Using In Silico Click Chemistry and Molecular Modeling Methods

An integrated approach including the click chemistry methodology, molecular docking, quantum mechanics, and molecular dynamics was used to computer-aided design of potential HIV-1 inhibitors able to block the membrane-proximal external region (MPER) of HIV-1 gp41, which plays an important role in the fusion of the viral and host cell membranes. Evaluation of the binding efficiency of the designed compounds to the HIV-1 MPER peptide was performed using the methods of molecular modeling, resulting in nine chemical compounds exhibiting high-affinity binding to this functionally important site of the trimeric “spike” of the viral envelope. The data obtained indicate that the identified compounds are promising for the development of novel antiviral drugs, HIV fusion inhibitors blocking the early stages of HIV infection.

Peptide-based Fusion Inhibitors for Preventing the Six-helix Bundle Formation of Class I Fusion Proteins: HIV and Beyond

: A number of different viral families have developed convergent methods to infect cells. Class I fusion proteins are commonly used by members of Arenaviridae, Coronaviridae, Filovirdae, Orthomyxoviridae, Paramyxoviridae, and Retroviridae. Class I viral fusion proteins are trimers that are involved in recognizing the cellular receptor, with a region that is responsible for fusing the viral and target cell membranes. During the fusion process, the fusion region folds into a six-helix bundle (6HB) which approximates the two membranes leading to fusion. For human immunodeficiency virus (HIV), the gp41 subunit is responsible for the formation of this 6HB. The fusion inhibitor drug enfuvirtide, or T20, is the only US Food and Drug Administration and European Medicines Agency approved drug which targets this crucial step and has been widely used in combination regimens for the treatment of HIV since March 2003. In this review, we describe the current state of peptide-based fusion inhibitors in the treatment of HIV, and review how the field of HIV research is driving advances in the development of similar therapeutics in other viral systems, including the severe acute respiratory syndrome (SARS) coronaviruses.

Discovery of highly potent pancoronavirus fusion inhibitors that also effectively inhibit COVID-19 variants from the UK and South Africa

We report the discovery of a series of benzoic acid-based inhibitors that show highly potent pancoronavirus activity. Some compounds also show complete inhibition of CPE (IC100) at 1.25 μM against an authentic SARS-CoV-2 (US_WA-1/2020). Furthermore, the most active inhibitors also potently inhibited variants initially identified in the UK and South Africa. We confirmed that one of the potent inhibitors binds to the prefusion spike protein trimer of SARS-CoV-2 by SPR. Besides, we showed that they inhibit virus-mediated cell-cell fusion. The ADME data show druglike characteristics, and in vivo PK in rats demonstrated excellent half-life (t½) of 11.3 h, mean resident time (MRT) of 14.2 h, and orally bioavailable. Despite the presence of ene-rhodamine moiety, we conclusively demonstrated that these inhibitors target the viral spike protein and are not promiscuous or colloidal aggregators. We expect the lead inhibitors to pave the way for further development to preclinical and clinical candidates.