Identification and characterization of 7-azaindole derivatives as inhibitors of the SARS-CoV-2 spike-hACE2 protein interaction

Abstract

The COVID-19 pandemic, caused by SARS-CoV-2, has become a global public health crisis. The entry of SARS-CoV-2 into host cells is facilitated by the binding of its spike protein (S1-RBD) to the host receptor hACE2. Small molecule compounds targeting S1-RBD-hACE2 interaction could provide an alternative therapeutic strategy sensitive to viral mutations. In this study, we identified G7a as a hit compound that targets the S1-RBD-hACE2 interaction, using high-throughput screening in the SARS2-S pseudovirus model. To enhance the antiviral activity of G7a, we designed and synthesized a series of novel 7-azaindole derivatives that bind to the S1-RBD-hACE2 interface. Surprisingly, ASM-7 showed excellent antiviral activity and low cytotoxicity, as confirmed by pseudovirus and native virus assays. Molecular docking and molecular dynamics simulations revealed that ASM-7 could stably bind to the binding interface of S1-RBD-hACE2, forming strong non-covalent interactions with key residues. Furthermore, the binding of ASM-7 caused alterations in the structural dynamics of both S1-RBD and hACE2, resulting in a decrease in their binding affinity and ultimately impeding the viral invasion of host cells. Our findings demonstrate that ASM-7 is a promising lead compound for developing novel therapeutics against SARS-CoV-2.

Introduction

As of the end of May 2023, the coronavirus disease 2019 (COVID-19) pandemic has resulted in >766 million confirmed cases and over 6.9 million fatalities worldwide, as reported by the World Health Organization [1]. Extensive research has established that the spike protein receptor-binding domain (S1-RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts with the human angiotensin-converting enzyme 2 (hACE2) receptor on the cellular surface [2]. This interaction is critical in the virus's ability to invade and replicate within host cells [3]. Hence, the development of small molecules capable of inhibiting the binding of S1-RBD and hACE2 has become a critical avenue for the creation of coronavirus treatments [4]. The elucidation of the intricate structure of the SARS-CoV-2 S1-RBD and hACE2 full-length protein complex has uncovered the mechanism behind the virus's infiltration of host cells and furnished an essential foundation for developing targeted new drugs [5]. However, the vast and featureless contact surface shared by the S1-RBD and hACE2 proteins poses a formidable challenge in identifying a suitable small molecule drug capable of stably binding and disrupting this regulatory pathway [6].

Of particular note, the discovery of the Omicron variant in November 2021 has presented a significant challenge, as it features over 14 mutations in the spike protein RBD and has rapidly spread worldwide. In the mutated Omicron variants, several amino acid positions in S1-RBD have been identified as being altered compared to the wild-type strain. These include G339D, S371L, S373P, S375F, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H [7,8]. The S477N mutation is a crucial concern, as it has emerged numerous times in various SARS-CoV-2 lineages and has been demonstrated to enhance the virus's affinity for the host receptor at the S1-RBD-hACE2 interface [9]. Consequently, the effectiveness of current COVID-19 vaccines and antibody therapies may be compromised due to the substantial number of mutations, including S477N [10].

Currently, small molecule drugs targeting the disruption of S1-RBD and hACE2 binding hold promise in mitigating antibody and vaccine resistance resulting from viral mutations. The growing body of research has identified an expanding repertoire of small-molecule drugs capable of inhibiting the interaction between S1-RBD and hACE2. All-trans retinoic acid (ATRA) that binds directly in a deep hydrophobic pocket of the S1-RBD leads to an “all-down” conformations [11], thereby blocking the interaction between RBD and hACE2. The ceftazidime binds to the interface of S1-RBD with ACE2 and blocks the binding of the complex [12]. Compounds such as nilotinib might induce significant conformational changes in the ACE2-RBD complex, intervene with the hydrogen bonds, and destabilize the complex of S1-RBD-hACE2 [13], thus potentially reducing the SARS-CoV-2 infection risk. Nevertheless, the need to conduct live SARS-CoV-2 experiments in biosafety level III laboratories has impeded research and drug development for COVID-19. Conversely, the utilization of SARS-CoV-2 spike protein (SARS2-S) pseudoviruses devoid of specific gene sequences is significantly safer and can be investigated in biosafety level II laboratories, providing a valuable tool for studying SARS-CoV-2 virology [14].

This study employed a combination of virtual and high-throughput screening techniques to identify the hit compound G7a, which can inhibit SARS-CoV-2 entry into host cells by obstructing the interaction between S1-RBD and hACE2. Building on the binding mode of G7a with the S1-RBD-hACE2 protein, we designed and synthesized a collection of 7-azaindole derivatives called ASMs, and conducted preliminary structure-activity relationship (SAR) studies on the compounds using the SARS2-S pseudovirus assay. Subsequently, we tested the most active compound, ASM-7, in vitro through live virus experiments. We employed molecular docking and molecular dynamics (MD) simulation techniques to further elucidate the interaction modes between the compounds ASMs and the S1-RBD-hACE2 protein.