The outbreak of coronavirus disease 2019 (COVID-19) signified the commencement of a global health crisis [1], [2]. Originating in late 2019, this disease was caused by a novel coronavirus known as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Up to January 30th, 2024, it had resulted in over 774 million confirmed cases and more than 7.01 million deaths worldwide (https://covid19.who.int/) [3]. Following HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, and MERS-CoV, SARS-CoV-2 is the seventh coronavirus capable of infecting humans, and it exhibited unprecedented transmissibility and a prolonged incubation period [4], [5], [6]. Respiratory droplets and direct or indirect contact with infected individuals are the primary modes of transmission [7]. Research indicated that SARS-CoV-2 initially infects the respiratory tract, entering alveolar epithelial cells, rapidly replicating, and triggering robust immune responses. Simultaneously, other organs are also affected [8]. Patients might manifest mild symptoms to severe respiratory failure, with severe cases potentially leading to fatality [9], [10]. Despite the development of several preventive vaccines, the emergence of highly pathogenic SARS-CoV-2 variants like Delta and Omicron, characterized by immune-evading properties, has compromised the efficacy of vaccines [11], [12]. Consequently, there is a pressing need to develop more effective antiviral drugs capable of combating current highly transmissible variants and future coronaviruses.

SARS-CoV-2 is an enveloped β-coronavirus possessing a positive-sense single-stranded RNA genome [13], [14]. Its lifecycle encompasses several key steps: infection, cell entry, synthesis of the viral genome and proteins, and the assembly of new virus particles (Fig. 1). Initially, the virus attaches to the host cell by engaging with ACE2 receptors on the cell surface, facilitating its entry through fusion or endocytosis [15]. Once inside the host cell, the spike (S) protein is activated by cathepsin L, releasing positive-sense RNA containing the viral genetic information into the cytosol [16]. Alternatively, the transmembrane protease serine 2 (TMPRSS2) can also trigger the S protein, leading to fusion at the plasma membrane [17]. These fusion events facilitate the release of single-stranded RNA into the cytosol for the synthesis of polyproteins (pp1a and pp1b) [18]. The polyproteins are then cleaved by papain-like protease (PLpro) and main protease (Mpro), resulting in the production of 16 nonstructural proteins, including RNA-dependent RNA polymerase (RdRp) and helicase [19], [20]. RdRp replicates the viral RNA, generating both genomic RNA and subgenomic RNA. Viral proteins and RNA are transported to the endoplasmic reticulum and Golgi apparatus, where new virions are assembled. The viral RNA is packaged with the nucleocapsid protein and assembled with structural proteins within the membrane of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) to form complete viral particles. Finally, these particles are released from the plasma membrane [21], [22], [23], [24].

In the development of small-molecule drugs, selecting the targeted protein is crucial for the design of the drug molecule. Mpro has emerged as an ideal target for the development of antiviral drugs against SARS-CoV-2 due to its high conservation and the absence of homologous proteases [25], [26]. Currently, four inhibitors (Fig. 2) targeting Mpro have been approved and various inhibitors developed based on this target are undergoing clinical trials [27], [28], [29], [30]. Structurally, these inhibitors can be categorized into two types. The first type is peptidomimetic inhibitors, which typically contain an electrophilic reactive warhead, to attack the thiol group of Cys145 in the active cavity of Mpro and form a covalent bond [31], [32]. The second type is non-peptidic inhibitors, which enter the active cavity and interact with the key amino acid residues through covalent or non-covalent bonds. Compared with peptidomimetic inhibitors, non-peptidic inhibitors exhibit reduced off-targeted toxicity and improved metabolic stability, and can also achieve outstanding selectivity and high affinity [33], [34]. These noteworthy advantages have positioned non-peptidic SARS-CoV-2 Mpro inhibitors as a focal point of attention. In addition, most non-peptide inhibitors have a smaller molecular weight and a more stable structure. This aspect renders them more suitable for oral administration, thereby enhancing patient convenience and adherence to treatment [35], [36]. Furthermore, their enhanced stability in the body prevents premature degradation by metabolic enzymes, prolonging half-life and reducing dosing frequency. Moreover, non-peptidic inhibitors are typically easier to design and synthesize, thereby enhancing drug development efficiency and lowering medication production costs [37], [38]. It's worth noting that some non-peptidic inhibitors exhibit enhanced cell membrane permeability, allowing them to effectively traverse biological membranes and reach target cells or tissues. Therefore, precise drug design can achieve high specificity for the target protein, and reduce the risk of off-target effects. Furthermore, non-peptidic inhibitors are less likely to trigger an immune response, providing an advantage in terms of safety and minimizing adverse reactions [39], [40], [41]. Based on the aforementioned advantages, non-peptidic Mpro inhibitors hold promising prospects for the future treatment of COVID-19 and possible emerging coronavirus diseases. Reflecting on these aspects, we have reviewed non-peptidic inhibitors of SARS-CoV-2 Mpro since 2020 to offer a valuable reference for the development of anti-coronavirus drugs targeting Mpro.