Serine proteases are characterized by a highly conserved Ser-His-Asp catalytic triad (41), as exemplified by TMPRSS2 ( Figure 1A ). The structural basis for their functional divergence lies in the diverse stereoconformations of substrate-binding pockets. For instance, the catalytic groove of TMPRSS2 precisely accommodates the receptor-binding domain (RBD) of coronavirus spike proteins (e.g., human coronavirus HKU1 (HCoV-HKU1)), inducing conformational changes that trigger membrane fusion (42). Conversely, furin recognizes multibasic cleavage motifs (e.g., Arg-X-X-Arg↓) within the polyproteins of diverse pathogens through its distinctive substrate-binding cleft, mediating their maturation and infectivity (40). During viral infections, serine proteases are extensively exploited to activate both surface glycoproteins (e.g., influenza HA, CoVs S, and HIV gp160) and internal precursor polyproteins—serving as critical rate-limiting steps in viral entry and maturation.

Metalloproteases are characterized by an active center coordinated through specific metal ions (e.g., Zn²⁺) coordinated by surrounding amino acid residues (e.g., histidine, glutamate, or aspartate) (43), as exemplified by a disintegrin and metalloproteinase 17 (ADAM17) ( Figure 1B ). Their functional diversity arises from the structural plasticity of the metal-binding and catalytic domains. For example, matrix metalloprotease-2 (MMP-2) and matrix metalloprotease-9 (MMP-9) exemplify this diversity, utilizing catalytic domains to degrade collagen networks with high efficiency (44), while ADAM17 precisely cleaves transmembrane signaling molecules using its zinc-finger motif (45). Under physiological conditions, metalloproteases maintain homeostasis by regulating extracellular matrix remodeling, growth factor release, and the migration of inflammatory cells. Viruses disrupt this balance through dual mechanisms: (1) inducing aberrant overexpression of metalloproteases in the inflammatory microenvironment, such as HBV infection upregulating MMP-9 to promote liver fibrosis (46), or (2) hijacking proteolytic functions to facilitate dissemination routes, as evidenced by HIV exploiting metalloprotease-mediated degradation of extracellular matrix components to enhance cell-to-cell spread (47).

Cysteine proteases feature a cysteine residue at their active site, functioning through a Cys-His catalytic dyad (48), exemplified by cathepsin L (CTSL) ( Figure 1C ). Their activity is tightly regulated by subcellular localization (e.g., pH, redox status). For instance, cathepsin B (CTSB) and CTSL activate viral fusion proteins in the acidic environment of endosomes (15), whereas caspase-3 executes apoptotic cascades in the cytoplasm (49). These enzymes dominate critical host processes—including lysosomal antigen processing, irreversible initiation of programmed cell death, and dynamic cytoskeletal remodeling—all precisely driven by the redox-sensitive thiol activity of their cysteine residues. Viruses hijack these mechanisms through spatiotemporally precise strategies. In the acidic endosomal microenvironment, activated CTSB/CTSL promotes viral entry [e.g., EBOV (14–17)]; in the cytoplasm, they regulate apoptosis-related proteases such as caspases to inhibit or promote cell death, creating a metabolic environment conducive to viral replication [e.g., adenovirus delays apoptosis by inhibiting caspase-3 activity (27)]. The mechanisms by which these microenvironmental molecular interactions determine infection outcomes will be dissected in subsequent mechanistic investigations.

In addition to the above, the host protease network also includes aspartic proteases and threonine proteases. Aspartic proteases utilize an active center formed by two conserved aspartic acid residues (Asp) (50), which enables the specific recognition and cleavage of viral polyprotein substrates in acidic microenvironments. For example, cathepsin D (CTSD) mediates the conformational rearrangement of HIV gp120, thereby facilitating direct interaction with coreceptors and enhancing the efficiency of viral membrane fusion (51). Threonine proteases employ threonine residues as nucleophilic attack centers to drive proteolytic cascades (52). The threonine hydrolase activity of the proteasome plays dual regulatory roles in the rotavirus life cycle, mediating viral capsid uncoating for genome release during invasion, and optimizing viral particle maturation through degradation of host restriction factors during assembly (53, 54). Through substrate-specific cleavage, these two protease classes cooperatively regulate key nodes of the viral replication cycle alongside previously described proteases.

From a molecular evolutionary perspective, the functional diversity of host proteases reflects a dynamic equilibrium forged through protracted virus-host coevolution. For example, the acquisition of furin cleavage sites (e.g., PRRA insertion in SARS-CoV-2) by coronavirus spike proteins likely reflects the adaptive evolution of viruses to exploit furin, which is highly expressed in the respiratory mucosa. In response, hosts have evolved defense mechanisms, such as the serine protease inhibitor (serpin) family, to continuously counteract the viral hijacking of proteases. This persistent evolutionary pressure drives the structural plasticity of protease substrate-binding domains, enabling specific viral families to utilize distinct protease subtypes to complete their life cycles, while also shaping the complexity of host defense networks (elaborated in Section 3) ( Table 1 ).

The process of viral invasion into host cells constitutes a dynamic interplay between pathogens and the host protease system. Conformational activation of viral surface glycoproteins, driven by host protease-mediated site-specific proteolytic cleavage, constitutes the essential initial step in the infection cascade. This regulatory strategy exhibits remarkable viral specificity and evolutionary adaptability.

Host proteases precisely regulate viral replication and assembly through specific cleavage events, exhibiting multidimensional coordination and dynamic evolutionary characteristics. During the viral replication phase, host proteases play a central role in regulating key processes, such as the activation of viral precursor proteins and the formation of replication complexes ( Figure 3 ). For instance, in RNA viruses, the dengue virus NS3 serine protease requires cooperation with the host serine protease acyl-CoA thioesterase 2 (ACOT2) to cleave polyproteins for functional replication complex formation (128). HEV initiates genome replication through thrombin-mediated cleavage at conserved sites of the ORF1 polyprotein (129). The caspase-6 cleavage motif (DTTD/272) in the non-structural protein NS5A of classical swine fever virus (CSFV) further confirms its regulatory role in viral replication (130). In DNA virus systems, caspase-mediated cleavage events exhibit bidirectional regulation of viral replication. Cleavage of adenovirus 2/12 (Ad2/12) early region 1A (E1A) protein by caspase-3 results in the loss of transcriptional activation function, impairing the transcriptional program necessary for efficient replication (27). In contrast, removal of the nuclear localization sequence from the NS1 protein of Aleutian mink disease parvovirus (AMDV) by caspase-3 promotes the cytoplasmic transport of ribonucleoprotein complexes, facilitating replication-related processes (131). These findings reveal the multi-target regulatory characteristics of host proteases in the viral replication process.

During virus assembly, host proteases primarily exert precise regulation by mediating the modification and maturation of viral structural proteins. The nucleocapsid protein (N) of transmissible gastroenteritis virus (TGEV) and IAV loses its genome-binding capacity after caspase-6/7 cleavage, resulting in a dramatic decrease in infectious virus particle yield (132, 133); the human astrovirus type 8 (HAstV-8) ORF2 polyprotein is specifically cleaved by trypsin to generate functional fragments that participate in capsid assembly and replication, respectively (134). Notably, protease processing strategies are viral species-specific. HIV-1 promotes the exposure of the gp41 fusion domain and the correct assembly of Env proteins through TL2 serine protease-mediated cleavage of the gp120 V3 loop (79). While furin/PC protease processing of the Crimean-Congo hemorrhagic fever (CCHF) glycoprotein precursor produces the GP38 glycoprotein, which may optimize the viral assembly environment through membrane remodeling mechanisms (135, 136). Furin cleavage of the RSV F protein is not essential for its transport but can significantly enhance viral particle assembly efficiency (137), highlighting their precise regulation of viral morphogenesis. In addition, caspase-3 mediates the nucleocytoplasmic transport of the influenza virus ribonucleoprotein complex (RNP), and its inhibition leads to RNP retention in the nucleus and triggers assembly defects. The enzyme cleaves the nuclear lamina protein Lamin A/C via a non-apoptotic pathway, remodels the nuclear membrane structure to facilitate RNP transport to the cytoplasm, and provides key components for viral particle assembly (138), further confirming the multifaceted regulatory mechanism of host proteases in viral morphogenesis.

The host-virus interaction network at the protease level is characterized by dynamic interplay and coevolution. Host factors, such as TRAB domain-containing protein 2A (TRABD2A), can inhibit viral assembly by degrading the Gag protein of HIV-1 (139), while the primate-specific restriction factor FAM111B inhibits the replication of mouse cytomegalovirus (MCMV) in human cells by enriching in viral replication regions—a restriction stemming from the fact that MCMV has not evolved strategies against family with sequence similarity 111 member B (FAM111B) in its natural hosts (rodents) (140). Studies on cross-species transmission reveal that viruses can actively utilize host proteases to break through barriers. The conserved cleavage of HEV pORF1 by thrombin and FXa is a key basis for its ability to cross host boundaries (141). Tick-borne encephalitis virus (TBEV) reorganizes the membrane system by relocating host ADAM15 protease to its replication region, thereby optimizing its own replication environment (142). Such adaptive strategies frequently drive systematic mutations in the cleavage sites of viral proteases.

Host proteases profoundly enhance viral particle release and transmission efficiency through precise regulation of viral maturation and microenvironment remodeling ( Figure 3 ). The core function of furin is evolutionarily conserved across viral families during terminal maturation. For example, furin-mediated cleavage of HIV gp160 enhances viral particle infectivity and induces conformational rearrangements to evade neutralizing antibodies (28, 29); flaviviruses (e.g., DENV and ZIKV) require furin cleavage of prM to M protein for mature particle formation, though incompletely cleaved immature particles retain infectivity via non-canonical entry pathways that exacerbate disease severity (30). While Borna disease virus (BDV) glycoprotein (GP, encoded by ORF-IV) strictly depends on site-specific furin cleavage at Arg249 to maintain bioactivity (143). These collective mechanisms underscore how proteolytic processing fine-tunes viral dissemination strategies across diverse species.

Viral cell-to-cell transmission involves a more extensive proteolytic regulatory network. Although furin cleavage of pseudorabies virus glycoprotein B (gB) is dispensable for in vitro viral replication, it remains critical for mediating membrane fusion and syncytium formation (144, 145). MMP-9 significantly enhances HIV cell-to-cell transmission by degrading extracellular matrix components (47). Additionally, caspase family members play pivotal roles in facilitating viral release. They cleave the human astrovirus capsid precursor VP90 to form mature capsids, thereby promoting viral release (146); while they dissolve cytoplasmic inclusion bodies maintained by Avian reoviruses (ARVs) mμNS protein to expel mature particles (147). Collectively, these diverse mechanisms highlight the intricate deep coevolutionary relationship between viruses and the host protease system.

Host proteases construct multidimensional regulatory networks spanning molecular cleavage to systemic immunity during viral immune responses and evasion strategies ( Table 2 ). Viruses achieve immune evasion by hijacking the protease activity. For instance, influenza viruses employ TMPRSS2 not only to enhance viral membrane fusion efficiency, but also to promote vascular permeability by activating the “influenza virus-cytokine-trypsin” cycle. The upregulated trypsin and pro-inflammatory cytokines exacerbate tissue destruction and immune suppression, enabling the virus to evade immune clearance and continue to replicate (148). Similarly, cathepsin G (CTSG) recruits monocytes/macrophages to inflammatory sites during HIV-1 infection and heightens their viral susceptibility, establishing a positive feedback loop (25). Conversely, the host has evolved protease-based antiviral defenses—neutrophil serine proteases (NE/PR3/CTSG) directly cleave the SARS-CoV-2 spike protein to block viral entry (149), while myeloid-specific serine proteases interfere with NF-κB activation by processing its p65 subunit, thereby inhibiting critical HIV replication processes (24). This bidirectional protease warfare underscores the evolutionary arms race at the host-pathogen interface.

The precise regulation of host protease activity by host factors constitutes a critical mechanism in antiviral defense. Interferon-induced guanylate binding proteins (GBPs, such as GBP2 and GBP5) inhibit furin and PCSK family activity, impeding the maturation of viral envelope glycoprotein precursors, including HIV-1 gp160, and significantly reducing the infectivity of various viruses, including HIV-1, ZIKV, MV, and IAV (150). Alpha-soluble NSF attachment protein (α-SNAP) binds to the P-domain of furin, inhibiting cleavage of the SARS-CoV-2 spike protein and other furin-dependent viral glycoproteins (151). Members of the serine protease inhibitor superfamily (SERPIN) also play essential roles, alpha (1)-antitrypsin (A1AT) inhibits TMPRSS2 and ADAM17, blocking SARS-CoV-2 spike protein activation and ACE2 shedding (152–154), while plasminogen activator inhibitor-1 (PAI-1) regulates fibrinolysis and additionally inhibits the enzymatic activity of proteases including FXIIa and TMPRSS2 (155). Furthermore, cystatin C (CST3) competitively inhibits the activity of CTSB/CTSL through its N-terminal region, while its dimer form enhances CTSB activity by binding to a structure-specific allosteric pocket of CTSB (156). Interleukin-1β (IL-1β) activates ADAM17 through phosphorylation (157). Collectively, these regulatory networks of host factors over protease activities profoundly influence viral infection processes.

The functions of host proteases frequently exhibit tissue- and cell-compartment specificity, and profoundly influence systemic immune responses. In renal cells, SARS-CoV-2 evades the inhibitory effect of the host restriction factor nuclear coactivator 7 (NCOA7) via a TMPRSS2-mediated non-endosomal pathway (158). Conversely, when SARS-CoV-2 infects human bronchial epithelial cells, it induces ST14/TMPRSS11D to activate prothrombin, triggering acute fibrin deposition (159). At the systemic level, SARS-CoV-2 activates the NETs-PAD-4 pathway to induce lung epithelial cell death (22), whereas dengue virus is directly linked to imbalances in the coagulation-fibrinolytic system through a metalloproteinase-mediated vascular leakage mechanism (160). Moreover, the degree of coagulation and fibrinolytic activation induced by it is positively correlated with disease severity (161).

The matrix metalloproteinase (MMP) family exhibits complex and differentiated functions in viral immunomodulation. In HBV infection, MMP-9 promotes viral replication and hepatic fibrosis by inhibiting interferon signaling (46). RSV infection efficiently stimulates MMP-9 expression in vivo and in vitro (162), while disruption of the MMP-9/TIMP-1 balance drives airway remodeling—a key pathogenic feature of chronic pulmonary fibrosis (163). In a neuroinvasive model, mouse adenovirus (MAV-1) activates microglial MMP-2/MMP-9 to disrupt the blood-brain barrier, representing a critical pathological mechanism underlying encephalitis development (164). Studies on MHV infection further revealed that increased viral replication during lethal infection is closely associated with significantly elevated expression levels of MMPs, TIMPs, and chemokine genes (165). Conversely, MMP-3 exerts broad-spectrum antiviral activity against vesicular stomatitis virus (VSV), H1N1, and HSV-1 through NF-κB signaling potentiation via nuclear translocation, while simultaneously enhancing anti-dengue immune responses (23, 166).

Complement system regulation represents another critical battleground for viral immune evasion. Aberrant interactions between the mannose-binding lectin-associated serine protease 2 (MASP-2) and the SARS-CoV-2 N protein drive complement hyperactivation, fueling cytokine storms and multiorgan damage—a mechanism particularly prominent in severe COVID-19 (167). Conversely, the high-temperature requirement protein A2 (HtrA2/Omi) effectively limits cytomegalovirus (CMV) spread by triggering apoptotic pathways through cleavage of key viral or host proteins (26). Long-term host-virus coevolution has forged dynamic equilibria in proteolytic cleavage sites. ADAM17, a pivotal immunoregulatory node, plays essential roles in host defense against pestiviruses (168), while being targeted by cytomegaloviruses to remodel the cell surface proteome (169). Neuroinvasive viruses exploit MMP-8 to degrade tight junctions in the blood-brain barrier (170), whereas RSV infection induces MMP-10 expression in nasal epithelial cells and modulates the immune microenvironment through NF-κB/JAK-STAT crosstalk (171). These mechanisms collectively demonstrate how viruses achieve immune microenvironment remodeling via multidimensional regulation of proteolytic networks.

Currently, research on single-target inhibitors targeting key host proteases continues to deepen. Small-molecule inhibitors remain the primary focus due to their favorable drugability and high maturity in development. In the field of targeting transmembrane serine proteases, the TMPRSS2 inhibitor nafamostat ( Figure 4A ) blocks 93% of SARS-CoV-2 plasma membrane invasion but exhibits limited inhibitory effects on TMPRSS4-dependent MERS-CoV (20, 172). The new-generation inhibitor N-0385, with low nanomolar potency, effectively inhibits the invasion of variants, including Omicron (173, 174). While nafamostat mesylate can reduce viral load in murine lungs (175), its clinical efficacy is limited by rapid cleavage and inactivation by TMPRSS11D (176). Other broad-spectrum serine protease inhibitors, such as 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) and N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), can inhibit RSV infection, among which AEBSF acts primarily at the early stage of viral entry (177). Structurally optimized HAT serine protease inhibitors (e.g., compound 15, K_i = 15 nM) enhance selectivity through novaricine modification, thereby effectively inhibiting influenza virus replication (178). Additionally, basic phenylalanine analogs reduce the titers of West Nile virus (WNV) and DENV by 10,000-fold via plasmin inhibition (179).

Classical cysteine protease inhibitors targeting CTSL demonstrate broad-spectrum antiviral efficacy. In vitro models, inhibitors E64d ( Figure 4B ) and Z-FY-CHO effectively suppress SARS-CoV-2 pseudovirus infection (20, 83, 180, 181), while the broad-spectrum inhibitor K11777 exhibits potent inhibitory activity against HEV, with an EC₅₀ of approximately 0.02 nM (127). In vivo, rotavirus capsid disassembly strictly depends on CTSL, and treatment with the inhibitor Z-Phe-Tyr(t-Bu)-diazomethyl ketone causes drastic viral yield reduction (121). Additionally, amantadine, an anti-influenza drug, blocks SARS-CoV-2 infection by inhibiting CTSL activity (87), while the natural product gallinamide A and its analogs also exhibit potent antiviral activity (182).

Furin inhibitor development has achieved significant advances across multiple fronts. The competitive inhibitor dec-RVKR-cmk can block F protein cleavage and viral budding of RSV (137), while exerting anti-SARS-CoV-2 activity by inhibiting spike protein cleavage and syncytium formation (183), though it is ineffective against filoviruses (184). This compound additionally suppresses flavivirus release without affecting RNA replication (185). In terms of allosteric inhibitors, cypermethrin exhibits broad-spectrum activity against drug-resistant SARS-CoV-2 by binding to a novel allosteric pocket (186), while the reversible inhibitor MI-1851 reduces viral load by 190-fold (83, 187). It is worth noting that protease inhibitors designed based on the (3,5-dichlorophenyl) pyridine skeleton ( Figure 4C ) exhibit high potency and antiviral activity against SARS-CoV-2 at the cellular level (188), providing new insights for broad-spectrum antiviral treatment. The natural product luteolin inhibits furin in a non-competitive manner and significantly reduces DENV viremia in vivo (189). Emerging approaches explore novel paradigms. Targeting the off-state of furin opens new avenues for the design of selective inhibitors (190). Decanoyl-RVKR-CMK effectively blocks E3E2 precursor cleavage by CHIKV (30). And furin conformation provides new opportunities for structure-based drug discovery (186), demonstrating the feasibility of developing customized inhibitors for specific viruses.

In addition to small-molecule inhibitors, peptide-based compounds, endogenous regulatory factors, and biologics serve as vital complements to single-target inhibition strategies due to their targeting specificity and biocompatibility advantages. In peptide inhibitor research, polyarginine repeat sequences function as competitive inhibitors of furin substrate cleavage, effectively inhibiting HIV infection by blockade of gp160 protein processing (191). Peptides P9 and P9R significantly reduce SARS-CoV-2 viral load in hamster models by impairing CTSL activity (192, 193). The TMPRSS13 peptidomimetic inhibitor N-0430 blocks SARS-CoV-2 pseudovirus entry (194), while the caspase-6 inhibitor Z-VEID-FMK alleviates pathological damage in SARS-CoV-2 and MERS-CoV animal models (195). Recombinant applications of endogenous inhibitors demonstrate substantial progress. Interferon-induced GBP5 protein inhibits furin activity, markedly reducing infectivity of multiple pathogens, including HIV-1, Zika virus (ZIKV), MV, and IAV (150). Serpin family B member 8 (Serpin B8, also known as PI8 and CAP2) binds to and inhibits the proprotein convertase furin (28). Furthermore, α-SNAP suppresses furin-dependent viral glycoprotein cleavage through binding to the furin P-domain (149). In the field of biologics, nanobodies exhibit exceptional advantages due to their high specificity. The anti-TMPRSS2 nanobodies inhibit the enzymatic activity of TMPRSS2 and hinder HKU1 pseudovirus entry using S441A TMPRSS2 (101). Dromedary heavy-chain-derived nanobodies specifically inhibit the catalytic activity of furin, blocking its cleavage of two critical substrates, transforming growth factor beta (TGFβ) and glypican-3 (GPC3) (196).

Multi-target synergistic strategies are overcoming the limitations of single-inhibitor therapies. Clinical studies have shown that the TMPRSS2 inhibitor N-0385, when combined with the antiviral drugs remdesivir or nirmatrelvir, exhibits broad-spectrum synergistic activity against Omicron subvariants (174). In chronic hepatitis B treatment, entecavir coupled with furin inhibitors concurrently suppresses viral replication and hepatitis B e antigen (HBeAg) secretion (197). Spironolactone enhances antiviral effects by antagonizing TMPRSS2/ADAM17 to reduce soluble ACE2, synergizing with DPP-4 inhibitors to improve clinical outcomes in COVID-19 patients (198). Additionally, xanthan gum combined with camostat significantly enhances anti-influenza virus potency (199). Mechanistic studies further confirmed that non-toxic furin inhibitors combined with TMPRSS2 inhibitors block 95% of lung cell infections (200), demonstrating the translational potential of inhibitor synergy strategies in multistep blockade of viral invasion.

Dual- and multi-target therapeutics are spearheading novel antiviral strategies. Compound BAPA exhibits an EC₅₀ of 0.3 μM against H1N1 by inhibiting HAT/TMPRSS2 (201). The tri-targeting peptidomimetic MM3122 simultaneously inhibits TMPRSS2, matriptase, and hepsin, maintains sub-nanomolar potency against the SARS-CoV-2 EG.5.1 variant, and significantly attenuates pulmonary edema in mice (202). Delivery system innovations propel the development of bispecific compound 212-148, which simultaneously inhibits TMPRSS2 and CTSL/CTSB (203), with nanoerythrocyte carriers substantially enhancing delivery efficiency (204). Diazoxide inhibits TMPRSS2/furin (IC₅₀=1.35/13.2 μM), while compound MI-1148 blocks transmission of highly pathogenic avian influenza (HPAI) and canine distemper virus by targeting PC1/3 (205). Notably, the mechanisms of action of protease inhibitors nafamostat and camostat may extend beyond TMPRSS2 inhibition itself, involving coagulation cascade-induced cleavage of spike proteins. Given the centrality of anticoagulation management in COVID-19 therapy, early intervention may provide synergistic benefits by blocking viral entry (89).

Structure-guided design has achieved pivotal breakthroughs. The α-ketoamide inhibitors 14a/14b exhibit potent broad-spectrum anti-coronaviral activity through covalent binding to CTSL and calpain-1 (CAPN1), achieving exceptional potency against SARS-CoV-2 variants (EC₅₀ as low as 0.80 nM) (206). The natural product omicsynin B4 demonstrates pan-coronaviral activity against human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), and SARS-CoV-2 prototype/variants by dual blockade of CTSL/TMPRSS2 (207). At the level of respiratory protease regulation, influenza HA activation mediated by human eosinophils and DESC1 (but not TMPRSS11A) is specifically inhibited by hepatocyte growth factor activator inhibitor 1 (HAI-1) (66). The endogenous regulator serine protease inhibitor Kazal‐type 6 (SPINK6) inhibits HAT/KLK5 to restrict influenza virion maturation (208), while dichlorobiphenyl-containing matriptase inhibitors achieve ultrahigh potency (K_i < 3 nM) through chemical optimization, demonstrating exceptional thrombin selectivity and concentration-dependent inhibition of H9N2 viral replication in MDCK(II) cells (209). Among matriptase/TMPRSS2 inhibitors evaluated by Gamba, D. et al., MI-463 and MI-1900 exhibit antiviral effects against H1N1/H9N2 at concentrations of 20-50 µM, suggesting that they block viral entry by inhibiting host protease-mediated cleavage (210). The oral dual-target drug olgotrelvir, which simultaneously inhibits SARS-CoV-2 M^pro and CTSL, has emerged as a new paradigm for clinical translation (211).

Despite extensive development of host protease inhibitors demonstrating antiviral potential in preclinical models, few have successfully transitioned to clinical application. The current translational bottlenecks primarily stem from three key challenges. First, host compensatory escape—viruses can not only bypass inhibition by activating functionally redundant host proteases, such as SARS-CoV-2 switching from TMPRSS2-dependent entry to CTSL-mediated invasion pathways (85–87); they can also utilize their own encoded proteases to compensate for critical functions. For instance, HCV relies solely on its NS3/4A serine protease with NS4A as a cofactor to independently cleave viral polyproteins (212). Similarly, DENV requires its NS3 protease—an essential component for nonstructural protein hydrolysis—which functions with its own NS2B cofactor (213). Meanwhile, SARS-CoV-2 processes polyproteins pp1a and pp1ab through its M^pro to generate 16 mature nonstructural proteins (nsp1-nsp16), which collectively form the replication/transcription complex that provides core support for viral replication (214). Second, off-target toxicity—broad-spectrum inhibitors (e.g., camostat) inhibit TMPRSS2 while interfering with proteases involved in coagulation, inflammation, and blood pressure regulation, significantly increasing the risk of serious adverse events in clinical treatment groups (215); third, tissue delivery obstacles—small-molecule inhibitors struggle to penetrate specific compartments (e.g., inactivation in the acidic lysosomal environment, blockage by the blood-brain barrier).

It is noteworthy that multi-target synergistic strategies aimed at enhancing antiviral efficacy, such as dual-target proteolysis-targeting chimera (PROTAC) degraders, may increase off-target risks due to the expanded range of target molecules. Current research seeks breakthroughs through two precision-optimized design approaches. One leverages spatial precision by confining activity release ranges using tissue-microenvironment-responsive carriers (216), while another employs conformational precision through allosteric site engineering to selectively engage inactive states of host proteases, thereby enhancing specificity (217). These approaches pave a critical pathway for balancing synergistic potency and safety. Thus, developing novel inhibitors with high selectivity, resistance barriers, and microenvironmental adaptability has become an urgent need to address the challenges of viral evolution. Cutting-edge strategies focus on three breakthroughs. One is the exploration of allosteric inhibitory sites (e.g., targeting furin exosite-III). Another is the design of dual-target PROTAC degraders (e.g., simultaneous degradation of TMPRSS2/CTSB). The third is the development of smart responsive nanocarriers (e.g., pH-sensitive liposomes loaded with cystatin C targeting endosomes).