"Flucovid" refers to a disease caused by the coinfection with influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presenting with both influenza and corona virus disease 2019 (COVID-19) symptoms including fever, cough, fatigue, headache, dyspnea, and loss of taste or smell, but excluding common cold symptoms such as sneezing, rhinorrhea/nasal congestion, and sore throat [
1]. A recent meta-analysis employing a random-effects model within 95% confidence intervals revealed that the prevalence of influenza coinfection among COVID-19 patients was 14% (95% confidence interval (CI): 8%–20%) [
2]. Notably, pediatric and geriatric populations demonstrate higher incidence rates of coinfection [
3]. Both SARS-CoV-2 and influenza viruses can directly attack pulmonary tissues, potentially inducing viral pneumonia. Coinfection may exacerbate pulmonary inflammation and injury, thereby increasing risks of respiratory failure and acute respiratory distress syndrome (ARDS). Simultaneous immune response to dual viral pathogens might compromise host defenses, elevating susceptibility to secondary bacterial infections (
e.g., Streptococcus pneumoniae). Coinfection with influenza A virus enhances SARS-CoV-2 infectivity [
4-
7]. Current therapeutic strategies for coinfection recommend combination therapy incorporating anti-influenza agents (
e.g., oseltamivir) and anti-COVID-19 medications (
e.g., paxlovid) [
1]. To date, no therapeutic agents specifically targeting the coinfection have received regulatory approval. More seriously, the overlapping epidemic seasons of the influenza virus and SARS-CoV-2 create an urgent need for novel therapeutic strategies to treat SARS-CoV-2 and influenza virus coinfection.
Pyrimidines, essential components of intracellular bioactive molecules, participate in the biosynthesis of DNA, RNA, polysaccharides, and phospholipids, playing a pivotal role in human metabolism [
7]. Cellular pyrimidine nucleotide synthesis occurs
via two distinct pathways: the salvage pathway and
de novo synthesis. In quiescent or fully differentiated cells,
de novo synthesis exhibits low activity, with pyrimidine demands predominantly met by the salvage pathway. However, proliferative cells (
e.g., tumor cells) and viruses necessitate
de novo pyrimidine biosynthesis to sustain their heightened requirements for nucleic acid precursors and cellular components [
8-
11]. Human dihydroorotate dehydrogenase (DHODH) is the rate-limiting enzyme in the fourth step of
de novo pyrimidine synthesis, catalyzes the oxidation of dihydroorotate to orotate. In virus-infected cells, rapid viral replication imposes an extraordinary demand for nucleosides to sustain high metabolic turnover, rendering
de novo biosynthesis more critical than the salvage pathway for viral propagation. As the key enzyme in this pathway, DHODH has emerged as a compelling broad-spectrum host antiviral target, with its inhibitors displaying antiviral potential [
12-
19]. Although these inhibitors exhibit substantial antiviral activity, but still face limitations.
In our previous studies [
20], we found that the DHODH inhibitor BAY2402234 exhibits anti-influenza virus and SARS-CoV-2 activity with considerably low half maximal effective concentration (EC
50) values. But, the 90% maximal effective concentration (EC
90) and 95% maximal effective concentration (EC
95) values exhibited a 10-fold or more increase compared to the EC
50. This indicates that while DHODH inhibitor possesses inhibitory activity against viruses, it fails to achieve complete viral clearance, thereby compromising its clinical efficacy. Furthermore, the inhibitors block the catalytic function of DHODH by binding to its active site, but the protein itself still exists, which may retain the non-enzymatic functions or restore activity through other pathways. In addition, long-term use of inhibitors may lead to target mutation or the activation of compensatory pathways, which may induce drug resistance [
21,
22]. Hence, a novel strategy targeting DHODH is required to remedy the limitations of inhibitors and thereby achieve complete viral eradication.
Proteolysis-targeting chimeras (PROTACs) have emerged as a robust strategy for selectively degrading disease-associated proteins, garnering significant momentum in drug discovery [
23-
28]. PROTAC degraders comprise two interconnected recruitment modules: one binds to the protein of interest (POI), while the other engages an E3 ubiquitin ligase. These bifunctional compounds facilitate E3 ligase recruitment, enabling POI ubiquitination and subsequent degradation
via the ubiquitin-proteasome system (UPS) (
Fig. 1A). A hallmark of PROTACs lies in their catalytic capability to eliminate entire POI, thereby potently inhibiting both catalytic and non-enzymatic functions [
29-
32]. BAY2402234 is a novel and potent DHODH inhibitor with antiviral activity [
13,
33,
34]. Preclinical studies revealed BAY2402234′s superior efficacy and pharmacokinetic profile compared to other DHODH inhibitors, prompting its selection as the DHODH ligand in this study. We systematically conjugated BAY2402234 to cereblon (CRBN) E3 ligase ligands using diverse linker systems to construct DHODH PROTACs, enabling structure-degradation relationship analysis (
Fig. 1B). The synthesis of PROTACs
1–
23 followed the routes outlined in
Scheme 1. Boc-protected linkers underwent nucleophilic substitution with pomalidomide derivatives to yield intermediate conjugates. Then, Boc-deprotection and coupling with BAY2402234 (synthesized in-house) afforded the final compounds
1–
23.
Studies have demonstrated that the introduction of conformationally rigid linkers in PROTAC degraders can improve the degradation efficacy, selectivity, and pharmacokinetic properties [
35,
36]. Therefore, we first employed linkers with varying rigidities, including monocyclic, spirocyclic, and bicyclic structures possessing comparable spatial spans, to conjugate BAY2402234 to the 5′-position of pomalidomide, thereby designed and synthesized a series of DHODH PROTAC degraders. Subsequently, we use the HEK293T cell line to evaluate the degradation efficiency of these PROTACs. Following 24 h treatment with 1 μmol/L PROTACs in HEK293T cells, the DHODH protein level was determined
via Western blot (WB) analysis (
Figs. 2A and B), the degradation efficiency was summarized in Table S1 (Supporting information). The results revealed that this compound series (
1–
13) exhibited limited DHODH degradation activity. This diminished efficacy may be attributed to the rigid linker architecture, which potentially reduces molecular flexibility and restricts critical spatial parameters (
e.g., distance and orientation between interacting moieties), thereby impeding optimal ternary complex formation. Furthermore, such structural rigidity might compromise cellular permeability of the molecules.
The flexibility of the linkers enables PROTAC molecules to achieve greater spatial adjustability when simultaneously binding to both the target protein and E3 ligase, thereby optimizing molecular activity and reactivity. This characteristic facilitates enhanced ubiquitination efficiency and degradation efficacy of the target protein [
36,
37].
8 and
9 in previous series share an identical linker but differ in their attachment site to the CRBN ligand.
8 attached at the 4′-position of pomalidomide, exhibits superior degradation activity compared to
9 attached at the 5′-position. This suggests that linkage at the 4′-position of the CRBN ligand may be more favorable for ternary complex formation. Furthermore, literature reports [
38] indicate that derivatives linked at the 4′-position of pomalidomide demonstrate greater structural stability and enhanced degradation potency compared to those linked at the 5′-position. Subsequently, we selected flexible alkyl chains and PEG chains with different lengths, and switched the connection point from the 5′-position of pomalidomide to the 4′-position to further prepare PROTACs
14–
23. These PROTACs demonstrated superior DHODH degradation activities compared to previous series (
Figs. 2A and B). The degradation efficiency was summarized in Table S2 (Supporting information). These results indicate that PROTACs have flexible linkers of appropriate length, and the flexible molecular conformation may perform better in recruiting DHODH and CRBN to form a ternary complex, thus making the better degradation activity.
In the preliminary anti-SARS-CoV-2 and degradation efficiency screening, all of these PROTACs demonstrated anti-SARS-CoV-2 activities at the same level (Table S3 in Supporting information). Among them,
17 and
19 consistently maintained stable and significant degradation activities and were identified as the most effective molecules (Figs. S1–S3 in Supporting information). The results showed that
17 and
19 effectively degraded DHODH at 0.3 and 1.1 μmol/L, respectively (
Fig. 3A) and both showed "hook effect". Similarly,
17 and
19 could also effectively degrade the DHODH in MDCK cells (Fig. S4 in Supporting information). We further investigated the mechanism of
17 and
19 induced DHODH degradation. Pretreatment with the proteasome inhibitor MG132 and CRBN ligand pomalidomide rescued DHODH degradation mediated by
17 and
19, demonstrated that
17 and
19 induced DHODH degradation through the proteasome and CRBN-dependent mechanisms (
Fig. 3B and Fig. S5 in Supporting information).
Since PROTACs
17 and
19 exhibit promising degradation activities, we further validated their broad-spectrum antiviral activity against influenza A and B viruses in the MDCK cell line. The results showed that
17 and
19 exhibited higher EC
50 values against B/Russia/69 (B/Russia), A/Hong Kong/8/68 (HK68) and A/California/07/2009 (CA07) strain than BAY2402234 (
Fig. 4A), but their 70% maximal effective concentration (EC
70), EC
90 and EC
95 values were very close to the EC
50 level. Notably,
17 and
19 demonstrated lower EC
90 and EC
95 values compared to BAY2402234, demonstrating superior viral clearance capacity against influenza virus than BAY2402234 (
Table 1). Meanwhile, the PROTACs
17 and
19 did not exhibit cytotoxicity at the concentration of 50 μmol/L (half maximal cytotoxic concentration (CC
50) > 50 μmol/L) in MDCK cells. Furthermore, we evaluated their anti-SARS-CoV-2 efficacy through indirect immunofluorescence assays and real-time quantitative PCR experiments. The results demonstrated that in SARS-CoV-2/BJ01 infected 293T-ACE2 cells,
17 and
19 exhibited anti-SARS-CoV-2 activities comparable to BAY2402234 at the concentration of 1 μmol/L, significantly reducing viral RNA copies and inhibiting viral protein expression (
Figs. 4B–D). Collectively, these findings suggest the potential of
17 and
19 for treating influenza virus and SARS-CoV-2 infection.
It is common knowledge that PROTAC molecules belong to "beyond Rule of 5″ (bRo5) chemical space. Their large and flexible structure is responsible for drug metabolism and pharmacokinetics limitations [
39]. We further evaluated the
in vivo pharmacokinetics (PK) properties of
17 and
19 through single-dose intravenous administration (1 mg/kg, i.v.) in Sprague Dawley rats. Animal research has been approved by the Animal Care Committee of the Beijing Institute of Biotechnology. All operations were following the Animal Care and Use Committee Guidelines of China. As shown in
Table 2, both
17 and
19 showed a moderate
t1/2 (3.08 and 3.20 h, respectively). However, the clearance rate indicates that the metabolic profiles of
17 and
19 in rats are less favorable. Potentially, the complex spatial structure of PROTACs precisely provides the binding sites required by the enzymatic active center, which facilitates their binding and catalyzes the metabolic process [
39].
In summary, we reported the discovery of novel PROTAC degraders with broad-spectrum anti-influenza virus and anti-SARS-CoV-2 activities by degrading DHODH. The structure-degradation relationship analysis revealed that PROTACs connected by flexible linkers with appropriate lengths demonstrated enhanced degradation efficacy compared to those with rigid linkers, due to their superior conformational adaptability. Mechanistic studies revealed that 17 and 19 selectively induced DHODH degradation in a proteasome- and CRBN-dependent manner. Significantly, 17 and 19 exhibit enhanced capacity for complete viral eradication against influenza virus compared to BAY2402234 and comparable anti-SARS-CoV-2 activities to BAY2402234, demonstrating therapeutic potential for influenza virus and SARS-CoV-2 infection. Moreover, 17 and 19 achieved moderate t1/2 in rats. Collectively, PROTACs 17 and 19 serve as viable initial lead compounds for further optimization, offering a new strategy for treating SARS-CoV-2 and influenza virus coinfection.
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