Microbiota-derived succinic acid boosts antiviral activity of AKT targeting in coronaviruses

Coronavirus infection in humans may cause not only pulmonary infection but also secondary intestinal infection. The AKT inhibitors have a significant inhibitory effect on various coronavirus infections, and they can effectively alleviate the intestinal barrier damage induced by the SARS-CoV-2. However, it is unknown how the AKT inhibitors exert their anti-viral effects through the intestinal tract. HCoV-OC43-infected mice were treated with MK-2206 or vehicle control via oral gavage. The body weight, viral load in the lungs, and pathological changes in the lung-intestine tissues were measured. Fecal samples were collected for 16S rRNA gene sequencing and non-targeted GC/LC-MS/MS metabolic profiling to determine the characteristics of the intestinal microbiota and metabolic profile. MK-2206 treatment significantly reduced the viral load in the lungs of infected mice and the damage to the lung-intestinal tissues. The analysis of the intestinal microbiota showed that MK-2206 treatment restored the levels of the Firmicutes and Actinobacteria phyla, increased the abundance of probiotic bacteria such as Lactobacillus, and decreased the abundance of Acinetobacter and Desulfobacter. Metabolomics analysis revealed an increase in the abundance of succinic acid, and the combination of succinic acid and MK-2206 exhibited a stronger antiviral effect. By integrating multi-omics methods, we discovered that succinic acid can enhance the antiviral efficacy of MK-2206, and clarified the related interactions between the intestinal microbiota and metabolites, revealing the crucial role of the intestinal microenvironment in the host’s response to MK-2206 treatment for coronavirus infection.

To date, a total of seven types of coronaviruses (1) that can infect humans have been identified. Among them, SARS-CoV, MERS-CoV and SARS-CoV-2 pose a significant threat to global health due to the potential for causing severe complications and high mortality rates. The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a persistent public health issue (1). HCoV-229E, HCoV-NL63 and HCoV-HKU1 tend to cause mild, self-limiting respiratory infections, and are collectively referred to as common cold coronaviruses. (2). Although HCoV-OC43 mainly causes mild symptoms, it can lead to severe complications in infants, the elderly, and individuals with weakened immune systems, such as pneumonia, bronchopneumonia, and even encephalitis. (3). Currently, there are no approved antiviral therapies or vaccines for HCoV-OC43. It is worth noting that, unlike SARS-CoV-2 which requires BSL-3 protection, HCoV-OC43 can be handled under standard laboratory conditions, making it a valuable model for studying the pathogenesis of coronaviruses and screening potential antiviral drugs. (4).

The coronavirus infection activates the PI3K/AKT signaling pathway in host cells to create a cellular environment conducive to virus replication. As a result, there is research attention in antiviral approaches against key host signals pathways which can be used in broad spectrum applications. The AKT signaling pathway has been shown to be generally antiviral against HCoV-229E, SARS-CoV-2, and SADS-CoV (5–7). MK-2206 is a strong AKT inhibitor and it has significant anti-viral activities. Studies have shown that it inhibits viral replication through the mTOR signaling pathway (8, 9), and suppresses SARS-CoV-2 infection in the intestine by regulating the SNAP29-dependent autophagic flux, while also helping to maintain the integrity of the intestinal barrier (10).

The human microbiome is a key determinant of health and disease mechanisms. (11); More and more evidence indicates that the gut microbiota plays a crucial role in regulating the host’s immune response and susceptibility to respiratory virus infections. (12). A healthy gut microbiome promotes host defense through multiple mechanisms, including strengthening the mucosal barrier, producing antimicrobial peptides, and competitively eliminating pathogens. (13). Pathogenically, Enterobacteriaceae enrichment concomitant with depletion of commensal Lactococcus and Lactobacillus may trigger pro-inflammatory cytokine cascades (14). SARS-CoV-2 infection alters patient gut microbiota, while commensal bacteria (e.g., Prevotella, Bacteroides) and microbial metabolites (especially short-chain fatty acids, SCFAs) critically modulate disease outcomes (15, 16). SCFAs bind to lung immune receptors and enhance mucosal antiviral defense through “metabolic reprogramming”. (17). This intestinal-lung axis regulation is not limited to coronaviruses: sub-lethal influenza (H3N2/H1N1) infections induce transient dysbiosis and result in impaired production of SCFAs (18); Lactobacillus plantarum supplementation augments pulmonary type I IFN responses and reduces viral load (19); Severe RSV infection correlates with increased Clostridium, Lactobacillaceae, and Actinobacteria abundance (20). In the case of COVID-19, it is particularly crucial that the metabolic processes of amino acids, carbohydrates, and neurotransmitters mediated by the gut microbiota directly regulate the dynamics of the cytokine storm (21). To date, there have been no public reports on the impact of HCoV-OC43 infection on the gut microbiota.

Although MK-2206 can effectively alleviate intestinal barrier damage induced by coronavirus infection, its impact on the intestinal has not yet been explored. This study systematically evaluated the changes in the intestinal microbial community and fecal metabolome in mice infected with HCoV-OC43 after treatment with MK-2206. Through correlation analysis, we further explored the interactions between the differentially abundant microbial groups and the dysregulated metabolites to elucidate the potential mechanism of MK-2206’s action. Our data indicate that the combination of succinic acid significantly enhanced the antiviral efficacy of MK-2206.

Mouse modeling, sample collection, and processing

BALB/c juvenile mice completed a 7-day acclimatization period under controlled environmental conditions to minimize physiological stress and ensure experimental reproducibility. Animals were stratified into three groups: blank controls (n = 8), HCoV-OC43-infected model controls (n = 12), and infected and MK-2206-treated mice (n = 12). Mice in the infected groups were inoculated intranasally with HCoV-OC43 at a dose of 1 × 10 6 PFU, and MK-2206-treated mice received daily oral gavage of MK-2206 (120 mg/kg; Cayman Chemical, 11593) post-infection. Daily records of body weight and food intake were maintained. At predetermined endpoints (days 2, 3, and 4 post-infection), mice were humanely euthanized while under isoflurane anesthesia. Lung tissues colonic tissues and fecal samples were aseptically collected, with body weights documented prior to tissue harvesting. Fresh fecal specimens were transferred to sterile cryovials, Samples were rapidly frozen using liquid nitrogen and subsequently stored at -80 °C until further analysis. All experimental procedures were in strict compliance with the ARRIVE guidelines and received approval from the Institutional Animal Care and Use Committee (IACUC) [Jinan Microecological Biomedicine Shandong Laboratory] (Protocol #2025060).

Quantitative real-time PCR analysis

Lung tissue was homogenized in TRIzol™ reagent (Thermo Fisher Scientific, Waltham, United States) using a mechanical disruptor. Total RNA was extracted, and RNA was reverse transcribed to cDNA using HiScript IV RT SuperMix (Vazyme Nanjing, China) (cycling: 37 °C 15 min, 85 °C 5 s). The quantification of HCoV-OC43 viral load was performed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) with SYBR Select Master Mix (Thermo Fisher Scientific, Waltham, United States). Primers targeted the viral N gene forward: 5′-GCTCAGGAAGG TCTGCTCC-3′, reverse: 5′-TCCTGCACTAGAGG CTCTGC-3′) and the endogenous reference gene β-actin (forward: 5’-CCAGCCTTCCTTCTTGGGTAT-3’, reverse: 5’-GGGTGTAAAACGCAGCTCAG-3’). All reactions were conducted in triplicate to ensure reproducibility.

Histopathological analysis

The collected lung and colonic tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated through a graded series of ethanol (70%, 80%, 95%, and 100%), and embedded in paraffin. Serial sections of 4–5 μm thickness were prepared using a rotary microtome and stained with hematoxylin and eosin (H&E). Two investigators blinded to the group allocation independently examined and imaged the sections under a Leica DMR 3000 light microscope.

Cell culture and processing

The human colon adenocarcinoma cell line Caco-2 (ATCC ® HTB-37™) was cultured in Dulbecco’s Modified Eagle Medium (Gibco, Grand Island, USA) supplemented with 20% heat-inactivated fetal bovine serum (Gibco, Grand Island, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, USA). Cells were maintained at 37 °C in a humidified 5% CO 2 /95% air atmosphere with medium changes every 48 h. After infecting cells with HCoV-OC43 (MOI = 1) for 2 hours, the viral supernatant was removed followed by PBS washing. Treatments were then applied for 24 hours: MK-2206 (5 μM), succinic acid (1 mM) (MedChemExpress, Monmouth Junction, USA), or their combination. Viral RNA copies were quantified by qRT-PCR using extracted total RNA. Viral protein expression in the different treatment groups was analyzed by immunofluorescence.

Fecal microbiota analysis

Genomic DNA was extracted from fecal samples using the Omega Mag-Bind Soil DNA Kit. DNA concentration and purity were measured with a NanoDrop 2000 spectrophotometer. The V3-V4 regions of the bacterial 16S rRNA gene were PCR-amplified using primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) under the following cycling conditions: 95 °C for 30 s; 40 cycles of 95 °C for 10 s, 55 °C for 30 s, 72 °C for30 s; final extension 72 °C for 10 min. PCR products were verified on 2% agarose gels. Verified amplicons were used to construct DNA libraries with the TruSeq ® DNA PCR-Free Sample Preparation Kit. Library concentration (Thermo Fisher Scientific, Waltham, USA) and size distribution (Agilent Technologies, Santa Clara, USA) were assessed; successful libraries underwent paired-end sequencing on an Illumina NovaSeq 6000 platform.

Fecal metabolomics analysis

Fecal samples were homogenized in cold methanol, incubated on ice for 5 min, and centrifuged. The supernatant was diluted with water to 53% methanol. An equal volume of supernatant from each sample was pooled to generate quality control (QC) samples. Untargeted metabolomic profiling utilized UHPLC-HRMS (Vanquish system coupled to Orbitrap Q Exactive™ HF-X mass spectrometer; Thermo Fisher Scientific). Separation occurred on a Hypersil Gold C18 column (100 × 2.1 mm, 1.9 µm; 40 °C) at 0.2 mL/min. Mobile phases were: positive mode - A: 0.1% formic acid (aqueous); B: methanol; negative mode - A: 5 mM ammonium acetate (pH 9), B: methanol. A 17-min linear gradient was applied for elution.

Statistical analysis

Paired-end reads were merged, quality-filtered, and denoised to generate Amplicon Sequence Variants (ASVs) for taxonomic profiling. Differentially abundant microbial taxa were identified using LEfSe (Kruskal-Wallis P < 0.05, pairwise Wilcoxon; LDA score > 3.0). Untargeted metabolomic data were preprocessed (peak alignment, batch normalization, missing value imputation). Significant metabolites were selected based on OPLS-DA VIP scores > 1.0 and univariate significance (P < 0.05), visualized via volcano plots and heatmaps, and functionally interpreted using KEGG pathway enrichment analysis. All analyses used R (4.4.3).

MK-2206 demonstrates antiviral efficacy on infected mice

To evaluate the therapeutic potential of MK-2206 against HCoV-OC43 infection, we monitored body weight changes in infected mice and infected/MK-2206-treated mice throughout the experimental period. Subsequent analyses focused on viral load quantification and histopathological evaluation in lung tissues collected at 2 and 4 days post-infection (dpi). MK-2206 administration demonstrated significant protective effects (Figure 1A). Infected mice exhibited progressive weight loss (>10% by 4 dpi), whereas treated animals showed only transient reduction followed by recovery (4.4% vs. 12.5% in controls).Treatment with MK-2206 significantly reduced pulmonary viral loads. At 2 days postinfection (dpi), viral titers in treated mice were reduced by 1.0 copies/mg relative to infected controls. By 4 dpi, viral titers were 3.36 copies/mg in the treated group versus 4.59 copies/mg in the infected controls (P < 0.0001; Figure 1B). We next examined the pathological changes in tissue samples from different groups of mice (Figure 1C). MK-2206 treatment protected the lungs and intestines of mice from damage induced by HCoV-OC43 infection. Lungs infected with HCoV-OC43 exhibit marked histopathological changes characterized by extensive consolidation, with observed alveolar space obstruction and significant thickening of alveolar septa. Significant improvement in lung tissue was observed following MK-2206 treatment. Most alveoli demonstrated patent lumens and thin alveolar septa. Some areas showed minimal inflammatory cell infiltration and residual consolidation (Figure 1D). In the intestinal tissues, the control group showed significant ulcerative necrotic foci in the mucosal layer, accompanied by marked degeneration and necrosis of the epithelial architecture, loss of structural integrity, disorganization of the lamina propria, disappearance of crypt structures, evident fibroblast proliferation. In the treatment group, the extent of ulcerative necrosis and fibroblast proliferation was markedly reduced (Figure 1E). Histopathological analysis demonstrated that MK-2206 treatment significantly ameliorated the pathological changes induced by HCoV-OC43 infection. (Figures 1F, G). MK-2206 treatment significantly mitigated weight loss, reduced pulmonary viral load, and ameliorated histopathological damage in both lung and intestinal tissues of HCoV-OC43-infected mice.

Impact of MK-2206 treatment on gut microbiota composition

To further evaluate the impact of MK-2206 treatment on the changes in the intestinal microbial composition following viral infection, we conducted a 16S rRNA gene sequencing analysis. The flatness of the dilution curve indicates that the sequencing data can reflect the species abundance in the sample (Supplementary Figure 1A). Alpha-diversity analysis revealed that the loss of the diversity of the microbes due to the effect of the virus was reversed to the level as in the control group by treatment with MK-2206 (Figure 2A). Principal coordinate analysis revealed that there were significant differences in beta-diversity in the three groups (R = 0.6942, P = 0.001). After treatment, some of the sample communities returned to nearly the blank control group, but some remained similar to the infected group, indicating that there is significant individual heterogeneity in the response to drug treatment. (Figure 2B).

The diversity of the main microbial taxa showed that Firmicutes, Bacteroidota, or even Verrucomicrobiota had the highest abundance in all groups (Supplementary Figure 1B). It is important to note, though, that Bacteroidota abundance did not differ significantly between the inter-group groups, whereas Verrucomicrobiota was significantly enriched in infected mice (Figure 2C). At the phylum level, the treatment of MK-2206 resulted in a considerable change in the composition of gut microbiota. In particular, the relative amounts of Lactobacillus and Bifidobacterium were significantly improved. Many investigations have proved that these genera have the ability to relieve pneumonia caused by viruses through the gut-lung axis (22). At the same time, the prevalence of Lachnospiraceae NK4A136_ group a bacterial group linked with the production of short-chain fatty acid (SCFA) was increased. The major product of this group, butyrate, is important in the implementation of anti-inflammatory activities, as well as in the maintenance of intestinal barrier integrity (23). In addition, Desulfovibrio which is a pro-inflammatory genus was decreased after MK-2206 treatment. This sulfate-reducing bacterium produces hydrogen sulfide by reduction of sulfate and when excessive, can damage intestinal epithelial lining and facilitate inflammation to occur (24). The subsequent change, decreased Desulfovibrio, could therefore be allied with an improvement of the intestinal inflammatory associated milieu (Figures 2D, E) These compositional variations suggest that MKgenera have the ability to relieve pneumonia caused by viruses through the gut-lung axis (22). At the same time, the prevalence of Lachnospiraceae NK4A136_ group a bacterial group linked with the production of short-chain fatty acid (SCFA) was increased. The major product of this group, butyrate, is important in the implementation of anti-inflammatory activities, as well as in the maintenance of intestinal barrier integrity (23). In addition, Desulfovibrio which is a pro-inflammatory genus was decreased after MK-2206 treatment. This sulfate-reducing bacterium produces hydrogen sulfide by reduction of sulfate and when excessive, can damage intestinal epithelial lining and facilitate inflammation to occur-2206 partially improved HCoV-OC43-induced dysbiosis in the intestine of mice.

To examine the effects of MK-2206 treatment on the microbiota, PICRUSt2 was used. This comparison showed that there was a substantial change in KEGG pathways at Level 2 of the HCoV-OC43-infected, MK-2206-treated and blank control groups (Figure 2F). Pathways in Genetic Information Processing and Cofactor and Vitamin Metabolism were increased and those in Immune System Diseases, Enzyme Families and Transcription were decreased with infection. This observation is largely consistent with the observations in COVID-19 patients, where the intolerance of production of beneficial immunomodulatory metabolites by the gut microbiota, including SCFAs, (15) are accompanied by the reprogramming (as adaptive) of microbial basic material and energy metabolic pathways (25), which is essentially the same pattern of gut microbial ecological response to host infection by viruses. The Enzyme Families, Replication and Repair, and Nucleotide Metabolism and Translation pathways increased, whereas the Cofactor and Vitamin Metabolism and Cellular Processes and Signaling were reduced following MK-2206 treatment. These alterations indicate that MK-2206 alleviates intestinal inflammation and viral injury by changing microbial activity to prioritize stress defense mechanisms with proliferation mechanisms. The treatment with MK-2206 could heal the core metabolic activities and decrease stress-related cofactor expenditure, thus serving as an induction of microbiota homeostasis.

Succinic acid enhanced antiviral capacity of MK-2206

To validate the PICRUSt2-predicted alterations in microbial function at the metabolic level and to directly assess the host-microbiome co-metabolic disturbances induced by HCoV-OC43 infection and MK-2206 treatment, we further sought to identify key metabolites potentially contributing to the antiviral efficacy. Accordingly, untargeted liquid chromatography–mass spectrometry (LC–MS) metabolomic profiling of fecal samples was performed to identify differentially abundant metabolites across experimental groups. Permutation testing confirmed a robust model fit (R 2 Y = 0.8542) and predictive capability (Q 2 Y = 0.7686) for the partial least squares-discriminant analysis (PLS-DA) model, which validated its reliability with no indication of overfitting (Supplementary Figures 2A, B). The PLS-DA score plot demonstrated distinct clustering of samples by group, indicating that metabolite profiles were significantly separated between groups. Variable Importance in Projection (VIP) scores derived from the validated PLS-DA model were employed to rank metabolites according to their contribution to group discrimination.

Differentially abundant metabolites (DAMs) in the infected and treated groups relative to blank controls were mapped to biochemical pathways using hypergeometric enrichment analysis. Volcano plot analysis identified 268 significantly upregulated and 140 downregulated DAMs following infection (Figure 3A). Compared to the infected group, treatment yielded 87 significantly upregulated and 245 downregulated DAMs (Figure 3B). Applying significance thresholds of P < 0.05 and fold change > 1.5, significantly elevated abundance of α-Solanine, Cyanidin 3-rutinoside, and 6′′-Acetylhyperin 7-rhamnoside was detected in the infected group, whereas Docosahexaenoic acid, Eicosapentaenoic acid, and Palmitoylethanolamide displayed significantly reduced abundance. Following treatment, significantly increased abundance was observed for trans-Resveratrol 4′-sulfate, Isoplumbagin, and Succinic acid, while significantly decreased abundance was observed for 4-Methyl-2-phenyl-1,3-dioxolane, DL-2-Aminooctanoic acid, and Hypotaurine (Figures 3C, D). Metabolomic analysis reveals that both HCoV-OC43 infection and MK-2206 treatment induce significant alterations in the host metabolic profile, with particularly pronounced changes in the levels of characteristic metabolites such as succinic acid.

Furthermore, pathway enrichment analysis based on KEGG annotation was performed. The differential metabolites after MK-2206 treatment are mainly enriched in specific biological pathways, which play a crucial role in microbial proliferation and the metabolic balance of the host. The differential metabolites mainly participate in metabolic processes such as ABC transporters and nucleotide metabolism. (Figure 3E). This finding aligns with the functional shifts in the microbiota predicted earlier by PICRUSt2 (Figure 2F). The agreement between predicted microbial functions and measured metabolite profiles provides strong validation for the computational results. It also points to the recovery of core biosynthetic processes as a key therapeutic mechanism. Succinic acid, as a key metabolic intermediate, participates in physiological processes such as immune regulation, inflammatory responses, and antiviral defense. (26, 27). For example, research shows succinate salts can inhibit influenza A virus infection by modifying the viral nucleoprotein through succinylation (28). To test how well succinate and MK-2206 work together, we used a succinate salt to avoid the drop in pH that comes with succinic acid itself. This let us study succinate’s real biological effect without interference. This approach isolated the biological activity of succinate from nonspecific pH artifacts on cell viability and viral replication. Cell-based assays demonstrated that combined MK-2206 (5 μM) and Di-succinate (1 mM) treatment reduced viral N gene expression by 99.9% compared to the infection control, significantly surpassing the efficacy of either monotherapy (MK-2206 alone: 75.0% reduction; Di-succinate alone: 96.7% reduction) (Figure 3F). Together, these findings demonstrate that the potent inhibition of viral gene expression by the combination treatment translates to a profound reduction at the protein level, as evidenced by a 75% decrease in N protein expression via immunofluorescence, significantly surpassing the effects of individual treatments (40% and 60% for MK-2206 and Di-succinate, respectively). (Figures 3G, H). Metabolomics analysis identified succinic acid as a potential biomarker. In vitro experiments confirmed that its combination with MK-2206 synergistically enhances antiviral effects, significantly suppressing HCoV-OC43 viral gene and protein expression.

Integrated analysis of gut microbiota and metabolites

Due to the complex interactions between gut microbiota and metabolites, we investigated the connections between the different bacterial genera and the different metabolites before and after treatment. A correlation heatmap was constructed based on Pearson’s correlation coefficients to illustrate the primary interactions between microbial communities and microbial metagenomes (r > 0.6, P < 0.05).From the heat map, we found that the Akkermansia genus was mainly positively correlated with the differential metabolites. On the contrary, the Lactobacillus genus was mainly negatively correlated with the metabolites including linolenic acid, xanthine, and hypoxanthine. The significantly different metabolites (such as xanthine, N-acetyl-β-D-galactosamine, and hypoxanthine) showed a strong negative correlation with the increase in the abundance of Lactobacillus and Ruminococcus genera (Figure 4). The results indicate that the alteration of specific gut microbial groups is associated with the MK-2206 treatment during HCoV-OC43 infection, and may be related to the antiviral response.

This study demonstrates that MK-2206 exhibits antiviral activity both in vitro and in vivo. Using an integrated approach combining 16S rRNA gene sequencing and liquid chromatography–mass spectrometry (LC–MS)-based untargeted metabolomics, we investigated the effects of MK-2206 treatment on the gut microbiota ecosystem in mice infected with HCoV-OC43. The results revealed that MK-2206 reduced the relative abundances of Akkermansia and Desulfovibrio, while increasing the abundance of Lactobacillus. It also elevated the levels of several metabolites, such as succinate. The alteration in the abundance of the differential metabolite succinic acid may suggest a potential association with the microbiota-metabolite axis, which may have a biological correlation with the host’s antiviral response and is parallel to the direct antiviral activity of MK-2206.

We found a complicated interrelation between AKT inhibitor MK-2206, HCOV-OC43 infection, and intestinal microbiota. HCOV-OC43 has both similar biological properties with other β- coronaviruses, including SARS-CoV-2, including the same host cell entry pathways. In line with our findings, SARS-CoV-2 infection has also been associated with a decrease in the relative abundance of Firmicutes and Bacteroidota and an increase in the abundance of Akkermansia (29). In addition, obligate anaerobic genera of the sort Parabacteroides, Anaerotignum, Ruminococcus and Mucispirillum had highly decreased relative abundances after treating with MK-2206. The dysbiosis is in line with the gut microbiota imbalance with ritonavir boosted protease inhibitor therapy, in HIV patients (30).

The relative abundances of Lactobacillus and Ligilactobacillus showed significant changes after the use of MK-2206. There is strong evidence suggesting Lactobacillus or colonization administration alleviates the viral pneumonia severity (31). Research shows that giving Lactobacillus preparations through the nose to mice activates the nasal immune receptor Nod2. This then activates granulocyte-macrophage colony-stimulating factor, which provides direct protection against respiratory tract infection (32). Therefore, the enrichment of intestinal Lactobacilli may be related to the systemic host response during HCoV-OC43 infection, possibly through metabolic or immune-related pathways, although no direct evidence was evaluated in this study. Other non-classical genus immunomodulatory lactobacilli have been found to be also mentioned in our results. Also, the recently re-classified Ligilactobacillus, formered from Lactobacillus, undergoes extensive probiotic potential. Ligilactobacillus salivarius have been studied to generate strong tumor necrosis factor (TNF) inhibitors, which underlines their immunomodulatory properties (33). Ligilactobacillus, in terms of its functional similarity to Lactobacillus, shares some of the most important metabolic pathways with Lactobacillus, such as: to metabolize and stabilize beneficial metabolites such as β-carotene and vitamin K (34) In other words, changes in the abundance of both Ligilactobacillus, as a result of the therapeutic effect, may change the home pool of the beneficial metabolites of the host.

Our metabolomic analysis in the HCoV-OC43 model shows disrupted energy metabolism. This matches findings from severe coronavirus infections. Several studies have characterized alterations in the serum metabolome following COVID-19. it is noted that 16 metabolites change significantly, such as amino acids (phenylalanine, tyrosine, valine, methionine), ketone bodies (3-hydroxybutyrate, acetoacetate, and acetone), isoleucine, mannose, lactate, and acetate (35). Increased concentrations of Krebs cycle intermediates, such as succinate and fumarate have been identified in the serum of post-COVID-19 patients as well (36). Our results in the mouse model show that MK-2206 treated lead a better gut microbiota function. This means that microbial metabolic functions, like energy and nucleotide metabolism, improved after treatment. The imbalances of the central carbon metabolism indicated in pyruvate, succinate, and glutamate dysregulation have been previously implicated in COVID-19 pathogenesis (37). The restoration of the levels of NAD and succinic acid following administration of MK-2206 therefore suggests a slow restoration of energy metabolism in gut microbial ecosystem. Also, the augmented concentration of a number of microbially produced metabolites such as agmatine (an arginine metabolite), indolelactic acid (a tryptophan metabolite), and N-acetylputrescine (proline metabolite), as well as indoleacetic acid, in fecal samples following treatment with MK-2206, which may profoundly influence the host-microbial co-metabolic circumstance.

The role of Succinic acid varies with the specific pathogen and the host’s microenvironment. For example, Succinic acid can suppress influenza A virus replication by directly modifying the viral nucleoprotein through succinylation (28). In contrast, it may worsen vesicular stomatitis virus infection by inhibiting MAVS signaling (38). Our study shows that in the context of HCoV-OC43 infection, succinate likely has a protective effect. MK-2206 treatment increased intestinal succinate levels. In vitro experiments revealed a strong synergistic antiviral effect between succinate and MK-2206. A recent study found that the fermentation supernatant of Lactobacillus salivarius is enriched with succinate. The succinate-activated receptor SUCNR1 significantly enhances the activity and proliferation of intestinal stem cells, thereby improving the intestinal mucosal absorption function and barrier integrity. (39). Based on these findings, we propose a working model: MK-2206 remodels the gut microbiota to promote the proliferation of bacteria that produce succinate, thereby increasing the local succinate concentration. Elevated succinate levels may be involved in host systemic responses, potentially through gut–lung axis–related signaling or other regulatory pathways.

It is worth noting that this study has some limitations. Although the controlled animal experiment design minimizes the influence of individual differences, lifestyle, and diet and other confounding factors, the relatively small sample size limits the statistical power based on correlation analysis. Although such a sample size is acceptable for exploratory, hypothesis-generating multi-omics studies, future research needs a larger and well-matched cohort to improve statistical robustness and verify the observed associations. Secondly, intestinal microbiota clearance, fecal microbiota transplantation (FMT), and in vivo succinate supplementation experiments are key subsequent steps for formally establishing the causal relationship between intestinal microbiota remodeling, elevated succinate levels, and antiviral protection. This study lacks direct immunological indicators, so it is impossible to determine whether changes related to the intestinal microbiota and succinate are directly regulating antiviral immunity or are secondary metabolic consequences of reduced viral load. Further experiments should further verify the causal relationship.

This integrated multi-omics study demonstrates that MK-2206 administration restructures the gut microbial community and metabolome in HCoV-OC43-infected mice. This shift is primarily characterized by an altered abundance of Akkermansia and corresponding fluctuations in succinate levels. These findings establish the gut microbiota-metabolite axis as a critical determinant of the drug’s therapeutic efficacy, providing a vital mechanistic insight for developing optimized antiviral strategies.