Acid sphingomyelinase inhibition as an antiviral strategy: mechanisms, functional inhibitors, and therapeutic potential against SARS-CoV-2 and beyond

1 Introduction

In a global systematic analysis of 85 pathogens, viral infections were noted to be associated with approximately 178 million disability-adjusted life years (DALYs) or 25.3% of the total DALYs in 2019, showing a significant contribution to global morbidity and premature mortality (1). In parallel, major viral diseases such as HIV (2), chronic viral hepatitis (3), seasonal influenza (4), and COVID-19 (5) continue to be responsible for millions of deaths annually worldwide. The COVID-19 pandemic caused 8.7 million deaths in 2021, which confirmed that such outbreaks lead to fatal results (6). Findings from large clinical signals (7,230 and 83,500 participants) and biomarker changes [extracellular vesicle (EV) tissue factor ~10xhigher levels in severe COVID-19] underscore the need for host-directed antiviral approaches (7–9). Severe viral infections can cause pathogenic hyperinflammatory immune responses, which are commonly called cytokine storms or cytokine release syndrome (10, 11). Consistent with this, inpatient COVID-19 patients have been reported to have elevated serum levels of interleukin (IL)-6, interleukin (IL)-8, and tumor necrosis factor alpha (TNF-α) (10). Additionally, higher concentrations of circulating inflammatory cytokines in serum such as IL-6, IL-8 and TNF-α measured at time of hospitalization were found to independently predict disease severity and mortality (10), highlighting that excessive host immune activation is a key driver of organ damage and death in viral disorders. Multiple viruses in humans which require host sphingolipid metabolism to transform cell membranes, thus enabling them to complete their life cycle. The network consists of acid sphingomyelinase (ASM) that breaks down sphingomyelin into ceramide which is critical to form platforms that link receptors to endocytic machinery for viral attachment, fusion, and intracellular transport (12–17). Modulation of the ASM-Ceramide axis is shown to affect viral infectivity across multiple viral families like coronavirus (18), filovirus (19), retrovirus (20, 21), and rhinovirus (14, 15, 22). Studies suggests that SARS-CoV-2 host cellular binding causes ASM activation, leading to ceramide platform formation that can cluster receptors responsible for viral entry, such as ACE2 and TMPRSS2 (12, 18, 23–25).

The evidence shows that ASM-blocking drugs including FIASMs reduce viral entry in vitro, but ceramide addition to the system enables viral entry to occur, which confirms the critical causal function of this pathway (18). Most approved antiviral drugs focus on rapidly mutating viral proteins but fail to target essential host lipid pathways, which creates an opportunity for developing broad-spectrum treatments that can withstand viral resistance. Indeed, many antivirals show limited efficacy because of the emerging resistance or having a narrow therapeutic spectrum. For example, extensive oseltamivir-resistant influenza strains occurred during the 2008–2009 season, which made these antivirals ineffective in many cases (26). Similarly, even use of a widely employed antiviral such as remdesivir has been reported to fail to significantly reduce mortality in hospitalized COVID-19 patients with similar 28-day mortality in the WHO Solidarity trial(14.5% vs 15.6%) (27). These clinical findings highlight the shortcomings of many current antiviral options. The current review addresses this knowledge gap through the integration of three main points: (i) provides evidence that ASM activation leads to ceramide platform formation, which enables viral entry and early trafficking processes. (ii) Shows how sphingolipids control different stages of viral life cycles, including replication, egress, and endosomal escape across multiple distinct viral families. (iii) Discusses how blocking the ASM function creates new translational opportunities for therapeutic applications. This review examines FIASMs, the FDA-approved drugs acting as ASM inhibitors by detaching ASM from lysosomal membranes to disrupt ceramide domains, and preventing SARS-CoV-2 entry and microvesicle (MVP/large EV) release in experimental models (18, 28–37). The review also evaluates observational clinical information together with preliminary trial results showing host-directed FIASM-based treatment strategies while discussing their advantages and challenges (dose/Pharmacokinetics, off-target pharmacology, trial design). To summarize, this manuscript will also outline two possible future directions that involve using engineered EV decoys and biomarker strategies that utilize sphingolipid signatures and MVP cargo to monitor drug effects and identify suitable patients.

2 Sphingolipids and viral life cycle: mechanisms and platforms

2.1 General roles of sphingolipids in viral infections

Sphingolipids are key plasma membrane components exploited by viruses throughout their life cycle:

• Viral Attachment: Viral entry often depends on glycosphingolipids, which can act as receptors or co-receptors. For example, rotavirus binds to gangliosides on enterocytes, and influenza A virus (IAV) binds GM3 on target cells (38, 39). These sphingolipid–protein interactions help the virus dock onto the cell membrane during its initial contact with cells.
• Entry and Internalization: Lipid rafts are known to concentrate the entry receptors. The viral contact activates the ASM enzyme to produce ceramide from sphingomyelin. This sphingomyelin-to-ceramide process results in lipid rafts reorganizing into larger ceramide-rich platforms that cluster receptors and endocytic machinery to facilitate viral internalization (12–14). In enveloped viruses, the membrane fusion is facilitated by ceramide’s property of having a conical geometry and a small polar head. Whereas in the non-enveloped viruses, this property causes destabilization of endosomes to allow escape (15–17). On the other hand, if ceramide is formed prematurely, like by application of an exogenous sphingomyelinase, this can restrict entry for viruses that require fluid membranes like HIV-1 via clustering and immobilization of CD4 receptors (40). Hence, the viral entry may be enhanced or inhibited due to ceramide, and this depends on the particular entry mechanism used by the virus.
• Intracellular Replication: Viruses are known to remodel sphingolipid metabolism to favor their replication. In response to various viruses, ceramide and other sphingolipids accumulate at replication organelles. For example, the Zika virus causes the redistribution of ceramide to replication sites and disturbing sphingolipid biosynthesis causes impaired RNA synthesis (41). Some viruses use sphingolipid signaling to help with survival in hosts: RSV activates sphingosine kinase-1 to make sphingosine-1-phosphate (S1P), which was found to delay host cell apoptosis to prolong the replication time (42). The intracellular balance between ceramide, sphingosine, and S1P regulates autophagy, inflammation, and apoptosis processes, which directly affects the replication efficiency (43).
• Egress (Virus Release): During the late stages of infection, some enveloped viruses bud from lipid rafts microdomain of the host plasma membrane rich in cholesterol and sphingolipids to acquire their envelope (44). Sphingomyelin present in the host and viral cell membranes is vital for maintaining virion structural integrity and infectivity. For example, the IAV envelope is rich in sphingomyelin, and depleting it from either host or virion greatly reduces the infectivity (45). Viruses also exploit the sphingomyelinase enzyme that converts sphingomyelin to ceramide and drive budding or scission (46). As such, the inhibitors of sphingomyelin, like D609, were reported in the 1980s to inhibit sphingomyelin synthesis/turnover and cause a reduction in the release of herpes simplex virus (HSV), SV40, HIV-1, respiratory syncytial virus (RSV), and rhinovirus (47–50). Thus, modulating sphingolipid composition influences virion budding and infectivity.

2.2 Viral examples exploiting sphingolipids

Over time, many diverse viruses have evolved specific ways to exploit the sphingolipid pathway for their own benefit.

• Rhinovirus (Picornavirus, non-enveloped): Rhinovirus entry requires ASM activation. ASM first binds to its receptor called ICAM-1 on respiratory epithelial cells, after which they are able to translocate to the surface (51, 52). There, ceramide is produced and creates large ceramide-rich domains that can concentrate viral particles (53, 54). It has been reported that an ASM deficiency or using FIASMs like imipramine can block these platforms to prevent infection. These findings establish that ceramide-rich microdomains are very important for viral uptake (14).
• Measles Virus (Paramyxovirus, enveloped): Dendritic cells use the DC-SIGN receptors to capture measles virus (55). The interaction activates sphingomyelinases (both acidic and neutral), leading to a ceramide wave. This wave can redistribute the CD150/SLAM to the cell surface in clusters that contain the activated ASM (22). Theon-demand platform increases viral uptake as shown by the finding that sphingomyelinase inhibition prevented CD150 recruitment to the cell membrane and reduced infection (22). Moreover, blocking sphingomyelinase activation with drugs [e.g., amitriptyline for ASM, or GW4869 for neutral sphingomyelinase (nSMase)] or siRNA (e.g., NSM2/SMPD3 siRNA)prevented CD150 from reaching the surface, which significantly reduced measles entry (22). Thus, measles viruses are highly dependent on ceramide signaling.
• Ebolavirus (Filovirus, enveloped): Ebolavirus’s infection efficiency is also dependent on ASM activity and its substrate sphingomyelin in the plasma membrane. Inhibition of ASM activity with ASM inhibitors (imipramine, desipramine and amitriptyline) or genetic reduction of ASM (SMPD1 targeting siRNAs) and ASM-deficient human fibroblast significantly decrease viral susceptibility even when viral receptors were present. Mechanistically it was found that ebolavirus particles bind with sphingomyelin-rich regions of cell membrane and ebolavirus like particle colocalizes with surface localized ASM at cell binding sites as early as in 30 minutes. This is consistent with local ceramide production which promotes membrane invagination and downstream fusion events (19).. Thus, like rhinovirus and measles, Ebola also exploits the ASM/ceramide system for cell entry.
• Coronaviruses (e.g., SARS-CoV-2, enveloped): Some coronaviruses including SARS-COV-2 use the ACE2 receptor for entry, but the receptor behavior is highly modulated by sphingolipids. It was found that SARS-CoV-2 cell binding causes ASM activation to form ceramide. The ceramide clusters ACE2 on ceramide-rich platforms that enhance fusion (18). In vitro functional inhibitors of ASM (FIASMs)including amitriptyline, imipramine, desipramine, fluoxetine, sertraline, escitalopram and maprotiline have been found to reduce spike-mediated entry. While adding exogenous ceramide restored infectivity to confirming dependence of SARS-CoV-2 on the ASM-Ceramide pathway (18). We will detail this mechanism further in Section 3.
• Influenza A Virus (Orthomyxovirus, enveloped): Sphingomyelin presence in the plasma membrane was found to be important for IAV cellular entry, and its depletion via sphingomyelinase decreased viral uptake. Consistent with this notion, IAV virions with low sphingomyelin have lower viral infectivity (56). However, contrary to most viruses, IAV is not dependent on ASM activation during entry. In contrast, IAV infection reduces ASM activity in later viral stages. Hence, IAV virus budding favors intact sphingomyelin-rich rafts (56). As a result, IAV utilizes sphingolipids structurally than via signaling.
• This pattern is in parallel with the findings of IAV’s dependence on the biophysical properties of raft-like membranes during entry and fusion. For example, hemagglutinin undergoes a low-pH conformational change to drive membrane fusion (57). The IAV hemagglutinin mediated fusion is noted to be sensitive to both target-membrane lipid composition and membrane order/fluidity (58). Specifically, cholesterol, which is known to preferentially associate with sphingolipids to form liquid-ordered raft microdomains promotes the hemaglutin -driven hemi fusion and fusion-pore expansion (58). This gives us a plausible basis for why intact sphingomyelin-rich domains support influenza entry, but the ASM-driven sphingomyelin hydrolysis/ceramide platform signaling is not required at this step.
• Human Immunodeficiency Virus (Retrovirus, enveloped): HIV-1 behaves differently from most viruses in that it avoids ceramide platforms. For its entry into CD4⁺ T cells, this retrovirus needs a heterogenous host plasma membrane with ordered (raft-like) and fluid (disordered) lipid domains so that its envelope can fuse at ordered/disordered domain boundaries (21). If artificial ceramide is used by treating cells with bacterial sphingomyelinase, the resultant membrane rigidly clusters CD4 and CCR5. Since HIV-1 needs mobility of receptors, this hinders fusion (20). This is a unique dependence and suggests that ceramide modulators may serve as anti-HIV microbicides. Sphingolipids not only affect the starting viral entry phase, but also later stages. The neutral sphingomyelinase activity in virus-producing (infected/transfected) cells influences endosomal sorting complexes required for transport (ESCRT)-dependent budding and virion release/maturation (59).
• Calciviruses (Caliciviridae, non-enveloped): These viruses are dependent on ASM for endosomal escape during entry. In porcine enteric caliciviral infections, the bile acids were found to use ASM activation and ceramide formation in endosomes to facilitate cytosolic virion release from endosomes. Blocking ASM via pharmacologic strategies or gene silencing caused retention of viral particles in endosomes (15). Both feline calicivirus and murine norovirus induce their own ceramide generation, which is blocked using ASM inhibition. Hence, for non-enveloped viruses, ceramide-driven membrane destabilization and fusion is a common strategy to escape from endosomal barriers (15).
• Rubella Virus (Togavirus, enveloped): For the Rubella virus, its entry is strongly dependent on the lipid composition of the host membrane (60). The fusion steps need both sphingomyelin and cholesterol. The viral envelope protein also require Ca²⁺ that support interaction with sphingomyelin/cholesterol-rich membrane domains (61). When sphingomyelin is absent, [e.g., sphingomyelin synthase-1(SGMS/SMS1) knockout cells, the virions can bind to the host cell and undergo hemifusion (lipid mixing) but cannot form full pores needed for nucleocapsid/genome penetration into the cytoplasm (60). Thus, sphingomyelin is regarded as a critical cofactor for rubella fusion, and it demonstrates that sphingolipids, even apart from ceramide, are important for infectivity.

2.3 Ceramide-induced microvesicle shedding

In this section, the ASM/ceramide-dependent vesicle biogenesis will primarily refer to plasma membrane–derived microvesicles (MVPs/large EVs) while the exosomes/small EVs discussed will be nSMase2/endosomal-dependent unless stated otherwise. Products of ceramide not only aid in viral entry, but they also promote the shedding of subcellular particles including MVP. This shedding helps with viral spread and the bioactive contents of MVP can modulate the host responses. The ASM activation at the plasma membrane has been found to induce MVP budding in the outward direction. Extracellular particles like MVP are also known by other names, such as microparticles or large EVs, depending on their size and origin within the host cell (62). The mechanism suggested is that when ceramide accumulates in the outer leaflet, this process enhances the negative curvature of the cell membrane and causes budding (63). A seminal study by Bianco and colleagues showed that ATP stimulated ASM translocation in glial cells, leading to ceramide generation and the rapid release of 100–500 nm microvesicles (62). The ASM inhibition was found to inhibit shedding as well as IL-1β release. These findings demonstrated that ceramide-driven vesicles also carry inflammatory cargo (62). These studies provide compelling evidence for ASM-generated ceramide being a key driver of microvesicle biogenesis.

Ceramide-driven microvesicles (MVPs) as cargo carriers: Virus-infected cells release more EVs containing both host and viral components:

• Cytokines and inflammatory mediators. The microvesicles that are triggered by ASM are known to often encapsulate cytokines such as IL-1β under stress conditions (64, 65), and can serve as vehicles for the propagation of inflammatory signals to the recipient (66) (67, 68). Under ASM activation, endothelial cells don’t just release inflammatory microvesicles, but the latter display tissue factor (TF) on their surface to spread pro-coagulant activity and inflammation (69, 70). Such elevation of TF-bearing MVPs in severe COVID-19 cases further connects ceramide-dependent vesicle release to systemic inflammatory and coagulation effects (71).
• Viral Proteins: Viruses can use EVs (including exosomes/small EVs) to spread viral proteins without the need to release full virions. In the case of Epstein-Barr virus (EBV), the oncoprotein LMP1 is packaged into exosomes through a process that is dependent on ceramide. When neutral sphingomyelinase is blocked, cells cannot make sufficient ceramide, and this causes mislocalization of LMP1 and reduction of exosome secretion (72, 73). These LMP1-containing exosomes can fuse with neighboring uninfected cells to transfer functional LMP1, which activates NF-κB in recipient B cells and remodels the microenvironment, favorable for B cell survival and proliferation (72, 74). Also, HIV-1 and HTLV-1 are known to release vesicles containing proteins like Nef or Tax, respectively, which are also linked to ceramide-driven vesicle formation (75, 76).
• Viral Nucleic Acids: Ceramide-triggered vesicles can also pack viral genomes to provide viruses with an alternative infection route other than classical virion release. In the case of hepatitis B virus (HBV), the infected liver cells secrete vesicles containing HBV DNA. These vesicles can infect new cells without production or release of new virions (77, 78). This transmission is also dependent on vesicles formed via ceramide-promoted inward budding of endosomal membranes (i.e., a ceramide-triggered exosome/small EV pathway linked to nSMase2) and vesicle release. Also, since these particles do not have hepatitis B surface antigen (HBsAg), they are able to escape recognition by neutralizing antibodies (78). Thus, HBV can hide its DNA cargo inside host-derived vesicles and can indirectly use the cell’s own communication system as “Trojan horses”. This concept, used by other viruses as well such as HIV-1 and human T- cell leukemia virus (HTLV)-1, has been termed as “Trojan exosome” hypothesis (79).
• MicroRNAs and Regulatory RNAs: A viral infection affects host-derived miRNA and frequently generates virus-coded miRNAs. Many of these bioactive agents get packaged into EVs, especially smaller exosomes (80). Such ceramide-enriched exosomes can fuse with uninfected cells to deliver these miRNAs and modulate gene expression in recipient cells. In the case of EBV, when the viral miRNAs enter uninfected immune cells, these miRNAs can suppress specific host genes to weaken immune defenses (81). The exosome biogenesis relies on neutral sphingomyelinase 2, which produces ceramide, and blocking ceramide reduces the release of miRNA-loaded vesicles (82). This ceramide-dependent miRNA transfer highlights another viral strategy for reducing antiviral responses or promoting host cell survival (83).

Thus, we can draw a two-sided picture of ceramide-induced microvesicle shedding during infection. This process can either act as a stimulator of host defense, as these vesicles can carry pro-inflammatory signals like IL-1β to alert neighboring cells and enhance immune signaling. Or the viruses can use the same ceramide-microvesicles pathway by loading these vesicles with antigens like viral proteins, nucleic acids, etc., and attempt to escape detection from the host immune system without the need to expose free virions. The ASM–ceramide–MVP axis is therefore central not just for entry of viruses but also for plasma membrane–derived microvesicle (MVP/large EV) release, which can disseminate bioactive cargo and enhance inflammation. Therapeutic targeting with ASM inhibitors (FIASMs) may limit ASM-dependent MVP release that otherwise spreads viral cargo, limit inflammation, such as the COVID-19 cytokine storm. In parallel, a few viruses use ceramide-dependent small EV/exosome pathways (usually nSMase2/endosomal) and thus the impact of ASM inhibition on ‘Trojan exosome’–type mechanisms will require direct supporting evidence.

3 ASM/ceramide axis in SARS-CoV-2 infection and pathogenesis

3.1 Ceramide-enriched platforms promote ACE2 clustering

For SARS-CoV-2 to fuse with and enter the cell, the host plasma membrane needs to have lipid organizational changes, which depend on ceramide. When the viral spike proteins binds to host ACE2, it activates ASM that hydrolyzes the sphingomyelin lipid to hydrophobic ceramide. The latter tends to self-cluster to form platforms in the plasma membrane (18). These molecules self-associate into microdomains due to non-polar character, to create larger platforms (23). This changes the membrane curvature and fluidity to make the membrane more fusogenic for viruses. This clusters SARS-CoV-2 receptors, ACE2, molecules and cofactors like TMPRSS2 (12, 24). These platforms enhance viral attachment and fusion as they bring multiple ACE2 molecules receptors in close distance from associated proteases like TMPRSS2 (25). This process is not unique to SARS-CoV-2, but also Measles and Ebola that use ceramide-rich rafts for bringing together receptors and reducing the fusion energy barrier (19, 22).

3.2 Experimental evidence of ASM inhibition reducing infection

Multiple studies have confirmed the ASM/ceramide pathway’s key role in SARS-CoV-2 entry. Using experimental approaches like pharmacological inhibition of ASM or silencing ASM expression via shRNA has led to similar results of decreased viral uptake and replication in cultured cells (18, 84). FIASMs such as amitriptyline, imipramine, fluoxetine, and sertraline, were shown to inhibit SARS-CoV-2 infection in both Vero E6 and human Caco-2 cells epithelial cells in a dose-dependent manner (18).

Please see Figure 1 for proposed pathways by which ASM is involved in SARS-CoV-2 infection and subsequent pathologies. The loss of function (blocking ceramide) and gain of function (adding ceramide back) have experimentally shown that ASM activation and ceramide production have causality with SARS-CoV-2 entry. For example, the infection itself activates ASM to create a ceramide surge. However, using interventions that block ceramide, like anti-ceramide antibodies or neutralize ceramide, such as using ceramidase, prevents viral entry (85). When exogenous C16-ceramide was added, it restored infectivity. The same protective function is also seen in freshly isolated primary human nasal epithelial cells (from healthy volunteers). When these were pretreated with ASM inhibitors like amitriptyline or anti-ceramide antibodies, the cells became resistant to infection. However, this protection was lost when ceramide was deliberately added, and cells again became susceptible to infection (18). Hence, SARS-CoV-2 activates as well as exploits the ASM-ceramide axis for entry, suggesting that if this pathway is blocked it can prevent infection.

3.3 Microvesicles in COVID-19 inflammation and pathology

Ceramide not only facilitates SARS-CoV-2 infection via fusion platforms but it is also associated with increased EV from SARS-CoV-2- infected cells and from stressed/damaged bystander cells (86). During infections and associated cellular stress, these cells can release vesicles enriched in sphingolipids as well as inflammatory cargo like cytokines, viral RNA, or proteins like Spike (86, 87). The exosomes associated with SARS-CoV-2 have been reported to exert pathological consequences. For example, plasma from COVID-19 patients has been reported to contain exosomes packed with viral RNA. These exosomes are then internalized by immune cells. These viral RNAs are recognized as danger signals by endosomal Toll-Like-Receptors (TLRs) such as TLR3/7/8. The TLR activation then initiates an inflammatory signaling cascade resulting in the release of cytokines like IL-6, IL-8, and TNF-α (87). Treatment of peripheral blood mononuclear cells (PBMCs) from healthy donors with exosomes from acutely ill COVID-19 patients triggered significantly higher cytokine release than those from recovered or uninfected individuals (87, 88). Given that these vesicles were reported as plasma exosomes/small EVs and their specific biogenesis pathway (ASM-dependent MVP shedding vs nSMase2/endosomal) was not directly tested, we describe them here as exosomes/small EVs and avoid attributing their formation to ASM inhibition.

Microvesicles also contribute to COVID-19–related endothelial dysfunction. Normally, endothelial cells are relatively resistant to direct SARS-CoV-2 infection, but they may be indirectly activated by inflammatory signals and exposure to circulating vesicles. The exosomes from the plasma of COVID-19 patients undergoing complicated infections were found to activate endothelial cells, resulting in the formation of NLRP3 inflammasome assembly inside them. These exosomes led to the release of IL-1β and other pro-inflammatory mediators (32). The pathological outcome is endothelitis, vascular leakage, and thrombosis. Thus, ceramide-induced EVs act as messengers that can disseminate viral and inflammatory signals throughout the body systematically to enhance cytokine storm and endothelial dysfunction. Since ceramide is key to their formation, using strategies to target ceramide pathways may help alleviate severe complications of COVID-19. Please see Figure 2 for the proposed schematic of the ASM -ceramide -extracellular vesicle axis and its downstream contributions to endothelial dysfunction, cytokine amplification, thrombosis and vascular leak in COVID-19.

4 Functional inhibitors of ASM and translational implications

4.1 Overview of FIASMs: drug classes and mechanisms

FIASMs are indirect inhibitors of ASM. They detach ASM from lysosomal membranes to make them functionally inactive. Many FDA-approved drugs with varying therapeutic classes are FIASMs, like antidepressants, antipsychotics, and antihistamines. Specific examples include SSRIs, antidepressants like fluoxetine, fluvoxamine, escitalopram, and sertraline; tricyclic antidepressants like amitriptyline and imipramine; H1 antagonists like hydroxyzine and some cationic amphiphilic drugs like calcium channel blockers also exert anti-ASM effects (28, 30, 31). Chemically, these drugs are lipophilic weak bases having a tertiary amine group. This allows them to accumulate inside acidic lysosomes by a process called lysomotropism and disrupt ASM function (32).

ASM is usually bound to the inner lysosomal membrane. The FIASMs detach the enzyme to destabilize it, leading to proteolysis. The lesser ASM activity thus leads to decreased production of ceramide (89). FIASMs thus prevent the formation of ceramide-rich cell membrane domains (18, 33). Such domains cluster ACE2 and thus promote the entry of SARS-CoV-2. So, FIASM-based inhibition of the ASM/ceramide signaling is proposed to have dual therapeutic effects of reduction of viral entry and potentially affecting the dangerous runaway inflammation that can be associated with this infection (34, 35). While not all the drugs that are lipophilic amines function as FIASMs, those identified as FIASMs have good oral bioavailability, established safety profiles due to being FDA-approved marketed drugs, and low cost to making them attractive candidates for repurposing for COVID-19 (90).

4.2 Preclinical and In silico data

There is experimental proof of concept for FIASMs against SARS-CoV-2. In vitro confirmation comes from antidepressant FIASMs (e.g., amitriptyline, imipramine, fluoxetine, sertraline, escitalopram) tested in Vero cell models, where inhibition of ASM reduced surface ceramide generation and thereby prevented SARS-CoV-2 spike–mediated viral entry (18). Adding exogenous ceramide reversed protection against SARS-CoV-2 spike mediated) infection to prove that ceramide blockade was key to reduced infection. In the ex vivo human translational validation proof, amitriptyline was given orally to healthy individuals, and nasal epithelial cells were collected. These nasal epithelial-derived cells were found to be resistant to SARS-CoV-2 infection ex vivo (18). Similarly, fluoxetine also inhibited infection in human epithelial cells (36). The antiviral effect was found to hold up across variants. For example, fluoxetine(10 μM) reduced entry of SARS-CoV-2 spike pseudovirus carrying RBD mutation(e.g., N501Y and E484K) in ACE2/TMPRSS2- expressing HEK293T cells, and it also reduced replication of live Alpha(B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) viruses in Calu-1 human lung epithelial cells (35). Since these variants that differ in spike sequences were all blocked, it suggests that FIASMs act on the host pathway of the ASM-Ceramide-dependent entry process rather than the virus itself.

As ASM is a key host target, investigators have used in-silico molecular docking to see how different FIASMs bind to ASM to potentially identify new candidates. Alkafaas and team found that several drugs had higher predicted ASM binding affinity than amiodarone, which is a known FIASM. These included dilazep (vasodilator), emetine (antiparasitic), pimozide (antipsychotic), carvedilol (beta-blocker), mebeverine (antispasmodic), and cepharanthine (a natural alkaloid with antiviral history) (91). Another computational study examined 257 compounds with FIASM activity and found that, in particular, cepharanthine and zafirlukast (a leukotriene antagonist) were the top hits, having very strong binding affinities (ΔG ≈ –9.5 kcal/mol) and favorable drug-like properties (7). Thus, together, the computational and lab studies have indicated that diverse drug classes can act as FIASMs and support ASM as a plausible druggable host target. Therefore, these docking analyses are more of hypothesis-generating and can help prioritize candidates for experimental validation rather than confirming target engagement or mechanism in vivo. Since SARS-CoV-2 also relies on the ASM-ceramide pathway for entry and vesicle-mediated spread, FIASM use appears to be a highly promising antiviral strategy in COVID-19.

4.3 Clinical observations

Real-world clinical data evidence also complements the laboratory-based data. Retrospective clinical data from a large multicenter cohort study (n = 7230 hospitalized COVID-19 patients looked at outcomes in patients already on antidepressants, many of them being FIASMs. It was found that using antidepressants was associated with a 44% lower risk of intubation or death. The adjusted hazard ratio was 0.56 (95% CI 0.43–0.73) after controlling for illness severity and comorbidities (92). This protective effect was shown across various FIASM antidepressants like escitalopram, fluoxetine, and paroxetine (p < 0.05) compared to patients not being exposed to antidepressants. The class-wide protective association is consistent with their shared pharmacologic property of FIASMA/ASM–ceramide modulation. However, given that we have substantial off-target actions of SSRIs and tricyclics, the ASM inhibition should be interpreted as one plausible mechanism among several instead of being as a sole driver of outcomes.

The beneficial association is not limited to antidepressant FIASMs. It also extends to non-psychiatric FIASMs, showing that the benefit is not tied to mood disorder treatments but may relate to shared FIASMA-associated properties, while remaining compatible with additional off-target mechanisms. In an observational study, hospitalized COVID-19 patients who took amlodipine, a calcium channel blocker with antihypertensive properties, had lower mortality (8). These findings suggest that cardiovascular drugs having the FIASM property were potentially protective. In another large cohort study of more than 15,000 inpatients, of those receiving hydroxyzine (n = 1.1%), which is an H1 antihistamine with strong FIASM properties, experienced a ~49% lower 28-day mortality (adjusted OR ~ 0.51, p = 0.016) compared to controls (28). This finding was consistent with earlier French observational studies that noted reduced hospitalization among antihistamine users to suggest that the signal wasn’t limited to one study or population, showing that FIASM effects extend to non-psychiatric drugs.

Use of SSRIs, which are one of the most commonly utilized antidepressants, have been linked to reduced COVID-19 severity and mortality. A large US cohort study of ~83,500 patients compared SSRI users against non-users during COVID-19 hospitalizations. It was found that SSRI use had an 8% lower mortality risk compared to controls who were not prescribed a SSRI within the defined exposure window. Use of fluoxetine alone had ~ 28% lower mortality risk, while combined fluoxetine/fluvoxamine was found to provide a 26% lower mortality risk (93). The study also suggested that not all SSRIs were equally or significantly protective, with fluoxetine and fluvoxamine as the most effective. These real-world findings have mechanistic alignment with their role as potent FIASMs to block ceramide-dependent viral entry and vesicle pathways. Prospective clinical validation comes from a placebo-controlled fluvoxamine trial reporting prevention of clinical deterioration in COVID-19 patients (94) and a large, prospective trial called TOGETHER, where fluvoxamine intervention reduced hospitalization and prolonged ER observation by 30% (95). This unified body of data from a) retrospective cohorts showing broad associations between antidepressants, cardiovascular, etc., and b) prospective clinical studies validating protective effects holds when formally tested; collectively support the consistent clinical signal while the relative contributions of ASM inhibition versus off-target effects remain to be resolved. Yet it is not clear whether the protective effects of FIASM use were due to blocking viral transmission alone, or the widespread pro-inflammatory effects, or both.

4.4 FIASMs and MVP suppression

Apart from the antiviral property, FIASMs may also help with decreasing microvesicle (MVP) release that can spread viral particles and contribute to inflammation. During the SARS-CoV-2 infection, the infected or stressed host cells release EVs carrying viral RNA, proteins, and inflammatory mediators like cytokines (86). This release depends on ASM activity leading to ceramide generation at the cell membrane (37). In glial cell experiments, simply activating ASM without viral infection was sufficient to trigger microvesicle shedding. When the ASM was pharmacologically inhibited or genetically knocked out, the cells no longer produced the vesicles. Pharmacologic inhibition of ASM also stopped IL-1β secretion, potentially showing that inflammatory signaling directly depends on ASM activity (62). Thus, ASM acts like a master switch for stress-induced microvesicle release, which, when turned on, causes vesicle budding and inflammatory cytokine secretion, while switching it off with FIASMs shuts down both processes.

Given this connection, FIASMs would be expected to reduce pro-inflammatory MVP release during the SARS-CoV-2 infection, and this is supported by studies with skin models. For example, vesicle secretion has been reported to be triggered by stressors such as UV radiation and exposure to pro-inflammatory mediators in keratinocyte cultures, human skin explants, mice, and in humans (67). Further, the use of FIASMs like imipramine and amitriptyline blocked MVP shedding to prevent ceramide buildup at cell membranes (29). If we apply this to COVID-19, this suggests that FIASMs may block viral entry as well as limit any vesicle-mediated spread of viral or pro-inflammatory cargos. During SARS-CoV-2 infection, stressed cells may release more vesicles enriched in inflammatory mediators and coagulation factors, which circulate systemically to drive cytokine storm and coagulopathy; ASM inhibition could mitigate this (96, 97). Thus, FIASMs have a dual antiviral mechanism. First, ASM inhibition would prevent the formation of ceramide platforms at the cell membrane to disrupt ACE2/TMPRSS2 clustering and thereby block viral entry and fusion. Secondly, blocking ASM should decrease ceramide-driven vesicle release to limit the spread of viral RNA and particles and reduce vesicle-mediated inflammatory and coagulopathy signaling. This is consistent with the clinical evidence we have discussed.

5 Therapeutic applications and antiviral strategies

Building on the mechanistic insights of ASM-ceramide signaling in viral infections, there are considerable therapeutic opportunities. In this section, we will address these for both SARS-CoV-2 as well as for other viral infections.

5.1 FIASM repurposing for COVID-19

The findings we have discussed so far support the FIASM repurposing as a potential strategy to alleviate SARS-CoV-2 infection and COVID-19 severity, with ASM inhibition representing one plausible mechanism among several. However, there are important challenges in achieving desirable repurposing of FIASMs as COVID-19 therapeutics:

• Trial Design: Much of the current evidence comes from observational or small trials. They lack the scientific rigor and power of larger multisite randomized clinical trials. Conducting the latter can be challenging as one has to carefully select the right patient population (early vs. severe disease), ensure ethics (repurposing of psychiatric drugs for non-psychiatric use), and logistics (timely enrollment as the COVID-19 pandemic wave varies). The studies that showed fluvoxamine’s benefits highlighted these barriers, as despite there being successful placebo-controlled trials, it did not lead to a regulatory approval (85).
• Dosing/Pharmacokinetics(PK): The optimal doses for FIASMs for antiviral actions remain uncertain. These drugs tend to concentrate in tissues, suggesting that standard approved doses may already reach the therapeutic threshold in lungs. For example, amitriptyline reaches ~ 50-fold higher concentration in lungs than in plasma (98, 99). This suggests that supra-therapeutic dosing may not be required in plasma. However, no study has established the drug distribution in cases of acute systemic illness (98, 99,). Hence, underdosing carries a risk of ineffectiveness, while overdosing may lead to unknown adverse events, especially in vulnerable multi-morbid COVID-19 patients. Therefore, carefully designed pharmacology studies are needed to establish a safe and effective dosing regimen of FIASMs for viral indications.
• Off-Target Effects: Beyond ASM inhibition, FIASMs also act on additional pathways like serotonin reuptake inhibition, sigma-1 agonism, and immune pathways (100–102). These off-target activities can be beneficial. For example, Fluvoxamine’s sigma-1 agonism may attenuate cytokine production and decrease inflammation (103). However, such off-target activity can also cause undesirable effects like sedation, QT interval prolongation, and drug-drug interactions, especially in hospitalized COVID-19 patients already receiving multiple drugs. Hence, this requires a cautious approach in the interpretation of clinical data and safety monitoring (104, 105).
• It is to be noted that many FIASMs are repurposed psychoactive/sedating agents. Given that clinically important off-target effects (independent of ASM inhibition) have the potential to confound efficacy signals and raise safety issues in acutely ill patients. As an example, tricyclic antidepressants have been noted to have conduction/QTc effects at therapeutic concentrations (PR/QRS/QTc prolongation with imipramine) (106). The Torsades-de-pointes risk profile observed in published case reports was noted to be enriched by classic modifiers such as hypokalemia, metabolic disease, co-prescribed QT-prolonging drugs, etc. (107). Hydroxyzine was reported to have a conditional risk of TdP signal from pharmacovigilance data and ion-channel (hERG) inhibition, with most cases happening in the presence of additional risk factors (108). Selective serotonin reuptake inhibitors also contribute some non-ASM risks which are relevant to the hospitalized care, such as gastrointestinal intolerance (109) and differential hyponatremia/SIADH risk noted across common antidepressants in a large EHR cohort (110). Additionally, bleeding risk was linked to platelet serotonin/aggregation changes (111) with meta-analytic evidence of increased bleeding (112) and higher major bleeding when it was combined with oral anticoagulants (113). It is also vital to note the drug–drug interactions such as fluvoxamine inhibits CYP2C19 and increases omeprazole exposure (114). Thus, in order to be able to distinguish ASM-mediated antiviral effects from these off-target mechanisms, the future trials should (i) firstly pre-specify the target engagement (ASM activity and ceramide/SM indices in plasma/EVs) and then test whether biomarker shifts will correlate with virologic/clinical endpoints, (ii) Secondly, these should evaluate pharmacokinetic–pharmacodynamic exposure–target engagement relationships in the relevant disease stage and (iii) then whenever possible should use mechanism-separating comparators (such as class-matched non-FIASMs or FIASMs with divergent non-ASM pharmacology but having similar ASM inhibition strength). It is also important to consider that ASM dependence is virus and stage specific. For example, influenza A entry is sphingomyelin-sensitive but not ASM-dependent in experimental systems (45). This highlights that ASM inhibition may be ineffective in some contexts and therefore, we should be careful with timing and patient selection.
• Site of intervention: Given that FIASM could be applied topically, FIASM formulations for intranasal or even via inhaler use could have potential. For example, intranasal use of the mucolytic ambroxol with FIASM activities has been demonstrated to block pp-VSV-SARS-CoV-2-spike entry into freshly isolated human nasal epithelial cells ex vivo (84). This strategy could be useful for prophylaxis or even for immediate post-exposure treatment. Topical use of FIASM imipramine has been shown to block ultraviolet B radiation-induced MVP release and subsequent erythema in human subjects, suggesting that agents could be used to treat/prevent cutaneous viral infections such as HSV (115).

Hence, FIASMs offer us a promising, low-cost, widely available therapeutic strategy for SARS-CoV-2 and potentially other viruses with well-established safety and efficacy data. However, work needs to be done to address the challenges discussed.

Table 1 gives the summary of these virus specific roles and corresponding therapeutic implications for ASM targeted studies.

Table 1. Virus-specific ASM/ceramide roles and therapeutic implications.

5.2 Targeting microvesicle release as therapy

Beyond blocking viruses, another strategy is to interrupt the host cell processes that exacerbate disease by inhibiting pro-inflammatory microvesicle (MVP) release. ASM activation drives ceramide-dependent budding of plasma membrane–derived microvesicles (MVPs/large EVs) (69) carrying cytokines, tissue factor, and viral remnants, which promote inflammation and coagulation during infection (125–127). In COVID-19, Spike-induced ASM activation in macrophages decrypts tissue factor and triggers TF-loaded vesicle shedding, enhancing coagulation; these vesicles are elevated in severe cases (128). FIASMs such as tricyclic antidepressants blocked this process, reducing TF activation and procoagulant EV release, highlighting a therapeutic approach to mitigate COVID-19 coagulopathy (129).

Targeting the MVP release may curb cytokine storm and tissue injury in viral infections. Vesicles carry interleukins, proteases, and viral antigens, amplifying inflammation (130, 131). Limiting their formation could restrain immune overactivation and organ damage (132, 133). In endothelial cells, ASM inhibition may stabilize membranes and reduce export of adhesion molecules and cytokines that drive ARDS (134–137). This aligns with reports that FIASM continuation in COVID-19 correlated with lower inflammation and better outcomes (138).

While direct causal evidence is still being gathered, the therapeutic rationale is clear: by halting ceramide-driven vesiculation, we remove a fuel source for systemic inflammation. Importantly, this strategy targets the host response, not the virus. Combining ASM blockers with antivirals or steroids may both lower viral load and blunt immune damage. Agents that stabilize membranes or inhibit ceramide synthesis are under study, potentially extending MVP-targeted therapy to COVID-19, sepsis, or autoimmune flare-ups.

5.3 Engineered MVPs as decoys or drug carriers

Recent evidence shows that EVs are now being explored as therapeutic platforms particularly for viral neutralization and delivery of antiviral cargo. One of the approaches is the formation of viral decoys where the vesicles have viral receptors to act as traps. In COVID-19, it was reported that the circulating ACE2-expressing EVs increased in patients with the ACE2-carrying EVs neutralizing SARS-CoV-2 infection through competition with cellular ACE2 (139). Another approach is engineering the EVs as delivery system (140). For example, the SARS-CoV-2 spike receptor binding domain (RBD)-tagged EVs can recognize ACE2 on target cells to accumulate in ACE2-expressing tissues (141). These delivered antiviral siRNA cargo leading to reduction of SARS-CoV-2 pseudovirus infection in vivo in human ACE2-transgenic (hACE2)mice (141). In addition, red blood cell–derived EVs were shown to have endogenous antiviral effects against SARS-CoV-2 (140). They were reported to increase the delivery of antiviral oligonucleotide therapy with noted viral entry and replication inhibition in experimental systems (140). These approaches are very early-stage and are not direct extensions of ASM/ceramide-dependent vesiculation. Hence, these should be viewed as adjacent therapeutic directions.

6 Biomarkers and translational considerations

Recent studies highlight biomarkers of sphingolipid dysregulation and microvesicle dynamics that can support stratified medicine by reflecting disease severity and guiding therapy. These are measurable with current assays such as mass spectrometric-based lipidomics, ELISA, and flow cytometry (142, 143). Two main classes – sphingolipid metabolites and MVP cargo – show promise, including for monitoring responses to FIASMs.

6.1 Sphingolipid-based biomarkers

Sphingosine-1-Phosphate (S1P): S1P is a lipid that can act as a signaling messenger. It is abundantly found in plasma in healthy conditions (144). When severe infections happen, such as in COVID-19, the S1-P levels have been noted to fall sharply. This was associated with disease severity worsening (145). In one study of 111 COVID-19 patients, it was found that serum S1P was inversely correlated with COVID-19 severity. It correlated with other markers of severity, such as elevated C-reactive protein, LDH, ferritin, and D-dimer. Also, patients having low S1P were noted to be at higher risk of needing ICU care or mortality (146). This identifies S1-P as a negative prognostic biomarker. Hence, restoration of S1-P or preventing its reduction has been proposed as a potential therapeutic direction (146). S1-P can be quantified in blood using ELISA or mass spectrometry, making it a measurable biomarker (143).

Ceramides: Ceramides are lipids of the sphingolipid family. They are pro-apoptotic and are found to rise with the severity of the disease. For example, in COVID-19, ceramides and related sphingolipid molecules like dihydroceramide and sphingosine were found to increase in blood (143). Other ceramide species like C16:0, C18:0, and C24:1 were found to significantly increase in patients with severe disease. These showed high predictive accuracy for disease progression when they were tested statistically via the area under the ROC curve. Hence, serum “ceramide signature” was noted as a biomarker of poor prognosis (143). Conversely, falling ceramide levels correspond to improvements and were noted as a potential indicator of positive response to therapy (147). Ceramides can be measured using targeted lipidomics (LC-MS/MS) in patients (143).

Anti-Ceramide Antibodies: The immune system’s response to sphingolipids can also provide additional information beyond the measurement of lipid levels. A recent study done in non-small cell lung cancer (NSCLC) patients found that anti-ceramide IgM antibody levels were significantly higher compared to controls (278.7 ng/mL vs 178.6 ng/mL in controls, p = 0.007) (142). The same subset of patients also had about 10x S1-P levels compared to controls. Even though these did not predict survival in the lung cancer cohort, their presence confirmed that sphingolipid metabolism was altered in malignancy (142). This means that severe disorders like cancers can generate both lipid shifts and immune responses to lipids. Hence, the strategy to measure antibodies along with lipids could add diagnostic insight even if prognostic power is limited.

Collectively, Sphingolipids like ceramide and S1P are opposite signaling molecules. While ceramides are pro-death and pro-inflammatory, S1P is protective due to its pro-survival and pro-repair properties. Hence, they are very attractive options for therapeutic monitoring. Using a patient’s sphingolipid profile gives us a biological snapshot of the disease state. These biomarkers are measurable in blood using minimally invasive techniques and may be incorporated into future studies to support risk stratification and therapy monitoring once standardized and validated.

6.2 MVP cargo as biomarkers

In section 2.3, we discussed the ASM/ceramide-driven microvesicle (MVP/large EVs) shedding and how MVP/EV cargo can contribute to viral pathogenesis. In contrast, in section 6.2, we will focus on the biomarker utility of the MVP/EV measure. That is, the MVP/EV feature can be quantified in patient samples(counts, phenotypes, and cargo)and how they may correlate with disease severity or thrombotic risk.

Microvesicle Counts and Phenotypes: As discussed, microvesicles are released during stress conditions, and these carry surface markers from parent cells from(e.g.; monocytes or platelets derived). etc. As they can reflect cell damage, microvesicles are real-time indicators of tissue injury or damage. In COVID-19, researchers found that patients had higher levels of both platelet-derived and monocyte-derived microvesicles in plasma (148). Severe patients also had higher levels of annexin-V-positive platelet microvesicles, indicating extensive cell activation and apoptosis. The platelet microvesicles expressing the platelet-specific marker of CD41a were found to strongly correlate with D-dimer (148). This suggested that microvesicle release was linked to coagulopathy and endothelial injury commonly seen in severe COVID-19 cases. Hence, measuring microvesicles by flow cytometry using markers like CD41a, CD42b for platelets and CD14 for monocytes can serve as a research/prognostic indicator of disease severity and thrombotic risk (148, 149).

miRNAs in EVs: EVs, particularly exosomes/small EVs, often have microRNAs (miRNAs) that can regulate gene expression in recipient cells. When enclosed in EVs (including exosomes/small EVs), the miRNAs remain protected from any degradation by RNases to enable their stability and measurability in blood. The severe COVID-19 patients have very distinct miRNA patterns in EVs compared to healthy controls. It was noted that miR-145-5p levels were significantly lower in COVID-19 patients compared to controls. The levels were found to inversely correlate with inflammatory markers like CRP and fibrinogen (9). Other miRNAs like miR-27b-3p, miR-320a/b, and miR-424-5p have been found to be increased in patients and linked to coagulopathy pathways (9). This pattern suggests that microvesicle miRNAs can act as biosignatures of severe infection. Importantly, these are not just biomarkers but also linked to pathology. For example, downregulated exosome miR-145 and miR-885 were linked to higher thrombotic events and mortality in COVID-19 patients (150). The endovascular miRNAs can be analyzed by RT-qPCR and Next-generation sequencing. Hence, they can serve as diagnostic or prognostic biomarkers for severe infection and monitoring them can help identify patients at risk for thrombosis or death.

Inflammatory or Viral Proteins in MVPs: Apart from lipids or RNAs, microvesicles also carry proteins from their parent cells that can act as signaling molecules of inflammation, coagulation, or infection to give a convenient window into disease processes. For example, endovascular Tissue Factor (TF), which is a pro-coagulant protein, was found to be 10 x higher in severe COVID-19 patients (median = 231 fM) compared to moderate disease severity (median = 25 fM) (151). The endovascular TF activity was strongly correlated with leukocyte numbers, indicating immune activation, with D-dimer to reflect clot burden and with inflammatory markers to indicate systemic response. Hence, endovascular TF is a risk biomarker for thrombotic events in COVID-19. The endovascular TF can be measured using factor Xa generation assays and may help risk stratification in research settings and inform anticoagulation hypotheses in prospective studies (151). For broader relevance, other than TF, other proteins inside microvesicles can act as useful biomarkers, such as inflammatory mediators to reflect immune activation and viral components to indicate persistent infection.

Hence, microvesicles carry cargo that can reflect what is happening inside the stressed or injured cells as they bud off directly from them. This makes the circulating fingerprints of pathology accessible using a simple blood test. The microvesicle biomarkers can show numbers, size distribution to indicate tissue injury or inflammation, such as increasing endothelial-derived MVPs may suggest ongoing vascular injury. The micro vesicle biomarkers also show cargo content that can reflect disease processes such as pro-inflammatory proteins, indicating worsening disease. Hence, clinical application can include serial tracking over time to show if patients are improving or deteriorating. This makes microvesicles especially useful for monitoring disease trajectory and determining if the treatment response is working.

Translational limitations. It is of note that most sphingolipid and EV/microvesicle biomarkers still remain very much in the research phase. Interpretation is sensitive to pre-analytical variables (sample type, timing, storage), assay methodology (lipidomics platform and normalization; EV isolation strategy) and measurement heterogeneity (flow cytometry gating/size thresholds; TF activity assay conditions). Hence, these markers should be in current state viewed as promising translational candidates which requires standardization, external validation and prospective testing before routine clinical adoption.

6.3 MVPs in monitoring treatment response (FIASM context)

Sphingolipid Level Shifts: We know FIASMs inhibit ASM activity, and this leads to reduced ceramide levels to indicate lower pro-apoptotic/pro-inflammatory signaling, higher sphingomyelin, and potentially higher S1-P to indicate protective signaling due to altered sphingolipid flux. These lipid changes can be measured in plasma to support pharmacodynamic monitoring/target engagement hypothesis. For example, by looking at ceramide levels before and during therapy, clinicians can see if FIASM worked as intended. Supporting evidence comes from a preclinical Alzheimer’s study where treatment with the FIASM imipramine significantly reduced ceramide buildup in the brain. The EVs derived from the brain exhibited less ceramide and less pathogenic Aβ cargo in comparison to the control (152). This showed that FIASMs can remodel sphingolipid metabolism in disease. Applying the analogy to COVID-19, a patient on FIASM therapy may show reduced ceramide and rising S1-P levels to show a balance shift towards a less inflammatory and more protective state. Such lipid changes can be accurately measured by mass spectrometry-based lipidomics, which is feasible in clinical laboratory settings (143).

Microvesicle Release and Content: As discussed, sphingomyelinase activity (ASM or neutral, depending on vesicle subtype) is known to cause more ceramide release and subsequently higher vesicle shedding (153–156). FIASMs can thus alter MVP release to reduce ceramide at the cell surface. For example, in vitro studies have indicated that 25 μM imipramine was found to reduce total EV release by 77% in cancer cells (157). This showed that patients having pathologically high MVP levels, such as in COVID-19, may have their microvesicles trend downwards to normal once FIASM treatment is underway. The clinical relevance can be seen in COVID-19. Patients with severe disease showed high levels of prothrombotic EVs rich in ceramide. FIASMs, in theory, could therefore reduce EV generation and reduce prothrombotic risk. Hence, EV count and cargo can be tracked during therapy as biomarkers of response, as a reduction in TF activity can signal lower coagulopathy risk, while restoration of protective exosomal miRNAs like miR-145-5p, which are usually low in severe COVID-19, can indicate decreased cellular stress and better vascular health.

Clinical Outcome Correlation: Biomarkers will only be clinically useful if they track or correlate with patient outcomes as well to give real-world clinical value. Evidence regarding COVID-19 and FIASMs comes from a retrospective study of hospitalized COVID-19 patients on FIASM psychotropics, who had 58% lower risk of progressing to intubation or mortality compared to controls. The hazard ratio was 0.42, showing risk reduction by more than half (158). This suggests that ASM inhibition via FIASMs has clinically meaningful benefits. In the future, trials can integrate monitoring of biomarkers along with clinical outcomes. For example, patients who show the highest reductions in ceramides or EVs -TF may be those who experienced the most clinical improvement. These types of studies can further validate these biomarkers as surrogates of treatment response. These findings will move the field towards more stratified therapy. For example, if sphingolipid and EVs profiles are measured at baseline levels, then patients can be organized into groups such as high ceramide/TF, which are likely to benefit most from FIASM treatments, and lower risk profiles that may not need aggressive intervention. As therapies progress, biomarker trajectory tracking can guide adjustments in therapy.

In summary, sphingolipid-based and microvesicle-based biomarkers form a new frontier in how we may diagnose, monitor, and treat complex diseases. They provide direct molecular signals of what is happening in the body. Biomarkers such as ceramides, S1-P, microvesicle counts, and EV cargo can glean some insight into how sick a patient may be and what their likely trajectory is. For example, higher ceramide levels reflect a higher risk. It can also help with therapeutic monitoring in real time as they are measurable using blood tests, where clinicians can track changes during treatment. Since ceramides and others can be quantified, we can measure FIASM impact directly in patients. Clinically, these biomarkers support a personalized medicine approach, which can stratify by baseline biomarker profiles and can individualize therapy based on biomarker shift to make treatment more effective and precise.

7 Conclusion

Sphingolipids are involved in various viral stages. ASM-generated ceramide platforms and ceramide-driven microvesicle shedding play a role from attachment to fusion to replication and egress. A large diversity of viruses uses or avoid this axis. A few examples include rhinovirus, measles virus, Ebola virus, SARS-CoV-2, influenza A virus, HIV, noroviruses, and rubella virus. Specifically in COVID-19, the ceramide platforms lead to enrichment of ACE2/TMPRSS2 clustering at the cell membrane and enhance microvesicle-mediated cytokine and coagulation cascades. FIASM, which are ASM inhibitors and come in various forms like FDA-approved SSRIs, TCAs, etc., have convergent mechanistic, preclinical, and clinical real-world signals. They show dual promise of blocking viral entry and reducing microvesicle-driven inflammation and coagulopathy. However, it remains essential that dosing is rigorous, trial design is optimum, and off-target management is addressed. Apart from inhibition. engineered vesicles such as ACE-2 decoys, EVs, deliver drugs, give us novel antiviral delivery and neutralization pathways. Translationally, the sphingolipid and microvesicle biomarkers, such as reduction in S1-P, increasing ceramide, TF-positive microvesicles, and endovascular miRNA signatures, make severity stratification and pharmacodynamic monitoring feasible and effective. This supports adaptive, personalized treatment strategies. Hence, targeting the ASM-ceramide-Microvesicles axis offers us a broad antiviral spectrum and great immunomodulatory potential that extends beyond COVID-19.

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Footnotes

Edited by: Matloob Husain, University of Otago, New Zealand

Reviewed by: Chee Keng Mok, Changping National Laboratory, China

HuiFeng Qian, Shaoxing Second Hospital, China