key: cord-0288465-qn6wawxk
authors: Cecon, Erika; Fernandois, Daniela; Renault, Nicolas; Coelho, Caio Fernando Ferreira; Wenzel, Jan; Bedart, Corentin; Izabelle, Charlotte; Wimez, Sarah Gallet; Poder, Sophie Le; Klonjkowski, Bernard; Schwaninger, Markus; Prevot, Vincent; Dam, Julie; Jockers, Ralf
title: Melatonin drugs inhibit SARS-CoV-2 entry into the brain and virus-induced damage of cerebral small vessels
date: 2022-01-03
journal: bioRxiv
DOI: 10.1101/2021.12.30.474561
sha: fdfeed734d640494c9f972e896212f1e2092a228
doc_id: 288465
cord_uid: qn6wawxk

COVID-19 is a complex disease with short- and long-term respiratory, inflammatory and neurological symptoms that are triggered by the infection with SARS-CoV-2. Invasion of the brain by SARS-CoV-2 has been observed in humans and is postulated to be involved in post COVID condition. Brain infection is particularly pronounced in the K18-hACE2 mouse model of COVID-19. Here, we show that treatment of K18-hACE2 mice with melatonin and two melatonin-derived marketed drugs, agomelatine and ramelteon, prevent SARS-CoV-2 entry in the brain thereby reducing virus-induced damage of small cerebral vessels, immune cell infiltration and brain inflammation. Brain entry of SARS-CoV-2 through endothelial cells is prevented by melatonin through allosteric binding to human angiotensin-converting enzyme 2 (ACE2), which interferes with the cell entry receptor function of ACE2 for SARS-CoV-2. Our findings open new perspectives for the repurposing of melatonergic drugs in the prevention of brain infection by SARS-CoV-2 and COVID-19-related long-term neurological symptoms.

As of 20 December 2021, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is estimated to have infected globally approximately 270 Mio. people, including more than 5 Mio. deaths and more than 500,000 new confirmed infected cases per 24h, reported to WHO (https://covid19.who.int/). Although COVID-19, the disease caused by the SARS-CoV-2 virus, targets primarily the lungs leading to an acute respiratory disease, increasing evidence indicates a more widespread infection of other organs including the heart and blood vessels, kidneys, gut, and brain 1 . Among the secondary symptoms, various neurologic signs and symptoms have been reported such as headache, nausea, anosmia, myalgia, hemorrhage, syncope, seizure, and stroke 2 . Many of these symptoms remain long after the acute illness has passed, a phenomenon described as "long COVID", post COVID-19 condition 3 or Post-Acute Sequelae of SARS-CoV-2 infection (PASC) 4 5 . The estimated incidence of a neurological or psychiatric diagnosis in the following 6 months of infection is of 33% with no apparent correlation with severeness of COVID-19 in the acute phase, using electronic health records of over 236,000 patients with COVID-19 6 . Several possible mechanisms explaining the neurological symptoms and the neuro-invasive potential of SARS-CoV-2 have been discussed 7 . Mechanisms by which the virus accesses the brain include a direct viral infection of host endothelial cells, altering the tight junction proteins forming the blood-brain barrier (BBB) resulting in a leaky BBB (paracellular migration), or a phagocytosis by circulating immune cells followed by brain infiltration of these cells ("Trojan horse" strategy, by transcellular migration) 7, 8, 9 . Another entry gate into the central nervous system is located at the neuralmucosa interface in the olfactory mucosa 10 . Much attention has been given to identify potential treatments for lung infection and to control the cytokine storm, but only very few efforts to treat or prevent symptoms of post COVID condition have been made so far.

Melatonin is a natural hormone produced by the pineal gland during the night with a wide range of effects on the central nervous system (CNS) including the regulation of the biological master clock in the hypothalamus and sleep on-set. Additional favorable effects are its neuroprotective, anti-inflammatory and anti-oxidant actions 11 . Melatonin acts through a variety of target proteins 12 of which the two high-affinity G protein-coupled receptors, MT 1 and MT 2 , are best-described 13 . Currently marketed drugs (ramelteon, agomelatine, tasimelteon and slow-release melatonin) acting on MT 1 and MT 2 are indicated for insomnia, « jet-lag », and depression 14 and have been proven to show a good safety profile displaying few or no side-effects 15, 16 . Recent systems-network-pharmacology studies revealed melatonin as one of the top-scoring molecules with potential anti-COVID-19 action 17, 18 .

Based on this large spectrum of action of melatonin, its potential beneficial effects to treat COVID-19 have been postulated in several review articles 19, 20, 21, 22 . However, only very few experimental data from animal models or humans are currently available. We have recently shown that melatonin treatment of K18-hACE2 mice (expressing the human ACE2 receptor) delayed the occurrence of severe clinical outcome and improved survival, associated with a dampening of virus-induced type I and type III interferon production in the lungs 23 .

Here, we show that daily injection of melatonin and melatonergic compounds largely diminishes SARS-CoV-2 infection of the brain in the K18-hACE2 COVID-19 mouse model, which displays high level of viral brain penetrance, by reducing viral entry through brain endothelial cells and damage of cerebral small vessels. This goes along with a concomitant reduction of neuroinflammatory markers and of markers of immune cell infiltration.

Furthermore, we identified a new binding target of melatonin, as melatonin reduces the entry of SARS-CoV-2 into brain cells by binding to ACE2, the SARS-CoV-2 cell entry receptor.

To evaluate the potential beneficial effect of melatonin and the two clinically used melatonin receptor ligands agomelatine (AgoMLT) and ramelteon (RML) on SARS-CoV-2 infection in the brain, we chose the K18-hACE2 mice, a robust model of brain infection by SARS-CoV-2 24, 25 . For melatonin, two doses were chosen, 10 mg/kg (MLT10) and 50 mg/kg (MLT50), aiming to maintain levels high over time as melatonin has a short plasma half-life of 20-30 minutes 26 . Intra-peritoneal treatment of mice with compounds started two days before intranasal infection of mice and was repeated daily until sacrifice at day 7 post infection (DPI-7) at the latest. Lungs and brain were collected for biochemical and histological analysis.

The course of development of COVID-19 in infected K18-hACE2 mice was followed daily by evaluating body weight, activity and piloerection, respiration, lethargy and eye closure and a clinical score ranging from 0 to 14 was determined according to previously established guidelines with a score of 0 being perfectly healthy and a score of 14 being severely ill 27 .

Clinical scores worsened markedly from DPI-5 to DPI-6 in vehicle-treated animals, as expected, while a significant improvement on DPI-6 for MLT10 and MLT50 groups and a tendency for improvement for AgoMLT and RML was observed (Fig. 1A) . At DPI-7 the beneficial effect was only maintained for MLT50 (Fig. 1A) . This differential clinical evolution in treated groups is further evidenced when analyzing the frequency distribution of mice with high clinical score within each group, with a persistent beneficial effect at DPI-7, with the exception of MLT10 (Fig. 1B) . These data suggest that treatment with melatonin and melatonergic drugs delay the onset of clinical symptoms and slow down disease progression.

We then compared the viral load in the lungs and in the cerebral cortex by monitoring the transcripts for the SARS-CoV-2 nucleocapsid (or N-protein) by RT-PCR using a set of FDA-approved primers used to diagnose COVID-19 patients. In accordance with previous observations, the level of viral RNA in the lungs showed strong inter-individual heterogeneity in this model (Fig. 1C) 23, 25 . Overall, the treatments did not seem to affect pulmonary viral load (Fig. 1C) . The N-protein transcript was also detected in the cortex of 100 % of the mice in the vehicle group, although at overall levels approximately three orders of magnitude lower than those in the lungs (Fig. 1D) . Despite the inter-individual heterogeneity, treatments with MLT10, AgoMLT and RML showed a tendency to lower viral load in the cortex, while MLT50 significantly decreases cortical viral load, an observation that was confirmed with two other FDA-approved N-protein primer pairs ( Fig. 1D ) (Supplemental Figure S1A ,B).

The effect of the treatment is confirmed when analyzing the distribution of mice into three categories according to their viral RNA load in the cortex classified per tercentiles (Fig. 1E) .

Whereas the majority of the mice in the vehicle-treated group falls into the category with the highest virus load, the treatment decreases the contingency in this category, and the frequency distribution was significantly different among the groups (Chi 2 -test, p=0.0065). When analyzing the contingency distribution of mice for each treatment compared to the vehicle group, MLT50 (Chi 2 -test, p=0.0357, Supplemental Figure S1C ) was the most efficient treatment, as MLT50 largely suppressed the category of mice with the highest viral load and conversely increased the number of animals with low viral load in the cortex ( Figure 1E ; Supplemental Figure S1C ). To a lower extent, MLT10 and RML also improved the distribution of mice (Chi 2 -test, p=0.0498 and 0.0357, respectively, Supplemental Figure   S1C ) increasing the intermediate category and decreasing the category of high cortical virus load. Decreased expression of the N-protein in treated mice was confirmed by microscope imaging of brain slices and the difference was even more pronounced in the heavily infected hypothalamic region of the brain (Fig. 1F -G) (Supplemental Figure S2, 3) . These results indicate that treatment with melatonin drugs decreases infection of the brain by SARS-CoV-2, even in the K18-hACE2 mouse model highly susceptible to brain infection.

The well-documented anti-inflammatory effect of melatonin has been hypothesized to be beneficial in COVID-19 treatment (see 28 for review). In cortex samples of SARS-CoV-2infected K18-hACE2 mice, MLT50 treatment tends to decrease the virus-induced mRNA expression levels of the proinflammatory cytokines Tnf (p=0.0514), Il1b (p=0.1191) and Il6 (p=0.0864) compared to vehicle-treated mice ( Fig. 2A-C) . Interestingly, the MLT50 treatment also tended to decrease mRNA levels of the chemokines Cxcl1 (p=0.1607) and

Cxcl2 (p=0.0690) ( Fig. 2D-E) and of markers of infiltrating macrophages Trem1 (p=0.0964) and Ms4a8a (p=0.0223) (Fig. 2F-G) . When considering the contingency distribution of infected mice in different treatment categories according to the tercentile classification of inflammatory markers in low, intermediate and high levels, a significant effect of MLT50 compared to vehicle was observed ( Fig. 2H-N) . MLT50 significantly decreased the number of mice with high levels and conversely increases the number of mice with low levels of Il1b (p=0.0384), Il6 (p=0.0350), Cxcl1 (p=0.0472), Cxcl2 (p=0.0221), Ms4a8a (p-=0.0357) with tendencies for Tnf (p=0.0989) and Trem1 (p=0.0578) ( Fig. 2H-N) . The other treatments (MLT10, AgoMLT and RML) had either no effect or a weaker effect than the MLT50 condition. Taken together, in particular MLT50 treatment had a favorable effect on preventing SARS-CoV-2-induced brain inflammation in K18-hACE2 mice, in accordance with the lowest levels of viral brain infection observed in this group.

Recent evidence indicated that COVID-19 damages cerebral small vessels in humans and K18-hACE2 mice by infecting brain endothelial cells 29 , which might favor brain infection by disrupting the blood-brain barrier. When staining cortical sections of our SARS-CoV-2infected K18-hACE2 mice for the basement membrane component collagen IV and the endothelial marker caveolin-1, we were able to replicate these results by showing the presence of string vessels that are formed when endothelial cells die (Fig. 3A) . The analysis showed increased string vessel number and length upon SARS-CoV-2 infection (Fig. 3B-C) .

Interestingly, both parameters were significantly decreased in MLT50, AgoMLT and RML treated animals ( Fig. 3A-C) , while MLT10 showed only a tendency for decrease ( Fig. 3A-C) .

Interestingly, the overall brain vascular density was found to be decreased in brains of mice infected with SARS-CoV-2, and MLT50 as well as RML and AgoMLT treatments protected the brains from capillary rarefaction ( Fig. 3D-E) . The mechanism by which SARS-CoV-2 damages cerebral small vessels involves the cleavage of the host protein NF-kappa-B Essential Modulator (NEMO), shown to be required for the anti-viral immune response 30 , by M pro , the main protease of SARS-CoV-2 29 . As melatonin has been shown to inhibit NFkappa-B activation 31, 32 , we evaluated whether melatonin and melatonergic ligands have an effect on M pro -induced cell death by investigating cell survival of cultured brain endothelial hCMEC/D3 cells transfected with a plasmid encoding for M pro . Treatment of hCMEC/D3 cells with melatonin, AgoMLT or RML (100µM, 48h) did not abrogate the effect of M pro on cell death (Fig. 3F-G) . Expression of the catalytically inactive C145A-M pro mutant 33 was used as a control and was without effect on cell survival, as expected ( Fig. 3F-G) .

Collectively, these data indicate that all treatments prevent SARS-CoV-2-induced string vessel formation and protected the vascular system density. These effects could not be explained by inhibition of apoptosis of endothelial cells induced by the viral M pro protease, suggesting thus that melatonin and its derivative act upstream of M pro , likely at the level of SARS-CoV-2 cell entry.

To explore an action of melatonin in endothelial cells occurring upstream of the M pro viral protease activity, such as SARS-CoV-2 cell entry mechanisms, we analyzed the effect of the treatments on the expression levels of the major components of the SARS-CoV-2 entry machinery: ACE2, neuropilin 1 (NRP1) and TMPRSS2 34, 35 . SARS-CoV-2 infection or compound treatments did not change the global mRNA levels of human ACE2 (under the control of the K18 promoter), Nrp1 or Tmprss2 neither in the cortex or in the lungs (Supplemental Figure S4) . Similarly, no effect was observed on the expression of mouse Ace2, which does not participate in the virus entry in this model but could indicate an effect of the ligands on the regulation of the endogenous ACE2 promoter (Supplemental Figure S4) .

Knowing that the expression of melatonin receptors can be restricted to specific cell subpopulations in the mouse brain 36 , we analyzed their expression on brain slices of the cortical vasculature of mice by RNAscope. Expression of MT 1 and MT 2 melatonin receptors (encoded by the Mtnr1a and Mtnr1b genes, respectively) were specifically expressed in collagen IV-positive endothelial cells in the brain (Fig. 4A) . These results indicate that vascular endothelia might be the target of melatonin and its derivatives mediating their protective effects against brain infection.

We next isolated the CD31 + endothelial cell population by FACS sorting from the cortex of K18-hACE2 mice treated with vehicle or MLT50 for 9 days to further evaluate the effect of melatonin on gene expression. Successful enrichment of endothelial cells in the CD31 + fraction vs. neurons in the CD31fraction was confirmed by the strong reciprocal expression of Pecam (CD31) in CD31 + cells and Elavl4 (HuD, neuronal marker) in CD31cells ( Fig. 4B-C) . Consistent with the notion that brain endothelial cells are SARS-CoV-2 entry cells, expression of Nrp1 and murin Ace2, two SARS-CoV-2 entry receptors, was particularly prominent in CD31 + cells with an enrichment of 3 and 34 times, respectively, over CD31cells ( Fig. 4D-E) . Expression of human ACE2 (under the control of the K18 promoter) was similar in both fractions (Fig. 4F) , while Tmprss2 was predominantly expressed in CD31cells (Fig. 4G) . Collectively, these results show that brain endothelial cells are potential melatonin target cells for receptor-mediated melatonin effects and that melatonin decreases the expression of the endogenous ACE2 and NFκB pathway genes in these cells thus potentially contributing to the protective effect of melatonin on cerebral small vessels. However, downregulation of mouse Ace2 cannot explain the reduction of virus entry in K18-hACE2 mice, which is dependent on human ACE2 expressed under the control of the K18 promoter and the latter is not downregulated by melatonin, suggesting another inhibitory mechanism by melatonin.

To address whether melatonin interferes directly with viral entry into host cells we used a SARS-CoV-2 pseudovirus luciferase reporter assay in HEK293 cells. Naïve HEK293 cells are resistant to infection but become highly susceptible for infection upon ectopic expression of human ACE2 (Fig. 5A) . Melatonin inhibited ACE2-dependent virus entry in a concentrationdependent manner, with an estimated EC 50 of 20 µM and a maximal inhibition of 60-70% at the highest concentration (Fig. 5B) . These results were recapitulated in human brain endothelial hCMEC/D3 cells, in which pseudovirus cell entry is also shown to be dependent on the level of ACE2, and melatonin significantly inhibited the entry of the SARS-CoV-2 pseudovirus by 40% in hCMEC/D3 cells (Fig. 5C) . These data show that melatonin directly interferes with ACE2-dependent SARS-CoV-2 entry.

We then determined the effect of melatonin on the spike/ACE2 interaction in an in vitro assay measuring the binding of recombinant biotinylated receptor binding domain (RBD) of spike to purified ACE2 immobilized on the plate. Melatonin was unable to interfere with RBD binding to ACE2 whereas an excess (400 nM) of non-biotinylated S1 significantly diminished RBD binding, as expected, indicating that melatonin does not directly compete with RBD binding to ACE2 (Fig. 5D) . We then employed a second, time-resolved FRET (TR-FRET)based cellular spike-ACE2 binding assay 37 . The assay measures the binding of RBD to ACE2 in a cellular context, by following the energy transfer between the N-terminal SNAP-tagged human ACE2 labelled with the energy donor terbium (Tb) and RBD labelled with the energy acceptor d2 (RBD-d2) (Fig. 5E ). The energy transfer reports on the molecular proximity between the two proteins coupled to the energy donor and acceptor if they are within a maximal distance of less than 10 nm 38 . Binding of the RBD-d2 tracer was readily detectable in this assay and was partially inhibited by micromolar concentrations of melatonin with maximal inhibition levels reaching 20 to 30%, as compared to an excess of unlabeled RBD defining the specific TR-FRET signal (Fig. 5F ). This reduction in TR-FRET signal can be either due to a direct competition of RBD binding to ACE2 by melatonin or to an allosteric effect of melatonin modifying the conformation of the RBD-ACE2 complex that moves the energy donor and acceptor apart. To address the question of whether melatonin modifies the affinity of RBD for ACE2 we performed competition TR-FRET binding assays with increasing concentration of unlabeled RBD in the absence and presence of melatonin. The pIC 50 value of 7.80 ± 0.06 (n=3) confirmed the high-affinity binding of RBD which was, however, not modified by 10 or 100 µM of melatonin (pIC 50 7.77 ± 0.05, and 7.59 ± 0.08, respectively; n=3, data not shown). Taken together, the partial inhibition of viral entry and RBD binding to ACE2 by melatonin and the absence of any competitive response of melatonin on the RBD-ACE2 interaction strongly suggests an allosteric behavior and conformational modifications within the RBD-ACE2 complex induced by melatonin.

To gain insights in the potential binding mode of melatonin to the RBD-ACE2 complex, we performed a computational blind docking of melatonin using an exploration grid comprising the entire RBD and the same volume counterpart of ACE2 of three solved crystal structures of RBD-ACE2 complex, 6M17, 6M0J and 6VW1 39, 40 . None of the docking solutions revealed the existence of a melatonin binding pocket on RBD. The top-ranked solution revealed a previously unknown melatonin binding cavity on ACE2 that was located in close proximity to but not directly at the RBD-ACE2 interface (Fig. 5G) . Molecular dynamics simulations confirmed the melatonin binding site depicting the stability and the specificity of melatonin binding to ACE2 in the N-glycosylated 6VW1 PDB structure through three replicates of 500 ns (Supplemental Figure S5) . The stable binding site identified in the replicates, R1 and R2, is located close to the two helices of ACE2 (helix 19-52 and helix 53-89) interacting with RBD. Melatonin binds into a site delimited by Phe40, Trp69, Leu73, Ala99, N-glycoslyated Asn103 and Lys562 as observed in two of the three replicates. The third replicate shows a rapid dissociation of melatonin from 100 ns. This new binding site is distinct from the central catalytic site of ACE2, an observation which is consistent with the absence of any effect of melatonin on the enzyme activity of ACE2, as assessed in an in vitro enzymatic assay (Fig.   5H ).

The location of the binding site close to the two helices of ACE2 interacting with RBD indicates that melatonin might modulate the conformation of the ACE2 interface thus impacting on the spike-ACE2 interaction. To test this hypothesis experimentally, we developed an intra-molecular TR-FRET assay to probe the movement of ACE helix 19-52 by measuring the distance between a Flag-tag introduced in front of ACE helix 19-52, and the SNAP-tag of ACE2 (Fig. 5I) . As expected, unlabeled RBD, which modifies the position of ACE2 helix 19-52 compared to the apo-ACE2 state, changed the TR-FRET signal, reflecting RBD-induced conformational changes in ACE2 (Fig. 5J) . Melatonin also triggered a change in TR-FRET signal, confirming its binding to ACE2 and its impact on the position of ACE2 helix 19-52 (Fig. 5J) .

Taken together, our data indicate that melatonin binds to an allosteric binding site at ACE2 and modulates the RBD-ACE2 complex by modifying the position of helix ACE2 19-52 helix that contacts RBD, directly impacting on viral cell entry.

SARS-CoV-2 infects several tissues including the brain, an observation which has been largely neglected as considered irrelevant for the acute phase of COVID-19. The description of persisting symptoms, several months after the acute phase, like increased incidence of headache, fatigue and neurological symptoms, point to a potential role of the brain as target of the virus. Strategies to protect the brain might be therefore of interest to prevent long-term consequences of SARS-CoV-2 infection. We show here in the K18-hACE2 COVID-19 mouse model that treatment with melatonin, specially at a high dose, protects the brain by inhibiting SARS-CoV-2 entry into this organ. Melatonin binds to an allosteric binding site at ACE2, discovered in this study, that is connected to the molecular interface interacting with the viral spike protein thus preventing ACE2 to serve as an efficient SARS-CoV-2 entry receptor.

Additional beneficial effects of melatonin may arise from the fact that melatonin drives endothelial cells to a less reactive inflammatory state due to lower expression of NFkB pathway genes. Additionally, reduced endogenous expression of Ace2 could also participate in the decrease of viral entry in models where endogenous receptors are sensitive to SARS-CoV-2. We identified brain endothelial cells as the possible main melatonin target cells expressing ACE2 and MT 1 and MT 2 receptors. Our data indicate that melatonin has a major effect on the protection of cerebral small vessels by preventing SARS-CoV-2 entry, SARS-CoV-2-induced string vessel formation and reduction in vascular density. The protective effect of melatonin and its clinically used derivatives in K18-hACE2 mice, a model of high SARS-CoV-2 infection compared to humans, puts them in a privileged position as repurposing candidates for long COVID studies in humans.

Several reviews suggest the beneficial use of melatonin to treat COVID-19 contrasts with the only few experimental evidences 19, 22, 41 , a scenario that prompted us to evaluate the overall effect of melatonin and agomelatine and ramelteon, two highly potent clinically-approved melatonin analogs. Despite the fact that all three compounds act on high-affinity melatonin receptors 13, 42 , their effects were not identical in our study. Clearest effects were observed in the MLT50 group, followed by AgoMLT and RML, and finally MLT10. The difference between MLT10 and MLT50 argues rather against the involvement of high-affinity receptors which should be saturated at both doses. The similar pattern of effects observed for MLT50, AgoMLT, and RML for most parameters would suggest the involvement of a common, most likely low-affinity target, which could be ACE2. The need for the higher dose of melatonin (MLT50) compared to AgoMLT and RML could be explained by the shorter plasma half-life of melatonin compared to the synthetic drugs. The superior effect of MLT50 compared to AgoMLT and RML in many parameters could be either a dose effect or due to additional antioxidant properties of melatonin at this high dose that are not observed with AgoMLT and RML 11 .

We identified brain endothelial cells as the possible main melatonin target cells expressing Finally, treatment with fluvoxamine, one of the mechanisms of which is to increase plasma melatonin levels, has reduced the need for hospitalization in high-risk ambulatory patients with COVID-19 60 . Altogether, these first clinical results highlight a therapeutic potential of melatonin in COVID-19. Considering the reduction in brain infection and the decrease in the central inflammatory response with melatonin treatment, it is predicted that long-term neurological disorders could be avoided/attenuated with melatonin treatment. Melatonin is a medicine that can be obtained without a prescription. Unfortunately, retrospective studies, seeking to determine the efficacy of drugs against neurological disorders in COVID-19, and which would be based on cohorts of already existing patients taking or not taking melatonin supplements, do not allow a well-controlled analysis because the dose and the time of taking melatonin-derived medicines are usually not known. In addition, the selection of melatonin patient group included in the retrospective analyzes may be initially biased as patients with previous neurological disorders (fatigue, depression, memory loss…) are more inclined to use alternative supplements such as melatonin. Thus, an analysis directly devoted to studying the effects of melatonin treatment, specially at high doses, in relieving long-term neurological effects would be required.

When considering to use melatonin, agomelatine or ramelteon in the context of COVID-19, the chronobiological and sleep initiating effects of these three molecules have to be managed 14 . Application at the right time, in the evening before bedtime, and for a restricted period (probably during and shortly after the acute phase of infection), will minimize the risk of dysregulation of the circadian system and of sleep induction. Additional strategies include the chemical optimization of these melatonergic compounds to improve the specificity and affinity for ACE2 vs. MT 1 and MT 2 receptors.

Our study describes the first allosteric binding site of ACE2 and melatonin is the first allosteric modulator of ACE2 which negatively regulates ACE2 binding to the SARS-CoV-2 spike protein. This opens additional opportunities for the development of ACE2-specific drugs that interfere with the spike interaction 61 . Importantly, binding of melatonin to this allosteric binding site of ACE2 did not interfere with ACE2 enzyme activity thus avoiding potential severe side effects as ACE2 activity is essential for the proper function of the reninangiotensin system.

In conclusion, we disclosed ACE2 as a new binding target of melatonin, which leads to allosteric negative modulation of the spike/ACE2 interaction. This effect on the SARS-CoV-2 entry receptor function of ACE2, combined with the inhibition of NFKB expression and endogenous ACE2 expression in endothelial cells, are most likely the predominant mechanisms for impaired SARS-CoV-2 brain invasion by melatonin and its derivates. 

Melatonin, agomelatine and ramelteon were purchased from abcr GmbH (Karlsruhe, Germany). Compounds were reconstituted in vehicle solution (5% ethanol in sterile saline solution). K18-hACE2 transgenic mice were randomly divided into the following groups (6 mice/group): vehicle, melatonin 10mg/kg (MLT10), melatonin 50mg/kg (MLT50), Ramelteon (RML, 10 mg/kg), and Agomelatine (AgoMLT, 20 mg/kg), and were intraperitoneally (i.p.) injected with vehicle or melatonergic ligands daily, one hour before lights off (to minimize the risk of disturbing the natural daily rhythm of endogenous melatonin production). The treatment started 2 days before virus inoculation and continued until the end of the experiment (7 days post-infection, DPI-7). A group of non-infected mice was housed under the same conditions and similarly monitored during the whole experiment.

Mice were uniquely identified using ear tags and provided an acclimation period of 1 week before the experiment. Mice were supplied nutrient gel when weights began to decrease. Mice that met the human endpoint criteria were euthanized to limit suffering. The study was ended at DPI-7 and surviving mice were sacrificed at that time for comparative analysis. Lung and brain samples were taken directly after sacrifice and stored at -80°C until analysis.

All mice were weighed and examined for the presence of clinical symptoms daily, or twice per day after DPI-4, when symptoms were evident. The score for clinical symptoms was attributed following an IACUC approved clinical scoring system, and included the following criteria: body weight, posture/fur, activity/ mobility, eye closure, respiratory rate. Scoring was performed according to standard guidelines 27 with a maximal score of 14. Mice died either naturally from the disease or were sacrificed for ethical reasons when reaching a clinical score of 5 for 2 parameters and for 2 consecutive observation periods, or if weight loss was equal to or greater than 20%.

15 non-infected K18-hACE2 male mice were injected ip either with vehicle (ethanol 5% in saline) or melatonin 50mg/Kg (MLT50) daily for 9 consecutive days. The morning of the 10 th day, brain was extracted and cortex immediately submerged in the cellular dissociation buffer. 

For infected mice, total RNA was extracted from cortex by using the ReliaPrep™ 

Ace2

Mouse angiotensin I converting enzyme (peptidyl-dipeptidase A) 2

Human angiotensin I converting enzyme 2

Cxcl1 chemokine (C-X-C motif) ligand 1

Cxcl2

Elavl4 ELAV-like protein 4 (Hu-antigen D, HuD)

Il1b

Interleukin-1 beta 

For string vessel measurements, pepsin antigen retrieval was performed for 5 min at 37 °C (0.1 mg ml −1 pepsin in PBS, 0.2 N HCl) on 50 μ m thick brain sections. For vascular density, an antigen retrieval procedure (20 min at 95°C in 10 mM sodium citrate solution) was performed before the staining. Sections were blocked with 3% BSA in PBS containing 0.1% Triton X-100 for 6h at room temperature, and incubation with primary antibodies (collagen IV: Bio-Rad, #134001, 1:200; caveolin-1: Cell Signaling Technology, #3267, 1:400) was performed at 4 °C overnight, while incubation with secondary antibodies was performed in blocking solution at room temperature for 2h. Images were taken using a confocal laser scanning microscope (Leica, SP5). For all analyses, we imaged four fields from at least two sections per mouse and analysis was performed blinded using ImageJ.

In situ hybridization was carried out using the protocol from Advanced Cell Diagnostics 

Recombinant RBD protein were purchased from SinoBiological (Beijing, China) and reconstituted in water according to provider's instructions. Fluorescently labelled spike RBD proteins were obtained by custom labelling of the recombinant proteins with d2 fluorophore on lysines with a N-hydroxysuccinimide activated d2 dye in 100mM PO4 buffer (pH8) by Cisbio Bioassays. SNAP-tagged human ACE2 construct was designed as previously reported 37 .

SNAP-tagged ACE2, expressed in HEK293 cells, were fluorescently labelled by incubating cells with a SNAP suicide substrate conjugated to the long-lived fluorophore Terbium cryptate (Tb; Lumi4-Tb, 100 nM; Cisbio Bioassays) in Tag-lite labelling medium (1h, on ice) 64 . After several washes, cells were collected using enzyme-free cell dissociation buffer Data is expressed as TR-FRET ratio (acceptor/donor). When indicated, TR-FRET ratio was normalized to % of basal or % of control group.

ACE2 conformational changes were assessed by performing intramolecular TR-FRET assay, as previously described 37 . Briefly, Lumi4-Tb-labelled ACE2 cells were incubated with d2labelled anti-FLAG tag antibody (2 μ g/mL, 1h at room temperature; 61FG2DLF, Cisbio Bioassays), followed by addition of non-labelled RBD (5 nM) or melatonin (100 µM) and

TR-FRET signal was immediately read during 1h. Data is expressed as delta-TR-FRET ratio, corresponding to the difference in TR-FRET ratio signal between vehicle-and compoundstreated groups.

In vitro binding of spike S1 to ACE2 in the absence or presence of melatonin was performed using a commercially available ACE2:SARS-CoV-2 spike S1 inhibitor screening assay kit (BPS Bioscience, San Diego, USA). The assay is based on Streptavidin-HRP detection of spike S1-Biotin protein bound to ACE2-coated wells in a 96-well plate. Compounds were preincubated with spike S1-Biotin (10 nM, 1h, RT) and then added to the ACE2-coated wells.

After several washes, streptavidin-HRP solution is added (1h, RT) followed by addition of HRP substrate to produce chemiluminescence, which is then measured using a chemiluminescence reader.

The exopeptidase activity of ACE2 was measured using commercially available kit (BPS Bioscience, San Diego, USA). The assay is based on fluorescent detection of a fluorogenic ACE2 substrate, incubated with recombinant ACE2 (1h, RT) in the absence or presence of competitors (melatonin or DX600 (10 µM) competitor as positive control).

SARS-Cov-2 cell entry was assessed using a pseudovirus luciferase reporter assay (BPS Bioscience 

Data shown as the means ± SEM. Sample sizes were designed to give statistical power while minimizing animal use. All statistical comparisons were performed using Prism 9

(GraphPad). The specific statistical tests used for each experiment are indicated in the figure legends. One-or Two-way ANOVA with two-stage linear step-up procedure of Benjamini,

Krieger and Yekutieli as post-test for multiple comparisons or uncorrected Tukey test was applied and statistical significance was determined as p-value <0.05 (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

The authors declare that all data supporting the findings of this study are available within the paper and its supplementary information files. 

A rampage through the body

Central nervous system outcomes of COVID-19

Post-acute COVID-19 syndrome

Burdens of post-acute sequelae of COVID-19 by severity of acute infection, demographics and health status

6-month neurological and psychiatric outcomes in 236 379 survivors of COVID-19: a retrospective cohort study using electronic health records

COVID-19-Associated Neurological Disorders: The Potential Route of CNS Invasion and Blood-Brain Relevance

Potential neurological impact of coronaviruses: implications for the novel SARS-CoV-2

Nervous system involvement after infection with COVID-19 and other coronaviruses

Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19

Melatonin Target Proteins: Too Many or Not Enough?

Update on Melatonin Receptors. IUPHAR Review

MT1 and MT2 Melatonin Receptors: A Therapeutic Perspective

Agomelatine, the first melatonergic antidepressant: discovery, characterization and development

Safety of higher doses of melatonin in adults: A systematic review and meta-analysis

In-silico drug repurposing study predicts the combination of pirfenidone and melatonin as a promising candidate therapy to reduce SARS-CoV-2 infection progression and respiratory distress caused by cytokine storm

Network-based drug repurposing for novel coronavirus 2019-nCoV/SARS-CoV-2

COVID-19) and Its Neuroinvasive Capacity: Is It Time for Melatonin?

Can Melatonin Be a Potential "Silver Bullet" in Treating COVID-19 Patients? Diseases 8

Melatonin multifaceted pharmacological actions on melatonin receptors converging to abrogate COVID-19

Does the melatonin supplementation decrease the severity of the outcomes in COVID-19 patients? A mini review of observational data in the in vivo and in vitro studies

Therapeutic potential of melatonin and melatonergic drugs on K18-hACE2 mice infected with SARS-CoV-2

Human angiotensin-converting enzyme 2 transgenic mice infected with SARS-CoV-2 develop severe and fatal respiratory disease

Lethality of SARS-CoV-2 infection in K18 human angiotensinconverting enzyme 2 transgenic mice

Pharmacokinetics of oral and intravenous melatonin in healthy volunteers

Evaluation of K18-hACE2 Mice as a Model of SARS-CoV-2 Infection

The Coronavirus Disease 2019 (COVID-19): Key Emphasis on Melatonin Safety and Therapeutic Efficacy

The SARS-CoV-2 main protease M(pro) causes microvascular brain pathology by cleaving NEMO in brain endothelial cells

Innate immune sensing and signaling of cytosolic nucleic acids

Melatonin inhibits expression of the inducible isoform of nitric oxide synthase in murine macrophages: role of inhibition of NFkappaB activation

Melatonin inhibits LPSinduced NO production in rat endothelial cells

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity

Neuropilin1 as a new potential SARSCoV2 infection mediator implicated in the neurologic features and central nervous system involvement of COVID19

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C-terminal domain

SARS-COV-2 spike binding to ACE2 in living cells monitored by TR-FRET

HTRF: A technology tailored for drug discovery -a review of theoretical aspects and recent applications

Structural basis of receptor recognition by SARS-CoV-2

Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2

Clinical trial to test the efficacy of melatonin in COVID-19

International Union of Basic and Clinical Pharmacology. LXXV. Nomenclature, classification, and pharmacology of G protein-coupled melatonin receptors

ACE2 and AT4R are present in diseased human blood vessels

Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: A report of five cases

Cerebrovascular melatonin MT1-receptor alterations in patients with Alzheimer's disease

Melatonin and Nacetylserotonin inhibit leukocyte rolling and adhesion to rat microcirculation

Melatonin effect on endothelial cells reduces vascular permeability increase induced by leukotriene B4

Effect of melatonin in the rat tail artery: role of K+ channels and endothelial factors

Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures

SARS-CoV-2 infection induces the dedifferentiation of multiciliated cells and impairs mucociliary clearance

Heterogeneous expression of the SARS-Coronavirus-2 receptor ACE2 in the human respiratory tract

Expression of the SARS-CoV-2 cell receptor gene ACE2 in a wide variety of human tissues

Long-lasting priming of endothelial cells by plasma melatonin levels

GRL-0920, an Indole Chloropyridinyl Ester, Completely Blocks SARS-CoV-2 Infection

Melatonin and other indoles show antiviral activities against swine coronaviruses in vitro at pharmacological concentrations

A network medicine approach to investigation and population-based validation of disease manifestations and drug repurposing for COVID-19

Melatonin effects on sleep quality and outcomes of COVID-19 patients: An open-label, randomized, controlled trial

Efficacy of a Low Dose of Melatonin as an Adjunctive Therapy in Hospitalized Patients with COVID-19: A Randomized, Double-blind Clinical Trial

The Effect of Melatonin on Thrombosis, Sepsis and Mortality Rate in COVID-19 Patients

Effect of early treatment with fluvoxamine on risk of emergency care and hospitalisation among patients with COVID-19: the TOGETHER randomised, platform clinical trial

ACE2, the Receptor that Enables Infection by SARS-CoV-2: Biochemistry, Structure, Allostery and Evaluation of the Potential Development of ACE2 Modulators

Molecular Detection of SARS-CoV-2 Infection in FFPE Samples and Histopathologic Findings in Fatal SARS-CoV-2 Cases

Comparison of Abbott ID Now, DiaSorin Simplexa, and CDC FDA Emergency Use Authorization Methods for the Detection of SARS-CoV-2 from Nasopharyngeal and Nasal Swabs from Individuals Diagnosed with COVID-19

A general method for the covalent labeling of fusion proteins with small molecules in vivo

AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading

UCSF Chimera--a visualization system for exploratory research and analysis

High performance molecular simulations through multi-level parallelism from laptops to supercomputers

CHARMM36m: an improved force field for folded and intrinsically disordered proteins

CHARMM general force field: A force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields

Drs Morgane Bomsel, Fernando Real (Institut Cochin) and Francisco Foriestiero (Sao Paulo) for the discussion at the initial phase of the project and Dr. Lara Jehi and her team (Cleveland Clinic) for assistance. We thank Wiebke Brandt and Beate Lembrich (both Lübeck) for expert technical assistance

The authors thank Dr Pierre Olivier Couraud for the human hCMEC/D3

BBB cell line. The authors thank the Mesocenter of Lille University for computational resources

Centre National de la Recherche Scientifique (CNRS), the Deutsche Forschungsgemeinschaft (WE 6456/1-1 to J.W.). RJ was supported by the Fondation de la Recherche Médicale (Equipe FRM DEQ20130326503) and La Ligue Contre le Cancer N/Ref: RS19/75-127 and E.C. by the Association France Alzheimer

Molecular docking and simulations

Most hydrophobic interactions are exhibited in a zoomed view centered onto the binding site (right panel). H. ACE2 enzyme activity in the absence or presence of melatonin at 0.5 and 10 μ M; DW600 inhibitor (10 μ M) is shown as a positive control. I. Scheme illustrating the assay probing conformational change within ACE2 based on intramolecular TR-FRET between Lumi4-Tb-labelled SNAP-ACE2 and d2-labelled anti-FLAG tag antibody

01: ***p<0.005: *****p<0.0005 by one-way ANOVA

The authors declare no competing interest.