Vincent Berry

François Chevenet

Jean-Philippe Doyon

Emmanuelle Jousselin
email: e.jousselinemmanuelle.jousselin@inra.fr

J-P Doyon

A geography-aware reconciliation method to investigate diversification patterns in host/parasite interactions

Keywords: Ancestral trait, biogeography, cophylogeny, host/parasite, software, reconciliation, tree visualization

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

1) INTRODUCTION

Biotic interactions play a prominent role in species diversification. Interactions that result into longterm associations persisting over evolutionary time scales can sometimes lead to cospeciation, i.e. the concomitant occurrence of speciation in lineages that are ecologically associated [START_REF] Brooks | Hennig's parastological method: a proposed solution Systematic[END_REF][START_REF] Page | Temporal congruence and cladistic analysis of biogeography and cospeciation[END_REF][START_REF] Page | Clocks, clades, and cospeciation: comparing rates of evolution and timing of cospeciation events in host-parasite assemblages[END_REF]. The idea that such a pattern can occur first stemmed from parasitological studies suggesting that parasite classifications reflect the phylogenetic relationships of their hosts [START_REF] Fahrenholz | Ectoparasiten und Abstammungslehre[END_REF]. Hafner & coll. (Hafner & Nadler 1988;[START_REF] Hafner | Disparate rates of molecular evolution in cospeciating hosts and parasites[END_REF] were the first authors to thoroughly test this assertion. They used the association between pocket gophers and their lice as a model system and provided a clear demonstration that the phylogenies of the two interacting lineages were parallel.

This study spurred further research on cospeciation. The developments of specific methods that aimed at testing the congruence of the phylogenetic histories of interacting organisms have since played an important role in the study of cospeciation. It is indeed these methods that moved cospeciation studies beyond visual comparisons of phylogenetic trees and ad-hoc narratives for these visualizations. It soon became apparent that the study of the concordance between phylogenetic trees could be applied to reconciling gene trees and species trees [START_REF] Page | From gene to organismal phylogeny: reconciled trees and the gene tree/species tree problem[END_REF][START_REF] Page | Trees within trees: phylogeny and historical associations[END_REF]) which further enhanced the interest of evolutionary biologists for methodological developments in this field.

Reviews on cospeciation methods [START_REF] Brooks | Ending a decade of deception: a valiant failure, a not so-valiant failure, and a success story[END_REF][START_REF] De Vienne | Cospeciation vs host-shift speciation: methods for testing, evidence from natural associations and relation to coevolution[END_REF][START_REF] Doyon | Models, algorithms and programs for phylogeny reconciliation[END_REF][START_REF] Johnson | Untangling coevolutionary history[END_REF]Martínez-Aquino 2016;[START_REF] Paterson | Analytical approaches to measuring cospeciation of host and parasites: through a glass, darkly[END_REF][START_REF] Stevens | Computational aspects of host-parasite phylogenies[END_REF]) all emphasize the diversity and the complexity of the scenarios that must be explored when testing for the congruence of speciation events in two interacting lineages. To compare host and parasite phylogenies, Brooks & coll. (Brooks 1981;[START_REF] Brooks | Phylogeny, Ecology, and Behavior[END_REF] first developed a parsimony method (the Brooks Parsimony Analysis, BPA). In this method the associations between hosts and their parasites are transformed into a matrix of host characters and the parsimony tree reconstructed from such a matrix is then compared to the host phylogeny. A decade later, Page and collaborators developed a fundamentally different method, called "tree reconciliation", a term first coined in the work of [START_REF] Goodman | Fitting the Gene Lineage into its Species Lineage, a Parsimony Strategy Illustrated by Cladograms Constructed from Globin Sequences[END_REF] that compared gene and species trees. This method attempts to reconcile the phylogenetic history of the parasite with that of their hosts: the parasite phylogeny is "mapped" onto the host phylogeny (i.e. each node in the parasite tree is assigned to a node or a branch in the host phylogeny). In such a map, the diversification events of the parasites are linked to their host phylogenetic history and four types of events are considered: cospeciation events, host switches, sorting events and duplication events (Page 1994a;Page 1994b) (see material and method for a description of each event). When graphically displayed, reconciliation maps greatly ease our understanding of the evolution of biotic interactions.

Algorithms to optimize reconciliations are numerous. One of the first reconciliation software, TreeMap 2, uses an algorithm called "Jungles" [START_REF] Charleston | Jungles: a new solution to the host/parasite phylogeny reconciliation problem[END_REF] where each event is assigned a cost: the chosen reconciliations are the ones that have minimum costs. However it generates in the process an exponential number of scenarios. Recent methods have proposed algorithms that are more efficient and can also just search for an optimal reconciliation: e.g. Tarzan, [START_REF] Merkle | Reconstruction of the cophylogenetic history of related phylogenetic trees with divergence timing information[END_REF]; Jane, [START_REF] Conow | Jane: a new tool for the cophylogeny reconstruction problem[END_REF] ; Core-PA [START_REF] Merkle | A parameter-adaptive dynamic programming approach for inferring cophylogenies[END_REF], Mowgli, [START_REF] Doyon | An Efficient Algorithm for Gene/Species Trees Parsimonious Reconciliation with Losses, Duplications and Transfers[END_REF], COALA [START_REF] Baudet | Cophylogeny Reconstruction via an Approximate Bayesian Computation[END_REF], ecceTERA [START_REF] Jacox | ecceTERA: comprehensive gene tree-species tree reconciliation using parsimony[END_REF], Notung [START_REF] Stolzer | Inferring duplications, losses, transfers and incomplete lineage sorting with nonbinary species trees[END_REF], EUCALYPT [START_REF] Donati | EUCALYPT: efficient tree reconciliation enumerator[END_REF] and ILPEACE (van Iersel et al. 2014). Recently, the RASCAL software proposed to infer suboptimal scenarios to reduce computing times [START_REF] Drinkwater | RASCAL: A Randomized Approach for Coevolutionary Analysis[END_REF]. Cospeciation is witnessed on a reconciliation map whenever a speciation node in the parasite phylogeny is mapped onto a speciation on the host phylogeny. Another requirement for demonstrating that two interacting lineages have cospeciated is to provide evidence of the temporal congruence of the cospeciation event in the host and parasite phylogenies [START_REF] Page | Clocks, clades, and cospeciation: comparing rates of evolution and timing of cospeciation events in host-parasite assemblages[END_REF]. Though reconciliation algorithms do not strictly enforce the simultaneity of cospeciation events, they can enforce time consistency in the sequence of . CC-BY-NC 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under

The copyright holder for this preprint (which was not this version posted January 16, 2018. ; https://doi.org/10.1101/166215 doi: bioRxiv preprint 3 evolutionary events, meaning that the parasite cannot switch back in time onto a host that no longer exists (i.e. transfers cannot occur towards a node in the host phylogeny that has already split into child species at the time of the transfer event) [START_REF] Merkle | Reconstruction of the cophylogenetic history of related phylogenetic trees with divergence timing information[END_REF]Nojgaard et al. 2017). This constraint is explicit in Mowgli [START_REF] Doyon | Models, algorithms and programs for phylogeny reconciliation[END_REF][START_REF] Doyon | An Efficient Algorithm for Gene/Species Trees Parsimonious Reconciliation with Losses, Duplications and Transfers[END_REF], ecceTera [START_REF] Jacox | ecceTERA: comprehensive gene tree-species tree reconciliation using parsimony[END_REF] and RASCAL [START_REF] Drinkwater | RASCAL: A Randomized Approach for Coevolutionary Analysis[END_REF]. Hence, reconciliation methods have greatly improved in the last decade; algorithms are now efficient and some have solved the time consistency issue that affected some of the first methodological developments in the field. However, interpreting the scenarios that emerge from these inferences remains a difficult task. It is generally challenging to identify biologically realistic reconciliations. Much remains to be done to improve these inferences and translate them into evolutionary scenarios that give insights into the biological factors that govern the evolution of interspecific associations.

Some key information that could significantly improve our inferences but are overlooked in codiversification methods are the geographic locations of extant and ancestral nodes. Indeed, the biogeographic histories of interacting lineages necessarily constrain their common part of evolutionary history [START_REF] Martinez-Aquino | Do the Historical Biogeography and Evolutionary History of the Digenean Margotrema spp. across Central Mexico Mirror Those of Their Freshwater Fish Hosts (Goodeinae)?[END_REF][START_REF] Nieberding | The use of co-phylogeographic patterns to predict the nature of host-parasite interactions, and vice versa[END_REF]. Obviously, a cospeciation event can only happen between taxa that co-occur in the same area.. The geographic context of both hosts and parasites also influences host switch events. In biotic interactions where the parasites can undergo long dispersal events, transfers can happen between allopatric hosts (i.e. hosts that do not live in the same geographic area). However, they are only possible if the geographic locations of the "sending host" (the host from which the switch is initiated) and the "receiving host" coincide with a dispersal event along the corresponding branch in the parasite phylogeny. Therefore, a more accurate mapping of cospeciation and host switch events can be obtained if the geographic locations of both hosts and parasites are known prior to conducting the reconciliation.

Methods for inferring historical biogeography from phylogenetic reconstructions have greatly improved in the last two decades. Early developments in historical biogeography aimed at reconstructing "area cladograms" that reflected the history of connections between areas of endemism for the group of organisms under study and used analytical tools that were very similar to the tools developed for the study of cospeciation using parsimony as the optimization criterion (e.g., BPA, see [START_REF] Morrone | Evolutionary biogeography. An integrative approach with case studies[END_REF] for a review on cladistic biogeography and its methodological developments). More recent probabilistic methods in the field of historical biogeography aim at reconstructing ancestral geographic range of focal lineages from current species distribution and a dated phylogenetic tree.

They model the evolution of geographic areas on a phylogenetic tree using Maximum Likelihood optimization or Bayesian inference and incorporate divergence times into the inference process: the longer the phylogenetic branch, the higher the probability of geographic range shifts and the larger the uncertainty in the ancestral range estimates. Geographic areas can be treated as simple categorical characters that are reconstructed on the tree using for instance a stochastic Markov model of evolution.

More biologically realistic and widely applied methods in historical biogeography, such as DEC (Dispersal, Extinction, Cladogenesis) [START_REF] Ree | A likelihood framework for inferring the evolution of geographic range on phylogenetic trees[END_REF][START_REF] Ree | Maximum likelihood inference of geographic range evolution by dispersal, local extinction, and cladogenesis[END_REF], model range evolution using different parameters for each biogeographic process (dispersal, range expansion or extinction).

In addition to modelling these key processes in range evolution, the main innovation of DEC consists in incorporating a time-dependent transition matrix that defines the movements between geographic areas, at different time intervals in order to reflect how dispersal opportunities changed through time (e.g. changes in the continents configuration for instance) (seeRee & Sanmartin 2009; [START_REF] Ronquist | Phylogenetic methods in biogeography[END_REF], for reviews on parametric biogeography). Fossil distribution and information on the climatic preferences of ancestral lineages can also be incorporated as constraints to improve biogeographic inferences [START_REF] Meseguer | Integrating Fossils, Phylogenies, and Niche Models into Biogeography to Reveal Ancient Evolutionary History: The Case of Hypericum (Hypericaceae)[END_REF]. Several conceptual and computational improvements have been [START_REF] Reddy | An Extended Maximum Likelihood Inference of Geographic Range Evolution by Dispersal, Local Extinction and Cladogenesis[END_REF]. Different biogeographic models have also been proposed (GeoSSE, Goldberg et al. 2011;BayArea Landis et al. 2013). As a result, robust biogeographic scenarios are now available for numerous lineages. Ancestral areas inferred by these methods can then serve as input for reconciliation analyses. In this paper we build on these advances to provide a geography-aware reconciliation method, pushing further the realism of scenarios proposed by such methods.

We first describe the constraints we enforce to ensure geographic consistency in reconciliations and how they were implemented in the Mowgli reconciliation software [START_REF] Doyon | An Efficient Algorithm for Gene/Species Trees Parsimonious Reconciliation with Losses, Duplications and Transfers[END_REF]. We also updated the SylvX reconciliation viewer [START_REF] Chevenet | SylvX: a viewer for phylogenetic tree reconciliations[END_REF], in order to integrate and visualize annotations (e.g. geographic areas) at ancestral nodes for the host and parasite phylogenies and highlight inconsistent zones in the reconciliation. We then test these new developments on a mock dataset and on a 'textbook' example of cospeciation, namely the interaction between figs (Ficus) and their pollinating fig wasps [START_REF] Cruaud | An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps[END_REF][START_REF] Rønsted | 60 million years of co-divergence in the fig-wasp symbiosis[END_REF][START_REF] Wiebes | Co-evolution of figs and their insect pollinators[END_REF]).

2) METHODS

Extending Mowgli to account for geographic information

In this section we first recall the reconciliation model followed by Mowgli [START_REF] Doyon | An Efficient Algorithm for Gene/Species Trees Parsimonious Reconciliation with Losses, Duplications and Transfers[END_REF].

Only rooted parasite and host trees are considered; their leaf nodes (tips) are each labelled by a taxon name. The host tree is dated, meaning that either each branch length represents an amount of time (the tree is thus ultrametric) or that the age of each internal node is provided (e.g. in million years). Internal nodes usually have two descendants, but an internal node can also have a single child also when the evolution of an ancestral lineage living a relatively long period of time is decomposed into a set of consecutive time periods called slices (see Fig. 3 of Mowgli Manual). This slicing of branches is a transparent artefact that allows reconciliation methods to achieve fast computing times while still ensuring time consistency of host switches (see [START_REF] Doyon | An Efficient Algorithm for Gene/Species Trees Parsimonious Reconciliation with Losses, Duplications and Transfers[END_REF][START_REF] Jacox | ecceTERA: comprehensive gene tree-species tree reconciliation using parsimony[END_REF]Libeskind-Hadas & Charleston 2009).

Let P and H denote respectively a parasite and a host tree, x and x p are nodes (or extant species, i.e., leaves) of H and u and u p are nodes (or extant species) of P. Reconciliation algorithms usually consider each current and ancestral host to be associated with one or several specific parasites at any time (e.g. in Mowgli, TreeMap, Jane). However, the identity of the host can vary over time, e.g. after a host switch. This evolutionary event is one of the four types of events considered in cospeciation studies:

-a host switch, also known as a transfer (T event), occurs when a parasite lineage from a source host is transferred to a destination host. The transfer of the parasite must be time consistent, that is the "sending" branch (x p ,x) and the "receiving" branch (x' p, x'), where the host switch is mapped, must belong to the same time slice; -a cospeciation (S event) happens when the speciation of a parasite shortly follows or coincides with the speciation of its host. This is considered by Mowgli as a joint speciation of both parasite and its host;

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The copyright holder for this preprint (which was not this version posted January 16, 2018. ; https://doi.org/10.1101/166215 doi: bioRxiv preprint 5 -a within host speciation also known as duplication (D event), models a speciation of a parasite u of P, where both descendant species continue to live on the host that u lived on. This is represented by u evolving along a (x p, x) branch of H and then splitting into two new lineages in (x p, x); -a parasite loss (L event) occurs when a parasite lineage goes extinct while its host persists.

An illustration of these events can be found the Mowgli Manual.

Mowgli also sometimes considers combinations of events in order to speed up computations. A SL event occurs shortly after a cospeciation (S): one of the parasite child lineages is quickly lost (L) in the host phylogeny on a child lineage of the involved speciation node. A TL event occurs when a parasite u evolving on a branch (x p, x) is lost (L) on this branch shortly after having switched (T) to another host (x' p, x').

As explained above, accounting for geographic information can lead to more realistic diversification scenarios. We first integrate such information by assigning a set of areas to each node of P and H. For an extant taxon this means that a population of the corresponding species is reported to live in each of the assigned areas. In contrast, when an internal taxon is assigned to one or several areas, this means that populations of this now extinct taxon are inferred to have lived back in time in one or several of these geographical zones.

In order to compute biogeographically meaningful reconciliations between the P and H trees, specific constraints have to be implemented in reconciliation algorithms. We detail below how we model these constraints in the context of the four D/T/L/S events or combinations thereof. First, note that areas of a node and its parent in the host or parasite tree can be different, due to dispersal and vicariance events. During the reconciliation process, the time period represented by a branch between nodes x p and x of the host tree is considered to be assigned the union of the areas of x p and x. If a species changes area along the branch from one area assigned to x p to a different one assigned to x, we do not know exactly when it happened, so we consider that at any time between x p and x, part of the population of the evolving species can live in any area proposed for x p or x.

Considering nodes of the trees, we denote by area(x) the set of geographic areas where an extant species x is observed (at the tip of a tree). Areas proposed for an internal node x, that is for an extinct species, are also denoted area(x). However, as indicated above, the meaning is somewhat different as area(x) represents in this case the set of areas where x could have lived. Because of the incertitude in the historical biogeography inferences, we do not enforce that x lived in each of these areas. Similarly, considering branches (x p ,x) of the H tree, area(x p ,x) denotes the set of areas where the species might have lived during this period: this is the union of areas(x p ) and area(x). Note that each area in which exactly one of the two species x and x p is present corresponds to a migration or extinction event that has occurred along this branch. In addition, only (x p ,x) branches being one slice higher are considered for H, as Mowgli operates on this level of detail.

We now detail which geographic constraints apply so that the reconciliation between a parasite tree P and a host tree H is geographically consistent. Recall that a reconciliation is a mapping of P's nodes and branches onto those of H.

-An extant parasite u can be mapped onto an extant host x, only if area(u) ⊆ area(x) (Fig. 1 A). If this constraint is not fulfilled then Mowgli cannot compute a reconciliation.

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The copyright holder for this preprint (which was not this version posted January 16, 2018. ; https://doi.org/10.1101/166215 doi: bioRxiv preprint 6 -We allow the mapping of an ancestral parasite u at a speciation node x in the host tree, only if area(u) and area(x) have a non-empty intersection, i.e. when there is at least one area where the parasite and the host were able to meet (Fig. 1 B).

-A parasite node u can be mapped into a branch (x p ,x) of H due to a duplication or host switch event (Fig. 1 C), and in those cases, we also require that area(u) ∩ area(x p ,x) ≠ ∅. Note, that this constraint does not prevent parasite dispersal events during host switches.

-If a branch (u p ,u) of the parasite tree is mapped for all or part of it onto a host branch (x p ,x) (Fig. 1 D), then we also require that area(u p ,u) ∩ area(x p ,x) ≠ ∅.

-Last, if a branch (u p ,u) of the parasite is going through a node x of the host tree (which happens when the host speciates into two descendant hosts but the parasite sticks to only one of them -an SL event), then the area(x) and area(u p ,u) must have common elements (Fig. 1 E).

Note that when part of the reconciliation mapping traverses an artificial node x in H, then no particular constraint applies locally: the possibility of such a scenario is directed by constraints ensured with respect to the branch (x p ,x) of H to which x belongs.

When respecting the above constraints, Mowgli will propose a scenario that is geographically consistent. This scenario can have a higher cost than those obtained when not accounting for geographic information. This simply results from the fact that the search space contains geographically inconsistent scenarios that are possibly less costly. Mowgli's extension described above, allows choosing the less costly scenario among those that are geographically consistent.

SylvX's new functionalities

We extended the SylvX editor in order to visualize current and ancestral geographic areas of hosts and symbionts. Pie charts can be used to display alternative areas for each node of the tree and/or the reconciliation. Area colour sets can be dynamically updated and tuned using the Hue, Saturation and Value scales. Thresholds are available to simplify views. SylvX also contains a new tool in the Annotation panel to highlight reconciliation parts that do not respect geographical constraints (when such constraints have not been enforced when computing the reconciliation). This is done by loading an annotation file generated by Mowgli (constraintsPBM.csv).

Implementation

Mowgli takes as input a "host tree" and a "parasite tree" stored in files in a Newick format. A list of nodes with their geographic areas (or other annotations) can be given in the same files. Biogeographic inferences typically generate probability or likelihood values for each character state (area) at each node. Mowgli can accept a single area or a set of areas at each node. To run Mowgli and obtain a reconciliation respecting geographical constraints, the -a flag must be added in the command launching the program. Adding the -y flag instead computes a reconciliation independently of the indicated constraints but pinpoints the places where the mapping violates these constraints (in mapping.mpr and constraintPBMs.csv files, see the provided manual for details). This allows users to identify inconsistencies between the most parsimonious reconciliations and the hosts and parasites respective biogeographic histories.

SylvX takes a host tree in Newick format with node id numbers and a reconciliation (with symbiont tree node id.). The host tree (outputSpeciesTree.mpr) and reconciliation obtained with Mowgli can be directly imported into SylvX. The latter also supports input files from other reconciliation . CC-BY-NC 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under

The copyright holder for this preprint (which was not this version posted January 16, 2018. ; https://doi.org/10.1101/166215 doi: bioRxiv preprint software, e.g., ecceTERA [START_REF] Jacox | ecceTERA: comprehensive gene tree-species tree reconciliation using parsimony[END_REF]. Annotation files for the host and parasite phylogenies giving node information can be imported in a CSV format. As many annotations as needed can be added in the annotation files and it is up to the user to choose which ones to plot onto the species tree and the reconciliation map through SylvX's interface.

In order to seamlessly pass a user annotation file in csv format into both Mowgli and SylvX, we provide a Perl script that can be run through the command line in order to: 1) obtain tree node identifiers that will be used by both programs and 2) merge input trees and corresponding annotations files into Mowgli's input format. Files can be generated so that a single (most likely) ancestral range can be specified or alternative geographic areas can be assigned to all nodes (see Supplementary Material 1 for a description of the full procedure to generate files, set a threshold value above which to keep alternative areas and perform a complete analysis).

3) WORKED EXAMPLE

Datasets

To demonstrate the method and its utility, we tested it on two datasets. We first created a mock dataset: two phylogenetic trees with nine tips for a hypothetical host/parasite interaction in which extant and ancient geographic areas for each lineage are informed. The dataset was generated by hand so that: 1) present-day geographic areas of associated taxa are consistent (i.e., hosts and associated parasites live in the same area); 2) the two phylogenies are not perfectly parallel but show some cospeciation events; 3) some geographic locations at nodes that we would like to cospeciate do not coincide in the parasite and host phylogenies. We ran Mowgli on this dataset successively with and without enforcing geographic constraints using in both cases the default parameters (cost 0 for a cospeciation, 1 for a loss and 1 for a host switch, 1 for duplication, not enforcing the root of the parasite tree to be mapped on the root of the species tree). In order to measure the impact of cost settings on the reconciliation scenarios, we ran this dataset using alternative costs for host switches and losses.

As a second dataset, we used a subset of the data from the latest phylogenetic investigation of figs (Ficus) and their pollinating wasps [START_REF] Cruaud | An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps[END_REF]. For both partners of the association, biogeographic scenarios were available for phylogenies of 200 taxa. From the complete phylogenetic trees (available in http://datadryad.org, doi: 10.5061/dryad.hr620), we derived two trees of 23 taxa each, that included a couple of representative species for each Ficus main taxonomic subdivision. We excluded one of the fig subgenera (Pharmacosycea) and its associated pollinators (Tetrapus spp.) whose phylogenetic positions are still debated. We have not tested our method on the total dataset presented in [START_REF] Cruaud | An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps[END_REF] as some uncertainties remain concerning the root of the phylogenetic trees, which could lead to spurious interpretations. The most likely ancestral geographic areas of each node were directly derived from the biogeographic reconstructions of Cruaud et al. 2012, obtained with Maximum Likelihood Optimization in Mesquite [START_REF] Maddison | Mesquite: a modular system for evolutionary analysis[END_REF]). We ran Mowgli on this dataset with and without enforcing geographic constraints (using default event costs and not enforcing the root of the parasite tree to be mapped on the root of the species tree), and explored how these reconstructions shed light on the biogeographic history of the association. In order, to investigate how incertitude on ancestral geographic ranges impacts the reconciliation, we ran the reconciliation on the dataset including alternative ancestral areas for both Ficus and their associated pollinators. For each node of the pollinator and the Ficus phylogenies, the geographic areas which proportional likelihood was above 0.15 were kept and assigned to their respective nodes.

Results

Figure 2 represents the reconstruction obtained on the mock dataset. When not taking geographic constraints into account (Fig. 2A), a cospeciation event at a node where the two associates do not live in the same area was retrieved (node S1 of the host tree in Fig. 2A). The transfer T1 preceding this cospeciation event is also geographically impossible as it suggests a dispersal (the donor host lives in Asia or Africa, and the receiving host lives in America) while the parasite actually stays in Africa. The scenario obtained when enforcing geographic constraints is more costly (Fig. 2B): it entails one more transfer and consequently one less cospeciation event but is biologically more realistic. When using different cost vectors (i.e. using a cost of 3 for losses), the reconciliation where geographic constraints are not taken into account includes additional transfers to avoid losses (Fig. S2A); those are all geographically inconsistent. The reconciliation with geographic constraints also changes (Fig. S2C) and necessitates 5 transfers to ensure geographic consistency of the diversification events in hosts and parasites without inferring any parasite losses. When we increased the cost of transfers (cost T=10, Fig. S2B), the reconciliation without geographic constraints infers several early duplications and losses in order to avoid a costly transfer. On the other hand the results of the reconciliation under constraints (Fig. S2D) did not change comparatively to the one obtained with default cost settings. Hence, in this particular case, adding biological constraints into the reconciliation process stabilizes the reconciliation and makes it less dependent on cost settings.

On the fig/fig wasp dataset (Fig. 3), not accounting for geographical constraints leads to geographic inconsistency in one node (cospeciation S1 in the host tree of Figure 3A). The transfer that precedes it (T5) is impossible and the mapping of the fig wasp phylogeny onto the fig phylogeny from node S1 to event T6 is geographically inconsistent ((Fig. 3A). Enforcing geographic constraints when a single (most likely) area is specified for each node generates a reconciliation scenario that is more costly (Fig. 3B, one more transfer is necessary to reconcile the two phylogenies) but coherent with the [START_REF] Cruaud | An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps[END_REF]. The annotation of ancestral geographic areas on the reconciliation map also shows that host switches occurred both in "sympatric" settings (within the same geographic areas as broadly defined in our dataset) and allopatric settings (i.e. host switches occur between two geographically distant hosts). Overall, four switches out of seven occurred in sympatry (T1, T2, T4, T5) while the remaining three switches (T3, T6, T7) correspond to long distance dispersal events (Fig. 3B). Adding incertitude in ancestral geographic range, generates a reconciliation that matches the one obtained without constraint (Fig. 3C

DISCUSSION

We provide here significant extensions for a reconciliation tool (Mowgli) and a visualization tool (Sylvx) to infer co-diversification scenarios that, for the first time, can take the historical biogeographies of the associated lineages into account. The extension of the Mowgli software precludes geographic inconsistency during the reconciliation process. The resulting reconciliations can then be visualized and edited in the SylvX updated graphical interface that now integrates annotations of ancestral nodes. Mowgli is already one of the few time-consistent efficient methods that build optimal reconciliations. With the integration of geographic constraints in its algorithm, this tool now provides more realistic codiversification scenarios than other reconciliation methods. Producing . CC-BY-NC 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under

The copyright holder for this preprint (which was not this version posted January 16, 2018. ; https://doi.org/10.1101/166215 doi: bioRxiv preprint biologically realistic scenarios can ease their interpretation. In addition, geography-aware reconciliations can reveal whether host switches occur in sympatry or whether they are associated with dispersal events of the symbionts/parasites: this helps unravelling the evolutionary processes underlying host switches According to the inference conducted in [START_REF] Cruaud | An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps[END_REF] the most likely area for the common ancestor of Neotropical figs (belonging to the Americana section) and Afrotropical figs (belonging to the Galoglychia section) is Africa while the proposed cospeciating pollinators lived in Asia (S1; Fig. 3A). In order to respect geographic consistency (when only the most likely area is kept for each ancestral species, Fig. 3B), our geography-aware reconciliation suggests that the current association of figs wasps with Galoglychia in Africa, resp. Americana in the Neotropics, is the result of two independent switches (Fig. 3B, events T6 and T7) of the pollinators from an Asian fig ancestor (the ancestor of the Conosycea figs). However, the biogeographic analysis of [START_REF] Cruaud | An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps[END_REF] also suggested that the node S1 of Ficus could be situated in Asia (though with a much lower likelihood than the Afrotropics). When specifying alternative geographic areas (Fig. 3C), including Asia for the conflicting node in the Ficus phylogeny, we obtain a reconciliation that matches the one obtained without constraints (therefore entailing one less transfer and one more cospeciation event). This result suggests that the common ancestor of the African figs of section Galoglychia and the new world figs from the section Americana could have been located in Asia. Under this latter scenario most of the host switches observed happen in sympatric settings. We will not conclude on the biogeographic history of the fig/fig wasp association as the purpose of our study is not to explore alternative scenarios for this association. The above discussion mainly demonstrates the utility of our method in revealing inconsistency between biogeographic scenarios and a cospeciation hypothesis and therefore proposing alternative scenarios that conciliate both. As in all ancestral character state inferences that rely on present day data, biogeographic reconstructions entail some incertitude. In particular, they are highly sensitive to missing data (species that have not been sampled and/or extinct species). It is therefore important to compute reconciliations with alternative ancestral ranges to investigate biogeographic scenarios.

Perspectives

The tools developed in this study can be applied to all interspecific interactions for which biogeographic scenarios are available for both partners. Fast developments in sequencing technologies generate more accurate and more exhaustive phylogenies and methods in historical biogeography have also improved. Therefore, we can hope that numerous datasets will be available in the near future and that cospeciation could be tested on more systems [START_REF] Cruaud | Testing cospeciation through large-scale cophylogenetic studies[END_REF]. For instance, robust phylogenies and biogeographic scenarios are now available for groups of lice that have been model systems in coevolutionary studies [START_REF] Boyd | Phylogenomics using Target-Restricted Assembly Resolves Intrageneric Relationships of Parasitic Lice (Phthiraptera: Columbicola)[END_REF]. Once comprehensive phylogenies of the hosts are available, our method could be used to better understand the geographic context of host switches in this model system. Geography-aware reconciliation could also be applied to explore the diversification history of the numerous parasitic wasps that are part of the microfauna exploiting figs: several lineages of parasitic wasps have been shown to partly cospeciate with their host figs [START_REF] Jousselin | One Fig to Bind Them All: Host Conservatism in a Fig Wasp Community Unraveled by Cospeciation Analyses among Pollinating and Nonpollinating Fig Wasps[END_REF][START_REF] Jousselin | Patterns of diversification of Afrotropical Otiteselline fig wasps: phylogenetic study reveals a double radiation across host figs and conservatism of host association[END_REF]) and biogeographic scenarios for some lineages are available [START_REF] Cruaud | Out of Australia and back again: the world-wide historical biogeography of non-pollinating fig wasps (Hymenoptera: Sycophaginae)[END_REF]. These developments could also be applied to specific sections of the genus Ficus in order to shed light on their complex biogeographic histories (e.g. section Urostigma that has experienced several dispersal events between Africa and Asia, [START_REF] Chantarasuwan | Palaeotropical intercontinental disjunctions revisited using a dated phylogenetic hypothesis with nearly complete species level sampling of Ficus subsect. Urostigma (Moraceae)[END_REF]. Other nursery . CC-BY-NC 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under

The copyright holder for this preprint (which was not this version posted January 16, 2018. ; https://doi.org/10.1101/166215 doi: bioRxiv preprint 10 pollination/mutualisms such as the interaction between Yucca and their pollinating moths are also good candidates for including geographic constraints into coevolutionary scenarios, as some studies have questioned the respective role of geography and host-plant association in driving the diversification of Yucca moths [START_REF] Althoff | Geographic isolation trumps coevolution as a driver of yucca and yucca moth diversification[END_REF]. Plant/pollinator systems [START_REF] Hutchinson | Cophylogenetic signal is detectable in pollination interactions across ecological scales[END_REF], parasitoid/host insect associations [START_REF] Deng | Cophylogenetic relationships between Anicetus parasitoids (Hymenoptera: Encyrtidae) and their scale insect hosts (Hemiptera: Coccidae)[END_REF]Wilson et al. 2012) , herbivorous insect/plant interactions (e.g. [START_REF] Mcleish | Parallel diversification of Australian gallthrips on Acacia[END_REF][START_REF] Percy | Plant-insect interactions: double-dating associated insect and plant lineages reveals asynchronous radiations[END_REF]) and various vertebrate/parasite associations (e.g. [START_REF] Badets | Correlating Early Evolution of Parasitic Platyhelminths to Gondwana Breakup[END_REF][START_REF] Bentz | Historical biogeography of amphibian parasites, genus Polystoma (Monogenea: Polystomatidae)[END_REF][START_REF] Weckstein | Biogeography explains: cophylogenetic patterns in toucan chewing lice[END_REF]) for which researchers have investigated the relative role of geography and biotic interactions in shaping cophylogenetic signals could also be studied.

Furthermore, the approach presented in this paper does not only apply to geographic information and could be extended to other biological traits. For instance in systems where the species are partitioned into different habitats (e.g. forest canopy species vs savannah species), geographic areas could be replaced by traits related to the ecological niches; constraints that are similar to the ones applied for geography could then be easily transferrable. Informing ancestral characters for habitats on the host and the symbiont phylogenetic trees and using "Mowgli with constraints" would result in constraining cospeciation and host switches to associates sharing the same ecological habitats. In a similar way, the respective climatic niches of associated organisms could also be used when parasite (or symbiont) distributions are known to be strongly constrained by thermal tolerance (see [START_REF] Singh | Fungal-algal association patterns in lichen symbiosis linked to macroclimate[END_REF], for a recent study showing that climatic conditions influences the patterns of association between fungi and their algal partners). In many specialized interactions, such as host/obligate bacterial endosymbionts (e.g. Jousselin et al. 2009[START_REF] Rosenblueth | Evolutionary relationships of flavobacterial and enterobacterial endosymbionts with their scale insect hosts (Hemiptera: Coccoidea)[END_REF] or host/viruses associations [START_REF] Ramsden | Hantavirus Evolution in Relation to Its Rodent and Insectivore Hosts: No Evidence for Codivergence[END_REF][START_REF] Garamszegi | Patterns of co-speciation and host switching in primate malaria parasites[END_REF], inferring ancestral character states for some ecological traits for the "symbiotic" lineages (the parasite) independently of their hosts is not always straightforward. However, the evolution of these obligate associations and their maintenance are still governed by some phenotype matching between the partners. For instance in host/bacterial symbiont associations, the metabolic complementarity of the host and the symbiont (Zientz et al. 2004) could be reconstructed and used to constrain the reconciliations. In host/virus associations, information about the host immune system and viruses adaptations could be used [START_REF] Longdon | The Evolution and Genetics of Virus Host Shifts[END_REF]. The extension of Mowgli proposed here could probably be adapted to fit the biological properties of these associations Independently of the new functionality implemented in Mowgli, the concomitant update of SylvX allows the comparison of ancestral states for any character of the hosts and/or the symbionts. This can help interpreting reconciliations by replacing them in their biological context. One of the most useful functionalities of SylvX is now to be able to visualize whether host switches are associated with evolutionary transitions in character states in both the parasite and/or the host. It can therefore help understanding the biological processes that are associated with these transfers. Mapping characters of the associates throughout the reconciliation can also help investigating whether there is correlated trait evolution in host and parasites. Until now, such correlations could only be investigated on one of the associate phylogeny (e.g. [START_REF] Sorci | Correlated evolution between host immunity and parasite life histories in primates and oxyurid parasites[END_REF][START_REF] Jousselin | Convergence and coevolution in a mutualism: Evidence from a molecular phylogeny of Ficus[END_REF]. Looking at simultaneous transitions in character states in both partners throughout a host/parasite reconciliation might help identifying co-adapted traits that constrain the association.

In conclusion, we provide here a framework that can integrate the character histories of the associates into the reconciliation process. It can take into account incertitude in the character states and allows recovering biologically realistic scenarios. It can also shed light on character history inferences by pointing out inconsistencies between the character states of the two associates on the reconciliation map. The new developments made in SylvX facilitate these interpretations. A more integrative . CC-BY-NC 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under

The copyright holder for this preprint (which was not this version posted January 16, 2018. ; https://doi.org/10.1101/166215 doi: bioRxiv preprint approach than the one presented here, would consist in co-optimizing the reconciliation and the biogeographical inference simultaneously. However this would require using the same optimization criterion for both inferences and setting adequate parameters for these very different processes in a single model. When conducted, this work should probably rely on Maximum Likelihood optimization as in the ALE reconciliation software [START_REF] Szöllősi | Phylogenetic modeling of lateral gene transfer reconstructs the pattern and relative timing of speciations[END_REF]. For now, we believe that the use of "constraint-aware" reconciliations is preferable to current practices that consist in elaborating ad-hoc narratives once the reconciliations are obtained and compared with the character histories of the associates.

Fig. 1: Description of how geographical constraints are handled by the Mowgli software. Plain lines and nodes represent branches and nodes of the parasite tree, while cylindres and dashed ellipses represent branches and nodes of the host tree. The colours of a node correspond to geographical areas, these areas are observed (hence enforced) for extant taxa but inferred for ancestral nodes. A) The parasite tip u can be mapped to a tip x of the host tree if the areas of the host contain all areas of the parasite. B) Mowgli accepts that a parasite node u cospeciates with a host at a node x, if the two nodes share an area. Here, it was inferred that the ancestral parasite u lived in green and/or red areas, but cospeciated with a host x that lived in blue and/or red areas, we conclude that u lived only in the red area at this time and that x lived at least in the red area. C) To map an ancestral parasite inside a branch (x p ,x) -to represent the source of a host switch (upper part of the figure), or a duplication of the parasite (lower part) --Mowgli requires that the parasite has potentially lived in an area of x p or x. In this example, u shares an area with x p and v shares an area with x. D) To map a branch (u p ,u) of the parasite tree inside a branch (x p ,x) of the host tree, Mowgli requires that the parasite mapped on the host branch (u in the left part of the figure showing the destination of a switch and v p in the right part showing the departure due to a switch) has potentially lived in any area assigned to node x p or to node

x. This is the case here for node u that was indicated as having lived in the red area (also assigned to x) and for v p assigned to the green area, also proposed for x p . Note that mapping v p into (x p ,x) would have also been correct if v p had been assigned to the red area, indicating that it changed from the green to the red area, together with its host, before switching to another host. E) When a parasite lineage (u p ,u) living on an ancestral host remains with one descending child of this host after its speciation at node x: Mowgli requires that the area at which the host speciation occurred is also found among the areas inferred for u p or u. The mapping in this example indicates that the parasite changed area with its host, before the host speciation event.

Fig. 2: Results of the reconciliations obtained on a simulated data set with default cost settings: A) without enforcing geographic constraints (7 coSpeciation events, 1 Transfer, 1 Loss), purple dashed branches indicate parts of the reconciliation where geographic constraints are not fulfilledB) when enforcing geographic constraints (6 S, 2 T, 1 L). In both cases, the big pie charts correspond to the host ancestral geographic areas while small pie charts correspond to the symbiont ancestral geographic areas. The leaves of the species tree are also coloured according to the current geographic distribution of the associates. Annotations files given for the host tree and the symbiont tree specified a single most likely area at each node.

  figs and the fig wasp biogeographic histories. This scenario suggests that the fig wasps independently colonized figs in the Neotropics and in the Afrotropics through two distinct host switches from Asia rather than accompanied the speciation of their hosts, as was suggested by Figure 3A (and node 29 of Fig. S12 in Cruaud et al. 2012). The annotation of ancestral geographic areas on the reconciliation

  ), as geographic areas of node S1 of the host figs now includes Asia among its potential geographic areas. This matches the ancestral geographic area of the inferred associated fig wasps. In that scenario a single host switch is associated with a long dispersal event of the fig wasps (T3: from Asia to Australasia ), all other host switches occur in sympatric settings (within Asia) and fig wasp geographic range evolution merely mirrors the one of their hosts.

  In the particular example of the fig/fig wasp interaction presented here, the geographic inconsistency revealed at one of the cospeciating nodes in the analysis (Fig 3A) ran without constraints might actually point out some ambiguity in the biogeographic history of the Ficus hosts.

Fig. 3 :

 3 Fig. 3: Results of the reconciliations inferred by Mowgli on the fig/fig wasp data set with default cost settings: A) using a single most likely area for ancestral species and without enforcing geographic constraints (events: 17 S, 6 T, 1 L), purple dashed branches indicate parts of the reconciliation where geographic constraints are not fulfilled; B) using a single most likely area when enforcing geographic constraints (events: 16 S, 7 T, 1 L).The leaves of the phylogenetic trees are coloured according to current geographic distribution of the associates. In both cases, big pie charts correspond to the Ficus ancestral geographic areas, small pie charts correspond to the pollinator ancestral geographic areas; C) Reconciliation obtained when alternative ancestral areas are considered (namely those with probability>0.15). Sections of the pies are proportional to the probability of the associated states.

Ficus

  taxonomic subdivisions are reported on the right inside of the figure.

  Figure1

ACKNOWLEDGMENTS

Funding: ANR Phylospace. We thank A. Cruaud, JY Rasplus & coll. for sharing the fig/figwasp dataset.

AUTHOR CONTRIBUTION

VB, JPD and EJ designed the study. VB and JPD developed Mowgli, FC developed SylvX. VB and EJ wrote the manuscript with contributions of FC.

SOFTWARE AVAILABILITY

Mowgli is available on http://www.atgc-montpellier.fr/Mowgli/ with manual, GeoRecHelper and example files. It runs on OSX (Mac) and Linux systems.

SylvX is available on www.sylvx.org with manual and example files and can be installed on any


Pharm D Pascal Le Corre
email: pascal.le-corre@univ-rennes1.fr

MD, PhD Gwenolé Loas

Pr Pascal Le Corre

Repurposing Functional inhibitors of Acid sphingomyelinase (FIASMAs) drugs: an opportunity against SARS-CoV-2 infection ?

Keywords: Acid sphingomyelinase, Functional inhibitors of Acid sphingomyelinase, ABCB1 transporter, repurposing, in vitro and in silico, antiviral activity, SARS-CoV-2

What is known and objective: SARS-CoV-2 outbreak is characterized by the absence of active compounds for the treatment. The purpose of this study was to present an hypothesis on the prophylactic and/or therapeutic use in SARS-CoV-2 infections of functional inhibitors of acid sphingomyelinase (FIASMAs) that are mostly molecules approved drugs for various diseases.

Comment:

We present and discuss the potential interest of FIASMAs presenting a lysosomotropism that may explain in vitro activities against viral infections, and more specifically in infections mediated by coronaviruses the SARS-CoV-2. A successful in vitro to in vivo translation of FIASMA requires a particular attention to drug pharmacokinetics (dosing regimen, drug-drug interactions) and to viral kinetics.

What is new and conclusion:

The drug repurposing approach with the hope to bring patients effective treatment faster has gathered much attention in the SARS-COV-2 pandemic era. The current knowledge suggests that FIASMAs, as small molecules having a lysosomotropism and clinically approved indications in various diseases, should be paid more attention for their repurposing as potential drugs against SARS-COV-2.

-What is known and objective

The current situation of the SARS-CoV-2 outbreak poses major therapeutic problems given the absence to date of a specific antiviral agent to treat this infection, decrease viral shedding and subsequent transmission. Numerous clinical trials are in progress with different disease severity at enrollment with a high majority of studies for treatment purpose compared to prevention purpose (1,628 trials versus 289 trials consulted on october, 28 2020, cf Living mapping and living systematic review of Covid-19 studies at https://covid-nma.com/dataviz). Efforts have to be maintained since it is likely that we will have to coexist in the future with pathogenic coronavirus type viruses (SARS-Cov-2 or other strains). Moreover, initial trials in the search for effective therapy have led somewhat disappointing results [START_REF] Cao | A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19[END_REF][START_REF] Baden | Covid-19 -The Search for Effective Therapy[END_REF].

Given the difficulties and costs in rapidly bringing up treatments with a favorable benefit-risk balance, in particular with the aim of large-scale treatment either for treatment of patient population or for prevention in healthy subjects, it seems relevant to investigate whetheramong the drugs marketed to date -some of them could have a therapeutic or a prophylactic potential. This so-called "drug repurposing" strategy is based on different methodological approaches, and to date the most successful examples of drug reuse have not involved a systematic approach but rather serendipity [START_REF] Pushpakom | Drug repurposing: progress, challenges and recommendations[END_REF]. In the repositioning strategy the first step which consists in identifying the candidate molecule (s) for a given indication is critical. The potential approaches are either experimental and / or computational [START_REF] Pushpakom | Drug repurposing: progress, challenges and recommendations[END_REF]. Among these, the use of new data sources -including clinical data repositories -offer new opportunities. Some organic molecules, including currently marketed drugs, have the potential to functionally inhibit, from reversible and additive manner, the activity of acid sphingomyelinase (ASM) which is a lysosomal glycoprotein involved in a wide range of disorders (4) including virus infections. These molecules identified by the acronym FIASMA (i.e. functional inhibitors of acid sphingomyelinase) have the potential to inhibit this enzyme, and therefore disrupt the entry of viruses into cells. The purpose of this study was to present an hypothesis on the prophylactic and/or therapeutic use of FIASMAs (Functional Inhibitors of ASM) in SARS-CoV-2 infections through a literature search in PubMed, and medRxiv.org preprint server for Health Sciences.

-Comment

-FIASMA's (Table 1)

Acid sphingomyelinase (ASM) is a lysosomal glycoprotein anchored to the inner lysosomal membrane which is translocated to the external leaflet of the cell membrane under specific stimuli where it catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Shingomyelin is the most abundant sphingolipid component of the mammalian plasma membrane where it is associated with cholesterol to form lipid rafts. ASM inhibitors work either by direct inhibition or by functional inhibition (FIASMA). Unlike FIASMA, there are few direct inhibitors, by these including biphosphonates, in particular zoledronic acid [START_REF] Roth | Potent and selective inhibition of acid sphingomyelinase by bisphosphonates[END_REF]. Functional inhibitors of ASM (FIASMAs) are cationic amphiphilic molecules relatively heterogeneous in terms of chemical structure but which have the characteristic of being polycyclic molecules, with at least 1 basic nitrogen atom (pKa > 4 which corresponds to a partially protonated functional group at acidic pH) and moderate to high lipophilicity (log P> 3) (6).

The internal wall of the lysosomes is negatively charged due to the abundant presence of phospholipids, and the positively charged proteins form electrostatic interactions with this internal wall of the lysosome which protects them from lysosomal degradation; ASM is thus attached to the lysosomal membrane. FIASMA's which are lipophilic molecules integrate into the lysosomal membrane with their lipophilic part, and present their positively charged part in the lysosomal lumen, which leads to detachment of the ASM from the lysosomal membrane and its subsequent proteolytic degradation in the lysosomal lumen. Hence, ASM activating stimuli cannot lead to the translocation of ASM to the external leaflet of the cell membrane, leading to perturbation in the downstream of the signaling cascade and in lipid raft formation (Figure 1). However, there remains residual ASM activity necessary for cell viability. There are significant differences in intra-lysosomal capture speed depending on lipophilicity (logP) and degree of ionization (pKa) (from minutes to hours in cell culture systems). Indeed, desipramine, fluoxetine, maprotiline, paroxetine and protriptyline have rapid lysosomal capture (<30 min) with moderate lysosomal accumulation (lysosome / extracellular concentration ratio <100: 1) (6). Drugs known for their significant FIASMAs properties (residual activity <50% at a concentration of 10 µM) have been described [START_REF] Kornhuber | Identification of new functional inhibitors of acid sphingomyelinase using a structure-property-activity relation model[END_REF][START_REF] Kornhuber | Functional Inhibitors of Acid Sphingomyelinase (FIASMAs). Erich Gulbins • Irina Petrache Editors Sphingolipids: Basic Science and Drug Development[END_REF][START_REF] Kornhuber | Identification of novel functional inhibitors of acid sphingomyelinase[END_REF][START_REF] Hoehn | Melatonin Acts as an Antidepressant by Inhibition of the Acid Sphingomyelinase/Ceramide System[END_REF] and are listed in Table 1.

The interest in FIASMA drugs relies on their potential interest in treating pathologies in which there is an increased ASM activity such as neuro-psychiatric pathologies, and in particular in major depression [START_REF] Mühle | Sphingomyelin Synthases in Neuropsychiatric Health and Disease[END_REF] where the inhibition of ASM by modifying the sphingomyelin-ceramide balance could contribute significantly to their mechanism of action [START_REF] Gulbins | Antidepressants act by inducing autophagy controlled by sphingomyelin-ceramide[END_REF][START_REF] Rhein | Derivatization of common antidepressant drugs increases inhibition of acid sphingomyelinase and reduces induction of phospholipidosis[END_REF]. Other diseases include ischemic strokes (14), alzheimer's disease [START_REF] He | Deregulation of sphingolipid metabolism in Alzheimer's disease[END_REF] and chronic obstructive pulmonary disease [START_REF] Chakinala | Sphingolipids in COPD[END_REF] as well as intra-cellular infections [START_REF] Cockburn | Functional inhibition of acid sphingomyelinase disrupts infection by intracellular bacterial pathogens[END_REF]. In addition, the production of ceramide via ASM plays a key role in the production of pulmonary edema in acute pulmonary insufficiency, and ASM inhibitors may be of therapeutic value in diseases with chronic and acute pulmonary injury (6).

-FIASMA's and viral infections

Viruses usually take benefit of the endocytosis mechanisms to penetrate in the cytosol of cells, and different mechanisms of internalization are involved. These are clathrin-mediated endocytosis, macropinocytosis, caveolar/lipid raft-mediated endocytosis, as well as several less well characterized clathrin-and caveolin/lipid raft-independent mechanisms [START_REF] Mercer | Virus entry by endocytosis[END_REF]. Viruses using lipid raft-dependent pathways depends on cholesterol, lipid rafts and a complex signaling pathway and use glycosphingolipids as receptors. The role of ASM inhibition in intracellular infectious pathologies, and in particular by enveloped viruses, is due to the fact that the life cycle of enveloped RNA viruses (e.g., Sars-Cov-2) is critically dependent of the lipid biosynthesis of the host [START_REF] Yager | Sphingolipids as Potential Therapeutic Targets against Enveloped Human RNA Viruses[END_REF]. Binding and entry of the virus involves interactions between the envelope's viral glycoproteins and cellular receptors concentrated in micro-domains rich in sphingolipids present on the membranes of host cells, and the sphingolipid itself is able to serve as an alternative receptor for certain viruses (i.e., HIV). The subsequent infection leads to a reorganization of cell membranes and lipid metabolism to support the production of new viral particles [START_REF] Yager | Sphingolipids as Potential Therapeutic Targets against Enveloped Human RNA Viruses[END_REF]. Beyond the role in the entry of the virus, sphingolipids also play an important role in modulating the replication of the virus and in shaping the innate antiviral immune response [START_REF] Bezgovsek | Sphingolipids in early viral replication and innate immune activation[END_REF]. More specifically, the role of FIASMA's in infection by intracellular bacteria has been shown [START_REF] Cockburn | Functional inhibition of acid sphingomyelinase disrupts infection by intracellular bacterial pathogens[END_REF], and ASM activity has been found essential for optimal cyle progression of intracellular bacterial pathogens. This study established the importance of ASM for infection by several intracellular bacteria and suggested FIASMAs (namely desipramine, amitriptyline, and nortriptyline) as potential therapies for diseases caused by intracellular pathogens. The role of sphingomyelin and ASM in the intra-cellular penetration of an alpha-herpes virus has been shown [START_REF] Pastenkos | Role of Sphingomyelin in Alphaherpesvirus Entry[END_REF]. Imipramine and amitriptyline (FIASMA's) have reduced cellular infection with porcine alphaherpesvirus pseudorabies virus (PRV) by intracellular sphingomyelin depletion. However, not all herpes viruses were dependent on sphingomyelin and ASM for cell infection. Several drugs, not considered as FIASMA drugs, may likely act via intracellular sequestration and increase in endosomal and/or lysosomal pH to explain their antiviral activity. Azithromycin, as well as chloroquine and hydroxychloroquine, may share this mechanism of action, and act in a synergistic manner [START_REF] Damle | Clinical Pharmacology Perspectives on the Antiviral Activity of Azithromycin and Use in COVID-19[END_REF]. A screen of 2,600 approved drugs with further evaluation of antiviral activity using in vitro virus-like particle entry assay and a murine model of EBOLA disease showed the cationic molecules had strong in vitro and in vivo antiviral activities [START_REF] Johansen | A screen of approved drugs and molecular probes identifies therapeutics with anti-Ebola virus activity[END_REF]. Viral entry assays showed that these compounds blocked a late stage of viral entry. The drugs with protective ability included sertraline and bepridil which are known as FIASMA's. Hence, drug targeting the lysosomes (lysosomotropic drugs) may have different mechanisms of action at the lysosomal level. The role of the Niemann-Pick C1 (NPC1) protein as an essential entry receptor was studied in a mouse model of EBOLA disease and showed that imipramine inhibited the entry of Ebola virus (EBOV) in vitro and protected against EBOV infection in a mouse model [START_REF] Herbert | Niemann-pick C1 is essential for ebolavirus replication and pathogenesis in vivo[END_REF]. Additionally, the increase in intralysosomal pH may impair the activity of cathepsin-L that is a key protease for membrane fusion as shown for SARS-CoV-2 [START_REF] Ballout | The lysosome: A potential juncture between SARS-CoV-2 infectivity and Niemann-Pick disease type C, with therapeutic implications[END_REF]. Very recently, as part of a high throughput screening using a genomic approach to reposition old molecules (DrugBank target database), amitriptyline has shown a reduction in cell death in a model of A549 infected human lung carcinoma cells by the lethal avian influenza A H5N1 virus [START_REF] Huang | Identification of amitriptyline HCl, flavin adenine dinucleotide, azacitidine and calcitriol as repurposing drugs for influenza A H5N1 virus-induced lung injury[END_REF]. In a mouse model infected with H5N1, amitriptyline showed a reduction in the number of infiltrating cells, decreased the score of pulmonary lesions on the basis of pulmonary histopathology, and reduced pulmonary edema and improved the survival of mice infected with the H5N1 virus. Analysis of RNA-seq data from the lung tissues of mice infected with H5N1 shows that the genes significantly influenced by amitriptyline were mainly grouped in those corresponding to immune responses, neurophysiological processes and processes related to apoptosis. As a whole, drugs that target the lysosomes (lysosomotropic drugs) and especially FIASMA's alter its functions and create an unfavorable host cell environment for viruses. [START_REF] Choi | Murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release[END_REF] and clathrinmediated pathway [START_REF] Inoue | Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic tail deleted[END_REF] have been identified in the Coronoviridae suggesting potential target for drugs acting at the internalization step (i.e., fusion and entry). However, targeting the late compartments of the endocytic pathway after delivery of the SARS-CoV genome in the cytoplasm should also considered as potential target for disruption of the replication, as shown with amiodarone [START_REF] Stadler | Amiodarone alters late endosomes and inhibits SARS coronavirus infection at a post-endosomal level[END_REF]. Hence different targets are to be considered against SARS-CoV-2, and those targeting directly the virus may be considered as most effective [START_REF] Li | Updated Approaches against SARS-CoV-2[END_REF] than targeting the host cells. However, targeting the biology of the host cells, instead of targeting the biology of the virus, has the advantage to be less influenced by genetic variations (much less frequent in host cells compared to the viruses (e.g., mutations). As lysosomotropic drugs with specific activity, attention should be paid to FIASMA's interfering via ASM inhibition to lipid-raft mediated endocytosis, and for some by interfering with other steps in the endocytic pathways.

-FIASMA's and coronaviruses (

In a docking study using two crystal structures from SARS-CoV main protease (M pro ), 4 drugs including the FIASMA dug promazine were identified used as templates for the rational designed of anti-SARS proteinase drugs [START_REF] Zhang | Old drugs as lead compounds for a new disease? Binding analysis of SARS coronavirus main proteinase with HIV, psychotic and parasite drugs[END_REF]. However, negative results with promazine were obtained using in vitro and in vivo (mouse lung model) methods [START_REF] Barnard | Is the anti-psychotic, 10-(3-(dimethylamino)propyl)phenothiazine (promazine), a potential drug with which to treat SARS infections? Lack of efficacy of promazine on SARS-CoV replication in a mouse model[END_REF]. High-throughput screening through a library of current or previously FDA-approved drugs made it possible to identify on cell culture systems several molecules having antiviral activity against MERS-CoV and/or SARS-CoV viruses [START_REF] Dyall | Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection[END_REF]. Among the 27 compounds identified as active against both MERS-CoV and SARS-CoV, 9 of them were FIASMAs (i.e., emetine, tamoxifene, benztropine, fluphenazine, promethazine, astemizole, chlorpromazine, triflupromazine, clomipramine). A library screening of 348 FDA-approved drugs for anti-MERS-COV activity in cell culture identified 4 compounds (chloroquine, chlorpromazine, loperamide and lopanivir) with activity in the low micromolar range [START_REF] De Wilde | Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture[END_REF]. These drugs also inhibited the replication of SARS coronavirus of and human coronavirus 229E. Among this set of compounds two of them (i.e., chlorpromazine and loperamide) are FIASMAs. In a mechanistic study on drugs. Taking into account previous results [START_REF] Dyall | Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection[END_REF][START_REF] De Wilde | Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture[END_REF], as part of a mechanistic study on drug ability to block virus entry, the ability of a panel of drugs including 7 FIASMA's (i.e., chlorpromazine, promethazine, fluphenazine, astemizole, triflupromazine, clomipramine and tamoxifen) to inhibit MERS-CoV was confirmed [START_REF] Liu | Testing of Middle East respiratory syndrome coronavirus replication inhibitors for the ability to block viral entry[END_REF].

Very recently, several studies have been directed towards the search of drugs potentially inhibiting Sars-CoV-2. SARS-CoV-2 proteins have been cloned, tagged and expressed in human cells and the human proteins associated with each have been identified [START_REF] Gordon | A SARS-CoV-2 protein interaction map reveals targets for drug repurposing[END_REF]. SARS-CoV-2 human protein-protein interactions were identified (n = 332) and 66 of them were targeted by 69 compounds (including 29 FDA-approved drugs) whose antiviral activity was investigated using 2 viral assays. Among this panel of drugs clinically usable for targeting the human proteins, two are FIASMAs (i.e., clemastine and cloperastine). A protein-protein interaction network containing the genes co-expressed with the angiotensin converting enzyme 2, a main cell receptor of SARS-COV and SARS-COV-2, has been set up [START_REF] Cava | In Silico Discovery of Candidate Drugs Against Covid-19[END_REF]. These authors found that 36 drugs were associated with the gene network, including one FIASMA (i.e., amiodarone). In a screen of 3,000 FDA-approved drugs against SARS-COV, tested in vitro on Vero cells, 24 drugs were retained including two FIASMAs (i.e., loperamide and cepharanthine) [START_REF] Jeon | Identification of antiviral drug candidates against SARS-CoV-2 from FDA-approved drugs[END_REF]. Using a systems pharmacology-based network medicine platform, the quantification of interplay between the human coronaviruses interactome and drugs targets found 16 potential drugs including four FIASMAs (i.e., paroxetine, melatonine, quinacrine, carvedilol) [START_REF] Zhou | Network-based drug repurposing for novel coronavirus 2019-nCoV/SARS-CoV-2[END_REF]. Four studies using docking, in vitro or in vivo studies reported that emetine could be a potential inhibitor of SARS-CoV-2 [START_REF] Choy | Remdesivir, lopinavir, emetine, and homoharringtonine inhibit SARS-CoV-2 replication in vitro[END_REF][START_REF] Das | An investigation into the identification of potential inhibitors of SARS-CoV-2 main protease using molecular docking study[END_REF][START_REF] Ianevski | Potential Antiviral Options against SARS-CoV-2 Infection[END_REF][START_REF] Yang | Repurposing old drugs as antiviral agents for coronaviruses[END_REF]. Using artificial intelligence technology to identify marketed drugs with potential activity against SARS-CoV-2, the FIASMA drug clofazimine was identified, in a subset of 8 drugs, as a potential candidate based on in vitro cell activities against a feline coronarovirus (44).

Studies currently in a review process, have evaluated either in silico [START_REF] Arya | Potential inhibitors against papain-like protease of novel coronavirus (SARS-CoV-2) from FDA approved drugs[END_REF] or in vitro [START_REF] Weston | FDA approved drugs with broad anti-coronaviral activity inhibit SARS-CoV-2 in vitro[END_REF] the potential of approved drugs. A docking was carried out with the papain-like protease (PLpro) of SARS-CoV-2 using a panel (n = 2,525) of molecules approved by the FDA (45 ) in order to find inhibitors. This work made it possible to select those who had the best affinity including chloroquine, formoterol and a FIASMA (i.e., amitriptyline). In an in vitro study, a subset of 17 drugs with activity as inhibitors of SARS-CoV-2 were identified [START_REF] Weston | FDA approved drugs with broad anti-coronaviral activity inhibit SARS-CoV-2 in vitro[END_REF]. This subset of drugs included 5 FIASMAS (i.e., fluphenazine, chlorpromazine, clomipramine, promethazine, and emetine), and also highlighted chloroquine derivatives (e.g., hydroxychloroquine) as potential SaS-CoV-2 antiviral drugs. About 30 FDA approved drugs have been selected by computational analysis to inhibit the SARS-CoV-2 main protease (M pro ) (47). Among 7 drugs, the 3 more potent were ebastine and 2 FIASMA's (i.e., pimozide and bepridil). Using quantitative high-throughput screen coupled with artificial intelligence based machine learning from a library of 1,425 FDA-approved drugs, a subset of 16 compounds with in vitro antiviral effects were identified including amiodarone as a FIASMA drug [START_REF] Mirabelli | Morphological Cell Profiling of SARS-CoV-2 Infection Identifies Drug Repurposing Candidates for COVID-19[END_REF]. In a cell culture model 3 FIASMAs (amiodarone, fluoxetine, and imipramine) inhibited the entry and propagation of SARS-CoV-2 without cytotoxic adverse effects and had also antiviral activity against two currently circulating influenza A virus subtypes [START_REF] Schloer | Targeting the endolysosomal host-SARS-CoV-2 interface by clinically licensed functional inhibitors of acid sphingomyelinase (FIASMA) including the antidepressant fluoxetine[END_REF]. In the study, fluoxetine was associated with increased cholesterol storage as well as dysregulated acidification. A very recent paper, showed that amitriptyline, desipramine, escitalopram, fluoxetine, imipramine, maprotiline, and sertraline displayed an almost complete ex vivo inhibition of the infection of human epithelial cells and different human cell lines with SARS-CoV-2 and pp-VSV-SARS-CoV-2 spike particles [START_REF] Carpinteiro | Pharmacological inhibition of acid sphingomyelinase prevents uptake of SARS-CoV-2 by epithelial cells[END_REF].

Based on a potential prophylactic activity of the anti-psychotic chlorpromazine against Sars-CoV-2, and as a result of a lower prevalence of COVID-19 infections in psychiatric patients (4%) comparatively to health care professionals (14%), suggestions of a prophylactic effect of with psychoactive compounds have emerged [START_REF] Plaze | Repurposing chlorpromazine to treat COVID-19: the reCoVery study[END_REF][START_REF] Javelot | Towards a pharmacochemical hypothesis of the prophylaxis of SARS-CoV-2 by psychoactive substances[END_REF][START_REF] Villoutreix | Prevention of COVID-19 by drug repurposing: rationale from drugs prescribed for mental disorders[END_REF], and a clinical trial (i.e., The reCoVery study) is ongoing. The antiviral activity of chlorpromazine involves the inhibition of clathrinmediated endocytosis and, once again, the status of FIASMA was not mentioned.

As a whole, numerous in vitro or in vivo study have been identified 28 FIASMA's as potential antiviral drug candidates against SARS-COV, MERS-COV or SARS-COV-2. In this subset of drugs, 6 can be identified as active against both the 3 coronaviruses (chlorpromazine, clomipramine, emetine, fluphenazine, loperamide, promethazine, see Table 2). It should be noticed that there are many unknowns in SARS-CoV-2 cell penetration. Apart from caveolar/lipid raft-mediated endocytosis, clathrin-mediated endocytosis and micropinocytosis potentially involved in cell entry may be modulated by some drugs with FIASMA properties such as chlorpromazine, sertraline and promethazine as inhibitors of clathrin-mediated endocytosis, and imipramine as inhibitors of micropinocytosis [START_REF] Glebov | Understanding SARS-CoV-2 endocytosis for COVID-19 drug repurposing[END_REF]. Furthermore, the endocytic mechanisms for coronaviruses can vary along the level in the respiratory tract. Nasal epithelial cells express a wide variety of endocytic markers while pneumocytes have a more restricted pattern with micropinocytosis as a predominant endocytic pathway [START_REF] Glebov | Understanding SARS-CoV-2 endocytosis for COVID-19 drug repurposing[END_REF]. Type II alveolar pneumocytes as preferential targets of SARS-CoV-2 should explain the late alveolar damage observed in some patients.

-In vitro-to-in vivo translation

Solid evidence of antiviral activity based on in vitro and on pre-clinical studies is a prerequisite to further studies in humans. However, in vivo translation of antiviral activity requires that the drugs have suitable profiles of drug exposure (concentration and duration) at the site(s) of action. Hence, drug pharmacokinetics and drug regimens are essential parameters for a successful translation.

FIASMA's are lipophilic basic amine drugs and as such have a very high volume of distribution. Indeed, steady-state volume of distribution (Vdss) of amitriptyline and of chlorpromazine is 14.4 L per kg (55) and 8.9 L per kg [START_REF] Yeung | Pharmacokinetics of chlorpromazine and key metabolites[END_REF]. As lipophilic drugs, they are highly metabolized with significant first-pass effect. Indeed, the systemic clearance of amitriptyline and chlorpromazine in quite high (0.9 L/min and 1.3 L/min), and they have a low and variable oral bioavailability: ranging from 4 to 38 % for chlorpromazine [START_REF] Yeung | Pharmacokinetics of chlorpromazine and key metabolites[END_REF] and from 33 to 62 % for amitriptyline [START_REF] Schulz | Amitriptyline disposition in young and elderly normal men[END_REF]. Even though they have a high volume of distribution, as a result of a high clearance, their apparent elimination half-life of is not too long (approx. 11 h and 18.5 h for amitriptyline and chlorpromazine, respectively). However, reaching steady-state to obtain a maximal effect would require a delay (approximately 7-times the half-life) that may prove unsuitable in an epidemic context either for prevention or for a curative use. Alternatively, a loading dose may be used to reach more rapidly the steady state provided that the tolerance profile is not a limiting factor. Furthermore, as illustrated for these two prototypic drugs, the variable oral bioavailability is a factor of inter-individual that should be considered. Other variability factors that may impact dosing such renal and hepatic impairment as well as drug-drug interactions with concomitant medications should also be considered with these drug candidates as lipophilic drugs usually have significant first-pass effect. Furthermore, a particular attention should be paid to FIASMA interacting with P-gp either as substrate and/or as inhibitor (Table 2). P-gp is present in the lysosomal membrane [START_REF] Yamagishi | P-glycoprotein mediates drug resistance via a novel mechanism involving lysosomal sequestration[END_REF] and P-gp substrates may have a higher lysosomal distribution compared to those driven only by passive diffusion [START_REF] Scherrmann | Intracellular ABCB1 as a Possible Mechanism to Explain the Synergistic Effect of Hydroxychloroquine-Azithromycin Combination in COVID-19 Therapy[END_REF]. Hence, co-administration of strong P-gp inhibitor (e.g., clarithromycin, itraconazole, verapamil and some HIV protease inhibitor) with FIASMAs may decrease their lysosomal accumulation. Such feature should be considered as part of drug combination therapy in the management of SARS-CoV-2 infection. As part of prophylaxis use in individual contacts, knowledge of the pharmacokinetics in different sub-populations (including pediatrics and elderly people) should be estimated. Besides drug pharmacokinetics, viral kinetics has to be considered to optimize dosing regimens given the limited window to initiate an antiviral therapy in a curative setting. The viral profile kinetics (i.e., time to peak viral load and duration of viral shedding) varies among viruses and is not yet well defined for SARS-CoV-2. These elements are mandatory for the definition of the initiation and of the duration of an antiviral treatment [START_REF] Smith | Dosing will be a key success factor in repurposing antivirals for COVID-19[END_REF]. These elements highlight the fact that drug pharmacokinetics and viral kinetics has to be known with precision in order not to be lost in translation.

-Framework of future investigations

Firstly, retrospective investigations on COVID-19 confirmed studied could be conducted to explore the prevalence and the potential effects of FIASMAs. Indeed, it seems worthwhile to investigate if patients infected with Sars-Cov-2 virus and exposed to FIASMA's and have a more favorable evolution of the disease. Indeed, a recent retrospective study [START_REF] Smith | Dosing will be a key success factor in repurposing antivirals for COVID-19[END_REF] on 65 inpatients tested positive for SARS-CoV-2 and treated or not by calcium channel blockers (CCB, i.e., nifepidine or amlodipine as a FIASMA) for hypertension found that patients treated by CCB were significantly more likely to survive than those not treated by CCB (50 % survived in the CCB group versus 14.6 % survived in the No-CCB group). As a retrospective study involving few patients, these data have to be considered with caution. Moreover, a submitted study [START_REF] Hoertel | Association between SSRI Antidepressant Use and Reduced Risk of Intubation or Death in Hospitalized Patients with Coronavirus Disease 2019: a Multicenter Retrospective Observational Study[END_REF] has explored the association between antidepressant use and the risk of intubation or death in inpatients with COVID-19. Significant association was reported between the use of any antidepressant and reduced risk of intubation or death. When individual antidepressant was taken into account, significant associations were found for escitalopram, fluoxetine and venlafaxine. Among the 3 antidepressants one was a FIASMA (fluoxetine). Data available in the clinical hospital data warehouses may help us testing this assumption and to identify marketed drugs to be repositioned. For example, in the Erasme hospital from Brussels, 370 (27 %) of the 1373 hospital admission for COVID-19 (March to October 2020) took at least one FIASMA. Alternatively, in the Rennes University Hospital, the prevalence of FIASMA in patients admitted for COVID-19 was 8.5 % (55 on 642 until May 5, 2020). Secondly, pharmaco-epidemiological studies could be carried out exploring in general population if the chronic prescription of particular FIASMAs would be associated with a low prevalence of severe symptoms of the COVID-19 infection. Thirdly, all the compound identified as FIASMAs could be tested in vitro to explore their activity against SARS-COV-2. Fourthly, the FIASMAs having either a protective effect in general population or a significant activity in vitro could be tested in vivo using notably randomized double blinded study against placebo.

-What is new and conclusion

The drug repurposing approach with the hope to bring patients effective treatment faster has gathered much attention in the SARS-COV-2 pandemic era. The current knowledge suggests that FIASMAs, as small molecules having a lysosomotropism and clinically approved indications in various diseases, should be paid more attention for their repurposing as potential drugs against SARS-COV-2. We strongly suggest to explore the potential protective effect of the FIASMAs in subjects treated chronically for various diseases, and in case of Left : ASM is anchored to the inner leaflet of the lysosomal membrane by electrostatic forces so that the enzyme is protected from proteolytic degradation. Specific stimuli allow the translocation of ASM from the inner lysosome to the external leaflet of the cell where ASM catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Shingomyelin is the most abundant sphingolipid component of the mammalian plasma membrane where it is associated with cholesterol to form lipid rafts.

Right : FIASMA are cationic drugs with lipophilic properties that diffuse in the lysosome by passive diffusion and potentially via an additional mechanism using ABCB1 transporter located on the lysosomal membrane. These drugs become protonated in the intra-lysosomal acidic environment and increase the intra-lysosomal pH so that ASM is detached from the inner leaflet of the lysosomal membrane, and is further degraded by proteolysis. ASM translocation is no longer effective and the formation of lipid rafts is altered. Hence, different mechanisms of internalization used by viruses (i.e., fusion and entry) to penetrate in the cytosol of cells are altered.

Functional acid sphingomyelinase (ASM)

anchored to the inner lysosomal membrane via electrostatic forces.

FIASMA interference with ASM membrane binding FIASMA diffusion in the lysosome via passive diffusion or transportermediated diffusion ASM membrane detachment and further inactivation

Figure 1 .

 1 Figure 1. Mechanisms of action of functional inhibitors of acid sphingomyelinase (ASM).

Table 2 )

 2 As mentioned above, different mechanisms of internalization are used by viruses to penetrate in the cytosol of cells, and lipid-raft mediated endocytosis

	F
	o r
	P
	e e
	r
	R
	e v i
	e w
	6

Table 1 :

 1 Functional inhibitors of acid sphingomyelinase with their ATC classification (functional inhibition is defined by a reduction to ASM activity below 50 % at a 10 μM concentration,[START_REF] Kornhuber | Functional Inhibitors of Acid Sphingomyelinase (FIASMAs). Erich Gulbins • Irina Petrache Editors Sphingolipids: Basic Science and Drug Development[END_REF][START_REF] Hoehn | Melatonin Acts as an Antidepressant by Inhibition of the Acid Sphingomyelinase/Ceramide System[END_REF]).

	Denomination	ATC-5	ATC-4
	Alverine	A03AX08	Drugs for functional gastrointestinal disorders
	Amiodarone	C01BD01	Class 1 and 3 antiarrhythmic drugs
	Amitriptyline	N06AA09	Antidepressant drugs
	Amlodipine	C08CA01	Selective calcium channel blockers
	Astemizole	R06AX11	Antihistamines for systemic use
	Benztropine	N04AC01	Antiparkinson drugs
	Bepridil	C08EA02	Non-selective calcium channel blockers
	Camylofine	A03AA03	Drugs for functional gastrointestinal disorders
	Carvedilol	C07AG02	Beta blocking agents
	Cepharanthine	(1)
	Chlorpromazine	N05AA01	Antipsychotic drugs
	Chlorprothixene	N05AF03	Antipsychotic drugs
	Cinnarizine	N07CA02	Antivertigo preparations
	Clemastin	R06AA04	Antihistamines for systemic use
	Clofazimine	J04BA01	Drugs for treatment of lepra
	Clomiphene	G03GB02	Gonadotropins and other ovulation stimulants
	Clomipramine	N06AA04	Antidepressant drugs
	Cloperastine	R05DB21	Cough suppressants
	Cyclobenzaprine	M03BX08	Muscle relaxants, centrally acting agents
	Cyproheptadine	A15AA01	Appetite stimulants
	Desipramine	N06AA01	Antidepressant drugs
	Desloratadine	R06AX27	Antihistamines for systemic use
	Dicyclomine	A03AA07	Drugs for functional gastrointestinal disorders

Journal of Clinical Pharmacy and Therapeutics

No funding was received for this work.

ACCEPTED MANUSCRIPT

10 positive results to test the corresponding FIASMAs in off-label therapy using notably largesized, randomized and double-blinded controlled clinical trials.

Author Contributions : All authors performed the literature search and wrote the manuscript.

Conflict of interest :

All the authors (PLC & GL) disclose any financial and personal relationships with other people or organizations that could inappropriately influence their work.

ACCEPTED MANUSCRIPT

o Clofazimine 

 

Emetine 



 (40-43, 46) + + Fluoxetine  (49, 50)

  [START_REF] Dyall | Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection[END_REF][START_REF] Liu | Testing of Middle East respiratory syndrome coronavirus replication inhibitors for the ability to block viral entry[END_REF])





  [START_REF] Dyall | Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection[END_REF][START_REF] Liu | Testing of Middle East respiratory syndrome coronavirus replication inhibitors for the ability to block viral entry[END_REF] + + Foot note :

In bold : drugs active against the 3 coronaviruses in silico (), in vitro ( ), ex vivo () and negative result ( †).

P-gp interaction : no information (o), positive information (+), negative information (-)