Aurélie Chazot

Claire Zimberger

Mikael Feracci

Adel Moussa

Steven Good

Jean-Pierre Sommadossi

Karine Alvarez

François Ferron
email: francois.ferron@univ-amu.fr

Bruno Canard
email: bruno.canard@univ-amu.fr

The activation chain of the broad-spectrum antiviral bemnifosbuvir at atomic resolution

Bemnifosbuvir (AT-527) and AT-752 are guanosine analogues currently in clinical trials against several RNA viruses. Here we show that these drugs require a minimal set of 5 cellular enzymes for activation to their common 5'-triphosphate AT-9010, with an obligate order of reactions. AT-9010 selectively inhibits essential viral enzymes, accounting for broad spectrum antiviral potency. Functional and structural data at atomic resolution decipher N 6 -purine deamination compatible with metabolic activation by human ADALP1. Crystal structures of human HINT1, ADALP1, GUK1, and NDPK at 2.09, 2.44, 1.76, and 1.9 Å resolution, respectively, with cognate precursors of AT-9010 illuminate the activation pathway from the orally available bemnifosbuvir to AT-9010, pointing to key drug-protein contacts along the activation pathway. Our work provides a framework to integrate the design of antiviral nucleotide analogues, confronting requirements and constraints associated with activation enzymes along the 5'-triphosphate assembly line.

Introduction

The recent COVID19 crisis due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evidenced the need for safe and potent antivirals, in conjunction with accurate and rapid diagnostics. First, at the public level, prophylaxis around index cases may significantly curb emergence by cutting viral load and transmissibility. Second, at the individual level, early therapy may limit virus spread to vital organs, avoiding complications.

Nucleoside/nucleotide analogues (NAs) represent the first family of compounds which have been used as direct-acting antivirals several decades ago [START_REF] Adamson | Antiviral drug discovery: preparing for the next pandemic[END_REF][START_REF] Seley-Radtke | The evolution of nucleoside analogue antivirals: A review for chemists and non-chemists. Part 1: Early structural modifications to the nucleoside scaffold[END_REF][START_REF] Yates | The evolution of antiviral nucleoside analogues: A review for chemists and non-chemists. Part II: Complex modifications to the nucleoside scaffold[END_REF] against DNA viruses such as herpesvirus, hepatitis B virus (HBV), and human immunodeficiency virus type 1 (HIV-1), and RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Upon reaching their target cells, antiviral drugs often act after intracellular activation. Early biologically active nucleoside analogues (NAs) such as ara-C or cytarabine penetrate the target cell efficiently and are transformed into the nucleoside analogue 5'-triphosphate (NA-TP) by a series of kinase reactions (reviewed in [START_REF] Seley-Radtke | The evolution of nucleoside analogue antivirals: A review for chemists and non-chemists. Part 1: Early structural modifications to the nucleoside scaffold[END_REF][START_REF] Yates | The evolution of antiviral nucleoside analogues: A review for chemists and non-chemists. Part II: Complex modifications to the nucleoside scaffold[END_REF][START_REF] Geraghty | Broad-Spectrum Antiviral Strategies and Nucleoside Analogues[END_REF] ).

ProTide prodrug technology has been implemented to deliver NA monophosphates [START_REF] Slusarczyk | Phosphoramidates and phosphonamidates (ProTides) with antiviral activity[END_REF] . Aryloxy phosphoramidate prodrugs of eg., tenofovir disoproxil fumarate (TDF) for HIV-1 and HBV, sofosbuvir (SOF) for HCV, and remdesivir (RDV) for SARS-CoV-2 have met clinical success 5- 7 . They achieve cell penetration through shielding the phosphate charges, and preferential hydrolysis at the P-N bond by-passes the first -often limiting-nucleoside kinase reaction, allowing significant building up of NA-TP pools. NA-TPs poison viral RNA synthesis specifically, provoking premature viral RNA chain termination or chemical/genetic corruption of the viral nucleic acid [START_REF] Shannon | Kill or corrupt: Mechanisms of action and drug-resistance of nucleotide analogues against SARS-CoV-2[END_REF] .

Two main parameters determine NA potency: the concentration ratio of NA-TP over its natural NTP competitor, and the selectivity of the viral RdRp for use of the NA-TP [START_REF] Johnson | Mechanisms of inhibition of viral RNA replication by nucleotide analogs[END_REF] . NA-TP pools must be formed efficiently upon intracellular activation, and the differences of the NA-TP scaffold relative to its natural NTP counterpart must remain 'below the radar' of the viral RdRp.

General knowledge, structural and functional data about cellular enzymes along a given NA activation pathway are fragmented. Numerous NAs have been designed showing appropriate poisoning of viral RNA synthesis through their 5'-TP in vitro, but clinical development has failed for lack of transmembrane permeability, intracellular metabolic activation, and/or cellular toxicity. Clearly, nucleotide analogue drug-design needs an integrated view (ie., structural, functional, and mechanistic) from the delivered NA up to the ultimate inhibited viral enzyme reaction accounting for antiviral effect, as well as the molecular basis for (non)interaction with cellular enzymes.

Such a global picture is emerging for two FDA-approved NA prodrugs directed against RNA viruses, SOF and RDV (Fig. S1). The activation pathway of RDV has been elucidated and profiled in a variety of tissues [START_REF] Li | Key Metabolic Enzymes Involved in Remdesivir Activation in Human Lung Cells[END_REF] . RDV is an aryloxy phosphoramidate prodrug (GS-5734, Veklury) converted in two steps to the 5'-monophosphate NA by esterases belonging to two families (CatA and CES1), followed by the cleavage of the P-N bond by a histidine triad nucleotide (HINT) phosphoramidase [START_REF] Li | Activation of Tenofovir Alafenamide and Sofosbuvir in the Human Lung and Its Implications in the Development of Nucleoside/Nucleotide Prodrugs for Treating SARS-CoV-2 Pulmonary Infection[END_REF] . The resulting monophosphate NA is then converted to RDV-TP by subsequent action of two cellular phosphotransferases. However, although detailed studies at the atomic level exist to understand accommodation of the 1'-CN group at the SARS-CoV-2 RdRp active site [START_REF] Wang | Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase[END_REF][START_REF] Kokic | Mechanism of SARS-CoV-2 polymerase stalling by remdesivir[END_REF] , structural insight of the relevant activation intermediates is lacking for all enzymes in the activation pathway.

SOF (PSI-7851, Sovaldi) undergoes the same CatA/CES1 deprotection pathway as RDV, but is activated to the 5'-triphosphate by the UMP-CMP kinase and the nucleoside diphosphate kinase (NDPK) sequentially [START_REF] Murakami | Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977[END_REF] . Here again, although the mechanism of chain-termination at the HCV NS5b RdRp has been elucidated at atomic resolution [START_REF] Appleby | Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase[END_REF] , the structural basis of activation remains uncharacterized.

Bemnifosbuvir (AT-527 (hemisulfate salt), AT-511 (free base)) is a guanosine analogue currently in clinical trials against SARS-CoV-2 and HCV. It showed in Phase II clinical trials a 71% risk reduction in out-patients with moderate COVID-19 (MORNINGSKY; NCT04396106) [START_REF] Toussi | Therapeutics for COVID-19[END_REF][START_REF] Horga | Oral bemnifosbuvir (AT-527) vs placebo in patients with mild-tomoderate COVID-19 in an outpatient setting (MORNINGSKY)[END_REF] , and is currently under investigation in a global phase III clinical trial in outpatients at high risk for disease progression (SUNRISE-3; NCT05629962). It is also being evaluated as an anti-HCV drug [START_REF] Good | Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus[END_REF] in combination with the NS5A inhibitor ruzasvir (NCT05904470). Its epimer AT-752 is currently in clinical phase II against Dengue virus (NCT05466240) [START_REF] Good | Evaluation of AT-752, a Double Prodrug of a Guanosine Nucleotide Analog with In Vitro and In Vivo Activity against Dengue and Other Flaviviruses[END_REF][START_REF] Feracci | AT-752 targets multiple sites and activities on the Dengue virus replication enzyme NS5[END_REF] .

Both bemnifosbuvir and AT-752, once processed by the CatA/CES1 pathway, converge to the same precursor AT-551 (Fig. 1A) [START_REF] Good | Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus[END_REF] . These two analogues are amongst the few antiviral purine nucleotide analogues devoid of significant cellular toxicity. Once the P-N bond of AT-551 is hydrolyzed (presumably) by HINT1 [START_REF] Shah | Caught before Released: Structural Mapping of the Reaction Trajectory for the Sofosbuvir Activating Enzyme, Human Histidine Triad Nucleotide Binding Protein 1 (hHint1)[END_REF] , giving rise to the monophosphate AT-8003, the diamino purine base is (presumably) converted to a natural guanosine base through specific N [START_REF] Mehellou | The ProTide Prodrug Technology: From the Concept to the Clinic: Miniperspective[END_REF] deamination carried out by the ADALP1 enzyme [START_REF] Schinkmanová | Human N 6 -Methyl-AMP/DAMP Aminohydrolase (Abacavir 5'-Monophosphate Deaminase) is Capable of Metabolizing N 6 -Substituted Purine Acyclic Nucleoside Phosphonates[END_REF][START_REF] Murakami | Adenosine Deaminase-like Protein 1 (ADAL1): Characterization and Substrate Specificity in the Hydrolysis of N 6 -or O 6 -Substituted Purine or 2-Aminopurine Nucleoside Monophosphates[END_REF] . This reaction is believed to skirt a cellular step responsible for the toxicity of unprotected, 'natural' guanosine analogues [START_REF] Good | Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus[END_REF][START_REF] Good | AT-527, a double prodrug of a guanosine nucleotide analog, is a potent inhibitor of SARS-CoV-2 in vitro and a promising oral antiviral for treatment of COVID-19[END_REF] . The resulting 2'-F-2'-C-methyl guanosine 5'-monophosphate (AT-8001) is (presumably) consecutively phosphorylated twice to yield AT-9010 by means of guanylate kinase 1 (GUK1) [START_REF] Khan | Solution structure and functional investigation of human guanylate kinase reveals allosteric networking and a crucial role for the enzyme in cancer[END_REF] and nucleotide diphosphate kinase (NDPK) [START_REF] Georgescauld | Structure, Folding and Stability of Nucleoside Diphosphate Kinases[END_REF][START_REF] Attwood | The actions of NME1/NDPK-A and NME2/NDPK-B as protein kinases[END_REF] . AT-9010 accumulates in various cell types [START_REF] Good | Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus[END_REF] . The HCV RNA synthesis is likely halted through RNA chain termination [START_REF] Good | Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus[END_REF] . SARS-CoV-2 RNA synthesis is halted through dual targeting of the replicase complex [START_REF] Shannon | A dual mechanism of action of AT-527 against SARS-CoV-2 polymerase[END_REF] , and the Dengue virus RNA synthesis is halted through dual targeting of the viral protein NS5 [START_REF] Good | Evaluation of AT-752, a Double Prodrug of a Guanosine Nucleotide Analog with In Vitro and In Vivo Activity against Dengue and Other Flaviviruses[END_REF][START_REF] Feracci | AT-752 targets multiple sites and activities on the Dengue virus replication enzyme NS5[END_REF] .

Most information relative to activation pathways has been obtained through measurements of intracellular concentrations of NA intermediates [START_REF] Li | Key Metabolic Enzymes Involved in Remdesivir Activation in Human Lung Cells[END_REF][START_REF] Li | Activation of Tenofovir Alafenamide and Sofosbuvir in the Human Lung and Its Implications in the Development of Nucleoside/Nucleotide Prodrugs for Treating SARS-CoV-2 Pulmonary Infection[END_REF][START_REF] Murakami | Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977[END_REF][START_REF] Good | AT-527, a double prodrug of a guanosine nucleotide analog, is a potent inhibitor of SARS-CoV-2 in vitro and a promising oral antiviral for treatment of COVID-19[END_REF] . Studies described herein aim to ascertain which individual enzymes are involved in the activation pathway, as well as clarify their structural and functional mode of interaction with activation intermediates. Interaction maps of NAs with both activating partners and viral enzymes at atomic resolution would guide improved design of novel NAs.

In this work, we have identified a minimal set of 5 human enzymes involved in the activation of bemnifosbuvir and AT-752 to their active 5'-TP form AT-9010. We demonstrate distinct stereo-preference of CatA and CES1 for AT-527 and its epimer AT-752, respectively. We elucidate an ordered activation pathway with potential limiting steps, including substrate specificity requirements at the N 6 -purine position for ADALP1. Structural analysis with key human enzymes HINT1, ADALP1, GUK1, and NDPK in complex with the bemnifosbuvir/AT-752 intermediate NA at 2.09, 2.44, 1.76, and 1.9 Å resolution informs on both the N 6 -purine and 2'-ribose chemo-steric requirements leading to active NAs targeting viral RdRps.

Results

The putative metabolic pathway of AT-527 (bemnifosbuvir) and AT-752 is shown in Fig. 1A.

Cathepsin A (CatA) and/or carboxylesterase 1 (CES1) presumably precede a spontaneous decomposition yielding the L-alanine phosphoramidate prodrug AT-551, common to both AT-752 and AT-527 pathways. AT-551 is then subjected to four enzyme-mediated reactions leading to the active 5'-triphosphate form, AT-9010. Apart from the N 6 -deamination reaction, this pathway has been inferred from that of the anti-HCV drug SOF, which carries a uracil base instead of the N 6 -methyl diamino purine base (a guanine precursor) of either AT-752 or AT-527. The SOF pathway, however, has been determined indirectly, by measuring intermediates in cells treated with SOF [START_REF] Li | Activation of Tenofovir Alafenamide and Sofosbuvir in the Human Lung and Its Implications in the Development of Nucleoside/Nucleotide Prodrugs for Treating SARS-CoV-2 Pulmonary Infection[END_REF][START_REF] Murakami | Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977[END_REF] .

Five enzymes are needed to convert AT-527 (bemnifosbuvir) or AT-752 to their active form AT-9010

We expressed, purified and crystallized four of the human enzymes widely distributed in human tissues (https://proteinatlas.org) and supposedly involved in the prodrug activation pathway (Fig. 1A, Fig. S2). Together with human enzymes CatA and CES1, we challenged HINT1, ADALP1, GUK1, and NDPKb with their anticipated substrates depicted in Fig. 1A and Fig. S3. We made use of an HPLC-UV method to follow the conversion of the substrate, together with a kinetic monitoring relative to known standards (Fig. 1B).

Both AT-527 and AT-752 can be enzymatically converted to AT-9010 by an ordered series of reaction involving non-specific esterases CatA/CES1, HINT1, ADALP1, GUK1, and NDPK (Table 1). For AT-511, non-specific esterases CatA and CES1 exhibit a ~300-fold difference in activity whereas hydrolysis efficiency is quite similar for AT-281 (< 3-fold difference). Both for control and AT compounds, the early steps of the pathway are the slowest, with di-and triphosphate formation showing the fastest turnovers.

The phosphate stereo-selectivity of CatA/CES1 usage

The phosphorus atom of the aryloxy phosphoramidate moiety is chiral, resulting in two possible epimers. AT-511 (SP isomer) is the free base form of AT-527, and AT-281 (RP isomer) is the free base form of AT-752 (Fig. S3). As shown for SOF, the difference of antiviral activity between the two stereoisomers could be due to the activation of the prodrug and more specifically to the stereo-selectivity of the enzymes involved in the first hydrolytic step.

CatA or CES1 were incubated with either AT-511 or AT-281 and comparative velocities of AT-551 formation were measured (Fig. S4). CatA always shows higher activity than CES1. CatA prefers by 14-fold the SP isomer AT-511 over the RP isomer AT-281 (9.8 ± 0.3 vs. 0.7 ± 0.1 nmol/min/nmol protein). Interestingly, however, a reversed stereo-selectivity is observed for CES1 relative to CatA: CES1 hydrolyses AT-281 ~10-fold faster than AT-511 (0.27 ± 0.02 vs. 0.033 ± 0.004 nmol/min/nmol protein, respectively). These results align with those obtained with a variety of clinically relevant prodrugs; for SOF, TAF, and RDV, CES preferentially hydrolyzes the RP isomer whereas CatA prefers the SP isomer of the aryloxy phosphoramidate moiety [START_REF] Li | Key Metabolic Enzymes Involved in Remdesivir Activation in Human Lung Cells[END_REF][START_REF] Li | Activation of Tenofovir Alafenamide and Sofosbuvir in the Human Lung and Its Implications in the Development of Nucleoside/Nucleotide Prodrugs for Treating SARS-CoV-2 Pulmonary Infection[END_REF][START_REF] Murakami | Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977[END_REF] . We conclude that non-specific esterases of the CatA/CES1 type are able to convert bona fide McGuigan ProTides into HINT1 substrates from both AT-752 and AT-527. The respective abundance of each enzyme in cells or tissues might be useful as a predictor of ester hydrolysis efficiency in these given cells or tissues.

Specificity and order of the reactions

AT-551 could not act as a substrate for ADALP1 (Fig. S5). This result indicates that the Lalanine phosphoramidate precludes efficient either binding or catalytic conversion of AT-551 with ADALP1 (see structural data below). AT-8003 could neither be a substrate for GUK1, indicating that the N 6 position must be either unsubstituted or only an O 6 to allow binding and phosphorylation of the NMP (Fig. S5). Unless other enzymes exist that could use these substrates and by-pass the proposed activation sequence, the absence of reactivity reported here is consistent with the order of the reaction pathway reported in Fig. 1A.

Structural and functional analysis of HINT1 substrate specificity

HINT1 belongs to the HIT (histidine triad) protein superfamily, and has been shown to hydrolyse the P-N bonds in SOF and RDV [START_REF] Li | Key Metabolic Enzymes Involved in Remdesivir Activation in Human Lung Cells[END_REF][START_REF] Li | Activation of Tenofovir Alafenamide and Sofosbuvir in the Human Lung and Its Implications in the Development of Nucleoside/Nucleotide Prodrugs for Treating SARS-CoV-2 Pulmonary Infection[END_REF][START_REF] Murakami | Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977[END_REF] . HINT1 is able to hydrolyze AMP-NH2 (control) into AMP, and AT-551 into AT-8003 with specific activities of 101 ± 8 and 0.7 ± 0.2 nmol/min/nmol protein, respectively (Fig. 1B and Table 1). HINT1 is thus ~140-fold faster with the control compound than with AT-551.

We crystallized HINT1 in complex with AT-551 or AT-8003 (Fig. 2).

For AT-551, reaction occurred in the crystal resulting in AT-8003 bound to the active site on one monomer (Fig. 2A). The crystal structure (2.09 Å resolution) shows a homodimer, each monomer being almost perfectly superimposable (RMSD ~0.18 Å on the visible 126 aa).

The bound product pose is significantly different from all existing HINT1 structures 21 in complex with AMP, GMP, or NMP-phosphoramidate ligands (Fig. 2B). In all available structures so far, the nucleotide analogue is buried in a pocket, contacting conserved amino acids Ile 44 and Asp 43 involved in purine base stacking and ribose 2',3'-OH ribose bi-dentate binding, respectively. Here, the methylated diamino purine base is bound outside the nucleobase binding pocket, shifted by 90° and making a single hydrogen bond with Ser 45 that could occur with any purine or pyrimidine base. Remarkably, the two parts of the AT-8003 which differentiate it from guanosine are not engaged in any amino acid contact. Both the N 6amino purine and the 2'-C-methyl, 2'-F are projecting into the solvent.

The phosphate is closely superimposable to that observed in other published structures [START_REF] Shah | Caught before Released: Structural Mapping of the Reaction Trajectory for the Sofosbuvir Activating Enzyme, Human Histidine Triad Nucleotide Binding Protein 1 (hHint1)[END_REF] . The catalytic His 112 nucleophilic nitrogen is 3.1 Å away from the phosphate, and the hydrolytic water molecule is positioned opposite to the putative leaving amine and general acid His 114 and/or His 51. In a similar complex (PDB: 5IPE), the O 6 -guanine of GMP is located at 3.3 Å from the Ile 18 side chain, and there is space to accommodate an N 6 -methyl amino group at the solvent-protein interface. The AT-8003 ribose pucker is of Northern (N) configuration: C3' up and C2' down (the Southern (S) pucker has the C3' down and C2' up), as this conformation must be imposed by the presence of its 2'-C-methyl. The ribose pucker of GMP and AMP analogues in all other structures being S, we surmise the 2'-C-methyl is driving this alternate preferential NA binding mode seen in all structures reported here.

Structural and functional analysis of ADALP1 substrate specificity

ADALP1 is a deaminase acting on N 6 -substituted purine nucleoside monophosphates [START_REF] Murakami | Adenosine Deaminase-like Protein 1 (ADAL1): Characterization and Substrate Specificity in the Hydrolysis of N 6 -or O 6 -Substituted Purine or 2-Aminopurine Nucleoside Monophosphates[END_REF] . The activity of purified ADALP1 was tested on N 6 -Me-AMP as a control compound before testing on the anticipated substrate AT-8003. Because the anti-HIV drug abacavir bears an N 6cyclopropyl modification [START_REF] Schinkmanová | Human N 6 -Methyl-AMP/DAMP Aminohydrolase (Abacavir 5'-Monophosphate Deaminase) is Capable of Metabolizing N 6 -Substituted Purine Acyclic Nucleoside Phosphonates[END_REF] , we tested several N 6 -modified analogues: -NH(nPr) (AT-8010), -N(CH3)2 (AT-8004) and -NH2 (AT-8002). The nucleoside versions of AT-8003 and AT-8010 were also tested, ie., AT-229 and AT-259, respectively. ADALP1 is able to convert N 6 -Me-AMP into IMP (42 ± 2 nmol/min/nmol protein), as well as AT-8003, AT-8004 and AT-8010 into AT-8001. AT-8003, AT-8004 and AT-8010 are converted 3 to 8-fold slower than N 6 -Me-AMP (9.4 ± 0.4 nmol/min/nmol protein, 11.9 ± 0.1 nmol/min/nmol protein and 5.21 ± 0.07 nmol/min/nmol protein, respectively, Fig. 1B and Table 1). ADALP1 is thus able to accept the 2'-C-methyl, 2'-F modifications on the ribose part, as well as several substituents on the N 6 -position. Extension of the N 6 -substituent, not its bulk immediately close to the nitrogen atom, seems to negatively influence activity (Fig. 3A).

Under our experimental conditions, ADALP1 converts only nucleoside monophosphate substrates: no activity was detected with the nucleosides AT-229 and AT-259.

Interestingly, the reaction is ~45-fold slower with AT-8002 as substrate compared to the natural substrate N 6 -Me-AMP, indicating that at least one substituent on the nitrogen atom may enhance the leaving group ability.

We co-crystallized ADALP1 and AT-8003. The solved ADALP1 structure adopts an α/βbarrel architecture similar to that of ADAs 29 , with the essential Zn 2+ ion tightly bound in the vicinity of the catalytic center (Fig. 3B and Fig. S6A). Fourteen α-helices surrounding eight parallel β-strands constitute a TIM-barrel fold [START_REF] Romero-Romero | Evolution, folding, and design of TIM barrels and related proteins[END_REF] . The asymmetric unit contains eight copies of the protein. Since ADALP1 was purified as a homogeneous monomer (Fig. S6B), the octamer likely appeared during crystallogenesis. Although ADALP1 was co-crystallized with the substrate AT-8003, the product AT-8001 was found bound at the active site. The compound is found in all eight chains and all the chains found in the asymmetric unit closely superimpose (r.m.s.d. of ~0.26 Å) except for the minor loop and N-and C-terminal ends (Fig. S6C). The structural zinc is coordinated by side chains of four residues (His 24, His 26, His 208 and Asp 293) and a water molecule, in a trigonal bipyramidal geometry [START_REF] Zheng | Validation of metal-binding sites in macromolecular structures with the CheckMyMetal web server[END_REF] . Water bridges His 232 and the resulting O 6 of the purine base suggesting that both His 293 and the water molecule did play a role in the hydrolysis of the amine. AT-8001 is buried in a closed hydrophobic pocket that can only accommodate a monophosphate compound checked and stabilized by Asn 28, His 74, Ser 106, and Thr 107.

Unlike in the HINT1:AT-8003 complex, there is no ambiguity in substrate nor product binding pose [START_REF] Romero-Romero | Evolution, folding, and design of TIM barrels and related proteins[END_REF] . Once the substrate is bound, the catalytic water attacks the purine C [START_REF] Mehellou | The ProTide Prodrug Technology: From the Concept to the Clinic: Miniperspective[END_REF] which likely stays at this location whilst the methylamine is leaving in the opposite exit channel.

Structural and functional analysis of GUK1 specificity

GUK1 is a nucleoside monophosphate kinase able to transfer a phosphate group from ATP to a guanosine 5'-monophosphate, yielding GDP and ADP [START_REF] Khan | Solution structure and functional investigation of human guanylate kinase reveals allosteric networking and a crucial role for the enzyme in cancer[END_REF] . Simultaneous binding of monophosphate substrate and triphosphate donor precedes conformational closure and phosphate transfer. This enzyme metabolizes several purine analogues such as abacavir and ganciclovir [START_REF] Barbarino | PharmGKB summary: abacavir pathway[END_REF][START_REF] Miller | Phosphorylation of acyclovir (acycloguanosine) monophosphate by GMP kinase[END_REF] . Expectedly, GUK1 is able to phosphorylate GMP into GDP, but also AT-8001 into AT-8500 (Fig. 1B and Table 1). GUK1 is much faster with GMP (control) than with AT-8001 (respectively 6800 ± 300 nmol/min/nmol protein vs. 34 ± 5 nmol/min/nmol protein). GUK1 exhibits the highest turnover in the activation chain (Table 1), but the 2'-C-methyl, 2'-F modification slows it down ~200-fold.

GUK1 was crystallized and its structure solved at 1.76 Å resolution in complex with AT-8001 (Fig. 4, Table 2).

The overall fold of GUK 1 closely resembles that of the yeast and mouse guanylate kinases [START_REF] Blaszczyk | Crystal structure of unligated guanylate kinase from yeast reveals GMP-induced conformational changes[END_REF][START_REF] Sekulic | Structural characterization of the closed conformation of mouse guanylate kinase[END_REF] . Two β-sheets and eight α-helices, the last two separated by a π-helix, define three structurally and functionally distinct sub-domains: the core, the NMP-binding site, and the lid [START_REF] Sekulic | Structural characterization of the closed conformation of mouse guanylate kinase[END_REF] . The core is defined by residues 5-31, 97-123, and 165-194 (helices α1, α4, α7, and α8; strands β1, β7, β8, and β9). The NMP-binding site is a mobile structural element defined by residues 37-89 (starting with a protruding loop and followed by helices α2 and α3; strands β3, β4, β5, and β6). The lid where the phosphate donor (eg., ATP) binds, is also mobile and defined by residues 126-156 (helices α5 and α6). These parts are interconnected with four hinges that allow conformational changes from an open to closed state [START_REF] Blaszczyk | Crystal structure of unligated guanylate kinase from yeast reveals GMP-induced conformational changes[END_REF] . The structure presented here is an open conformation (Fig. 4), with the AT-8001 substrate binding through extensive contact with both the phosphate moiety and the nucleobase. Thus, on the one hand, Tyr 81, Tyr 53 and Arg 41 share hydrogen bonding with the oxygen of the phosphate, while Arg 44 makes a contact through a water molecule. On the other hand, Glu 72 and Ser 37 engage hydrogen bonds with the base while Tyr 53 contributes to positioning the base through π-stacking. The protein structurally checks the substrate by contacting both the purine base and the presence of a monophosphate, with no significant check on the ribose at this stage.

We modeled AT-8001 into a closed GUK1 conformation based on an energy-minimized model of hGUK1 (Fig. S7). The base is constrained by interactions with Ser 37, Glu 72, and Thr 83. The major difference observed with bound NMP substrates is the ribose pucker conformation. Again, in the structure with bound GMP, the 3'-OH ribose is in S pucker position to accommodate the closed conformation of the active site, but not for AT-8001, as described for HINT1 above. To avoid a steric conflict of the 2'-C-methyl, the compound has to slide into the core where the base is π-stacked by Tyr 81 and stabilized by Ser 37 and Glu 72. Both Arg 41 and Arg 44 contact the phosphate; Arg 137 and Arg 148 are expected to catalyze phosphate transfer from ATP bound in the lid, but the repositioning creates distances not compatible for optimal transfer. This positional adjustment likely explains the ~200-fold discrimination of AT-8001 versus the natural substrate GMP (Table 1).

Structural and functional basis of NDPK specificity

NDPK is able to transfer a phosphate group from a triphosphate nucleoside donor to a nucleoside diphosphate acceptor [START_REF] Georgescauld | Structure, Folding and Stability of Nucleoside Diphosphate Kinases[END_REF][START_REF] Chen | Nucleotide binding to nucleoside diphosphate kinases: X-ray structure of human NDPK-A in complex with ADP and comparison to protein kinases[END_REF] , to yield the active triphosphate form of bemnifosbuvir AT-9010. The reaction proceeds through a so called 'ping-pong' mechanism in which a triphosphate nucleoside transfers covalently its g-phosphate to the enzyme, and the diphosphate nucleoside acceptor is subsequently bound to the same site to accept the phosphate. NDPK is known to phosphorylate a wide range of nucleotide analogues bearing various structural modifications. The activity of purified NDPK chain b (NDPKb) [START_REF] Attwood | The actions of NME1/NDPK-A and NME2/NDPK-B as protein kinases[END_REF] ,is able to phosphorylate GDP (control) into GTP and also AT-8500 into AT-9010, the expected product (Fig. 1B and Table 1). NDPKb shows a high conversion rate and is only 4-fold slower with AT-8500 as substrate than GDP (1300 ± 100 nmol/min/nmol protein vs. 5400 ± 800, respectively).

We crystallized the NDPKb with AT-9010 (Fig. 5).

The crystal structure was solved at 1.9 Å resolution. A hexamer defines the asymmetric unit with six extra densities corresponding to six AT-8500 diphosphate. Interestingly, only the α and β-phosphates of the 5'-triphosphate moiety are visible. A new density is observed near the NE2 of His 118 of each monomer, which correspond to the histidine phosphorylated state His 118-P in Fig. 5 (inset). The AT-9010 g-phosphate is transferred to the latter due to the protein ability to catalyze the reaction in both directions via a ping-pong mechanism. Structural features of the nucleotide binding site are consistent with the required NDPKb substrate promiscuity. Direct comparison with NDPKb in complex with GDP (PDB: 1NUE) shows a conserved basestacking between the guanine and Phe 60. The hydrogen bond between Lys 12 and the 3'-OH of the ribose is no longer present because of a displacement of the ribose, probably due to the 2'-C-methyl, 2'-F modification. This shift allows the 3'-OH to make direct contact with the phosphate group of His 118-P (3Å). The β-phosphate is engaged in hydrogen bonds with Arg 88 and is facing the catalytic site of the enzyme.

Discussion

The journey of a drug begins with cellular uptake up to expression of its antiviral activity into a virus-infected cell. There is a need to better anticipate unexpected complexities. A number of NA prodrugs (eg., SOF, TAF, REM) need CatA/CES1 [START_REF] Li | Key Metabolic Enzymes Involved in Remdesivir Activation in Human Lung Cells[END_REF][START_REF] Li | Activation of Tenofovir Alafenamide and Sofosbuvir in the Human Lung and Its Implications in the Development of Nucleoside/Nucleotide Prodrugs for Treating SARS-CoV-2 Pulmonary Infection[END_REF][START_REF] Murakami | Metabolism and pharmacokinetics of the anti-hepatitis C virus nucleotide prodrug GS-6620[END_REF][START_REF] Li | Contributions of Cathepsin A and Carboxylesterase 1 to the Hydrolysis of Tenofovir Alafenamide in the Human Liver, and the Effect of CES1 Genetic Variation on Tenofovir Alafenamide Hydrolysis[END_REF] -mediated activation. However, CatA is inhibited by the antiviral drug telaprevir (an HCV protease inhibitor) [START_REF] Li | Key Metabolic Enzymes Involved in Remdesivir Activation in Human Lung Cells[END_REF][START_REF] Murakami | Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977[END_REF] , complicating potential combination therapies relying on this esterase. Likewise, the ribavirin antiviral effect results both from cellular inosine monophosphate dehydrogenase and viral growth inhibitions (reviewed in [START_REF] Thomas | The application and mechanism of action of ribavirin in therapy of hepatitis C[END_REF] ).

Here we cast light on the activation pathway of bemnifosbuvir and its epimer AT-752. Our work quantitates the differential activation of these substrates relative to their natural counterpart GTP. We visualize and measure essential interactions these two compounds and their metabolites engage with specific enzymes involved in their sequential activation.

Turnover measurements (Table 1) show that the rate-limiting steps are generally occurring early in the pathway (CatA/CES1, HINT1, and ADALP1) rather than at the level of the kinases. On these slow, early steps, we note a de-esterification rate ~14-fold higher for AT-511 than AT-281 into the common intermediate AT-551 by CatA. Comparatively, CES1 reacts ~300fold slower than CatA with AT-511, but shows an opposite stereo-preference with AT-281 being preferred ~8-fold over AT-511. These results are aligned with those obtained with PSI-7851, a racemic mixture of PSI-7976 and PSI-7977 (SOF) which shows both the same CatA and stereoisomer preference [START_REF] Murakami | Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977[END_REF] .

Murakami et al. [START_REF] Murakami | Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977[END_REF] have shown that HINT1 is the rate-limiting step along the SOF activation pathway. Its metabolic intermediate PSI-352707 (Fig. S1) binds poorly to HINT1, precluding saturating substrate conditions in infected cells and determination of kcat and Km in enzyme assays. In the case of AT-551, HINT1 is also the rate-limiting step (Table 1). Our HINT1:AT-8003 complex structure provides a structural basis for the kinetic bottleneck along the SOF activation line [START_REF] Murakami | Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977[END_REF] . We propose that the higher HINT1 conversion activity of AT-551 as compared to the corresponding intermediate metabolite of SOF, PSI-352707, which differ only by their nucleobase, results from the higher stacking power of purines versus uracil onto Ile 44. GS-6620 (Fig. S1) is a C-nucleoside combining a purine-like base with a 2'-hydroxy, 2'-C-methyl and a 1'-CN group. Murakami et al. have shown that GS-6620 has a comparable HINT1 turnover to that of AT-551 [START_REF] Murakami | Metabolism and pharmacokinetics of the anti-hepatitis C virus nucleotide prodrug GS-6620[END_REF] suggesting that GS-6620 likely adopts the same binding mode as AT-551 rather than fitting into the pocket able to tightly accommodate natural purines. The HINT1 limiting step, however, does not seem to impact accumulation of the SOF 5'triphosphate form (nor AT-9010) in cells. The compound pools are then readily available for selective use by the NS5B viral RdRp in HCV-infected liver cells.

These results indicate that cell types and their relative expression of these 'early step' enzymes CatA/CES1 and HINT1 are expected to play a major role in drug activation: i) the esterase-mediated deprotection step may well govern the overall rate of conversion of the drug into its active metabolite; ii) the relative abundance of CatA-and CES1-like enzymes should influence these early steps; and iii) further optimization in antiviral activity might be possible through optimization of the prodrug part in the 5' portion of the NA rather than focusing on optimizing the kinase steps. The choice of targeted infected cells may ultimately point to the most appropriate epimer and prodrug. In the case of ADALP1, unlike HINT1, the substrate and products are superimposable in the crystal structure, indicating that this complex should be reliable and useful for drug design.

Ideally, the NA activation line must be proven functional in cells, organs, or animal models supporting pre-clinical tests before commitment to full clinical trials in humans. AlphaFold2 40generated structural models of homologous animal enzymes did not point to obvious polymorphisms potentially deleterious for activation (not shown). The very high degree of structural conservation along species suggests a vital role in mammals for these activation enzymes. As such, monitoring differential expression of HINT1 should remain a sufficient and essential asset in predicting P-N hydrolysis in a given tissue or cell. The situation is somewhat similar for ADALP1, which shows significant polymorphisms across organisms, although mapping them invariably points to external, solvent exposed loops, with no direct impact on the NA binding site.

In the case of exposition of cells to bemnifosbuvir, its corresponding 5'-triphosphate AT-9010 has been demonstrated in cellular concentrations up to ~700 µM in human primary cells and cell types incubated in vitro with AT-511, including bronchial and nasal epithelial cells [START_REF] Good | Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus[END_REF] , hepatocytes and Huh7 cells [START_REF] Good | AT-527, a double prodrug of a guanosine nucleotide analog, is a potent inhibitor of SARS-CoV-2 in vitro and a promising oral antiviral for treatment of COVID-19[END_REF] , and peripheral blood mononuclear cells (data not shown). Thus, none of the enzymes studied here limit the formation of the active compound AT-9010 in these cells. Bemnifosbuvir, though, shows reduced antiviral activity in some cell lines, eg., in VeroE6, HeLa, or MRC-5 cells [START_REF] Good | Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus[END_REF] . We note that cynomolgus monkey hepatocytes have been reported to activate bemnifosbuvir to levels 50-fold lower than Huh7 cells [START_REF] Good | Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus[END_REF] . The superimposition of human and cynomolgus HINT1 and ADALP1 structures does not point to any polymorphism that could substantiate differences in substrate binding or catalysis (not shown).

Bemnifosbuvir shares two activation enzymes (HINT1 and NDPKb) with SOF and RDV. Hence, the HINT1 structure should represent the most relevant enzyme to optimize the prodrug part of such antivirals. Two other enzymes ADALP1 and GUK1 are specific for diamino purine-and guanine-containing NAs, respectively. With the abacavir success in mind [START_REF] Schinkmanová | Human N 6 -Methyl-AMP/DAMP Aminohydrolase (Abacavir 5'-Monophosphate Deaminase) is Capable of Metabolizing N 6 -Substituted Purine Acyclic Nucleoside Phosphonates[END_REF] , the ADALP1 structure presented here should guide further exploration of chemical diversity at the N 6 -amino purine group.

A wealth of structural data already exists regarding nucleotide analogues in complex with RNA virus replicases, eg., HCV 15 ; picornavirus 41 ; Reovirus 42 ; SARS-CoV-2 [START_REF] Kokic | Mechanism of SARS-CoV-2 polymerase stalling by remdesivir[END_REF][START_REF] Shannon | A dual mechanism of action of AT-527 against SARS-CoV-2 polymerase[END_REF][START_REF] Malone | Structural basis for backtracking by the SARS-CoV-2 replicationtranscription complex[END_REF] ; for a repository of stacked RdRp structures, see [START_REF] Peersen | A Comprehensive Superposition of Viral Polymerase Structures[END_REF] . Much like along the NA activation line, analysis of selectivity of viral polymerase in ternary complexes also identifies proximal amino acid side chains potentially able to accommodate chemical decorations without impacting the NA-5'triphophate incorporation efficiency.

Finally, a NA drug potential is also conversely determined by its innocuity towards host enzymes that could misuse their 5'-triphosphate form and alter cell metabolism or host nucleic acid. Human mitochondrial RNA polymerase (POLRMT) inhibition accounts, in part, for cellular toxicity of NAs [START_REF] Freedman | A computational approach for predicting off-target toxicity of antiviral ribonucleoside analogues to mitochondrial RNA polymerase[END_REF] . Comparative examination of ternary complexes of viral RdRps and POLRMT [START_REF] Schwinghammer | Structure of human mitochondrial RNA polymerase elongation complex[END_REF] with their RNA and NA-TP substrates may further document the strategy to rationally integrate NA drug design from chemistry to selective inhibition of viral growth [START_REF] Freedman | A computational approach for predicting off-target toxicity of antiviral ribonucleoside analogues to mitochondrial RNA polymerase[END_REF] .

Our work casts light on a whole set of previously ill-defined reactions involved in drug activation, and contributes to the integration of nucleotide analogue drug-design from bioavailability to precise and selective mechanism of action as direct-acting antivirals.

Tables

Materials and Methods

Materials

Recombinant human cathepsin A (CatA), cathepsin L (CatL), carboxylesterase 1 (CES1) and trans-epoxy-succinyl-L-leucylamino(4-guanidino)butane (E-64) were purchased from R&D Systems. The respective genes coding for the human recombinant enzymes Histidine triad nucleotide binding protein 1 (HINT1), Adenosine deaminase-like protein 1 (ADALP1), Guanylate kinase 1 (GUK1) and Nucleoside diphosphate kinase (NDPKB) were coded into a pNC-ET28 expression vector with an N-terminal His-tag and TEV cleavage site, purchased from Twist Bioscience. Control compound AMP-NH2 was from Biosynth, N 6 -Me-AMP was purchased from BioLog Life Science Institute and AMP, IMP, GMP, GDP and GTP were from Sigma Aldrich. Other compounds with a name starting with "AT" were synthesized by TopPharm, Inc., USA.

Protein expression

The DNA sequence encoding for Adenosine deaminase-like protein 1 (ADALP1), Nucleoside diphosphate kinase B (NDPKB), Guanylate kinase 1 (GUK1) and Histidine triad nucleotide binding protein 1 (HINT1) were synthesized and cloned into a pET-28a (+) vector for HINT1 and GUK1, into a pNC-ET28 vector for NDPKB and into a pET-28 for ADALP1 (Twist Biosciences). The final construct encoded for HINT1, ADALP1, GUK1 and NDPKB with a Nterminal hexahistidine tag (6xHis) and a TEV protease recognition site.

All proteins were expressed in E. coli NEB C2566 cells (New England Biolabs). The cells were grown at 37°C in TB medium for HINT1, GUK1 and NDPKB, LB Borth for ADALP1 containing 50µg/mL Kanamycin until the absorbance at 600nm reached 0.6 -0.8. At this stage induction was started with 0.5mM IPTG and the cells were grown overnight at 16°C. Then the cells were harvested and the pellets were suspended in lysis buffer (50mM Tris pH = 8.0, 300mM NaCl, 10mM imidazole, 1mM PMSF, 0.25mg/mL lysozyme and 10µg/mL DNase) for HINT1, GUK1 and NDPKB, before storing them at -80°C. For ADALP1 the pellet was freezedried and stored at -80°C.

HINT1, GUK1 and NDPKb purification

The lysate was disrupted and cleared by centrifugation at 12,000g for 30min at 10°C. Then the supernatant was loaded onto a Ni-NTA-beads (ThermoFisher) batch and washed with 50 mM Tris pH = 8.0, 300 mM NaCl, 20 mM imidazole buffer. The recombinant protein was then eluted with 50 mM Tris pH = 8.0, 300 mM NaCl, 250 mM imidazole buffer. 10% glycerol was added for HINT1. TEV protease was used to remove the N-terminus His-tag during an overnight dialysis in a buffer (50 mM Tris pH = 8.0, 150 mM NaCl, 1 mM DTT) at 4°C for HINT1, at 37°C for GUK1and at room temperature for NDPKB.

A second purification step on Ni-NTA beads was performed to remove any uncleaved protein before to achieve a SEC purification onto a Superdex 75 16/600 GE column (GE Healthcare) in a final buffer containing 10 mM Tris pH = 8.0, 50 mM NaCl for HINT1, 20 mM Tris pH = 8.0, 100 mM NaCl, 1 mM DTT for GUK1 and 50 mM Tris pH = 8.0, 150 mM NaCl, 1 mM DTT for NDPKb. The fractions were analysed on SDS-PAGE and the ones containing the pure target protein were pooled. The purified protein was then concentrated until 10 mg/mL, aliquoted and stored at -80°C. Same protocol was used to express and purify uncleaved NDPKb for activity assays.

ADALP1 purification

Bacterial pellets were lysed in half buffer (50 mM HEPES pH = 7.5, 300 mM NaCl, 10% glycerol, 0.5 mM TCEP) and half Master Mix Bug Buster (Merck) supplemented with a tablet of EDTA-free antiprotease cocktail (Roche) per 50 ml of lysate before sonication. The lysate was cleared by centrifugation at 12,000g for 30min at 10°C and the supernatant was applied onto a Talon Superflow beads batch (Merck). The immobilized proteins were washed with 50 mM HEPES pH 7.5, 300 mM NaCl, 12.5 mM Imidazole, 10% glycerol, 0.5 mM TCEP, then washed with 50 mM HEPES pH 7.5, 1.5M NaCl, 10% glycerol, 0.5 mM TCEP and eluted in buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 150 mM Imidazole, 10% glycerol 0.5 mM TCEP).

TEV protease was used to remove the N-terminus His-tag during an overnight dialysis in a buffer 50 mM HEPES pH = 7.5, 300mM NaCl, 10% glycerol, 1mM DTT. The untagged ADALP1 was further purified by a Talon Superflow beads batch (Merck). Finally, ADALP1 was purified by size exclusion chromatography using a S75 16/60 GE Column (GE Healthcare) equilibrated in buffer containing 10 mM Tris pH = 8.0, 40 mM NaCl, 1 mM TCEP. The single ADALP1 peak corresponded to the monomeric form. It was concentrated to 10 mg/mL, flash-frozen in liquid nitrogen, and stored at -80°C.

CatA enzyme assay

Human recombinant CatA was first activated by following the manufacturer's instructions (R&D Systems), incubating at 37°C 10 µg/mL CatA with 1 µg/mL CatL in an activation buffer (25 mM MES pH = 6.0, 5 mM DTT) during 30 min. A). The substrates and reaction products were eluted using a non-linear gradient of acetonitrile in buffer A, detailed in supplementary data.

Crystallization of HINT1

Crystallization conditions were adapted from [START_REF] Shah | Caught before Released: Structural Mapping of the Reaction Trajectory for the Sofosbuvir Activating Enzyme, Human Histidine Triad Nucleotide Binding Protein 1 (hHint1)[END_REF] . Briefly, co-crystals with AT-8003 were grown at 293.15 K, using a 1:1 ratio of HINT1 at 10 mg/mL with AT-8003 (final concentration 25 mM) to precipitant solution 0.1M MES pH 6.1-6.5, 27%-30% PEG 8000. Crystals grew in a few days and were cryo-protected with reservoir solution supplemented with 20% PEG 400, and flash-frozen in liquid nitrogen at 100 K.

Crystallization of ADALP1

Crystallization assay were set up using the sitting drop vapor diffusion method, using a 2:1 ratio of protein solution to the reservoir solution at 293.15K. The co-crystals with AT-8001 were obtained by mixing ADALP1 at 17 mg/mL with AT-8003 (final concentration 10 mM) and 1:10000 w/w dilution a-Chymotrypsin prior to crystallization and were grown from 1.1-2.1 M ammonium sulfate, 0.1 M sodium cacodylate/HCl pH 5.3-6.3, 0.2 M sodium chloride for 2 months. Crystals were cryo-protected with reservoir solution supplemented with 20% glycerol, and flash-frozen in liquid nitrogen at 100 K.

Crystallization of GUK1

Large co-crystals of GUK1 (17 mg/mL) with AT-8001 (2 mM) supplemented with 5 mM MgCl2 were grown using the sitting drop method with a 3:1 ratio of protein solution to precipitant solution consisting of 66 mM Sodium Cacodylate pH 6.5-7.5, 12.2-22.2% PEG 3350, 8.3% PEG 4000, 33 mM MES pH6.5 and 66 mM magnesium chloride over a period of 10 days at 293.15K. Crystals were cryo-protected with reservoir solution supplemented with 20% glycerol, and flash-frozen in liquid nitrogen at 100 K.

Crystallization of NDPKb

Crystallization conditions were adapted from 47 using the sitting-drop vapor diffusion method using crystallization buffer. All crystals were grown at 277.15K, using a 1:1 ratio of protein (5 mg/mL) mixed with AT-9010 (final concentration 1 mM) to precipitant solution (12% PEG 4000, 50 mM Tris pH = 8.4, 16% glycerol, 1 mM DTT). Crystals grew in 3 days and were flashfrozen directly in liquid nitrogen at 100 K.

Structure Determination of the Human HINT 1: AT-8003 complex

The dataset of HINT1 in complex with AT-8003 was collected on the Proxima-1 beamline at the Synchrotron SOLEIL. Dataset was processed using AUTOPROC [START_REF] Vonrhein | Data processing and analysis with the autoPROC toolbox[END_REF] . The phase was obtained using Molecular Replacement using MOLREP [START_REF] Vagin | MOLREP : an Automated Program for Molecular Replacement[END_REF] with the PDB entry 6N3V as a model. The ligand AT-8003 and geometry description files were generated using the GRADE2 server [START_REF] Smart | Grade2 version 1[END_REF] .

Structure handling and refinement were done using COOT 51 and BUSTER [START_REF] Smart | Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER[END_REF] .

Structure Determination of the Human ADALP 1 : AT-8001 complex

The dataset of ADALP 1 in complex with AT-8001 was collected on the Proxima-2 beamline at the Synchrotron SOLEIL. Dataset was processed using AUTOPROC [START_REF] Vonrhein | Data processing and analysis with the autoPROC toolbox[END_REF] and processed data were sent to the CCP4 cloud suite [START_REF] Krissinel | CCP4 Cloud for structure determination and project management in macromolecular crystallography[END_REF] . The pipeline is fully automatic. The phase was obtained using Molecular Replacement -MORDA 53 using PDB entry 6IV5 as a model. The ligand AT-8001 and geometry description files were generated using the GRADE2 server [START_REF] Smart | Grade2 version 1[END_REF] . Minor correction on the structure and further refinement were done using COOT 51 and REFMAC5 [START_REF] Murshudov | REFMAC5 for the refinement of macromolecular crystal structures[END_REF] , respectively.

Structure Determination of the Human GUK 1 : AT-8001 complex

The dataset of GUK1 in complex with AT-8001 was collected on the ESRF ID23-1 beamline. Dataset was processed using AUTOPROC [START_REF] Vonrhein | Data processing and analysis with the autoPROC toolbox[END_REF] . The phase was obtained using Molecular Replacement -MORDA 53 using PDB entry 4F4J as a search model and optimized with ARP/WARP [START_REF] Langer | Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7[END_REF] . Structure refinement was done using COOT [START_REF] Emsley | Features and development of Coot[END_REF] and BUSTER [START_REF] Smart | Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER[END_REF] , respectively.

Structure Determination of the Human NDPK : AT-8500 complex

The dataset of hNDPKb in complex with AT-8500 was collected on the Proxima-2 beamline at the Synchrotron SOLEIL. Dataset was handled using the CCP4 suite [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF] . Images were processed by XDS 57 and AIMLESS [START_REF] Evans | How good are my data and what is the resolution?[END_REF] . The phase was obtained using Molecular Replacement -PHASER [START_REF] Mccoy | Phaser crystallographic software[END_REF] with the PDB entry 1NUE as a model. The ligand AT-8500 corresponding to the diphosphate form of AT-9010 was generated using the AceDRG program [START_REF] Long | AceDRG: a stereochemical description generator for ligands[END_REF] . Structure handling and refinement were done using COOT 51 and REFMAC5 54 software, respectively. All electron-density maps were inspected using COOT [START_REF] Emsley | Features and development of Coot[END_REF] . Extra density accounting for ions, and/or compounds were observed for all complexed structures. The structures were evaluated using MOLPROBITY 61 and PROCHECK [START_REF] Laskowski | PROCHECK: a program to check the stereochemical quality of protein structures[END_REF] . Structural analysis and high resolution figures were done with UCSF ChimeraX [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF] . Facilities, statistics of data collections, refinements and PDB deposition code are given in Table 1.

Modeling of GUK 1 closed conformation

Sequence of human GUK 1 was submitted to an homology modeling process using modeler V10 and the structure of mouse GUK complexed with GMP and ADP (PDB 1LVG) as template. The two sequence have 88 % identity, which makes us confident that the resulting closed conformation model is reasonable.

Docking of GMP and AT-8001 in hGUK 1 closed conformation

The 3D model of hGUK 1 was energy minimized by the steepest gradient method of energy minimization followed by conjugate gradient minimization, using the MMTK63 and AMBER64 packages. Mol2 and PDB files format of the ligands and receptor were converted to PDBQT format using chimera prior to docking. All the water and solvent atoms of the protein were removed and the polar hydrogen and polar charge were added onto the ions and ligand prior to docking. The protein was kept rigid while the ligand was allowed to rotate and explore more flexible binding pockets. Docking of the respective ligands into the cavity were performed iteratively using AUTODOCK VINA65. The best poses from the first round of docking were used as seed for the second round. The resulting first round of docking were carefully analyzed to retained the best poses. The grid box size dimensions were first 40X40X40, to verify that our ligands will preferentially bind in the catalytic site. The grid box size was further optimized to 23.2X25.6X21.2 thus covering the binding pockets, the default scoring function was used for docking. As control for the procedure GMP was docked following the same protocol and the final pose is virtually identical to the one measure in the experimental structure (PDB 1LVG). Binding modes of the docked complexes were obtained and sorted based on their binding energy, ions and amino acid residues present at a distance less of 3 Å were considered as the binding partners of the ligands. Binding modes were compared to theses of the native structure. The interaction figures representing the docked complexes have been generated using ChimeraX [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF] .

Table 1 :

 1 Activities of enzymes involved in AT-527/AT-511 and AT-752/AT-281 activation

	Enzyme	Substrate	Specific activity
			(nmol.min -1 .nmol -1 ) a
	CatA	AT-511	9.8 ± 0.3
		AT-281	0.7 ± 0.1
	CES1	AT-511	0.033 ± 0.004
		AT-281	0.27 ± 0.02
	HINT1	AMP-NH2	101 ± 8
		AT-551	0.7 ± 0.2
	ADALP1	N 6 -Me-AMP	42 ± 2
		AT-8003	9.4 ± 0.4
		AT-8010	5.21 ± 0.07
		AT-8004	11.9 ± 0.1
		AT-8002	0.94 ± 0.03
		AT-551	No activity
		AT-229	No activity
		AT-259	No activity
	GUK1	GMP	6800 ± 300
		AT-8001	34 ± 5
		AT-8003	No activity
	NDPKB	GDP	5400 ± 800
		AT-8500	1300 ± 100

a Results were obtained from at least 3 independent experiments and reported as means ± standard deviations

Table 2 :

 2 Data-collection and refinement statistics for native human enzymes of the bemnifosbuvir activationpathway complexed with substrate or product compounds.

		HINT 1 complexed with	ADALP 1 complexed with	GUK 1 complexed with	NDPK complexed
		AT-8003	AT-8001	AT-8001	with AT-9010
		-8PWK	-8QCH	-8PTS	-8PIE
	Wavelength (Å)	0.97856	0.980	0.8856	0.978
	Resolution range	39,8 -2,09 (2,13-2.09)	105.53 -2.44 (2.48 -2.44)	62.85 -1.76 (1.79 -1.76)	49.68 -1.90 (1.97 -1.9)
	Space group	C2	P 21212	C 2	P21
	Unit cell
	a,b,c	77.96, 46.45, 63.77	142.7, 142.9, 156.4	132.7, 54.3, 58.9	54.2, 120.3, 71.9
	α,β,γ	90.00, 94.98, 90.00	90.0, 90.0, 90.0	90.0, 108.8, 90.0	90.0, 110.2, 90.0
	Total reflections	89090	1216890	158084	480674
	Unique reflections	12964	119069	37848	67263
	Multiplicity	6.9 (6.4)	10.2 (10.5)	4.2 (4.3)	7.1 (7.2)
	Completeness (%)	95.8 (96.8)	100 (100)	96 (96)	98.89 (98.00)
	Mean I/sigma(I)	10.6 (3.3)	7,2 (1,0)	9.7 (2.2)	11.03 (1.21)
	Wilson B-factor	23,3	40,6	27.8	21.5
	R-merge	0.116 (0.545)	0.363 (3.973)	0.055 (0.69)	0.2401 (0.7668)
	R-meas	0.125 (0.592)	0.383 (4.180)	0.063 (0.79)	0.2596 (0.8257)
	CC1/2	0.995 (0.904)	0.988(0.350)	0.99 (0.82)	0.958 (0.745)
	No. of reflexions used in refinement (free reflections)	12960 (646)	118763 (5715)	364600 (1823)	67261 (6623)
	R-work	0,19	0.21	0.24	0,17
	R-free	0.23	0.24	0.26	0,21
	Total atoms in structure	1929	23207	3232	7869
	macromolecules	1756	44365	3019	7287
	Ligands	44	320	80	180
	RMS (bonds)	0.007	0.0151	0.008	0.030
	RMS (angles)	0,095	2.253	1.86	2.28
	Ramachandran	99.55	97.27	98,4	97.95
	favoured (%)
	allowed (%)	1.06	2.0	1,6	1,4
	outliers (%) *	0.0	0.69	0.0	0.68
	Average B-factor	48.60	50.00	34.0	26.16
	Beamline	Soleil Proxima-1	Soleil Proxima 2	ESRF ID23-1	Soleil Proxima-2
	Statistics for the highest-resolution shell are shown in parentheses.

*Outliers are in highly flexible regions of the protein.

  10 µM E-64 (CatL inhibitor) was then added before aliquoting and storage of activated CatA at -80°C. AT-511 and AT-281 hydrolysis by activated CatA was measured by incubating 100 µM compound in a reaction buffer containing 25 mM MES pH 6.5, 100 mM NaCl, 1 mM DTT, 0.1% NP-40 and 20 nM enzyme at 37°C for 45 min. The reaction was started by adding the enzyme. At various time points, 10 µL aliquots were collected from the reaction mixtures mixed with EDTA 10 mM (final concentration) and stopped by heating the samples at 95°C for 5 min. The reaction was started by adding the enzyme. At various time points, 10 µL aliquots were collected from the reaction mixtures mixed with EDTA 10 mM (final concentration) and stopped by heating the samples at 95°C for 5 min. The sample are filtered on AcroPrep Advance 96-Well Filter Plates with 3K Omega membrane (Pall). The filtrates were mixed with TEAB 1M and injected onto a C18 reverse phase column (3 µm, 3 by 150 mm, Acclaim Polar Advantage II, ThermoFischer) equipped with a guard column, equilibrated with TEAB 50 mM pH = 7 (buffer A). The substrates and reaction products were eluted using a non-linear gradient of acetonitrile in buffer A, detailed in supplementary data. NaCl and 1 mM DTT. Reactions were incubated at 37°C for 2h. 1 µM ADALP1 was also tested with AT-8002, AT-551, AT-229 and AT-259. The reaction was started by adding the substrate. At various time points, 10 µL aliquots were collected from the reaction mixtures diluted with H2O and stopped by heating the samples at 95°C for 5 min. The sample were filtered on AcroPrep Advance 96-Well Filter Plates with 3K Omega membrane (Pall). The filtrates were mixed with TEAB 1M and injected onto a C18 reverse phase column (2.5 µm, 4.6 by 100 mm, Xbridge C18 BEH Premier, Waters) equipped with a guard column, equilibrated with TEAB 50 mM pH = 7 (buffer A). The substrates and reaction products were eluted using several non-linear gradients of acetonitrile in buffer A, detailed in supplementary data.GUK1 enzyme assay GUK1 (40 nM) was assayed on GMP and AT-8001 (200 µM) in a reaction buffer containing 50 mM Tris buffer pH = 8.0, 50 mM KCl, 5 mM MgCl2, 1 mM ATP and 1 mM DTT. Reactions were incubated at 37°C for 1h. The reaction was started by adding the enzyme. At various time points, 10 µL aliquots were collected from the reaction mixtures, mixed with EDTA 10 mM (final concentration) and stopped by heating the samples at 95°C for 5 min.

	The sample were filtered on centrifugal filters Microcon® -10 (Sigma Aldrich). The filtrates were mixed with TEAB 1M (1:1) and injected onto a C18 reverse phase column (2.5 µm, 4.6 by 100 mm, Xbridge C18 BEH, Waters) equipped with a guard column, equilibrated with 98:2 TEAB 50 mM pH = 7 (buffer A):MeCN. The substrates and reaction products were eluted using a non-linear gradient of acetonitrile in buffer A, detailed in supplementary data. CES1 enzyme assay AT-511 and AT-281 hydrolysis by human recombinant CES1 (100 nM) was assayed by incubating at 37°C the enzyme with 100 µM compound in 50 mM Tris buffer pH = 7.5, 0.1% NP-40 and 1 mM DTT for 2h. The reaction was started by adding the substrate. At various time points, 20 µL aliquots were collected from the reaction mixtures mixed with EDTA 10 mM (final concentration) and stopped by heating the samples at 95°C for 5 min. The sample were filtered on centrifugal filters Microcon® -10 (Sigma -Aldrich). The filtrates were mixed with TEAB 1M (1:1) and injected onto a C18 reverse phase column (2.5 µm, 4.6 by 100 mm, Xbridge C18 BEH, Waters) equipped with a guard column, equilibrated with TEAB 50 mM pH = 7 (buffer A). The substrates and reaction products were eluted using a non-linear gradient of acetonitrile in buffer A, detailed in supplementary data. HINT1 enzyme assay The hydrolytic reactions with HINT1 (100 nM) were performed on AMP-NH2 and AT-551 (200 µM) in 20 mM HEPES buffer pH 7.2, 20 mM KCl, 1 mM MgCl2 and 1 mM DTT, at 37°C ADALP1 (100 nM) activity was measured on several substrates (N 6 -Me-AMP, AT-8003, AT-8002 AT-8004, AT-551, AT-259, AT-229) at 200 µM concentration in a reaction buffer containing BTP pH = 6.8, 100 mM The sample were filtered on AcroPrep Advance 96-Well Filter Plates with 3K Omega membrane (Pall). The filtrates were mixed with TEAB 1M and injected onto a C18 reverse phase column (3 µm, 3 by 150 mm, Acclaim Polar Advantage II, ThermoFischer) equipped with a guard column, equilibrated with TEAB 50 mM pH = 7 (buffer A). The substrates and reaction products were eluted using a non-linear gradient of acetonitrile in buffer A, detailed in supplementary data. NDPKB enzyme assay NDPKB (20 nM) was assayed on GDP and AT-8500 (200 µM) in a reaction buffer containing 50 mM Tris buffer pH = 8.0, 50 mM KCl, 5 mM MgCl2, 1 mM ATP and 1 mM DTT. Reactions were incubated at 37°C for 1h. The reaction was started by adding the enzyme. At various time points, 10 µL aliquots were collected from the reaction mixtures mixed with EDTA 10 mM and stopped by heating the samples at 95°C for 5 min. The sample were filtered on AcroPrep Advance 96-Well Filter Plates with 3K Omega membrane (Pall). The filtrates were mixed with TEAB 1M and injected onto a C18 reverse phase column (3 µm, 3 by 150 mm, Acclaim Polar Advantage II, ThermoFischer) equipped with a guard column, equilibrated with TEAB 50 mM for 2h. ADALP1 enzyme assay pH = 7 (buffer

Acknowledgments:

We thank Veronique Fattorini, Camille Falcou, and Pierre Gauffre for synthetic gene design and help in protein purification. We thank Nancy Agrawal, Alex Vo, and Qi Huang for critical reading of the manuscript, together with Ashleigh Shannon and numerous colleagues for helpful suggestions along the project. The authors acknowledge SOLEIL and ESRF for provision of synchrotron radiation facilities and we would like to thank beamline teams of Proxima-1, Proxima-2 and ID23-eh1 for their assistance during data collection. This work was supported by the 'Agence Nationale pour la Recherche' through the French Infrastructure for Integrated Structural Biology (FRISBI) [ANR-10-INSB-05-01].

Funding:

This project has received funding through the 2023 Louis Pasteur Bicentenary Prize and the Grand Prix Scientifique de la Fondation Simone et Cino Del Duca -Institut de France awarded to BC. Funding acquisition: BC Writing: FF, BC

Author contributions:

Conceptualization: KA, FF, BC Methodology: KA, FF, BC Experiments: AC, CZ, MF, AM, FF Analysis: AC, MF, AM, SG, JPS, KA, FF, BC Competing interests: S.G., A.M. and J.P.S. are employees of ATEA Pharmaceuticals, Inc. The other authors declare no competing interests.

Data and materials availability:

The coordinates and structure factors for HINT1, ADALP1, GUK1, and NDPKb structures have been deposited in the PDB with accession codes 8PWK, 8QCH, 8PTS, and 8PIE, respectively.


G Repetto

T Glantz

G Guillard

B Bruyère

Q Grando

CORE COOLABILITY IN LOSS OF COOLANT ACCIDENT: THE COAL EXPERIMENTS INVESTIGATING THE THERMAL HYDRAULICS OF A ROD BUNDLE WITH BLOCKED AREA DURING THE REFLOODING

Keywords: Coolability, blockage, LOCA, Fuel relocation, DRACCAR

During an accident causing loss of the coolant of the primary circuit (LOCA) in a pressurized water reactor, partial or even complete drying of the fuel assemblies may lead to the fuel temperature increase, significant deformation and, consequently the rupture of the fuel rod claddings. In addition to the restriction of the flow area, the fragmented fuel relocation within the ballooned zone leads to an increase of the local residual power. The COAL experiments will focus on the outstanding issue of the coolability of a partially deformed fuel assembly, in particular the study of the thermo hydraulic behavior during the cooling phase by water injection with the safety systems. Competing influences due to the presence of a blockage will be evaluated through integral experiments using a 7x7 bundle of electrically heated rods. In one hand, the reduction of the cross-section between the deformed fuel rods will increase the flow velocity and consequently the heat transfer. On the other hand, the flow bypass of the blocked region, towards the periphery of the fuel assembly will lead to a reduction of mass flow in the sub channels of the blockage and therefore the cooling capacity. This paper presents the scenario and the pre-calculations of the COAL experiments using the 3D DRACCAR code coupled to the CESAR thermal hydraulics module developed by IRSN. The effect of the flow blockage (intact geometry up to long ballooning with different blockage ratios 80 to 90%) will be evaluated for various mass flow rates and different pressures representative to Large (LBLOCA) and Medium break size (MBLOCA) configuration.

INTRODUCTION

During an accident causing loss of the primary coolant (LOCA) in a pressurized water reactor, partial or even complete dryout of the fuel assemblies may occur. In these conditions, the fuel temperature increase and the coolant pressure drop in the reactor core may lead to significant deformation and rupture of the fuel rod cladding. The results, which illustrate this phenomenon, come from a LOCA experimental program performed, in the 1980's by the Institut de Protection et de Sûreté Nucléaire now IRSN [START_REF] Bourdon | Analyses of the Phebus-LOCA tests 215-R and 2018 using FRETA-F V1.1 code[END_REF], in 5x5 bundle including fresh fuel rods. In case of irradiated fuel rods, fuel fragmentation may occur, leading to fuel relocation within the ballooned area. Depending on the size and the distribution of the ballooned areas within the fuel bundle, the cooling flow might be impaired. All the LOCA experiments performed with irradiated fuel rods (in particular 3 examples, at 35 GWd/t for the FR2 experiments (Germany) in 1983 [START_REF] Karb | LWR fuel rod behaviour in the FR2 in-pile tests simulating the heat up phase of a LOCA[END_REF], in the ORNL Tests (USA) [START_REF]Fuel fragmentation, relocation and dispersal during the loss-of coolant-accident[END_REF] for various burnups and, more recently, in Halden (Norway) [START_REF] Kekkonen | LOCA testing at Halden, the fourth experiment IFA-650.4[END_REF] at 90 GWd/t in 2006), have shown such an accumulation of the fragmented fuel pellets in the ballooned region. The resulting increase of the power density in the ballooned area may challenge the fuel assembly coolability and, by extension, the whole core coolability. So, the consequences of both deformations and fuel relocation on the cooling efficiency when reflooding the core by the ECCS (Emergency Core Cooling Systems) are important safety issues, and have never been experimented with representative rods ballooning with irradiated fuels (simulated by electrical rods). The knowledge generated by these new experiments will answer long shared concerns by the international community (such as the effect of fuel relocation in blocked areas in the core), and will allow to validate the DRACCAR code [START_REF] Repetto | DRACCAR: a new 3D-thermal mechanical computer code to simulate LOCA transient on Nuclear Power Plants -Status of the development and the validation[END_REF] developed and used by IRSN since 2006 to simulate any prototypical LOCA transient. As the previous experiments (as FEBA [START_REF] Ihle | FEBA "Flooding Experiments with Blocked Arrays[END_REF], SEFLEX [START_REF] Ihle | SEFLEX -Fuel Rod Simulator Effects in Flooding Experiments[END_REF], THETIS [START_REF] Pearson | Reflooding Experiments on a 49-Rod Cluster Containing a Long 90% Blockage[END_REF][START_REF] Cooper | The THETIS 80% Blocked Cluster Experiment[END_REF] and ACHILLES [START_REF] Denham | ACHILLES Un-ballooned Cluster Experiments -Part 1 : Description of the ACHILLES Rig, Test section and Experimental Procedures[END_REF]) were not representative enough (rod thermal inertia and fuel relocation was not taken into account), the need of such complementary experiments have been proposed in 2005 by IRSN [START_REF] Grandjean | Coolability of blocked regions in a rod bundle after ballooning under LOCA conditions, Main findings from a review of the past experimental programmes[END_REF][START_REF] Grandjean | Coolability of blocked regions in a rod bundle after ballooning under LOCA conditions Main findings from a review of past experimental programs[END_REF]. DRACCAR [START_REF] Bascou | Computational Analysis of Multi-pin Ballooning during LOCA and Post LOCA Transient using the Multi-physics Code DRACCAR[END_REF][START_REF] Ricaud | Multi-pin Ballooning during LOCA transient: A Three Dimensional Analysis[END_REF], developed by IRSN with the support of EDF ,is a simulation tool for fuel assembly mechanical behavior and coolability assessment during a LOCA transient. Its aim is to simulate the 3D thermomechanical deformation and reflooding of a fuel rod assembly including its coolability as well as structure embrittlement. The DRACCAR code is based on a 3D non-structured meshing able to model a simple fuel rod, a partial or a full assembly, as well as surrounding shrouds. It is based on an axial discretization of the rod which leads to analyze quasi-independent 2D thermal mechanical problems.

Important features such as pellet eccentricity, heat transfers (within the solid and through the fluid) or material properties evolution (oxidation layer, phase changing,...) can be taken into account and the cladding integrity during a LOCA transient can be addressed even in case of contact between the structural elements. In that case, the geometry is strongly changed (flattened zone contact) as well as the loading nature (mixed stress-displacement loading) and so the rupture is more difficult to model than a threshold criteria used in most of the multi-rod codes: with DRACCAR, non-linear geometrical effects are added to non-linear behaviour laws in the modeling. Also important is the possibility to get a better knowledge on the system's capability to cool structures whatever are the evolutions of the deformation of the rods and the blockage of the sub-channels. Obviously these two critical issues which are essential to treat in modeling LOCA transient effects, can only be dealt with in a realistic manner with a multi-pin code coupled to an efficient 3D thermal hydraulics code, and that's why DRACCAR V2.3 is currently coupled to the two phase flow module CESAR of the ASTEC code [START_REF] Trégoures | Reactor Cooling Systems Assessment of the ASTEC V1.3 code in Support to the French IRSN PSA-2 on the 1300 MWe PWR[END_REF], able to compute deformed geometry evolutions thus actualizing the coolant flow passage within the different sub-channels. In this case DRACCAR is able to calculate the heat exchanges between the wall and the fluid which have been validated against PERICLES and ACHILLES tests.

THE COAL EXPERIMENTS

The COAL experiments, in the frame of the PERFROI project [START_REF] Repetto | Core coolability in loss of coolant accident: the PERFROI project[END_REF][START_REF] Repetto | The R&D PERFROI project on Thermal mechanical and thermal hydraulics behaviors of a fuel rod assembly during a Loss Of Coolant Accident[END_REF] will concern thermal hydraulics experiments on electrically heated rods, with simulation of large blockage and fuel relocation.

The test section design and the bundle geometry

The COAL bundle [START_REF] Repetto | The core coolability in a Loss Of Coolant Accident: the COAL experiments[END_REF] will consist of rods (electrically heated), some with a pre-deformed zone (Fig. 1a) with local overheating representing fuel relocation. The bundle will be made of 49 (7 x 7) rods (including in the center 16 deformed rods, 30 non-deformed rods and 3 guide tubes (Fig. 1b). The presence of 3 guide tubes will increase the representativity of the thermal hydraulics behavior for the reactor case (Fig. 3 of [START_REF] Repetto | The R&D PERFROI project on Thermal mechanical and thermal hydraulics behaviors of a fuel rod assembly during a Loss Of Coolant Accident[END_REF]) part of the PWR 17x17 fuel assembly. The test section will also include 6 spacer grids (4 mixing vanes grids plus 2 holding grids at the bottom and at the top of the bundle) each 500 mm apart. For these experiments, the cladding of the electrical rods was proposed to be constructed with Hastalloy material to prevent oxidation. To avoid any chemical interaction, the grids should be in Inconel alloy and including mixing vanes that would have an effect of thermal hydraulic behavior. For these experiments, the cladding of the electrical rods was proposed to be constructed with Hastalloy material to prevent oxidation. To avoid any chemical interaction, the grids should be in Inconel alloy and including mixing vanes that would have an effect of thermal hydraulic behavior. The presence of guide tubes is also necessary for instrumentation and for mechanical reasons. The heated zone is about 3m high (close to the real PWR geometry). The bundle will be inserted in 94mm square electrically heated shroud (Fig. 1c) with roughly 4-5 mm thickness to be representative of the surrounding fuel rods regarding their thermal inertia. The test section will be included in a pressure vessel, (4m high, 220 mm diameter -Fig. 1d) with two lower and upper plenums for the electrical and instrumentation connections and the fluid supply. The COAL experiments will be performed in the AREVA BENSON facility at Erlangen (Germany) which allows running high system pressure tests to 3 MPa [START_REF] Repetto | The core coolability in a Loss Of Coolant Accident: the COAL experiments[END_REF].

The geometry of the blockage and the axial power distribution

The experimental parameters of the test campaigns will be defined in order to investigate the bundle geometry related phenomena (blockage ratio, balloon length) and the thermal hydraulic related phenomena (local overheating of the balloon, injected water temperature, coolant flow rate and system pressure to simulate various break sizes). The geometrical parameters are reported in Table I below. The choice of geometries and the associated axial power distributions were designed to obtain a gradual evolution of the pressure drop in the ballooned zone and a gradual increase of the maximum linear power in the ballooned area of the fuel rods which constitute the different bundles (B0, B1 and B2). So, the configuration for the B2 bundle would be the worst conditions for the coolability concern. The axial power distributions were obtained by DRACCAR calculations which can simulate with some parameters the fuel relocation in the balloon (fuel filling ratio in the balloon) according the cladding deformation (Fig. 5 of [START_REF] Repetto | Thermal hydraulics behavior of a rod bundle with partially blocked area during the reflooding phase of the Loss Of Cooling accident[END_REF]) and the corresponding power profile deformation (Fig. 2a).

Some modifications were done on the axial power profile, in particular for the bundle B2, as with the previous axial power profile, temperature in the balloons were too high compared to the technical limits of the electrical rods (roughly 1000°C) (cf § 3.3.1). For both the cases (B1 and B2), the powers in the connection from the non-deformed part to the part of the maximum deformation were reduced to take into account the real fuel inventory. In the balloon of the rods for the B2 bundle, the axial peaking factor was significantly reduced by assuming a density of 60% for the fuel relocation instead of 70% (both values were observed for irradiated fuels (Fig. 5 of [START_REF] Repetto | Thermal hydraulics behavior of a rod bundle with partially blocked area during the reflooding phase of the Loss Of Cooling accident[END_REF]). The new proposition is illustrated by the Fig. 2b. Thus, we will change only one parameter between B1 and B2 bundles.

The test instrumentation plan

For these kinds of experiments, the major physical variables for model qualification are as follows:  Inlet and outlet water and steam mass flow rates (other two phase flow measurements such as void fraction, droplet size will be not available),  Temperature of the fluid at different points (type K thermocouples),  Temperature inside the fuel rods (thermocouples at different elevations and different radial positions in the bundle) in an adequate distribution, temperature of the shroud, grids and guide tubes,  Total pressure and pressure drop sensors at the periphery of the rod bundle.

Up to 184 thermocouples (4 each 46 rods) will be positioned every 10 cm to follow the quench front propagation (QFP). Additional thermocouples are foreseen, in some fluid channels, on the 6 grids and in the 3 guide tubes. The shroud will be also equipped with thermocouples on each face in order to accurately set the boundary thermal conditions during the reflooding process. The outlet mass flow rate of steam and water is foreseen to be evaluated by a specific techniques developed by AREVA (vaporization of the droplets and measurement of the outlet steam flow by mean of a "venturi type" device. A thermal balance will allow the evaluation of water remaining at the outlet of the section.

The test scenario and the thermal hydraulics parameters

The scenario is divided in two phases as for classical "Reflooding experiments" performed in the 80's:

1. Heat up phase in dry steam atmosphere (the depressurization is not simulated), 2. Reflooding by water injection with different thermal hydraulic conditions (see test matrix). The bundle will be preheated up to 300°C by hot steam injection (step 1). As the shroud temperature has to be as close as possible to the rod temperatures at the end of the first phase in order to avoid perturbation during the reflooding, the rods and the shroud will be heated up to 600°C (step 2 and 3) before the water injection phase (step 4). Rod power and water flow will be stopped for Step 5. The main thermal hydraulics parameters foreseen are in the range below:

 inlet water flow velocity: 1,5 to 8 cm/s (15 to 80 kg/s/m 2 ),  sub-cooling water temperature: -60°C/0 to saturation conditions,  coolant pressure: 0.2 to 3 MPa,  power 3.3 kW  , respectively 2 kW, per rod for Large Break (low pressure) scenario, respectively for Intermediate Break (high pressure) scenario. The T/H conditions come from recent NPP CATHARE calculations (Table 1 of [START_REF] Repetto | The core coolability in a Loss Of Coolant Accident: the COAL experiments[END_REF]) with different break sizes (1 to 40 inches) and conditions that were used for past programs such as PERICLES experiments.

The test matrix

The COAL test matrix referring to (Q, P) diagram where Q is the inlet water flow rate in kg/s/m 2 and P, the system pressure in bar is given in Fig. 3 below. As the targeted number of experiments was initially too ambitious, optimization of the test matrix was necessary. 24 thermal hydraulics conditions were proposed to develop a test matrix for the reference bundle B0 (46 intact rods + 3 guide tubes "GT"). Some thermal hydraulics conditions (circle in Fig. 3) are similar to those of PERICLES experiments for comparison tests for past reference cases. Tests 10 to 12,  in order to simplify the test procedure, the power will be kept constant during the transient, nevertheless some tests could be performed with a reduction of power representative of the decrease of the residual power such as ACHILLES Tests [START_REF] Denham | ACHILLES Un-ballooned Cluster Experiments -Part 1 : Description of the ACHILLES Rig, Test section and Experimental Procedures[END_REF]. One of their conditions will be reproduced with COAL Test 14 (0.2 MPa, 20 kg/s/m 2 ). Tests 25 and 26 will be focused to explore different thermal hydraulics boundary conditions, such as subcooling of the coolant (at low and high pressures), shroud temperature and initial temperature of the rods. Six tests (25%) will be repeated in order to investigate the reproducibility of the results. About 16 experiments (+4 repetitions) would be sufficient to test the effect of the pressure and the injection water flow rate with the bundles including the deformed rods (B1 and B2). The total number of tests will depend on the reliability of the rods and the thermocouples. As peripheral rods will be used for two experimental campaigns, these intact rods need to resist to roughly 50 (30+20) refloodings. So, the prototype of electrical rod, fabricated by the Stern Lab. Company (from Canada) has been tested successfully 55 times for various initial temperatures (from 600 to 900°C) at different flow rates.

PRECALCULATION USING THE DRACCAR CODE

The simulation tool DRACCAR, was used, at this stage, to define the parameters of the experiments such as the blocked bundle geometry, the deformed axial power profile and the heat-up scenario. Most of the tests of the test matrix have been simulated with DRACCAR code in order to prepare technical aspects for the COAL experiments (adaptation of the BENSON facility, to support the design of some equipment GT/rods and the test section design). All the physical models, used in these calculations, are described in detail in [START_REF] Glantz | DRACCAR: a multi-physics code for computational analysis of multi-rod ballooning and fuel relocation during LOCA transient. Part one: General modeling description[END_REF]. Thermal mechanical models are not activated, as the geometry is fixed (Table I).

DRACCAR code modeling

The bundle is simulated on a height equivalent to the rod heated length, with a square housing (metallic shroud in a similar material as the cladding). One boundary condition at the inlet and one at the outlet of this portion close the system. DRACCAR is able to model each 46 electrical rods of the bundle and 3 guide tubes (Fig. 4a). Taking into account the symmetry of the test section, half a bundle is modeled (Fig. 4b). The ballooned bundle includes 16 rods with a local ballooning (Fig. 4c). In the balloon, the fuel fragments are simulated by a porous material which has a density, a heat capacity and a conductivity leading to properties (thermal inertia and conductivity) close to those of the UO 2 fragments. The calculation presented here, is composed of balloons that have a height of 100 (B1) and 300 mm (B2), at the middle of the bundle, combined to 80 (B1) and 90% (B2) blockage of the test section. The axial power profile imposed in the intact rods has a cosine shape (Fig. 3). The relocation in the balloon has been simulated by setting a specific axial deformation profile in the ballooned rods (Fig. 3). These deformed axial power profile has been determined by a DRACCAR calculation after fuel relocation in a pre-deformed rod. The meshes

Intact rod Deformed rod Guide Tube Shroud

height never exceeds 100mm (smaller in the ballooned area to simulate the two cones up and downstream the balloon: 35 mm each: two "50 mm" meshes for the 100 mm balloon and eight "37,5 mm" meshes for the 300 mm long balloon. Six grids are taken into account by setting a singular pressure loss at their positions.

Results of the DRACCAR simulation

The results will concern the main thermal hydraulic parameters governing the reflooding such as:  the temperature of the rods during the transient,  the quench front propagation,  and the duration of the cooling of the fuel rods.

Temperature of the rods

The temperature evolution for B0 (intact geometry) and B1 bundles (moderate partially blockage area) were largely detailed in [START_REF] Repetto | Thermal hydraulics behavior of a rod bundle with partially blocked area during the reflooding phase of the Loss Of Cooling accident[END_REF]. The results for reference cases (Test1 at low pressure: 0,3 MPa, 36 kg/m 2 /s and Test 21 at high pressure: 3 MPa, 20 kg/m 2 /s) are illustrated by the Figs. 12 a), b), c) and d) reported in the appendix A. Rod temperatures were roughly limited to 800°C during the whole transient, for which the residual power was maintain constant, in order to simplify the test procedure (3.3 kW for LBLOCA and 2 kW for IBLOCA conditions). The Test7 at low flow velocity (1,5 cm/s) and low pressure (0.3 MPa) for which the reflooding would be the less efficient has been simulated for the partially blocked (80%) bundle B1 (short balloon with moderate power increase).

Temperature in the balloon was increased by about 60°C (Fig. 6). This test that reached the technology limit of the electrical rod (1050°C) will be surely performed at the end of the experimental campaign. ) that will be performed at the lowest water flow rate (i.e. velocity at 1,5 cm/s). In these calculations, the real decrease of residual power is simulated and the maximum rod temperatures never exceed the limit of 1000°C. These two simulations performed at the same T/H conditions except the pressure show the effect of this parameter (0.3 and 3 MPa) on the efficiency of the reflooding process, by decreasing the time for a complete cooling of the fuel bundle by roughly 30% (9 minutes down to 6 minutes).

Some preliminary simulations were performed with the B2 bundle configuration representing the worst conditions for the coolability concern: the so-called long-ballooning (370mm), the largest blocked area (90% restriction of the fluid channel) and factor roughly 2 for the local power increase to simulate the fuel relocation in the balloon, with the largest rate of fragmented pellets observed for irradiated fuel rods.

The results for reference cases (Test 1 at low pressure: 0,3 MPa, 36 kg/m 2 /s and Test 21 at the maximum pressure: 3 MPa, 20 kg/m 2 /s) are illustrated by the Figs. 12 g) and h) reported in the appendix A.

The maximum temperature of the rods reached 900°C for the high pressure test whereas for the low pressure test the maximum temperature at the level of the balloons over past the limit of 1000°C for the electrical rods, even for moderate flow velocity (v = 3,6 cm/s): it means that for all the cases at lower flow rate, internal rods will also over past this limit (Test 1, Test 2, Test 6, Test 7 and Test 19).

In the particular test for very low flow rate 15 kg/m 2 /s (i.e. V = 1,5 cm/s), with the nominal protocol, the maximum rods temperature exceeds the LOCA criteria (Table II). So, it was decided to decrease the specific power in the blocked area for the bundle G2 and to change the increase of the power from factor of 2 down to factor of 1,5 (cf § 2.2). The results (Test 1-0,3 MPa and Test 21-3MPa) for the new configuration for B2 bundle, are given by the Fig. 12 e) and f) reported in the appendix A. The decrease of the power in the balloon leads to lower temperature of about 80°C, nevertheless, maximum temperatures remain over 1000°C for all the cases at rather low flow rate (table IIcolumn 4) for which we have to test the coolability of the partially blocked area. As, the so-called Test Protocol provides the power shutdown during the transient when the rod temperatures exceed 1000°C (to protect the electrical wires), those results need to change some parameters of the transient. One possibility could be to inject the water for lower initial temperature (500°C instead of 600°C). Another solution could be to modify the initial power. Fig. 7 summarizes temperatures at the middle elevation of the ballooned area for representative tests of the test matrix, as function of the geometry of the partially blocked area (B0, B1, B2 and B2+). The red curves indicate temperature for internal rod (i.e n°25) in the ballooned zone, whereas the blue curves the corresponding temperature for external intact rod (i.e n° 1). The temperatures of the inner rod are increasing with the increase of the local power and the blockage, whereas the temperature at the periphery remain at rather lower temperature for both the cases (around 700 to 800°C) due to the bypass of the flow.

Table II. Maximum clad temperature as a function of the blockage ratio

Figure 7. DRACCAR temperature for different flow blockage (low and high pressures)

For low pressure and velocity above 2 cm/s (respectively high pressure tests) the power is kept constant at 3,3 kW/rod i.e. 1 kW/m average (respectively 2 kW/rod) in order to simplify the test scenario ( §2.4). The decrease of the power (representative of the residual power) is necessary for low flow rate (1,5 and 2 cm/s) at low pressure. In that case, when the water front reaches the blocked area, the power is roughly 2,4 kW. To reduce maximum temperatures and for simplification, this power transient could be simulated by a constant power fixed at 2,4 kW. So, to simplify the test procedure, it is proposed to perform all the tests for the LBLOCA conditions (P < 0,5 MPa) at 2,4 kW and IBLOCA conditions (at 2 kW).

DRACCAR was also used for the design of the equipment of the test section. As an example, Fig. 8 illustrates the maximum temperature evolution of the fluid for different T/H conditions and shows that very high temperature (from 600 up to 900°C) are obtained during the reflooding phase, in particular for low water flow velocity (v = 1,5 cm/s). This means that noble materials such as Inconel were chosen for the structure of the test section in order to have a corrosion resistance for running numerous experiments and for several test campaigns. For the highest pressure (3 MPa), we observe a slight delay for the cooling of the blocked zone, but the complete reflooding is obtained earlier (Fig. 10 b) due to the acceleration of the QFP after the balloons (lowest power in the upper part of the bundle due to fuel motion in the fissile column). Whereas, for low pressure (0.2 MPa), due to the reduced efficiency of the heat exchanges, the reflooding of the partially blocked area is largely delayed (Fig, 10a) in particular for the so-called long ballooning (bundle B2) with the largest blocked area (90%). The reflooding of the blocked area (located in the middle part) takes roughly 300 s more. In that case, the maximum temperature reached 1200°C (Fig. 9 a) with 200°C more compared to the smallest balloon situation (bundle B1). This configuration seems to be the limit for the The time for a complete reflooding is decreasing as a function of the increase of the inlet water flow velocity. The red curve indicates the duration of the partial reflooding when the quench front propagation reached the level 1.65 m (just above the blockage area). The duration of the reflooding of the blocked area is significantly increased in particular for the largest blockage (Bundle B2) for which the partial and the complete reflooding are roughly similar (Fig. 12 g in appendix A).

Figure 11. DRACCAR simulation -duration of reflooding

The partial reflooding occurs later than the complete reflooding for the so-called B2+ bundle (Test1 : Q = 36 kg/m 2 /s); it means that the quench front arrives at the top of the bundle whereas the partially blocked area remains not completely quenched due to the flow diversion and the high value of the local power simulating the fuel relocation.

The reflooding appears to be more efficient at high pressure. The pressure effect is mainly observed between 0.3 and 1 MPa (Table III). Above this pressure limit (1 MPa), as well as for the increase of the water flow rate (Fig. 11), the gain seems to be negligible.

CONCLUSION

This paper presents the new COAL experiments on the reflooding of a deformed fuel bundle with simulation of the fuel fragment relocation inside the balloons. We have described the test section, the thermal hydraulic parameters for the experiments and pre-calculations (B0, B1 and B2 bundles) using the DRACCAR code developed by IRSN, in order to prepare the experiments planned from 2018 to 2020. The preliminary calculations have shown the limit of the coolability of the bundle for a flow velocity ranging from 1,5 to 2 cm/s, at least for the second bundle configuration (moderate power increase in the balloons which simulates the fuel relocation). The pre-calculation of the largest blocked bundle (90% and long ballooning with the B2 bundle) shows that in some cases, in particular for low water flow rates, the maximum clad temperature is close or even greater than the LOCA criteria. The reflooding appears to be more efficient at high pressure above a certain limit: this "finding" has to be compared to to experimental results. The thermal hydraulics behavior (temperature evolution, quench front propagation…) observed with the numerical simulation has to be compared with the future experimental COAL results in order to bring more comprehensive understanding of these phenomena: those data will help to improve and validate the heat exchange models implemented in the code studying the reflooding process and the behavior of the code in such a configuration to be used to nuclear power plants for safety applications. Specific separate-effect-tests at the sub-channel scale are in progress in order to improve our understanding on different processes involved in the reflooding of a deformed fuel assembly, in particular the evaluation of the heat transfers taking into account steam charged with droplets [START_REF] Pena Carrillo | Experimental study on heat transfer inside a blocked region of a rod bundle during LOCA[END_REF][START_REF] Pena Carrillo | Study on heat transfer inside a blocked region of a rod bundle during LOCA -Model and experimental approach[END_REF].

In addition to the COAL experiments, experiments at atmospheric pressure and in air conditions including droplet fields are foreseen to be performed in order to get information of the flow map around the partially blocked area, in particular the flow diversion, data which are not possible, at least more difficult, to get from an integral experiment.

Figure 1 .

 1 Figure 1. Experimental device (deformed rod, bundle, shroud, test section)

Figure 2 .

 2 Figure 2. Axial power profile for inner rods (cosine profile for outer rods)

Figure 3 .

 3 Figure 3. COAL test matrix in a (Q, P) diagram.

Figure 4 .

 4 Figure 4. DRACCAR nodalization of the bundle

Figure 5 .Figure 6 .

 56 Figure 5. DRACCAR simulation for a low flow rate situation (low and high pressure)

Fig. 5 ,

 5 Fig.5, gives the results of the simulation for the two tests (Test 7 and Test 19) that will be performed at the lowest water flow rate (i.e. velocity at 1,5 cm/s). In these calculations, the real decrease of residual power is simulated and the maximum rod temperatures never exceed the limit of 1000°C. These two simulations performed at the same T/H conditions except the pressure show the effect of this parameter (0.3 and 3 MPa) on the efficiency of the reflooding process, by decreasing the time for a complete cooling of the fuel bundle by roughly 30% (9 minutes down to 6 minutes).

Figure 8 . 3 . 2 . 4 .Figure 9 .

 83249 Figure 8. Maximum steam temperature calculated by the DRACCAR code

Figure 10 .

 10 Figure 10. DRACCAR simulationquench front propagation (low and high pressure)

NOMENCLATURECOAL:Figure 12 .

 12 Figure 12. Synthesis of DRACCAR simulationsrod temperatures for low and high pressures tests

Table III . Time for complete/partial reflooding of the bundle versus water flow rate and pressure

 III Time (s)coolability concern to respect the LOCA criteria for the maximum cladding temperature (i.e. 1204°C: 2200°F). On the basis of the numerous DRACCAR calculations, TableIIIsummarizes the duration of the complete (and partial) reflooding for different tests as function of T/H conditions (pressure and water flow rate) and different bundles geometries (B0, B1, B2 and B2+ (maximum power in the balloons).

		Test 4	Test 3	Test 1	Test 2	Test 14	Test 23	Test 20 Test 21
	Pressure (MPa)		0,3 (3 bar)		0.2	1	2.5	3
	Water flow ( kg/s/m 2 )	80	50	36	25	20	20	20	20
	Bundle B0 (comp/part.)	112/77	183/109	254/138	440/189	606/249	242/131	238/130	238/130
	Bundle B1 (comp/part.)	150	176/120	256/155	434/224	587/300	248/129	235/117	235/114
	Bundle B2 (comp/part.)			350/285	484/437	704/545		226/142	225/139
	Bundle B2+(comp/part.)			227/329					227/143

ACKNOWLEDGMENTS

This work is completed within the framework of RSNR Project from a French State aid managed by the National Agency of Research under the program of Investments for the Future carrying the reference n° ANR-11-RSNR-0017. Particular acknowledgments are also given to EDF and more recently, to US-NRC for their financial supports.