Yan Lin

Hyeim Jung

Christina A Bulman

James Ng

Robin Vinck

Cillian O'beirne

Shuai Zhong

Matthew S Moser

Nancy Tricoche

Ricardo Peguero

Robert W Li

Joseph F Urban

Patrice Le Pape

Fabrice Pagniez

Marco Moretto

Tobias Weil
email: tobias.weil@fmach.it

Sara Lustigman
email: slustigman@nybc.org

Kevin Cariou
email: kevin.cariou@chimieparistech.psl.eu

Makedonka Mitreva
email: mmitreva@wustl.edu

Judy A Sakanari
email: judy.sakanari@ucsf.edu

Gilles Gasser
email: gilles.gasser@chimieparistech.psl.eu.

Discovery of New Broad-Spectrum Anti-Infectives for Eukaryotic Pathogens Using Bioorganometallic Chemistry

Drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat especially neglected tropical diseases (NTDs) caused by eukaryotic parasitic pathogens including fungal infections. Herein, we show that simple modification of one of the most well-known antifungal drugs, fluconazole, with organometallic moieties not only improves the activity of the parent drug but also broadens the scope of application of the new derivatives. These compounds were highly effective in vivo against pathogenic fungal infections and potent against parasitic worms such as Brugia, which causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths that infects millions of people globally. Notably, the identified molecular targets indicate a mechanism of action that differs greatly from the parental antifungal drug, including targets involved in biosynthetic pathways that are absent in humans, offering great potential to expand our armamentarium against drug-resistant fungal infections and NTDs targeted for elimination by 2030.

INTRODUCTION

The need for new broad-spectrum anti-infective agents is becoming urgent considering the modest number of compounds available for treatment and the problem of drug resistance observed with many of them. For example, the discovery of new drugs to treat neglected tropical diseases (NTDs) [START_REF] Weng | Innovation in Neglected Tropical Disease Drug Discovery and Development[END_REF] and fungal infections [START_REF]Candida auris[END_REF][START_REF] Fisher | Tackling the Emerging Threat of Antifungal Resistance to Human Health[END_REF][START_REF] Berman | Drug Resistance and Tolerance in Fungi[END_REF] is a top priority as many of these pathogens are developing resistance to the available drugs. Crucially, in order to be accessible to the entire world's population and not only for the more developed countries, these newly repurposed or derivatized agents should be, if possible, easy-toprepare to allow for commercialization based on their low cost [START_REF] Wouters | Estimated Research and Development Investment Needed to Bring a New Medicine to Market, 2009-2018[END_REF] .

To tackle this problem for NTDs, our earlier approach used highly annotated nematode genomic information to computationally prioritize genes that are essential for the survival of parasitic helminths and integrated these data with experimental screening studies using repurposed drugs against filarial worms in vitro [START_REF] Tyagi | An Integrated Approach to Identify New Anti-Filarial Leads to Treat River Blindness, a Neglected Tropical Disease[END_REF] . By integrating computationally prioritized compounds with the list of experimentally active hits, we identified several repurposed compounds, which are amenable to structural optimization and could be further explored as potentially new lead compounds. One of the major groups of compounds that was identified using this strategy was the azoles, which are fivemembered heterocyclic compounds that incorporate a nitrogen atom and at least one other non-carbon atom in its ring and have antifungal activity that targets membrane sterol synthesis [START_REF] Shafiei | History of the Development of Antifungal Azoles: A Review on Structures, SAR, and Mechanism of Action[END_REF] .

In our earlier reports, we have studied azoles, in particular fluconazole (FCZ), a first generation triazole approved for use as a broad-spectrum antifungal agent. FCZ has a clinically favorable pharmacokinetic profile and has been used as a safe and effective treatment for several fungal infections since the 1990s 8,9 . A successful strategy for developing low-cost, alternative therapies to improve the potency and therapeutic scope of an organic anti-infective drug is the use of metal complexes [START_REF] Ong | Metal Compounds against Neglected Tropical Diseases[END_REF][START_REF] Lin | Recent Developments of Metal-Based Compounds against Fungal Pathogens[END_REF] or the addition of a ferrocenyl moiety [START_REF] Patra | The Medicinal Chemistry of Ferrocene and Its Derivatives[END_REF] to the parent drug, as exemplified with the antimalarial ferroquine, [START_REF] Biot | Synthesis and Antimalarial Activity in Vitro and in Vivo of a New Ferrocene-Chloroquine Analogue[END_REF][START_REF] Biot | The Antimalarial Ferroquine: From Bench to Clinic[END_REF] which successfully completed phase II clinical trials [START_REF] Abd-Rahman | Scoping Review of Antimalarial Drug Candidates in Phase I and II Drug Development[END_REF][START_REF]ZY19489+ferroquine[END_REF] . Importantly, we demonstrated that the replacement of one of the triazoles of FCZ by an organometallic moiety (e.g., ferrocene and ruthenocene) allowed unveiling of a subset of FCZ analogues (1a-4a, Fig. 1A) more effective than the parent FCZ against various fungal strains [START_REF] Rubbiani | In Vivo Active Organometallic-Containing Antimycotic Agents[END_REF] . In the present study, we expanded on our previous work and assessed the efficacy of eight newly synthesized FCZ analogs (5a-12a; Fig. 1B) that are metabolically more stable than the previous series tested, on a panel of clinical fungal isolates, including highly drug-resistant strains. We then tested all twelve FCZ analogs (1a-12a) on two phylogenetically divergent parasitic nematodes, Brugia pahangi and Trichuris muris. We demonstrate that structural modification of FCZ not only improves the activity of the parent drug but also allows us to broaden the scope of application of these derivatives. Here, we unveil several easy-to-prepare agents that are not only effective in vivo against fungal pathogens but also are potent against parasitic worms: Brugia that causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths, that infects millions of people globally. This broadspectrum activity reveals new avenues for the treatment of various infections by eukaryotic organisms that cause major human diseases, including other NTDs and newly emerging diseases.

RESULTS

Synthesis and characterization of 8 novel derivatives based on the parent compound FCZ

Ruthenocenylmethanamines (16 and 17) were obtained in excellent yields from formylruthenocene [START_REF] Sanders | The Lithiation of Ferrocene and Ruthenocene: A Retraction and an Improvement[END_REF] (13) through a two steps reductive amination sequence using similar procedure as the ones previously reported for ferrocenylmethanamines. [START_REF] Rubbiani | In Vivo Active Organometallic-Containing Antimycotic Agents[END_REF] Primary amine 16 was obtained through the reduction of oxime 14, while reduction of methyl imine 15 gave secondary amine 17 (Scheme 1A).

(Metallocenylmethyl)trimethylammonium iodides 18 and 19 were used for the homologation of the carbon chain. It is worth noting that what renders these (metallocenylmethyl)trimethylammoniums electrophilic, i.e. the leaving group in in a benzylic position, might also impact the stability of the FCZ analogs bearing an ammonium such as 1a-6a, as a nucleophilic attack could cleave the metallocenyl moiety. Homologation of the carbon chain, would thus mitigate the risk of the metallocenyl moiety to act as a leaving group. 2-(Ferrocenyl)ethyl-amine 22 was prepared in two steps, by first reacting ferrocenylmethyltrimethylammonium iodide 6 with sodium cyanide in a water reflux setup, [START_REF] Yi | Probing the Tolerance of Cucurbit[7]Uril Inclusion Complexes to Small Structural Changes in the Guest[END_REF] resulting in the formation of compound 20 as orange solid with a 74% isolated yield. Compound 20 was then converted into amine 22 by reduction using LiAlH 4 . The N-methyl amine 26 was synthesized by protecting amine 22 using di-tert-butyl dicarbonate. Full reduction with LiAlH 4 led to the successful formation of the expected compound 36 in good yields. Likewise, the ruthenocenyl analogs 23 and 27 were synthesized using the identical procedures applied to compounds 22 and 26, respectively. (Scheme 1B) Key epoxide 29 was synthesized through Corey-Chaykovsky epoxidation of the commercially available ketone 28 by treatment with a solution of sulfoxonium ylide, which was generated in situ by deprotonating trimethylsulfoxonium iodide with sodium hydride in DMSO. [START_REF] Corey | Dimethyloxosulfonium Methylide ((CH 3 ) 2 SOCH 2 ) and Dimethylsulfonium Methylide ((CH 3 ) 2 SCH 2 ). Formation and Application to Organic Synthesis[END_REF] The subsequent epoxide ring-opening with primary amines or N-methyl amines were carried out in the presence of Et 3 N in ethanol or THF under reflux to give the derivatives 32-39 in high yields except for the ruthenocene derivatives with a shorter chain 32 and 33 (Scheme 1C). To enhance the water solubility and hence the bioavailability of tested compound, FCZ derivatives 32-39 were converted into the corresponding hydrochloride salts 5a-12a. This transformation was achieved by treating the derivatives with 37% aq.

HCl in deoxygenated acetone the hydrochloride salts were easily isolated on the gramscale (from 0.31 g for 6a to 1.40 g for 10a) through precipitation followed by filtration after 30 minutes of stirring (Scheme 1D). Of note, in order to obtain initial insights into their biological activity, we synthesized these compounds as racemates to avoid an asymmetric synthesis. All newly synthesized compounds were unambiguously characterized by 1 H, [START_REF] Biot | Synthesis and Antimalarial Activity in Vitro and in Vivo of a New Ferrocene-Chloroquine Analogue[END_REF] CNMR, [START_REF] Yi | Probing the Tolerance of Cucurbit[7]Uril Inclusion Complexes to Small Structural Changes in the Guest[END_REF] F, 2D NMR, HRMS, IR. The >95% purity of the tested compounds was confirmed through a combination of microanalysis, mass spectrometry and NMR.

Details on synthesis and characterization of the complexes can be found in the Supporting Information.

We then studied the stability of potential drug candidates, including the 4 from previous work (Fig. 1A; 1a-4a) 1a-12a in DMSO-d 6 and D 2 O using 1 H-NMR spectroscopy and found that the majority of compounds were stable in DMSO-d 6 and D 2 O over 48 h (except for 4 and 6, which contain a methyl chain), and slightly decompose in D 2 O after 48 h (Fig. S1-24). Both compounds were also found to be stable in RPMI culture medium (Fig. S25-29). To further assess metabolic stability, two representative compounds (2a and 8a) were incubated with human liver microsomes. After 48 h incubation of both compounds with human liver microsomes, samples were analyzed via HPLC.

n = 0 M = Ru, 10% n = 0 M = Ru, 15% n = 1 M = Fe, 65% n = 1 M = Fe, 70% n = 1 M = Ru, 40% n = 1 M = Ru, 85% D: N N OH N N F F R •HCl n N N OH N N F F R n HCl/acetone 32 -39 5a -12a Fe = Ru n = 1, R = H, 7a, 63% n = 1, R = Me, 8a , 70% n = 0, R = H, 5a, 65% n = 0, R = Me, 6a, 60% n = 1, R = H, 9a, 73% n = 1, R = Me, 10a, 65% n = 1, R = Me, 11a, 46% n = 1, R = Me, 12a, 44%
Scheme 1. Synthesis of the analogues of FCZ.

Compound 2a was shown to undergo 43% metabolism, while compound 8a, containing the longer linker between ferrocene and the central structure, remained stable throughout (Fig. S30-40). This investigation has verified that compound 8a which features a longer alkyl linker between the FCZ body and the ferrocenyl moiety, exhibits significantly improved metabolic stability compared to compound 2a.

New derivatives have potent activity in in vitro antifungal susceptibility screens

The novel compounds were first screened for their antifungal activity against a panel of clinical fungal isolates including FCZ-resistant (MIC 50 > 100 µM) and mycotoxin-producing fungi (Fig. 2A). A comparison of the novel drug candidates (5a-12a) with the previously published compound 2a [START_REF] Rubbiani | In Vivo Active Organometallic-Containing Antimycotic Agents[END_REF] and the parent drug FCZ revealed that all compounds showed activity against isolates resistant to FCZ. Here, especially the incorporation of either a ferrocenyl (2a, 7a, 8a) or a phenyl moiety (11a) led to potent antifungal activity, with remarkable activity against all tested isolates highly resistant to FCZ (MIC 50 > 100 µM). Also, the introduction of a ruthenocenyl (5a, 6a, 9a, 10a) or adamantyl moiety (12a) conferred high antifungal activity against some FCZ-resistant isolates while showing comparable or weaker (5a) antifungal activity compared to FCZ isolates not resistant to FCZ.

The 8a compound prolongs mouse survival in an in vivo model of murine invasive candidiasis

Since compound 8a was the most potent analog tested against various clinical fungal isolates in vitro, it was also tested in a model of invasive candidiasis in the immunocompromised Balb/c mouse model.

After infection with C. albicans CAAL28, an FCZ-resistant strain by overexpression of CDR1 and 2 Erg11 mutations (IC 50 against this strain was > 100 and 13.2 ± 0.8 µM for FCZ and 8a, respectively), mice were treated for 3 days with 10 mg/kg of FCZ, or compound 8a per os. Survival curves indicated that in contrast to the recognized antifungal drug FCZ (50% survival), compound 8a significantly (P=0.0247) prolonged mice survival compared to the vehicle control group (survival of 87.5% vs 25%, respectively by day 4) (Fig. 2B).

A B

FCZ derivatives have potent activity against parasitic nematodes in in vitro assays

We next assessed the efficacy of the FCZ and analogs on two groups of phylogenetically diverse species of parasitic nematodes to determine the potential use of these compounds for treatment against a broader range of human pathogens. The lymphatic filarial parasite Brugia pahangi and the soiltransmitted gastrointestinal parasite Trichuris muris, were selected as representatives of taxonomic groups with very different modes of parasitism (Clade III and I, respectively) [START_REF] Blaxter | A Molecular Evolutionary Framework for the Phylum Nematoda[END_REF] . We hypothesized that any drug with efficacy against both of these nematode species may have broad potential against other parasitic organisms. B. pahangi adult females (4 replicates) and T. muris adults (4 replicates) were tested in a whole-worm in vitro motility assay [START_REF] Marcellino | WormAssay: A Novel Computer Application for Whole-Plate Motion-Based Screening of Macroscopic Parasites[END_REF][START_REF] Bulman | Repurposing Auranofin as a Lead Candidate for Treatment of Lymphatic Filariasis and Onchocerciasis[END_REF][START_REF] Jasmer | De Novo Identification of Toxicants That Cause Irreparable Damage to Parasitic Nematode Intestinal Cells[END_REF][START_REF] Tyagi | Identification of Small Molecule Enzyme Inhibitors as Broad-Spectrum Anthelmintics[END_REF] in the presence of the test compounds over 6 days in culture.

Table 1. In vitro whole-worm assays reveal FCZ analogs are potent against both parasite species.

Most of the compounds outperformed FCZ in both the B. pahangi and T. muris assays (Table 1).

Particularly, 4a, 8a, 10a, and 12a were identified as best hits and were then tested to determine the IC 50 s in both species. Overall, the range of activity of the 4 compounds against both species was comparable, and based on IC 50 values, the compounds were somewhat better against B. pahangi (2.2-23.2 µM) compared to T. muris (9.0-25.7 µM). The IC 50 values were lowest for 12a against the filarial nematode and for 4a against the whipworm (Table 2 and Fig. S41).

Brugia female worm fecundity is reduced after treatment with FCZ analogs in vivo

With the promising results obtained for B. pahangi with 8a, 10a and 12a (IC 50 <10 µM), we next tested these compounds in vivo. Male Mongolian jirds infected with B. pahangi were given oral doses of 8a, 10a or 12a (10 mg/kg twice a day for 5 days) following reported protocols [START_REF] Bulman | Repurposing Auranofin as a Lead Candidate for Treatment of Lymphatic Filariasis and Onchocerciasis[END_REF][START_REF] Hübner | Short-Course Quinazoline Drug Treatments Are Effective in the Litomosoides Sigmodontis and Brugia Pahangi Jird Models[END_REF][START_REF] Gunderson | Pyrvinium Pamoate and Structural Analogs Are Early Macrofilaricide Leads[END_REF] . Drug efficacy was evaluated based on: 1) number of adult worms recovered, 2) microfilarial (MF) output to evaluate a female worm's ability to transmit its progeny, 3) embryogram analyses to determine the effects of the compounds on the developing embryonic stages within the reproductive tract and 4) transmission electron microscopic analyses of the structures in the uteri from recovered female worms. Animals necropsied 5 days after the last dose did not have a significant decrease in worm burden after treatment with 8a, 10a nor 12a (Fig. S41) which suggests that longer-term studies are necessary to observe efficacy of the derivatives to eliminate adult worms. However, female worm fecundity and reproductive output were affected by these derivatives compared to female worms recovered from the vehicle control group (Fig. 3A and Fig. S42). Female worms recovered from animals treated with 8a, 10a or 12a had fewer number of eggs in their reproductive tracts (Fig. 3A and Fig. S42), with 12a causing significantly lower number of eggs (Fig. S43A) and pre-MF stages (Fig. S42C). Notably, both 10a and 12a had significantly higher numbers of deformed embryos (Fig. S42E) and there were significantly fewer numbers of MF released ex vivo from female worms removed from animals treated with 12a compared to the other groups (P < 0.01) (Fig. 3B). Ultrastructural analyses of treated worms support the drug's effect on the reproductive fitness of

Brugia pahangi adult female worms recovered from infected jirds after treatment with FCZ analogs

The impact of the FCZ analogs on the embryonic development within the worms' uteri, from eggs to stretch microfilariae (Fig. 3A), is supported by the ultrastructural analyses of adult female B. pahangi recovered from treated infected jirds. The worms collected from animals treated with 8a, 10a and 12a had major tissue damage in their reproductive tracts (Fig. 4). Specifically, female worms from the group treated with 8a had electron dense, globular material throughout the deformed embryos (Fig. 4G).

Notably, disturbances to nuclear ultrastructure were frequently detected in 10a treated worms (Fig. 4I-L), including several deformed and damaged nuclei within a developing microfilaria (Fig. 4I-L), and an apparent large rupture in the nuclear envelope as well as the appearance of abnormal vacuoles (Fig. 4K).

Remarkably, the damage was more profound in worms collected from 12a treated animals (Fig. 4M-P)

as it also showed deformed embryos containing considerable amounts of globular material (Fig. 4M) and punctate, vacuolated spaces adjacent to embryos having deformed and damaged nuclei (Fig. 4N). In addition, many developing embryos also had numerous autophagolysosomes or mature lysosomes (Fig. pahangi genes were less pronounced than those in T. muris but they were distinct from those induced in T. muris. In 12a treated T. muris worms, 1501 (10.0 %) and 1275 (8.5 %) genes were significantly upand down-regulated, respectively (Fig. 6A). Interestingly, 9 of the top 10 most significantly up-regulated genes encode proteins involved in mitochondrial respiratory chain complexes and ATP synthesis (Fig. 6A). The functional enrichment analysis indicated that spliceosome, ribosome biogenesis and mitochondria biogenesis pathways were significantly enriched in the up-regulated genes in 12a treated T. muris (Fig. 6C). In addition, we found a significant induction of 7 ABC transporter genes in the 12a treated worms out of a total of 25 ABC transporters identified in T. muris (Fig. 6B and6D). On the other hand, the expression levels of genes belonging to various signaling pathways (Hippo, mTOR, AMPK, and Wnt), transcription factors, and the fatty acid biosynthesis were significantly decreased (Fig. 6C). Of the 13 genes in the fatty acid biosynthesis pathway, 6 genes including fatty acid synthase, acyl-CoA synthetase, and mitochondrial acyl carrier reductase were significantly decreased in the 12a treated T. muris (Fig. 6E).

4O-P, respectively).

Chemogenomic studies identify new drug targets

Analogues of FCZ that showed elevated activity against fungi and parasitic nematodes (8a, 10a, 11a and 12a) were selected to investigate their mechanism of action using a chemogenomic assay. Most interestingly, the genome wide screen revealed that the main targets of the new analogs are different from the main target of FCZ, which is the lanosterol 14α-demethylase encoded by the gene ERG11. While for the previously synthesized compound 2a, [START_REF] Rubbiani | In Vivo Active Organometallic-Containing Antimycotic Agents[END_REF] ERG11 was identified as the main target, the elongation of the linker between the FCZ body and the moiety in the new compounds led surprisingly to a series of new drug targets. These include top targets involved in transcription and translation, such as an acetyl-CoA synthetase with function in histone acetylation (ACS2), a snoRNA binding protein (ENP1) and a translation initiation factor (HYP2), as well as subunits of the RSC-type chromatin remodeling complex (RSC9), the RNA polymerase II mediator complex (SRB2), the RNA polymerase (RPC19) and the small subunit processome complex (UTP14) (Table 3 and Fig. S44). Furthermore, identified deletion strains that are highly sensitive to the new compounds were heterozygous for genes involved in calcium homeostasis and biofilm formation (PMC1), cytoskeleton organization, cell wall deposition and endocytosis (INP52), the biosynthesis of branched-chain amino acids (ILV6) and in activation of the plasma membrane and segregation of phosphatidylserines (PMP1) (Table 3 and Fig. S44).

Table 3. The 5 S. cerevisiae heterozygous deletion mutants showing highest sensitivity to the compounds 8a, 10a, 11a and 12a based on the chemogenomic screen. Values are log 2 ratios (control intensity/treatment intensity) calculated as a function of gene (P<0.01).

We then compared the results for down-regulated genes of the chemogenomic screen of 12a-treated S. (chaperones, glycosyltransferases and protein phosphatases) and degradation (ubiquitin system, peptidases, proteasome) (Fig. 7). Together with the KEGG terms DNA repair, chromosome, cytoskeleton and exosome, we can assume a severe impact of 12a on core biological processes suggesting abnormal cell division and proliferation similar to cancer cells.

DISCUSSION

We report the discovery of novel FCZ derivatives, including organometallic-containing ones, that are uniquely active against not only a panel of clinical fungal isolates, including highly drug-resistant strains, but also on two phylogenetically distant species of parasitic nematodes, Brugia (Clade III) and Trichuris (Clade I). Significantly, the most potent analog of our in vitro antifungal screen (e.g., 8a) was found to be extremely effective in a model of invasive candidiasis in the immunocompromised Balb/c mouse (i.e., an infection with C. albicans CAAL28, an FCZ-resistant strain by overexpression of CDR1 and 2 ERG11 mutations). More surprisingly, chemogenomic studies showed that 2a and 8a, which have only a CH 2 difference in their structures, had different targets. While ERG11 is the main target of 2a, 8a has other top targets such as the acetyl-CoA synthetase ACS2, required for histone acetylation and the regulatory subunit of the acetolactate synthase ILV6. In yeast ACS2p is notable because it produces acetyl-coA required for histone acetylation, consequently impacting global gene expression [START_REF] Takahashi | Nucleocytosolic Acetyl-Coenzyme A Synthetase Is Required for Histone Acetylation and Global Transcription[END_REF] . Acetolactate synthase (ALS), on the other hand, catalyzes the first steps of branched-chain amino acid biosynthesis (isoleucine, leucine, and valine). Since mammals cannot synthesize branched-chain amino acids, this pathway, and especially the ALS, represents an interesting target for antifungal drugs and other inhibitors such as herbicides [START_REF] Richie | Identification and Evaluation of Novel Acetolactate Synthase Inhibitors as Antifungal Agents[END_REF] . Furthermore, C. albicans ALS mutants showed significant reduced virulence and in vivo viability [START_REF] Kingsbury | Cytocidal Amino Acid Starvation of Saccharomyces Cerevisiae and Candida Albicans Acetolactate Synthase (ilv2Δ) Mutants Is Influenced by the Carbon Source and Rapamycin[END_REF] Resistance to azoles is mainly due to increased drug efflux and alteration of the drug target. Our genome wide screen revealed that the analogues 10a, 11a and 12a also seem to act on cellular mechanisms that are distinct from the parental antifungal, desirable features that may help to decrease tolerance and resistance against FCZ. For example, both blocking Ca 2+ sequestration accompanied by accumulation of intracellular Ca 2+ (PMC1) and epigenetic alteration of gene expression via chromatin regulation (RSC9), can potentiate antifungal activity [START_REF] Oyama | Deletion of the Golgi Ca2+-ATPase PMR1 Gene Potentiates Antifungal Effects of Dodecanol That Depend on Intracellular Ca2+ Accumulation in Budding Yeast[END_REF] . Ribosome biogenesis (UTP14) and translation initiation and elongation (HYP2), dependent or independent of TOR (target of rapamycin) signalling, are thought to be required for adaptation to azoles [START_REF] Berman | Drug Resistance and Tolerance in Fungi[END_REF][START_REF] Weil | Adaptive Mistranslation Accelerates the Evolution of Fluconazole Resistance and Induces Major Genomic and Gene Expression Alterations in Candida Albicans[END_REF][START_REF] Khandelwal | Azole Resistance in a Candida Albicans Mutant Lacking the ABC Transporter CDR6/ROA1 Depends on TOR Signaling[END_REF] . Moreover, phosphatidylserine secreting membrane proteins (PMP1) were discussed as an interesting drug target as phosphatidylserines are among others involved in virulence, mitochondrial function and cell wall thickness of C. albicans [START_REF] Cassilly | It's Complicated: The Roles of Phosphatidylserine and Phosphatidylethanolamine in the Pathogenesis of Candida Albicans and Other Microbial Pathogens[END_REF][START_REF] Wolf | Lipid Biosynthetic Genes Affect Candida Albicans Extracellular Vesicle Morphology, Cargo, and Immunostimulatory Properties[END_REF] . Interestingly, it was shown that the symmetry in the membrane and the exposure of phosphatidylserines as well as their impact on extracellular vesicle formation do not only play an important role in virulence and pathogenicity of pathogenic fungi but also for parasites [START_REF] Cassilly | It's Complicated: The Roles of Phosphatidylserine and Phosphatidylethanolamine in the Pathogenesis of Candida Albicans and Other Microbial Pathogens[END_REF] .

Since the new FCZ analogs were highly potent against several clinical fungal isolates, we next evaluated the broader application of these compounds on two parasitic helminths, B. pahangi and T. muris. Both the ferrocenyl analogs 8a and 10a were potent against both species of parasitic worms in vitro as was the N-methyl-β-adamantylethylamine analogue 12a. In particular, analog 12a had the greatest effect on the overall reproductive capacity of female B. pahangi in vivo, i.e., female worms produced more deformed embryonic stages and released significantly fewer progeny (microfilariae). The structural cellular abnormalities seen in the developing embryos and multifocal damage in the various embryonic stages within the worm's uterus support the embryogram analyses that revealed reduced fitness of fecundity in these treated worms. Taken together, the in vivo data show that the FCZ analogs were highly effective in reducing female worm fecundity and reproductive output. This is an important finding since transmission of the parasites depends upon the female worms' ability to produce microfilariae that are transmitted to the insect vector, thus perpetuating the life cycle of the parasite. Further in vivo studies, however, are needed to optimize the potency and increase the efficacy of these new leads. For example, treatment of infected people with antibiotics that target Wolbachia, a mutualistic symbiont of filarial worms, affects worm burden only after 18 months or more post-treatment [START_REF] Johnston | Anti-Wolbachia Drugs for Filariasis[END_REF] . Importantly, the Target Drug Profile for candidate macrofilaricidal drugs includes evidence of impacting adult worms, killing adults or impeding fecundity [START_REF]Target product profile for filarial diseases | DNDi[END_REF] . Our study showed that the ferrocenyl and adamantyl analogs are potential new leads for further development not only as antifungals but also as anthelmintics to treat

NTDs that can impact the transmission and prevalence of new infections. The FCZ analogs also show promise as a low-cost treatment for developing countries whose communities suffer from years of disabilities from parasitic infections.

By analyzing transcriptional profiles of 12a-treated B. pahangi and T. muris, we were able to identify DEGs and molecular pathways disturbed by the in vitro treatment. Although the number of DEGs in B.

pahangi was relatively lower than the ones in T. muris, one of the significantly enriched pathways in the up-regulated genes was the chaperones and folding catalysts, and 7 of top 20 up-regulated genes were associated with heat shock protein (HSP) functions. A total 12 hsp genes were significantly induced in 12a treated B. pahangi and most of them were highly conserved in other species, from nematodes to yeast: for example, BPAG_139801 (orthologous to HSP90 in C. albicans), BPAG_1021901 (STI1), and BPAG_1149201 (MSI3/HSP70). In addition to important functional roles in protein refolding and maturation, the Hsp90-Hsp70 complex interacts with client proteins such as calcineurin via another cochaperone, a stress-inducible phosphoprotein 1 (STI1) which regulates various cellular signaling pathways [START_REF] Cowen | Hsp90 Orchestrates Stress Response Signaling Governing Fungal Drug Resistance[END_REF][START_REF] Cruz | Calcineurin Is Essential for Survival during Membrane Stress in Candida Albicans[END_REF][START_REF] Singh | Hsp90 Governs Echinocandin Resistance in the Pathogenic Yeast Candida Albicans via Calcineurin[END_REF][START_REF] Lafayette | PKC Signaling Regulates Drug Resistance of the Fungal Pathogen Candida Albicans via Circuitry Comprised of Mkc1, Calcineurin, and Hsp90[END_REF][START_REF] Gong | Candida Albicans Heat Shock Proteins and Hsps-Associated Signaling Pathways as Potential Antifungal Targets[END_REF][START_REF] Gu | The Hsp90 Co-Chaperones Sti1, Aha1, and P23 Regulate Adaptive Responses to Antifungal Azoles[END_REF] . In C. albicans, the calcineurin signaling pathway has been shown to confer tolerance to antifungal azoles including fluconazole [START_REF] Cowen | Hsp90 Orchestrates Stress Response Signaling Governing Fungal Drug Resistance[END_REF][START_REF] Cruz | Calcineurin Is Essential for Survival during Membrane Stress in Candida Albicans[END_REF] . It is possible that the immediate transcriptional induction of chaperones and co-chaperones in B. pahangi could be part of an adaptative mechanism responding to stress induced by the FCZ derivative 12a as well.

In T. muris, 9 of the top 10 DEGs that encode proteins involved in mitochondrial respiratory chain complexes and ATP synthesis were significantly up regulated. Mitochondrial functions are directly or indirectly related to efflux pump-mediated azole resistance in various fungal species [START_REF] Song | Mitochondria-Mediated Azole Drug Resistance and Fungal Pathogenicity: Opportunities for Therapeutic Development[END_REF] . Mitochondria are the major cellular source of ATP, and increased activity of the mitochondrial respiratory metabolism can produce and transport more ATP to drug efflux pumps, enhancing the efflux of intracellular azole drugs (32). In addition, defects in mitochondrial functions can affect cytoplasmic Ca 2+ homeostasis via the calcineurin signaling pathway followed by activation of transcription factors which regulate expression of drug efflux transporter genes [START_REF] Li | Mitochondrial Dysfunctions Trigger the Calcium Signaling-Dependent Fungal Multidrug Resistance[END_REF][START_REF] Li | Calcium Signaling Pathway Is Involved in Non-CYP51 Azole Resistance in Aspergillus Fumigatus[END_REF][START_REF] Xu | RNA Sequencing Reveals an Additional Crz1-Binding Motif in Promoters of Its Target Genes in the Human Fungal Pathogen Candida Albicans[END_REF] . Remarkably, the significant induction of 7 ABC transporter genes in the 12a-treated T. muris in addition to the upregulated genes associated with mitochondrial biogenesis, suggest that although phylogenetically distant from each other, T. muris seemed to display similar responses to azole drugs that have been observed in fungal species. On the other hand, fatty acid biosynthesis was one of the significantly enriched pathways that were downregulated in the 12a-treated T. muris, including fatty acid synthase (TMUE_2000008856). Fatty acids whose synthesis is mediated by fatty acid synthase provide energy during oxidation in mitochondria, peroxisomes and the ER, and are precursors of the building blocks such as sterols, phospholipids, and sphingolipids [START_REF] Riezman | The Long and Short of Fatty Acid Synthesis[END_REF][START_REF] Wei | Fatty Acid Synthesis Configures the Plasma Membrane for Inflammation in Diabetes[END_REF] . Therefore, the alteration observed may broadly impact important metabolic processes, membrane fluidity/permeability and signaling cascades.

In the in vitro motility screening on B. pahangi and T. muris, FCZ showed no direct effect but the FCZ analogs such as 4a, 8a, 10a, and 12a effectively inhibited worm motility, which eventually resulted in worm death. Considering the loss of some genes involved in de novo cholesterol synthesis in nematode species (unlike fungal species which have Erg11, the target of FCZ), the modifications on the FCZ analogs likely altered other molecular target(s), consequently improving their potencies against the parasitic nematodes as well as FCZ-resistant fungal strains. In consideration of these observations, we interfaced the chemogenomic result from S. cerevisiae and the transcriptomic profiles from the two parasitic nematode species to examine affected targets and/or pathways shared among them.

The results suggest that similar pathways are down-regulated when fungal and parasitic nematode species are treated with our new FCZ analogs. In particular, we can assume a severe impact of 12a on core biological processes suggesting abnormal cell division and proliferation similar to cancer cells; especially because, next to the KEGG categories DNA repair and cytoskeleton, the vast majority of KEGG categories shared by the 3 species are related to protein biosynthesis, modification and degradation.

Moreover, membrane trafficking was the most enriched of the shared KEGG categories. Membrane trafficking is a key process for maintaining vital cellular activities by transporting micro-and macromolecules via endo-and exocytosis into, within and out of the cell, including trafficking of pharmacological agents and their intracellular delivery, and is therefore of great interest for drug development [START_REF] Kumar | Membrane Trafficking and Subcellular Drug Targeting Pathways[END_REF] . In this context, GPI-anchors are crucial for the transport of proteins to the plasma membrane and their function, as well as for the endocytosis of cell surface proteins that are recycled in the endosome. GPI-anchored proteins, another shared KEGG category, are often associated with lipid rafts of the membrane that are rich in sterols and sphingolipids [START_REF] Sabharanjak | GPI-Anchored Proteins Are Delivered to Recycling Endosomes via a Distinct Cdc42-Regulated, Clathrin-Independent Pinocytic Pathway[END_REF][START_REF] Muñiz | Sorting of GPI-Anchored Proteins from Yeast to Mammals -Common Pathways at Different Sites?[END_REF] . They are also involved in modifications of the cell wall and have been discussed not only for their role in antifungal susceptibility [START_REF] Weil | Adaptive Mistranslation Accelerates the Evolution of Fluconazole Resistance and Induces Major Genomic and Gene Expression Alterations in Candida Albicans[END_REF][START_REF] Plaine | Functional Analysis of Candida Albicans GPI-Anchored Proteins: Roles in Cell Wall Integrity and Caspofungin Sensitivity[END_REF] but also for germline development and eggshell formation in nematodes which may explain the increase in number of deformed embryos and ultrastructural damage observed in the developing stages of microfilariae in treated Brugia 51 . Hence, the manifold interactions between lipid biosynthesis, vesicle production, cell wall homeostasis, mitochondrial function and immune activation represent a fascinating area for future studies. Overall, these novel FCZ analogs showed broad-spectrum efficacy across two phylogenetically distinct groups of eukaryotic organisms and may also show promise against other pathogens of medical and veterinary importance in future studies.

CONCLUSIONS

The search for new anti-infective drugs, notably for the treatment of helminth and fungal infections is a race against the clock, as clearly highlighted by the World Health Organization, that published in October 2022 its first-ever list of health-threatening fungi and its overreaching global targets for control or elimination, including filarial infections, by 2030 [START_REF]The road map targets for[END_REF] . In this work, we demonstrated that the facile structural modulation of a well-established antifungal drug, fluconazole (FCZ), by some organic but also organometallic moieties, led to the identification of new compounds with not only high activity in vivo against a broad range of resistant fungi but also, more surprisingly, the phylogenetically distant parasitic worms such as Brugia and Trichuris, which cause NTDs that infect millions of the poorest people around the globe. As demonstrated in this work, the newly identified targets for our derivatives are distinctive from the ones of the parent drug FCZ, a vital attribute to circumvent resistance. The discovery of targets that are unique to pathogens, as described herein, is of high value and we are confident that our new family of easy-to-make compounds with broad-spectrum activity offers new hope to treat multiple eukaryotic infectious agents as well as other major human diseases, including newly emerging diseases.

The discovery of these advanced hit compounds and the characterization of the mode of action provide the groundwork for highly promising lead optimization in either antifungal or antiparasitic anti-infective development.

EXPERIMENTAL SECTION

Study design

A new series of (metal-containing) FCZ analogs or azoles were prepared and characterized by 1 H, [START_REF] Biot | Synthesis and Antimalarial Activity in Vitro and in Vivo of a New Ferrocene-Chloroquine Analogue[END_REF] CNMR, 19 F, HRMS, IR and their purity were confirmed by microanalysis. The (metabolic) stability of the compounds were then assessed. This series of compounds was then screened to identify compounds that were active in vitro against a panel of Candida strains, B. pahangi and T. muris. After the identification of several promising hits, these compounds were tested on infected mice (invasive candidiasis) and Mongolian jirds (B. pahangi). In order to ascertain tissue damage in the reproductive tracts of worms collected, ultrastructural analyses of B. pahangi adult females was performed using TEM microscopy. To further assess the effects of the compounds at the molecular level, chemogenomic studies and RNA-seq analyses of B. pahangi and T. muris adult worms were conducted.

Chemistry

Compounds 1a-4a were prepared as previously described [START_REF] Rubbiani | In Vivo Active Organometallic-Containing Antimycotic Agents[END_REF] . The synthesis and characterization of compounds 5a-12a is described in-depth in the supplementary materials. All compounds prepared and used in biology were >95% pure, as confirmed by elemental analysis.

Metabolic Stability

Compounds 2a and 8a were subjected to metabolic studies using human microsomes (Gibco TM ) and compared with caffeine (Sigma-Aldrich) as an internal standard to ascertain the stability of the novel compounds in a human metabolic environment. Samples were prepared in an Eppendorf tube with 175 µL PBS solution, 5 µL of compound stock (1 mg/ 100 µL biological grade DMSO) and 5 µL of Human microsomes (20 mg/mL). Samples were pre-incubated at 37 o C and 500 rpm for 5 minutes. The reaction was then initiated with the addition of 10 µL NADPH solution (20 mM, prepared by dissolving 33 mg of NADPH in 2 mL PBS solution) incubated at 37 o C and 500 rpm for the respective time: 0 h, 6 h, 24 h, 48 140-bp UPTAG and DNTAG libraries were purified using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel), quantified using the Qubit dsDNA HS kit (ThermoFisher Scientific) and the libraries were sequenced separately on an Illumina NextSeq 2000 using standard methods, including the use of the standard Illumina sequencing primer (5'-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3'). The qseq files are available from the European Nucleotide Archive (ENA) with the accession number PRJEB57349. Read matching was performed using an in-house developed Python script (https://github.com/marcomoretto/Bar-seq) as previously described [START_REF] Tocci | Potent Antifungal Properties of Dimeric Acylphloroglucinols from Hypericum Mexicanum and Mechanism of Action of a Highly Active 3′Prenyl Uliginosin B[END_REF] . The statistical analysis was performed with the voom [START_REF] Law | Precision Weights Unlock Linear Model Analysis Tools for RNA-Seq Read Counts[END_REF] transformation package that estimates the mean-variance relationship of log counts, generating a precision weight for each observation that is fed into the limma [START_REF] Ritchie | Limma Powers Differential Expression Analyses for RNA-Sequencing and Microarray Studies[END_REF] empirical Bayes analysis pipeline. A double threshold based on both p-value (≤0.01) and expression log 2 fold change (≥1.5) was imposed to identify barcodes differentially abundant through pairwise comparison.

The Fig. 7 has been obtained using the R language and the package circlize 0.4.15. DEGs from the three species (S. cerevisiae, T. muris and B. pahangi) obtained as described above, have been annotated using KEGG BRITE database (https://www.genome.jp/kegg/brite.html). The relative abundance of the species DEGs are represented in each KEGG category, for example, for all the genes in the three species annotated as membrane trafficking ~30% are coming from S. cerevisiae, ~30% from T. muris and ~40% from B. pahangi. In order to reduce the total number of KEGG categories, only the ones shared among all three species have been retained.

Fig. 1 .

 1 Fig. 1. (A) Structure of FCZ and previously reported ferrocenyl derivatives 1a-4a 17 . (B) Structure of the FCZ analogs 5a-12a that have been developed in this study.

Fig. 2 .

 2 Fig. 2. (A) Antifungal susceptibility testing on clinical fungal isolates treated with fluconazole (FCZ) and derivatives (2a, 5a -12a). MIC 50 values are given in µM. Superscript letter r indicates FCZ-resistant isolate (MIC 50 > 100 µM). (B) Mice (8 per group) were infected with FCZ-resistant C. albicans CAAL28 and treated immediately with FCZ or 8a (10 mg/kg) for 3 days per os, once daily.

Fig. 3 .

 3 Fig. 3. FCZ analogs had a significant effect on the reproductive output and fecundity of female worms recovered from in vivo treated infected jirds. (A) Embryogram analyses were conducted on female B. pahangi worms recovered from control and treated infected jirds (N = 12-36 per group). Bar graphs indicate the relative mean frequency of each of the developmental stages for each treatment group. (B) The number of MF released ex vivo from the recovered female worms were counted after worms were incubated overnight in media (**P< 0.01).

Fig. 4 .

 4 Fig. 4. Ultrastructure of embryonic stages within the uteri of female B. pahangi worms collected from infected jirds treated with vehicle (A-D), 8a (E-H), 10a (I-L) and 12a (M-P). (A-D) Worms from the vehicle treated worms display healthy ultrastructure of the cuticle and varying embryonic stages. (E and F) Female worms treated with 8a had both deformed (boxed regions) and healthy embryos. (G) A deformed embryo from 8a-treated worm shown in higher magnification that contains electron dense, globular material (H, white arrows) throughout the deformed embryo. (I-L) Worms treated with 10a show frequent disturbances to nuclear ultrastructure, including deformed and damaged nuclei (J-K, asterisks) within a developing microfilaria. (L) A high magnification image of the boxed cell in (K) with an apparent large rupture in the nuclear envelope (white arrow). (M-P) Diverse effects on the ultrastructure of developing embryos were observed in 12a treated worms; including (M) considerable amounts of globular material (white arrows) within deformed embryos, (N) punctate, vacuolated spaces (boxed regions) between developing embryos having deformed and damaged nuclei (asterisks), (O) numerous autophagolysosomes (white arrow heads), and (P) mature lysosomes (white arrows) within the developing embryos. Scale bars: A-D & H, 2 microns; E-G, 10 microns; I-K & M-P, 3 microns; L, 1 micron.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Early transcriptional responses of 12a treated B. pahangi adult worms. (A) The volcano plot was generated using differentially expressed genes (DEGs) in 12a treated B. pahangi versus DMSO with absolute log 2 fold change > 0 and FDR-adjusted p-value (padj) ≤ 0.05 for significance. B. pahangi genes related to chaperones and folding catalysts pathway were annotated with KEGG orthology symbol in parentheses. (B) 12 B. pahangi genes enriched in chaperones and folding catalysts pathway were significantly up-regulated after in vitro treatment with 12a. Sequence-based alignment was performed to identify orthologs of the B. pahangi genes to Caenorhabditis elegans, Saccharomyces cerevisiae, Candida albicans and Aspergillus fumigatus using reciprocal best blast hit search. Log 2 fold change values were relative to DMSO control. For significance, FDR-adjusted p-values were represented as defined *padj ≤ 0.05, **padj ≤ 10 -2 , ***padj ≤ 10 -3 , ****padj ≤ 10 -4 , *****padj ≤ 10 -5 .

  cerevisiae with those obtained by RNA-seq for the nematode species B. pahangi and T. muris treated by 12a to determine which pathways and associated functions are affected and identify the ones that are shared among them. Membrane trafficking was the most abundant KEGG category shared by the 3 species, and several of the KEGG categories were related to protein biosynthesis (transcription machinery, transcription factors, spliceosome, and mRNA, tRNA and ribosome biogenesis), modification

Fig. 7 .

 7 Fig. 7. FCZ analog 12a affects similar KEGG pathways among fungal and parasitic nematode species. The relative abundance of the down-regulated genes in three species (S. cerevisiae, B. pahangi and T. muris) are represented in each KEGG category. In order to reduce the total number of KEGG categories, only the ones shared among all three species are depicted.

  which might explain the prolonged survival of mice infected with FCZresistant C. albicans under treatment with 8a. The NH-(CH 2 -Ferrocene) bond in 2a was not completely stable, contrary to the NH-(CH 2 -CH 2 -Ferrocene) bond in 8a which may explain this difference in their respective targets.

Table 2 .

 2 IC 50 of the top 4 hits with B. pahangi and T. muris on Day 6.

		B. pahangi		T. muris
	Compound	females	R 2 Value	males & females	R 2 Value
		IC 50 (µM)		IC 50 (µM)
	4a	23.2	0.99	9.0	0.85
	8a	9.1	0.80	23.9	0.78
	10a	4.6	0.70	25.7	0.80
	12a	2.2	0.64	20.4	0.90

h. The reaction is then ceased by the addition of 200 µL HPLC grade MeOH and 5 µL caffeine (2 mg in 1 mL Mili-Q water) was added as an external standard. The samples are then centrifuged at 4 o C and 3000 rpm for 10 minutes. The supernatant is withdrawn and filtered through a 0.2 µm membrane and then analyzed by HPLC by direct injection. Fluconazole (FCZ) (5 mg / 100 µL biological grade DMSO) and a 0 h and 48 h control (no NADPH) were run as controls for 0 h and 48 h timepoints.

HPLC method: C18 Column A, flow rate 1 mL/min: 0 min, 5 % MeCN (0.1% HCOOH) -95% H 2 O (0.1% HCOOH); 17 min, 100 % MeCN (0.1% HCOOH); 25 min, 5 % MeCN (0.1% HCOOH) -95% H 2 O (0.1% HCOOH).

In vitro antifungal susceptibility assay

Clinical isolates of

Candida strains (C. albicans MFB005FS3, C. albicans YMS 102_2, C. albicans YMS 102-6, C. parapsilosis MFB005FS5, C. parapsilosis MFB070 N1, C. glabrata MFB005FS4, C. glabrata RTT199_3 and C. tropicalis RTT35-1, C. tropicalis RTT35-3, Penicillium paneum MFB042 N1, Aspergillus glaucus MFB027 N1, Trichosporon asahii MFB034 N1 and the C. albicans reference strain SC5134 were tested for their susceptibility to FCZ and the novel compounds. Fungal isolates were grown on Sabouraud agar medium (Sigma Aldrich, St. Louis, MO, USA) for 48 h at 30°C and resuspended in distilled water at a concentration of 1-5 x 105 CFU/ml before testing. Minimal inhibitory concentration (MIC) values for FCZ and derivates were determined following the European Committee for Antimicrobial Susceptibility

Testing protocol (EUCAST Definitive Document EDef 7.3.2 Revision, 2020). Briefly, cells were grown in RPMI-1640 medium supplied with 2.0% glucose, counted and inoculated at a concentration of 1-5 x 105 CFU/ml. MIC 50 values were detected using a spectrophotometer (at 530 nm) after 48 h of incubation, as the lowest concentration of the drug that resulted in a ≥ 50% inhibition of growth, relative to the control.

In vivo studies of murine invasive candidiasis

The compound was tested in a model of invasive candidiasis in the immunocompromised Balb/c mouse 55 . Mice were immunosuppressed by subcutaneous injection of 15 mg/kg dexamethasone (Dexamedium ® ) one day before challenge. On day 0, mice, 8 per group, were infected intravenously (100 µL) with a suspension of blastopores (3. 10 6 /mL) of C. albicans strain, CAAL28, FCZ-resistant by overexpression of CDR1 and 2 Erg11 mutations. One hour after infection, mice were treated per os for 3 days once daily with 10 mg/kg of compound 8a or fluconazole solubilized in distillated water/5% DMSO.

The control group received 100 μL of distillated water/5% DMSO. Survival differences in cohorts were analyzed by the log-rank test. A P value of <0.05 was considered statistically significant. Swiss mice (Janvier Labs, Le Genest-Saint-Isle, France) with a body weight of ∼25 g were obtained and allowed to acclimate for 7 days prior to use. Environmental controls for the animal room were set to maintain a temperature of 16 to 22°C, a relative humidity of 30 to 70%, and a 12:12 hourly light-dark cycle. All the experimental protocols were prior approved by the Ethic Committee on Animal Testing (comité d'éthique en expérimentation animale N°006) and was authorized by the French Ministry of Higher Education, Research and Innovation (APAFIS#29252-2021011815391956 v4).

In vitro motility assays with adult female Brugia pahangi and adult Trichuris muris

The efficacy of the FCZ derivatives were tested on adult female B. pahangi and T. muris in in vitro inhibition assays [START_REF] Marcellino | WormAssay: A Novel Computer Application for Whole-Plate Motion-Based Screening of Macroscopic Parasites[END_REF][START_REF] Bulman | Repurposing Auranofin as a Lead Candidate for Treatment of Lymphatic Filariasis and Onchocerciasis[END_REF][START_REF] Jasmer | De Novo Identification of Toxicants That Cause Irreparable Damage to Parasitic Nematode Intestinal Cells[END_REF][START_REF] Tyagi | Identification of Small Molecule Enzyme Inhibitors as Broad-Spectrum Anthelmintics[END_REF] . Briefly, each well in a 24-well plate contained one adult female B. pahangi (4 replicate wells per compound) and 500 µL of complete media (RPMI-1640 with 25 mM HEPES, 2.0 g/L NaHCO 3 , 5% heat inactivated FBS, and 1X antibiotic/antimycotic solution), and the plates were incubated for 6 days at 37°C with 5% CO 2 . The motility of the worms was measured using the Worminator instrument on days 0, 1, 2, 3, and 6 [START_REF] Marcellino | WormAssay: A Novel Computer Application for Whole-Plate Motion-Based Screening of Macroscopic Parasites[END_REF] . Each plate of worms was recorded for 60 s to determine the number of pixels displaced by the worms per second. The percentage of inhibition of motility was calculated by dividing the mean movement units (MMUs) of treated worms by the average MMUs of the control worms (treated with 1% DMSO), then subtracting that value from 1. The values were floored to zero and multiplied by 100%. IC 50 assays were conducted for 4a, 8a, 10a and 12a using 3-and 6-point dilutions and were calculated using Prism v6.0. Adult T. muris were assayed in the same manner as B. pahangi except each well contained 2 worms in culture media (4 replicates wells per compound).

Animal infections and dosing for Brugia pahangi/jird model of infection

Animal studies were approved under the New York Blood Center (NYBC) Institutional Animal Care and Use Committee Protocol 369 and adhered to the guidelines set forth in the NIH Guide for the Care and Use of Laboratory Animals and the USDA Animal Care Policies. Mongolian jirds (Meriones unguiculatus), 5-7 week old male weighing 50-60 grams, were injected intraperitoneally with 200 B. pahangi thirdstage larvae and then shipped to the NYBC from the NIH/NIAID Filariasis Research Reagent Resource Center, Athens, GA. Five months post-infection, when larvae have matured into fertile adult worms, animals were given oral doses of 10 mg/kg of 8a, 10a and 12a twice a day for 5 days. Compounds were formulated with 5% glucose and the vehicle group received 5% glucose only. Five days after the last dose, animals (6 animals per treatment group and 9 animals in the vehicle group) were euthanized following NIH Animal Guidelines (NYBC) and necropsies were performed.

B. pahangi microfilarial output and embryogram analyses

Individual adult B. pahangi female worms (n = 12-24 females per group) from each group were collected during necropsies and maintained overnight in 24-well plates with 2 mL of RPMI-1640 supplemented with 1X GlutaMAX, 1X Antibiotic/Antimycotic (Life Technologies, Grand Island, NY), and 10% heat inactivated-FBS (R&D Systems, Flowery Branch, Georgia).. The MF released ex vivo from individual females after 18 hours in culture were counted using a dissecting microscope. For the embryogram analyses, individual adult female worms were finely chopped in 1 mL of PBS (Life Technologies, Grand Island, NY) to disrupt the cuticle and release the reproductive content. The various developing embryonic stages released from the two gonads (eggs, embryos, pre-MF, and stretched MF, deformed embryos) were assessed in a blinded fashion for their developmental stages using an inverted microscope and hemocytometer. A minimum of 200 developmental stages were counted from each female and the relative proportions of each stage as well as the % of each developmental stage per female worm in 6-9 worms per treatment group were calculated [START_REF] Gunderson | Pyrvinium Pamoate and Structural Analogs Are Early Macrofilaricide Leads[END_REF][START_REF] Ford | Functional Analysis of the Cathepsin-Like Cysteine Protease Genes in Adult Brugia Malayi Using RNA Interference[END_REF] . Release of MF ex vivo were analyzed using a Kruskal-Wallis test followed by Dunn's multiple comparison test. For the embryogram analyses, a two-way ANOVA was conducted with a Dunnett's multiple comparisons test where significance levels were determined based on comparisons with worms from vehicle treated animals. All statistical analyses were determined using Prism 8 version 8.2.0.

Transmission electron microscopy of female Brugia.

B. pahangi adult females from necropsy were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde buffered with 0.1 M sodium cacodylate, worms were cut into small pieces while in the fixative and stored at 4°C until processed as described [START_REF] Gunderson | Pyrvinium Pamoate and Structural Analogs Are Early Macrofilaricide Leads[END_REF] . Samples were washed with 0.1 M sodium cacodylate, and a secondary fixation was done with 1% osmium tetroxide for 1 hour. Following another wash with 0.1 M sodium cacodylate, samples were dehydrated in increasing ethanol concentrations (30-100%) and washed two times with propylene oxide for complete desiccation. Dehydrated samples were embedded in BEEM capsules using Spur low viscosity embedding kit (Electron Microscopy Sciences). Blocks were polymerized at 70°C for 8 hours. Ultrathin sections were collected on nickel formvar/carbon coated 100 mesh grids and contrasted with Uranyless (Electron Microscopy Sciences) and lead citrate. Sections were imaged on a Tecnai G2 Spirit TEM equipped with an AMT camera.

RNA-seq of Brugia pahangi and Trichuris muris adult worms treated in vitro with 12a

Adult female B. pahangi and adult female T. muris were treated with 100 µM of 12a and 1% DMSO (vehicle control) for 2-3 hours in vitro and stored at -80 ˚C. Four replicates of B. pahangi (2-3 individual worms per sample) and T. muris (1 worm per sample) were then subjected to RNA extraction. Worms were homogenized using 1.5 mm zirconium beads (Benchmark scientific, D1032-15) and BeadBug6 (Benchmark scientific, D1036, Sayre-ville, NJ, USA) and total RNA was isolated using Trizol (Invitrogen, Waltham, MA, USA). cDNA libraries were prepared from RNA samples with poly(A) enrichment and the processed cDNA was sequenced on the Illumina NovaSeq S4 platform (paired-end 150bp reads).

RNA-seq analysis and functional enrichment tests

Sequencing produced an average of 42 million reads per sample. An average of 93.1 % and 94.9 % of reads were mapped to the B. pahangi genome assembly (PRJEB497, WBPS13) and T. muris (PRJEB126, WBPS16) respectively using STAR and quantified per gene using featureCounts. Relative gene expression was calculated using "fragments per kilobase per million reads" (FPKM) with normalization per gene per sample. Fold changes and differential expression significance values (relative to DMSO controls) were calculated using DESeq2 (version 1.34.0; default settings, FDR ≤ 0.05). Functional annotations for all B. pahangi and T. muris genes were assigned using annotations from InterProScan v5.42 to identify gene ontology classifications and InterPro functional domains, and GhostKOALA v2.2 to identify KEGG annotations. Enriched KEGG pathways among differentially expressed gene (DEG) sets were identified using WebGestaltR (minimum 2 genes per pathway, FDR ≤ 0.05 threshold for significance).

Protein sequence-based comparison to identify orthologous genes

Protein sequence data were downloaded from WormBase Parasite: B. pahangi (PRJEB497), T. muris (PRJEB126) and Caenorhabditis elegans (WBcel235). In addition, for protein sequence comparison, yeast genomes were also downloaded from Ensembl release 55: S. cerevisiae (R64-1-1), C. albicans (GCA000182965v3) and A. fumigatus (Af293). Best bi-directional hits were identified based on protein sequence alignment of the longest isoforms per gene of parasitic nematodes (B. pahangi and T. muris, each) against 4 comparative species including the 3 yeast species (S. cerevisiae, C. albicans, and A. fumigatus) and C. elegans using Diamond blastp (v2.0.6.144). This alignment was performed twice for each pairwise combination of the two parasitic nematodes and the 4 comparative species. Once with the parasitic nematode being the query and the comparative species being the subject, and again with the comparative species being the query and the parasitic nematode being the subject. Cases in which a pair of proteins were each other's top scoring hit were flagged as being reciprocal hits. Gene symbols and descriptions were harvested from the BioMart instance hosted at WormBase Parasite and were used to annotate the results.

Chemogenomic Studies

Frozen aliquots of Yeast Deletion Heterozygous Diploid Pools (Cat. no.95401.H4Pool) were recovered for 10 generations and logarithmically growing cells were diluted to a final OD600 of 0.06 (=10^5 cells/ml) in YPD containing 1% DMSO or compound. The compound was applied at a dose of 1 µM corresponding to 10%-20% wild-type growth inhibition. Cells were harvested after 20 generations of growth and frozen at -20°C for subsequent preparation of DNA. Genomic DNA was purified according to the Qiagen supplementary protocol 'Purification of total DNA from yeast using the DNeasy® Blood &Tissue Kit' (Qiagen). DNA quality was assessed via agarose gel electrophoresis and UV-Vis spectroscopy. For each compound three biological replicates were generated.

A two-step PCR protocol for efficient multiplexing of Bar-seq libraries was applied as previously described with the following modifications. In a first step, UPTAGs and DNTAGs from a single sample were amplified using the primers Illumina UPTAG Index and Illumina UPkanMX and Illumina DNTAG Index and Illumina DNkanMX in separate PCR reactions. Illumina UPTAG and Illumina DNTAG primers contain a 5bp sequence that uniquely identifies the sample. A complete list of primer sequences is provided in Table S1. Genomic DNA was normalized to 20ng/ml and 100ng were used as template for amplification of barcodes using KAPA Hifi HotStart Ready Mix (Roche), applying the following PCR program: 4 min at 95°C followed by 25 cycles of 10 sec at 95°C, 10 sec at 50°C, 10 sec at 72°C, and a final extension step of 2 min at 72°C. PCR products were confirmed on 2% agarose gels and purified using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel). Purified PCR products were quantified using the Quant-iT dsDNA kit (Invitrogen) and 60ng from each of the 15 different UPTAG libraries and, in a separate tube, 60 ng from each of the 15 different DNTAG libraries were combined. The multiplexed UPTAG libraries were then amplified using the primers P5 and Illumina UPkanMX, and the combined DNTAG libraries were amplified using the P5 and IlluminaDNkanMX primers (Table Sx) and the PCR program: 4 min at 95°C followed by

Supporting Information

The Supporting Information is available free of charge at XXXX. All data associated with this study are present in the paper or the Supporting Information.

-Stability studies (Fig. S1-S40), IC 50 graphs for B. pahangi and T. muris, worm burden, embryogram results and chemogenomic screen (Fig. S41-44), cytotoxicity results at 25, 50 and 100 µM on RPE-1 cells (Figure S45), List of primer sequences (Table S1). Complete synthetic route (Fig. S46), procedures and characterization of the compounds, copies of the IR (Fig. S47-S54) and NMR (Fig. S55-S128) spectra.

-Molecular formula strings.

Acknowledgments:

The


Florian Brunier-Coulin

Diogo C Barros
email: diogo.camello-barros@univ-amu.fr

Alexander Piqué

Marcus Hultmark

Pierre Dupont

Thermal response of a nanoscale hot-wire in subsonic and supersonic flows

Keywords: Hot-wire anemometry, Frequency response, Thermal response

A comprehensive characterization of the thermal response of nanoscale hot-wire probes is performed in both subsonic and supersonic flows. A constant current anemometer was designed for the measurement of the intrinsic thermal inertia of hot-wire probes. In particular, the nanoscale probe is considered with the effect of gold-plating on supporting structure of the targeted sensing element. Gold-plated nanoscale probes present a response time one order of magnitude smaller than conventional cylindrical hot-wire probes. Heat transfer simulations show that the temperature profile is considerably modified by the addition of a conductive metal layer, hence increasing the sensor's frequency response in both subsonic and supersonic flows. The increase of frequency response is finally exemplified by the numerical computation of the power spectral density of a turbulent flow signal without any electric compensation of the hot-wire signal.

Introduction

Hot-wire anemometry has become an ubiquitous tool for the investigation of turbulent flows since the pioneering work of [START_REF] King | On the convection of heat from small cylinders in a stream of fluid: Determination of the convection constants of small platinum wires with applications to hot-wire anemometry[END_REF]. The small dimension of the probes is associated with high spatial and temporal resolution, making them appropriate for the characterization of unsteady flows both in subsonic and supersonic applications [START_REF] Kovasznay | The hot-wire anemometer in supersonic flow[END_REF][START_REF] Comte-Bellot | Hot-wire anemometry[END_REF].

Conventional hot-wire probes consist of cylindrical tungsten or platinum wires with active length and diameter on the order of 1 mm and 5 µm, respectively. Novel fabrication methods in microelectromechanical systems (MEMS) have paved the way for the design of nanoscale thermal anemometry probes (NSTAP) targeting highly resolved measurements in a variety of flow conditions [START_REF] Bailey | Turbulence measurements using a nanoscale thermal anemometry probe[END_REF][START_REF] Vallikivi | Fabrication and characterization of a novel nanoscale thermal anemometry probe[END_REF][START_REF] Kokmanian | Development of a nanoscale hot-wire probe for supersonic flow applications[END_REF][START_REF] Le-The | Fabrication of freestanding pt nanowires for use as thermal anemometry probes in turbulence measurements[END_REF]. The deposition of controlled thin metal layers makes possible the design of smaller probes with a well defined geometry to increase both time and spatial resolution of turbulent flow measurements [START_REF] Bailey | Turbulence measurements using a nanoscale thermal anemometry probe[END_REF].

While the frequency response of the NSTAP probes operated in constant temperature anemometry (CTA) has been investigated in previous experiments [START_REF] Hutchins | A direct measure of the frequency response of hot-wire anemometers: temporal resolution issues in wall-bounded turbulence[END_REF][START_REF] Kokmanian | Heat transfer measurements of a nanoscale hot-wire in supersonic flow[END_REF], the intrinsic thermal inertia, and corresponding open loop response of such hot-wire probes have not been characterized systematically. In CTA anemometers, the probe resistance, hence its temperature, is kept constant by a feedback loop in the circuit that imply current variations to compensate for the flow unsteadiness and the probe's thermal inertia. In constant current (CCA) systems, a targeted electric current traverses the wire, whose electric resistance varies due to flow unsteadiness. A step change of flow velocity or temperature, for example, would imply a voltage response across the wire. The time lag of this response is associated with the intrinsic wire thermal inertia when no electrical compensation is applied within the circuit.

The possibility to combine materials with different thermal and electrical properties to modify the local response of the NSTAP probes has never been explored. In this work, this approach is implemented to optimize the intrinsic ther-mal response of nanoscale probes in both subsonic and supersonic flow conditions. The probes studied here are an extension of the nanoscale probes examined in [START_REF] Kokmanian | Heat transfer measurements of a nanoscale hot-wire in supersonic flow[END_REF], which have been proved robust to supersonic flow conditions. The time constant of the probes was measured by the use of square-wave tests in a CCA system specifically designed for this study. In section 2, we present the electronic characteristics of the CCA system, the details about the NSTAP probes and the employed aerodynamic flow configurations. Section 3 describes the square-wave tests and the time constant results. A heat transfer analysis is performed in section 4 to explain the probe temperature profile and main results. Finally, section 5 provides an example of a turbulent flow measurement and associated numerical compensation using a theoretical model employing the measured time response, followed by the concluding remarks in section 6.

Experimental setup

Anemometer system

A constant current anemometer (CCA) system was designed to measure the thermal response of the nanoscale probes. Figure 1 presents a sketch of all electrical components of the device. The hot-wire sensor, represented here by the resistance R w , is connected within a Wheatstone bridge composed of two top resistances R 1 = R 2 = 100 Ω and a third variable resistance R 3 in the range 0-100 Ω . This resistance was employed to set a target value of R w when the bridge is perfectly balanced. The bridge is supplied by a constant current generator connected to the main resistance R s = 12 Ω .

In order to generate periodic variations of the electrical current traversing the bridge, another resistance R s ′ = 3.5 Ω was installed with a MOSFET (metal oxide semiconductor field effect transistor) transistor. Supplying the transistor with a square-wave signal, thus short-circuiting the resistance R s ′ , allows one to modify periodically the current I o traversing the bridge. This method has been widely recognized to simulate a step change in flow velocity to determine the frequency response of hot-wire probes [START_REF] Kovasznay | The hot-wire anemometer in supersonic flow[END_REF][START_REF] Bruun | Hot-wire anemometry: principles and signal analysis[END_REF]. Indeed, the thermal response of the sensing element is driven by the time needed for saturation following a step change of velocity. This duration corresponds to the time required to reach the equilibrium between the heat transfer from the Joule effect in the sensing element and the heat exchange with the flow. This approach differs from the frequency response test in a CTA system, in which the wire signal is intrinsically compensated to maintain a constant probe temperature [START_REF] Hutchins | A direct measure of the frequency response of hot-wire anemometers: temporal resolution issues in wall-bounded turbulence[END_REF]).

For each measurement, by varying R 3 and balancing the bridge, we set the value of R w to obtain a known overheat resistance ratio a w = (R w -R e )/R e . Here, R e is the equilibrium resistance of the unheated wire in the flow. The procedure consists in adjusting the supply current while the equilibrium of the Wheatstone bridge is maintained by adjusting R 3 . The bridge voltage E xy is proportional to the variation of the probe resistance R w . In this configuration, the variation of the voltage corresponding to the response of the sensing element is measured along the power input of the hot wire. It implies a lower signal-to-noise ratio (SNR) when compared to the direct measurement of E w variations, but the equilibrium condition of the Wheatstone bridge allows a direct amplification of E xy with a differential amplifier, limiting the number of electronic components. In addition, as described in section 3.1, a phase average is conducted to highlight only the periodic variation of R w . During the square wave tests, E xy was monitored to record the time response of the sensor due to the step changes in the bridge traversing current.

It is important to note that the present anemometer does not apply any electrical compensation to the bridge voltage. Hence, the measured signal response corresponds to the intrinsic thermal lag of the probes.

Hot-wire probes

The nanoscale thermal anemometry probes (NSTAP) investigated in the current experiments are an extension of the sensors explored in [START_REF] Kokmanian | Heat transfer measurements of a nanoscale hot-wire in supersonic flow[END_REF]. In figure 2, we present a microscope image of the sensor's tip. It consists of a platinum nanoscale ribbon sensing element with length 30 µm and 2 µm wide. This sensing element is supported by two triangular platinum structures, which are held by longer platinum prongs with a total length of 4 mm supported by a silicon base. The thickness of the entire platinum structure is 300 nm. Further details about the fabrication steps and the full geometry of the sensor can be found in [START_REF] Kokmanian | Development of a nanoscale hot-wire probe for supersonic flow applications[END_REF]. The design of the sensors takes into account its operation in high-speed flows. Indeed, the continuous variation of the width of the triangular supports and the location of the sensing element at the most upstream position limit the effects of the existing shock waves in supersonic flow conditions.

Given that the entire platinum structure represents a unique electrical resistance, it is natural to inquire to what extent the triangular supports impact the thermal lag of the entire sensor. To quantify the role of this structure on the sensor's thermal response, layers of gold (Au) were superposed specifically over the triangular supports of the sensing element. The gold layer starts at the end of the sensing element and covers the supports until they reach a width of approximately 50 µm, where the heat generation is negligible due to the large cross section (see figure 3). In addition, the end of the gold layer coincides approximately with the silicon substrate that plays the role of a heat sink. In the following, two gold-plated configurations with gold thickness 200 nm and 360 nm will be considered together with the original platinum sensor. The gold was added on top of the 300 nm thick platinum structure.

The thermal response of a hot-wire probe depends directly on the electrical resistance of the sensor's body. Due to the more complex geometry of the NSTAP when com-pared to a conventional cylindrical hot-wire probe, it is difficult to estimate precisely the resistance of the entire platinum structure. Also, measuring the electrical resistance of the sensing element is very challenging due to its small dimension. An estimation of the sensor's resistance (see [START_REF] Kokmanian | Heat transfer measurements of a nanoscale hot-wire in supersonic flow[END_REF] for the details of the entire geometry) based on the resistance of each section R i = χ 0 l i /S i gives R w = 16 Ω , while the measured probe resistance at 20 • C was R w = 24.3 Ω . Here, χ 0 is the electrical resistivity at 20 • C, S i is the local cross-section and l i the corresponding length of the element. In the presence of 200 and 360 nm gold thicknesses, the estimated theoretical resistances are 13.7 and 12.9 Ω , while the measured resistances are R w = 22.3 Ω and R w = 21.2 Ω , respectively.

The addition of gold reduces the electrical resistance given the lower value of the gold electrical resistivity when compared to the platinum (see table 3). The difference between the estimated and measured values of R w is presumably due to the choice of the platinum resistivity χ 0 = 1.1 × 10 -7 Ω m. This value corresponds to the bulk resistivity of the platinum for relatively thick metal sheets. It is known that the electrical resistivity of platinum increases exponentially as the nanoscale thickness decreases. [START_REF] Féry | Etude de la variation de la résistivité des couches minces de platine en fonction de leur épaisseur et de l'influence des corps oxygénés sur de semblables resistances[END_REF] measured the origin of the resistivity growth for a thickness t = 348 nm, indicating that χ 0 should have a larger value for the current studied thickness t = 300 nm. This could explain the differences between the estimated and the measured values of R w . In the following, the values of both R e and R w used for the calculation of a w correspond to the resistance of the entire metallic structure (not only the sensing element).

Finally, a conventional cylindrical hot-wire probe was employed to provide a comparative basis of the thermal response. This probe is a tungsten wire of diameter 5 µm and length 800 µm soldered on classical Dantec prongs.

Flow configurations

Measurements of the NSTAP's thermal response time were performed in both subsonic and supersonic flow conditions. The subsonic configuration is a low-speed axisymmetric jet with exit nozzle diameter D 1 = 30 mm and jet maximum velocity equal to 5 m/s. Details about the flow field of this jet facility can be found in the experiments of [START_REF] Schreyer | Development of a dual-PIV system for high-speed flow applications[END_REF]. The probes were placed in the potential core of the jet at the centerline exit location.

Experiments in supersonic flow were performed in the continuous wind-tunnel at the IUSTI laboratory (Institut Universitaire des Systèmes Thermiques Industriels, Marseille). Measurements of the time constant were taken in the freestream of a 170 mm x 105 mm test section following a Mach 2 nozzle. The very low turbulence level in the free-stream together with controlled gas drying conditions are suitable for the operation of hot-wire probes. The flow stagnation pressure was varied from 0.2 to 0.8 bar.

Results

Response time measurements

Figure 4(a) presents a typical response of the NSTAP sensor to the square wave test, and the methodology to compute the response time τ. Due to inherent flow unsteadiness from one period to another, a phase-average response was computed over more than 1000 periods to obtain the exponential growth corresponding to the increase of the sensor resistance. The phase-averaged response is exemplified in figure 4(b).

It was clear from all the experiments that the wire's voltage follows a first order linear system response:

E = C 1 -e -t τ , (1)
where the time constant τ represents the thermal inertia of the probe. To determine this thermal response, an exponential fit of the phase-averaged signals was implemented using equation 1, as exemplified in figure 4(b). The difference between the fit obtained either by the growth or the decrease of the sensor's resistance gives a good estimation of the error on τ. In both subsonic and supersonic conditions, the errors were found to be 3-5%. Time constant measurements were performed for various overheat ratios between 0.06 and 0.8. Figure 5 depicts the evolution of the time constant for all sensors investigated. Noticeably, the response time of the nanoscale sensors is at least five times lower than the conventional cylindrical 5 µm-tungsten wire in both subsonic and supersonic flow configurations. Interestingly, the addition of gold layers monotonically decreases the NSTAP's response time down to τ = 20 µs. This lowest value of τ was obtained for the gold-plated nanoscale probe with a gold layer of 360 nm, corresponding to a time constant reduction by a factor of 30 when compared to a classical 5 µm cylindrical probe.

In table 1, we summarize the values of τ averaged over a w according to both the sensor and the flow configurations. We note that τ generally reduces for higher flow speeds, a fact that can be predicted from the unsteady heat equation over the probe [START_REF] Bruun | Hot-wire anemometry: principles and signal analysis[END_REF]. This reduction, however, is negligible for the gold-plated NSTAP sensors. A detailed knowledge of the sensing element's resistance would be crucial to understand why the response time remains unchanged for subsonic and supersonic flow conditions when the gold layers are added.

Another point of interest is the effect of the flow condition associated with the overheat ratio on the response time. Although limited measurements were performed for supersonic flow conditions, figure 6 depicts the response time for both the conventional cylindrical and a gold-plated NSTAP for varying flow speed (i.e. total pressure P 0 ) and overheat ratio a w . The 5 µm-probe presents a linear growth with a w for both flow speeds. It is interesting to note that for increasing a w , there is a decrease of the response time when the flow speed rises. Both the linear growth of τ with a w and the nonlinear decrease of τ with flow speed can be predicted using a simple heat transfer model of a cylindrical hot-wire probe [START_REF] Bruun | Hot-wire anemometry: principles and signal analysis[END_REF]. On the other hand, the gold-plated NSTAP probe presents no a w dependence within the measurement uncertainties, but a decrease of τ with flow speed. While this behavior would be beneficial for experimental purposes and robustness to a w variations, the physical reasons behind the difference between the cylindrical and the NSTAP probes remains to be elucidated.

Heat transfer analysis

In CCA operation without electronic compensation, the time constant is governed only by the thermal inertia of the sensor. The effect of the gold plating on the probe temperature profile is explored here using a heat transfer model and corresponding numerical simulations. In particular, we attempt to characterize how the temperature gradient changes in the vicinity of the sensing element.

Theoretical model

We consider a one-dimensional model along the sensor's length axis x following [START_REF] Bruun | Hot-wire anemometry: principles and signal analysis[END_REF]. The equilibrium between the electrical heat generated by the Joule effect Qe and the heat exchange by conduction Qc and convection Qh will be expressed for an incremental sensor element. The heat balance reads

d Qe = d Qh + d Qc + d Qs , ( 2
)
where Qs is the heat stored by the sensor. The generated heat by the traversing electric current I is given by: In this example, the wire was placed in supersonic flow conditions: M = 2 and P 0 = 0.8 bar, the time constant was found to be τ = 45.4 µs for the conventional platinum NSTAP sensor. d Qe = dR w I 2 = χ w (x, T (x))I 2 A(x) dx.

(3)

Here, χ w (x, T ) and A(x) are the local electrical resistivity and cross section of the sensor, respectively, while T (x) is the local wire temperature. The local resistivity χ w is modelled by introducing the thermal coefficient of resistance α 0 and the resistivity χ 0 at T O = T 293K :

χ w (x) = χ 0 {1 + α 0 [T (x) -T 0 ]}. (4)
The heat convection from the wire to the flow is governed by the heat transfer coefficient, h, acting on the external surface S w :

d Qh = h(x)S w [T (x) -T e ] dx.
(5)

We consider S w = 2[w(x) + t] which takes into account both the upper and lower surfaces of the rectangular structure of the sensor. Thermal conduction along the sensor is considered in the axial direction:

d Qc = -k w A(x) ∂ 2 T (x) ∂ x 2 dx (6)
and the heat storage is written as

d Qs = ρ w C p A(x) ∂ T (x) ∂t dx. (7)
In the equations above, the density and heat capacity of the sensor's material are respectively ρ w and C p ; the thermal conductivity is k w . The equilibrium temperature of the unheated wire is T e . Finally, the local width, the local crosssection and the sensor's thickness are w(x), A(x) and t, respectively

Numerical simulation

The stationary solution of equations (2-7) provides the temperature profile across the sensing element and its vicinity. The problem consists in solving the heat transfer equations accounting for the geometry and materials of both a conventional cylindrical wire and the NSTAP sensors, which are sketched in figure 7. A Neumann condition is applied at the center of the wire, and the equilibrium temperature T e is imposed at both extremities of the sensor. The equations were solved using the Finite Element Method (FEM), where the probe geometry was discretized in 1300 nodes. The sensor is surrounded by a low-speed air flow with free-stream velocity u = 5 m/s.

The convective heat transfer coefficient is modeled using a local Nusselt number:

h(x) = Nu k f ℓ(x) , (8)
where k f is the fluid thermal conductivity and ℓ(x) the characteristic length scale in the flow direction. For a cylindrical wire, we set ℓ(x) = d. For the ribbon shape of the NSTAP, we follow [START_REF] Kokmanian | Heat transfer measurements of a nanoscale hot-wire in supersonic flow[END_REF] who considered both the width w(x) and the thickness t. As the width w can be much larger than t and parallel to the fluid flow, we expect the most appropriate length scale ℓ(x) = w(x), which varies from 2 µm at the tip until approximately 50 µm at the vicinity of the sensing element (see figure 3 for the sensor's geometry).

The Nusselt number employed here follows the King's law Nu = A √ Re + B, with coefficients A = 0.43 and B = 0.32 empirically obtained for cylindrical hot-wires [START_REF] Mcadams | Measurements of the single-point and joint turbulence statistics of high sub-sonic jets using hot-wire anemometry[END_REF]. Here, the Reynolds number Re = ul(x)/ν is computed with the length scale ℓ(x) and the mean flow speed u of air with kinematic viscosity ν. Table 2 Thermal and physical properties of materials at 293 K. The numerical model was first validated with the cylindrical 5 µm-tungsten wire. The temperature distribution along the wire axis T (x) is shown in figure for an arbitrary overratio and electric power. The temperature profile agrees well with previous theoretical predictions examined [START_REF] Bruun | Hot-wire anemometry: principles and signal analysis[END_REF]. The mean temperature of the wire obtained numerically was ⟨T w ⟩ num = 516K. while the estimated temperature from the experiments was ⟨T w ⟩ exp = 478K. This difference might come from the experimental estimation that uses the measured wire resistance to compute the wire temperature:

Mat. C p [J.kg -1 .K -1 ] ρ w [kg.m -3 ] k w [W.m -1 .K -
⟨T w ⟩ exp = R w -R e R 0 α 0 + T e (9)
The model uses the resistivity of the wire to calculate the wire temperature that is integrated over the sensor's length.

To understand the effect of gold-plating on the heat transfer across the wire, simulations were performed for the NSTAP sensors matching the experimental conditions I = 10 mA for the platinum probe and I = 12 mA for the 360 nm gold plated sensor. These values correspond to the experiments conducted in the potential core of a jet with maximum flow speed u = 5 m.s -1 , coresponding to R w I 2 ≃ 3.8 mW and a w ≃ 0.2. For simplicity, only one half of the sensing element is solved as sketched in figure 7.

Figure 9 depicts the temperature profile T (x) along the center-axis of the NSTAP probes. To better contrast the ef-

NSTAP body

Fig. 9 Comparison of the numerical profile T (x) -T e normalized by the mean temperature of the sensing element ⟨T w ⟩ -T e along the half length of the sensor with and without gold plating. The dashed line represent the limit between the ribbon-shaped sensing element and the supporting triangular structure. fects of gold on the temperature profile, we normalize T (x)-T e by the average temperature of the sensing element (i.e. for 0 ≤ x ≤ 15 µm for the half-sensor) subtracted by T e . Without the presence of the gold layer, the curve presents a substantial sensor heating along the triangular structure (15 ≤ x ≤ 100 µm), which is highly attenuated by the presence of the 360 nm-metal layer. gold-plating mostly focuses the temperature gradient the sensing element which directly reduces the response time of the sensor.

It is interesting to inquire to what extent this temperature profile is associated with end-conduction effects of the probe. For a cylindrical hot-wire, these effects can be quantified using the Γ parameter proposed by [START_REF] Hultmark | A new criterion for end-conduction effects in hot-wire anemometry[END_REF], which is defined based on the length-to-diameter ratio, the resistance ratio a = R w /R e , the Nusselt number Nu, the wire and the fluid thermal conductivity k w and k f , respectively. For the nanoscale ribbon geometry investigated here, the parameter assumes the following form [START_REF] Byers | Theoretical and experimental investigations of similarity solutions in turbulent flows[END_REF]:

Γ = l w 2a k f k w w + t t Nu (10)
where l, w and t stand for the length, the width and the thickness of the ribbon sensing element. Preliminary estimations of the Nusselt number reported by [START_REF] Kokmanian | Heat transfer measurements of a nanoscale hot-wire in supersonic flow[END_REF] indicate Nu ∼ O(50), which would lead to a Γ pa-rameter insufficiently small to avoid end-conduction effects.

An extensive characterisation of the end-conduction effects for the present nanoscale geometry is hence necessary in future work, taking into consideration the exact values of the sensing element resistance, in spite of the entire probe impedance.

To localize the heat transfer on the sensing element, it is necessary to reduce the electrical heat production of the ribbon structure. As the heat production is proportional to R w I 2 , one would consider to reduce the local resistivity χ by adding a parallel conductor, here the gold-layer with thickness similar to the platinum structure. The gold-layer reduces the global resistivity by a factor 2χ Au /(χ Pt + χ Au ) ≃ 0.33. In addition, the thermal conductivity of the sensor is increased by a factor of almost 3 reducing the temperature variation between both extremities of the triangular supports at a constant heat production. The properties of all materials are presented in tables 2 and 3.

Numerical compensation of a turbulent flow signal

The addition of gold to the supporting structure focuses the temperature gradient into the sensing element, lowers the temperature of the stubs, hence reducing the response time of the probe. The decrease of the response time by an order of magnitude is associated with an equivalent increase of the sensor's frequency response. While in conventional operation of hot-wire probes in both CTA and CCA systems the signals are compensated with a parallel electronic circuit, one could inquire to what extent numerical compensation using the analytical first-order transfer function would provide reliable power spectral density results for the use of nanoscale probes in turbulent flows.

Given that the phase-averaged response signals show unambiguously the first-order nature of the sensor's response, the probe transfer function can be modeled as a low-pass filter. This is governed by the well-known transfer function:

H( jω) = 1 1 + j ω ω o . ( 11
)
ω is the angular frequency and ω 0 is the cut-off angular frequency defined as ω 0 = 1/τ. The resulting cut-off frequency is given by f 0 = ω 0 /2π. The transfer function gain G is the magnitude ∥H( jω)∥. Using the conventional frequency variable f = ω/2π, the frequency-dependent gain is computed as:

G( f ) = 1 1 + ( f / f 0 ) 2 (12)
For a given power spectral density of a turbulent flow signal acquired without compensation, the compensated power spectral density S i is computed by the ratio between the measured power spectral density S o and the squared gain G( f ) 2 . To experimentally test the numerical compensation of a turbulent flow signal, the gold-plated nanoscale probe was placed in a high-subsonic jet issuing from a nozzle with an exit internal diameter D i = 4 mm. The estimated jet flow speed based on static and stagnation pressure is v = 105 m/s. During the measurements, the probe was placed within the jet shear layer approximately four diameters downstream of the nozzle's outlet.

The power spectral density of hot-wire signals in this location should exhibit the universal k -5/3 decay for more than one decade (Proenc ¸a et al, 2019). Figure 10 presents the power spectral density (PSD) of both the original and the numerically compensated signals. It is important to note that the original signal was acquired using the CCA anemometer system described in section 2.1 without any compensation. Hence, in the PSD of the original signal, the rapid decay starting at approximately 4 kHz is due to the intrinsic thermal response of the nanoscale probe. This cut-off frequency agrees well with the response time τ = 30 -40 µs presented in figure 5 for the 200 nm gold-plated probe. The compensated PSD shows a compelling k -5/3 decay until 30 kHz where turbulent flow dissipation increases the decay rate until 100 kHz, which is the cut-off frequency of the compensated signal limited by the signal's noise originated from a digital converter of N = 16 bits.

Summary and conclusions

A constant current anemometer was built to characterize systematically the response time of nanoscale probes under multiple aerodynamic and overheat configurations. The experimental results show that the NSTAP probes present a response time at least one order of magnitude smaller than conventional cylindrical hot-wire probes. The associated frequency response shows similar features whether the probe is placed in subsonic or supersonic flow conditions, thus confirming the possibility of using the nanoscale probes in a variety of Mach number flows. It was shown that the time constant of the NSTAP sensors is insensitive to the overheat ratio, which is in contrast to the behavior of conventional hot-wire probes.

To explore the effects of the supporting structure on the sensor's frequency response, a gold layer was added at the vicinity of the sensing element over the original platinum structure. It demonstrates the possibility to control the response time by gold-plating, which focuses heat transfer along the nanoscale sensing element. The addition of gold-plating at the vicinity of the sensing element deserves further detailed investigations to find an optimal configuration considering both the response time and the end-conduction effects.

Finally, numerical compensation of acquired CCA signals without any electric compensation was successfully implemented in a high-speed turbulent flow. Due to the higher frequency response of the probe when compared to conventional hot-wires, the power spectral density of a turbulent flow signal can be computed over a large range of frequencies, which are limited only by the intrinsic noise of the acquisition system. In the tested experiments, a gold-plated nanoscale probe covers a spectral range until 100 kHz, paving the way for its application in a variety of flow conditions without the need of any sophisticated anemometer system with electric compensation.

Fig. 1

 1 Fig. 1 Sketch of the constant current anemometer system.

Fig. 2

 2 Fig. 2 Microscope photography of the NSTAP probe's tip consisting of a 300 nm-platinum layer (Pt) deposited on a silicon (Si) wafer.

Fig. 3

 3 Fig. 3 Gold-plated NSTAP probe. The gold layer thicknesses considered are 200 nm and 360 nm.

  Fig. 4 a) Response of the NSTAP sensor (blue) to the square wave signal (yellow) due to step electrical current changes of 3.3 mA around the mean current of I o = 25.1 mA. b) Phase-averaged wire voltage response and first-order exponential fit to compute the time constant τ using equation 1.In this example, the wire was placed in supersonic flow conditions: M = 2 and P 0 = 0.8 bar, the time constant was found to be τ = 45.4 µs for the conventional platinum NSTAP sensor.

Fig. 5 Fig. 6

 56 Fig.5Time constant in subsonic (left) and supersonic (right) flow conditions as a function of the overheat ratio a w for the conventional 5 µm cylindrical probe (•), the conventional platinum NSTAP (+), the NSTAP with a 200 nm gold layer (△) and the NSTAP with a 360 nm gold layer (▽).

Fig. 7

 7 Fig. 7 Geometry of the cylindrical tungsten wire (d =5 µm, top) and the nanoscale probe (bottom) considered in the numerical simulations.

Fig. 8

 8 Fig.8Temperature profile along the 5 µm-tungsten wire computed from the numerical model (red curve) and its corresponding mean value ⟨T w (dashed). The black dashed line represents the temperature estimated from the experimental data. For both cases, a w = 0.5 and R w I 2 = 24 mW.

Fig. 10

 10 Fig. 10 Spectrum of NSTAP with a gold layer of 200 nm. Red dashed lines represent the dissipation range and the cut-off frequency after compensation.

Table 1

 1 Averaged τ (µs) over a w in subsonic and supersonic flows.

	Sensor	Subsonic Supersonic
	5 µm cylindrical wire	678.7	526.3
	NSTAP platinum	98.3	54.6
	NSTAP platinum + 200 nm of gold 35.9	34.2
	NSTAP platinum + 360 nm of gold 19.6	19.5

  1 ]

	Pt	130	21450	71.6
	Au	128	19300	320
	W	203	15630	174

Table 3

 3 Electric properties of the sensor's materials at 293 K.Mat. χ 0 10 -6 [Ω .m] α 0 10 -3 [K -1 ]

	Pt	0.1111	3.92
	Au	0.0222	3.71
	W	0.1124	3.68

Acknowledgements The authors gratefully thank Dr. Katherine Kokmanian for her advices during the preparation of the NSTAP probes.

Funding

This work was supported by AFOSR/EOARD under the grant award FA8655-20-1-7040 monitored by Dr. Douglas R. Smith.

Availability of data and materials

Not applicable.

Ethical Approval

Not applicable.

Competing interests

The authors declare no conflict of interest.

Authors' contributions

All authors wrote and reviewed the manuscript.