Promising Drug Candidates and New Strategies for Fighting Against the Emerging Superbug Candida Auris

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Review Promising Drug Candidates and New Strategies for Fighting against the Emerging Superbug Candida auris

Muriel Billamboz 1,2,*, Zeeshan Fatima 3, Saif Hameed 3 and Samir Jawhara 4,*

1 Inserm, CHU Lille, Institut Pasteur Lille, Université Lille, U1167—RID-AGE—Facteurs de Risque et Déterminants Moléculaires des Maladies liées au Vieillissement, F-59000 Lille, France 2 Junia, Health and Environment, Laboratory of Sustainable Chemistry and Health, F-59000 Lille, France 3 Amity Institute of Biotechnology, Amity University Haryana, Manesar, Gurugram 122413, India; [email protected] (Z.F.); [email protected] (S.H.) 4 UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Centre National de la Recherche Scientiﬁque, INSERM U1285, University of Lille, F-59000 Lille, France * Correspondence: [email protected] (M.B.); [email protected] (S.J.)

Abstract: Invasive fungal infections represent an expanding threat to public health. During the past decade, a paradigm shift of candidiasis from Candida albicans to non-albicans Candida species has fundamentally increased with the advent of Candida auris. C. auris was identiﬁed in 2009 and is now recognized as an emerging species of concern and underscores the urgent need for novel drug development strategies. In this review, we discuss the genomic epidemiology and the main virulence factors of C. auris. We also focus on the different new strategies and results obtained during the past decade in the ﬁeld of antifungal design against this emerging C. auris pathogen yeast, based on a medicinal chemist point of view. Critical analyses of chemical features and physicochemical descriptors will be carried out along with the description of reported strategies.

Citation: Billamboz, M.; Fatima, Z.; Keywords: Candida auris; antifungal drugs; repurposed drugs; combinatorial therapy; natural Hameed, S.; Jawhara, S. Promising compounds; nanoparticles Drug Candidates and New Strategies for Fighting against the Emerging Superbug Candida auris. Microorganisms 2021, 9, 634. https://doi.org/10.3390/ microorganisms9030634 1. Introduction First identified in 2009 from the ear canal of a patient in Japan [1], C. auris is now recog- Academic Editor: Eric Dannaoui nized as an emerging species of concern [2,3], and more and more publications are related to C. auris studies (Figure1). When searching for “ Candida auris” key words on Scifinder, Received: 25 January 2021 fewer than 1000 reports have been compiled, most of them during the two last years, Accepted: 8 March 2021 proving the acceleration of research and clinical studies around this emerging pathogen. Published: 18 March 2021 Its emergence is defined by the occurrence of C. auris infections in a dozen of countries all around the world. In Europe, since 2015, sporadic epidemics have been reported in Publisher’s Note: MDPI stays neutral Spain [4], United-Kingdom [5], Germany, or Norway [6], for example. Simultaneously, with regard to jurisdictional claims in candidiasis caused by C. auris were reported in Korea [7], South Korea [8], India [9–11], published maps and institutional affil- South-Africa [12], or Kowait [13]. In the same period, the United States were also af- iations. fected [14–16]. This human pathogen is associated with severe invasive infections with high mortality rates ranging from 35 to 72% [8,12,17,18]. Since June 2016, governmental in- stitutions (Centers for Disease Control and Prevention (CDC), European Centre for Disease Prevention and Control (ECDC), World Health Organization (WHO), Pan American Health Copyright: © 2021 by the authors. Organization (PAHO), National institute for Communicable Diseases (NICD) ... ) have Licensee MDPI, Basel, Switzerland. issued clinical alerts to health care facilities and provided interim guidelines for clinical This article is an open access article management, laboratory testing and infection control of C. auris [19,20]. This fungus poses distributed under the terms and significant challenges to microbiologists and clinicians because of its frequent multidrug conditions of the Creative Commons resistance; high transmissibility and severe outcomes coupled with misidentification by Attribution (CC BY) license (https:// standard biochemical identification systems such as Vitek 2 [21]. creativecommons.org/licenses/by/ 4.0/).

Microorganisms 2021, 9, 634. https://doi.org/10.3390/microorganisms9030634 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 634 2 of 40 Microorganisms 2021, 9, x 2 of 42

350

300

250

200 year 150

100

Number of publications per of publications Number 50

0 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020

FigureFigure 1.1. Evolution of of annual annual reports reports dealing dealing with with C.C. auris auris studiesstudies (Scifinder (Sciﬁnder search, search, keykey word word Candida auris, Data Compiled on 31 December 2020). Candida auris, Data Compiled on 31 December 2020).

Moreover,Moreover, the the widespread widespread and and prolonged prolonged use use of of available available antifungal antifungal drugs drugs has has given given riserise to to multidrug multidrug resistance resistance that that alters alters the therapeuticthe therapeutic available available options. opti Itons. has It been has reported been re- thatported nearly that 90% nearly of C .90%auris ofisolates C. auris exhibit isolates resistance exhibit resistance to fluconazole, to fluconazole, around 30% around resistance 30% toresistance amphotericin to amphotericin B, and less B, thanand less 5% than resistance 5% resistance to echinocandins to echinocandins [22,23]. [22,23]. Under Under such compellingsuch compelling circumstances, circumstances,C. auris C. displaysauris displays all the all features the features of a “superbug”of a “superbug” and effortsand ef- areforts ongoing are ongoing to identify to identify newtherapeutic new therapeutic alternatives alternatives to fight to againstfight againstC. auris C. infections.auris infec- Aftertions. introducing After introducingC. auris C.genomic auris genomic epidemiology epidemiology and virulence and viru factors,lence thisfactors, review this aims review at compilingaims at compiling the different the strategiesdifferent strategies and results and obtained results during obtained the pastduring ten the years past in ten the fieldyears ofin antifungal the field of research antifungal against research emerging againstC. emerging auris from C. a medicinalauris from chemista medicinal point chemist of view. point To summarize,of view. To summarize, six complementary six complementary strategies have strate beengies developed, have been developed, from the most from attractive the most toattractive the less reported:to the less (i) reported: repurposing (i) repurposin of drugs;g (ii) of evaluation drugs; (ii) ofevaluation combination of combination drugs; (iii) discoverydrugs; (iii) of discovery new drug-candidates; of new drug-candidates; (iv) traditional (iv) traditional medicines medicines and natural andcompounds; natural com- (v)pounds; metal, (v) metalloids metal, metalloids and complexes; and complexes; (vi) others (vi) approaches others approaches including including nanoparticles nanopar- and irradiation.ticles and irradiation. Results and Results data will and be data analyzed will be from analyzed a chemical from approach a chemical based approach on chemical based featureson chemical and physicochemicalfeatures and physicochemical descriptors suchdescriptors as lipophilicity such as lipophilicity (expressed by(expressed logP) and by topologicallogP) and polartopological surface polar area (TPSA)surface inarea order (TPSA) to establish in order a comparison to establish between a comparison classes be- of compoundstween classes and of definecompounds potential and common define potential pharmacophoric common moieties.pharmacophoric moieties.

2.2.Candida Candida aurisaurisGenomic Genomic Epidemiology Epidemiology and and Virulence Virulence Factors Factors 2.1. Global View 2.1. Global View C. auris is taxonomically placed as a close relative to the Candida haemulonii since its C. auris is taxonomically placed as a close relative to the Candida haemulonii since its discovery in 2009 from the ear canal of a patient in Japan [1]. C. auris was isolated in all discovery in 2009 from the ear canal of a patient in Japan [1]. C. auris was isolated in all continents except Antarctica. Munoz et al. found that four of the five clades of C. auris are continents except Antarctica. Munoz et al. found that four of the five clades of C. auris are genetically related to other Candida species including C. haemulonii, C. duobushaemulonii, genetically related to other Candida species including C. haemulonii, C. duobushaemulonii, and C. psuedohaemulonii [24]. These five genetically distinct clades of C. auris correspond to: Cladeand C. I frompsuedohaemulonii India and Pakistan [24]. These (South five Asian), genetically Clade distinct II from Japanclades (Eastof C. Asian),auris correspond Clade III fromto: Clade South I from Africa India (African), and Pakistan Clade IV (South from VenezuelaAsian), Clade (South II from American) Japan (East and most Asian), recently Clade aIII potential from South Clade Africa V from (African), Iran [22 Clade,25]. OverIV from the Venezuela past ten years, (SouthC. Am auriserican)has been and isolatedmost re- acrosscently all a majorpotential continents, Clade V including from Iran elsewhere [22,25]. Over in Asia, the Africa, past ten North years, and C. South auris America,has been Australia,isolated across Europe, all andmajor the continents, Middle East. including elsewhere in Asia, Africa, North and South America,Outbreaks Australia, in United Europe, States and have the Middle been reported East. to different clades of C. auris that are introducedOutbreaks from in other United continents. States have In terms been ofreported the analysis to different of whole-genome clades of C. auris sequencing that are studies,introduced Chow from et al. other found continents. that the 133 In clinicalterms of isolates the analysis of C. auris of whole-genomeidentified in United-States sequencing betweenstudies, 2013Chow and et al. 2017 found were that related the to133 South clinical Asian, isolates South of American,C. auris identified African, in and United- East AsianStates isolatesbetween but 2013 unexpectedly, and 2017 were only related 7% of to theseSouth clinical Asian, SouthC. auris American,isolates wereAfrican, identi- and fiedEast from Asian patients isolates with but unexpected clear evidencely, only of being 7% of acquired these clinical through C. auris health-care isolates exposureswere iden- abroadtified from [16]. patients Outbreaks with in clear Europe evidence were also of being attributed acquired to recent through spread health-care from other exposures continents.abroad For [16]. example, Outbreaks the firstin Europe case of wereC. auris alsoinfection attributed in Franceto recent has spread been reportedfrom other in conti- 2018 nents. For example, the first case of C. auris infection in France has been reported in 2018

Microorganisms 2021, 9, 634x 3 3of of 42 40

fromfrom a a patient patient who who travelled travelled in in India and Iran two months before his hospitalization in FranceFrance [26]. [26]. C.C. auris auris isis believed believed to to have have been been misidentified misidentified as as C.C. haemulonii onon several occasions, occasions, suggestingsuggesting that that C.C. auris auris hashas likely been circulating as a human fungal pathogen before 2009.2009. However, However, retrospective retrospective analysis of the SENTRY Antifungal Surveillance collection ofof over over 15,000 15,000 CandidaCandida isolatesisolates from from 135 135 participating participating medical medical centers centers in in North North America, America, Europe,Europe, Latin America,America, andand the the Asia-Pacific Asia-Pacific regions regions since since 1997 1997 shows shows no misidentificationsno misidentifica- tionsof C. of auris C. aurisbefore before 2009 [200927]. [27].

2.2.2.2. Virulence Virulence Factors Factors of of C. C. auris auris LikeLike C.C. albicans albicans,, C.C. auris auris expressesexpresses several several virulence virulence fact factorsors that that contribute contribute to to patho- patho- genesisgenesis including including the the transition transition between between blas blastoconidiatoconidia and and filamentous filamentous forms, hydrolytic enzymeenzyme production, production, thermotolerance, biofilm/ad biofilm/adhesionhesion to to host host cells, cells, osmotolerance, osmotolerance, fila- fila- mentation,mentation, and and phenotypic phenotypic switching (Figure 22).).

FigureFigure 2. 2. SchematicSchematic overview overview of ofC.C. auris auris virulencevirulence factors. factors. Stress Stress resistance, resistance, hydrolytic hydrolytic enzyme enzyme production,production, thermotolerance, bioﬁlm/adhesionbiofilm/adhesion toto hosthost cells,cells, osmotolerance,osmotolerance, ﬁlamentation, filamentation, and and white whiteand opaque and opaque switching switching are important are important virulence virulence traits intraitsC. auris. in C. auris.

InIn contrast contrast to to other other close close relatives relatives including including C.C. haemulonii haemulonii or orC. C.pseudohaemulonii pseudohaemulonii, C., aurisC. auris is ableis able to togrow grow at at42 42 °C◦ C[[1].1 ].The The thermotolerance thermotolerance of of C.C. auris auris contributescontributes to to persist againstagainst the the high high fever fever response response and and causes causes an an invasive invasive candidemia candidemia [28]. [28]. Casadevall Casadevall et et al. al. suggestedsuggested that that the the thermotolerance thermotolerance of ofC. C.auris auris is relatedis related to climate to climate change change and global and global tem- peraturetemperature changes changes supporting supporting the thehypothesis hypothesis that that C. C.auris auris is isthe the first first example example of of a a new new pathogenicpathogenic fungus emerging from human-induced global warming [[29].29]. C.C. auris auris isis also also osmotolerant osmotolerant and and known known to to survive survive at at high high salt salt concentrations, concentrations, which which mightmight enable itit toto survivesurvive environmentally environmentally including including hypersaline hypersaline desert desert lakes, lakes, salt-evaporat- salt-evap- oratinging ponds, ponds, or tidalor tidal pools pools (Figure (Figure2)[ 2)28 [28].]. C. C. auris aurisis is able able to to survive survive onon humanhuman skinskin and environmentalenvironmental surfaces surfaces for for several several weeks weeks and and can can even tolerate being exposed to some commonlycommonly used used disinfectants disinfectants [30]. [30]. InIn terms terms of of filamentation filamentation of of C.C. auris auris, Yue, Yue et al. et showed al. showed that thatC. aurisC. auriscan switchcan switch to three to distinctthree distinct cell types cell including types including typical typical yeast, yeast,filamentation-competent filamentation-competent (FC) yeast, (FC) and yeast, a fila- and mentousa filamentous form form[31]. [Of31 ].note, Of note, typical typical yeast yeast cells cells are filamentation-incompetent are filamentation-incompetent while while FC yeastFC yeast and and filamentous filamentous cells cells are are filamentation-competent filamentation-competent under under specific specific inin vitro vitro culture conditionsconditions [31]. [31]. Furthermore, Furthermore, the the difference difference between between the typical typical yeast yeast and and filamentous filamentous cell typestypes was was variable variable among among the the expression expression levels levels of of metabolism genes. In In particular, particular, both

Microorganisms 2021, 9, 634 4 of 40

Krebs cycle- and glycolytic pathway-associated genes were downregulated in filamentous C. auris cells while fatty acid metabolism-related genes were upregulated, supporting that C. auris employs different metabolic modes to adapt to its host to reap the overall benefits of its commensal and pathogenic lifestyle [31]. Additionally, some strains of C. auris do not produce pseudohypae and show a decreased ability to assimilate different carbon sources (galactose, l−sorbose, cellobiose, l−arabinose, ethanol, glycerol, salicin, or citrate) [1]. Another morphological switch implicates the interconversion between white and opaque forms of C. auris [32]. Opaque yeast cells are more frequent colonizers in skin infections than white cells, whereas white yeast cells are much virulent in systemic infections than opaque cells [33,34]. Bentz et al. showed that C. auris phenotypic switching is regulated by WOR1 that control phenotypic switching in C. auris [33]. Like C. albicans, C. auris can produce the extracellular hydrolytic enzymes that are recognized as an important virulence trait including proteinases, hemolysins, lipases and phospholipases. Regarding secreted aspartyl proteinases (SAPs), that are one of the most significant extracellular enzymes produced in Candida species [35], SAPs contribute to degradation of host tissue by providing nutrients for pathogen propagation and promote the inactivation of host antimicrobial peptides, the evasion of the immune responses and the induction of inflammatory mediator release from host cells [36]. In terms of the difference of SAP activity between C. auris and C. albicans, C. auris is able to maintain high SAP activity at 42 ◦C when compared to that of C. albicans supporting that C. auris maintains its pathogenicity at higher temperatures [36]. Another important group of lytic enzymes are the lipases and phospholipases that are involved in the host damage, immune evasion and biofilm formation [37,38]. In contrast to C. albicans, the production of C. auris lipases or phospholipases appears to be decreased and strain-dependent although C. auris and C. albicans share the same quantity of lipase encoding genes in the genome [39]. Hemolysin is an exotoxin that is capable of lysing red blood cells as well as nucle- ated cells and different pathogenic Candida species including C. auris display hemolysin activity [40]. C. auris strains isolated from hospital infections exhibit a high production of hemolysin when compared to those from environmental sources suggesting that hemolysin activity is involved in C. auris virulence factors (Figure2)[41]. In terms of stress sensitivities of C. auris, Hog1-related stress-activated protein kinase is an important virulence trait for fungal survival against host-imposed stresses and are highly required for the pathogenicity of many fungal pathogens during infection [42]. Day et al. showed that Hog1 is involved in regulating stress resistance, cell morphology, aggregation, and virulence in C. auris [43]. With regard to biofilm formation, in contrast to C. albicans which forms the heteroge- neous architecture of biofilms combined with blastoconidia and hyphae embedded within the extracellular matrix, C. auris produces thin biofilms composed mostly of blastoconidia and occasionally pseudohyphae embedded within very limited extracellular matrix [40]. In- terestingly, these C. auris biofilms display lower susceptibility against antifungals including polyenes, azoles, echinocandins and chlorhexidine when compared to those of C. albicans suggesting other mechanisms to be more important for this antifungal-resistant biofilm than the reduced biomass of C. auris or limited extracellular matrix [43–45]. Additionally, adhesion plays a key role in C. auris virulence and biofilm formation. Agglutinin-like sequence (ALS) proteins, in particular Als3, are involved in C. auris adherence [46]. Singh et al. showed that sera containing anti-Als3 antibodies prevent C. auris biofilm formation supporting an important role of Als3 in biofilm formation [47]. Knowing the specificity of C. auris and its virulence factors, different strategies are conducted to fight this emerging superbug. MicroorganismsMicroorganisms2021 2021,,9 9,, 634 x 55 of 4042

3.3. Repurposing Drugs 3.1.3.1. Drugs: In Vitro Screening RepurposingRepurposing ofof drugs drugs is is an an attractive attractive pathway pathway to discoverto discover new new applications applications to already to al- developedready developed and filed and drugs. filed Drugdrugs. repositioning Drug repositioning could help could spare help substantial spare substantial costs linked costs to newlinked drug to discoverynew drug anddiscovery development. and development. It is also highly It is interestingalso highly because interesting it involves because the it useinvolves of de-risked the use compounds,of de-risked avoidingcompounds, high avoidi attritionng high rate [attrition47,48]. Therefore, rate [47,48]. this Therefore, strategy hasthis beenstrategy applied has been for repurposing applied for ofrepurposing drugs against of drugsC. auris against. It is notableC. auris. that It is the notable reported that studiesthe reported have beenstudies mainly have conductedbeen mainly in conducted vitro unless in specified. vitro unless specified. InIn thatthat context,context, thethe PrestwickPrestwick ChemicalChemical librarylibrary®®,, aa repurposingrepurposing librarylibrary ofof 12801280 smallsmall mainlymainly off-patentoff-patent molecules,molecules, hashas been screened several times by different research groups. Microorganisms 2021, 9, x 9 of 42 InIn 2018,2018, WallWall etet al.al. identified,identified, fromfrom thisthis library,library, ebselenebselen asas aa repositionablerepositionable moleculemolecule [[49].49]. EbselenEbselen isis aa synthetic synthetic organoselenium organoselenium drug drug molecule molecule with with antioxidant, antioxidant, anti-inflammatory, anti-inflamma- andtory, cytoprotective and cytoprotective activity activity (Figure (Figure3). It exhibited 3). It exhibited 100% inhibition 100% inhibition of growth of of growthC. auris ofat C. a concentrationauris at a concentration of 2.5 µM. of In 2.5 addition, µM. In addition, ebselen also ebselen inhibited also inhibited the formation the formation of biofilm of andbio- N wasfilm definedand was as defined a broad-spectrum as a broad-spectrum antifungal, antifungal, not only activenot only on activeCandida onspecies, Candida but species, also againstbut also a against variety a of variety medically of medically important important fungi, including fungi, including yeasts and yeasts molds. and molds. O Zotepine Antipsychotic 59 63 56

Cl S

FigureFigureGowri 3.3. StructuresStructures et al. studied ofof ebselenebselen sertraline, andand alexidinealexidine a repurposing dichloride.dichloride. drug classically used for the treatment of depression, against three different isolates of C. auris (Figure 4). Sertraline demon- stratedMamoueiMamouei to effectively etet al.al. recentlyrecently kill C. auris reportedreported but also thethe to resultsresults inhibit issuedissued the formation fromfrom theirtheir of ownown biofilm. screeningscreening Its mode ofof thethe of samesameaction chemicalchemical was elucidated collectioncollection by against againstin silico C.C. studies aurisauris [[50].50 and]. TheyTheyrevealed focusedfocused the bindingonon inhibitorsinhibitors nature ableable of tosertralineto blockblock thethe to fungalfungalthe sterol growthgrowth 14 α-demethylase and and to to kill kill preformed preformed which is biofilms. involvedbiofilms. The inThe bis-biguanideergosterol bis-biguanide biosynthesis alexidine alexidine dihydrochloride[52]. dihydrochlo- (AXD)ride (AXD)Taurolidine (Figure (Figure3), aand potent 3), aits potent and derivatives selective and selective PTPMT1have PTPMT1been (Protein recently (Protein Tyrosine patented Tyrosine Phosphatase by Phosphatase Diluccio Localized andLo- tocalizedReidenberg the Mitochondrion to the for Mitochondrion the treatment 1) inhibitor, 1) of inhibitor, blood exhibited infection exhibited the highest by theC. auris antifungalhighest [53]. antifungal Taurolidine and antibiofilm and (Figure antibiofilm activity 4) is againstactivityan antimicrobial aagainst panel ofa thatpanel pathogens, is ofused pathogens, to including try to preventincludingC. auris infections. C. auris. in catheters. ® InInOctenidine parallel,parallel, fromfromdihydrochloride thethe PrestwickPrestwick is LibraryLibrarya cationic®,, a agemini-surfactant, majormajor workwork dealingdealing derived withwith repurposing repurposingfrom pyridine ofof compoundscompounds(Figure 4). More toto fightfight than specificallyspecifically two decades againstagainst ago, C.C.it was aurisauris designed hashas beenbeen for conductedconducted skin, mucous byby ZagarozaZagaroza membrane etet al.al. and inin 20192019wound [[51].51 antisepsis.]. From this It is massive currently screening, used as antiseptic 27 drugsdrugs provedprovedin a large toto field inhibitinhibit of applications thethe growthgrowth ofofas threealter-three differentdifferentnative to strainsstrainschlorhexidine, ofof C.C. aurisauris polyvidone-iodine withwith differentdifferent geographicalgeographical or triclosan origin: origin:[54]. In CL-10093,CL-10093, 2019, Ponnachan JCMJCM 15448,15448, et al. andand re- KCTCKCTCported 1781017810 the ability (Table(Table of1 ).1). octenidine From From this this pre-selection,dihydrochloride pre-selection, the the scientiststo scientists kill C. auris carried carried strains complementary complementary [55]. studies stud- onies 10onFiftydrugs, 10 drugs, commercially namely namely MK MKavailable 801 801 hydrogen hydrogen herbicides, maleate, maleate, targeting ciclopirox ciclopirox acetohydroxyacid ethanolamine, ethanolamine, trifluoperazinesynthase, trifluopera- were dihydrochloride,zinerecently dihydrochloride, evaluated suloctidil, by suloctidil,Guddat ebselen, et ebselen, al. tamoxifen against tamoxifen C. citrate, auris citrate,CBS10913 rolipram, rolipram, thiethylperazinestrain. thiethylperazine Among these dimalate, com- di- guanadrelmalate,pounds, guanadrel bensulfuron sulfate, andsulfate, methyl pyrvinium and (B pyrviniumSM), pamoate, belonging pamoate, and to also the and recentlysulfonylurea also recently confirmed chemical confirmed the describedsubfamily the de- repositionable compounds such as alexidine and ebselen. scribedwas the repositionablemost potent discovered compounds antibiofilm such as alexidineformation and and ebselen. antifungal agent (Figure 4) [56].

Table 1. Active repositionable drugs against C. auris according to Zagaroza et al.

Growth Inhibition (%) at 50 µM

Structure Name Class CL JCM 15448 KCTC 17810 10093

FigureFigure 4.4. StructuresStructures ofof iodoquinol, miltefosine,Antibacterial sertra sertraline, line, taurolidine, 97 octenidine octenidine 97 dihydrochloride dihydrochloride 97 andand bensulfuronbensulfuron methyl.methyl.

3.2. Drugs: In Vitro Screening and In Vivo Validation Using medicines for malaria venture’s pathogen box, Wall et al. recently confirmed iodoquinol and miltefosine as potent inhibitors of C. auris strain 0390, both under planktonic and biofilm growing conditions (Figure 4) [57]. Both compounds possess broad- spectrum of activity against Candida spp., including multiple strains of the emergent C. auris, irrespective of their resistance profiles. Miltefosine (MFS) was also reported in combination with amphotericin B [58]. In the last few months, Barreto et al. confirmed the interest of MFS as an alternative approach to fight against the emerging fungus C. auris [59]. They reported its fungicidal activity against planktonic cells of C. auris clinical isolates, and antibiofilm ability. They also studied the encapsulation of MFS in alginate nanoparticles (MFS-AN). Using a Galleria mellonella larvae infected by C. auris model, they demonstrated that both MFS and MFS-AN were able to increase the survival rate. The main advantage of MFS-AN over MFS was its reduced toxicity.

Microorganisms 2021, 9, x 5 of 42

3. Repurposing Drugs 3.1. Drugs: In Vitro Screening Repurposing of drugs is an attractive pathway to discover new applications to already developed and filed drugs. Drug repositioning could help spare substantial costs linked to new drug discovery and development. It is also highly interesting because it involves the use of de-risked compounds, avoiding high attrition rate [47,48]. Therefore, this strategy has been applied for repurposing of drugs against C. auris. It is notable that the reported studies have been mainly conducted in vitro unless specified. In that context, the Prestwick Chemical library®, a repurposing library of 1280 small mainly off-patent molecules, has been screened several times by different research groups. In 2018, Wall et al. identified, from this library, ebselen as a repositionable molecule [49]. Ebselen is a synthetic organoselenium drug molecule with antioxidant, anti-inflammatory, and cytoprotective activity (Figure 3). It exhibited 100% inhibition of growth of C. auris at a concentration of 2.5 µM. In addition, ebselen also inhibited the formation of biofilm and was defined as a broad-spectrum antifungal, not only active on Candida species, but also against a variety of medically important fungi, including yeasts and molds.

Figure 3. Structures of ebselen and alexidine dichloride.

Mamouei et al. recently reported the results issued from their own screening of the same chemical collection against C. auris [50]. They focused on inhibitors able to block the fungal growth and to kill preformed biofilms. The bis-biguanide alexidine dihydrochloride (AXD) (Figure 3), a potent and selective PTPMT1 (Protein Tyrosine Phosphatase Lo- calized to the Mitochondrion 1) inhibitor, exhibited the highest antifungal and antibiofilm activity against a panel of pathogens, including C. auris. In parallel, from the Prestwick Library®, a major work dealing with repurposing of compounds to fight specifically against C. auris has been conducted by Zagaroza et al. in 2019 [51]. From this massive screening, 27 drugs proved to inhibit the growth of three different strains of C. auris with different geographical origin: CL-10093, JCM 15448, and KCTC 17810 (Table 1). From this pre-selection, the scientists carried complementary studies on 10 drugs, namely MK 801 hydrogen maleate, ciclopirox ethanolamine, trifluoperazine dihydrochloride, suloctidil, ebselen, tamoxifen citrate, rolipram, thiethylperazine dimalate, guanadrel sulfate, and pyrvinium pamoate, and also recently confirmed the de- Microorganisms 2021, 9, x scribed repositionable compounds such as alexidine and ebselen. 6 of 42

Microorganisms 2021, 9, x Table 1. Active repositionable drugs against C. auris according to Zagaroza et al. 6 of 42

Microorganisms 2021, 9, x 6 of 42 Alexidine dihydrochloride MicroorganismsMicroorganisms 2021, 20219, x , 9, 634 Growth Inhibition (%) at 506 µM of 426 of 40

Microorganisms 2021, 9Alexidine, x dihydrochloride 6 of 42

StructureAlexidine dihydrochloride Name Class CL Microorganisms 2021Alexidine, 9, x dihydrochlorideTable 1. Active repositionable drugs against C. auris according to Zagaroza et al. 6 of 42 Artemisinin Antimalarial 65 JCM56 15448 KCTC61 17810 Alexidine dihydrochloride Microorganisms 2021, 9, x Growth10093 Inhibition (%) at 50 µ6M of 42 Structure Name Class Microorganisms 2021, 9, x Artemisinin Antimalarial CL 1009365 JCM56 15448 KCTC616 of 1781042 Alexidine dihydrochloride Artemisinin Antimalarial 65 56 61 Artemisinin Antimalarial 65 56 61 Alexidine dihydrochloride Antibacterial 97 97 97 Antibacterial 97 97 97 Alexidine dihydrochlorideArtemisinin Antimalarial 65 56 61

Alexidine dihydrochloride Antibacterial 96 97 100 Artemisinin Antimalarial 65 56 61

Benzethonium chloride Antibacterial 96 97 100 Artemisinin Antimalarial 65 56 61 Antibacterial 96 97 100 ArtemisininArtemisinin AntimalarialAntimalarial 65 65 56 5661 61 Benzethonium chloride Antibacterial 96 97 100

Benzethonium chloride Antibacterial 96 97 100

Benzethonium chloride Benzethonium chloride Antibacterial 96 97 100 Antibacterial 98 99 98 Antibacterial 96 97 100 Antibacterial 96 97 100 Benzethonium chloride AntibacterialAntibacterial 9698 97 99 100 98 BenzethoniumChlorhexidine chloride Antibacterial 98 99 98 Benzethonium chloride Benzethonium chloride Antibacterial 98 99 98 Chlorhexidine Antibacterial 98 99 98 Chlorhexidine Antibacterial 98 99 98 Chlorhexidine Chloroxine AntibacterialAntibacterial 9598 97 99 98 98 Chlorhexidine

Chlorhexidine Chloroxine Antibacterial 9598 97 99 98 Chlorhexidine Chloroxine Antibacterial 9895 9997 98

Chloroxine Antibacterial 95 97 98 Chlorhexidine ChloroxineChloroxine Antibacterial/Antifunga AntibacterialAntibacterial 95 95 97 9798 98 Ciclopirox ethanolamine 94 97 94 Chlorhexidine l Chloroxine Antibacterial/AntifungaAntibacterial 95 97 98 Ciclopirox ethanolamine 94 97 94 Antibacterial/Antifungal Ciclopirox ethanolamine 94 97 94 Chloroxine Antibacterial/AntifungaAntibacteriall 95 97 98 CiclopiroxCiclopirox ethanolamine ethanolamineAntibacterial/Antifungal 9494 97 9794 94 Chloroxine Antibacterial/AntifungaAntiamebic/AntibacteriAntibacteriall 95 97 98 CiclopiroxClioquinol ethanolamine 9489 9793 9493 al

Antibacterial/AntifungaAntiamebic/Antibacteri CiclopiroxClioquinol ethanolamine 8994 9397 9394 Antiamebic/Antibacterial ClioquinolClioquinol Antiamebic/Antibacteria 8989 93 9393 93 Antibacterial/Antifungaa Ciclopirox ethanolamine Antiamebic/Antibacteri 94 97 94 Clioquinol Antibacterial/Antifungal 89 93 93 Ciclopirox ethanolamine Antiamebic/Antibacteria 94 97 94 Clioquinol l 89 93 93 a Antibacterial 81 86 89 Antiamebic/Antibacteri Clioquinol 89 93 93 Antibacteriala 81 86 89 Dequalinium dichloride Antibacterial 81 86 89 Antiamebic/Antibacteri Clioquinol Antibacterial 8189 8693 8993 Dequalinium dichloride Antiamebic/Antibacteria Dequalinium dichloride Clioquinol Antibacterial 8981 93 86 93 89 a Dequalinium dichloride Antibacterial 81 86 89

Dequalinium dichlorideDimethisoquinDimethisoquin AntipruriticAntipruritic 59 59 65 6551 51 Dequalinium dichloridehydrochloridehydrochloride Antibacterial 81 86 89

Dimethisoquin Antipruritic 59 65 51 hydrochloride Antibacterial 81 86 89 Dequalinium dichlorideDimethisoquin AntibacterialAntipruritic 8159 8665 8951 Dimethisoquinhydrochloride Antipruritic 59 65 51 Dequalinium dichlorideDimethisoquinhydrochloride Antipruritic 59 65 51 Dequalinium dichloridehydrochloride

Dimethisoquin Antipruritic 59 65 51 hydrochloride Dimethisoquin Antipruritic 59 65 51 Dimethisoquinhydrochloride Antipruritic 59 65 51 hydrochloride

Microorganisms 2021, 9, x 7 of 42

MicroorganismsMicroorganisms 20212021, 9,, x9 , 634 Dyclonine hydrochloride Local anesthetic 63 63 547 of7 42 of 40 Dyclonine hydrochloride Local anesthetic 63 63 54 Microorganisms 2021, 9, x 7 of 42

Microorganisms 2021, 9, x Dyclonine hydrochloride Local anesthetic 63 63 547 of 42

Microorganisms 2021, 9, x Dyclonine hydrochlorideTable 1. LocalCont. anesthetic 63 63 547 of 42

DyclonineEbselen hydrochloride Anti-inflammatory Local anesthetic 6387 6391 54 92 Growth Inhibition (%) at 50 µM Structure DyclonineNameEbselen hydrochloride Anti-inflammatory LocalClass anesthetic 87 63 91 63 9254 DyclonineEbselen hydrochloride Anti-inflammatory Local anesthetic CL 6387 10093 JCM 6391 15448 KCTC 5492 17810 DyclonineEbselen hydrochloride Anti-inflammatory Local anesthetic 6387 6391 54 92 Dyclonine DyclonineEbselen hydrochloride Anti-inflammatoryLocal anesthetic 87 63 63 91 63 63 9254 54 hydrochloride FipexideEbselen hydrochloride Anti-inflammatory Anti-fatigue 87 68 91 63 92 54 FipexideEbselen hydrochloride Anti-inflammatory Anti-fatigue 6887 6391 5492 FipexideEbselenEbselen hydrochloride Anti-inﬂammatoryAnti-inflammatory Anti-fatigue 87 68 87 91 63 91 9254 92

FipexideEbselen hydrochloride Anti-inflammatory Anti-fatigue 87 68 91 63 92 54 Fipexide hydrochloride Anti-fatigue 68 63 54

FipexideGuanadrel hydrochloride sulfate Antihypertensive Anti-fatigue 68 97 63 97 54 97

FipexideFipexideGuanadrel hydrochloride hydrochloride sulfate Antihypertensive Anti-fatigue 9768 68 9763 63 9754 54 FipexideGuanadrel hydrochloride sulfate Antihypertensive Anti-fatigue 6897 6397 5497 FipexideGuanadrel hydrochloride sulfate Antihypertensive Anti-fatigue 68 97 63 97 54 97

Guanadrel sulfate Antihypertensive 97 97 97 Hexachlorophene Antiseptic 97 98 86 GuanadrelGuanadrel sulfate sulfate Antihypertensive 97 97 97 97 97 97 Hexachlorophene Antiseptic 97 98 86 Guanadrel sulfate Antihypertensive 97 97 97 Hexachlorophene Antiseptic 97 98 86 Guanadrel sulfate Antihypertensive 97 97 97 Hexachlorophene Antiseptic 97 98 86 Guanadrel sulfate Antihypertensive 97 97 97 HexachloropheneHexachlorophene Antiseptic 97 97 98 98 86 86 Methyl benzethonium Hexachlorophene AntibacterialAntiseptic 9798 98 97 86100 Methyl benzethoniumchloride Hexachlorophene AntibacterialAntiseptic 98 97 9798 100 86 Methylchloride benzethonium Hexachlorophene AntibacterialAntiseptic 9798 9897 100 86 MethylMethyl benzethoniumchloride benzethonium Hexachlorophene AntibacterialAntibacterialAntiseptic 97 9898 98 97 97 86100 100 Methylchloride benzethoniumchloride Antibacterial 98 97 100 Methylchloride benzethonium Antibacterial 98 97 100 MethylMethiothepinchloride benzethonium maleate Antipsychotic 74 72 64 Antibacterial 98 97 100 MethylMethiothepinchloride benzethonium maleate Antipsychotic 74 72 64 Antibacterial 98 97 100 MethiothepinMethylMethiothepinchloride benzethonium maleate maleate Antipsychotic Antipsychotic 74 74 72 72 64 64 Antibacterial 98 97 100 Methiothepinchloride maleate Antipsychotic 74 72 64 Methiothepin maleate Antipsychotic 74 72 64 MK 801 hydrogen Methiothepin maleate AntipsychoticAnticonvulsant 7498 72 98 64 97 MKMK 801 801maleate hydrogen hydrogen Methiothepin maleate Anticonvulsant Antipsychotic 98 74 98 9872 98 9764 97 MKmaleate 801maleate hydrogen Anticonvulsant 98 98 97 Methiothepinmaleate maleate Antipsychotic 74 72 64 MK 801 hydrogen Methiothepin maleate AntipsychoticAnticonvulsant 7498 72 98 64 97 MK 801maleate hydrogen Anticonvulsant 98 98 97 MK 801maleate hydrogen Anticonvulsant 98 98 97 MK 801maleate hydrogen AnticonvulsantAnthelmintic 98 40 98 76 97 61 maleate Microorganisms 2021, 9, x MK 801 hydrogen Anthelmintic 40 76 618 of 42 Anticonvulsant 98 98 97 MK 801maleate hydrogen Anthelmintic 40 76 61 Anticonvulsant 98 98 97 maleate Anthelmintic 40 76 61

Pyrvinium pamoate Anthelmintic 40 76 61 Pyrvinium pamoate

Pyrvinium pamoate Anthelmintic 40 76 61

Pyrvinium pamoate Anthelmintic 40 76 61 ProchlorperazineProchlorperazine Antiemetic/Antipsychot Antiemetic/AntipsychoticAnthelmintic 407171 7666 66 6172 72 Pyrvinium pamoate dimaleatedimaleate ic

Pyrvinium pamoate Anthelmintic 40 76 61

Pyrvinium pamoate

Pyrvinium pamoate Pyrvinium pamoate Tamoxifen citrate Antineoplastic 98 98 90

Thiethylperazine Antiemetic 68 90 86 dimalate

Antiseptic 98 98 98

Thonzonium bromide

Trifluoperazine Antiemetic 54 88 61 dihydrochloride

Rolipram Antidepressant 98 97 91

Sertraline Antidepressant 59 88 56

Suloctidil Antiplatelet 96 99 78

Microorganisms 2021, 9, x 8 of 42

Microorganisms 2021, 9, x 8 of 42 Prochlorperazine Antiemetic/Antipsychot Microorganisms 2021, 9, x 71 66 728 of 42 Prochlorperazinedimaleate Antiemetic/Antipsychotic Microorganisms 2021, 9, x 71 66 72 8 of 42 Prochlorperazinedimaleate Antiemetic/Antipsychotic 71 66 72 Prochlorperazinedimaleate Antiemetic/Antipsychotic 71 66 72 dimaleate ic

Microorganisms 2021, 9, 634 Prochlorperazine Antiemetic/Antipsychot 8 of 40 71 66 72 Prochlorperazinedimaleate Antiemetic/Antipsychotic 71 66 72 TamoxifenProchlorperazinedimaleate citrate Antiemetic/Antipsychot Antineoplasticic 98 98 90 71 66 72 Tamoxifendimaleate citrate Table 1. AntineoplasticCont. ic 98 98 90

Tamoxifen citrate Antineoplastic 98 98 90 Growth Inhibition (%) at 50 µM Structure TamoxifenName citrate AntineoplasticClass 98 98 90 CL 10093 JCM 15448 KCTC 17810

Tamoxifen citrate Antineoplastic 98 98 90 TamoxifenThiethylperazine citrate Antineoplastic 98 98 90 Antiemetic 68 90 86 TamoxifenThiethylperazineTamoxifendimalate citrate citrate Antineoplastic Antineoplastic 98 98 98 98 90 90 Antiemetic 68 90 86 Thiethylperazinedimalate Antiemetic 68 90 86 Thiethylperazinedimalate Antiemetic 68 90 86 dimalate

Thiethylperazine Antiemetic 68 90 86 ThiethylperazineThiethylperazinedimalate Antiemetic 68 68 90 90 86 86 Thiethylperazinedimalatedimalate AntiemeticAntiseptic 9868 9890 9886 dimalate Antiseptic 98 98 98 Antiseptic 98 98 98 Thonzonium bromide Antiseptic 98 98 98 Thonzonium bromide

Thonzonium bromide Antiseptic 98 98 98 Antiseptic 98 98 98 Thonzonium bromide Antiseptic 98 98 98 Trifluoperazine Thonzonium bromide AntiemeticAntiseptic 5498 8898 6198 Thonzonium bromideTrifluoperazine dihydrochloride Antiemetic 54 88 61 Thonzonium bromidedihydrochlorideTrifluoperazine Antiemetic 54 88 61 Thonzonium bromideTriﬂuoperazinedihydrochlorideTrifluoperazine Antiemetic 54 54 88 88 61 61 dihydrochloridedihydrochloride

Trifluoperazine Antiemetic 54 88 61 TrifluoperazinedihydrochlorideRolipram Antidepressant 98 97 91 Antiemetic 54 88 61 dihydrochlorideTrifluoperazineRolipram Antidepressant 98 97 91 Antiemetic 54 88 61 RolipramdihydrochlorideRolipram Antidepressant 98 98 97 97 91 91

Rolipram Antidepressant 98 97 91

RolipramSertraline AntidepressantAntidepressant 9859 9788 9156 SertralineSertralineRolipram AntidepressantAntidepressant 9859 59 9788 88 9156 56

SertralineRolipram AntidepressantAntidepressant 5998 8897 5691

Microorganisms 2021, 9, x Sertraline Antidepressant 59 88 569 of 42

SuloctidilSertralineSuloctidil Antidepressant AntiplateletAntiplatelet 965996 998899 56 78 SertralineSuloctidil AntidepressantAntiplatelet 5996 8899 5678

SuloctidilSertraline AntiplateletAntidepressant 9659 9988 7856 N Suloctidil Antiplatelet 96 99 78

O ZotepineZotepine AntipsychoticAntipsychotic 59 59 63 63 56 56 Suloctidil Antiplatelet 96 99 78 Cl S Suloctidil Antiplatelet 96 99 78

Suloctidil Antiplatelet 96 99 78

GowriGowri et etal. al. studied studied sertraline, sertraline, a repurposing a repurposing drug drug classically classically used used for for the the treatment treatment of ofdepression, depression, against against thre threee different isolatesisolates ofofC. C. auris auris(Figure (Figure4). Sertraline4). Sertraline demonstrated demon- stratedto effectively to effectively kill C. kill auris C.but auris also but to also inhibit to theinhibit formation the formation of bioﬁlm. of Itsbiofilm. mode Its of actionmode of was elucidated by in silico studies and revealed the binding nature of sertraline to the sterol 14 action was elucidated by in silico studies and revealed the binding nature of sertraline to α-demethylase which is involved in ergosterol biosynthesis [52]. the sterol 14 α-demethylase which is involved in ergosterol biosynthesis [52]. Taurolidine and its derivatives have been recently patented by Diluccio and Reidenberg for the treatment of blood infection by C. auris [53]. Taurolidine (Figure 4) is an antimicrobial that is used to try to prevent infections in catheters. Octenidine dihydrochloride is a cationic gemini-surfactant, derived from pyridine (Figure 4). More than two decades ago, it was designed for skin, mucous membrane and wound antisepsis. It is currently used as antiseptic in a large field of applications as alternative to chlorhexidine, polyvidone-iodine or triclosan [54]. In 2019, Ponnachan et al. reported the ability of octenidine dihydrochloride to kill C. auris strains [55]. Fifty commercially available herbicides, targeting acetohydroxyacid synthase, were recently evaluated by Guddat et al. against C. auris CBS10913 strain. Among these compounds, bensulfuron methyl (BSM), belonging to the sulfonylurea chemical subfamily was the most potent discovered antibiofilm formation and antifungal agent (Figure 4) [56].

Figure 4. Structures of iodoquinol, miltefosine, sertraline, taurolidine, octenidine dihydrochloride and bensulfuron methyl.

Microorganisms 2021, 9, 634 9 of 40

Taurolidine and its derivatives have been recently patented by Diluccio and Reiden- berg for the treatment of blood infection by C. auris [53]. Taurolidine (Figure4) is an antimicrobial that is used to try to prevent infections in catheters. Octenidine dihydrochloride is a cationic gemini-surfactant, derived from pyridine (Figure4). More than two decades ago, it was designed for skin, mucous membrane and wound antisepsis. It is currently used as antiseptic in a large ﬁeld of applications as alternative to chlorhexidine, polyvidone-iodine or triclosan [54]. In 2019, Ponnachan et al. reported the ability of octenidine dihydrochloride to kill C. auris strains [55]. Fifty commercially available herbicides, targeting acetohydroxyacid synthase, were recently evaluated by Guddat et al. against C. auris CBS10913 strain. Among these compounds, bensulfuron methyl (BSM), belonging to the sulfonylurea chemical subfamily was the most potent discovered antibioﬁlm formation and antifungal agent (Figure4)[56].

3.2. Drugs: In Vitro Screening and In Vivo Validation Using medicines for malaria venture’s pathogen box, Wall et al. recently confirmed iodoquinol and miltefosine as potent inhibitors of C. auris strain 0390, both under planktonic and biofilm growing conditions (Figure4)[ 57]. Both compounds possess broad-spectrum of activity against Candida spp., including multiple strains of the emergent C. auris, irrespective of their resistance profiles. Miltefosine (MFS) was also reported in combination with amphotericin B [58]. In the last few months, Barreto et al. confirmed the interest of MFS as an alternative approach to fight against the emerging fungus C. auris [59]. They reported its fungicidal activity against planktonic cells of C. auris clinical isolates, and antibiofilm ability. They also studied the encapsulation of MFS in alginate nanoparticles (MFS-AN). Using a Galleria mellonella larvae infected by C. auris model, they demonstrated that both MFS and MFS-AN were able to increase the survival rate. The main advantage of MFS-AN over MFS was its reduced toxicity.

3.3. Vaccines: In Vivo Evaluation Ibrahim et al. reported the efﬁcacy of NDV-3A vaccine to protect mice from multidrug resistant Candida auris infection [43,60]. NDV-3A vaccine, harboring the N-terminus of Als3p formulated with alum, has been developed initially against C. albicans. The authors proved that it generated cross-reactive antibodies against C. auris clinical isolates and protected neutropenic mice from C. auris infection. Moreover, this repositioning vaccine displayed an additive protective effect in neutropenic mice when combined with micafungin. This vaccine alternative has been recently ﬁled [46].

3.4. Critical Analysis of Repurposing Drugs from a Chemical/Physicochemical Point of View From a chemical point of view, it has to be mentioned that the chemical diversity of reported antifungal active against C. auris is quite low. Only 30 repositionable compounds have been described for their activity against C. auris and most of them can be gathered based on common chemical features: quinoline/isoquinoline, guanidine, phenothiazine/benzothiepine or amide-based cores. These cores are decorated with protonable/polarizable nitrogen groups (pyridine, ternary amine, ammonium, piperazine) and lipophilic moieties (fatty chain or halogenated aromatics). This could be summarized as followed in Figure5. The protonable nitrogen could be part of the core moiety as in quinoline/isoquinoline derivatives. Microorganisms 2021, 9, x 11 of 42

MW < 250, XLOGP > 2 2.86 33.12 214.05 Yes High 3.5 R = I = Clioquinol

R = Cl = Chloroxine

Microorganisms 2021, 9, x 10 of 42

3 O 3.66 25.36 272.39 XLOGP > 3.5 Yes High 3.3. Vaccines: In Vivo Evaluation N DimethisoquinIbrahim et al. reported the efficacy of NDV-3A vaccine to protect mice from multidrug resistant Candida auris infection [43,60]. NDV-3A vaccine, harboring the N-terminus of Als3p formulated with alum, has been developed initially against C. albicans. The authors proved that it generated cross-reactive antibodies against C. auris clinical isolates and protected neutropenic mice from C. auris infection.MW Moreover, > 350 Rotors this repositioning> 7 vaccine displayed an additive protective effect in neutropenic mice when combined with mi- 4 4.14 59.80 456.67 No High cafungin. This vaccine alternative has been recently filed [46]. XLOGP > 3.5 Dequalinium dichloride 3.4. Critical Analysis of Repurposing Drugs from a Chemical/Physicochemical Point of View From a chemical point of view, it has to be mentioned that the chemical diversity of reported antifungal active against C. auris is quite low. Only 30 repositionable compounds have been described for their activity against C. auris and most of them can be gathered based on common chemical features: quinoline/isoquinoline,MW > 350guanidine, phenothia- 5 zine/benzothiepine or amide-based4.29 12.05 cores. These382.52 cores are decorated with protonable/po-Yes High larizablePyrvinium nitrogen active groups (pyridine, ternary amine, ammonium,XLOGP > 3.5piperazine) and lipophilic moieties (fatty chain or halogenated aromatics). This could be summarized as fol- Microorganisms 2021, 9, 634 compound 10 of 40 lowed in Figure 5. The protonable nitrogen could be part of the core moiety as in quinoline/isoquinoline derivatives. 1. LogP = octanol/water partition coefficient calculated by SwissADME; 2 TPSA = topology polar surface area by Swis- sADME; 3 Druglikliness = criteria which should be problematic for drug development according to SwissADME; 4 BBB permeant: blood brain barrier permeant as predicted by SwissADME; 5gastro-intestinal permeation ability as predicted by SwissADME.

Concerning the guanidine derivatives, the same features are also represented (Figure 6). The guanidine core serves also as the protonable part. From a descriptor point of view, these compounds are not blood brain barrier (BBB) permeants. The bis-biguanidines Figure(chlorhexidineFigure 5. Proposed 5. Proposed scaffold and scaffold alexidine) of reported of reportedC. are auris C. quiteaurisantifungals. antifungals. lipophilic with logP equal to 2.79 and 4.67, respectively.The reportedHowever,reported repositionable repositionable guanadrel compounds compoundsis quite have balanced have been been gathered (log gatheredP in = Tables 0.59). in2 Tables– The4 and threeFigures2–4 and drugs5 Fig-and6 exhibited high withuresTPSA analysis5 and (82.86 6 with of theirto analysis 167.58 composition ofÅ). their according composition to the according proposed to scaffold. the proposed scaffold. Concerning the quinoline/isoquinoline sub-group, composed of five molecules, it is notable than the simplest drugs—clioquinol and chloroxine—differed in only one halogen atom. Indeed, the iodine in clioquinol is replaced by a chlorine in chloroxine. This slightly impacted the lipophilicity of the compound as chloroxine is a little more hydrophilic than clioquinol. However, these two compounds could be referred as analogs (Table 2, Entries 1 and 2). In dimethisoquin, the quinoline core is substituted by an additional dimethylamine group which is easily protonable and a fatty butyl chain to enhance the lipophilicity (Table 2, entry 3). Dequalinium dichloride is a bis-quinoline derivative than perfectly fit the proposed scaffold (Table 2, entry 4). In the same manner, pyrvinium pamoate respects the defined features (Table 2, entry 5). On the whole, the quinoline/isoquinoline series is composed of lipophilic compounds (2.86 < logP < 4.29). FigureFigure 6. 6.Critical Critical analysis analysis for the for guanidine the guanidine derivatives. derivatives. Table 2. ConcerningCritical analysis the of quinoline/isoquinoline quinoline/isoquinoline repositionable sub-group, composeddrugs. of five molecules, it is notableHeterocyclic than the simplest sulfur drugs—clioquinol compounds and bearing chloroxine—differed a phenothiazine in only or one a benzothiepine halogen core dis- atom.played Indeed, good the activities iodine in TPSA clioquinolagainst 2 MC. is auris replacedDruglikeliness. All by are a chlorine highly 3 in absorbed chloroxine. in This the slightlyGI gastrointestinal tract impacted the lipophilicityLog P of the compound as chloroxine is a littleBBB more Permeant hydrophilic than Entry Structuresand can pass the BBB.1 The five reported compounds could4 be divided into two sub-classes clioquinol.and differed However, only these by twothe(Å) compoundsnature (g/mol) of couldthe R-substitution(Alert) be referred as analogs on the (Table aromaticAbsorption2, Entries moiety 1 5 (Table 3). and 2). In dimethisoquin, the quinoline core is substituted by an additional dimethylamine group which is easily protonable and a fatty butyl chain to enhance the lipophilicity 1 (Table2 , entry 3). Dequalinium 2.96 33.12 dichloride 305.50 is a XLOGP bis-quinoline > 3.5 derivative Yes than perfectly High fit the proposed scaffold (Table2, entry 4). In the same manner, pyrvinium pamoate respects the defined features (Table2, entry 5). On the whole, the quinoline/isoquinoline series is composed of lipophilic compounds (2.86 < logP < 4.29). Concerning the guanidine derivatives, the same features are also represented (Figure6 ). The guanidine core serves also as the protonable part. From a descriptor point of view, these

compounds are not blood brain barrier (BBB) permeants. The bis-biguanidines (chlorhexidine and alexidine) are quite lipophilic with logP equal to 2.79 and 4.67, respectively. However, guanadrel is quite balanced (logP = 0.59). The three drugs exhibited high TPSA (82.86 to 167.58 Å). Heterocyclic sulfur compounds bearing a phenothiazine or a benzothiepine core displayed good activities against C. auris. All are highly absorbed in the gastrointestinal tract and can pass the BBB. The five reported compounds could be divided into two sub-classes and differed only by the nature of the R-substitution on the aromatic moiety (Table3). Two benzothiepine derivatives (methiothepin and zotepine) were reported. Both are lipophilic (logP > 3.9) and exhibited a protonable N-methylpiperazine ring as the side-chain. The same N-methylpiperazine moiety is kept in the phenothiazine derivatives (prochlorperazine, thiethylperazine, trifluoperazine) with logP ranging from 3.47 to 4.53. The introduction of more lipophilic substituents—ethylsulfur and trifluoromethyl—increased the activity against C. auris JCM 15448 strain particularly. Microorganisms 2021, 9, x 11 of 42

Microorganisms 2021, 9, x 11 of 42

Microorganisms 2021, 9, 634 11 of 40

Microorganisms 2021, 9, x 11 of 42 Table 2. Critical analysis of quinoline/isoquinolineMW < 250, XLOGP > repositionable drugs. 2 2.86 33.12 214.05 Yes High 2 3.5 3 1 TPSAMW < 250, XLOGPM > Druglikeliness BBB GI Entry2 R = StructuresI = Clioquinol 2.86 Log33.12P 214.05 Yes High 4 5 (Å) 3.5 (g/mol) (Alert) Permeant Absorption MW < 250, XLOGP > 2 R = I = Clioquinol 2.86 33.12 214.05 Yes High R = Cl = Chloroxine 3.5 High 1 R = I = Clioquinol 2.96 33.12 305.50 XLOGP > 3.5 Yes R = Cl = Chloroxine R = Cl = Chloroxine MW < 250, XLOGP > High 2 2.86 33.12 214.05 Yes High 2RN = I = Clioquinol 2.86 33.123.5 214.05 MW < 250, XLOGP > 3.5 Yes RR = = Cl I = = Clioquinol Chloroxine 3 O 3.66 25.36 272.39 XLOGP > 3.5 Yes High N

3 RO = ClN = Chloroxine 3.66 25.36 272.39 XLOGP > 3.5 Yes High N Dimethisoquin 3 3 O N 3.66 25.363.66 272.39 25.36 XLOGP > 3.5 272.39 Yes XLOGP High > 3.5 Yes High Dimethisoquin N

N Dimethisoquin MW > 350 Rotors > 7 34 O 3.664.14 25.3659.80 272.39456.67 XLOGP > 3.5 YesNo High MW > 350 Rotors > 7 XLOGP > 3.5 MW > 350 Rotors > 7 4 4 N 4.14 59.804.14 456.67 59.80 456.67 No High No High DequaliniumDimethisoquin dichloride MW > 350 Rotors > 7 XLOGP > 3.5 XLOGP > 3.5 4 Dequalinium dichloride 4.14 59.80 456.67 No High XLOGP > 3.5 Dequalinium dichloride MW >MW 350 >Rotors 350 > 7 MW > 350 5 4 4.14 59.804.29 456.67 12.05 382.52 No High Yes High 5 4.29 12.05 382.52 YesXLOGP High > 3.5 XLOGPMW > 350> 3.5 Pyrvinium Pyrviniu activem active XLOGP > 3.5 5 Dequalinium dichloride 4.29 12.05 382.52 Yes High compound MW > 350 Pyrvinium active XLOGP > 3.5 1. LogP =5 octanol/water partition coefﬁcient calculated by SwissADME;4.292 TPSA 12.05 = topology 382.52 polar surface area by SwissADME;Yes3 Druglikliness High = criteria which should be problematic for drug development compound according to1. SwissADME; LogP = octanol/water4 BBB permeant: partition bloodPyrvinium coefficient brain barrier active calculated permeant as by predicted SwissADME; by SwissADME; 2 TPSAXLOGP 5=gastro-intestinal topology > 3.5 polar permeation surface area ability by as Swis- predicted by SwissADME. sADME; 3 Drugliklinesscompound = criteria which should be problematic for drug development according to SwissADME; 4 BBB 1. 2 MW > 350 permeant:LogP = octanol/water blood brain barrier partition permea coefficientnt as predicted calculated by SwissADME;by SwissADME; 5gastro-intestinal TPSA = topology permeation polar abilitysurface as area predicted by Swis- by 5 sADME; 3 Druglikliness = criteria which should be problemati4.29 12.05c for382.52 drug development according to SwissADME;Yes 4 HighBBB 1.SwissADME. LogP = octanol/water partition coefficient calculated by SwissADME; 2 TPSA = topology polar surface area by Swis- permeant: blood brain barrier permeant as predicted by SwissADME; 5gastro-intestinalXLOGP permeation> 3.5 ability as predicted by sADME; 3 Druglikliness = criteriaPyrvinium which should active be problematic for drug development according to SwissADME; 4 BBB SwissADME. permeant: blood braincompound barrier permea Concerningnt as predicted the guanidine by SwissADME; derivatives, 5gastro-intestinal the same features permeation are ability also represented as predicted (Figureby SwissADME. 6). The guanidine core serves also as the protonable part. From a descriptor point of view, 1. Concerning the guanidine derivatives,2 the same features are also represented (Figure LogP = octanol/water partitionthese coefficientcompounds calculated are not by blood SwissADME; brain barrieTPSAr =(BBB) topology permeants. polar surface The areabis-biguanidines by Swis- sADME; 3 Druglikliness = criteria6). The which guanidine should core be problematiserves alsoc foras thedrug protonable development part. according From a todescriptor SwissADME; point 4 BBB of view, (chlorhexidineConcerning and the alexidine) guanidine are derivatives, quite lipophilic the same with features logP equal are also to 2.79 represented and 4.67, (Figurerespec- permeant: blood brain barrierthese permea compoundsnt as predicted are bynot SwissADME; blood brain 5gastro-intestinal barrier (BBB) permeation permeants. ability The as bis-biguanidines predicted by 6).tively. The However,guanidine guanadrel core serves is also quite as balanced the protonable (logP = part. 0.59). From The threea descriptor drugs exhibited point of view, high SwissADME. (chlorhexidine and alexidine) are quite lipophilic with logP equal to 2.79 and 4.67, respec- theseTPSA compounds(82.86 to 167.58 are Å). not blood brain barrier (BBB) permeants. The bis-biguanidines tively.(chlorhexidine However, and guanadrel alexidine) is arequite quite balanced lipophilic (log Pwith = 0.59). logP The equal three to 2.79drugs and exhibited 4.67, respec- high Concerning the guanidine derivatives, the same features are also represented (Figure TPSAtively. (82.86 However, to 167.58 guanadrel Å). is quite balanced (logP = 0.59). The three drugs exhibited high 6). The guanidine core serves also as the protonable part. From a descriptor point of view, TPSA (82.86 to 167.58 Å). these compounds are not blood brain barrier (BBB) permeants. The bis-biguanidines (chlorhexidine and alexidine) are quite lipophilic with logP equal to 2.79 and 4.67, respectively. However, guanadrel is quite balanced (logP = 0.59). The three drugs exhibited high TPSA (82.86 to 167.58 Å).

Figure 6. Critical analysis for the guanidine derivatives. Figure 6. Critical analysis for the guanidine derivatives. Heterocyclic sulfur compounds bearing a phenothiazine or a benzothiepine core dis- Figureplayed 6. good Critical activities analysis against for the guanidineC. auris. All derivatives. are highly absorbed in the gastrointestinal tract Heterocyclic sulfur compounds bearing a phenothiazine or a benzothiepine core dis- and can pass the BBB. The five reported compounds could be divided into two sub-classes played good activities against C. auris. All are highly absorbed in the gastrointestinal tract and differedHeterocyclic only sulfur by the compounds nature of the bearing R-substitution a phenothiazine on the oraromatic a benzothiepine moiety (Table core dis- 3). andplayed can good pass activitiesthe BBB. The against five C.reported auris. All compounds are highly could absorbed be divided in the gastrointestinal into two sub-classes tract Figure 6. Critical analysis for the guanidine derivatives. and candiffered pass theonly BBB. by theThe naturefive reported of the compoundsR-substitution could on bethe divided aromatic into moiety two sub-classes (Table 3). and differed only by the nature of the R-substitution on the aromatic moiety (Table 3). Heterocyclic sulfur compounds bearing a phenothiazine or a benzothiepine core displayed good activities against C. auris. All are highly absorbed in the gastrointestinal tract and can pass the BBB. The five reported compounds could be divided into two sub-classes

and differed only by the nature of the R-substitution on the aromatic moiety (Table 3).

Microorganisms 2021, 9, x 12 of 42

Two benzothiepine derivatives (methiothepin and zotepine) were reported. Both are lipophilic (logP > 3.9) and exhibited a protonable N-methylpiperazine ring as the side-chain. The same N-methylpiperazine moiety is kept in the phenothiazine derivatives (pro- Microorganisms 2021, 9, x 12 of 42 chlorperazine, thiethylperazine, trifluoperazine) with logP ranging from 3.47 to 4.53. The introduction of more lipophilic substituents—ethylsulfur and trifluoromethyl—increased the activity against C. auris JCM 15448 strain particularly. Two benzothiepine derivatives (methiothepin and zotepine) were reported. Both are lip- Table 3.ophilic Critical (log analysisP > 3.9) of andbenzothiepine/ exhibitedphenothiazine a protonable repositionableN-methylpiperazine drugs. ring as the side-chain. The same N-methylpiperazine moiety is kept in the phenothiazine derivatives (prochlorperazine, thiethylperazine, trifluoperazine) with logP ranging from 3.47 to 4.53. The M introduction of more lipophilic substituents—ethylsulfurTPSA and trifluoromethyl—increased Entry Structure R LogP 1 Druglikeliness (Alert) 3 (Å) 2 the activity against C. auris JCM 15448 strain particularly.(g/mol)

Table 3. Critical analysis of benzothiepine/phenothiazine repositionable drugs.

-S-CH3 Microorganisms 2021, 9, 6341 3.94 57.08 356.55M MW > 350, XLOGP > 3.5 12 of 40 TPSA Entry Structure (Methiothepin) R LogP 1 Druglikeliness (Alert) 3 (Å) 2 (g/mol) Table 3. Critical analysis of benzothiepine/phenothiazine repositionable drugs. -Cl 2 4.37 37.77 331.86 XLOGP > 3.5 Entry Structure-S-CH3 R LogP 1 TPSA (Å) 2 M (g/mol) Druglikeliness (Alert) 3 1 (Zotepine) 3.94 57.08 356.55 MW > 350, XLOGP > 3.5 (Methiothepin)-S-CH 1 3 3.94 57.08 356.55 MW > 350, XLOGP > 3.5 (Methiothepin) -H 3 -Cl 3.47 35.02 339.50 XLOGP > 3.5 XLOGP > 3.5 2 -Cl 4.37 37.77 331.86 2 (Prochlorperazine)(Zotepine) 4.37 37.77 331.86 XLOGP > 3.5 (Zotepine) -H 3 3.47 35.02 339.50 XLOGP > 3.5 -S-CH(Prochlorperazine)2CH3 4 -H 4.43 60.32 399.62 MW > 350, XLOGP > 3.5 -S-CH CH MW > 350, XLOGP > 3.5 4 (Thiethylperazine)2 3 4.43 60.32 399.62 3 (Thiethylperazine) 3.47 35.02 339.50 XLOGP > 3.5 (Prochlorperazine) Microorganisms 2021, 9, x -CF 13 of 42 5 3 4.53 35.02 407.50 MW > 350, XLOGP > 3.5 (Triﬂuoperazine) -CF3 1. Microorganisms 2021, 9, x 2 3 13 of 42 Log P = octanol/water5 partition coefﬁcient calculated by SwissADME;-S-CH2CH3TPSA = topology4.53 polar surface 35.02area by SwissADME; 407.50 Druglikliness MW > 350, =XLOGP criteria which> 3.5 should be problematic for drug development according to SwissADME. 4 (Trifluoperazine) 4.43 60.32 399.62 MW > 350, XLOGP > 3.5 Microorganisms 2021, 9, x 13 of 42 (Thiethylperazine)Table 4. Critical analysis of amide-based repositionable drugs. 1. LogP = octanol/water partition coefficient calculated by SwissADME; 2 TPSA = topology polar surface area by Swis- sADME; 3 Druglikliness = criteria which should be problematiTPSA 2c for drug development according to DruglikelinessSwissADME. 3 GI 5 Entry Structures LogP 1 M (g/mol) BBB Permeant 4 1 2.38 42.23 207.27(Å) MW < 250 Yes (Alert)High Absorption -CF3 5 The last sub-group which4.53 could be 35.02 addressed 407.50is based on an MW amide > 350, fragmentXLOGP > 3.5. The 1 2.38 42.23 207.27 MW < 250 Yes High Ciclopirox following(Trifluoperazine) repositionable drugs have been also analyzed (Table 4). In that case, the polar 1 amide bond2.38 could replace 42.23the nitrogen-protonable 207.27 part. However, lipophilic MW < 250 moieties are Yes High 1 1. Ciclopirox 2.38 42.23 207.27 MW <2 250 Yes High LogP = octanol/water partitionclearly coefficient present, calculatedsuch as para by-chlorobenzyl SwissADME; orTPSA cyclohexyl = topology groups. polar surfaceIt is notable area by that Swis- tauroli- 3 sADME;Ciclopirox Druglikliness = criteriadine, whicha bis-sulfonamide should be problemati drug, isc forthe drug only development repositionable according drug, towhich SwissADME. is hydrophilic, with Ciclopirox low GI absorption. 2 The1.75 last sub-group22.0 274.18 which could Nobe addressedalert is based Yes on an amide High fragment . The 2 Ebselen followingTable 4. Critical1.75 repositionable analysis of drugs 22.0amide-based have been repositionable also 274.18 analyzed drugs. (Table 4). In No that alert case, the polar Yes High 2 amide 1.75bond could22.0 replace 274.18 the nitrogen-protonable No alert part. However, Yes lipophilic High moieties are Ebselen clearly present, such as para-chlorobenzyl or cyclohexyl groups. It is notable that tauroli- Ebselen TPSA 2 Druglike-Liness 3 GI 5 2 dine, a1.75 bis-sulfonamide 22.0 274.18 drug, is the only No alert repositionable drug, Yes which is hydrophilic, High with Entry Structures LogP 1 M (g/mol) BBB Permeant 4 low GI absorption. 3 3 Ebselen 2.442.44 47.56 (Å) 275.34 47.56 No alert 275.34 (Alert) Yes No alert HighAbsorption Yes High Rolipram RolipramTable 4. Critical analysis of amide-based repositionable drugs. 3 2.44 47.56 275.34 No alert Yes High Rolipram 2 3 5 3 2.44 47.56 TPSA 275.34 No Druglike-Linessalert Yes High GI Entry Structures LogP 1 M (g/mol) BBB Permeant 4 Rolipram 4 2.82 51.24 (Å) 388.84 MW > 350(Alert) Yes HighAbsorption

4 Fipexide 2.82 51.24 388.84 MW > 350 Yes High

Fipexide 5 −1.53 115.58 284.36 0 alert No Low

5 −1.53 115.58 284.36 0 alert No Low Taurolidine

5 1. LogP = octanol/waterTaurolidine partition coefficient−1.53 calculated 115.58 by SwissADME; 284.36 2 TPSA 0 alert = topology polar surface No area by Swis- Low

sADME; 3 Druglikliness = criteria which should be problematic for drug development according to SwissADME; 4 BBB 1.permeant: blood brain barrier permeant as predicted by SwissADME; 5 gastro-intestinal2 permeation ability as predicted LogP = octanol/waterTaurolidine partition coefficient calculated by SwissADME; TPSA = topology polar surface area by Swis- sADME;by SwissADME. 3 Druglikliness = criteria which should be problematic for drug development according to SwissADME; 4 BBB 5 1.permeant: LogP = octanol/water blood brain barrier partition permeant coefficient as predicted calculated by by SwissADME; SwissADME; gastro-intestinal 2 TPSA = topology permeati polar onsurface ability area as predictedby Swis- All others repositionable compounds except artemisin and hexaclophene exhibited a sADME;by SwissADME. 3 Druglikliness = criteria which should be problematic for drug development according to SwissADME; 4 BBB permeant: blood brain barriernitrogen permeant protonable as predicted group by SwissADME; or a quaternary 5 gastro-intestinal ammonium permeati coupledon with ability a lipophilic as predicted moiety by SwissADME. evenAll if noothers common repositionable core can becompounds distinguished except (Figure artemisin 7). Their and hexaclophenelogP ranged from exhibited 2.98 toa nitrogen5.11, proving protonable their lipophilicity. group or a quaternary ammonium coupled with a lipophilic moiety evenAll if no others common repositionable core can be compounds distinguished except (Figure artemisin 7). Their and hexaclophenelogP ranged from exhibited 2.98 to a nitrogen5.11, proving protonable their lipophilicity. group or a quaternary ammonium coupled with a lipophilic moiety even if no common core can be distinguished (Figure 7). Their logP ranged from 2.98 to 5.11, proving their lipophilicity.

Microorganisms 2021, 9, x 13 of 42

1 2.38 42.23 207.27 MW < 250 Yes High

1 2.38 42.23 207.27 MW < 250 Yes High Ciclopirox Ciclopirox

2 1.75 22.0 274.18 No alert Yes High 2 1.75 22.0 274.18 No alert Yes High Ebselen Ebselen Microorganisms 2021, 9, 634 13 of 40

3 2.44 47.56 275.34 No alert Yes High 3 Rolipram 2.44 47.56 275.34 Table No4. alertCont. Yes High Rolipram TPSA 2 Druglikeliness 3 GI 5 Entry Structures LogP 1 M (g/mol) BBB Permeant 4 (Å) (Alert) Absorption

4 2.82 51.24 388.84 MW > 350 Yes High 4 4 2.822.82 51.24 388.84 51.24 MW > 350 388.84 Yes MW > 350High Yes High Fipexide Fipexide Fipexide

5 −1.53 115.58 284.36 0 alert No Low 5 −1.53 115.58 284.36 0 alert No Low

5 Taurolidine −1.53 115.58 284.36 0 alert No Low Taurolidine 1. LogP = octanol/water partition coefﬁcient calculated by SwissADME; 2 TPSA = topology polar surface area by SwissADME; 3 Druglikliness = criteria which should be problematic for drug development according to SwissADME; 4 BBBTau permeant:rolidine blood brain barrier permeant as predicted by SwissADME; 5 gastro-intestinal permeation ability as predicted by SwissADME. 1. LogP = octanol/water partition coefficient calculated by SwissADME; 2 TPSA = topology polar surface area by Swis- 3 4 sADME;1. LogP = Drugliklinessoctanol/water =partition criteria whichcoefficient should calculated be problemati by SwissADME;c for drug development2 TPSA = topology according polar to surface SwissADME; area by Swis-BBB 5 permeant:sADME; 3blood Druglikliness brain barrier = criteria permeant which as should predicted be problemati by SwissADME;c for drug gastro-intestinal development accordingpermeation to ability SwissADME; as predicted 4 BBB bypermeant: SwissADME. blood brain barrier permeant as predicted by SwissADME; 5 gastro-intestinal permeation ability as predicted by SwissADME. All others repositionable compounds except artemisin and hexaclophene exhibited a nitrogenAll protonableothers repositionable group or a compounds quaternary exceptammonium artemisin coupled and hexaclophenewith a lipophilic exhibited moiety a evennitrogen if no protonable common core group can or be a distinguishedquaternary ammonium (Figure 7). coupled Their log withP ranged a lipophilic from 2.98moiety to 5.11,even proving if no common their lipophilicity. core can be distinguished (Figure 7). Their logP ranged from 2.98 to 5.11, proving their lipophilicity.

Microorganisms 2021, 9, 634 14 of 40

The last sub-group which could be addressed is based on an amide fragment. The following repositionable drugs have been also analyzed (Table4). In that case, the polar amide bond could replace the nitrogen-protonable part. However, lipophilic moieties are clearly present, such as para-chlorobenzyl or cyclohexyl groups. It is notable that taurolidine, a bis-sulfonamide drug, is the only repositionable drug, which is hydrophilic, with low GI absorption. All others repositionable compounds except artemisin and hexaclophene exhibited a nitrogen protonable group or a quaternary ammonium coupled with a lipophilic moiety Microorganisms 2021, 9, x even if no common core can be distinguished (Figure7). Their log P ranged from14 2.98 of to42

5.11, proving their lipophilicity.

Figure 7. Critical analysis for repurposing drugs.

4. Combination Combination Drugs Drugs Because discovering co-drugsco-drugs capable capable of of overcoming overcoming resistance resistance to frontlineto frontline antifungals antifun- galsis of is prime of prime clinical clinical importance, importance, combination combination drugs weredrugs often were reported often reported during theduring last fivethe lastyears. five The years. potential The combinationspotential combinations could be between could be known between antifungal known agents antifungal (Section agents 4.1), (Sectionbetween 4.1), repurposing between drugrepurposing and one drug antifungal and one (Section antifungal 4.2) or(Section between 4.2) a or novel between active a novelcompound active and compound an “old” and antifungal an “old” (Section antifungal 4.3). (Section 4.3). 4.1. Combination of Antifungal Drugs 4.1. Combination of Antifungal Drugs To date, only in vitro results are described in this part. To date, only in vitro results are described in this part. In 2017, Fakhim et al. reported the effects of combination between echinocandins In 2017, Fakhim et al. reported the effects of combination between echinocandins and and triazoles against 10 multidrug-resistant C. auris isolates [61]. Using a microdilution triazolescheckerboard against technique, 10 multidrug-resistant they screened the C. inauris vitro isolatesinteractions [61]. Using of azoles a microdilution and echinocandins check- erboardin indian technique, clinical isolates they screened resistant the to in fluconazole vitro interactions and/or of micafungin. azoles and echinocandins They discovered in indianpromising clinical synergistic isolates resistant interactions to fluconazole for two couples: and/ormicafungin/voriconazole micafungin. They discovered and prom- mica- isingfungin/fluconazole. synergistic interactions This strategy for two has couples: also been micafungin/voriconazole demonstrated recently byand the micafun-in vitro gin/fluconazole.synergistic combination This strategy of isavuconazole has also been (azole demonstrated derivative) recently with colistin by the [62 in]. vitro syner- gisticIn combination 2020, O’Brien of etisavuconazole al. tested if two-drug (azole derivative) combinations with werecolistin effective [62]. in vitro against multidrug-resistantIn 2020, O’BrienC. et auris al. testedisolates if two-drug [63,64]. From combinations nine reference were antifungals effective in and vitro around against a multidrug-resistantthousand tested combinations, C. auris isolates they reported[63,64]. From that flucytosinenine reference (5FC) antifungals at 1.0 µg/mL and potenti-around aated thousand the most tested combinations, combinations, especially they reported in the case that of flucytosine amphotericin (5FC) B, at echinocandins 1.0 µg/mL poten- and tiatedvoriconazole the most resistant combinations, strains whereespecially its addition in the case allowed of amphotericin to restore the B, echinocandins fungicidal activity. and voriconazole resistant strains where its addition allowed to restore the fungicidal activity.

4.2. Combinations of a Repositioning Drug with an Antifungal Drug 4.2.1. In Vitro Screening In 2018, Seleem et al. studied the combinations between sulfa drugs and reference azole antifungals. Among the active sulfa drugs, the bacteriostatic antibiotic sulfamethoxazole exhibited the most potent in vitro synergistic interactions with voriconazole and itraconazole (Figure 8). The addition of sulfamethoxazole restored the activity of azole drugs against azole-resistant strains if the resistance originated from either overproduction of or decreased affinity for the azole target (ERG11p). Strains resistant because of efflux pump hyperactivity were not susceptible to these combinations [65].

Microorganisms 2021, 9, x 15 of 42 Microorganisms 2021, 9, 634 15 of 40

In a parallel manner, in 2020, the same team from Seleem, Mayhoub et al. evaluated 4.2. Combinations of a Repositioning Drug with an Antifungal Drug the ability of Pharmakon® 1600 drug library to sensitize an azole-resistant C. albicans to 4.2.1.the effect In Vitro of fluconazole Screening [66]. They discovered that pitavastatin, a relatively newly devel- opedIn cholesterol 2018, Seleem lowering et al. studiedagent, was the combinationsa potent azole between chemosensitizing sulfa drugs agent and (Figure reference 8). azoleFrom antifungals.a chemical point Among of theview, active pivastatin sulfa drugs, belongs the to bacteriostatic the quinoline antibiotic sub-group, sulfamethox- including azolesome exhibitedrepurposing the drugs. most potent Pivastatinin vitro displayedsynergistic broad-spectrum interactions withsynergistic voriconazole interactions and itraconazolewith both fluconazole (Figure8). even The against addition C. of auris sulfamethoxazole strains (MIC from restored 8 to 64 the µg/mL activity for ofcombina- azole drugstion against against 64 azole-resistant to 256 µg/mL strains for drugs if the alone). resistance Moreover, originated pitavastatin-fluconazole from either overproduction combi- ofnation or decreased significantly afﬁnity reduced for the the azole biofilm-forming target (ERG11p). abilities Strains of the resistant tested Candida because species of efﬂux by pumpup to 73%. hyperactivity were not susceptible to these combinations [65].

FigureFigure 8.8. StructureStructure ofof PivastatinPivastatin andand Sulfamethoxazole.Sulfamethoxazole.

InIn a2019, parallel the manner,team of inChowdhary 2020, the same reported team the from in Seleem,vitro effects Mayhoub of combination et al. evaluated between the abilitygeldanamycin of Pharmakon (Hsp90-inhibitor)® 1600 drug library with triazoles to sensitize and an echinocandins azole-resistant C.against albicans commonto the effect and ofemerging fluconazole Candida [66]. Theyspecies discovered (Figure that8) [67]. pitavastatin, Whereas a relativelysynergistic newly interactions developed between cholesterol gel- loweringdamycin agent,and antifungal was a potent drugs azole were chemosensitizing observed for C. agent albicans (Figure, C. glabrata8). From and a chemical C. parapsilosis point , ofno view, interesting pivastatin effect belongs was observed to the quinoline for any sub-group,combination including against C. some auris repurposing. drugs. PivastatinDannaoui displayed et al. broad-spectrum evaluated the in synergistic vitro interaction interactions between with bothcolistin fluconazole and two evenechi- againstnocandinsC. auris (caspofunginstrains (MIC and from micafungin) 8 to 64 µ g/mLagainst for 15 combination C. auris isolates against [68]. 64 Colistin, to 256 µ org/mL pol- forymyxin drugs E, alone). is a last-resort Moreover, antibiotic pitavastatin-fluconazole against multidrug combination resistant significantlygram-negative reduced infections the biofilm-forming(Figure 9). It displayed abilities ofno the activity tested withCandida MICspecies of >64 by µg/mL up to for 73%. all the isolates. However, whenIn colistin 2019, the was team combined of Chowdhary with caspofungin, reported the synergisticin vitro effects interactions of combination were observed between for geldanamycinall strains. (Hsp90-inhibitor) with triazoles and echinocandins against common and emergingVery Candidarecently,species Seleem (Figure et al. demonstrated8)[ 67]. Whereas that synergistic aprepitant, interactions an antiemetic between agent, gel-is a damycinnovel broad-spectrum and antifungal azole drugs chemosensitizing were observed for agentC. albicans (Figure, C. 9). glabrata The combinationand C. parapsilosis aprepi-, notant/itraconazole interesting effect is wasparticularly observed efficient for any in combination an in vivo C. against elegansC. model auris. of infection by C. aurisDannaoui. This synergistic et al. evaluated relationship the in could vitro beinteraction mediated between by interferences colistin and with two metal echinocan- ion ho- dinsmoestasis (caspofungin and subsequent and micafungin) impact on against ROS 15prodC.uction auris isolates [69]. The [68 ].same Colistin, team oralso polymyxin reported E,that is alopinavir, last-resort an antibiotic HIV protease against inhibitor, multidrug is able resistant to chemosensitize gram-negative azole infections drug. (Figure 9). It displayed no activity with MIC of >64 µg/mL for all the isolates. However, when colistin was4.2.2. combined In Vivo Confirmation with caspofungin, synergistic interactions were observed for all strains. Very recently, Seleem et al. demonstrated that aprepitant, an antiemetic agent, is a Following the report by Seleem et al., the synergy between sulfamethoxazole and novel broad-spectrum azole chemosensitizing agent (Figure9). The combination aprepi- voriconazole was confirmed in vivo in a C. elegans model of C. auris infection [65]. tant/itraconazole is particularly efﬁcient in an in vivo C. elegans model of infection by C. aurisMoreover,. This synergistic the same relationshipgroup demonstrated could be in mediated vivo that by the interferences lopinavir/itraconazole with metal com- ion homoestasisbination decreased and subsequent the fungal impact burden on ROSin C. productionauris-infected [69 ].C. The elegans same nematodes team also reportedby 88.5% thatand lopinavir,enhanced antheir HIV survival protease rate inhibitor, by 90% relati is ableve toto chemosensitizethat of the untreated azole control drug. [70]. Com- paring lopinavir and aprepitant, both studies reported by the same team on the same in vivo protocols, lopinavir seems to be more potent than aprepitant in the optimized combination with itraconazole.

Microorganisms 2021, 9, 634 16 of 40 Microorganisms 2021, 9, x 16 of 42

Microorganisms 2021, 9, x 16 of 42

FigureFigure 9.9. StructureStructure ofof Colistin,Colistin, LopinavirLopinavir andand Aprepitant.Aprepitant.

4.2.2.4.3. Combination In Vivo Confirmation of New Compounds and Old Drug: In Vitro Results FollowingRevie, Robbins the report et al. recently by Seleem described et al., thethe synergyability of between oxadiazolecontaining sulfamethoxazole macrocy- and voriconazoleclic peptides wasto potentiate confirmed the in vivoantifungal in a C. ac eleganstivity modelof fluconazole of C. auris [71].infection Few of [65 the]. newly obtainedMoreover, oxadiazole-containing the same group demonstrated macrocyclic peptidesin vivo that displayed the lopinavir/itraconazole activity against Candida com- binationspp on their decreased own, but the many fungal increased burden the in C. efficacy auris-infected of fluconazole,C. elegans resultingnematodes in a synergistic by 88.5% andcombination enhanced (Figure their survival10). rate by 90% relative to that of the untreated control [70]. Comparing lopinavir and aprepitant, both studies reported by the same team on the same inFigure vivo 9.protocols, Structure lopinavir of Colistin, seems Lopinavir to be and more Aprepitant. potent than aprepitant in the optimized combination with itraconazole. 4.3. Combination of New Compounds and Old Drug: In Vitro Results 4.3. Combination of New Compounds and Old Drug: In Vitro Results Revie,Revie, Robbins Robbins et al.et recentlyal. recently described described the ability the ofability oxadiazolecontaining of oxadiazolecontaining macrocyclic macrocy- peptidesclic peptides to potentiate to potentiate the antifungal the antifungal activity of fluconazoleactivity of [ 71fluconazole]. Few of the [71]. newly Few obtained of the newly oxadiazole-containingobtained oxadiazole-containing macrocyclic peptides macrocyclic displayed peptides activity displayed against Candida activity spp against on their Candida own,spp on but their many own, increased but many the efficacy increased of fluconazole, the efficacy resulting of fluconazole, in a synergistic resulting combination in a synergistic (Figurecombination 10). (Figure 10).

Figure 10. Oxadiazole-containing macrocylic peptides and azoffluxin.

The synthetic library created by Boston University’s Center for Molecular Discovery® (BU-CMD) was screened at 50 µM to identify azole-synergizing compound. Azoffluxin, due to its strong synergistic interaction with fluconazole against a resistant strain of C. auris Ci6684 was identified (Figure 10). Azoffluxin enhances azole efficacy in a Cdr1-dependent manner [72]. Seleem et al. tested nine stilbene compounds for their ability to interact synergistically with azole drugs, particularly against azole-resistant fungal isolates (Figure 11) [73]. Ospemifene displayed the most potent azole chemosensitizing activity and increased the potency of itraconazole against C. auris. Indeed, when MIC are superior to 256 µg/mL for FigurebothFigure ospemifene 10. 10.Oxadiazole-containing Oxadiazole-containing and itraconazole macrocylic macrocylic alone, peptides their peptides combination and azofﬂuxin. and azoffluxin. exhibited a MIC equal to 2

µg/mL. Moreover, the authors determined that ospemifene interfered directly with fungal® effluxTheThe systems, synthetic synthetic thus library allowinglibrary created created the byfungal Boston by Bostonintake University’s of University’s itraconazole. Center Center for Molecular for Molecular Discovery Discovery® (BU-CMD) was screened at 50 µM to identify azole-synergizing compound. Azoffluxin, (BU-CMD) was screened at 50 µM to identify azole-synergizing compound. Azoffluxin, due to its strong synergistic interaction with fluconazole against a resistant strain of C. auris Ci6684due to was its identifiedstrong synergistic (Figure 10 ).interaction Azoffluxin enhanceswith fluconazole azole efficacy against in a Cdr1-dependent a resistant strain of C. mannerauris Ci6684 [72]. was identified (Figure 10). Azoffluxin enhances azole efficacy in a Cdr1-de- pendentSeleem manner et al. tested [72]. nine stilbene compounds for their ability to interact synergistically withSeleem azole et drugs,al. tested particularly nine stilbene against compounds azole-resistant for fungal their isolates ability (Figure to interact 11)[73 synergisti-]. cally with azole drugs, particularly against azole-resistant fungal isolates (Figure 11) [73]. Ospemifene displayed the most potent azole chemosensitizing activity and increased the potency of itraconazole against C. auris. Indeed, when MIC are superior to 256 µg/mL for both ospemifene and itraconazole alone, their combination exhibited a MIC equal to 2 µg/mL. Moreover, the authors determined that ospemifene interfered directly with fungal efflux systems, thus allowing the fungal intake of itraconazole.

Microorganisms 2021, 9, x 17 of 42 Microorganisms 2021, 9, 634 17 of 40 Microorganisms 2021, 9, x 17 of 42

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Ospemifene displayed the most potent azole chemosensitizing activity and increased the potency of itraconazole against C. auris. Indeed, when MIC are superior to 256 µg/mL for both ospemifene and itraconazole alone, their combination exhibited a MIC equal Microorganisms 2021, 9, x to 2 µg/mL. Moreover, the authors determined that ospemifene interfered directly17 withof 42

fungal efﬂux systems, thus allowing the fungal intake of itraconazole.

Figure 11. Stilbene compounds tested by Seelem et al. in combination with itraconazole. Figure 11. Stilbene compounds tested by Seelem et al. in combination with itraconazole. Figure 11. StilbeneAhmad compounds et al. examinedtested by Seelem the effects et al. in of combination combination with of itraconazole. natural monoterpene phenols Figure 11. Stilbenewith Ahmadantifungal compounds et al.drugs examinedtested against by Seelem the 25 clinicaleffects et al. in ofisolates combination combination of C. withauris of itraconazole. [74].natural None monoterpene of the monoterpene phenols phenolswith Ahmadantifungal was activeet al.drugs examinedalone against against the 25 clinicalC.effects auris isolatesof (Table combination 5).of C.Carvacrol auris of [74].natural was None the monoterpene ofmost the activemonoterpene phenols phenol withphenolswith Ahmadmedianantifungal was activeetMIC al.drugs ofexamined alone 125 against µg/mLagainst the 25 clinicalandeffectsC. auris its ofisolatescombination (Table combination 5).of C.Carvacrol auriswith of [74].naturalamphotericin was None the monoterpene ofmost the B, activemonoterpenenystatin, phenols phenol flu- conazolewithphenols antifungalmedian wasand activeMICcaspofungin drugs of alone 125 against µg/mLagainst resulted 25 clinical andC. synergistic auris its isolatescombination (Table and 5).of additiveC.Carvacrol auriswith [74]. amphotericineffects was None thein 64%, ofmost the B, 96%, activemonoterpenenystatin, 68%, phenol andflu- Figure 11. Stilbene compounds tested 28%,conazolephenolswith by respectively.median Seelem was and activecaspofunginMIC et al. of in alone 125combination againstµg/mL resulted andC.with synergistic auris itsitraconazole. combination(Table and 5). additiveCarvacrol with amphotericineffects was thein 64%, most B, 96%, activenystatin, 68%, phenol andflu- 28%,withconazole respectively.median and MICcaspofungin of 125 µg/mL resulted and synergistic its combination and additive with amphotericin effects in 64%, B, 96%, nystatin, 68%, andflu- AhmadAhmadTable 5. et Antifungal al. examined conazole28%, activity respectively. andthe of effects caspofunginphenolic of compounds combinationcombination resulted against synergistic of natural C. auris and monoterpeneas reported additive by effects Ahmad phenols in et 64%, al. 96%, 68%, and with antifungal Table 5. Antifungal drugs 28%, against activity respectively. 25 of clinical phenolic isolates compounds of C. againstauris [74].[74 C.]. auris None None as of ofreported the the monoterpene monoterpene by Ahmad et al. phenolsTable was 5. active Antifungal alone activity against of C.ph aurisenolic(Table compounds5). Carvacrol against C. was auris the as mostreported active by Ahmad phenol et al. phenols was active alone against C. auris (Table 5). Carvacrol was the mostMIC activeValues phenol (µg/mL) (n = 3) with median Table 5. MICAntifungal of 125 activity µµg/mLg/mL of and andphenolic its its combination combination compounds againstwith with amphotericinamphotericin C. auris as reported B, B, nystatin, nystatin, by Ahmad flu- ﬂu- et al. conazoleTest and Agents caspofungin resulted synergistic Structure and additive effects in 64%,MIC 96%, Values 68%, (µg/mL) and (n = 3) conazole and caspofungin resulted synergistic and additive effects in 64%,MIC 96%, Values 68%, (µg/mL) and (n = 3) 28%, respectively. 28%, respectively.Test Agents Structure MIC ValuesMedian (µg/mL) (Range) ( n = 3) Test Agents Structure Median (Range) Table 5. Antifungal Table activity 5. AntifungalTest of Agents phenolic activity compounds of phenolic against compounds Structure C. aurisagainst as reportedC. auris by asAhmad reported et al. by Ahmad et al. Median (Range) MIC Values (µg/mL)Median (n = 3) (Range) Test Agents Structure Carvacrol MIC Values Median(µg/mL) (Range) (n = 3)125 (62–250) Test Agents Carvacrol Structure 125 (62–250) Carvacrol 125 (62–250) Median (Range) CarvacrolCarvacrol 125 (62–250) 125 (62–250)

Thymol 312 (156–625) CarvacrolC. auris Thymol 125 (62–250) 312 (156–625) Thymol Thymol 312 (156–625) 312 (156–625) C. auris C. auris C. auris Thymol 312 (156–625)

C. auris Eugenol 625 (312–1250) Eugenol Eugenol 625 (312–1250)625 (312–1250) Thymol Eugenol 312 (156–625) 625 (312–1250) C. auris Eugenol 625 (312–1250) Methyl Methyl eugenol 1250 (625–1250)1250 (625–1250) eugenol Methyl eugenol 1250 (625–1250) Methyl eugenol 1250 (625–1250) Eugenol 5. NovelMethyl Compounds eugenol 625 (312–1250) 1250 (625–1250) 5. Novel Compounds In this section, we reviewed series of novel compounds which have displayed a good 5. Novel Compounds to potent activity against C. auris. For each chemical class, a critical analysis based on its 5. Novel Compounds Methyl eugenol 5. Novel Compounds 1250 (625–1250)

5. Novel Compounds

Microorganisms 2021, 9, 634 18 of 40

Microorganisms 2021, 9, x chemicophysical descriptors will be performed for comparison purpose. The ﬁrst section19 of 42 is

devoted to compounds with potent in vitro activities, whereas the second section concerns chemical classes with proved in vivo efﬁcacy.

5.1. Small Small Molecules Molecules 5.1.1. In In Vitro Vitro Evaluation InIn 2018, in order to target virulence traits,traits, Selam et al.al. described the screening of a 698 small-molecule collection against the invasive hyphal growth of C. albicans [[75].75]. From theirtheir data, data, niclosamide, niclosamide, an FDA-approved FDA-approved anthelmintic inin humans and one of its analogue (N1-(3,5-dichlorophenyl)-5-chlor1-(3,5-dichlorophenyl)-5-chloro-2-hydroxybenzamide),o-2-hydroxybenzamide), an an halogenated salicylanilidesalicylanilide (1)—emerged as capable of inhibitinhibit both filamentationfilamentation and biofilmbiofilm formation.formation. Based on thesethese preliminary results,results, theythey extendedextended their their work work to to a a series series of of a dozena dozen of of compounds compounds to toimprove improve their their biological biological activities. activities. Therefore, Therefore, they they brought brought toto lightlight compoundscompounds 3 and 77,, which displayed both anti-filamentation anti-filamentation and biofilmbiofilm formation capacities (Figure 1212).). This series ofof compoundscompounds are are amide-based amide-based small small molecules molecules with with balanced balanced lipophilicity lipophilicity induced in- ducedby addition by addition of halogenated of halogenated substituents. substituents They could. They be could related be to related the previously to the previously described describedamide-based amide-based repurposing repurpos drugs.ing As example,drugs. As log example,P of Compound logP of Compound1 was calculated 1 was atcalcu- 4.10 latedand its at topological4.10 and its topological polar surface polar area surface equal area to 49.33Å, equal to which 49.33Å, is in which accordance is in accordance with the withvalues the observed values observed for repurposing for repurposing drugs active drugs against activeC. against auris. C. auris.

Figure 12. Series of derivatives from niclosamide and their activi activitiesties towards growth and ﬁlamentationfilamentation as reported by Selam et al.

InIn 2018, 2018, Genberg Genberg et et al. al. also also filed filed a patent a patent concerning concerning methods methods for fortreating treating fungal fungal infectionsinfections using using a cationic a cationic steroid steroid antimicrobial antimicrobial (CSA) (CSA) [76]. Some [76]. cationic Some cationic steroid steroidantimicro- an- bialstimicrobials have been have described been described and exhibited and exhibited a broad-spectrum a broad-spectrum of activity of against activity a against panel of a fungi,panel ofincluding fungi, including Candida Candidaspp. (Figure spp. 13). (Figure In such 13). Instructures, such structures, the steroid the steroidcore is highly core is lipophilichighly lipophilic and the and aminoalkyloxychains the aminoalkyloxychains could couldbring bringa little a hydrophilicity little hydrophilicity and also and pro- also tonableprotonable primary primary or secondary or secondary amino amino groups. groups. In 2019, Tetz et al. described the in vitro activity of a novel compound named MYC- 053 (Figure 13) against clinically significant multidrug resistant strains such as C. glabrata, C. auris, C. neoformans and Pneumocystis spp. [77,78]. Under their screening conditions, MYC-053 proved to be equally effective against both susceptible control strains, clinical isolates, or resistant strains and exhibited MICs ranging from 0.125 to 4.0 µg/mL. The profile of activity of MYC-053 is unique, because, unlike other antifungals such as azoles, polyenes, and echinocandins, MYC-053 was effective against Pneumocystis isolates. The

compound is currently under development by TGV-Therapeutics. MYC-053, as a sodium Figuresalt, is 13. hydrophilic CSA steroids (log as Ppatented= −0.60). by Genberg, Its topological Beus and polar Savage surface and MYC-053 area is equalas reported to 78.76 by Å.

Tetz.The pyrimidine dione core (in grey) is quite similar to amide-based derivatives and the 2-hydroxy-3,5-dichlorophenyl moiety helped to increase the lipophilicity. In 2019, Tetz et al. described the in vitro activity of a novel compound named MYC- 053 (Figure 13) against clinically significant multidrug resistant strains such as C. glabrata, C. auris, C. neoformans and Pneumocystis spp. [77,78]. Under their screening conditions, MYC-053 proved to be equally effective against both susceptible control strains, clinical

Microorganisms 2021, 9, x 19 of 42

5.1. Small Molecules 5.1.1. In Vitro Evaluation In 2018, in order to target virulence traits, Selam et al. described the screening of a 698 small-molecule collection against the invasive hyphal growth of C. albicans [75]. From their data, niclosamide, an FDA-approved anthelmintic in humans and one of its analogue (N1-(3,5-dichlorophenyl)-5-chloro-2-hydroxybenzamide), an halogenated salicylanilide (1)—emerged as capable of inhibit both filamentation and biofilm formation. Based on these preliminary results, they extended their work to a series of a dozen of compounds to improve their biological activities. Therefore, they brought to light compounds 3 and 7, which displayed both anti-filamentation and biofilm formation capacities (Figure 12). This series of compounds are amide-based small molecules with balanced lipophilicity induced by addition of halogenated substituents. They could be related to the previously described amide-based repurposing drugs. As example, logP of Compound 1 was calculated at 4.10 and its topological polar surface area equal to 49.33Å, which is in accordance with the values observed for repurposing drugs active against C. auris.

Figure 12. Series of derivatives from niclosamide and their activities towards growth and filamentation as reported by Selam et al.

In 2018, Genberg et al. also filed a patent concerning methods for treating fungal infections using a cationic steroid antimicrobial (CSA) [76]. Some cationic steroid antimicro- bials have been described and exhibited a broad-spectrum of activity against a panel of fungi, including Candida spp. (Figure 13). In such structures, the steroid core is highly Microorganisms 2021, 9, 634 lipophilic and the aminoalkyloxychains could bring a little hydrophilicity19 of 40 and also pro-

Microorganisms 2021, 9, x tonable primary or secondary amino groups. 20 of 42

isolates, or resistant strains and exhibited MICs ranging from 0.125 to 4.0 µg/mL. The profile of activity of MYC-053 is unique, because, unlike other antifungals such as azoles, polyenes, and echinocandins, MYC-053 was effective against Pneumocystis isolates. The compound is currently under development by TGV-Therapeutics. MYC-053, as a sodium salt, is hydrophilic (logP = −0.60). Its topological polar surface area is equal to 78.76 Å. The pyrimidine dione core (in grey) is quite similar to amide-based derivatives and the 2-hydroxy-3,5-dichlorophenylFigure 13. 13.CSA CSA steroids steroids as patented moietyas patented by helped Genberg, by to Genberg, Beusincrease and Savagethe Be uslipophilicity. andand MYC-053 Savage asand reported MYC-053 by Tetz. as reported by In 2020, Soliman et al. described the chemical association of the essential oil Tetz. cuminaldehydeIn 2020, Soliman and azole et al. moieties described (as the present chemical in sulconazole association of or the voriconazole) essential oil cuminalde-in order to takehyde advantage and azole moieties of their(as individual present in previously sulconazole reported or voriconazole) anti-candidal in order properties to take advantage (Figure 14)of their [79].In individual UoST2019, series Tetz previously was et al. evaluated described reported against anti-candidal the C. in albicans vitr propertieso activityand C. (Figureauris of. aOnly 14novel) [ 79UoST5,]. compound UoST UoST7, series named MYC- UoST8was053 evaluated(Figure and UoST11 13) against against displayedC. albicans clinically goodand activityC. significant auris against. Only multidrug UoST5,C. auris UoST7,(MIC resistant ranging UoST8 strainsfrom and UoST112 to such 15 as C. glabrata, µg/mL).displayedC. auris Obtained, goodC. neoformans activity by a againstsix-steps andC. Pneumocystispathway, auris (MIC Uo rangingST5, spp. their from [77,78].best 2 to designed 15 µUnderg/mL). hybrid their Obtained molecule screening by a conditions, six-steps pathway, UoST5, their best designed hybrid molecule exhibited a 2 µg/mL MIC exhibitedMYC-053 a 2proved µg/mL MICto be50 against equally C. auriseffective where against amphotericin both Bsusceptible displayed MIC control50 = 0.350 strains, clinical µg/mL.against C.Compound auris where UoST5 amphotericin was then Bformulated displayed into MIC 50PLGA= 0.3 nanoparticlesµg/mL. Compound (NPs) and UoST5 this formulationwas then formulated proved to into enhance PLGA nanoparticlesand prolong (NPs)the anti-candidal and this formulation activities proved of UoST5. to enhance OsST5 isand a very prolong lipopholic the anti-candidal triazole-based activities compound of UoST5. (log OsST5P = 7.11) is a verywith lipopholicmoderate triazole-basedpolar surface areacompound (TPSA (log= 56.01).P = 7.11) with moderate polar surface area (TPSA = 56.01).

Figure 14. Rational design of cuminaldehyde-azole hybrid compounds by Soliman et al.

Montoya et al. reported that derivatives of the anti-malarial drug mefloquinemefloquine have broad-spectrum antifungalantifungal activityactivity against against pathogenic pathogenic yeasts yeasts and and molds molds [80 ].[80]. These These deriva- de- rivativestives were were tested tested against against a panela panel of of fungal fungal strains, strains, including includingC. C. auris auris strainstrain 03810381 and they revealed MIC fromfrom 2–42–4 µµg/mLg/mL compared to MICMIC == 128 µµg/mLg/mL for mefloquine mefloquine itself (Figure(Figure 15).15). From From a achemical chemical point point of of view, view, mefloquine mefloquine and and its itsderivatives derivatives belongs belongs to the to quinolinethe quinoline chemical chemical class. class. They Theydisplayed displayed the three the features three features defined defined previously: previously: (i) quino- (i) linequinoline core; (ii) core; lipophilic (ii) lipophilic moiety moiety (halogenated (halogenated aromatics) aromatics) and and(iii) (iii)piperidine piperidine protonable protonable ni- trogennitrogen group. group. Mefloquine Mefloquine derivatives derivatives are are quite quite lipophilic lipophilic (log (logPP fromfrom 4.36 4.36 to to 4.72) with moderate TPSA. Orofino et al. reported in 2020 the potency of BM1, a macrocyclic amidinourea active against azole-resistant Candida strains [81]. The authors conducted a complete characterization of the in vitro and in vivo biological properties of BM1 and established its good ADME and biochemical characteristics so as its high activity towards several Candida species, including preliminary data against C. auris (Table6). BM1 exhibited a log P of 3.38 and a very high topological polar surface area of 116.13 Å. From a chemical point of view, it belongs to the guanidine derivatives.

Figure 15. Structure of the derivatives of mefloquine as reported by Montoya et al.

Orofino et al. reported in 2020 the potency of BM1, a macrocyclic amidinourea active against azole-resistant Candida strains [81]. The authors conducted a complete characterization of the in vitro and in vivo biological properties of BM1 and established its good

Microorganisms 2021, 9, x 20 of 42

Figure 14. Rational design of cuminaldehyde-azole hybrid compounds by Soliman et al.

Montoya et al. reported that derivatives of the anti-malarial drug mefloquine have broad-spectrum antifungal activity against pathogenic yeasts and molds [80]. These derivatives were tested against a panel of fungal strains, including C. auris strain 0381 and they revealed MIC from 2–4 µg/mL compared to MIC = 128 µg/mL for mefloquine itself (Figure 15). From a chemical point of view, mefloquine and its derivatives belongs to the quinoline chemical class. They displayed the three features defined previously: (i) quino- Microorganisms 2021, 9, 634 line core; (ii) lipophilic moiety (halogenated aromatics) and (iii) piperidine protonable20 of ni- 40 trogen group. Mefloquine derivatives are quite lipophilic (logP from 4.36 to 4.72) with moderate TPSA. Microorganisms 2021, 9, x 21 of 42

Microorganisms 2021ADME, 9, x and biochemical characteristics so as its high activity towards several Candida spe- 21 of 42 cies, including preliminary data against C. auris (Table 6). BM1 exhibited a logP of 3.38 and a very high topological polar surface area of 116.13 Å . From a chemical point of view, it belongs to the guanidine derivatives. ADME and biochemical characteristics so as its high activity towards several Candida spe- Table 6. Antifungal activitiescies, including of compound preliminary BM1 against data various against Candida C. auris speci (Tablees. 6). BM1 exhibited a logP of 3.38 and a very high topological polar surface area of 116.13 Å. From a chemical point of view, it belongs to the guanidine derivatives. Figure 15. Structure of the derivatives of meﬂoquinemefloquine as reported by MontoyaMontoya etet al.al.

TableTable 6.6. Antifungal activities of compound BM1 against variousCandida Candida species. AntifungalOrofino activities et al. reported of compound in 2020 BM1 the against potency various of BM1, a macrocyclicspecies. amidinourea active against azole-resistant Candida strains [81]. The authors conducted a complete characterization of the in vitro and in vivo biologicalBM1 properties of BM1 and established its good

MIC and MBC Rage (µg/mL) against Candida Species

BM1 C. albicans C. tropicalis C. parapsilosis C. glabrata C. krusei C. auris MIC and MBC Rage (µg/mL) against Candida Species MIC and MBC Rage (µg/mL) against Candida Species MIC MBCC. albicansMIC MBC C. tropicalisMIC MBC C. parapsilosisMIC MBC C.MIC glabrata MBC C.MIC krusei MBC C. auris MICC. albicans MBC MIC C. tropicalis MBC MIC C. parapsilo MBCsis MIC C. glabrata MBC MIC C. krusei MBC MIC C. auris MBC 0.125–2 0.125–22–4 2–42 2–4 2 8–16 2–4 32–64 8–16 32–64 32–64 8–64 32–64 8–64 8–6464–128 8–648–64 64–128128–256 8–64 128–256 MICMBC, minimumMBC bactericidalMBC,MIC minimum concentration;MBC bactericidal MIC MIC, concentration; minimumMBC MIC, inhibitoryMIC minimum MBCconcentration inhibitory MIC concentration.. MBC MIC MBC

0.125–2 2–4 Seleem, 2Mayhoub Seleem,2–4 et al. Mayhoub8–16 are widely32–64 et al. involved are32–64 widely in the involved8–64 development in8–64 the developmentof64–128 polysubstituted 8–64 of polysubstituted 128–256 thiazoles as novelthiazoles antibacterial as novel agents. antibacterial Based agents. on structure Based-activity on structure-activity relationships relationshipsstudies, studies, MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration. they recently describedthey recently oxadiazolylthiazoles described oxadiazolylthiazoles as novel and selective as novel antifungal and selective agents antifungal [82]. agents [82]. Via a five-stepsVia pathway, a ﬁve-steps the pathway,authors obtained the authors a series obtained of oxadiazolylthiazole a series of oxadiazolylthiazole OXA1-21 OXA1-21 Seleem, Mayhoub et al. are widely involved in the development of polysubstituted (Figure 16) and(Figure evaluated 16) and them evaluated against thema panel against of fungal a panel and of bacterial fungal andstrains. bacterial OXA11 strains. OXA11 thiazoles as novel antibacterial agents. Based on structure-activity relationships studies, proved to be provedselective to towards be selective fungal towards strains fungal and the strains most andactive the compound most active from compound this from this they recently described oxadiazolylthiazoles as novel and selective antifungal agents [82]. series, with MICseries, = 2– with4 µg/mL MIC against = 2–4 µC.g/mL auris strains. against Moreover,C. auris strains. OXA11 Moreover, exhibited OXA11a fun- exhibited a Via a five-steps pathway, the authors obtained a series of oxadiazolylthiazole OXA1-21 gistatic behavior,fungistatic a broad behavior,-spectrum a broad-spectrumactivity against fungal activity pathogens, against fungal with pathogens,capability of with capability (Figure 16) and evaluated them against a panel of fungal and bacterial strains. OXA11 disrupting biofilmsof disrupting but without bioﬁlms harming but withoutthe human harming normal the microbiota. human normal The OXA microbiota. deriv- The OXA proved to be selective towards fungal strains and the most active compound from this atives perfectlyderivatives respect the perfectly three common respect features: the three ( commoni) an original features: oxadiazolylthiazole (i) an original oxadiazolylthiazole core series, with MIC = 2–4 µg/mL against C. auris strains. Moreover, OXA11 exhibited a fun- (in grey) coupledcore with (in grey) (ii) a coupledlipophilic with moiety (ii) a(in lipophilic blue) and moiety (iii) a proton (in blue)able and amine (iii) agroup protonable amine gistatic behavior, a broad-spectrum activity against fungal pathogens, with capability of (cyclic or acyclic).group OXA11 (cyclic, the or most acyclic). potent OXA11, reported the molecule most potent from reported this series, molecule displayed from this series, disrupting biofilms but without harming the human normal microbiota. The OXA deriv- logP equal to displayed4.44, a high log topologicalP equal to surface 4.44, a area high of topological 118.10 Å and surface no alert area concerning of 118.10 its Å and no alert atives perfectly respect the three common features: (i) an original oxadiazolylthiazole core drug likeliness.concerning its drug likeliness. (in grey) coupled with (ii) a lipophilic moiety (in blue) and (iii) a protonable amine group (cyclic or acyclic). OXA11, the most potent reported molecule from this series, displayed logP equal to 4.44, a high topological surface area of 118.10 Å and no alert concerning its drug likeliness.

Figure 16.Figure Structures 16. Structures of OXA compounds of OXA compounds described described by Seleem, by Mayhoub Seleem, Mayhoub et al. et al.

Seleem et al. alsoSeleem described et al. also the describedsynthesis theand synthesis biological and evaluation biological of evaluationa small series of a small series

of new pyrazolofo[5 new,1-c pyrazolo[5,1-][1,2,4]triazinesc][1,2,4]triazines against a panel against of fungal a panel strains, of fungal including strains, C. auris including C. auris Figure 16. Structures of OXA compounds described by Seleem, Mayhoub et al. (Figure 17) [83]. Compound PYR4 was the most potent antifungal, with a moderate MIC around 15–20 µg/mL. The chemical feature of PYR4 is composed of a pyrazolotriazine Seleem et al. also described the synthesis and biological evaluation of a small series of new pyrazolo[5,1-c][1,2,4]triazines against a panel of fungal strains, including C. auris (Figure 17) [83]. Compound PYR4 was the most potent antifungal, with a moderate MIC around 15–20 µg/mL. The chemical feature of PYR4 is composed of a pyrazolotriazine

Microorganisms 2021, 9, 634 21 of 40 Microorganisms 2021, 9, x 22 of 42

Microorganisms 2021Microorganisms, 9, x 2021, 9, x 22 of 42 22 of 42

(Figure 17)[83]. Compound PYR4 was the most potent antifungal, with a moderate MIC core, a piperazine protonable nitrogen group and a lipophilic aromatic (see Figure 17). around 15–20 µg/mL. The chemical feature of PYR4 is composed of a pyrazolotriazine core, PYR4, the most potent reported molecule from this series, displayed logP equal to 2.16, a core,a piperazine a piperazine protonable protonable nitrogen nitrogen group group and a and lipophilic a lipophilic aromatic aromatic (see Figure (see Figure17). PYR4, 17). core,medium a piperazine topological protonable surface areanitrogen of 49.56 group Å andand noa lipophilicalert concerning aromatic its (see drug Figure likeliness. 17). PYR4,the most the potent most reportedpotent reported molecule molecu from thisle from series, this displayed series, displayed logP equal log toP 2.16,equal a to medium 2.16, a PYR4,However, the most compared potent to reported other series molecu developedle from this by series,Seleem displayed et al., PYR log derivativesP equal to are2.16, less a mediumtopological topological surface area surface of 49.56 area Å of and 49.56 no Å alert and concerning no alert concerning its drug likeliness. its drug likeliness. However, mediumpowerful topological than OXA surfaceor PHE areaseries. of 49.56 Å and no alert concerning its drug likeliness. However, comparedHowever,compared to othercompared to other series series to developed other developed series by developed Seleem by Seleem et al.,by et al.,SeleemPYR PYR derivatives et derivatives al., PYR are derivatives areless less powerful are less powerful thanpowerfulthan OXA OXA or PHEthan or PHE series.OXA series. or PHE series.

Figure 17. Series of pyrazolo[5,1-c][1,2,4]triazines described by Seleem et al.

Figure 17. SeriesLateFigure 2020,of pyrazolo[5,1-c][1,2,4]triazines 17.Ishida Series and of pyrazolo[5,1-c][1,2,4]triazinesStephani reported described 2-aryloxazoline by Seleem described et al. compounds, byby SeleemSeleem etet al.al.obtained from L-threonine and aromatic carboxylic acid as promising antifungal compounds (Scheme 1) Late 2020, IshidaIshida andand StephaniStephani reported reported 2-aryloxazoline 2-aryloxazoline compounds, compounds, obtained obtained from from L- [84].Late They 2020, discovered Ishida and ArOX-1 Stephani and reportedArOX-2, 2-aryloxazolinewhich exhibited compounds, a strong in vitroobtained antifungal from L-threoninethreonine and and aromatic aromatic carboxylic carboxylic acid acid as promising as promising antifungal antifungal compounds compounds (Scheme (Scheme1)[84 1)] . L-threonineactivity, with and MIC aromatic lower carboxylicthan 0.25 µg/mLacid as forpromising all tested antifungal Candida species.compounds Moreover, (Scheme these 1) They[84]. They discovered discovered ArOX-1 ArOX-1 and ArOX-2, and ArOX-2, which which exhibited exhibited a strong a instrong vitro inantifungal vitro antifungal activity, [84].two Theynon-toxic discovered compounds ArOX-1 were and particularly ArOX-2, which active exhibitedagainst C. a auris strong CBS10913 in vitro (MICs antifungal = 0.06 withactivity, MIC with lower MIC than lower 0.25 thanµg/mL 0.25 for µg/mL all tested for allCandida testedspecies. CandidaMoreover, species. Moreover, these two these non- activity,µg/mL) withand CBS12766MIC lower (MICs than 0.25= 2 µg/mL). µg/mL for all tested Candida species. Moreover, these two non-toxictwotoxic compounds non-toxic compounds were compounds were particularly particularly were active particularly active against against activeC. aurisC. against aurisCBS10913CBS10913 C. auris (MICs CBS10913 (MICs = 0.06 = 0.06(MICsµg/mL = 0.06) µg/mL) and CBS12766µg/mL)and CBS12766 and (MICs CBS12766 (MICs = 2 µg/mL). = (MICs 2 µg/mL). = 2 µg/mL).

Scheme 1. Synthesis of 2-aryloxazoline derivatives (ArOx) and the two most potent compounds

ArOX-1 and ArOx-2. Scheme 1.SchemeSynthesis 1. ofSynthesis 2-aryloxazolineScheme of 2-aryloxazoline1. Synthesis derivatives of 2-aryloxazoline derivatives (ArOx) and (ArOx) the derivatives two and most the potent(ArOx)two most compoundsand potent the two compounds ArOX-1 most potent and ArOx-2.compounds ArOX-1Ganesh and ArOx-2. ArOX-1et al. discovered and ArOx-2. a series of α-iodonitroalkenes as potential antifungal and α antitubercular compounds.Ganesh et Based al. discovered on a α-iodation a series on of β-nitroalkenes,-iodonitroalkenes the authors as potential obtained antifungal and Ganesh et al. discovered a seriesα of α-iodonitroalkenesβ as potential antifungal and a seriesGanesh of derivatives etantitubercular al. discovered (Scheme compounds. a series2) which of Based αexhibited-iodonitroalkenes on a interesting-iodation as on antifungalpotential-nitroalkenes, antifungalactivity the against authorsand obtained a antitubercular compounds. Based on a α-iodation on β-nitroalkenes, the authors obtainedC. auris antitubercularC. auris with seriesMIC compounds. lower of derivatives than Based 8 µg/mL (Scheme on a α for-iodation2) all which compounds. on exhibited β-nitroalkenes, It interesting is notable the antifungal than authors the furan activityobtained and against witha series MIC of lower derivatives than 8 µ(Schemeg/mL for 2) all which compounds. exhibited It isinteresting notable than antifungal the furan activity and thiophene against athiophene series of derivatives derivatives (Scheme were the 2) most which broad-sp exhibitedectrum interesting antifungals antifungal devoted activity of cytotoxicity against C.derivatives auris with were MIC the lower most than broad-spectrum 8 µg/mL for antifungalsall compounds. devoted It is of notable cytotoxicity than the [85 ].furan and C.[85]. auris with MIC lower than 8 µg/mL for all compounds. It is notable than the furan and thiophene derivativesthiophene were derivatives the most were broad-sp the mostectrum broad-sp antifungalsectrum devoted antifungals of cytotoxicity devoted of cytotoxicity [85]. [85].

Scheme 2. α-Iodonitroalkenes synthesis. Scheme 2. α-Iodonitroalkenes synthesis. SchemeAmong 2. α-Iodonitroalke Scheme40 testedAmong 2. hydrazoneαnes-Iodonitroalke 40 synthesis. tested derivatives, hydrazone nes synthesis. fl derivatives,uorinated aryl- ﬂuorinated and heteroaryl-substituted aryl- and heteroaryl-substituted hydrazone (FAH)hydrazone have (FAH)demonstrated have demonstrated broad-sprectrum broad-sprectrum fungicidal activities, fungicidal antibiofilm activities, antibioﬁlm Among 40 tested hydrazone derivatives, fluorinated aryl- and heteroaryl-substituted capacityAmong coupled 40capacity tested with hydrazone coupledlow cytotoxicity with derivatives, low and cytotoxicity did fluorinated not trigger and aryl- didthe and development not heteroaryl-substituted trigger the of resistance development of resis- hydrazone (FAH) have demonstrated broad-sprectrum fungicidal activities, antibiofilm hydrazonewhen exposed (FAH)tance to haveC. when auris demonstratedexposed (Scheme to 3)C. [86].broad-sprectrum auris Compounds(Scheme3)[ FAH-1fungicidal86]. Compounds to FAH-6 activities, exhibited FAH-1 antibiofilm toMIC FAH-6 < exhibited capacity coupledcapacity with coupledlow cytotoxicity with low and cytotoxicity did not trigger and did the not development trigger the developmentof resistance of resistance when exposedwhen to C. exposedauris (Scheme to C. auris 3) [86]. (Scheme Compounds 3) [86]. FAH-1Compounds to FAH-6 FAH-1 exhibited to FAH-6 MIC exhibited < MIC <

Microorganisms 2021, 9, x 23 of 42 Microorganisms 2021, 9, 634 22 of 40 Microorganisms 2021, 9, x 23 of 42

1 µg/mL against nine of the 10 different C. auris strains. From a chemical point of view, µ C. auris these compoundsMIC1 µg/mL < fit 1 theagainstg/mL previouslyagainst nine of ninedescribed the of10 thedifferent model 10 different forC. aurisC. auris strains. antifungals.strains. From Froma chemical a chemical point pointof view, of view, these compounds ﬁt the previously described model for C. auris antifungals. these compounds fit the previously described model for C. auris antifungals.

Scheme 3. Series of fluorinated hydrazone derivatives (FAH) as reported by Watt and Garneau- Tsodikova. SchemeScheme 3.3. Series of fluorinate fluorinatedd hydrazone hydrazone derivatives derivatives (FAH) (FAH) as as reported reported by by Watt Watt and and Garneau- Garneau- Tsodikova.Tsodikova. 5.1.2. In Vitro Screening and In Vivo Validation First described5.1.2.5.1.2. InIn VitroVitroin 2008 ScreeningScreening by Mitsuyama and and In In Vivo Vivoet al. Validation Validation [87], T-2307, an arylamidine, exhibited potent broad-spectrumFirstFirst describeddescribed activities inin against 20082008 byby the MitsuyamaMitsuyama majority et etof al. al.fungal[ 87[87],], T-2307, T-2307,pathogens an an arylamidine, arylamidine,[88], even exhibited exhibited against echinocandin-resistantpotentpotent broad-spectrum broad-spectrum C. albicans activities activities and against againstC. glabrata the majoritythe strains majority of [89,90]. fungal of fungal pathogensT-2307 pathogens is trans- [88], even [88], against even ported intoechinocandin-resistant againstC. albicans echinocandin-resistant by a high-affinityC. albicans spermine C.and albicansC. glabrataand and spermidine C.strains glabrata [89 carrier ,strains90]. T-2307 regulated [89,90]. is transported T-2307 by is trans- into Agp2 [91], andC.ported albicans causes into bycollapse C. a high-affinityalbicans of mitochondria by a spermine high-affinityl membrane and spermidinespermine potential and carrier inspermidine yeast regulated [92]. carrierIn by 2020, Agp2 regulated [91], and by Patterson etcauses Agp2al. reported [91], collapse and that ofcauses mitochondrialarylamidine collapse T-2307of membrane mitochondria as very potential activel membrane in in vitro yeast potential and [92]. in In vivo 2020,in yeast anti- Patterson [92]. In et2020, al. fungal againstreportedPatterson C. auris that et(Figure al. arylamidine reported 12) [93]. that In T-2307 vitroarylamidine asMIC very ranged T-2307 active from inas vitro0.125very and activeto 4 inµg/mL in vivo vitro atantifungal 100% and in vivo against anti- inhibition. Moreover,C.fungal auris against(Figure 3 mg/kg C. 12 auris)[ subcutaneous93 ].(FigureIn vitro 12)MIC [93].once ranged In daily vitro treatment from MIC 0.125ranged with to from 4 T-2307µg/mL 0.125 revealed atto 100%4 µg/mL inhibition. at 100% an improvedMoreover,inhibition. survival 3 rateMoreover, mg/kg and subcutaneousreduced 3 mg/kg kidney subcutaneous once fungal daily treatmentburden once daily compared with treatment T-2307 to control. revealed with T-2307 In an improved revealed 2019, T-2307survivalan was improved acquired rate and survival by reduced Appili rate Therapeu kidney and reduced fungaltics Inc. burden kidney for development compared fungal burden to and control. is compared now In called 2019, to T-2307 control. was In ATI-2307. acquired2019, T-2307 by Appili was acquired Therapeutics by Appili Inc. forTherapeu developmenttics Inc.and for development is now called and ATI-2307. is now called From a chemical point of view, T-2307 displayed the features defined in the previous From a ATI-2307.chemical point of view, T-2307 displayed the features defined in the previous Figure 18. It exhibited two benzimidamide groups which are linked by a lipophilic propyl Figure 18. It exhibitedFrom two a chemical benzimidamide point of view, groups T-2307 which disp arelayed linked the by features a lipophilic defined propyl in the previous chain to a protonable N-subtituted piperidine. From this structure, compound T-2307 is chain to a protonableFigure 18. NIt -subtitutedexhibited two piperidine. benzimidamide From this grou structure,ps which compound are linked byT-2307 a lipophilic is propyl rather like the guanidine sub-group. rather like thechain guanidine to a protonable sub-group. N-subtituted piperidine. From this structure, compound T-2307 is rather like the guanidine sub-group.

Figure 18. StructureFigure of 18. T-2307 Structure and SCY-078 of T-2307 as reportedand SCY-078 by Patterson as reported et al. by and Patterson Larkin et al.,al. and respectively. Larkin et Structure al., respec- evolution of second generationtively. fungerpStructureFigure SCY-247. evolution 18. Structure of second of T-2307generation and SCY-078fungerp SCY-247.as reported by Patterson et al. and Larkin et al., respectively. Structure evolution of second generation fungerp SCY-247. In 2017, LarkinIn 2017, et al. Larkin reported et al.the reported effects of the the effects orally of available the orally SCY-078, available a novel SCY-078, 1,3- a novel 1,3- β-D-glucan βsynthesis-D-glucanIn 2017, inhibitor, synthesis Larkin on et inhibitor, al.grow reportedth morphology on growththe effects morphology and of thebiofilm orally and formation available biofilm in formationSCY-078, C. auris a in novelC. auris 1,3- (Figure 18) (Figure[40].β-D-glucan SCY-078 18)[40 synthesis ].is SCY-078a triterpene inhibitor, is a triterpenecompound on grow compound whichth morphology differs which from differsand echinocandins biofilm from echinocandinsformation by in C. by auris its its oral bioavailabilityoral(Figure bioavailability 18) and [40]. its SCY-078 non-sensitivity and its non-sensitivityis a triterpene towards co towardsthempound most the common which most commondiffers mutations from mutations within echinocandins within the by the protein proteintargetits oral Fks targetbioavailability. SCY-078Fks. SCY-078 exhibited and exhibitedits non-sensitivityan MIC an90 MIC of 190 mg/liter towardsof 1 mg/liter against the most against C. commonaurisC. aurisby inter-mutationsby interrupting within rupting cell-division.cell-division.the protein Additionally, target Additionally, Fks. SCY-078 SCY-078 SCY-078 exhibited displayed displayed an powerful MIC powerful90 of antibiofilm1 mg/liter antibiofilm against activity, activity, C. by auris by reducing by inter- reducing metabolicmetabolicrupting activitycell-division. activity and and thickness Additionally, thickness comp compared aredSCY-078 to tothe displayed the untreated untreated powerful control. control. antibiofilmIn parallel, In parallel, activity, Berkow by Berkow et al.etreducing demonstrated al. demonstrated metabolic the the inactivity vitroin vitro activityandactivity thickness of SCY-078 of SCY-078 comp onared on a collection ato collection the untreated of of100 100 isolates, control. isolates, In represen- parallel, representativetativeBerkow of ofthe theet four al. four demonstrated clades clades of of C.C. auris auristhe .in .The Thevitro overall overall activity mode mode of SCY-078 of SCY-078SCY-078 on a waswas collection 1 1µ µg/mLg/mL of with 100 isolates, MIC50 with MIC50 andrepresentative MIC 90 equalequal of to to the 0.5 0.5 µg/mLfourµg/mL clades and and 1of 1µg/mL, µC.g/mL, auris respectively.. respectively.The overall SCY-078 SCY-078mode of also alsoSCY-078 showed showed was very 1 µg/mL little variation in activity between the clades compared to micafungin, caspofungin or anidula- very little variationwith MIC in50 activity and MIC between90 equal theto 0.5 clades µg/mL compared and 1 µg/mL, to micafungin, respectively. caspofungin SCY-078 also showed or anidulafunginfunginvery little[94]. [94]. variationDue Due to to its itsin first firstactivity encour encouraging betweenaging data, the data, cladesSCY-078 SCY-078 compared was was referred referred to micafungin, as asa prom- a promising caspofungin new ising new compoundcompoundor anidulafungin by by McCarthy McCarthy [94]. Due and and to Walsh Walshits first in in encour their their review aging data, about SCY-078 new strategies was referred and challenges as a prom- challenges againstagainstising new emerging emerging compound and and resistant resistant by McCarthy fungal fungal and pathogens pathogens Walsh [95]. [in95 their]. Since Since review its its discovery, discovery, about new SCY- SCY-078 strategies (named and Ibrexafungerp) has been integrated in a dozen of clinical trials studies and patented [96]. challenges against emerging and resistant fungal pathogens [95]. Since its discovery, SCY-

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Microorganisms 2021, 9, 634 23 of 40 078 (named Ibrexafungerp) has been integrated in a dozen of clinical trials studies and patented [96]. Concerning C. auris, one particular multicenter, open-label, non-comparator, single-arm study to evaluate the efficacy, safety, tolerability and pharmacokinetics of Concerning C. auris, one particular multicenter, open-label, non-comparator, single-arm oral SCY-078 as an emergency use treatment for patients with a documented C. auris in- study to evaluate the efficacy, safety, tolerability and pharmacokinetics of oral SCY-078 fection is still ongoing (ClinicalTrials.gov Identifier: NCT03363841). Recently, in 2020, Ar- as an emergency use treatment for patients with a documented C. auris infection is still endrup et al. investigated the in vitro activity of SCY-078 against C. auris by applying EU- ongoing (ClinicalTrials.gov Identifier: NCT03363841). Recently, in 2020, Arendrup et al. CAST methodology and compared its potential against C. albicans and C. glabrata and in investigated the in vitro activity of SCY-078 against C. auris by applying EUCAST method- reference with six control drugs (anidulafungin, micafungin, amphotericin B, fluconazole, ology and compared its potential against C. albicans and C. glabrata and in reference with six voriconazole, and isavuconazole) [97]. More than 150 strains with various resistance pro- control drugs (anidulafungin, micafungin, amphotericin B, fluconazole, voriconazole, and file were screened and proved to be uniformly susceptible to SCY-078. In parallel, Cha- isavuconazole) [97]. More than 150 strains with various resistance profile were screened andturvedi proved et al. to reported be uniformly that susceptibleIbrexafungerp to SCY-078. was efficient In parallel, against Chaturvedi pan-resistant et al. (defined reported as thatin vitro Ibrexafungerp resistance wasto two efficient or more against azoles, pan-resistant all echinocandins (defined and as in amphotericin vitro resistance B) C. to twoauris orisolates more azoles,from the all outbreak echinocandins in New-York and amphotericin [98] and Ghannoum B) C. auris isolateset al. demonstrated from the outbreak that it inwas New-York active to [ 98control] and Ghannoumskin infection et al.and demonstrated colonization thatof hospitalized it was active patients to control [99]. skin Re- infectioncently, a andreview colonization compiled ofdata hospitalized on Ibrexafungerp, patients [established99]. Recently, it is a a review promising, compiled new dataanti- onfungal Ibrexafungerp, agent to treat established C. auris infections, it is a promising, as patients new experienced antifungal a complete agent to response treat C. auris after infections,treatment as[100]. patients experienced a complete response after treatment [100]. FromFrom a a chemicalchemical pointpoint ofof view,view, SCY-078 SCY-078 is is based based on on a a lipophilic lipophilic tetracyclic-fused tetracyclic-fused core core withwith addition addition of of protonable protonable nitrogen nitrogen moieties moieties (pyridine, (pyridine, triazole triazole and and primary primary amine). amine). Its Its structurestructure is is in in accordance accordance with with the the common common defined defined scaffold. scaffold. SCY-247,SCY-247, a a second-generation second-generation fungerpfungerp whichwhich differsdiffers fromfrom SCY-078SCY-078 by by two two chemical chemical moietiesmoieties (the(the 4-(1H-1,2,4-triazol-5-yl)pyridine4-(1H-1,2,4-triazol-5-yl)pyridine and and 2,3,3-trimethylbutan-2-amine 2,3,3-trimethylbutan-2-amine moieties moieties inin SCY-078SCY-078 that that have have been been replaced replaced by by a a methylester methylester and and a a 2-methylpropan-2-amine 2-methylpropan-2-amine in in SCY-247,SCY-247, respectivelyrespectively (Figure(Figure 18 18)) hashas beenbeen recently recently reported reported by by Ghannoum Ghannoum et et al. al. andand provedproved similar similar activities activities against against a a panel panel of ofCandida Candidastrains strains and and enhanced enhanced potency potency against against C.C. aurisaurisstrains. strains.Moreover, Moreover, SCY-247 SCY-247 retained retained its its potential potential even even when when pH pH decreased decreased [101 [101].]. FirstFirst reportedreported in in 2018, 2018, APX001A APX001A (formely (formely E1210, E1210, Amplyx) Amplyx) is is an an antifungal antifungal agent agent that that inhibitsinhibits thethe fungal fungal enzyme enzyme Gwt1 Gwt1 in in the the glycosylphosphatidylinositol glycosylphosphatidylinositol (GPI) (GPI) biosynthesis biosynthesis pathway.pathway. APX001APX001 isis thethe prodrugprodrug ofof APX001A APX001A (Scheme (Scheme4 ).4). Both Both APX001A APX001A and and APX001 APX001 havehave beenbeen shownshown toto bebe effectiveeffective againstagainst aa variety variety of of fungal fungal species, species, including including Candida Candida spp.,spp., AspergillusAspergillus spp.spp. andand otherother filamentousfilamentous fungi. Compou Compoundnd APX001 APX001 has has emerged emerged as as a anovel novel potent potent antifungal antifungal agent agent against against C.C. auris auris. It. has It has been been successfully successfully tested tested against against 100 100geographically geographically distinct distinct clinical clinical isolates isolates and andexhibited exhibited high high MIC MIC50 and50 MICand90 MIC in the90 in nano- the nanogramgram per liter per literrange range [102,103]. [102, 103Extended]. Extended evalua evaluationtion of large of largepanels panels of strains of strains from differ- from differentent origins origins has confirmed has confirmed the interest the interest of APX001 of APX001 in the in treatment the treatment of infection of infection by C. by aurisC. auris[104].[104 The]. compound The compound APX001 APX001 has entered has entered clinical clinical trials and trials proved and proved good efficacy good efficacy in mice inmodel mice of model candidiasis of candidiasis [105,106]. [105 Fosmanogepix,106]. Fosmanogepix (APX001) (APX001) is currently is currently in Phase in II Phasetrials for II trialsinvasive for invasivefungal infections. fungal infections. Both fosmanogepix Both fosmanogepix and manogepix and manogepix (APX001A) (APX001A) are currently are currentlyunder investigation under investigation and proofs and of proofstheir in of vivo their efinficacy vivo againstefficacy C. against auris infectionC. auris infection accumu- accumulatelate [105–109]. [105 When–109]. Whenanalyzing analyzing its chemical its chemical structure, structure, it appeared it appeared that thatAPX001A APX001A dis- displayedplayed protonable protonable nitrogen nitrogen moieties moieties (primary (primary amine amine and andpyridine) pyridine) coupled coupled with with a lipo- a lipophilicphilic benzyl benzyl group. group. This This balance balance gave gave a log aP log equalP equal to 3.21 to 3.21for compound for compound APX001A APX001A with withTPSA TPSA of 87.06 of 87.06 Å which Å which is in is inthe the range range obse observedrved for for repurposing repurposing drugs. Its prodrugprodrug isis muchmuch moremore polar polar (TPSA (TPSA of of 176.84 176.84 Å) Å) and and hydrophilic hydrophilic (log (logPP= =1.70). 1.70).

SchemeScheme 4. 4.Structure Structure ofof prodrugprodrug APX001 APX001 and and active active moiety moiety APX001A. APX001A.

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GepinacinGepinacin is is a a pre-clinical pre-clinical Gwt-1 Gwt-1 inhibitor inhibitor which which is structurally is structurally different different from from manogepix. mano- Despitegepix. Despite high in high vitro inantifungal vitro antifungal activity, activity, gepinacin gepinacin failed failed in in vivoin intrials vivo trials due to due its to low its metaboliclow metabolic and and serum serum stability. stability. A series A series of analogues of analogues was was then then designed designed and and evaluated evaluated by Walshby Walsh and and Cowen Cowen in 2020 in 2020 (Scheme (Scheme5) [ 110 5) [110]]. A dozen. A dozen of compounds of compounds have have been been obtained obtained and exhibitedand exhibited MIC MIC in the in range the range 0.625–10 0.625–10µg/mL µg/mL against againstC. auris C. .auris However,. However, despite despite interesting inter- potencyesting potency and promising and promising single-dose single-dose pharmacokinetics, pharmacokinetics, the identified the identified lead (R lead1 = (R isobutyl;1 = iso- Rbutyl;2 = 4-chlorophenyl) R2 = 4-chlorophenyl) did not did reveal not any reveal activity any at activity the maximal at the dosemaximal in a neutropenic dose in a neutro- rabbit modelpenic rabbit of disseminated model of disseminated candidiasis. candidiasis.

SchemeScheme 5.5. Structure ofof gepinacingepinacin andand modulations.modulations.

InIn 2019,2019, thethe groupgroup headedheaded byby SeleemSeleem discovereddiscovered thatthat phenylthiazolephenylthiazole smallsmall moleculesmolecules possesspossess dualdual antifungal antifungal and and anti-biofilm anti-biofilm activities activities against againstC. albicans C. albicansand C.and auris C. [auris111]. [111]. They testedThey tested 85 synthetic 85 synthetic phenylthiazole phenylthiazole derivatives derivatives (Figure 19(Figure) and compound19) and compound PHE1 emerged PHE1 asemerged the most as the potent most molecule, potent molecule, with MIC with ranging MIC ranging from 0.25 from to 20.25µg/mL to 2 µg/mL against against a panel a ofpanelC. albicans of C. albicansand C. and auris C.strains. auris strains. The HIT The compound HIT compound also exhibited also exhibited anti-biofilm anti-biofilm ability, fungicidalability, fungicidal activity activity and ability and toability prolong to prolong survival survival in infected in infectedCaenorhabditis Caenorhabditis elegans model.elegans Frommodel. a chemicalFrom a chemical point of point view, of PHE1 view, perfectly PHE1 perfectly fit the previously fit the previously defined defined scaffold scaffold with a hydrazinecarboximidamidewith a hydrazinecarboximidamide moiety moiety (in red) (in which red) which is a guanidine is a guanidine isostere, isostere, a thiazole a thiazole core (incore grey) (in grey) and aand 4-butylphenyl a 4-butylphenyl lipophilic lipophili chainc chain (in blue). (in blue). PHE1 PHE1 displayed displayed a log Pa log= 3.64P = and3.64 TPSAand TPSA = 115.39 = 115.39 Å which Å which are in are accordance in accordance with with the other the other reported reported drugs. drugs.

FigureFigure 19.19. ModiﬁcationsModifications aroundaround thethe phenylthiazolephenylthiazole compoundscompounds asas reportedreported byby SeleemSeleem etet al.al.

The work by Wiederhold etet al.al. described the evaluation of VT-1598, a fungal CYP51- specificspecific inhibitorinhibitor againstagainst C.C. aurisauris infectionsinfections [[112].112]. TheThe tetrazoletetrazole VT-1598,VT-1598, developeddeveloped byby ViametViamet PharmaceuticalsPharmaceuticals (Figure 2020)) hashas demonstrateddemonstrated in vitrovitro activityactivity againstagainst variousvarious fungifungi [113,114] [113,114] and and has has also also shown shown promising promising results results in inexperimental experimental models models of invasive of invasivefungal fungal infections infections such suchas ce asntral central nervous nervous system system coccidioidomycosis coccidioidomycosis or cryptococcal or cryptococcal men- meningitisingitis [115,116]. [115,116 The]. authors The authors demonstrated demonstrated the in the vitrino vitroand inand vivoin vivoefficacyefficacy of VT-1598 of VT- C. auris 1598against against C. auris infections.infections. VT-1598 VT-1598 is currently is currently on Phase on Phase I clinical I clinical trials trialsfor coccidioido- for coccidioidomycosis in order to evaluate its safety and PK of single oral doses in healthy patients mycosis in order to evaluate its safety and PK of single oral doses in healthy patients (ClinicalTrials.gov Identifier: NCT04208321). From a chemical point of view, VT-1598 is (ClinicalTrials.gov Identifier: NCT04208321). From a chemical point of view, VT-1598 is a a tetrazole-based compound which exhibited a protonable pyridine link and a lipophilic tetrazole-based compound which exhibited a protonable pyridine link and a lipophilic aromatic chain (in blue). Its lipophilicity (logP = 5.12) and polar surface (TPSA = 109.74 Å) aromatic chain (in blue). Its lipophilicity (logP = 5.12) and polar surface (TPSA = 109.74 Å) are quite high and its gastrointestinal absorption is estimated as low. are quite high and its gastrointestinal absorption is estimated as low.

Microorganisms 2021, 9, 634 25 of 40 Microorganisms 2021, 9, x 26 of 42

FigureFigure 20. 20.Structures Structures of of VT-1598 VT-1598 and and PC945. PC945.

StillStill inin 2019,2019, RudramurthyRudramurthy etet al.al. describeddescribed thethe activityactivity ofof aa novelnovel topicaltopical triazoletriazole namednamed PC945PC945 againstagainstC. C. aurisauris(Figure (Figure 20 20))[ 117[117].]. PC945, PC945, optimized optimized forfor topical topical application, application, provedproved to to be be 7.4-fold 7.4-fold and and 1.5-fold 1.5-fold more more potent potent than voriconazolethan voriconazole and posaconazole and posaconazole against aagainst collection a collection of more of thanmore 50 than clinical 50 clinical isolates isolates from from India, India, UK, UK, USA, USA, Japan Japan and and South- South- Korea.Korea. Three isolates isolates were were found found to to be be cross-resistant cross-resistant to toPC945 PC945 and and other other azoles azoles but with but withno clear no clearevidence evidence of involved of involved mutations. mutations. The tolerability, The tolerability, efficacy, efficacy, and safety and safetyof PC945 of PC945were evaluated were evaluated by four by clinical four clinical trials in trials the frame in the of frame aspergillosis of aspergillosis and candidiasis and candidiasis recently recently(ClinicalTrials.gov (ClinicalTrials.gov Identifier: Identifier: NCT03870841; NCT03870841; NCT03905447; NCT03905447; NCT03745196 NCT03745196 andand NCT02715570).NCT02715570). Three Three studies studies have have been been interrupted interrupted because because of the of COVID19the COVID19 outbreak outbreak and resultsand results are not are available. not available. From From a chemical a chemical point ofpoint view, of PC945view, exhibitedPC945 exhibited a triazole a triazole ring— presentring—present in several in several antifungal antifungal references’ references’ compounds—and compounds—and an amide anlink. amide The link. integration The inte- ofgration a piperazine of a piperazine link resulted link inresulted easily protonable in easily protonable nitrogen groups nitrogen and groups fluorinated and fluorinated aromatics increasedaromatics the increased lipophilicity. the lipophilicity. Its lipophilicity Its lipophilicity (logP = 5.70) (log andP polar= 5.70) surface and polar (TPSA surface = 84.75 (TPSA Å) are= 84.75 quite Å) high are and quite its gastrointestinalhigh and its gastrointestinal absorption is estimatedabsorption as is low. estimated These values as low. and These the presencevalues and of athe 2,4-difluorophenyl presence of a 2,4-difluorophenyl moiety gave PC945 moiety some gave similarities PC945 some with similarities VT-1598. with VT-1598.Overall, a dozen novel antifungal chemical families have been reported during the last few yearsOverall, and a some dozen of novel the lead antifungal products chemical are currently families under have clinical been trialsreported investigations. during the Fromlast few a chemical years and point some of view,of the these lead compoundsproducts are exhibited currently the under same clinical features trials as discovered investiga- repurposingtions. From compounds,a chemical point divided of view, into three thes complementarye compounds exhibited parts: (i) the core; same (ii) protonablefeatures as nitrogendiscovered groups; repurposing (iii) lipophilic compounds, moieties divided (generally into halogenatedthree complementary aromatics parts: or fatty (i) chains).core; (ii) protonable nitrogen groups; (iii) lipophilic moieties (generally halogenated aromatics or 5.2. Echinocandins fatty chains). In 2017, CD101 (then named rezafungin) was patented for the treatment of fungal infec- tions5.2. Echinocandins (Figure 21)[118 ]. CD101 is a novel echinocandin with long-acting profile (Figure 21). In 2018, another patent was filed for the use of CD101 alone or in combination with another In 2017, CD101 (then named rezafungin) was patented for the treatment of fungal antifungal drug for the treatment of fungal infections [119]. Overall, CD101 shows encour- infections (Figure 21) [118]. CD101 is a novel echinocandin with long-acting profile (Fig- aging activity against the emerging pathogen C. auris. In late 2017, Berkow and Lockhart ure 21). In 2018, another patent was filed for the use of CD101 alone or in combination reported the activity of CD101 against 100 clinical isolates of C. auris [120]. with another antifungal drug for the treatment of fungal infections [119]. Overall, CD101 They observed that the MICs values ranged from 0.03 to 8 µg/mL, the average MIC90 wasshows 0.5 encouragingµg/mL. In particular, activity ag CD101ainst the was emerging investigated pathogen against C.C. auris auris. Instrains late 2017, resistant Berkow to otherand Lockhart echinocandins. reported The the results activity showed of CD101 a better against activity 100 clinical of CD101 isolates compared of C. auris to [120]. other echinocandins, excepted for isolates containing the S639P amino acid substitution in FKS1 hot spot 1 which is related to previously reported mutations linked to echinocandin resistance in other Candida spp. A few months later, Hager et al. examinated the efficacy of rezafungin (previously CD101) in the treatment of disseminated C. auris infection using an immunocompromised mouse model [121]. Rezafungin proved to have significantly lower average CFU/g of kidney tissue compared with amphotericin B, micafungin and vehicle after ten days of treatment. The pharmacodynamics of rezafungin was then evaluated by Lepak et al. in a neutropenic mouse invasive candidiasis model of C. auris infection [122]. In 2019, Majoros et al. extended the study on CD101 and determined the in vitro susceptibility of 689 clinical isolates of 5 common and 19 rare Candida species, as well as Saccharomyces cerevisiae [123]. Their results proved that rezafungin was an excellent antifungal against

Microorganisms 2021, 9, 634 26 of 40

common and non-common Candida species, with results similar to other echinocandins excepting caspofungin. In 2020, Helleberg et al. investigated the potency of rezafungin and comparators against 1293 Nordic yeast isolates and 122 Indian C. auris isolates proving that rezafungin had species-specific activity comparable to that of micafungin and anidulafungin. Even if rezafungin was highly active against the majority of common Candida species, it lacked activity against a significant proportion of C. auris isolates with mutations in fks target genes that conferred echinocandin cross-resistance. However, fks1 mutations increased rezafungin MICs notably less than micafungin and anidulafungin MICs in C. auris [124]. Gathering all these results, rezafungin entered a Phase III multicenter, prospective, randomized, double-blind, efficacy and safety clinical trial in 2018 (ClinicalTrials.gov Identifier: NCT03667690) which is still currently recruiting for comparative evaluation of Rezafungin Microorganisms 2021, 9, x 27 of 42 to caspofungin followed by optional oral fluconazole step-down therapy in subjects in subjects with candidemia and/or invasive candidiasis [125].

FigureFigure 21.21. StructureStructure ofof CD101.CD101.

5.3. SelvamycinThey observed Analogues that the MICs values ranged from 0.03 to 8 µg/mL, the average MIC90 was In0.5 2017, µg/mL. a patent, In particular, covering CD101 selvamicin was andinvestigated its analogues, against was C. ﬁled auris for strains the treatment resistant ofto fungalother echinocandins. infections (Figure The 22 )[results126]. Selvamicinshowed a be resemblestter activity the referenceof CD101 antifungals compared nystatinto other A1echinocandins, and amphotericin excepted B, butfor isolates bears several containing distinctive the S639P structural amino acid features, substitution impacting in FKS1 its pharmacokineticshot spot 1 which andis related cell target. to previously The data compiledreported mutations in the patent linked showed to echinocandin that selvamicin re- Microorganisms 2021, 9, x 28 of 42 andsistance its analogues, in other Candida compared spp. to A nystatin few months A1, are later, potent Hager antifungal et al. examinated against Candida the efficacystrains, of includingrezafunginC. (previously auris. CD101) in the treatment of disseminated C. auris infection using an immunocompromised mouse model [121]. Rezafungin proved to have significantly

R3 R4 lower average CFU/g of kidney tissueR9 compared with amphotericin B, micafungin and R O R vehicle after17 ten days of treatment. The pharmacodynamics10 of rezafungin was then eval- R R22 O O O R R R5 R6 R7 8 O R uated by LepakR et al. 1in2 a neutropenic mouse11 invasive candidiasis model of C. auris infec- 20 R18 tion [122]. In 2019, Majoros et al. extended the study on CD101 and determined the in vitro R16 R21 R12

susceptibilityR15 of 689 clinical isolatesO of 5 commonR13 and 19 rare Candida species, as well as Saccharomyces cerevisiae [123]. Their results proved that rezafungin was an excellent anti- Selvamycin derivatives O fungal against common and non-commonR14 Candida species, with results similar to other R echinocandins excepting caspofungin.19 In 2020, Helleberg et al. investigated the potency FigureFigureof rezafungin 22.22. StructureStructure and of ofcomparators selvamycinselvamycin derivatives. derivatives.against 1293 Nordic yeast isolates and 122 Indian C. auris isolates proving that rezafungin had species-specific activity comparable to that of mica- 5.4. Polymers 5.4.fungin Polymers and anidulafungin. Even if rezafungin was highly active against the majority of commonRecently,Recently, Candida AriasArias species, etet al.al. itreported lacked activity the in vitroagainst andand a significantinin vivovivo activityactivity proportion ofof chitosanchitosan of C. auris againstagainst iso- planktonicplanktoniclates with andandmutations sessilesessile formsinforms fks ofoftarget C.C. aurisauris genes.. InIn athata galleriagalleria conferred mellonellamellonella echinocandin modelmodel of C.C. cross-resistance. aurisauris infection,infection, chitosanchitosanHowever, decreaseddecreased fks1 mutations thethe killingkilling increased effectseffects ofofrezafungin C.C. auris infectioninfection MICs notably withoutwithout less anyany than toxicity micafungin to the larvae. and TheTheanidulafungin putativeputative mode mode MICs of of action inaction C. ofauris of chitosan chitosan [124]. couldGathering coul bed be mediated mediatedall these by results,by the the expression expression rezafungin of a of stress-likeentered a stress- a genelikePhase gene expression III expressionmulticenter, response, respon prospectiv conductingse, conductinge, randomized, to protection to protection double-blind, in the in larvae the larvaeef [ficacy127]. [127]. and safety clinical However, in 2017, Chauhan and Loonker described the modification of chitosan to trial However,in 2018 (ClinicalTrials.gov in 2017, Chauhan Identifier: and Loonker NCT03667690) described which the modification is still currently of chitosan recruiting to integrate N-methyl piperazine moieties and the biological evaluation of the resulting polymer integratefor comparative N-methyl evaluation piperazine of moietiesRezafungin and to the caspofungin biological evaluation followed byof theoptional resulting oral pol- flu- ymerconazole called step-down CE-MP [128]. therapy They in realizedsubjects thein su cross-linkagbjects withe candidemia of the naturally and/or occurring invasive linear can- polysaccharidedidiasis [125]. matrix of chitosan with epichlorohydrin and then incorporated N-methyl piperazine moieties (Scheme 6). The resulting artificial polymer was fully characterized and5.3. Selvamycinproved to be Analogues active against some fungi but unfortunately displayed no activity against C. aurisIn .2017, a patent, covering selvamicin and its analogues, was filed for the treatment of fungal infections (Figure 22) [126]. Selvamicin resembles the reference antifungals nystatin A1 and amphotericin B, but bears several distinctive structural features, impacting its pharmacokinetics and cell target. The data compiled in the patent showed that selvamicin and its analogues, compared to nystatin A1, are potent antifungal against Candida strains, including C. auris.

Scheme 6. Synthesis of CE-MP as reported by Chauhan and Loonker.

Taken together, these results demonstrated that naturally derivated polymers such as chitosan could be interesting alternatives to conventional antifungals.

5.5. Polyclonal Antibody In 2018, Bujdakova et al. reported the activity of anti-CR3-RP polyclonal antibody against biofilms formed by C. auris [129]. During biofilm formation, Candida species generally expressed the complement receptor 3-related protein (CR3-RP) as surface antigens. The authors proved the presence of CR3-RP in C. auris cells within biofilms and the subsequent activity of anti-CR3-RP polyclonal antibody to eradicate the formation of biofilm by C. auris. Polymers and polyclonal antibody could represent exciting therapeutic alternatives to small drugs but, for the moment, the number of works dealing with such strategies in the frame of fighting against C. auris is significantly restricted.

6. Traditional Medicines and Natural Compounds It is notable that the reported data mainly resulted from in vitro evaluation. Few compounds experimented in vivo efficacy, unless specified.

6.1. Small Natural Compounds Farnesol, an endogenous quorum sensing molecule, was reported in 2020 by Vartika and Aijaz [130] for its ability to modulate development of biofilms and drug efflux in C. auris. Farnesol also blocked efflux pumps and downregulated biofilm and efflux pump

Microorganisms 2021, 9, x 28 of 42

R3 R4 R9 R17 O R10

R R22 O O O R R R5 R6 R7 8 O R R 1 2 11 20 R18

R16 R21 R12

R15 O R13

Selvamycin derivatives O R14 R19 Figure 22. Structure of selvamycin derivatives.

5.4. Polymers Recently, Arias et al. reported the in vitro and in vivo activity of chitosan against planktonic and sessile forms of C. auris. In a galleria mellonella model of C. auris infection, chitosan decreased the killing effects of C. auris infection without any toxicity to the larvae. The putative mode of action of chitosan could be mediated by the expression of a stress- Microorganisms 2021, 9, 634 like gene expression response, conducting to protection in the larvae [127]. 27 of 40 However, in 2017, Chauhan and Loonker described the modification of chitosan to integrate N-methyl piperazine moieties and the biological evaluation of the resulting polymer called CE-MP [128]. They realized the cross-linkage of the naturally occurring linear polysaccharidecalled CE-MP [128 matrix]. They of realizedchitosan the with cross-linkage epichlorohydrin of the naturallyand then occurringincorporated linear N-methyl polysac- piperazinecharide matrix moieties of chitosan (Scheme with 6). epichlorohydrin The resulting andartificial then incorporatedpolymer wasN fully-methyl characterized piperazine andmoieties proved (Scheme to be6 active). The against resulting some artificial fungi polymer but unfortunately was fully characterized displayed no and activity proved against to be C.active auris against. some fungi but unfortunately displayed no activity against C. auris.

Scheme 6. Synthesis of CE-MP as report reporteded by Chauhan and Loonker.

Taken together,together, thesethese results results demonstrated demonstrated that that naturally naturally derivated derivated polymers polymers such such as aschitosan chitosan could could be be interesting interesting alternatives alternatives to conventionalto conventional antifungals. antifungals.

5.5. Polyclonal Polyclonal Antibody In 2018, Bujdakova et al. reported reported the the activity of anti-CR3-RP polyclonal antibody against biofilms formed byby C.C. aurisauris[ [129].129]. DuringDuring biofilm biofilm formation, formation,Candida Candidaspecies species gener- gen- erallyally expressed expressed the the complement complement receptor receptor 3-related 3-related protein protein (CR3-RP) (CR3-RP) as surface as surface antigens. antigens. The Theauthors authors proved proved the presence the presence of CR3-RP of CR3-RP in C. aurisin C.cells auris within cells within biofilms biofilms and the and subsequent the sub- C. auris sequentactivity ofactivity anti-CR3-RP of anti-CR3-RP polyclonal polyclonal antibody toantibody eradicate to theeradicate formation the offormation biofilm byof biofilm. Polymers and polyclonal antibody could represent exciting therapeutic alternatives to by C. auris. small drugs but, for the moment, the number of works dealing with such strategies in the Polymers and polyclonal antibody could represent exciting therapeutic alternatives frame of fighting against C. auris is significantly restricted. to small drugs but, for the moment, the number of works dealing with such strategies in the6. Traditional frame of fighting Medicines against and C. Natural auris is Compoundssignificantly restricted. It is notable that the reported data mainly resulted from in vitro evaluation. Few 6. Traditional Medicines and Natural Compounds compounds experimented in vivo efficacy, unless specified. It is notable that the reported data mainly resulted from in vitro evaluation. Few compounds6.1. Small Natural experimented Compounds in vivo efficacy, unless specified. Farnesol, an endogenous quorum sensing molecule, was reported in 2020 by Vartika 6.1.and Small Aijaz Natural [130] for Compounds its ability to modulate development of biofilms and drug efflux in C. aurisFarnesol,. Farnesol an also endogenous blocked efflux quorum pumps sensing and molecule, downregulated was reported biofilm in and 2020 efflux by Vartika pump andassociated Aijaz [130] genes for at aits concentration ability to modulate of 125 mM development (Figure 23). of In biofilms parallel, and the effectsdrug efflux of farnesol in C. auriswere. alsoFarnesol reported also by blocked Kovacs efflux et al. [131pumps,132 ].and The down authorsregulated studied biofilm the effects and of efflux 100–300 pumpµM farnesol on growth, biofilm production ability, production of enzymes related to oxidative stress, triazole susceptibility and virulence towards C. auris strains. After 24 h, farnesol was not able to inhibit the formation of biofilm but caused a significant growth inhibition against C. auris planktonic cells. Moreover, farnesol decreased the metabolic activity and increased the production of ROS. Used in combination with other antifungals, farnesol displayed a synergistic behavior. In vivo, using an immunocompromised murine model of disseminated candidiasis, daily 75 µM farnesol proved to lower the fungal burden and could be a benefit as therapeutics or adjuvant for the treatment of candidiasis.

−

Figure 23. Structures of farnesol, [6]-shogoal and (−)-drimenol.

[6]-Shogoal, one of the pungent constituents of ginger, exhibited antifungal and an- tibioﬁlm formation effects against C. auris [133]. The mode of action of [6]-shogoal was Microorganisms 2021, 9, x 29 of 42

associated genes at a concentration of 125 mM (Figure 23). In parallel, the effects of farnesol were also reported by Kovacs et al. [131,132]. The authors studied the effects of 100– 300 µM farnesol on growth, biofilm production ability, production of enzymes related to oxidative stress, triazole susceptibility and virulence towards C. auris strains. After 24 h, farnesol was not able to inhibit the formation of biofilm but caused a significant growth inhibition against C. auris planktonic cells. Moreover, farnesol decreased the metabolic activity and increased the production of ROS. Used in combination with other antifungals, farnesol displayed a synergistic behavior. In vivo, using an immunocompromised murine model of disseminated candidiasis, daily 75 µM farnesol proved to lower the fungal burden and could be a benefit as therapeutics or adjuvant for the treatment of candidiasis.

Figure 23. Structures of farnesol, [6]-shogoal and (−)-drimenol. Microorganisms 2021, 9, 634 28 of 40 [6]-Shogoal, one of the pungent constituents of ginger, exhibited antifungal and antibiofilm formation effects against C. auris [133]. The mode of action of [6]-shogoal was experimentallyexperimentally relatedrelated to to the the reduction reduction of of the the levels levels of of aspartyl aspartyl proteinases proteinases and and downregu- downreg- lationulation of of the the expression expression of of the the efflux efflux pump-related pump-relatedCDR1 CDR1-gene-gene in inC. C. auris auris.. However,However, thethe inin vitrovitroactivity activity displayeddisplayed byby [6]-shogoal,[6]-shogoal, eveneven higherhigher thanthan fluconazole,fluconazole, isis quitequite modestmodest withwith MICMIC8080 ranging from 32 to 64 µµg/mL.g/mL. EightEight drimanedrimane sesquiterpenoidssesquiterpenoids includingincluding ((−−)-drimenol and (+)-albicanol(+)-albicanol were syn-syn- thesizedthesized fromfrom (+)-sclareolide(+)-sclareolide andand evaluatedevaluated for for their their antifungal antifungal activities. activities. ( −(−)-Drimenol)-Drimenol (Figure(Figure 2323)) provedproved toto limitlimit thethe growthgrowth ofof C.C. aurisauris betterbetter thanthan fluconazolefluconazole inin thethe samesame con-con- centrationcentration [[134].134]. ThisThis naturalnatural compoundcompound alsoalso protectedprotectedC. C. eleganselegansfrom from deathdeathin in aa candiasiscandiasis modelmodel [[134].134]. TraditionalTraditional herbalherbal monomersmonomers (THMs),(THMs), mainlymainly composedcomposed ofof sodiumsodium houttuyfonatehouttuyfonate (SH),(SH), berberineberberine (BER),(BER), palmatinepalmatine (PAL),(PAL), jatrorrhizinejatrorrhizine (JAT),(JAT), andand cinnamaldehydecinnamaldehyde (CIN),(CIN), werewere evaluatedevaluated for for their their potential potential to induceto induce cell cell wall wall modulations modulations in C. in auris C. (aurisFigure (Figure 24)[135 24)]. The[135]. authors The authors proved proved that combination that combination of these of these herbal herbal monomers monomers induced induced good good antifun- an- galtifungal activity activity against againstC. auris C. aurisisolates isolates the the most most potent potent combinations combinations being being SH/CIN SH/CIN andand BER/PAL/JAT.BER/PAL/JAT.

FigureFigure 24.24. CompositionComposition ofof thethe fivefive mostmost common common traditional traditional herbal herbal monomers monomers as as stated stated by by Liu Liu et et al. al. Bark and leaf essential oils from Cinnamomum zeylanicum proved in vitro antifungal activityBark against and leafC. aurisessential, by damaging oils from theCinnamomum membrane zeylanicum and blocking proved the hyphaein vitro formation.antifungal Fromactivity a chemical against C. point auris of, by view, damaging the main the components membraneof andC. zeylanicumblocking theleaf hyphae is eugenol formation. (62%) andFromC. a zeylanicum chemical pointbark isoftrans view,-cinnamaldehyde the main components (66%), of the C. other zeylanicum components leaf is eugenol being present (62%) atand less C. than zeylanicum 7% [136 bark]. is trans-cinnamaldehyde (66%), the other components being present Evenat less if than the 7% use [136]. of natural compounds is of great interest, the activity of farnesol, [6]-shogoal,Even if (the−)-drimenol use of natural or common compounds traditional is of great herbal interest, monomers the activity is quite of low farnesol, compared [6]- toshogoal, synthetic (−)-drimenol drugs and or they common have to traditional be envisioned herbal as adjuvantsmonomers or is platformquite low molecules compared for to drugsynthetic chemical drugs development. and they have to be envisioned as adjuvants or platform molecules for drug chemical development. 6.2. Extracts of Natural Organisms Marine sponges are among the richest sources of bioactive ingredients from marine organisms. In 2017, Hasaballah et al. reported the biological evaluation of crude extracts of marine sponges, Negombata magnifica and Callyspongia siphonella towards a large panel of biological targets, including C. auris. In addition to their high insecticidal capacity, the two sponge extracts also displayed high antibacterial and antifungal activities, with growth inhibition of 15.3 ± 1.2 mm for N. magnifica and 13.7 ± 1.5 mm for C. siphonella compared to 19.8 ± 0.63 mm for amphotericin B [137]. In 2020, Zhang et al. reported the identification of turbinmicin, a lead antifungal which was identified from marine microbiome (Figure 25). Turbinmicin is highly interesting because it functions through a fungal-specific mode of action, targeting Sec14 of the vesicular trafficking pathway [138]. Moreover, the same team reported the striking ability of turbinmicin to block biofilm formation. Indeed, turbinmicin acts by disruption of extracellular vesicle delivery during biofilm growth [139]. Turbinmicin revealed as a promising anti-biofilm and antifungal drug [140]. Microorganisms 2021, 9, x 30 of 42

6.2. Extracts of Natural Organisms Marine sponges are among the richest sources of bioactive ingredients from marine organisms. In 2017, Hasaballah et al. reported the biological evaluation of crude extracts of marine sponges, Negombata magnifica and Callyspongia siphonella towards a large panel of biological targets, including C. auris. In addition to their high insecticidal capacity, the two sponge extracts also displayed high antibacterial and antifungal activities, with growth inhibition of 15.3 ± 1.2 mm for N. magnifica and 13.7 ± 1.5 mm for C. siphonella compared to 19.8 ± 0.63 mm for amphotericin B [137]. In 2020, Zhang et al. reported the identification of turbinmicin, a lead antifungal which was identified from marine microbiome (Figure 25). Turbinmicin is highly interesting because it functions through a fungal-specific mode of action, targeting Sec14 of the vesicular trafficking pathway [138]. Moreover, the same team reported the striking ability of turbinmicin to block biofilm formation. Indeed, turbinmicin acts by disruption of ex-

Microorganisms 2021, 9, 634 tracellular vesicle delivery during biofilm growth [139].29 Turbinmicin of 40 revealed as a promising anti-biofilm and antifungal drug [140].

FigureFigure 25. Structure25. Structure of turbinmicin. of turbinmicin. 6.3. Peptides and Derivatives In 2015, Cm-p5, an antifungal hydrophilic peptide derived from the coastal mollusk cenchritis6.3. Peptides muricatus andwas reported Derivatives by Lopez-Abarrategi et al. [141]. Cm-p5 proved to have a fungistaticIn activity2015, againstCm-p5,C. albicans an antifungalwith MIC around hydrophilic 10 µg/mL and moderatepeptide toxicity derived from the coastal mollusk toward mammalian cell lines. Following this first report, the same team obtained mutants ofcenchritis Cm-p5 and muricatus then designed was and synthetized reported a helical-stabilized,by Lopez-Abarrategi cyclic, and nontoxic et al. [141]. Cm-p5 proved to have analogue of Cm-p5. This analogue displayed moderate antifungal activity against C. aurisa fungistatic[142]. In 2020, activity Rosenau et against al. also reported C. albicans that the same with derivatives MIC around of the Cm-p5 10 µg/mL and moderate toxicity weretoward able to mammalian inhibit the development cell lines. of C. auris Followingbiofilms in vitro this [143 first]. report, the same team obtained mutants Ramachandran et al. evaluated the efficacy of three novel cyclic lipopeptides of theof classCm-p5Bacillomycin and tothen limit designed fungal infection and [144 synthetized]. The lipopeptide a analogues, helical-stabilized, bearing cyclic, and nontoxic an- thealogue same peptide of Cm-p5. sequence Asn-Pro-Tyr-Asn-Gln-Thr-Ser,This analogue displayed were purified moderate from a cell-freeantifungal activity against C. auris supernatant of Bacillus subtilis RLID 12.1. The three active lipopeptides exhibited MICs from[142]. 3.5 toIn 8.5 2020,µg/mL Rosenau against 10 C. auriset al.isolates. also reported that the same derivatives of the Cm-p5 were ableAntimicrobial to inhibit peptides the development (AMPs) constitute a keyof componentC. aurisof biofilms these innate in immune vitro de- [143]. fences and AMPs from a variety of sources have shown potent antifungal activities [145–148]. Since theyRamachandran induce no or minimal et MDR al. in evaluated target fungi, AMPs the are efficacy considered of strongly three attrac- novel cyclic lipopeptides of the tiveclass as potential Bacillomycin therapeutic to drugs limit [149]. fungal Pathirana etinfection al. have investigated [144]. salivary The lipopeptide Histatin analogues, bearing the 5 (Hst 5) for its ability to limit fungal infections [150]. Hst 5 is a well-studied salivary cationicsame peptide.peptide After sequence treatment at a Asn-Pro- 7.5 µM dose, 55Tyr-Asn-Gln-Thr-Ser, to 90% of all C. auris cells were killedwere purified from a cell-free su- bypernatant Hst 5, irrespective of Bacillus of the MDR-profile subtilis strains. RLID The 12.1. mode ofThe action three of Hst active 5 is based lipopeptides on its exhibited MICs from translocation to the cytosol and vacuole and the reported study suggested that it differed from3.5 theto one8.5 of µg/mL fluconazole. against Baso, Garcia 10 etC. al. auris recently isolates. reported the antifungal properties of θ-defensins,Antimicrobial macrocyclic peptides peptides expressed (AMPs) in tissues constitute of old world monkeys a key [ 151component]. The of these innate immune mode of action of Rhesus θ-defensin 1 (RTD-1), the prototype θ-defensin, occurred by cell permeabilization,defences and correlated AMPs with from ATP releasea variety and intracellular of sources accumulation have of shown killing potent antifungal activities reactive[145–148]. oxygen Since species. they Other naturalinduce defensins no or isoforms minimal were evaluated MDR andin thetarget most fungi, AMPs are considered promising proved to be more active than caspofungin and/or amphotericin B caspofun- ginstrongly against fluconazole-resistant attractive as potential organisms, including therapeuticC. auris. drugsθ-defensin [149]. activity Pathirana was et al. have investigated compared with Hst 5 and proved to be 200-fold more active and more stable to proteases. salivaryDal Mas Histatin et al. described 5 (Hst the effect 5) for of Crotamine, its ability a natural to limit antimicrobial fungal peptide infections [150]. Hst 5 is a well- (AMP)studied isolated salivary from a South cationic American peptide. rattlesnake onAfterC. auris treatment[152]. Crotamine, at a which 7.5 µM has dose, 55 to 90% of all C. auris structural similarities with human defensins, exhibited in vitro activity against most isolates tested,cells whereaswere thesekilledCandida byisolates Hst 5, showed irrespective resistance to amphotericin of the MDR-profile B and fluconazole. strains. The mode of action of HstCathelicidins 5 is based are on a class its of translocation epithelial antimicrobial to peptidesthe cytosol that are and expressed vacuole in the and the reported study sug- intestinal epithelium and kill microorganisms by membrane disruption. Haagsman et al. recentlygested obtained that it cathelicidin-inspired differed from antimicrobial the one of peptides flucon andazole. evaluated Baso, their Garcia anti- et al. recently reported the fungalantifungal capacity [153properties]. These novel of antimicrobial θ-defensins, peptides termedmacrocyclic ‘PepBiotics’, peptides containing expressed in tissues of old between 21 and 37 residues, have been patented and showed a strong inhibitory activity againstworld a largemonkeys panel of fungi [151]. and yeastsThe atmo lowde concentrations of action (≤ of1 µ M).Rhesus In particular, θ-defensin they 1 (RTD-1), the prototype exhibitedθ-defensin, MIC = 0.6–1.3 occurredµM against by C.cell auris pestrains.rmeabilization, correlated with ATP release and intracellular accumulation of killing reactive oxygen species. Other natural defensins isoforms were

Microorganisms 2021, 9, x 31 of 42

evaluated and the most promising proved to be more active than caspofungin and/or amphotericin B caspofungin against fluconazole-resistant organisms, including C. auris. θ- defensin activity was compared with Hst 5 and proved to be 200-fold more active and more stable to proteases. Dal Mas et al. described the effect of Crotamine, a natural antimicrobial peptide (AMP) isolated from a South American rattlesnake on C. auris [152]. Crotamine, which has structural similarities with human defensins, exhibited in vitro activity against most isolates tested, whereas these Candida isolates showed resistance to amphotericin B and fluconazole. Cathelicidins are a class of epithelial antimicrobial peptides that are expressed in the intestinal epithelium and kill microorganisms by membrane disruption. Haagsman et al. recently obtained cathelicidin-inspired antimicrobial peptides and evaluated their antifungal capacity [153]. These novel antimicrobial peptides termed ‘PepBiotics’, containing Microorganisms 2021, 9, 634 between 21 and 37 residues, have been patented and showed a strong inhibitory activity30 of 40 against a large panel of fungi and yeasts at low concentrations (≤1 µM). In particular, they exhibited MIC = 0.6–1.3 µM against C. auris strains.

6.4. Bioinspiration With the goalgoal toto mimicmimic AMPs,AMPs, SavageSavage etet al.al. recently developeddeveloped cerageninsceragenins (CSAs),(CSAs), aa class ofof non-peptide non-peptide molecules, molecules, based based on aon common a common bile acid,bile thatacid, conserve that conserve the amphiphilic the am- morphologyphiphilic morphology common to common AMPs (Figure to AMPs 26). (Figur They havee 26). then They evaluated have then the evaluated susceptibilities the sus- of C.ceptibilities auris isolates, of C. in auris bioﬁlm isolates, and planktonicin biofilm and forms, planktonic to CSAs. forms, In addition, to CSAs. they In alsoaddition, measured they thealso effectivenessmeasured the of effectiveness the most promising of the most CSAs promising in gel and CSAs cream in formulationsgel and cream in formula- treated infectedtions in treated tissue explants infected [tissue154]. Lead explants CSAs [154]. led toLead activity CSAs comparable led to activity with comparable reference drugs with andreference no cross-resistance drugs and no cross-resistance was observed. was In ex-vivo observed. mucosal In ex-vivo tissues, mucosal CSAs tissues, were able CSAs to signiﬁcantlywere able to significantly reduce the fungal reduce burden the fungal with 2%burden topical with application. 2% topical application.

Figure 26. Structures of selected ceragenins CSAs.

6.5. Necrotrophic Mycoparasitism Necrotrophic mycoparasitism describes the ab abilityility of a fungal species to killkill otherother Saccharomy- fungi [155]. [155]. In In 2018, 2018, Junker Junker et etal. al. examinated examinated the theuse useof a ofpredatory a predatory yeast, yeast, Saccharomycopsis copsis schoenii, as a potential biocontrol agent against C. auris [156]. They investigated the schoenii, as a potential biocontrol agent against C. auris [156]. They investigated the inter- interaction between the two species by seeding equal quantity of dimorphic S. schoenii and action between the two species by seeding equal quantity of dimorphic S. schoenii and ovoid C. auris NCPF8985#20 cells on minimal media agar on microscopy slides. Using ovoid C. auris NCPF8985#20 cells on minimal media agar on microscopy slides. Using specialized penetration pegs, S. schoenii attacked C. auris cells upon contact and killed 34% specialized penetration pegs, S. schoenii attacked C. auris cells upon contact and killed 34% of them within a period of 6 h. All isolates of Candida species tested, including several of them within a period of 6 h. All isolates of Candida species tested, including several drug resistant C. auris strains, were susceptible to predation by S. schoenii, opening new drug resistant C. auris strains, were susceptible to predation by S. schoenii, opening new possibilities to eradicate such multidrug resistant strains. possibilities to eradicate such multidrug resistant strains. 7. Metal, Metal Complexes and Metalloids 7. Metal, Metal Complexes and Metalloids Recently, Goldman et al. evaluated the potential of gallium, already known for its antibacterialRecently, activity, Goldman as antifungalet al. evaluated [157]. the They pote demonstratedntial of gallium, that already gallium known nitrate for had its a fungistaticantibacterial action activity, against as antifungal fungi. Regarding [157]. TheyC. auris demonstrated, several strains that gallium (473/2015, nitrate 490/2015, had a 501/2015,fungistatic andaction 502/2015) against fungi. were completelyRegarding C. inhibited auris, several by gallium strainsat (473/2015, concentrations 490/2015, of 128–256501/2015, mg/L and .502/2015) However, were others completely (strains inhibited 467/2015, by 470/2015, gallium at and concentrations 484/2015) survived of 128– the challenge at the higher concentration tested limiting the use of gallium as a broad- spectrum antifungal agent. In 2020, the team of Garneau-Tsodikova and Awuah explored the potential of linear and square-planar gold(I)−phosphine complexes as antimicrobial agents against a panel of 28 fungal strains [158]. Compounds GOLD1 and GOLD2 proved to be the most efficient against C. auris, with MIC similar to caspofungin. Moreover, these two gold-complexes displayed low cytotoxicity against a panel of cell lines (Figure 27). The amount of data gathered on the use of metal and metallic complexes towards C. auris is too low to ascertain the interest of such strategy, but the first data are encouraging. Therefore, medicinal chemists should go further with this alternative area of research in the fight against fungal resistant strains. Microorganisms 2021, 9, x 32 of 42

256 mg/L. However, others (strains 467/2015, 470/2015, and 484/2015) survived the challenge at the higher concentration tested limiting the use of gallium as a broad-spectrum antifungal agent. In 2020, the team of Garneau-Tsodikova and Awuah explored the potential of linear and square-planar gold(I)−phosphine complexes as antimicrobial agents against a panel of 28 fungal strains [158]. Compounds GOLD1 and GOLD2 proved to be the most efficient Microorganisms 2021, 9, 634 31 of 40 against C. auris, with MIC similar to caspofungin. Moreover, these two gold-complexes displayed low cytotoxicity against a panel of cell lines (Figure 27).

FigureFigure 27. Gol(I)-complexes 27. Gol(I)-complexes as antifu as antifungals:ngals: structure, structure, MIC valu MICes values range range and cytotoxicity. and cytotoxicity.

The amount8. of Others data gathered Approaches on the use of metal and metallic complexes towards C. auris is too low 8.1.to ascertain Probiotics the and interest Postbiotics of such strategy, but the first data are encouraging. Therefore, medicinalThe chemists review should by Zhang go further et al. described with this the alternative recent reports area of that research indicate in that probiotics the fight againstmay fungal also resistant contribute strains. to protect against fungal infections [159]. Even if some publications have evaluated the benefits of probiotics against fungal infections caused by C. albicans, 8. Others ApproachesC. glabrata , A. fumigatus, T. tonsurans, M. canis, M. gypseum, or E. flocossum, no reports on C. 8.1. Probiotics andauris Postbioticsare presented in that review published in 2019. Late 2019, Rao et al. exploited the probiotic properties of two novel, food-derived The review by Zhang et al. described the recent reports that indicate that probiotics yeasts, Saccharomyces cerevisiae (strain KTP) and Issatchenkia occidentalis (strain ApC). These may also contributealternative to protect approaches against to fungal combat infections widespread [159]. opportunistic Even if some fungal publications infections proved to be have evaluatedeffective the benefits to inhibit of probiotics virulence against traits such fungal as adhesion,infections biofilm caused formation by C. albicans and filamentation, of C. glabrata, A. fumigatusCandida, spp.,T. tonsurans especially, M.C. canis auris, M.[160 gypseum,161]. , or E. flocossum, no reports on C. auris are presentedIn in 2020, thatCampos-Junqueira review published in et 2019. al. described the pro- and postbiotic activity of Lacto- Late 2019, bacillusRao et paracaseial. exploited28.4 the against probiotiC. aurisc properties. It is notable of two that novel, they food-derived conducted an in vivo study yeasts, Saccharomyceswith G. cerevisiae mellonella (strainlarvae KTP) infected and Issatchenkia with C. auris occidentalis. Injections (strain of LPCE ApC). and These LPF1 (crude extract alternative approachesand fraction to combat 1 derived widespread from L. paracasei opportunistic28.4 supernatant, fungal infections respectively) proved prolonged to survival be effective to inhibitof these virulence insects compared traits such to as a adhesion, control group biofilm (p

with G. mellonella larvaeIn 2019, infected Philip with and C. Kuriakoseauris. Injections reported of LPCE thesynthesis and LPF1 of(crude superparamagnetic extract Fe2O3 and fraction 1 derivednanoparticles from L. stabilized paracasei 28.4 by biocompatible supernatant, respectively) supramolecular prolongedβ-cyclodextrin survival and their evalua- of these insectstion compared against to three a control strains group of C. auris(p < 0.05)but with and amodulated moderate MICthe host = 500 immuneµg/mL [163]. response [162]. Chapman, Truong et al. reported in 2020 the fabrication of long-term microbicidal silver nanoparticle clusters [164]. This silver nanoparticle cluster coating was constructed 8.2. Nanoparticleson and copper Coatings surface using an ion-exchange and reduction reaction. The resulting surface In 2019, Philipwas contaminatedand Kuriakose by reportedC. auris theand synthesis evaluated of at superparamagnetic extended periods. AfterFe2O3 sevenna- days, 90% of noparticles stabilizedC. auris byproved biocompatible to be non-viable supramolecular on the new β-cyclodextrin designed surface. and their evaluation against three strainsIn parallel, of C. auris using but a microwavewith a moderate assisted MIC synthetic = 500 µg/mL approach, [163]. Lara et al. reported the synthesis of pure round silver nanoparticles (AgNPs) and their use to inhibit C. auris biofilm formation on surfaces [165]. These AgNP-treated biofilms showed cell wall damage mostly by disruption and distortion of the outer surface of the fungal cell wall. Moreover, the AgNPs-functionalized fibers proved to be stable and kept their fungicidal potential even after repeated thorough washes. Very recently, Nosanchuck et al. described the effects of nanoparticles capable of generating nitric oxide (NO) to kill C. auris [166]. This NO-nanotherapeutics were incubated with six different C. auris strains and eradicated both planktonic and biofilm C. auris with a 10 mg/mL and emerged as a promising approach to fight against MDR-fungal strains. Microorganisms 2021, 9, 634 32 of 40

Bismuth nanoparticles (BiNPs) demonstrated strong antifungal activity against a panel of C. auris strains, with MIC ranging from 1 to 4 µg/mL. Moderate effects of these nanoantibiotics were also examined by scanning electron microscopy and showed the disruption of the C. auris cell morphology and the bioﬁlm structure [167].

8.3. Hydrogels In 2019, Shukla and Vera-Gonzalez patented an aspartic protease-triggered antifungal hydrogel able to locally deliver antifungal drugs that specifically respond to aspartic proteases secreted by virulent and pathogenic Candida species [168]. In 2020, Rosenau et al. described a novel anti-infective multicomponent gatekeeper hydrogel [169]. This bilayer hydrogel protected the patient from invasion by C. auris from infected wounds, by combining to complementary layers. The first layer reduced the loading of the pathogen C. auris by selective cell capturing within the affinity layer of the gel followed by subsequent inactivation within the therapeutic gel layer. In a similar manner, Gupta et al. reported similar antibacterial silver-nanoparticle hydrogel for wound dressing applications [170]. The particularity of their design relied on the green chemistry approach—based on natural curcumin as reducing agent-developed to obtain the nanoparticles.

8.4. Irradiation Candida spp. are the most common cause of fungal infections worldwide and the fifth most common cause of nosocomial infections [171]. In particular, C. auris resists disinfection with cleaning agents widely used in hospitals and long-term care facilities [31,172], is frequently drug resistant, and can persist in the environment for months [173]. As a consequence, new protocols to identify how to eliminate C. auris from surface and devices drove several studies. In 2018, Cadnum et al. stated about the relative resistance of C. auris to be eradicated by ultraviolet light [174]. Before this study, it was not clear if mobile ultraviolet-C (UV-C) light room decontamination devices efficiently cleaned healthcare facilities. From their data, it appeared that C. auris was significantly less susceptible to killing by UV-C than methicillin-resistant Staphylococcus aureus. This first statement brought to light the need in understanding how efficiently eradicate C. auris using UV lights. Consequently, in 2019, Ponnanchan et al. evaluated the antifungal activity of ultraviolet C (UVC) light using mercury vapour lamp with a peak emission of 254 ± 2 nm against 32 C. auris isolates. Contaminated plates were held parallel to the UVC source throughout the period of exposure. After 15 min of irradiation at this wavelength, all experiments demonstrated complete inhibition of growth up to 72 h incubation for C. auris [35]. In a parallel manner, in 2019, Maslo et al. reported the efficacy of pulsed-xenon ultraviolet light to eradicate C. auris [175]. After inoculation, each sample was exposed during uninterrupted periods of 5, 10 or 15 min at distances ranging from 1 to 2 m. The PX-UV mobile device killed 99.4% of C. auris CFU on the surface after 5 min irradiation at 1 m-distance. The time and distance of exposure have also been quantified by de Groot et al. [176]. The optimum for C. auris eradication was obtained after 30 min of decontamination at 2 m. Dividing time by two or double the distance decreased the activity by 10- and 50-fold, respectively. In 2020, Lemons et al. also demonstrated the efficiency of ultraviolet irradiation to kill C. auris [177]. Ultraviolet germicidal irradiation (UVGI) was investigated to inactivate ten clinically relevant strains of C. auris. In order to determine dose-response curves, each sample was exposed eleven times to range of UV-dose from 10 to 150 mJ/cm2. From these data, C. auris required more energy than C. albicans to be eradicated. As a consequence, the authors stated that UVGI could be applied against C. auris but the variation of susceptibility would have to be considered. Microorganisms 2021, 9, 634 33 of 40

From these reports, irradiation could be a powerful tool to eradicate C. auris in medical area. However, all parameters should be carefully estimated to avoid a lack of effectiveness and propose tailor-made solutions to eradicate C. auris.

9. Conclusions and Perspectives Through gathering all these data it appeared that the fight against C. auris is con- tinuously increasing for the last five years, in parallel of its emergence and definition as a fungus of concern. Even if most reports dealt with Candida species, some important works focused only on C. auris and took in consideration its own particularities. No perfect solution has been stated for the moment, but clear opportunities have been demonstrated whether from drug repositioning, combination of drugs, or the design of novel antifungal entities. From a medicinal chemist point of view, some molecular features seemed to emerge as required for the design of potent antifungals against C. auris, and there is no doubt that these preferred moieties should inspire the conception of novel effective drugs. As always, nature is a great provider of active compounds and bioinspiration also drives some exciting research works. Even if it is of great importance to treat patients, it is also absolutely required to clean devices and surfaces to avoid contamination and proliferation. Therefore, the emerging solutions based on the specific characteristic of C. auris in terms of irradiation or antimicrobial surfaces are of particular interest. Such an approach could be considered as tailor-made conception towards C. auris. To summarize, ongoing works are very encouraging, but there is still an urgent need to propose therapeutic alternatives to eradicate C. auris.

Author Contributions: Conceptualization: M.B., S.J., S.H. and Z.F.; writing—original draft prepara- tion, M.B. and S.J.; writing—review and editing, S.H. and Z.F. All authors have read and agreed to the published version of the manuscript. Funding: This work was partially funded by the Agence Nationale de la Recherche (ANR) in the setting of project “InnateFun”, promotional reference ANR-16-IFEC-0003-05, in the “Infect- ERA” program. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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