Anti-Malassezia Drug Candidates Based on Virulence Factors of Malassezia-Associated Diseases

Abstract

Malassezia is a lipophilic unicellular fungus that is able, under specific conditions, to cause severe cutaneous and systemic diseases in predisposed subjects. This review is divided into two complementary parts. The first one discusses how virulence factors contribute to Malassezia pathogenesis that triggers skin diseases. These virulence factors include Malassezia cell wall resistance, lipases, phospholipases, acid sphingomyelinases, melanin, reactive oxygen species (ROS), indoles, hyphae formation, hydrophobicity, and biofilm formation. The second section describes active compounds directed specifically against identified virulence factors. Among the strategies for controlling Malassezia spread, this review discusses the development of aryl hydrocarbon receptor (AhR) antagonists, inhibition of secreted lipase, and fighting biofilms. Overall, this review offers an updated compilation of Malassezia species, including their virulence factors, potential therapeutic targets, and strategies for controlling their spread. It also provides an update on the most active compounds used to control Malassezia species.

1. Introduction

Malassezia is a lipophilic unicellular fungus [1]. Many species of Malassezia can be found in the healthy skin microbiota of humans [2]. Under specific conditions, they are capable of causing severe cutaneous and systemic diseases in predisposed subjects [3]. Malassezia species can become pathogenic when the physical, chemical, or immunological properties of the skin are altered [3]. Many environmental and endogenous factors have been found to facilitate the transition of Malassezia yeasts from a commensal state to a pathogenic one such as high temperatures and humidity, greasy skin, sweating, heredity, and immunosuppressive conditions [4]. Malassezia species have been associated with a number of diseases affecting human skin, such as seborrheic dermatitis, pityriasis versicolor, Malassezia folliculitis, and catheter-associated sepsis [5].

Seborrheic dermatitis is caused by a non-specific immune response to Malassezia species, which trigger an inflammation in seborrheic areas such as the scalp, face, chest, back, axilla, and groin [6,7]. For instance, M. globosa and M. restricta strains that are commonly isolated from human skin have distinct profiles of proinflammatory cytokines production from epidermal cells. However, dandruff is a less severe form of seborrheic dermatitis, which is restricted to the scalp, and causes itchy, flaking skin without visible inflammation [8]. A pityriasis versicolor infection is characterized by the proliferation of Malassezia yeasts and the activation of hyphae formation, which lead to the superficial invasion of the stratum corneum [6]. Malassezia folliculitis is caused by Malassezia yeasts invading the pilo-sebaceous unit, leading to a dilation of the follicles [9]. In addition, inflammatory infiltrates, and clinical inflammation are caused by ruptures of the follicular walls [9]. Currently, ten Malassezia species have been isolated from human skin (

Table 1. Malassezia species isolated from human skin.

Malassezia Species	References
M. restricta	[13]
M. globosa	[14]
M. furfur	[15]
M. dermatis	[16]
M. arunalokei	[17]
M. slooffiae	[14]
M. sympodialis	[18]
M. obtusa	[13]
M. yamatoensis	[19]
M. japonica	[20]

). Culture and molecular analysis as well as mycobiome approaches led to the identification of M. restricta and M. globosa as the most predominant fungal species on human skin [10,11,12].

This review discusses how virulence factors contribute to Malassezia pathogenesis that triggers skin diseases. These virulence factors include Malassezia cell wall resistance, lipases, phospholipases, acid sphingomyelinases, melanin, reactive oxygen species (ROS), indoles, hyphae formation, hydrophobicity, and biofilm formation.

2. Malassezia Virulence Factors

2.1. Malassezia Cell Wall Resistance

In comparison to other yeasts, Malassezia’s cell wall is very thick in comparison with other yeasts, representing up 26 to 37% of the cell volume [21,22]. Most cell wall components consist of glycans (70%), protein (10%), and lipid (between 15 and 20%), with a small amount of nitrogen and sulfur [22,23]. Mittag et al. showed that an outer lamellar layer surrounds the cell wall [24]. This lamellar layer is involved in adhesion of Malassezia to the catheter and the human skin [24]. All Malassezia species with the exception of Malassezia pachydermatis are all lipid-dependent [25]. Thus, Malassezia species require exogenous lipids to grow, making lipids a critical component of their growth [24]. The high lipid content of Malassezia cell wall contributes to their mechanical stability, pathogenicity, resistance, and osmoresistance [26,27]. Consequently, Malassezia pathogens are protected from phagocytosis and can escape the immune response [26,27]. Additionally, fungal adhesion to host surfaces is further enhanced by the hydrophobicity of the lipid-rich cell wall [24].

2.2. Lipases, Phospholipases and Acid Sphingomyelinases

Malassezia species lack the genes encoding fatty acid synthase suggesting that they cannot synthesize de novo fatty acids [28]. As a compensatory survival mechanism, Malassezia species display a panel of genes that encode secreted hydrolases like lipases, phospholipases, and acid sphingomyelinases, which promote these yeasts to obtain fatty acids from outside their fungal cells [28].

Lipases are considered to be a major virulence factor for Malassezia pathogens due to their lipid predominance in the cell walls. The lipases catalyze the hydrolysis of triacylglycerols to yield free fatty acids, diacylglycerol, monoacylglycerol, and glycerol [29]. There has been evidence that M. furfur releases fatty acids from a variety of lipids [30,31]. Malassezia contains many genes encoding lipases, which indicates that the fungus is required to degrade host sebum triglycerides into fatty acids. These neoformed fatty acids not only support the growth of the yeasts, but also cause skin diseases [31,32]. Malassezia phospholipases hydrolyze glycerol-phospholipid ester bonds. Like Malassezia lipases, it has been shown that Malassezia produces a variety of extracellular phospholipases that are considered virulence factors [30,33]. In addition to lipases and phospholipases, Malassezia yeast contains acid sphingomyelinase that hydrolyzes membrane lipid sphingomyelin into ceramide and phosphorylcholine. It contributes to Malassezia growth and virulence [28].

2.3. Melanin, ROS and Indoles

The pigment melanin has been implicated in the virulence of several important fungal pathogens indicating that melanin promotes their survival in hosts and facilitates fungal infection [34]. Different studies showed that Malassezia yeasts produce melanin [35,36]. Additionally, the yeasts and hyphae of Malassezia in both hypo- and hyperpigmented pityriasis versicolor lesions are strongly reactive with melanin-specific antibody, supporting that both forms of pityriasis versicolor undergo in vivo melanization [35].

One of the virulence factors of Malassezia species relies on their ability to generate ROS [37]. Both M. furfur and M. pachydermatis exhibited reduced ROS production after terbinafine treatment evidencing that terbinafine antifungal agent acts as an oxygen species scavenger [37].

Malassezia yeasts produce indole alkaloids from tryptophan [38]. When tryptophan is the only nitrogen source, this pathway is most active [38]. Indoles derived from M. furfur, such as malassesin, indirubin, and indolo[3,2-b]carbazole (ICZ), act as potent ligands for the host aryl hydrocarbon receptor (AhR). Thus, the interaction between these indoles and AhR affects all epidermal cells that express this receptor [39,40,41,42]. In addition, tryptophan metabolites can cause melanocyte apoptosis or prevent respiratory bursts in neutrophils [43]. By engaging AhR pathway, Malassezia yeasts modulate the inflammatory response and lead to skin pathologies [44].

2.4. Hyphae Formation

The hyphal formation plays an important role in the pathogenesis of Malassezia species [45]. The scales of pityriasis versicolor lesions provide an ideal environment for hyphal growth, and these lesions often contain large numbers of hyphae [46]. Some environmental factors play a role in Malassezia filamentation. A high humidity level increases skin secretions, which, in turn, promotes the growth and filamentation of M. furfur that facilitates skin infection [45]. Youngchim et al. showed that M. furfur filamentation was induced most strongly at pH 4.6 supporting that an acidic skin pH promotes the attachment of human microbiota to the skin [45]. In addition, a higher level of sebum may induce the formation of Malassezia hyphae in individuals with hyperactive sebaceous glands [25].

2.5. Hydrophobicity and Biofilm Formation

Fungal cell wall architecture has been associated with cell surface hydrophobicity (CHS) status [47,48]. Hydrophobic cells have short and aggregated fibril structures of cell wall mannoproteins [48]. In contrast, hydrophilic cells have longer, evenly spaced, and radiating structures [47]. CSH enhances microbial adhesion enabling pathogens to produce a biofilm which is a microbial community protected by an extracellular polysaccharide matrix. Angiolella et al. showed that many Malassezia isolates possess hydrophobic properties, adhere to abiotic surfaces, and produce biofilms [49]. Malassezia is prone to biofilm formation, which is a serious health threat and may provide drug resistance and increase virulence. Pedrosa et al. showed that M. furfur from skin isolates form biofilms on epidermal surfaces in the in vitro model, and the amount of biofilm increases over time [50].

Figure 1 summarizes the main Malassezia virulence factors.

3. Antifungal Drugs against Malassezia Species

The prevalence of fungal infections worldwide has increased year after year due to the widespread use of immunosuppressive agents and the growing number of patients with critical infections [51]. Different studies evaluate the susceptibility of commercialized drugs to clinical Malassezia strains. Malassezia isolates are identified by morphological and biochemical criteria. The activity of reference drugs such as azoles (fluconazole, itraconazole, ketoconazole, voriconazole, luliconazole), or amphotericin B was assessed in vitro [52,53,54]. Rodrigues Silva et al. reported that Malassezia yeasts were susceptible to all tested drugs. However, around 1 in 10 strains exhibited high MIC for fluconazole and 1 in 100 strains showed resistance to several drugs [55]. Applying the same experiment design, Cafarchia et al. proved that fluconazole-resistant M. pachydermatis isolates exhibited cross-resistance to other azoles. This study underlined the importance of diversifying therapeutic options to fight fungal infections [56].

The purpose of this section is to describe active compounds that target identified virulence factors, such as thick membranes. Additionally, specific enzymes needed for yeast replication, like β-carbonic anhydrase or secreted lipases, are also interesting targets. Since Malassezia species are particularly lipophilic, this section will discuss how these drugs balance their hydrophilic/lipophilic properties.

Scheme 1 describes the different strategies used to tackle Malassezia-related infections.

3.1. Drugs Targeting Cell Wall and Membrane

Fungal cell walls are composed of glucans, chitin, mannans, glycoproteins and/or galactomannans, and chitosan. Their proportions and assembly depend on species. Therefore, drugs can impair the fungal cell wall and its biosynthesis.

Table 2. Targeting cell wall biosynthesis.

Biological Target	Active Compounds
Fungal ergosterol synthesis inhibitors	Azoles (ketoconazole, itraconazole, fluconazole, voriconazole, posaconazole…)
Squalene epoxidase inhibitors	Morpholines: amorolfine
	Allyamines: terbinafine, butenafine
Ergosterol disruptors (membrane permeabilization)	Polyenes antibiotics: amphotericin B or nystatin
Glucan synthesis Inhibitors	Echinocandins, caspofungine, micafungine, anidulafungin
Chitin synthesis inhibitors	Nikkomycin, Polyoxins

summarizes the different biological pathways that can be disrupted along with some active compounds.

3.1.1. Inhibition of the Cell Wall and Membrane Biosynthesis

Alkyl betaines are synthetic products obtained from betaine. Betaine 1, also known as trimethylglycine, is a natural compound used in the human diet and comes from sugar beet (Beta vulgaris). Alkyl betaines 2, or alkylamidopropylbetaines 3 are often introduced in shampoos as antistatic and viscosity-increasing agents (Figure 2). Hee Jung et al. tested the antifungal activity of alkyl betaines against M. restricta KCTC 27527 [57]. Among the six betaine derivatives, lauryl betaine 2a exhibited high activity with MIC = 1.5–3 mg/mL. Using Cryptococcus neoformans as a phylogenetically close model, the authors showed that 2a impaired cell membrane synthesis by affecting ergosterol production.

Amorolfine hydrochloride (AMF), terbinafine hydrochloride (TBF), butenafine hydrochloride (BTF), neticonazole hydrochloride (NCZ), and ketoconazole (KCZ) are filed drugs classified as ergosterol biosynthesis inhibitors and have been used for three decades for dermatomycoses (Figure 3). In their comparative study, Nimura et al. tested them against their different strains, such as Candida albicans, M. furfur, and Trichophyton spp. Surprisingly, even if all these antifungal agents were supposed to have the same biological target, they clearly exhibited different antifungal potency. The two azole compounds (NCZ and KCZ) were active against M. furfur, whereas allylamine TBF and benzylamine BTF failed to inhibit M. furfur. The morpholine derivative AMF was the only drug that was fully active against all strains and could be called broad-spectrum antifungal [58]. The susceptibility of the fungal strains against this panel of drugs is represented in Figure 4. As shown, the antifungal properties of AMF, TBF, BTF, NCF, and KCZ strongly differed. This could be due to the difference in their chemical structures but also to the specific targets within the sterol biosynthesis. Three sub-groups can be defined according to their mode of action. TBF and BTF specifically inhibit the activity of the squalene epoxidase enzyme that is crucial in the formation of sterols necessary for fungal cell membranes (represented in red and orange, respectively, in Figure 4). Fungi treated with those drugs accumulate squalene while becoming deficient in ergosterol. As squalene epoxidase is not an enzyme of the cytochrome P-450 type, there is no potential inhibition of this class of enzymes. The morpholine derivated drug AMF (blue in Figure 4) mode of action is different and relies on the inhibition of two fungal enzymes: D14 reductase and D7-D8 isomerase, which are implicated in sterols biosynthesis. NCZ and KTZ (pale and dark green in Figure 4) block the synthesis of ergosterol, a key component of the fungal cell membrane, through the inhibition of cytochrome P-450 dependent enzyme lanosterol 14α-demethylase. By observation of the MIC against the panel of fungal strains, it is obvious that targeting the squalene epoxidase enzyme is more efficient in Tricophyton spp. Than in C. albicans or M. furfur. In contrast, KCZ and NCZ are potent antifungal agents against C. albicans and M. furfur. As far as M. furfur is concerned, AMF seemed the best antifungal agent. Its structure and mode of action could inspire the development of next-generation antifungal drugs. Chimaphilin, also known as 2,7-dimethyl-1,4-naphthoquinone, is the principal ingredient in Chimaphila umbellata (L.) W. Bart (Pyrolaceae) ethanol extracts (Figure 3). Chimaphilin exhibited antifungal activity against M. globosa (MIC = 0.39 mg/mL) and M. restricta (MIC = 0.55 mg/mL). Chemical-genetic profiling of chimaphilin indicates that this drug targets cell wall biogenesis and transcription pathways [59].

3.1.2. Permeabilization of the Cell Membrane

Natural Compounds

M. restricta strains are becoming increasingly resistant to antifungal agents due to their resistance profile. Zanthoxylum schinifolium Siebold & Zucc. Essential oil (ZSEO) was investigated by Kan et al. [60]. Linalool (34.15%), limonene (21.24%), and sabinene (10.39%) were the main ingredients of ZSEO. ZSEO showed efficient antifungal activity against M. restricta with MIC and MFC values of 2.5 and 10 mg/mL, respectively. This result could be attributable to linalool, which is itself an antifungal agent (MIC = 2.5 mg/mL), more than to limonene or sabinene, which both displayed MIC values superior to 20 µg/mL. Low content components, such as α-pinene, β-ocimene, myrcene, terpinolene or hotrienol could also act as antifungals. To elucidate ZSEO’s mode of action, Kan et al. performed a series of experiments. Cell membrane damage assays revealed interactions between ZSEO and the cell membrane. This led to an increase in Zeta potential and depolarization of the cell membrane (Figure 5). ZSEO provoked a perturbation of membrane lipid organization, resulting in greater cell membrane fluidity (Figure 5). Moreover, the level of oxidative stress of M. restricta upon ZSEO treatment was measured by the fluorescent oxidative stress-sensitive probe DCFH-DA. The accumulation of intracellular ROS is characterized by the increasing green fluorescence intensity of the probe. This has been quantified by spectrophotometry, indicating a strong ROS accumulation (Figure 5). Overall, ZSEO interfered with cell metabolism, disturbed ROS homeostasis, and induced morphological and ultrastructural changes in M. restricta cells (Figure 5).

Scolopendin, a novel cationic antimicrobial peptide (AMP), was isolated and characterized from the centipede Scolopendra subspinipes mutilans [61]. Lee et al. evaluated its antimicrobial activity against a panel of pathogenic microorganisms, including M. furfur KCTC 7744. Scolopendin exhibited a MIC value of 25 µM, which was lower than the one of Mellitin used as reference (MIC = 3.1–6.3 µM). This AMP did not produce hemolyze human erythrocytes even at 100 µM whereas Mellitin fully hydrolyzed them at the same concentration. Through the combination of several assays, the mode of action of scolopendin was elucidated on a C. albicans model strain and relied on its ability to interact with the microbial membrane and create pores (Figure 6).

The nuclear entry inhibitory signal peptide of HIV-1 Rev protein (Rev-NIS) showed potent antifungal activity against M. furfur (MIC = 20 µM) without hemolytic effects. Gathering a panel of assays, its mode of action was elucidated and relied on the depolarization and disruption of fungal membranes [62].

Synthetic Compounds

The cationic antimicrobial polymer-polyhexamethylene guanidine hydrochloride (PHMGH) has been described as a potent antifungal against Malassezia furfur KCTC 7744 with MIC = 2.5 µg/mL, being as active as amphotericin B [63]. This compound is positively charged and highly hydrophobic (Figure 7). After PHMGH treatment, the cells changed size and granularity. The membrane permeabilization was confirmed by ion transition assays. This showed that PHMGH exerted fungicidal activity by producing pores through the cell membrane. PHMGH did not show any hemolytic activity against human erythrocytes even at concentrations up to 16 × MIC. In contrast, amphotericin B destroyed erythrocytes at the same concentration. The mode of action of PHMGH is based on the cationic guanidinium groups that bind to the oxoanion phosphate group of the bacterial phospholipid membrane in a two-point hydrogen bonding chelate motif [64,65].

In the last two decades, less than a dozen studies investigated drugs that interfere with cell membrane biosynthesis or disrupt membranes. However, this thick and highly hydrophobic layer is responsible for the attachment of the cell to surfaces and subsequent proliferation. The yeast’s hydrophobicity has been identified as a major virulence factor. Studies on lipophilicity management in drug development have been conducted. The impact of maintaining a hydrophilic/lipophilic balance will be demonstrated in the following sub-part.

3.2. Impact of Hydrophobicity in Drug Ability towards Malassezia spp.

It is crucial to optimize the hydrophilic/lipophilic balance (HLB) of a compound in order to optimize its intrinsic antifungal properties [66]. Zhou et al. demonstrated that an appropriate amphiphilicity balanced by the alkyl chain length, and the positive charge is an essential factor for the biocompatibility of cationic antimicrobial guanidine polymer. By studying four chemically related oligoguanidine polymer derivatives (Figure 8), the authors linked the alkyl carbon chain length with biocidal activity of the polymer and increased hemolytic activity, calcein dye leakage rate, and membrane interactions. Thus, intermediate compounds that disrupt the cytoplasmic phospholipid membrane of bacteria while having limited hemolytic effects on eucaryotic cells are preferred. Overall, polymers with optimal amphiphilicity, which are selected by varying the length of the alkyl chains, the positive charge, and their spatial arrangement, selectively target bacterial membranes instead of mammalian membranes [67,68].

The importance of lipophilicity has also been demonstrated by Supuran et al. for the design and evaluation of dithiocarbamate derivatives as β-class carbonic anhydrase inhibitors, as described in the title subpart [69].

Management of hydrophobicity helps to control drug delivery and efficacy. A number of recent studies have demonstrated that nanoparticles can increase antifungal activity against Malassezia species.

In 2020, Zhang, Lv et al. described the impacts of ketoconazole-lecithin-zein nanoparticles compared to free ketoconazole (KCZ) [70]. In the treatment of skin fungal diseases, KCZ is a widely used antifungal. However, it exhibits some limitations, such as low concentration, short efficacy duration, and severe systemic toxicity. Core-shell structures containing KCZ trapped in lecithin-zein nanoparticles (KLZ-NPs) allowed us to overcome these issues. Indeed, the in vitro penetration of KLZ-NPs in the stratum corneum and in deeper layers of skin increased. This improved skin distribution coupled with lower concentrations of KLZ-NPs in the spleen and liver, resulting in limited side-effects.

KCZ encapsulation was also reported by Kaur et al. one year later [71]. KC loaded solid lipid nanoparticles (KTZ-SLNs) were prepared and evaluated. Spherical nanoparticles, displaying a mean size of 292 ± 6.3 nm and an optimal zeta potential equal to—24.39 exhibited a strong antifungal ability against M. furfur. KTZ-SLNs showed a 75–95% reduction in MIC value compared to the free-drug suspension (KTZ-SUS). Moreover, adding the lipidic shell allowed a 12.6-fold higher penetration up to the dermal layer of the skin. In addition, such nanoparticles showed high biocompatibility and hemocompatibility profiles.

Following the same objective, carvacrol nano-based delivery systems were designed [72]. Altogether, nanoparticles increased hydrophobicity [73].

3.3. Impacting Nuclear Aryl Hydrocarbon Receptors by AhR Antagonists

The aryl hydrocarbon receptor (AhR) is a nuclear receptor and transcriptional regulator with multifaceted biological functions. Malassezia yeasts produce potent AhR ligands, such as indirubin (IND), indolo[3,2-b]carbazole (ICZ), tryptanthrin, and malassezin, has been linked with its pathogenesis. For example, malassezin induces apoptosis in primary human melanocytes [74]. Janosik, Begman et al. recently reviewed the chemistry and properties of indolocarbazoles, underlying their involvement in numerous biological pathways [75]. Therefore, proposed molecules able to block these activators may lower Malassezia spp. virulence.

Only a few studies on AhR antagonists have been conducted in Malassezia species to date. In 2022, Magiatis et al. described the capacity of Rosmarinus officinalis L. Leaf Extracts (ROE) and their metabolites to inhibit AhR activation. The measurements were performed both in vitro (guinea pig cytosol) and in human keratinocytes. The prototype ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and Malassezia ligands were used to activate AhR. In such assays, metabolites of R. officinalis L., as abietane derivatives carnosol, carnosic acid, and triterpene betulinic acid, exhibited dose-dependent antagonist activity against TCDD (Figure 9) [76].

3.4. Targeting Enzymes Implied in Cell Survival

Few strategies and treatments combat Malassezia species, most of which target fungal growth. Drug resistance has led to alternative approaches. Several alternative approaches have been suggested, including inhibition of enzymes, such as the β-carbonic anhydrase (CA, EC 4.2.1.1) or the lipase secreted by Malassezia species.

3.4.1. β-Class Carbonic Anhydrases

Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes belonging to seven genetically distinct families. The active sites of these enzymes are usually zinc-containing, but ferrous iron, cadmium, and zinc may also be present, depending on the organism. Many of these enzymes can be inhibited pharmacologically [77,78,79]. Over the years, synthetic and natural compounds have been reported.

Synthetic Compounds

Using dithiocarbamate derivatives (DTCs) as an inhibitor, Supuran et al. demonstrated that it was able to inhibit a β-class carbonic anhydrase (CA, EC 4.2.1.1) from M. globosa (MgCA) [69]. DTCs are bioactive compounds largely used in varied medicinal applications, due to their ability to form chelates and interact with metalloenzymes active sites [80]. All DTCs proved to be more effective than the reference drug acetazolamide AAZ with Ki ranging from 383–6235 nm compared to Ki = 74 µM for clinically used AAZ (Figure 10). For aliphatic DTC derivatives, lipophilicity was correlated to antifungal activity (Figure 10).

Surupan and Nocentini groups are actively involved in the design and evaluation of new compounds as β-carbonic anhydrases inhibitors. Following their preliminary work on N-nitrosulfonamides [81], in 2019, they synthesized a series of benzenesulfonamides bearing nitrogenous bases, such as uracil and adenine, and evaluated their activity against M. globosa carbonic anhydrase (MgCA). These compounds displayed low Ki with values ranging from 138.8 nM to 5012.8 nM. The most active compound was the adenine derivative AD (Figure 10). According to this study, designed compounds inhibited strongerβ-carbonic anhydrases from Candida glabrata (CgNce103) and Cryptococcus neoformans (Can2) [82].

Boron molecules are increasingly studied in medicinal chemistry, as antifungals [83]. In 2017, the same team described benzoxaboroles’ activity. As for benzenesulfonamides, all reported derivatives showed nanomolar inhibitory activities against Can2 and CgNce103 vs. micromolar inhibition against MgCA (Figure 11). The selectivity for fungal CA towards human CA was fair to excellent. MgCA’s active site interactions were predicted using molecular modeling (Figure 11). Chemically, modification of the ureido by a thioureido group did not impact inhibitory activity. However, changes in the substitution pattern on the phenyl ring of the ureido/thioureido group result in differences in inhibitory activity and selectivity compared to hCA I and hCA II [84]. Bortezomid BZ, a boron derivative, was also investigated previously by Supuran as a CA inhibitor [85]. BZ inhibited MgCA at low micromolar concentrations. This compound interacts with CAs by chelating the zinc cation present in the active site (Figure 11).

Recently, Surpuran et al. reported that seleno-containing molecules act as potent and selective antifungal agents against M. pachydermatis. Compared to oxygen or sulfur, the selenium atom increased antifungal activity. The sulfonamide moiety has been modified to be selective for M. pachydermatis compared to M. furfur and M. globosa from its related genus. A number of selenium-containing compounds displayed a safe toxicity profile and may be considered as promising antifungal agents (Figure 12) [86].

Natural Compounds

In the same period, Supuran et al. proved that natural polyphenols, such as flavones, flavonols, flavanones, flavanols, or isoflavones exhibited MgCA inhibitory activity in the micromolar range. In addition, they showed high selectivity towards the fungal isozyme compared to its off-target human isoforms. Thanks to molecular docking investigations, they proposed that the natural polyphenols binding mode is based on zinc interaction through the hydroxyl group [87]. Synthetic phenols were also investigated. The hydroxyl group in the inhibitors anchored to the zinc-water coordinated in the active site and created hydrogen bonds with amino acid residues. Changing the phenyl group altered the hydrophobic pocket within MgCA’s active site [88] (Figure 13).

In summary, research conducted by th Supuran team has led to the design and evaluation of half a dozen chemical classes that can successfully inhibit Malassezia β-carbonic anhydrases. These derivatives are categorized into two main groups: anions, such as dithiocarbamates and hetero-compounds, bearing sulfur, boron, or selenium moiety. Sulfonamide derivatives are most abundant (Figure 14). A comprehensive overview of all compounds is available in [77]. Based on these reports, the design of novel entities is still ongoing and could help defeat resistant fungal infections due to Malassezia species.

3.4.2. Quenching Secreted Lipases

Secreted lipase is an indispensable virulence factor to ensure fungal cell growth. The ability of Malassezia spp. to metabolize lipids and incorporate fatty acids into the cell wall is necessary for its pathogenicity. Therefore, quenching secreted lipases may be an effective method of stopping proliferation.

Karutha, Pandian et al. identified embelin as an antifungal that can inhibit lipase activity in Malassezia species [68]. A MIC of 400 µg/mL was observed for embelin, which resulted in a 65–89% reduction in lipase activity. Additionally, it showed synergistic effects when combined with ketoconazole [89].

The synergy between conventional drugs and natural products has already been described [90]. Additionally, embelin is highly lipophilic (logP = 5.70). Its long aliphatic chain (eleven carbons) may interact with the lipid membrane and disturb it. This long alkyl tail is like seleno-derivative 10c (Figure 15).

One approach to relieving the M. globosa induced diseases relies on blocking the secreted lipase activity. Malassezia globosa LIP1 (SMG1), a representative secreted lipase from M. globosa CBS 7966. It represents a potential target. Wang et al. reported a docking-based virtual screening based SMG1 crystal structure and lipase assay [91]. These experiments were conducted to identify the first HIT molecule against SMG1 called Cpd 1 (Figure 16). RHC 80267, a well-known inhibitor of mammals’ lipases, was selected as a positive control. RHC 80267 exhibited an IC₅₀ = 75.25 µM whereas Cpd 1 IC₅₀ was 4-fold higher (IC₅₀ = 20.09 µM). These results could serve as a starting point for the rational design of more potent inhibitors against SMG1.

Targeting secreted lipases to quell its activity is a novel approach to drug development against Malassezia spp.

3.5. Antioxidant Activities and ROS Attenuation

Pathogens from the Malasseziae genus cause persistent inflammation. To control diseases caused by Malassezia species, compounds that reduce ROS production and inflammation are of great interest. A number of routine assays can be performed, such as the DPPH assay and nitric oxide (NO) radical scavenging activity assay. A compound’s anti-inflammatory potential can also be determined by measuring the expression of interleukins or TLRs. Recently, only natural compounds, such as essential oils, fatty acids, and antimicrobial peptides, have been reported.

3.5.1. Extracts

In 2022, Chaiyana et al. reported the anti-inflammatory and antimicrobial activities of fermented Ocimum sanctum Linn. extract (FE) against a panel of microbial strains, including four different strains of M. furfur [92]. Nuclear factor kappa B (NF-kB) expression inhibition was measured using Western blot analysis.

Table 3. MIC₅₀ (mg/mL) and MFCs (mg/mL) of fermented O. sanctum extract (FE) against four strains of M. furfur.

Samples	M. furfur 133		M. furfur 656		M. furfur 6000		M. furfur 7966
	MIC50	MFCs	MIC50	MFCs	MIC50	MFCs	MIC50	MFCs
Ketoconazole	0.0078	0.0078	0.25	0.25	0.0625	0.0625	0.125	0.125
Eugenol (main component of FE)	0.125	0.125	0.25	0.25	0.125	0.5	0.125	0.5

and Figure 17A summarize the antifungal activities against M. furfur. Figure 17B shows the level of NF-kB after treatment of U937 cells. FE showed excellent activity, comparable to iodomethacin INC used as a positive control. Taken together, these data suggest that FE could be used as an alternative anti-inflammatory agent. In addition, it could be formulated as a topical antimicrobial agent against M. furfur to replace current chemical antimicrobial agents.

Kritzinger et al. reported the activity of nine plant ethanolic extracts against M. furfur. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging activities of these extracts were investigated. Among the tested panel, the root extract of Rhoicissus tridentata showed the highest antifungal activity against M. furfur with a 20.3 ± 4.7 mm zone of inhibition at a concentration of 2 mg/mL. R. tridentata extract displayed an IC₅₀ = 2.3 ± 1.9 µg/mL DPPH radical scavenging activity, being as potent as ascorbic acid used as a reference. It also showed a fair NO radical scavenging ability with an IC₅₀ = 306.2 ± 3.4 µg/mL, being 5-fold less active than vitamin C. Moreover, the absence of a cytotoxic effect on HaCat cells indicates that such extract could be explored for use as a topical treatment [93].

Chimaphilin (structure shown in Figure 3), also known as 2,7-dimethyl-1,4-naphthoquinone, is the principal ingredient in Chimaphila umbellata (L.) W. Bart (Pyrolaceae) ethanol extracts. Chimaphilin exhibited an antifungal activity against Malassezia globosa (MIC = 0.39 mg/mL) and Malassezia restricta (MIC = 0.55 mg/mL). C. umbellata crude extract showed strong antioxidant activity of measured using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. The IC₅₀ was determined at 142.1 ± 3.6 ppm, ten-fold less active than vitamin C (22.1 ± 0.1 ppm) or quercetin (17.2 ± 0.1 ppm). This assay suggests antioxidant ability of extract [59]. The diversity of cellular processes affected by chimaphilin suggests that the compound has multiple targets within the yeast cell.

According to Pedrosa et al. [94], seaweeds are a bioresource of active compounds. The antioxidant potential of S. muticum fractions was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, the ferric reducing antioxidant power (FRAP), the oxygen radical absorbance capacity (ORAC) and its total phenolic content (TPC) was determined by the Folin–Ciocalteu method (Figure 18).

3.5.2. Fatty Acids

The role of fatty acids in dietary supplementation is highly studied. Medium fatty acids have been widely reported for their metabolic features and cognition in humans [95,96,97] whereas long ones are fewer mentioned. Boulila et al. reported Dittrichia viscosa L. leaves lipid extract as an underestimated source of essential fatty acids and tocopherols with antifungal and anti-inflammatory activities [98]. The chemical composition of this lipid extract was determined by spectroscopic methods. Linoleic and linolenic acids were assessed as the main fatty acids while α- and δ-tocopherols were the most abundant tocopherols (Figure 19). All these compounds are highly lipophile with logP ranging from 6.5 to 11.9. This extract exhibited inhibition of a panel of Malassezia strains with ZOI ranging from 13 to 20 mm at 100 mg/mL even for fluconazole-resistant strains. This property of inhibiting elastase enzyme may be crucial to managing inflammation associated with yeast infections. This extract displayed anti-elastase activity (Figure 19B), increasing with concentration. Anti-elastase properties were similar to those of epigallocatechin gallate (EGCG) at a concentration of 10 mg/mL. The authors linked the richness of polyunsaturated fatty acids and tocopherols to this high anti-elastase capacity. However, the mechanism of elastase inhibition is still under-documented. These data must be related to those underlining the role of fatty acids and tocopherols in decreasing inflammation pathways [99,100,101] or the impact of additional fatty acids on Malassezia species’ growth [102]. In conclusion, this lipophilic extract may be effective in controlling inflammatory diseases and fungal infections.

3.5.3. Antimicrobial Peptides (AMPs)

Because M. furfur-related diseases are problematic to treat and associated with increasing inflammation, the antifungal and anti-inflammatory effects of the antimicrobial peptide cecropin A(1–8)–magainin 2(1–12) hybrid peptide analog P5 on M. furfur were investigated. P5’ s minimal inhibitory concentration against M. furfur was 0.39 mM. This made it 3–4 times more potent than commonly used antifungal agents such as ketoconazole (1.5 mM) or itraconazole (1.14 mM). P5 does not induce eukaryotic cytotoxicity at its fungicidal concentration. P5 decreased the expression of Toll-like receptor 2 and IL-8 in M. furfur-infected human keratinocytes (Figure 20). In addition, P5 significantly downregulated NF-kB activation and intracellular calcium fluctuation, two pathways related to increased responses to keratinocyte inflammation induced by M. furfur infection [103].

3.6. Apoptosis Inducers

Apoptosis is a form of programmed cell death in multicellular organisms. Tambjamines are natural products belonging to the group of alkaloids. They are composed of a bipyrrole ring bearing an enamine moiety. The diversity of this nitrogen-substituent led to a large variety of derivatives and biological applications. From 2010, Tambjamine derivatives were reported with potent antimicrobial, anticancer, and antimalarial bioactivities [104,105,106,107,108,109,110,111,112]. In 2010, Banwell, Pessoa et al. reported the first anti-Malassezia potential of some synthetically derived tambjamines [113]. Most of them were more active than amphotericin B to eradicate M. furfur colonies. Tambjamine I and Tambjamine J displayed a zone of inhibition against M. furfur strains equal to 17 mm at a concentration of 0.1 mg/mL. Tambjamines I and J exhibited significant apoptosis-provoking effects (Figure 21), which is slightly similar to those of the Doxorubicin (D) used as a positive control. According to reported works, the biological effects of Tambjamines to induce apoptosis could be related to their anion transport ability [114,115,116,117,118] and their lipophilicity is considered as a key factor for activity [119].

Coprisin is a 43-mer defensin-like peptide from the dung beetle, Copris tripartitus. Coprisin exhibited high antifungal activity against M. furfur with MIC = 5 µM without any hemolytic side-effects. Like C. albicans, its mode of action is likely related to apoptosis. Indeed, coprisin did not induce rupture of the cell membrane but accumulates in the nucleus, inducing programmed cell death [120].

3.7. Targeting Hyphae Growth

Rhizomes from the Zingiberaceae species are particularly popular in Thaï folk remedies to treat Malassezia-related skin infections [121]. In 2021, Juntachai and Laokor investigated the antifungal potency of Zingiberaceae extracts. Among the tested panels, A. galanta n-hexane extract containing 83% of (2,6-dimethylphenyl)borate was the most active fraction with MIC ranging from 0.04–0.08 mg/mL against a panel of 6 different M. furfur strains. In addition to the low content of hexadecenoic acid (4%) and 1,8-cineol (2.6%), other lower content components were also recorded (Figure 22). Due to the reported activity of long chain fatty acids, hexadecanoic acid could play a role in the observed antifungal activity. This is even at low concentrations. Its mode of action was elucidated using microscopic and macroscopic cell observation. It relies on an inhibitory effect on virulent hyphal growth (Figure 23).

3.8. Active Compounds without Known Biological Targets

Compounds, active against Malassezia species but without indication of mode of action, will be discussed in this subpart. Natural compounds, biosourced derivatives, and synthetic compounds are divided into three categories.

3.8.1. Synthetic Anti-Malassezia Compounds

A novel protocol for the N-acylation of sulfonamides and carbamates with carboxylic acid anhydrides under solvent-free conditions was reported by Kumar Thulam et al. [122]. The synthesized N-acyl drug candidates were successfully evaluated as antifungal agents against M. furfur and M. pachydermatis using ketoconazole as a reference. Compounds 2a, 4a exhibited MIC = 10 µM, being more active than ketoconazole (MIC around 95 µM for both strains) (Figure 24). The lipophilicity of compounds 2a and 4a, expressed by their logP, is lower than ketoconazole. It is notable that modification of the NH did not impact biological activity, whereas chemical modifications of the aryl or alkyl moieties dramatically decreased antifungal potency [122]. Structure–activity relationships are summarized in Figure 24. No mode of action for such compounds has been hypothesized but these compounds are structurally similar to β-carbonic anhydrase inhibitors.

In a similar manner, Gruzman, Cohen et al. described the 8-step synthesis of novel sulfamoylbenzoates and their subsequent inhibition of M. furfur growth. Methyl-3-bromo-2-nitro-5-(N-phenylsylfamoyl)benzoate SB-1 exhibited significant cytotoxic activity against M. furfur and was chosen as HIT for further development in the area of seborrheic dermatitis drugs (Figure 25) [123].

3.8.2. Bio-Inspired Compounds

Green silver nanoparticles (AgNPs) were synthesized using Coriandrum sativum leaf extract. Flavonoids present in the extract serve as reducing agents for nanoparticle formation. A particle size of 37 nanometers was measured. The AgNPs antifungal activity was measured against Malassezia furfur MTCC 1374 and displayed a MIC = 25 µg/mL [124]. Rao and Paria [125] also reported the antifungal activity of Aeglemarmelos leaf extract mediated AgNPs against M. furfur. They reported a 20.5 mm inhibition zone at 6.8 μg/mL concentration. A review on the use of natural-product-based nanomedicine has recently been published, which helps to rationalize such development [126].

3.8.3. Natural Compounds

Numerous reports and some reviews deal with natural compounds used as antifungals against Malassezia species without describing their mode of action [127,128]. The mode of action of natural compounds has already been hypothesized based on other yeasts, such as Candida spp. These include the permeabilization of the membrane, DNA, and mitochondrial damage [129]. In this subpart, selected examples will be detailed to show the richness of chemical structures from bioresources.

An investigation by Santomauro et al. reported that Artemisia annua essential oil had antifungal activity against a panel of Malassezia species, both in liquid and vapor phases [130]. Characterization of the essential oil revealed monoterpenes as the major compounds in both phases (Figure 26). Essential oil of A. annua, both in liquid and vapor phase, exhibited significant antimicrobial activity towards almost all the twenty strains of Malassezia tested. For most strains, the MIC ranged from 0.78 µL/mL to 1.56 µL/mL. Moreover, the MIC for the vapor diffusion assay was lower than those obtained by the liquid method.

The essential oils of four aromatic plants, e.g., Origanum vulgare subsp. hirtum, Mentha spicata, Lavandula angustifolia, and Salvia fruticose, exhibited antifungal properties against M. furfur without any mutagenic activity detected by the Ames test [131]. The chemical composition of each essential oil was determined.

Table 4. Composition of extracts and activity of the pure compound against M. furfur.

Entry		ZOI * (mm)	Origanum vulgare subsp. hirtum	Mentha spicata	Lavandula angustifolia	Salvia fruticose
1	Thymol	19	45.22	-	-	0.03
2	Carvacrol	15	33.05	-	-	-
3	Carvone	7	-	59.12	-	-
4	dihydrocarveol	9	-	6.87	-	-
5	1,8-cineole	2	0.21	5.42	13.10	43.10
6	Camphor	2	-	-	5.50	18.34
7	linalool	0	0.18	0.06	20.18	0.12
8	p-cymene	1	7.35	-	0.09	0.93
9	γ-terpinene	1	5.54	0.04	0.07	0.09
10	Linalyl acetate	0	-	-	18.60	0.07
11	Lavandulyl acetate	ND **	-	-	16.01	-
12	limonene	0	1.21	5.02	0.25	1.50
13	Essential oil ZOI (mm)		21	0	3.5	0

* Zone of inhibition measured in millimeters in the disk diffusion assay. ** ND is used for Not Determined.

summarizes the main components of each compound, along with the diameter of inhibition measured by disk diffusion against M. furfur. Among the 4 essential oils tested, Origanum vulgare subsp. Hirtum was the most effective growth inhibitor against M. furfur with a zone of inhibition of 21 mm. Based on its composition, thymol and carvacrol were identified as its main active ingredients. Interestingly, carvacrol and thymol share strong chemical properties (Figure 27), which means they are both natural phenols.

Celery essential oil at 1% and its volatile vapor diluted at 1 mL/800 mL air inhibited the M. furfur growth [132]. However, the exact composition of such celery essential oil was not determined. Usnea sp. methanol extract (1 mg/disc) was reported by Rukayadi et al. as fair antifungal against M. furfur ATCC 14521 [117]. These fruticose thalli lichen inhibited growth of M. furfur with inhibition zone diameter of 34 mm and MIC and minimum fungicidal concentration (MFC) values of 16 and 64 μg/mL, respectively [133,134]. Notably, all identified chemical components displayed high lipophilicity, as expressed by their logP.

Maboni et al. as reported that propolis extract was active against M. pachydermatis with a MBC₉₀ = 5.3 mg/mL [135]. In this study, the ethanol and butanol fractions of white rose petal extract were evaluated for their antifungal activities against M. furfur. They exhibited strong MICs of 1.5 mg/mL and 0.5 mg/mL for WRPE-EtOH and WRPE-BuOH, respectively [136].

Naeini et al. reported in 2011 the evaluation of essential oils (EO) derived from water-distillation from three Iranian medicinal plants against 29 different Malassezia strains. The chemical composition of Zataria multiflora, Pelargonium graveolens and Cuminum cyminum EOs was fully characterized and revealed that the main oil components were carvacrol (61.3%) and thymol (25.2%) for Z. multiflora, α-pinene (30%), limonene (21%) and 1,8-cifor C. cyminum and citronellol (28.2%) and geraniol (22.1%) for P. graveolens (

Table 5. Chemical structures and activities of essential oils from [137].

	Zataria multiflora	Pelargonium graveolens	Cuminum cyminum
Mean ZOI (mm)	28.1	26.1	48.3
Lower ZOI (mm)	20	10	30
Higher ZOI (mm)	>50	>50	>50

). The activity was measured by the disk diffusion method and expressed as the zone of inhibition (ZOI in mm). In comparison with ketoconazole as a reference, Malassezia spp. showed a high susceptibility to the three extracts [137]. No information was provided about the mode of action of these EOs.

A review by Bilia et al. detailed the biological activity of a variety of plant essential oils against a panel of Malassezia species [138].

Table 6. Main components and biological activity against M. furfur for a panel of essential oils.

Resource		Main Chemical Components	MIC	Reference
	Cinnamomun zeylanicum Blume		32 µg/mL	[139]
	Ocimum kilimandscharicum Gürke		128 µg/mL
	Malaleuca leucadendrun (L.) L.		64 µg/mL
	Syzygium aromaticum (L.)		0.625 µL/mL	[140]
	Thapsia villosa (L.)		2.5 µL/mL	[141]
	Deverra tortuosa subsp. arabica Chrtek, flowers		5.0 µL/mL	[142]
	Deverra tortuosa subsp. arabica Chrtek, stem		8.0 µL/mL
	Origanum vulgare L.		780 µg/mL	[143]
	Thymus vulgaris L.		920 µg/mL

summarizes the main chemical components identified in the essential oil towards their measured activity against M. furfur.

Table 7. Main components and biological activity against different Malassezia strains for a panel of essential oils. * is for µL/mL.

			M. furfur	M. sympodialis	M. slooffiae	M. globosa	M.obtusa	M. nana	M. restricta
Bioresource		Main Chemical Components	MIC (µg/mL or *µL/mL)							Reference
	Zataria multiflora Boiss.		35	30	80	50	60	30	40	[127]
	Thymus kotschyanus Boiss.		60	60	80	80	80	30	110
	Mentha spicata L.		125	100	100	250	85	65	85
	Artemisia sieberi Besser		250	85	150	50	155	110	100
	Salvia rosmarinus Schleid.		260	250	420	410	850	100	350
	Heracleum persicum Desf. ex Fisch.		125	250	480	250	560	75	320
	Myrtus communis L.		31 *	62	31	31	62	-	125	[144]

presents the same data but extended to seven different Malassezia strains. As described before, all active compounds exhibited high liphophilicity as expressed by logP.

All these data highlight the potential clinical applications of essential oils and natural resources against Malassezia species. In essential oils, active compounds come from different chemical families, such as terpenes and phenols, but they share some similarities, in particular their high lipophilicity. Indeed, the logP of each ingredient ranges from 1.90 for thujone to 6.78 for β-caryophyllene. For the highly lipophilic Malassezia yeast, this characteristic is essential.

To go further, in 2020, Rajput and Kumar reviewed medicinal plants as a source of bioactive ingredients against pathogens targeting hair and scalp [128]. Kowalski also reported earlier the antimicrobial activity against M. pachydermatis of essential oils and extracts of the roseweed plants used by American Indians. These oils had an inhibitory effect comparable to Amphotericin B [145]. Other bioresources have been reported such as: (a) roots of Asparagus racemosus Willd [146], (b) antimicrobial antibiotics from soil Streptomyces sp. SCA 7 [147], (c) leaf and rhizome of Hedychium coronarium, active thanks to its antioxidative capacity [148], (d) seed and leaf from Alpinia speciosa cultivated in Taiwan [149], (e) Ilex paraguariensis [150], (f) apricot seed [151], and (g) Vitis vinifera seeds [152].

In summary, nature is a great inspiration for the discovery and development of new antifungals to treat Malassezia species.

4. Critical Review, Conclusions, and Perspectives

This review studies and compiles around fifty different reports. Over the past two decades, little research has been conducted on new compounds to tackle Malassezia spp. with a maximal occurrence of six papers per year (Figure 28). A growing number of papers is observed from 2018, possibly due to the emergence of resistant Malassezia strains, but this tendency needs to be confirmed.

Among them, 37%, i.e., 18 reports, presented active compounds without any clue about their mode of action against Malassezia spp.

In terms of the source of active compounds, it is noteworthy that 63% of the drug candidates are bioresources such as essential oils or antimicrobial peptides. Over half of such research reports mention extracts from medicinal plants. Generally, the chemical composition of the extracts has been elucidated and the main components are known.

From this review, we can draw the following conclusions:

• Lipophilicity is the key. Indeed, natural active molecules are lipophilic, with logP ranging from 1.90 to 11.90, with a medium value around 3–4. Moreover, synthetic organic compounds also displayed high lipophilicity. This criterion can be adjusted by formulation if the drug is incorporated into lipidic nanoparticles, which favors skin penetration and decreases side-effects.
• Chemically speaking, two classes of molecules could be developed. These are (i) heteroatomic ones, including selenium or boron, with an adjusted HLB; and (ii) amphiphilic compounds with a high ability to disrupt the thick cell membrane.
• Biological targets remain underexploited. Based on the reported mode of action, it appears (Figure 29) that β-carbonic anhydrase inhibitors and antioxidant compounds are the most common. Drug candidates mainly target membranes. Three targets are under-valued in anti-Malassezia drug development, AhR antagonists, inhibitors of secreted lipase, and biofilm inhibitors.

4.

Nature is a source of inspiration for the development of bio-inspired optimized drugs to selectively target Malassezia spp.

Overall, the growth of resistant strains of Malassezia is of current interest, which calls for the development of new strategies, relying on the virulence factors of this organism, to prevent an uncontrollable spread of human pathogenic strains. Among the various strategies that could be employed, the development of AhR antagonists, the inhibition of the secreted lipase, as well as the fight against the development of biofilms sound particularly interesting. The hydrophily/lipophily balance of a drug candidate should also be considered within this context, as well as within lipophilic yeast being the dominant cause of skin infections within a skin disease context, in order for the drug candidate to be successful in skin penetration and killing lipophilic yeast.