Chemical modifications of diphenyl diselenide enhance its anti-Sporothrix spp. activity and exhibit synergism with itraconazole

TL;DR

This study shows that modified forms of an organoselenium compound are effective against Sporothrix fungi, both alone and when combined with itraconazole.

Contribution

The study introduces two novel organoselenium compounds with enhanced antifungal activity and synergistic effects with itraconazole.

Findings

BMD inhibited all Sporothrix isolates with a minimum inhibitory concentration of 2 to 16 µg/mL.

Combining BMD or CF3 with itraconazole showed synergistic effects against most isolates.

CF3 showed partial activity against 92% of isolates at similar concentrations.

Abstract

Sporotrichosis is a globally distributed subcutaneous mycosis caused by fungi belonging to the Sporothrix genus. The limitations of current treatment options for sporotrichosis have driven an urgent search for new antifungal drugs. Diphenyl diselenide (PhSe)₂, a stable organoselenium, has promising activity against Sporothrix spp. Since chemical modifications in molecules can enhance their antifungal properties, this study aimed to evaluate the anti-Sporothrix activity of two analogs of (PhSe)₂—p-methoxyl-diphenyl diselenide (BMD) and bis-(3-trifluoromethylphenyl) diselenide (CF3)—both alone and in combination with itraconazole (ITZ). A total of 25 Sporothrix spp. isolates (S. brasiliensis
n = 20; S. schenckii
n = 4 and S. globosa
n = 1) were included. The microdilution and checkerboard assays were used to assess its antifungal activity alone and in combination, respectively. BMD inhibited all isolates, with a total minimum inhibitory concentration ranging from 2 to 16 µg/mL. In contrast, CF3 exhibited only a partial minimum inhibitory concentration activity in 92% of isolates at concentrations ranging from 2 to 16 µg/mL. When combined with ITZ, both compounds predominantly exhibited a synergistic effect (100% for BMD and 73% for CF3). Our study presents pioneering data on two organoselenium compounds with significant in vitro activity against the main pathogenic Sporothrix species, both alone and in combination with ITZ. Further studies are needed to determine whether these in vitro findings translate into in vivo efficacy.

Figures

Diphenyl diselenide and their analogs used to test their anti-*Sporothrix* sp. activity: *p*-methoxyl-diphenyl diselenide (BMD), with a methoxyl group in the *para* position at the aromatic ring and bis-(3-trifluoromethyl phenyl) diselenide (CF3) with a trifluoromethyl group in the *meta* position at the aromatic ring

Results of the interaction between *p*-methoxyl-diphenyl diselenide (BMD) and itraconazole (ITZ) against 15 *Sporothrix* sp. isolates, showing fold decreases of ITZ and BMD minimum inhibitory concentration (MIC) values for each isolate when drugs were combined

Result of the interaction between bis-(3-trifluoromethyl phenyl) diselenide (CF3) and itraconazole (ITZ) against 15 *Sporothrix* sp. isolates, showing a fold decrease of ITZ and CF3 minimum inhibitory concentration (MIC) values for each isolate when drugs were combined

Full text

Introduction

Sporotrichosis is an emerging endemic mycosis caused by Sporothrix genus [1]. Three species—S. schenckii stricto sensu, S. globosa, and S. brasiliensis—are responsible for more than 90% of clinical cases in humans and animals, each exhibiting distinct epidemiological patterns [2]. S. schenckii and S. globosa are primarily transmitted through environmental sources, such as organic matter and thorns, whereas S. brasiliensis is mainly spread through bites or scratches from infected domestic cats. Additionally, S. schenckii has a global distribution, while S. globosa is more prevalent in Asian countries, including China, Japan, and India [1,3]. S. brasiliensis, on the other hand, is predominantly found in South America, particularly in Brazil, where it poses a significant public health concern [4–6].

Itraconazole (ITZ) is the drug of choice for treating human and animal sporotrichosis [7,8]. However, its use has several limitations, including a long treatment duration (ranging from 12 to 27 weeks), high cost, and a significant frequency of adverse effects [9,10]. In humans, ITZ has a high efficacy rate, achieving clinical cure in 90 to 100% of cases with dosages ranging from 100 to 400 mg/day [11,12]. However, in cats, its effectiveness varies widely (˂50% to 71%), with treatment success further hindered by the difficulty of administering an oral medication once daily for several months. This often leads to treatment discontinuation by pet owners [13–15]. Additionally, clinical resistance and non-wild-type Sporothrix spp. isolates have been reported in both human and feline cases [16–19].

Therefore, it is essential to evaluate new compounds with potential anti-Sporothrix activity. One promising area of research involves organoselenium compounds, particularly ebselen and diphenyl diselenide (PhSe)₂, due to their activity against yeasts, molds, and dimorphic fungi [20–22]. Notably, (PhSe)₂ demonstrates in vitro antifungal activity against S. schenckii, S. globosa, and S. brasiliensis, both alone and in combination with ITZ [20,23]. Additionally, (PhSe)₂ has shown promising in vivo results in treating experimental sporotrichosis caused by S. brasiliensis, both alone and in combination with ITZ [22].

Since molecular modifications of organoselenium compounds can enhance their antimicrobial activities [24], two analogs—p-methoxyl-diphenyl diselenide (BMD) and bis-(3-trifluoromethylphenyl) diselenide (CF3)—are notable for their beneficial effects on host cells, such as hepatoprotective, antinociceptive, and nephroprotective properties (BMD), or antioxidant and anti-inflammatory activities (CF3) [25–27]. Additionally, an *in vitro *screening study demonstrated that BMD inhibits Candida spp., Aspergillus spp., and Fusarium spp., while CF3 is active against Fusarium spp. [28]. Given the urgent need for new antifungal drugs to treat sporotrichosis, we aimed to evaluate the activity of BMD and CF3 against Sporothrix spp., both alone and in combination with ITZ.

Materials and methods

A total of 25 Sporothrix spp. isolates were included (*S. brasiliensis *n = 20, *S. schenckii *n = 4, and *S. globosa *n = 1). Of these, 15 were obtained from human sporotrichosis cases, 8 from infected cats (all S. brasiliensis), 1 from an environmental source (S. brasiliensis), and 1 was a reference strain (S. brasiliensis CBS 120339). All isolates were previously identified using species-specific polymerase chain reaction defined as Rodrigues et al. [29]. For the interaction assays, 15 isolates were randomly selected and included (*S. brasiliensis *n = 10, *S. schenckii *n = 4, and *S. globosa *n = 1). All strains were stored in the repository of the Medical Mycology Laboratory (Faculty of Medicine, Federal University of Rio Grande, Brazil) and were recovered from storage at − 20 °C in brain heart infusion broth supplemented with 30% glycerol prior to testing.

ITZ (Janssen^®^, São Paulo, São Paulo, Brazil) was obtained commercially. The compounds BMD and CF3 were synthesized via the Grignard reaction (final HPLC purity of 99% and 98%, respectively) in collaboration with the Department of Biochemistry and Molecular Biology at the Federal University of Santa Maria (Fig. 1). BMD was tested at concentrations ranging from 0.25 to 64 µg/mL, CF3 at concentrations from 1 to 64 µg/mL, and ITZ at concentrations from 0.03 to 8 µg/mL. The concentrations tested for the organoselenium compounds were initially based on previously published studies [20,23], and were further determined through preliminary solubility and dilution assays in RPMI 1640 medium, in order to establish the highest concentration that could be reliably diluted in this medium. All drugs were diluted in dimethyl sulfoxide and stored at -20 °C at a stock concentration of 6.400 µg/mL.

Fig. 1. Diphenyl diselenide and their analogs used to test their anti-Sporothrix sp. activity: p-methoxyl-diphenyl diselenide (BMD), with a methoxyl group in the para position at the aromatic ring and bis-(3-trifluoromethyl phenyl) diselenide (CF3) with a trifluoromethyl group in the meta position at the aromatic ring

The broth microdilution technique was used for in vitro antifungal susceptibility testing and was performed in 96-well microplates according to the M38-A2 protocol of the Clinical and Laboratory Standards Institute (CLSI). Briefly, the isolates were subcultured as young mold colonies in potato dextrose agar (PDA) tubes (Kasvi^®^, São José dos Pinhais, Paraná, Brazil) and incubated at 25 °C for 7 days. Each colony was covered with sterile distilled water and gently scraped to obtain a conidial suspension, which was then transferred to a separate tube containing only conidia. The solution was adjusted using a spectrophotometer at 530 nm to a final concentration of 0.8 × 10⁴ to 1 × 10⁵ colony-forming units (CFU)/mL and subsequently diluted in RPMI 1640 medium. The Pour-Plate technique was used to confirm the concentration for each isolate and in all tests was used the reference strain (S. brasiliensis CBS 120339). Drug concentrations were prepared by dilution in RPMI 1640 (1:50 ratio) and added to the microplate (100 µL), along with the inoculum (100 µL). For all tests, growth, and sterility controls were included in RPMI 1640, and *S. brasiliensis *CBS 120339 susceptibility testing was used as a control for drug activity. The microplates were incubated at 35 °C for 72 h. After incubation, visual readings were performed to determine the partial minimum inhibitory concentration (MIC_p_), defined as the lowest concentration that inhibits approximately 50% of fungal growth, and the total minimum inhibitory concentration (MIC_t_), defined as the lowest concentration that completely inhibits fungal growth, for each isolate against each compound.

Additionally, the minimum fungicidal concentration (MFC) was determined as the concentration capable of killing 100% of the fungal inoculum. This was assessed by plating 50 µL from wells without fungal growth onto PDA and incubating for seven days to evaluate fungal viability. Results were analyzed by determining the geometric mean (GM). Furthermore, the compounds were classified as fungistatic or fungicidal based on the MFC/MIC_t_ ratio for each strain, considering the values of 100% inhibition. A ratio > 2 indicated a fungistatic effect, whereas a ratio ≤ 2 indicated a fungicidal effect [31].

The interaction between organoselenium compounds and ITZ was assessed using the checkerboard method [32], following the same protocol and inoculum standardization described above. The MIC for the interaction was determined based on 100% inhibition of visible fungal growth for all drugs. The interaction between ITZ and BMD or CF3 was evaluated using the fractional inhibitory concentration index (FICi), calculated according to the following formula: (MIC_a_​ in combination / MIC_a​_ tested alone) + (MIC_b_​ in combination / MIC_b​_ tested alone), where MIC_a_ corresponds to BMD or CF3 and MIC_b_​ to ITZ. For each isolate and interaction, the results were classified as strong synergism (SS) when FICi < 0.5, weak synergism (WS) when 0.5 ≤ FICi < 1, additive (AD) when 1 ≤ FICi < 2, indifferent (IND) when FICi = 2, or antagonistic (ANT) when FICi > 2 [32]. Additionally, S. brasiliensis and S. schenckii isolates were classified based on ITZ susceptibility as wild-type (≤ 2 µg/mL) or non-wild-type (> 2 µg/mL) [33]. The fold decrease in ITZ MIC when combined with organoselenium compounds was also evaluated.

Results

BMD was active against all isolates, with MIC_p_ values ranging from 0.5 to 8 µg/mL (GM: 1.25 µg/mL) and MIC_t_ values ranging from 2 to 16 µg/mL (GM: 3.2). BMD killed all isolates, with MFC values ranging from 2 to 64 µg/mL (GM: 6.59 µg/mL). This compound exhibited fungicidal activity against 84% (n = 21/25) of the isolates, with an MFC/MIC_t_ ratio of 1 (n = 5/25) or 2 (n = 16/25) (Table 1). CF3 exhibited only MIC_p_ values against 92% (n = 23/25) of the isolates, including all S. brasiliensis isolates and three S. schenckii isolates. The MIC_p_ values ranged from 2 to 16 µg/mL (GM of 4.25 µg/mL) (Table 1).

Table 1. Result of the in vitro susceptibility of 25 Sporothrix spp. isolates to p-methoxyl-diphenyl diselenide (BMD) and bis-(3-trifluoromethyl phenyl) diselenide (CF3)FURG IDOrigin**SpeciesHostBMDCF3MIC_p_**MIC_t_^#^MFC^##^Ratio^###^MIC_pMIC_t_MFCRatioCBS120339- S. brasiliensis -12424> 64--716RS S. brasiliensis Feline12844> 64--1078RS S. brasiliensis Feline12422> 64--1878RS S. brasiliensis Human12422> 64--2691RS S. brasiliensis Environmental12424> 64--3357RS S. brasiliensis Human12422> 64--3952RS S. brasiliensis Human14418> 64--4559RS S. brasiliensis Feline24414> 64--4720RS S. brasiliensis Human0.54412> 64--4748RS S. brasiliensis Felina0.52428> 64--5150RS S. brasiliensis Human12842> 64--9011PY S. brasiliensis Human14824> 64--9012PY S. brasiliensis Human14828> 64--9013MG S. brasiliensis Feline14828> 64--9014MG S. brasiliensis Feline14824> 64--9015ES S. brasiliensis Feline24828> 64--9016PE S. brasiliensis Feline0.52212> 64--9018RJ S. brasiliensis Human24828> 64--9019RJ S. brasiliensis Human0.52424> 64--9020RJ S. brasiliensis Human12424> 64--1917RSS. scheckii s. str.Human48414> 64--2657RSS. scheckii s. str.Human24828> 64--5196RSS. scheckii s. str.Human288216> 64--7453RSS. scheckii s. str.Human816644> 64> 64--5823RJ S. globosa Human416644>> 64--*FURG ID: Isolate identification of Federal University of Rio Grande; **Origin (brazilian states our country): Rio Grande do Sul/RS, Minas Gerais/MG, Espírito Santo/ES, Pernambuco/PE, Rio de Janeiro/RJ, and Paraguay/PY; ***MIC_p: minimum inhibitory concentration capable of inhibiting 50% of the growth; ^#^MIC_t_: minimum inhibitory concentration capable of inhibiting 100% of the growth; ^##^MFC: minimum fungicidal concentration; ^###^MFC/MIC ration, considering the values of 100% inhibition

The combination of BMD and ITZ demonstrated a synergistic effect against all isolates, with 60% showing WS (n = 9/15) and 40% exhibiting SS (n = 6/15) (Table 2; Fig. 2). Similarly, the combination of CF3 and ITZ was predominantly synergistic (73%; n = 11/15), with 66% displaying SS (n = 10/15) and 7% showing WS (n = 1/15) (Table 2; Fig. 3). Regarding ITZ susceptibility among the isolates included in the interaction evaluation, the majority (60%; n = 9/15) were classified as non-wild type. BMD reduced the MIC values of all non-wild-type isolates by 1- to 7-fold, while CF3 reduced the MIC values of five non-wild-type isolates (55.5%; 5/9) by 3- to 6-fold (Figs. 2 and 3).

Table 2. Results of the interaction between p-methoxy-diphenyl diselenide (BMD) and bis(3-trifluoromethylphenyl) diselenide (CF3) with itraconazole (ITZ) against 15 Sporothrix spp. isolatesSpeciesFURG IDMIC alone**BMD + ITZ combinationCF3 + ITZ combinationBMDCF3ITZBMDITZFICi^#^IN^##^CF3ITZFICiIN S. brasiliensis 18782> 64> 810.250.52WS> 64> 82IND39524> 64> 820.250.52WS160.50.17SS45594> 64> 820.50.53WS> 64> 82IND47204> 640.520.060.62WS80.120.30SS90112> 64110.250.38SS80.250.31SS90124> 6410.50.250.38SS160.120.25SS90144> 64> 80.510.19SS160.120.13SS90154> 640.2520.060.74WS80.060.30SS90184> 64110.120.37SS80.120.18SS90202> 640.2510.060.74WS80.030.18SSS. schenckii* s. str.19178> 64> 840.50.53WS3210.31WS26574> 64> 8240.75WS> 64> 82IND51968> 64> 840.50.53WS160.50.19SS745316> 64> 840.50.28SS80.50.09SS S. globosa 5823160> 64> 840.50.28SS> 64> 82IND*FURG ID: Isolate identification of Federal University of Rio Grande; **MIC alone: minimum inhibitory concentration capable of inhibiting 100% of the growth; #FICi: fractional inhibitory concentration index; ##IN: interpretation: < 0.5 strong synergism (SS); 0.5 to < 1 weak synergism (WS); 1 to < 2 additive (AD); 2 indifferent (IND); > 2 antagonism (AN)

Fig. 2. Results of the interaction between p-methoxyl-diphenyl diselenide (BMD) and itraconazole (ITZ) against 15 Sporothrix sp. isolates, showing fold decreases of ITZ and BMD minimum inhibitory concentration (MIC) values for each isolate when drugs were combined

Fig. 3. Result of the interaction between bis-(3-trifluoromethyl phenyl) diselenide (CF3) and itraconazole (ITZ) against 15 Sporothrix sp. isolates, showing a fold decrease of ITZ and CF3 minimum inhibitory concentration (MIC) values for each isolate when drugs were combined

Discussion

BMD and CF3 are organoselenium analogs of (PhSe)₂, known for their pharmacological properties on mammalian cells and their inhibitory activity against various fungal species [28,34]. Our study expands on the antifungal potential of these organoselenium compounds against Sporothrix spp., being the first to demonstrate the inhibitory and/or fungicidal activity of BMD and CF3 against the three main clinical species of the genus, as well as their synergistic effect with ITZ, drug of choice for the treatment of sporotrichosis [8].

Although both (PhSe)₂ analogs evaluated in this study showed antifungal activity, BMD exhibited stronger anti-Sporothrix spp. effects than CF3. Similar findings were previously reported by Loreto et al. (2011), who demonstrated superior antifungal activity of BMD compared to CF3 against other fungal species, including Candida spp., Aspergillus spp., and Fusarium spp. Comparing the BMD MIC values presented here with those from previous studies conducted by our research group [20,23] we could show that BMD was able to enhance the (PhSe)2 anti-Sporothrix activity in almost four times (GM MIC of BMD 3.12 µg/mL versus (PhSe)2 12.12 µg/mL). Additionally, our study shows that S. brasiliensis was more sensitive to BMD than S. schenckii and S. globosa.

Considering that BMD and CF3 is an organoselenium compound, it is hypothesized that, similarly to its precursor (PhSe)₂, both compounds may exert antifungal effects through the induction of oxidative stress in fungal cells. Structurally, BMD contains a methoxyl group in the para position of the aromatic ring, whereas CF3 present a trifluoromethyl group in the meta position. The addition of the methoxyl substituent appears to enhance the antifungal activity of the (PhSe)2 scaffold, possibly by favoring fungal cell entry and oxidative stress in fungal cells. In contrast, the introduction of a trifluoromethyl group, despite increasing molecular stability, may limit cellular uptake or effective target engagement, resulting in reduced anti-Sporothrix activity [28,35]. However, further studies are needed to confirm these hypothesis and to elucidate the pharmacodynamics of both compounds.

Additional favorable characteristics of BMD and CF3 relate to their toxicity profiles in animal models (mice). Both compounds did not increase the toxicity previously described for their precursor, (PhSe)2, whose lethal dose (LD₅₀) is > 312 mg/kg; the LD₅₀ values of BMD and CF3 were 372 mg/kg and 448 mg/kg, respectively [36]. Moreover, when administered as a single acute dose (10–372 mg/kg for BMD or 10–448 mg/kg for CF3), neither compound altered lipid peroxidation, vitamin C levels, cerebral Na⁺/K⁺-ATPase activity, or biochemical parameters (aspartate aminotransferase, alanine aminotransferase, plasma urea, and creatinine levels), indicating low toxicity [36]. Importantly, the LD₅₀ values of CF3 and BMD (448 mg/kg and 372 mg/kg, respectively) are markedly higher than the concentrations showing antifungal activity against Sporothrix spp. (0.016 mg/L; highest MIC for BMD and MIC for CF3). Nevertheless, future studies evaluating the in vivo pharmacokinetics of these compounds are required to better characterize their toxicity and therapeutic potential.

The ability of BMD and CF3 to inhibit approximately 50% of fungal growth in vitro highlights their potential antifungal activity, following a criterion similar to that used for echinocandins, which are also evaluated based on approximately 50% growth inhibition in vitro [30]. Echinocandins are primarily used for the treatment of invasive mycoses, such as candidiasis and aspergillosis, due to their potent antifungal efficacy and favorable safety profile; in addition, it is important to highlight that their use for Sporothrix spp. inhibition has also been proposed based on the partial inhibition criterion [37–38].

Given the long duration of sporotrichosis treatment, BMD and CF3 may be considered promising compounds for future therapeutic development due to their partial inhibitory activity against Sporothrix spp. and their low toxicity observed in animal models. In combination with ITZ, BMD remained the most active compound, showing synergism with 100% of the tested isolates. Similarly, CF3 exhibited synergism against the majority of the strains (~ 70%). Previous studies on the interaction of (PhSe)₂ with ITZ demonstrated similar beneficial effects between the compounds (73% in S. brasiliensis and 87.5% in S. globosa and S. schenckii). By integrating the results of (PhSe)₂ and its analogs (BMD and CF3) against Sporothrix spp., we reaffirm that no antagonistic effects were observed with any of the compounds. The interaction between organoselenium compounds and ITZ was predominantly synergistic, with BMD being the most active molecule [20,23].

Furthermore, both compounds demonstrated the ability to reverse the non-wild-type status of all isolates (BMD) and more than half of the isolates (CF3). Our data suggest that BMD and CF3 have potential for use in combination with ITZ in the treatment of human and animal sporotrichosis. In this context, the hepatoprotective properties attributed to BMD may help mitigate the hepatotoxicity commonly associated with prolonged ITZ therapy. In addition, the anti-inflammatory effects described for CF3 could contribute to reducing tissue damage resulting from the host immune response, potentially attenuating inflammatory injury when used in association with ITZ [25–28] ,[39–42].

This study has some limitations. First, it was restricted to in vitro assays, which, although essential for initial antifungal screening, do not fully reproduce the complexity of host–pathogen interactions observed in vivo. Second, direct mechanistic investigations were not performed; therefore, the proposed modes of action of the organoselenium compounds remain hypothetical. Finally, the limited number of S. schenckii and S. globosa isolates restricts broader comparisons of organoselenium activity among different Sporothrix species.

Given the global burden of sporotrichosis and the urgent need for more effective therapeutic options for this emerging mycosis, the present study provides preliminary in vitro evidence of the antifungal activity of two organoselenium compounds, evaluated individually and in combination with ITZ. Among the compounds tested [BMD, CF3, and the precursor (PhSe)₂], BMD exhibited the highest in vitro anti-Sporothrix activity.