Abstract

Aspergillus genus causes many diseases, and the species Aspergillus flavus is highly virulent. Treatment of aspergillosis involves azole derivatives such as voriconazole and polyenes such as amphotericin B. Due to an increase in fungal resistance, treatments are now less effective; the search for new compounds with promising antifungal activity has gained importance. The aims of this study were to evaluate the effects of the synthetic amide 2-chloro-N-phenylacetamide (A₁Cl) against strains of Aspergillus flavus and to elucidate its mechanism of action. Thus, the minimum inhibitory concentration, minimum fungicidal concentration, conidial germination, associations with antifungal agents, cell wall activities, membrane activities and molecular docking were evaluated. A₁Cl presented antifungal activity against Aspergillus flavus strains with a minimum inhibitory concentration of between 16 and 256 μg/mL and a minimum fungicidal concentration between 32 and 512 μg/mL. The minimum inhibitory concentration of A₁Cl also inhibited conidial germination, but when associated with amphotericin B and voriconazole, it promoted antagonistic effects. Binding to ergosterol on the fungal plasma membrane is the likely mechanism of action, along with possible inhibition of DNA synthesis through the inhibition of thymidylate synthase. It is concluded that the amide 2-chloro-N-phenylacetamide has promising antifungal potential.

Key words
2-chloro-N-phenylacetamide; antifungal activity; Aspergillus flavus; resistance

INTRODUCTION

Aspergillus genus comprises more than 250 species; it is one of the largest genera of filamentous fungi that cause human disease (Rudramurthy et al. 2019RUDRAMURTHY SM, PAUL RA, CHAKRABARTI A, MOUTON JW & MEIS JF. 2019. Invasive aspergillosis by Aspergillus flavus: Epidemiology, diagnosis, antifungal resistance ang management. J Fungi (Basel) 5: 1-23.). Aspergillus species A. fumigatus, A. flavus, A. niger, A. terreus and A. versicolor are frequently involved in diseases, such as rhinosinusitis, cutaneous and subcutaneous aspergillosis, pulmonary infections, cardiac infections, otomycosis, among others (Barnes & Marr 2006BARNES PD & MARR KA. 2006. Aspergillosis: Spectrum of disease, diagnosis, and treatment. Infect Dis Clin North Am 20: 545-561., Sugui et al. 2014SUGUI JA, KWON-CHUNG KJ, JUVVADI PR, LATGÉ JP & STEINBACH WJ. 2014. Aspergillus fumigatus and related species. Cold Spring Harb Perspect Med 5: 1-17., Rudramurthy et al. 2019RUDRAMURTHY SM, PAUL RA, CHAKRABARTI A, MOUTON JW & MEIS JF. 2019. Invasive aspergillosis by Aspergillus flavus: Epidemiology, diagnosis, antifungal resistance ang management. J Fungi (Basel) 5: 1-23.). The genus, in general, presents low pathogenicity for humans, and it requires a large inoculum to infect immunocompromised individuals if their physiological performance is intact. However, once the infection is established, a variety of presentations are observed, with the lung being the most commonly affected organ (Castello Branco et al. 2014CASTELLO BRANCO BP, CARVALHO FA, ANDRADE AMV, CASTELLO BRANCO IT & CHAVES TSV. 2014. Colonização intracavitária pulmonar por Aspergillus: Análise de 190 casos. J Bras Med 12: 1-16.).

The infectious spectrum of aspergillosis is classified into four types: invasive disease, corresponding to life-threatening infections in immunocompromised patients; subacute or chronic infectious disease, in patients with structural lung abnormalities, pre-existing sinus or lung disease (or some subtle defect in innate immunity); allergic or eosinophilic disease, manifesting in various forms, such as allergic bronchopulmonary aspergillosis, eosinophilic rhinosinusitis, and extrinsic allergic alveolitis; and locally invasive infections resulting from trauma or surgery, such as keratitis or a postoperative infection (Rudramurthy et al. 2019RUDRAMURTHY SM, PAUL RA, CHAKRABARTI A, MOUTON JW & MEIS JF. 2019. Invasive aspergillosis by Aspergillus flavus: Epidemiology, diagnosis, antifungal resistance ang management. J Fungi (Basel) 5: 1-23.). A. fumigatus species is the agent most commonly involved in aspergillosis and it is one of the most commonly studied species.

Studies have revealed A. flavus as the most virulent species in terms of time and initial inoculum, which causes death in immunocompromised mice (Mosquera et al. 2001MOSQUERA J, WARN PA, MORRISSEY J, MOORE CB, GIL-LAMAIGNERE C & DENNING DW. 2001. Susceptibility testing of Aspergillus flavus: Inoculum dependence with itraconazole and lack of correlation between susceptibility to amphotericin B in vitro and outcome in vivo. Antimicrob Agents Chemother 45: 1456-1462.). The drugs used to treat aspergillosis include voriconazole (being the drug of choice) and amphotericin B (as a rescue therapy) when azole derivatives cannot be administered (Bellmann 2013BELLMANN R. 2013. Pharmacodynamics and pharmacokinetics of antifungals for treatment of invasive aspergillosis. Curr Pharm Des 19: 3629-3647., Patterson et al. 2016PATTERSON TF ET AL. 2016. Practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the Infectious Diseases Society of America. Clin Infect Dis 63: 1-60.). However, A. flavus is resistant to azoles (Paul et al. 2015PAUL RA, RUDRAMURTHY SM, MEIS JF, MOUTON JW & CHAKRABARTI A.2015. A novel Y319H substitution in cyp51C associated with azole resistance in Aspergillus flavus. Antimicrob Agents Chemother 59: 6615-6619., Ukai et al. 2018UKAI, Y, KUROIWA M, KURIHARA N, NARUSE H, HOMMA T, MAKI H & NAITO A. 2018. Contributions of yap1 mutation and subsequent atrF upregulation to voriconazole resistance in Aspergillus flavus. Antimicrob Agents Chemother 62: 1-11.) and polyenes (Taylor et al. 2017TAYLOR E, FERREIRA G, FREITAS G, FERREIRA R, DE ASSIS SANTOS D & DE RESENDE-STOIANO M. 2017. Screening of antifungal susceptibility in cave-dwelling aspergilli and report of an amphotericin B-resistant Aspergillus flavus. Int J Speleol 46: 369-378., Reichert-Lima et al. 2018REICHERT-LIMA F, LYRA L, PONTES L, MORETTI ML, PHAM CD, LOCKHART SR & SCHREIBER AZ. 2018. Surveillance for azoles resistance in Aspergillus spp. highlights a high number of amphotericin B-resistant isolates. Mycoses 61: 360-365.), which limits the use of these drugs. New therapeutic alternatives for aspergillosis are needed.

In a previous investigation carried out by our research group, an unprecedented antifungal activity of the synthetic amide 2-chloro-N-phenylacetamide (A₁Cl) (Figure 1) against A. flavus strains was demonstrated, with MICs ranging from 16 to 256 μg/ml. However, the mechanism of action against the microorganism under study is still unknown. Thus, our objective was to further evaluate the effects of the synthetic amide 2-chloro-N-phenylacetamide against various strains of Aspergillus flavus and to elucidate its probable mechanism of action.

MATERIALS AND METHODS

Microorganisms

To evaluate antifungal activity, eleven (11) strains of clinical origin and one (1) ATCC strain of A. flavus (shown in Table I) were obtained from the collection of the Research Laboratory Micoteca for Antibacterial and Antifungal Activity of Natural and/or Bioactive Synthetic Products. The strains on Aspergillus flavus were isolated and grown on potato dextrose agar for 7 days at 35°C. After growth, they were stored under refrigeration at 4°C. To prepare the inoculum, samples were taken from the grown cultures, suspended in sterile 0.85% saline and transferred to sterile tubes. The inoculum was standardized according to the 0.5 concentration of the McFarland scale (10⁶ CFU/mL). The concentration was confirmed by counting the conidia using a Newbauer chamber (CLSI 2008CLSI - CLINICAL AND LABORATORY STANDARDS INSTITUTE. 2008. Formerly NCCLS (National Committee for Clinical Laboratory Standards). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi, 2nd ed., CLSI Standart M38, Wayne 37: 11., Guerra et al. 2015GUERRA FSQ, ARAUJO RSA, SOUSA JP, PEREIRA FO, MENDONÇA-JUNIOR FJB, BARBOSA-FILHO JM & LIMA EO. 2015. Evaluation of Antifungal Activity and Mode of Action of New Coumarin Derivative, 7-Hydroxy-6-nitro-2H-1-benzopyran-2-one, against Aspergillus spp. Evid Based Complement Alternat Med 8: 1-8.).

Drugs and Reagents

The tested product was the synthetic amide 2-chloro-N-phenylacetamide, obtained by synthesis (Huang 2016HUANG MFN. 2016. Síntese de novos compostos organosselênicos com potencial atividade antimicrobiana e antitumoral. 2016. Dissertação (Mestrado em Quimica) - Centro de Ciências Exatas e da Natureza - Universidade Federal da Paraíba, João Pessoa, 1-129. (Unpublished).). The antifungals voriconazole and amphotericin B, and the ergosterol were obtained from Sigma-Aldrich® (São Paulo, São Paulo, Brazil). The sorbitol was obtained from INLAB® (São Paulo, Brazil). Potato dextrose agar was obtained from Difco Lab. (USA). All of the substances were dissolved in DMSO (dimethylsulfoxide – Sigma-Aldrich®, São Paulo, São Paulo, Brazil). and solubilized in sterile distilled water to obtain solutions at a concentration of 1024 μg/mL. For evaluation of the antifungal activity, RPMI 1640 with L-glutamine was used (Sigma-Aldrich®, São Paulo, SP, Brazil), and prepared according to the manufacturer’s instructions.

Determination of in vitro antifungal activity

Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of A₁Cl against strains of A. flavus

The MIC determination of A₁Cl against strains of A. flavus was performed using the broth microdilution technique recommended by the Clinical and Laboratory Standards Institute (CLSI 2008CLSI - CLINICAL AND LABORATORY STANDARDS INSTITUTE. 2008. Formerly NCCLS (National Committee for Clinical Laboratory Standards). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi, 2nd ed., CLSI Standart M38, Wayne 37: 11.). Initially, 100 μL of double-concentrated RPMI was added to the wells, followed by 100 μL of each product suspension (A₁Cl, voriconazole, and amphotericin B) at an initial concentration of 1024 μg/mL in the first row of the plate, and then the range of concentrations from 1024 to 4 μg/mL were obtained by means of serial dilution. Finally, 10 μL of each of the tested microorganism suspensions was added. Negative controls (without drugs) were used to assess the viability of the strains and the sterility of the medium, as well as the sensitivity controls (to DMSO). After 2 days of incubation, visual plate reading was performed, and the MIC was considered the lowest concentration inhibiting 100% of the strain growth (CLSI 2008CLSI - CLINICAL AND LABORATORY STANDARDS INSTITUTE. 2008. Formerly NCCLS (National Committee for Clinical Laboratory Standards). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi, 2nd ed., CLSI Standart M38, Wayne 37: 11.). The results are expressed as the arithmetic mean of the MICs obtained in three tests.

For the determination of the MFC, 100 μL of RPMI was added to the 96-well sterile plate, and 10 μL samples were removed from wells where there was no fungal growth on MIC plates and added to the new plate containing the culture medium (RPMI 1640, Sigma-Aldrich®, São Paulo, SP, Brazil). The plates were incubated at 35°C for 2 days. The fungicidal activity was considered the lowest concentration of the product capable of inhibiting visible growth on the plate (Ncube et al. 2008NCUBE NS, AFOLAYAN AJ & OKON AI. 2008. Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods and future trends. Afr J Biotechnol 7: 1797-1806., CLSI 2008CLSI - CLINICAL AND LABORATORY STANDARDS INSTITUTE. 2008. Formerly NCCLS (National Committee for Clinical Laboratory Standards). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi, 2nd ed., CLSI Standart M38, Wayne 37: 11.).

Evaluation of A₁Cl inhibition of conidial germination in A. flavus strains

The strains ATCC 16013 and LM 22 were used for this test. After reading the MIC plates, 10 μL of the MIC, MIC × 2 and MIC × 4 concentrations were removed from each product tested and seeded on sterile slides. A drop of lactophenol blue dye was then added to each preparation, homogenized, and covered with a coverslip. Conidia counting was performed under an optical microscope in 5 different fields to obtain an arithmetic mean. The results were expressed in graphs as the percentage of conidial germination as a function of the concentrations of the tested products (Adapted from Trajano et al. 2013TRAJANO VN, LIMA EO & SOUZA FS. 2013. Antifungal Activity of the Essential Oil of Cinnamomum zeylanicum Blume and Eugenol on Aspergillus flavus. J Essent Oil-Bear Plants 15: 785-793.).

Evaluation of the mechanism of action of A₁Cl against clinical strains of A. flavus

Cell wall assay

To assess the possible mechanisms of action involving A. flavus fungal cell wall, the MIC of A₁Cl was determined in the presence and absence of sorbitol (INLAB®, São Paulo, Brazil) (0.8 M) in the medium using the microdilution method and 96-well “U” plates (Alamar, Diadema, SP, Brazil). The plates were aseptically sealed and incubated at 35°C. Readings were taken at 2 days. A microorganism check and a sterility check were performed. The test was performed in triplicate and expressed as the arithmetic mean of the results (Escalante et al. 2008ESCALANTE A, GATTUSO M, PEREZ P & ZACCHINO S. 2008. Evidence for the mechanism of action of the antifungal phytolaccoside B isolate from Phytolacca tetramera Hauman. J Nat Prod 71: 1720-1725.).

Cell membrane assay

To assess the possible mechanisms of action involving A. flavus fungal cell membrane, the MIC of A₁Cl was determined in the presence and absence of ergosterol (400 µg/mL) (Sigma-Aldrich®) in the medium using the microdilution method with 96-well “U” plates (Alamar, Diadema, SP, Brazil). A microorganism check and a sterility check were performed. Amphotericin B was used as a positive control. For reading, the plates were sealed and incubated at 35°C for 2 days. The test was performed in triplicate and expressed as the arithmetic mean of the results (Escalante et al. 2008ESCALANTE A, GATTUSO M, PEREZ P & ZACCHINO S. 2008. Evidence for the mechanism of action of the antifungal phytolaccoside B isolate from Phytolacca tetramera Hauman. J Nat Prod 71: 1720-1725.).

Molecular docking

Using the ChemDraw® program, the A₁Cl structure was designed and saved as an MOL file. The enzymes investigated with their respective PDB IDs were C-14-α-demethylase (PDB ID: 4UYL) (Hargrove et al. 2015HARGROVE TY, WAWRZAK Z, LAMB DC, GUENGERICH FP & LEPESHEVA GI. 2015. Structure-Functional characterization of cytochrome P450 sterol 14-demethylase (CYP51B) from Aspergillus fumigatus and molecular basis for the development of antifungal drugs. J Biol Chem 290: 23916 -23934.), squalene epoxidase (PDB ID: 4 MAH) (Frost et al. 1995FROST DJ, BRANDT KD, CUGIER D & GOLDMAN R. 1995. A whole-cell Candida albicans assay for the detection of inhibitors towards fungal cell wall synthesis and assembly. J Antibiot (Tokyo) 48: 306-310.), delta-14-sterol reductase (PDB ID: 4QUV) (Standards 2002STANDARDS NCFCL. 2002. Reference methods for broth dilution antifungal susceptibility testing of yeast: approved standard. 2002: National Committee for Clinical Laboratory Standards.), 1,3-β-glucan synthase (PDB ID: 3A58) (RasoolI & Abyaneh 2004RASOOLI I & ABYANEH MR. 2004. Inhibitory effects of thyme oils on growth and aflatoxin production by Aspergillus parasiticus. Food Control 15: 479-483.) and thymidylate synthase (PDB ID: 5UIV) (Sinha & Rule 2017SINHA K & RULE GS. 2017. The structure of thymidylate kinase from Candida albicans reveals a unique structural element. Biochem 56: 4360-4370.). The ligand and its receptors were subjected to molecular coupling using the Molegro Virtual Docker, v. 6.0.1 (MVD) (Morris et al. 2009MORRIS GM, HUEY R, LINDSTROM W, SANNER MF, BELEW RK, GOODSELL DS & OLSON AJ. 2009. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem 30: 2785- 2791.). The proteins and ligand structures were prepared using the standard software package parameter settings (function Score: MolDock Score). The A₁Cl structures and the receptors used were prepared using PyRx 0.9 software (Wolf 2009WOLF LK. 2009. Digital briefs. Chemical & Engineering News 87: 31.), and the results are presented as the total ligand-receptor interaction energy values.

Assessment of A₁Cl with standard antifungal associations

The effects of the associations of the products with standard antifungals were determined using the microdilution - checkerboard technique for derivation of the FICI (Fractional Inhibitory Concentration Index). Solutions of the tested products were used in concentrations determined from their respective MICs. Initially, 100 μL of RPMI was added to the wells of a sterile 96-well microplate, and then 50 μL of each tested product was added at different concentrations (MIC ÷ 8, MIC ÷ 4, MIC ÷ 2, MIC, MIC × 2, MIC × 4, and MIC × 8) in the vertical (standard antifungal) and horizontal (test product) directions of the microplate. Finally, 20 μL of the fungal suspension was added. The assay was performed in triplicate, with the microplates incubated at 35°C for 2 days (Eliopoulos & Moellering 1991ELIOPOULOS GM & MOELLERING RC. 1991. Antimicrobial combinations, In: Antibiotics in laboratory medicine. Baltimore: Willians & Wikins, p. 434-441., Dutta et al. 2004DUTTA NK, DASTIDAR SG, ASOK KUMAR A, MAZUMDAR K, RAY R & CHAKRABARTY AN. 2004. Antimycobacterial activity of the anti-inflammatory agent diclofenac sodium, and its synergism with streptomycin. Braz J Micr 35: 316-323.).

For calculation of the FICI (Fractional Inhibitory Concentration Index), the method described by Bonapace et al. (2002)BONAPACE CR, BOSSO JA, FRIEDRICH LW & WHITE RL. 2002. Comparison of methods of interpretation of checkerboard synergy testing. Diagn Microbiol Infect Dis 44: 363-366. was used, and the lowest MIC was used (where there was no fungal growth) for the calculation performed by adding the FIC^A + FIC^B, where A represents the test product and B the standard antifungal. The FICI, in turn, is calculated using the combined FIC^A/FIC^A ratio alone, while the FIC^B is calculated using the formula FIC^B = combined FIC^B/FIC^B alone. The interpretation of this index was performed as follows: synergism = (FICI ≤ 0.5), antagonism = (FICI > 4.0), and indifference = (0.5 < FICI ≤ 4) (Odds et al. 2003ODDS FC, BROWN AJP & GOW NAR. 2003. Antifungal agents: mechanisms of action. Trends Microbiol 11: 272-279.).

Statistical analysis

The results obtained from the MIC, MFC, cell wall, and cell membrane assays are expressed as the arithmetic means. The results of the conidial germination percentage are expressed as the mean ± standard error of the mean (S.E.M.), which were statistically analyzed using one-way analysis of variance (ANOVA) followed by the Bonferroni post-test to compare more than two groups. The null hypothesis was rejected when p < 0.05, and the GraphPad Prism® program version 5.01 (GraphPad Software Inc., San Diego CA, U.S.A.) was used.

RESULTS

Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of A₁Cl against strains of A. flavus

The product A₁Cl presented MICs that varied from 16 to 256 μg/mL, as shown in Table II, inhibiting visible growth of A. flavus strains used in this study. At concentrations below 100 μg/mL, six strains (50%) were weakly sensitive to A₁Cl, with MICs established between 16 and 64 μg/mL.

In the assessment of the minimum fungicidal concentration (MFC) of A₁Cl, a variation of the MFC between 32 and 512 μg/mL was observed (Table II).

Regarding the standard antifungal amphotericin B, it was noted that the strains presented MICs that varied between 1 and 16 μg/mL, with 8 out of 12 presenting MICs of > 2 μg/mL, while the MFC of amphotericin B for the tested strains ranged from 2 to 32 μg/mL (Table II). For voriconazole, there was a variation in MIC between 0.5 and 2 μg/mL, with its MFC being equal to 2 μg/mL for all strains tested (Table II).

Inhibition of conidial germination: A₁Cl against strains of A. flavus

A₁Cl, when submitted to A. flavus strain ATCC 16013, reduced the germination of conidia at the three concentrations used, presenting results superior to those found for amphotericin B and voriconazole (Figure 2a). The clinical LM 22 strain, when exposed to A₁Cl, presented a decrease in conidial germination at all concentrations tested, similar to voriconazole, yet the conidial germination inhibition presented by amphotericin B was higher (Figure 2b).

Evaluation of the effect of A₁Cl on the fungal cell wall

To verify the possible action of A₁Cl on the fungal cell wall, sorbitol was used as an osmotic protector. In this study, the MIC values of A₁Cl in the presence of sorbitol remained the same in relation to the control (RPMI), suggesting that A₁Cl does not act at the level of the cell wall, as shown in Table III.

Evaluation of the effect of A₁Cl on the fungal cell membrane

Whether A₁Cl interacts with the fungal cell membrane through complex formation with ergosterol was also investigated. An increase in the MIC of A₁Cl was observed, from 128 to 1024, related to the ATCC 16013 strain and from 64 to 1024 related to A. flavus LM 22 strain in the presence of ergosterol, compared to the RPMI control, indicating a potential mechanism of action against the fungal cell membrane. When amphotericin B was used as a positive control, an increase in MIC from 2 μg/mL to 32 μg/mL was also observed in the two strains tested in the presence of ergosterol compared to the RPMI control, confirming a mechanism of action against the fungal cell membrane as already described in the literature (Table IV).

Evaluation of the mechanism of action of A₁Cl by molecular docking

In this assay, the interaction was demonstrated through binding energy, obtained by binding the A₁Cl test product with the enzymes C-14-α-demethylase, squalene epoxidase, delta-14-sterol reductase, 1,3-β-glycan synthase, and thymidylate synthase, as shown in Table V.

In the molecular docking assay, A₁Cl interacted with the enzyme C-14-α-demethylase through hydrogen bonds (blue dashes) between the enzyme’s tyrosine amino acid and the carbonyl oxygen of A₁Cl, which also interacted through steric interactions (red dashes) with the enzyme’s amino acid threonine, presenting a binding energy of −55.87 Kcal/mol. In addition, steric interactions of the enzyme’s amino acid glycine with the carbon ɑ1 of A₁Cl, as well as hydrogen bonds with the aromatic ring, were observed, as shown in Figure 3a.

Regarding the interaction of A₁Cl with the enzyme 1,3-β-glycan synthase (3A58), steric interactions (red dashes) were observed between the carbonyl oxygen of A₁Cl and the amino acid glutamic acid132(A) of the enzyme, presenting an energy of −58.82 Kcal/mol. The amino acid leucine77(B) of the enzyme interacts with the aromatic ring of A₁Cl by means of steric bonds and with carbon ɑ1 through hydrogen bonds (blue dashes), as shown in Figure 3b.

Squalene epoxidase interacted with A₁Cl by means of steric interactions (red dashes) with the amino acids asparagine194, proline173, proline193, phenylalanine192, and glutamine66 of the enzyme with the aromatic ring and the ɑ1 carbon of A₁Cl, presenting a binding energy of −60.28 Kcal/mol (Figure 3c).

The enzyme Delta-14-sterol reductase interacted with A₁Cl by means of steric bonds (red dashes) with the aromatic ring of A₁Cl, presenting a binding energy of −61.92 through the amino acid leucine306A (Figure 3d).

Regarding the interaction of the enzyme thymidylate synthase (5UIV) with A₁Cl, steric interactions (red dashes) were identified between glycine97 of the enzyme and the carbonyl oxygen of A₁Cl, as well as between the enzyme’s tyrosine amino acid and the A₁Cl methyl, with a binding energy of −83.36 Kcal/mol. Hydrogen interactions (blue dashed lines) were also observed between amino acid arginine 71 of the enzyme and the oxygen in the A₁Cl carbonyl (Figure 3e).

Assessment of A₁Cl – standard antifungal associations

The effect of A₁Cl in association with amphotericin B and voriconazole (Table VI) against A. flavus strains was investigated. It was observed that regardless of A. flavus strain, the association of A₁Cl with amphotericin B or with voriconazole resulted in the same antagonistic response pattern, considering that the FICI was > 4.0; see Odds et al. (2003)ODDS FC, BROWN AJP & GOW NAR. 2003. Antifungal agents: mechanisms of action. Trends Microbiol 11: 272-279..

DISCUSSION

The emergence of resistant Aspergillus strains, in addition to causing higher mortality rates, indicates the need for the identification of new compounds with promising antifungal activity (Sanglard 2016SANGLARD D. 2016. Emerging Threats in Antifungal-Resistant Fungal Pathogens. Front Med 3: 1-10.). Since A₁Cl presented promising antifungal activity in screening studies performed by our research group, this aroused our interest in investigating the molecule in depth and in verifying its antifungal potential.

MIC testing was performed for the A₁Cl compound, and a variation from 16 to 256 μg/mL was observed for A. flavus strain (Table II), Fifty percent of the A. flavus strains (Table II) which was considered in this case as the ideal concentration of a substance (Holetz et al. 2002HOLETZ FB, HOMES MJ, LEE CC & STEVENSON G. 2002. Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem Inst Oswaldo Cruz 97: 1027-1031.). The strong antimicrobial activity of A₁Cl has also been demonstrated by Katke et al. (2011)KATKE SA, AMRUTKAR SV, BHOR RJ & KHAIRNAR MV. 2011. Synthesis of biologically active 2-chloro-n-alkyl/aryl acetamide derivatives. Int J Pharma Sci Res 2: 148-156. against strains of Escherichia coli and Pseudomonas aeruginosa and by Aschale (2012)ASCHALE M. 2012. Synthesis and Antimicrobial Evaluation of Some Novel Substituted 2-chloroacetanalides. Int J Chemtech Res 4: 1437-1441. against strains of A. niger, demonstrating that substituted chloroacetamides present greater antifungal activity than unsubstituted chloroacetamides, regardless of the positions or nature of the substituent.

In the A₁Cl and MFC assays against A. flavus strains, a range of 32 to 512 μg/mL was observed (Table II). To explain the nature of the antimicrobial effect with regard to growth inhibition or death, Hazen (1998)HAZEN KC. 1998. The fungicidal versus fungistatic activity of terbinafine and itraconazole: An in vitro comparison. J Am Acad Dermatol 38: 1-5. established that a substance is to be considered fungicidal when the MFC/MIC ratio is ≤ 4, and when the ratio is > 4, the compound is to be considered fungistatic. In this study, A₁Cl was assayed as a fungicide for all strains tested, with MFC/MIC ratios ranging from 1 to 4 (Table II). The fungicidal activity of this compound is promising, since treatment of diseases that affect immunocompromised individuals, such as invasive aspergillosis, one of the prominent causes of death in these patients, requires drugs with fungicidal potential (Chowdhary et al. 2015CHOWDHARY A, SHARMA C, KATHURIA S, HAGEN F & MEIS JF. 2015. Prevalence and mechanism of triazole resistance in Aspergillus fumigatus in a referral chest hospital in Delhi, India and an update of the situation in Asia. Front Microbiol 6: 1-10.).

It is well known that there are great difficulties involved in correlating and interpreting microdilution test results together with clinical results, and the scientific literature has not yet defined breakpoints for assessing the susceptibility or resistance of clinical species of Aspergillus genus. However, Borman et al. (2017)BORMAN AM, FRASER M, PALMER MD, SZEKELY A, HOULDSWORTH M, PATTERSON Z & JOHNSON EM. 2017. MIC Distributions and Evaluation of Fungicidal Activity for Amphotericin B, Itraconazole, Voriconazole, Posaconazole and Caspofungin and 20 Species of Pathogenic Filamentous Fungi Determined Using the CLSI Broth Microdilution Method. J Fungi (Basel) 3: 1-14. presented a study with the objective of facilitating the development of cutoff points for several fungi, including Aspergillus. According to these authors, strains that present MIC values above 2 µg/mL are considered resistant to amphotericin B. The authors also observed that 13.7% of the tested A. flavus strains were thus considered resistant.

Reichert-Lima et al. (2018)REICHERT-LIMA F, LYRA L, PONTES L, MORETTI ML, PHAM CD, LOCKHART SR & SCHREIBER AZ. 2018. Surveillance for azoles resistance in Aspergillus spp. highlights a high number of amphotericin B-resistant isolates. Mycoses 61: 360-365., assessing the susceptibility of Aspergillus strains to antifungals used in the clinic, found that of 27 strains of A. flavus tested, (81%) 22 obtained MICs of ≥ 2 µg/mL, considered high MIC values. Garcia-Martos et al. (2005)GARCÍA-MARTOS P, GARCÍA-AGUDOA L, GUTIÉRREZ-CALZADA J, RUIZ-ARAGÓNA J, ABEL SALDARREAGA A & MARÍNA P. 2005. In vitro activity of amphotericin B, itraconazole and voriconazole against 20 species of Aspergillus using the Sensititre microdilution method. Enferm Infecc Microbiol Clin 23: 15-23. also evaluated the susceptibility of Aspergillus species against the antifungals amphotericin B, itraconazole, and voriconazole and observed that A. flavus presents little sensitivity to amphotericin B.

In this study, 8 of 12 A. flavus strains tested presented MICs of > 2 μg/mL (Table II) and are considered resistant to amphotericin B. The literature reports that the sensitivity profile of A. flavus presents elevated MIC values for amphotericin B and that values above 2 μg/mL are associated with failures in treating invasive aspergillosis (Hadrich et al. 2012HADRICH I, MAKNI F, NEJI S, CHEIKHROUHOU F, BELLA AJH, ELLOUMI M, AYADI A & RANQUE S. 2012. Amphotericin B in vitro resistance is associated with fatal Aspergillus flavus infection. Med Mycol 50: 829-834., Gonçalves et al. 2013GONCALVES SS, STCHIGEL AM, CANO J, GUARRO J & COLOMBO AL. 2013. In vitro antifungal susceptibility of clinically relevant species belonging to Aspergillus section Flavi. Antimicrob Agents Chemother 57: 1944-1951., Heo et al. 2015HEO MS ET AL. 2015. Molecular identification and amphotericin B susceptibility testing of clinical isolates of Aspergillus from 11 hospitals in Korea. Ann Lab Med 35: 602-610.). The mechanisms that confer resistance to amphotericin B are still not well understood; however, according to Chamilos & Kontoyiannis (2005)CHAMILOS G & KONTOYLANIS DP. 2005. Update on antifungal drug resistance mechanisms of Aspergillus fumigatus. Drug Resist Updat 8: 344-358., two mechanisms may be involved: decreasing ergosterol in the cell membrane (target of the drug) due to mutations in its biosynthetic pathway and an increase in the production of catalases, which provide resistance to fungal cells against oxidative stress caused by the drug.

Despite being used to treat many fungal diseases, such as aspergillosis, the appearance of nephrotoxicity (caused by amphotericin B) is common, even in lipid formulations. The mechanism by which this happens is mediated at the cellular level through apoptosis (Varlam et al. 2001VARLAM DE, SIDDIQ MM, PARTON LA & RUSSMANN H. 2001. Apoptosis contributes to amphotericin B-induced nephrotoxicity. Antimicrob Agents Chemother 45: 679-585.). The effect is common in approximately 65% of patients and can cause significant loss of renal function, even requiring dialysis (Falci & Pasqualotto 2015FALCI DR & PASQUALOTTO AC. 2015. Amphotericin B: uma revisão sobre suas diferentes formulações, efeitos adversos e toxicidade. Clin Biomed Res 35: 65-78.). The search for a substance with promising antifungal activity and low toxicity as a substitute for amphotericin B is critical.

Voriconazole presented MICs varying between 0.5 and 2 μg/mL for strains of A. flavus (Table II). According to Borman et al. (2017)BORMAN AM, FRASER M, PALMER MD, SZEKELY A, HOULDSWORTH M, PATTERSON Z & JOHNSON EM. 2017. MIC Distributions and Evaluation of Fungicidal Activity for Amphotericin B, Itraconazole, Voriconazole, Posaconazole and Caspofungin and 20 Species of Pathogenic Filamentous Fungi Determined Using the CLSI Broth Microdilution Method. J Fungi (Basel) 3: 1-14., strains that present MICs above 2 μg/mL are resistant to voriconazole. Wang et al. (2018)WANG HC, HSIEH IM, CHOI PC & WU CH. 2018. Comparison of the Sensititre YeastOne and CLSI M38-A2 microdilution methods in determining the activity of amphotericin B, itraconazole, voriconazole, and posaconazole against Aspergillus species. J Clin Microbiol 25: 56-66., in a study verifying resistance patterns, observed that all 23 strains of A. flavus tested were considered sensitive to voriconazole, with MICs ranging between 0.5 and 1 μg/ml. Based on these studies, all of A. flavus strains used in this study are sensitive to voriconazole.

In view of the aspergillosis resistance to current antifungal therapies, especially amphotericin B, and of the need to seek new therapeutic strategies, the A₁Cl molecule is promising for presenting antifungal effects. Knowing that in the field of organic synthesis, a molecule can undergo changes in its chemical structure to improve its activity and taking into account that A₁Cl is a synthetic molecule, modifications can be made to make it more potent.

With regard to the development of new drugs with antifungal potential, a major challenge due to the similarity between fungal cells and human cells is the difficulty of selecting targets. The targets for new antifungals must be exclusive to the fungus or sufficiently different from their human cell analogs to avoid adverse pharmacodynamic effects (Martinez-Rossi et al. 2008MARTINEZ-ROSSI M, PEREZ NTA & ROSSI A. 2008. Antifungal resistance mechanisms in dermathophytes. Mycopathology 166: 369-383., Guerra et al. 2015GUERRA FSQ, ARAUJO RSA, SOUSA JP, PEREIRA FO, MENDONÇA-JUNIOR FJB, BARBOSA-FILHO JM & LIMA EO. 2015. Evaluation of Antifungal Activity and Mode of Action of New Coumarin Derivative, 7-Hydroxy-6-nitro-2H-1-benzopyran-2-one, against Aspergillus spp. Evid Based Complement Alternat Med 8: 1-8.). Thus, to elucidate A₁Cl’s mechanism of action, tests were performed on the cell wall and membrane of the species studied.

Ergosterol is the main component of sterol present in the fungal plasma membrane and it plays the same role in fungal membranes as cholesterol plays in mammalian cell membranes (Bowman & Free 2006BOWMAN SM & FREE SJ. 2006. The structure and synthesis of the fungal cell wall. BioEssays 28: 799-808.), exhibiting qualitatively similar properties to cholesterol.

If A₁Cl activity is promoted through its binding to ergosterol of the fungal membrane, exogenous ergosterol, when added to the culture medium, will prevent this binding. Thus, an increase in MIC for A₁Cl in the presence of exogenous ergosterol will occur since only a higher concentration of the product in the medium will guarantee interactions with fungal membrane ergosterol (Lunde & Kubo 2000LUNDE CS & KUBO I. 2000. Effect of polygodial on the mitochondrial ATPase of Saccharomyces cerevisiae. Antimicrob Agents Chemother 44: 1943-1953., Escalante et al. 2008ESCALANTE A, GATTUSO M, PEREZ P & ZACCHINO S. 2008. Evidence for the mechanism of action of the antifungal phytolaccoside B isolate from Phytolacca tetramera Hauman. J Nat Prod 71: 1720-1725.).

In this study, an increase in the MIC of A₁Cl was observed in the presence of exogenous ergosterol for the two strains tested compared to the control (RPMI) (Table IV). As a positive control, amphotericin B, which acts at the level of the fungal cell membrane, was used. An increase in MIC was observed in relation to the control (RPMI) (Table IV), which was expected, considering that its mechanism of action is already well elucidated and involves binding with ergosterol, the consequential formation of pores in the membrane, and leakage of the intracellular content, culminating in death to the fungus (Filippin & Souza 2006FILIPPIN FB & SOUZA LC. 2006. Eficiência terapêutica das formulações lipídicas de amphotericin B. Braz J Pharm Sci 42: 1-28.). Thus, it is suggested that A₁Cl exerts its mechanism of action in A. flavus interfering with cell functions involving the membrane ergosterol.

Computational tools, such as docking, have been reported in the literature as essential for the development of computer-aided drugs (Brooijmans & Kuntz 2003BROOIJMANS N & KUNTZ ID. 2003. Molecular recognition and docking algorithms. Annu Rev Biophys Biomol Struct 32: 335-373.).

In the dockings performed in this work, the interactions of A₁Cl with important enzymes responsible for the synthesis of the fungal cell wall and membrane components, as well as for nucleic acid formation, were analyzed. The interactions were demonstrated through the binding energies obtained by binding the test product to C-14-α-demethylase, squalene epoxidase, delta-14-sterol reductase, 1,3-β-glycan synthase and thymidylate synthase.

C-14-α-demethylase (4UYL) is a microsomal enzyme associated with cytochrome P-450 (CYP), encoded by the ERG11 gene, which is involved in the final stages of ergosterol biosynthesis, the main sterol responsible for regulating fluidity and other functions of the fungal plasma membrane (Yang et al. 2015YANG H, TONG J, LEE CW, HA S, EOM SH & IM YJ. 2015. Structural mechanism of ergosterol regulation by fungal sterol transcription factor Upc2. Nat Commun 6: 1-13.). The enzyme 1,3-β-glycan synthase (3A58) is responsible for the synthesis of β-glycans, an important component of the fungal cell wall (Cortés & Russi 2011CORTÉS JAL & RUSSI JNA. 2011. Echinocandins. Rev Chil Infectol 28: 529-536.). Squalene epoxidase (4 MAH) is a monooxygenase that participates in ergosterol biosynthesis together with delta-14-sterol reductase and is responsible for catalyzing the conversion of squalene to 2,3-oxidosqualene (Ruckenstuhl et al. 2007RUCKENSTUHL C, LANG S, POSCHENEL A, EINDENBERGER A, BARAL PK, HAPALA I, GRUBER K & TURNOWSKY F. 2007. Characterization of Squalene Epoxidase of Saccharomyces cerevisiae by Applying Terbinafine Sensitive Variants. Antimicrob Agents Chemother 51: 275-284.). Inhibition of this enzyme promotes toxicity, affecting the structure and function of the cell membrane and leading to impairment of the fungal cell’s selective permeability and consequent cell lysis (Sueth-Santiago et al. 2015SUETH-SANTIAGO V, FRANKLIM TN, LOPES ND & LIMA MEF. 2015. Artigo CYP51 : Uma Boa Ideia ? CYP51: Uma Boa Ideia? Rev Virtual Quim 7: 539-575.). The enzyme thymidylate synthase (5UIV) is responsible for DNA synthesis and protein synthesis of the fungus, and of all of the enzymes studied, thymidylate synthase presented the lowest binding energy with A₁Cl (−83.36 Kcal/mol); the lower the binding energy, the greater the ligand’s affinity for the receptor (Qidwai 2017QIDWAI T. 2017. QSAR modeling, docking and ADMET studies for exploration of potential anti-malarial compounds against Plasmodium falciparum. In Silico Pharmacol 5: 1-13.) and thus a greater affinity for thymidylate synthase (Table V). It is possible that one of the mechanisms of action of A₁Cl is interference in DNA synthesis through inhibition of this enzyme, promoting the death of the fungus.

This study elucidating the mechanism of action of A₁Cl is unprecedented, and its mechanisms of action against strains of A. flavus have been demonstrated in the literature for the first time.

In addition to isolated antifungal activity, many compounds can alter the effects of the antifungals on the market. In this study, for the first time, it was shown through the checkerboard trial that A₁Cl interacts antagonistically with the two antifungals used (Table VI).

In clinical practice, drug interactions are common, the definition of which is the association of two or more drugs, such that the safety or efficacy of one drug is significantly altered in the presence of the other (Gaddis et al. 2002GADDIS GM, HOLT TR & WOODS M. 2002. Drug interactions in atrisk emergency department patients. Acad Emerg Med 9: 1162-1167.). In this study, it was observed that one of the mechanisms of action of A₁Cl occurs through binding to ergosterol present in the fungal cell membrane, probably leading to the formation of pores in the membrane and extravasation of cellular content, similar to amphotericin B. If associated with voriconazole, which acts by inhibiting C-14-ɑ-demethylase and decreasing ergosterol biosynthesis, the interaction with ergosterol promoted by A₁Cl would be impaired due to the deficiency of ergosterol synthesis promoted by voriconazole, thus compromising the antifungal activity of A₁Cl, an antagonistic association (Fillipin & Souza 2006).

The association A₁Cl and amphotericin B should be better studied and detailed, since, in the trial presented, the result was antagonism. Amphotericin B is chemically incompatible with some drugs in the clinic, such as sodium chloride 0.9%, meropenem, midazolan, calcium gluconate and vancomycin (Santos et al. 2011SANTOS L, MARTINBIANCHO J, TADIOTTO AL & KREUTZ LM. 2011. Perfil das interações medicamentosas solicitadas ao centro de informações sobre medicamentos de hospital universitário. Rev HCPA 31: 326-335.) generating the presence of precipitates, turbidity, changes in color and chemical reactions, and in association with some antifungals such as itraconazole, terbinafine and voriconazole, has an antagonistic effect when studied in strains of the genus Aspergillus (Vazquez 2008VAZQUEZ A. 2008. Combination antifungal therapy for mold infections: much ado about nothing? Clin Infect Dis 46: 1889-1901.). It is possible that there was chemical incompatibility between the two products, where a chemical reaction could have occurred, forming a substance different from the originals, which may not have the same pharmacological action as the initials, justifying the effect presented.

Given the above, it can be concluded that the synthetic amide 2-chloro-N-phenylacetamide (A₁Cl) presents antifungal activity against A. flavus being fungicidal. The results also suggest a mechanism of action related to the presence of ergosterol in the cell membrane and possibly action at the DNA level, yet further studies to prove this hypothesis will be required, yet further studies to prove this hypothesis will be required. This study’s findings strongly suggest that 2-chloro-N-phenylacetamide presents antifungal potential against A. flavus.

ACKNOWLEDGMENTS

Thanks to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001 for funding the study in part. This article is from the thesis of student Elba dos Santos Ferreira, who is in the process of graduating.

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