Azole Based Acetohydrazide Derivatives of Cinnamaldehyde Target and Kill Candida albicans by Causing Cellular Apoptosis

Abstract

Opportunistic fungal pathogens including Candida albicans are responsible for the alarming rise in hospital acquired infections and millions of deaths worldwide. The current treatment modalities are not enough to handle this situation, and therefore, new treatment modalities and strategies are desperately needed. In this direction, we synthesized a series of azole based acetohydrazide derivatives of cinnamaldehyde and subjected it to antifungal activity evaluation. Preliminary antifungal activity evaluation revealed tremendous antifungal potential of some of the derivatives against fluconazole susceptible and resistant clinical isolates of Candida albicans. Although all the compounds in the series are structurally similar except for the presence of different substituents on the phenyl ring of the acetohydrazide pendent, they sharply differed in their activity profile. Further mechanism of action studies revealed that these compounds have an apoptotic effect on C. albicans confirmed via Annexin V-FITC staining and TUNEL assay.

The discovery of new and effective human therapeutic agents is one of humanity’s most critical tasks. Equitable access to essential medicines for priority diseases is one of the most important requirements to healthy life. It has been estimated that over 300 million people are afflicted with a serious fungal infection and about 25 million are at a high risk, ended dead or doomed.¹ Among the fungal infections Candida alone is responsible for causing >350,000 deaths annually, and the number is expected to grow as the current treatment modalities are not sufficient enough to tackle the growing multidrug resistance menace.^2,3 Development of a new antifungal drug or identifying novel drug targets is challenging because there are many similarities between fungal and human cells. Despite this fact, different approaches and strategies have been pursued to prevent and combat this growing problem.⁴⁻⁶ Among the different strategies, modifications of the chemical structure of traditional antifungals have greatly improved their activity, and it is our premise that the structural modifications of the active scaffolds would help evade the fungal resistance mechanisms although not for a long time; albeit, it would at least augment the current treatment modalities until a new drug with a different mechanism of action would arrive.

Azoles are actually the most widely used drugs against fungal infections, and the most important azoles such as fluconazole, voriconazole, itraconazole, and posaconazole are used to treat invasive fungal infections (Figure1). Azoles are considered to be extremely well tolerated but at the same time they can interfere with a number of other drugs due to their ability to inhibit cytochrome P450.⁷ They inhibit the synthesis of sterols in fungi by inhibiting cytochrome P450-dependent 14α-lanosterol demethylase (P-45014DM), which removes the methyl group on C-14 of lanosterol, a key intermediate step in the formation of ergosterol in the fungal cell membrane.^7,8 Most of the azole based antifungal drugs are fungistatic, and their protracted use has hugely contributed to the development of drug resistance in C. albicans and other species.⁷

Figure 1.

Structures of the most common azole antifungal drugs.

Although huge efforts have been made to develop new compounds with antifungal activity, only a few have shown the mechanism of cell death.^9,10Candida albicans shows different patterns of cell death when responding to the external insults.¹¹ Numerous biochemical and morphological changes differentiate apoptotic cell death with that of necrosis. Among different mechanisms, studying the movement of phosphatidylserine and DNA fragmentation by terminal deoxynucleotidyl transferace mediated dUTP nick end labeling (TUNEL) assay is considered standard to study the apoptotic mode of cell death. Understanding these mechanisms while developing new antifungals can provide a strong basis for effective antifungal therapies.

Herein, we report on the synthesis and anticandida activity of a new series of azole based acetohydrizide derivatives obtained from cinnamaldehyde by a multistep reaction procedure as illustrated in Schemes S1 and S2 (Supporting Information) following our previously reported reaction methods.¹²⁻¹⁴ The structures of the intermediate compounds (1–5) and the target derivatives (6a–6j) were established by different physical and spectroscopic techniques (see Supporting Information). The in vitro susceptibility of all the 11 different isolates of C. albicans against the five intermediates (1–5) and the target compounds (6a–6j) were evaluated by determining MIC and MFC values. Table 1 summarizes the in vitro susceptibilities (MIC and MFC) of all the test compounds against 1 standard laboratory C. albicans SC5314, 7 clinically obtained FLC susceptible C. albicans, and 3 FLC resistant C. albicans isolates. MIC values of the intermediates (1–5) were comparatively higher (>125 μg/mL) than the MIC values of the target compounds (6a–6j), which ranged from 0.125–125 μg/mL, while MFC values varied from 1 to 125 μg/mL. MIC results reflected that compound 6f exerted the highest inhibitory activity against all the tested C. albicans isolates with a mean MIC value of 0.125 μg/mL and MFC value of 1 μg/mL against FLC susceptible isolates and these figures against FLC resistant C. albicans isolates reached to 0.5 μg/mL. Among all the target compounds 6a, 6b, and 6c possess moderate antifungal activity with a mean MIC value of 125, 64, and 64 μg/mL, respectively. On the basis of the MIC and MFC results, order of potency of the seven active compounds was 6f> 6h > 6i > 6j > 6g > 6e > 6d. As a positive control, FLC was used because of its common use in the treatment of Candida infections and as expected the mean MIC values for FLC against susceptible and resistant strains was 0.25 μg/mL and 16 μg/mL, respectively. The C. albicans strains with MIC values of ≥8 μg/mL for FLC were considered as FLC resistant strains following the CLSI interpretive guidelines for in vitro susceptibility testing of Candida species (CLSI 2012). 1% DMSO was used as a negative control because all the test compounds were prepared to different concentrations using this concentration of DMSO and it was observed to have no inhibitory activity against any of the tested isolates.

Table 1. Minimum Inhibitory Concentrations and Minimum Fungicidal Concentrations of the Intermediates (1–5^a) and Target Compounds (6a–6j) against Different Candida albicans.

		Mean MIC/MFC (μg/mL)
Comp.	R	FLC susceptible (n = 8)		FLC resistant (n = 3)
1	--	>125	>125	>125	>125
2	--	>125	>125	>125	>125
3	--	>125	>125	>125	>125
4	--	>125	>125	>125	>125
5	--	>125	>125	>125	>125
6a	H	125	>125	125	>125
6b	2-Cl	64	>125	125	>125
6c	4-Cl	64	>125	64	>125
6d	4-CH3	16	16	16	16
6e	4-OCH3	8	8	8	16
6f	4-NO2	0.125	1	0.50	1
6g	4-OC2H5	4	8	8	8
6h	3-NO2	0.25	1	0.50	1
6i	2-NO2	0.50	4	0.50	4
6j	3,4-dimet	2	8	4	8
FLC	--	0.25		16

^aFor structures see Supporting Information.

Due to the poor or moderate antifungal activity of the intermediates and compounds 6a, 6b, and 6c, these compounds were excluded for further studies.

To further confirm the antifungal activity of the most active compounds (6d–6j), we quantified the effect of these compounds on the cellular viability of C. albicans SC5314 at their respective MIC values. Cells at a density of 1 × 10⁷ were exposed to MIC values of the test compounds and cell viability was accessed by Muse count and viability kit. In comparison to the negative untreated cells, cellular viability decreased after the exposure of the C. albicans cells to the test compounds. The results showed that the decrease in cell viability after exposure to test compounds was at varying extents ranging from 55.8–22.8% (Figure 2). The results also depict that compound 6f is the most effective among all the tested compounds with a cell death of >75%. The viability results reflect the same pattern as observed in the MIC determination, which also advocates the reliability of this technique to be used in antifungal studies. To further differentiate the results between the cells and debris and/or non-nucleated cells, cell size index was also done in the same assay and it was confirmed that the results are due to live and dead cells (Figure S1).

Figure 2.

Cell Viability assay for C. albicans SC5314: Cells at a density of 1 × 10⁷ were exposed to MIC values of the test compounds (6d–6j) and cell viability assay was performed by the Muse Count & Viability kit (Millipore) following the manufacturer’s protocols. Percentage live and dead cells are shown in two different subsets. Negative and positive controls represent untreated and heat killed cells, respectively.

From the viability data shown above, it was clear that the target compounds (6d–6j) were able to kill the C. albicans cells within a maximum concentration of 16 μg/mL. To further assess if the killing occurred through induction of apoptosis and/or necrosis, phosphatidylserine externalization was studied using annexin V-FITC labeling and PI staining, by flow cytometry. The asymmetry of phospholipids varies during normal physiological conditions and during apoptosis. In the latter case, phosphatidylserine is externalized to the outer leaflet of the plasma membrane, which could be detected using annexin V-FITC dye.¹⁵ On the other hand, PI was used to detect necrotic cells as this dye can only permeate into necrotic cells with disintegrated membranes. The results of annexin V-FITC and PI staining are shown in a cytogram (Figure 3) and the percentage of apoptotic (early and late apoptosis) and necrotic cells were calculated in Table 2. As protoplasts were used for this assay, it is important to make sure that lyticase did not cause false positive annexin V or PI staining. Therefore, titration of lyticase was done and it was observed that a concentration of ≥1 μg/mL of lyticase did not cause any apoptosis or necrosis (data not shown). Exposure of C. albicans cells to 1/2 MIC values of the test compounds for 3 h resulted in higher percentage of cells in late apoptosis phase with a mean range of 15.60% (for 6d) to 48.30% (for 6f). Among all the tested compounds, 6f, 6h, and 6i, being the most active antifungal compounds, they exhibit higher percentage of apoptosis (Table 2). These results also revealed that these compounds induced early apoptosis in only 8–14%, while as complete necrosis was induced in 2–12%. Cells incubated with 2.5 mM H₂O₂ (positive control) showed mean early apoptosis of 27.5%, late apoptosis of 26.5% and necrosis of <3% (Table 2).

Figure 3.

Representative density plots showing phosphatidylserine externalization, an early stage apoptosis marker, shown by annexin V staining. The plots represent the alterations of phosphatidylserine externalization when cells were exposed to compounds (6d–6j) at their respective 1/2 MIC values. Untreated cells and cells exposed to H₂O₂ were used as negative and positive controls, respectively. Quadrant Q1 shows necrotic, Q2 shows late apoptotic, Q3 shows early apoptotic, and Q4 shows live cells.

Table 2. Mean Percentage of Cells Showing Early Apoptosis, Late Apoptosis, and Necrosis after Exposure to 1/2 MIC Values of the Most Active Compounds for 3 h^a.

	% cell population with different effects after 3 h exposure
Comp. (MIC)	Annexin V+/PI– (early apoptosis)	Annexin V+/PI+ (late apoptosis)	Annexin V–/PI+ (necrosis)
Negative control	3.47 ± 0.26	0.06 ± 0.01	0.15 ± 0.03
Positive control	27.60 ± 2.76	26.43 ± 0.54	2.31 ± 0.01
6d	10.18 ± 0.58	15.60 ± 0.65	3.70 ± 0.22
6e	13.90 ± 0.53	23.60 ± 0.97	1.78 ± 0.11
6f	8.21 ± 1.82	48.30 ± 1.97	9.80 ± 0.32
6g	10.49 ± 0.76	27.92 ± 2.13	2.44 ± 0.08
6h	8.23 ± 1.08	44.65 ± 2.76	11.70 ± 0.34
6i	13.37 ± 0.69	36.34 ± 2.97	1.98 ± 0.97
6j	9.69 ± 0.95	24.93 ± 2.11	4.55 ± 0.79

^a2.5 mM H₂O₂ was used as a positive control.

As observed in annexin V-FITC affinity labeling, it is evident that these compounds showed more late apoptosis than early apoptosis, and therefore, TUNEL assay was performed after exposing the cells to 1/2 MIC values of most active compounds. In late apoptosis, due to proteolysis of nuclear proteins chromatin damages which further results in DNA damage and chromatin condensation.¹⁶ In TUNEL assay, these cells with late apoptotic characteristics can be evaluated by DNA fragmentation at a single cell level which can be visualized with the help of a TUNEL assay by labeling 3′-OH ends of nicked DNA with fluorescent dUTP. DNA damage is the characteristic feature of late apoptosis.¹⁷ The apoptotic cells showed green fluorescence when treated with the test compounds, whereas blue fluorescence was observed in all cell populations (Figure 4). The C. albicans SC5314 cells exposed to 1/2 MIC values of the active test compounds exhibited a significant amount of nuclear DNA damage. The extent of apoptosis in the treated cells was approximately in the range of 25–55% cells. These results can be corroborated with the results observed with the phosphatidylserine externalization in flow cytometry. Comparatively, cells exposed to positive control (0.125 μg/mL amphotericin B) showed an increase of >90% in TUNEL-positive nuclei, identified as green fluorescence spots. It is important to note that all the cells were only exposed to 1/2 MIC values and not the MIC values of the test compounds in order to avoid the killing effects of these compounds. It is evident from the viability assay that these compounds have rapid antifungal action. At sub-MIC values of these compounds, the percentage of cells that exhibited TUNEL-positive nuclei was significantly greater than the percentage of untreated controls cells. These results are in corroboration with the previous findings where antifungal drugs such as amphotericin B and caspofungin were reported to cause late apoptosis.^11,16 In the same study, it was also reported that nuclear fragmentation associated with DNA damage was caspofungin concentration independent.¹⁶

Figure 4.

Representative confocal scanning laser fluorescence images of treated and untreated C. albicans cells, revealing the presence of (A) live or dead intact cells, stained by hoechst 33342 dye indicated by blue fluorescence; (B) apopototic cells with fragmented DNA by the test compound exposed cells, indicated by green fluorescence; and (C) merged image of both live and apoptotic cells. Negative control represents untreated cells with no apoptosis and positive control represents cells treated with 0.125 μg/mL of amphoterecin B. Wavelengths of Ex/Em: 488/515 nm and for Hoechst dye Ex/Em: 350/460 nm were used. The bar represents 10 μm.

To ensure that the active compounds do not show any cytotoxicity, hemolysis assay on horse red blood cells was done. Red blood cells provide a handy tool for toxicity studies of the compounds, because they are readily available, their membrane properties are well-known, and their lysis can easily be monitored by measuring the release of hemoglobin.¹⁸ The in vitro hemolytic assay is a possible screening tool for gauging in vivo toxicity to host cells. The %-age cell lysis caused by these compounds at MIC and MFC values is <10 and reached a maximum of 40% at higher concentrations (Figure 5). These results confirmed that all the test compounds are less toxic and can be tested for in vivo studies using animal models.

Figure 5.

% cell lysis caused by the test compounds against horse red blood cells. Hemolysis was determined by an absorbance reading at 450 nm and compared to hemolysis achieved with 1% Triton X-100 (reference for 100% hemolysis).

Despite the extensive studies in synthesizing new compounds to introduce new and effective antifungal drugs for the treatment of invasive fungal infections, only a few have reached clinical trials. The biggest challenge in developing novel antifungal agents is to identify unique drug targets that are not shared with human hosts. Therefore, new compounds are needed to be identified or synthesized that can cause fungal programmed cell death also called apoptosis to allow specific manipulation of cell death without costing human counterparts. The target compounds under study were obtained from a natural compound, cinnamaldehyde with inherent biological activities,¹⁹⁻²¹ and the aldehyde functionality of this molecule was converted to an azole which was further alkylated to furnish the different acetohydrazide derivatives (6a–6j). The target compounds bear a similar ring skeleton except for the presence of different substituents on the phenyl ring of the acetohydrazide pendent. The different substituents resulted in disparity in activity of the otherwise similar derivatives. Without any substituent 6a was the least active derivative, followed by 6b, 6c, and 6d which contain 2-Cl, 4-Cl, and 4-methyl groups as substituents respectively on the phenyl ring. The activity sharply increased (4- to 6-fold increase) when these substituents were replaced by 4-methoxy in 6e, 4-ethoxy in 6g, and 2,4-dimethoxy in 6j, respectively. The activity further increased by 9–11-fold when these substituents were replaced by a −NO₂ group at positions -4, -3, and -2 of the phenyl ring in 6f, 6h, and 6i, respectively. The effect of these substituents on the activity of the derivatives could be attributed to their different electronic effects. The nitro group can undergo enzymatic reduction inside the biological systems by both one- or two-electron mechanisms. The reduction of a NO₂ group by the two-electron mechanism generates amines through nitroso and hydroxylamine intermediates.^22,23 The radicals generated during the process could react with certain biomolecules to produce compounds having undesired effects. Reduction by the one-electron mechanism, on the other hand, generates a nitro radical anion, which is unstable. This unstable radical gets oxidized back to the nitro group by molecular oxygen, which in turn is reduced to a very reactive superoxide. The compounds under study were found to possess moderate to strong antifungal activity against different FLC susceptible and resistant C. albicans isolates. When the cells were exposed to these compounds, the number of viable cells decreased. Among all the tested compounds, only 7 (6d–6j) possessed high antifungal activity and were further investigated for the mode of cell death in C. albicans.

Induction of apoptosis in yeast is a complex process, which involves many pathways. There are several endogenous and exogenous factors which also regulate apoptosis in C. albicans and have been regulated both at biochemical and molecular levels.²⁴ Activation of endogenous factors results in apoptosis via increase in ROS production, mitochondrial dysfunction including mitochondrial depolarization, release of cytochrome c, increase in intracellular Ca²⁺, DNA fragmentation, etc. Therefore, targeting these mechanistic switches may lead to the development of novel antifungal agents that switch on endogenous cell suicide mechanisms. Flow cytometry analysis revealed that the C. albicans cells exposed to sub-MIC concentrations of the test compounds showed the movement of phosphatidylserine to the outer leaflet of the plasma membrane. The externalization of phospholipids results in the binding of annexin V, which was detected by flow cytometer. It has been extensively observed that fungicidal compounds at sub-MIC values show programmed cell death.^24,25 Similarly, the most active compounds in this study are fungicidal, and at sub-MIC values these compounds are able to cause apoptosis as can be seen in flow cytometry and confocal microscopy.

The gold standard drug for treating fungal diseases, amphotericin B, has also been reported to have fungicidal activity, causing apoptosis at sub-MIC values.²⁶ As from the results, it is evident that most of the compounds cause late apoptosis rather than early apoptosis, which could be due to the exposure time and concentrations used. It can also be hypothesized that at higher concentrations, these compounds show more necrosis than apoptosis leading to the fungicidal characteristics of these compounds. The other significance of targeting apoptosis relies in the metacaspases targeted in C. albicans during apoptosis. It has already been established that the only caspase identified as executing apoptosis in Candida is a metacaspase, which is unique to fungi and are represented by the other caspases in human hosts.²⁷ Furthermore, induction of apoptosis at lower concentrations can also be due to the oxidative insult caused by these compounds, and therefore, there is a scope for further studies identifying other mitochondrial factors activated during the course of mediating apoptosis in these cells. Like other lipophilic compounds, these compounds (cLogP = 2.97–3.21) can conjugate with a reduced form of glutathione and thereby disrupt the intracellular redox equilibrium and lead to cellular apoptosis. This selective targeting death process in C. albicans by these azole based acetohydrazide derivatives and their least off target toxicity may help find new antifungal agents that could switch on endogenous cell suicide mechanisms.

Glossary

Abbreviations

PI

propidium iodide

TUNEL

terminal deoxynucleotidyl transferase dUTP nick end labeling

DNA

deoxyribonucleic acid

MIC

minimum inhibitory concentration

MFC

minimum fungicidal concentration

FLC

fluconazole

CLSI

Clinical & Laboratory Standards Institute

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00030.

• Details on preparation and characterization of compounds 1–5 and 6a–6j, in vitro assays, mechanism of action, protoplast preparation, Annexin V and PI staining, and confocal microscopy (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

This work was funded by the University of Jeddah, Saudi Arabia, under grant No. UJ-45-18-DR. The authors therefore acknowledge with thanks the University technical and financial support.

The authors declare no competing financial interest.