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. 2021 Oct 14;6(42):27798–27813. doi: 10.1021/acsomega.1c03379

Development of Oxadiazole-Sulfonamide-Based Compounds as Potential Antibacterial Agents

Asghar Ali †, Phool Hasan ‡, Mohammad Irfan ‡, Amad Uddin ‡, Ashba Khan ‡, Juhi Saraswat §, Ronan Maguire ∥, Kevin Kavanagh ∥, Rajan Patel §, Mukesh C Joshi ⊥, Amir Azam #, Mohd Mohsin ∇, Qazi Mohd Rizwanul Haque †,*, Mohammad Abid ‡,*
PMCID: PMC8552329  PMID: 34722980

Abstract

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In this work, substituted 1,2,4-oxadiazoles (OX1–OX27) were screened against five bacterial strains, identified to be OX7 and OX11 as growth inhibitors with minimum inhibitory concentration (MIC) values of 31.25 and 15.75 μg/mL, respectively. The growth inhibitory property of OX7 and OX11 was further validated by disk diffusion, growth curve, and time kill curve assays. Both disrupted biofilm formation with 92–100% reduction examined by the XTT assay were further visualized by scanning electron microscopy analysis. These compounds in combination with ciprofloxacin also exhibit synergy against Escherichia coli cells. With insignificant cytotoxic behavior on HEK293 cells, human red blood cells, and Galleria mellonella larvae, OX11 was tested against 28 multidrug resistant environmental isolates of bacteria and showed inhibition of Kluyvera georgiana and Citrobacter werkmanii strains with 32 and 16 μg/mL MIC values, respectively. The synergistic behavior of OX11 with ampicillin showed many fold reductions in MIC values against K. georgiana and Klebsiella pneumoniae multidrug resistant strains. Further, transmission electron microscopy analysis of OX11-treated E. coli cells showed a significantly damaged cell wall, which resulted in the loss of integrity and cytosolic oozing. OX11 showed significant changes in the secondary structure of human serum albumin (HSA) in the presence of OX11, enhancing HSA stability. Overall, the study provided a suitable core for further synthetic alterations and development as an antibacterial agent.

1. Introduction

According to the 2016 UN Declaration on Antimicrobial Resistance, global strategies and campaigns are underway to combat the disease.1,2 The availability of inadequate treatment and extensive use of antimicrobial agents has resulted in antimicrobial resistance against the available drugs.2 Thus, the conspicuous absence of novel and effective antimicrobials to control the microbial growth and increasing resistance has encouraged us to develop novel antimicrobial entities. Among various factors, formation of a biofilm is one of the significant features adopted by bacteria to develop resistance toward antibiotics.3 Antibiotic resistance in bacteria may increase due to formation of a biofilm, which can elevate the rate of infection and cause morbidity and thus is important in clinical illnesses. In the presence of certain antimicrobials, growth in planktonic cells is increased to several folds as compared to biofilm, demonstrated by the susceptibility date literature.4 However, in most biofilm susceptibility studies, only the survival of cells in a preformed biofilm is recorded rather than the ability of a biofilm to grow. Several approaches have been identified for the search of efficient antimicrobials, which can disrupt biofilm formation, including the role of nitrogen containing heterocycles as a biofilm inhibitor/disruptor.5,6 Among them, oxadiazole, a five-membered azole, and its derivatives are best known for their wide spectrum of antimicrobial activity. Oxadiazole ring containing compounds have been discovered and studied for their various properties viz. antitumor, antioxidant, antibacterial, antiviral, anti-inflammatory, insecticidal, and antiparasitic activities.7−19 Besides enormous biological potential of oxadiazole derivatives, we preferred sulfonamide functionality with the aim to link it with the oxadiazoles to get anticipated improved antimicrobial motifs. Sulfonamides are well-known for their broad spectrum activity against Gram-positive and Gram-negative strains and also exhibit activity viz. carbonic anhydrase inhibition, insulin releasing, anti-tumor and anti-inflammatory effects, etc.20−23 Based on the dominant biological profile of oxadiazoles as well as sulfonamides, we screened our library of diversely substituted oxadiazoles (OX1–OX27) against five tested bacterial strains. Among all, OX7 and OX11 showed better potency against Streptococcus pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. Furthermore, growth kinetic as well as time kill curve studies were done on E. coli and S. pneumoniae treated with OX7 and OX11 confirming bactericidal character. Interestingly, both the compounds almost eradicated the formation of the biofilm in E. coli cells. Further, OX11 was studied for in vitro (cytotoxicity assays and hemolysis) and in vivo toxicity assays on Galleria mellonella larvae and revealed to be non-toxic. The effect of OX11 on resistant bacterial strains showed that it is a selective inhibitor of Kluyvera georgiana and Citrobacter werkmanii with a 32 μg/mL MIC value. The antibacterial efficacy of OX11 was considerably improved when it was combined with AMP against S. pneumoniae and K. georgiana.

Further, the interactions between human serum albumin (HSA) and OX11 were also studied. HSA has the ability to bind remarkably to a variety of drugs and impacts its delivery and efficacy. Mostly, concentration of an unbound drug in a tissue depends completely on the presence of an unbound drug in the plasma.24 The interaction of the HSA drug has become increasingly important to analyze the pharmacological as well as the pharmacokinetic effect of a drug.25−28 Various spectroscopic techniques viz. UV–visible, fluorescence, 3D fluorescence, synchronous fluorescence, and circular dichroism (CD) were employed, and the experimental results suggested that OX11 strongly quenches the intrinsic fluorophore of HSA. The thermodynamic parameters viz. ΔH, ΔS, and ΔG suggest the involvement of the hydrophobic interaction and spontaneity of the OX11-HSA complex formation. Additionally, synchronous fluorescence showed that amino acid tryptophan (Trp) is involved in the quenching process. Circular dichroism (CD) data and three-dimensional (3D) fluorescence revealed that OX11 stimulates the conformational changes in the secondary structure of HSA and enhances its stability.

2. Results

2.1. In Vitro Antibacterial Screening

2.1.1. Minimum Inhibitory Concentration (MIC) Determination

Preliminary antimicrobial screening of oxadiazole derivatives (OX1–OX27) resulted in OX7 and OX11 with better antibacterial activity against tested bacterial strains, and the data is presented in Table 1. OX7 showed an MIC value of 31.25 μg/mL against P. aeruginosa and 15.75 μg/mL against E. coli, whereas OX11 exhibited an MIC value of 15.75 μg/mL against P. aeruginosa and E. coli (Table 1). Both compounds inhibited S. pneumoniae with an MIC value of 15.75 μg/mL. However, no growth inhibition was observed against Salmonella typhimurium and Enterococcus faecalis with MIC values more than 500 μg/mL. Thus, it was concluded from the study that OX7 and OX11 are selective inhibitors of P. aeruginosa, S. pneumoniae, and E. coli bacterial cells with a better growth inhibitory property.

Table 1. MIC Values (in μg/mL) of all the Screened Compoundsa.

2.1.1.

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a

CIP, ciprofloxacin; AMP, ampicillin.

2.1.2. Disk Diffusion Assay

The antibacterial efficacies of the test compounds OX7 and OX11 were determined using the disk diffusion assay on a solid nutritional agar medium at concentrations corresponding to 1/2MIC, MIC, and 2MIC. The zones of clearance (dose-dependent) were observed in the assay when various concentrations of both test compounds were applied. On treatment with OX7, clear zone of inhibition (ZOI) values of 20, 23, and 24 mm were observed around the disk of 1/2MIC, MIC, and 2MIC, respectively, with the culture of Gram-negative E. coli and the same result was observed with the culture of Gram-positive S. pneumoniae cells, whereas the ZOI values with the culture of Gram-negative P. aeruginosa were 19 mm (1/2MIC), 22 mm (MIC), and 23 mm (2MIC) (Table 2).On treatment with OX11 the ZOI values obtained were 20, 22, and 28 mm with E. coli; 19, 22, and 25 mm with S. pneumoniae; and 19, 22, and 23 mm with P. aeruginosa around the disk of 1/2MIC, MIC, and 2MIC, respectively. Therefore, the largest ZOI is obtained with S. pneumoniaeandE. coli against both the compounds. The images of agar plates showing zone of inhibition are provided in the Supporting Information.

Table 2. Zone of Inhibition (ZOI) in mm Determined around the Disk of Various Concentrations of Compounds OX7 and OX11.
    ZOI at distinct concentrations of the test compound
compound bacterial strain 1/2MIC MIC 2MIC
OX7 P. aeruginosa 19 mm 22 mm 23 mm
S. pneumoniae 20 mm 23 mm 24 mm
E. coli 20 mm 23 mm 24 mm
OX11 P. aeruginosa 19 mm 22 mm 23 mm
S. pneumoniae 19 mm 22 mm 25 mm
E. coli 20 mm 22 mm 28 mm
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2.1.3. Growth Kinetics Assay

The effect of OX7 and OX11 on the growth of the test organisms was investigated by growth curve analysis using E. coli and S. pneumoniae bacterial strains. As a negative and a positive control, we took treated (CIP and AMP) cells and untreated cells, respectively. The result of the growth curve revealed an S-shape sigmoid curve for untreated bacterial cells. No growth was observed till 20 h in E. coli at 2 MIC and MIC of the test compound OX7. At 1/2MIC of OX7, the growth of E. coli cells was observed after 4 h in comparison to untreated cells; it was still delayed. However, OX11 completely prevented the growth of both strains of bacteria at all test concentrations. As a consequence, the data indicated that both compounds have antimicrobial effects against the tested strains. The study concluded that OX11 is a more efficient inhibitor of the tested bacterial strains than OX7 since no significant growth was detected at any of the test concentrations after 24 h (Figure 1).

Figure 1.

Figure 1

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Growth curve analysis of (a) E. coli in the presence of distinct concentration of OX7, (b) S. pneumoniae in the presence of distinct concentration of OX7, (c) E. coli in the presence of distinct concentration of OX11, and (d) S. pneumoniae in the presence of various concentrations of OX11.

2.1.4. Time Kill Curve Study

To establish whether the tested compounds are bactericidal or bacteriostatic in nature, a time kill curve study was conducted. The study was done at 4MIC and MIC concentrations of OX7 and OX11 against bacterial strains of E. coli and S. pneumoniae bacterial strains. The study demonstrated that there was only a slight difference in CFU (colony forming units) values for MIC and 4MIC concentrations. The killing activity was observed up to 24 h through a regular decrease in CFU with time intervals, which fallen up to <1 log 10 CFU. At the MIC concentration, there was a substantial drop in log 10 CFU/mL with time; however, no full eradication of the bacterial population was observed. At higher concentration, corresponding to 4MIC, significant eradication of tested bacterial cells was observed after 24 h. The observations explicitly indicated the bactericidal nature of both the test compounds against the tested bacterial strains (Figure 2a,b).

Figure 2.

Figure 2

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Time kill curve for (a) E. coli and (b) S. pneumoniae treated with OX7 and OX11 at different concentrations.

2.1.5. Synergistic Antibacterial Activity of Test Compounds (OX7 and OX11)

To investigate the synergistic antibacterial activity, the selected compounds OX7 and OX11 were further evaluated in combination with CIP and AMP against S. pneumoniaeandE. coli bacterial cells. The outcomes indicated a significant enhancement in the antibacterial activity of OX7 against the E. coli strain when used in combination with CIP or AMP, showing synergy, but in the case of S. pneumoniae, it was an indifferent mode of interaction with combination of CIP, whereas a syngerstic mode of interaction was observed with AMP. Similarly, OX11 also showed synergy with CIP or AMP against E. coli. Additionally, OX11 exhibits an indifferent mode of interaction with CIP and a synergistic mode of interaction with AMP against S. pneumoniae. The FICI value was 1 < FICI ≤ 4 in all the combinations (Table 3). FICI indices of ≤0.5 and >4 were used to chatagorize synergy and antagonism, respectively, whereas 1 < FICI ≤ 4 was used to indicate indifference.29

Table 3. Antibacterial Activity of OX7 and OX11 in Combination with CIP and AMP.
    MIC alone (μg/mL)
 MIC in combination (μg/mL)
    
combination bacterial strain OX7 OX11 CIP AMP OX7 OX11 CIP AMP FICIa interaction pattern
OX7 with CIP E. coli 16   0.25   2   0.062   0.375 synergistic
S. pneumoniae 16   0.25   2   0.5   2.125 indifferent
OX7 with AMP E. coli 16     16 0.5     0.078 0.343 synergistic
S. pneumoniae 16     31.25 1     2.5 0.1425 synergistic
OX11 with CIP E. coli   16 0.25     2 0.062   0.375 synergistic
S. pneumoniae   16 0.25     2 0.25   1.125 indifferent
OX11 with AMP E. coli   16   16   0.5   0.078 0.03 synergistic
S. pneumoniae   16   31.25   0.25   0.312 0.025 synergistic
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a

Fractional inhibitory concentration index.

2.2. Assessment of Biofilm Disruption

2.2.1. XTT (2,3-Bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide) Assay

Biofilm formation is a significantly important virulence factor of bacterial pathogens, which helps them to survive in stressed conditions within the host. The effect of OX7 and OX11 on biofilm disruption was evaluated in E. coli and S. pneumoniae bacterial strains using an XTT assay kit. Both the compounds showed significant disruption of biofilm formation in E. coli as well as in S. pneumoniae. Compound OX7 disrupted 92, 86, and 84% biofilm formation in E. coli in the presence of 4MIC, 2MIC, and MIC concentrations, respectively. In the case of S. pneumoniae, it disrupted 98, 95, and 92% biofilm formation at their 4MIC, 2MIC, and MIC concentrations, respectively. Similarly, compound OX11 disrupted 95, 90, and 81% of biofilm formation in E. coli at their 4MIC, 2MIC, and MIC concentrations, respectively, while completely disrupting biofilm formation at each concentration in S. pneumoniae. Thus, compounds OX7 and OX11 exert their antibacterial effect via disruption of biofilm formation (Figure 3).

Figure 3.

Figure 3

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Percent biofilm disruption by the XTT assay in E. coli and S. pneumoniae corresponding to (1) 4MIC, (2) 2MIC, and (3) MIC concentrations of OX and OX11.

2.2.2. Scanning Electron Microscopic (SEM) Analysis

The XTT test was used to detect biofilm disruption in E. coli cells, and the results were quantified using scanning electron microscopy (SEM). The concentrations of the 2MIC value of OX7 and OX11 were taken to study their effectiveness against biofilm development as compared to untreated cells. The cells in untreated samples appeared clustered and entrenched in the extracellular matrix under SEM analysis (Figure 4a). At 2MIC, the sample showed significant damage in biofilm (Figure 4b,c). The result clearly indicated that, in comparison with the untreated cells, both the test compounds disrupted the formation of biofilm in E. coli cells, but it was also observed that the effect was stronger in the case of OX11. Thus, we concluded that OX11 has the better ability to disrupt biofilm formation and its molecular mechanism can be further evaluated in this regard.

Figure 4.

Figure 4

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SEM images of biofilm formation in (a) untreated E. coli cells, (b) OX11-treated E. coli cells, and (c) OX7-treated E. coli cells.

2.3. Toxicological Studies

2.3.1. Hemolytic Assay

A hemolytic assay using human red blood cells (hRBCs) was used to screen any toxic effect of the test compounds (OX7 and OX11). The findings were compared to the conventional antibiotic CIP. Compounds OX7 and OX11 demonstrated 36 and 38% hemolysis of hRBCs, respectively, at a concentration of 400 μg/mL. At the same concentration, however, standard drug CIP showed 28% hemolysis. Our test compounds were likewise found to be non-toxic at concentrations between 25 and 12.5 μg/mL, indicating their non-toxic nature. Both the test compounds demonstrated less than 10% hemolysis, indicating their non-toxic character (Figure 5).

Figure 5.

Figure 5

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Hemolytic activity of compounds OX, OX11, and CIP on human red blood cells (hRBCs).

2.3.2. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) Assay

To be further sure about the non-toxic behavior of the test compounds, we evaluated the cytotoxic effect of the compounds on human embryonic kidney (HEK293) cells as the test model to check the cell viability. The screening was carried out for 48 h using the MTT assay in the concentration range of 0–250 μM. It was interesting to find that, even at 250 μM concentration of OX7 and OX11, the viability of HEK293 cells remained unaffected (Figure 6). These findings indicated that both compounds are non-toxic to HEK293 cells at the concentration ranges tested. Furthermore, based on the cell survivability tests, we can speculate that, among the two lead test compounds, OX11 may be taken as a promising lead molecule against selective bacterial strains since it did not induce harm to normal cells in the micromolar concentration levels tested but rather inhibited bacterial cells. So, based on the toxicity assays also, OX11 emerged as the lead compound as it was observed in the case of other biological assays.

Figure 6.

Figure 6

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Cytotoxic effect of OX and OX11 using HEK293 cells.

2.3.3. Evaluation of In Vivo Toxicity of OX11 on G. mellonella Larvae

To further evaluate the cytotoxic effect of our lead compound, we used G. mellonella as the model that exhibits strong correlation with the innate immunity response of mammals. An in vivo administration of 125 μg/mL for the test compound (OX11) did not induce any significant change in the larval viability, indicating its nontoxic behavior toward the larvae (Figure 7). However, hemocyte density of the larvae shows some variation, which is indicative of a stress response in larvae (Figure 8).

Figure 7.

Figure 7

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Survival of G. mellonella larvae injected with OX11 over 72 h at 30 °C.

Figure 8.

Figure 8

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Changes in hemocyte density following injection with OX11 over 72 h at 37 °C.

2.4. Transmission Electron Microscopic (TEM) Analysis

To assess the impact of OX11 on the morphology of E. coli cells, TEM analysis was performed. We used the E. coli cell culture treated with the MIC concentration of OX11 and untreated cells as a control. The treated cells had moderate to severe cellular abnormalities, but the untreated cells were normal in shape with undamaged cell walls (Figure 9). The treatment of bacterial cells with OX11 resulted in a substantial loss of cell wall integrity and cytoplasmic oozing. The degraded cell walls of bacteria observed in the TEM micrographs propose the bactericidal activity of OX11.

Figure 9.

Figure 9

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TEM images of (a) untreated E. coli cells and (b) OX11-treated E. coli cells.

2.5. Confocal Laser Scanning Microscopic (CLSM) Analysis

Using confocal laser microscopy, the cellular uptake of compound OX11 by E. coli bacterial cells was determined. The bacterial cells were stained with DAPI (4′,6-diamidino-2-phenylindole), a nucleic acid binding dye. DAPI is a fluorescent dye that binds strongly to the DNA’s A-T rich region and emits blue fluorescence as emission spectra at 461 nm after being excited at 358 nm. Untreated as well as treated cells (exposed to MIC of OX11 for 3 h) were stained and observed under confocal laser microscopy. Untreated cells were alive and did not emit blue fluorescence, as shown in Figure 10a. In the treated sample, the number of live cells decreases (Figure 10b). The presence of compound OX11 resulted in a large number of fluorescence emitting cells, clearly indicating cell lysis. As a result of this finding, it was determined that compound OX11 has significant antibacterial properties, which could be investigated further to develop better antibacterial agents.

Figure 10.

Figure 10

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Confocal laser microscopic images of E. coli cells (a) untreated and (b) treated with OX11 at MIC concentrations.

2.6. Effect of the Lead Compound (OX11) on Resistant Strains

2.6.1. MIC Determination

Based on the biological assays performed on both the selected compounds, OX11 was carried forward to further investigate its inhibitory effect on environmental resistant strains in terms of MIC against 19 different strains of E. coli, two strains of Klebsiella pneumoniae, and one each of Acinetobacter, K. georgiana, and C. werkmanii. All these strains were isolated from environmental wastewater samples from Delhi-NCR.30,31 The MIC values were calculated and compared to AMP and SMX as the reference antibacterial drugs. The results showed that OX11 exhibited better results than AMP against K. georgiana (with an MIC value of 32 μg/mL) and C. werkmanii (with an MIC value of 32 μg/mL) (Table 4). This MIC value is far less than as compared with that of the standard drug AMP (MIC, 500 μg/mL) in the case of K. georgiana strain. With SMX (MIC value, ∼1000 μg/mL), OX11 showed better result against E. coli strain (AE-31) also, in addition to K. georgiana and C. werkmanii. Therefore, we found that OX11 is more effective than SMX as the MIC values of the former are far less than those of the latter against selective resistant bacterial strains, rendering it a promising antibacterial agent.

Table 4. MIC Values of OX11 against Environmental Isolates of Bacteria (μg/mL).
bacterial isolates code strain OX11 AMP SMX
AE-9 E. coli 1000 1000 >1000
AE-27 E. coli 1000 1000 1000
AE-44 E. coli 1000 500 1000
AE-32 E. coli 1000 >1000 >1000
AE-17 E. coli 1000 1000 500
AE-23 E. coli 1000 1000 500
AE-42 E. coli 1000 1000 >1000
AE-21 E. coli >1000 1000 >1000
AE-31 E. coli 500 8 >1000
EC-36 E. coli 1000 1000 1000
EC-2 E. coli 1000 500 1000
EC-3 E. coli 1000 1000 1000
EC-6 E. coli 1000 1000 1000
EC-25 E. coli 1000 500 1000
OB-18 E. coli 1000 1000 1000
OB-6 E. coli 1000 500 >1000
AE-2 E. coli 1000 500 1000
OE-11 E. coli 1000 500 >1000
EE-2 E. coli 1000 500 >1000
EH-8 Acinetobacter sp. 1000 32 1000
KP K. pneumoniae 1000 1000 1000
EA-13 K. pneumoniae >1000 125 >1000
SH-14 K. georgiana 32 500 500
SH-52 C. werkmanii 32 8 500
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2.6.2. Combination Studies of OX11 against Selective Environmental Bacterial Isolates

The combination study of OX11 was performed with the standard drugs SMX and AMP as per the standard protocol. The results showed the synergistic effect of OX11 with SMX against KP, SH-14, AE-44 (FICI value, 0.282), and SH-52 (FICI value, 0.314). Meanwhile, in combination with AMP, OX11 showed synergy with KP, EH-8, and SH-14 (FICI value range between 0.032 and 0.314) (Table 5). An FICI value of less than 0.5 is considered to be in synergy. The combination studies proved that OX11 has effectively exhibited synergy against a wide range of resistant bacterial strains in combination with AMP and SMX.

Table 5. Antibacterial Activity of OX11 in Combination with SMX and AMP.
    MIC alone (μg/mL)
 MIC in combination (μg/mL)
    
combinationn bacterial strain OX11 SMX AMP OX11 SMX AMP FICIa interaction pattern
OX11 with SMX Acinetobacter sp. (EH-8) 1000 1000   500 32   0.532 indifferent
K. pneumoniae (KP) 1000 1000   250 32   0.282 synergistic
K. georgiana (SH-14) 32 500   8 16   0.282 synergistic
C. werkmanii (SH-52) 32 500   8 32   0.314 synergistic
E. coli (AE-44) 1000 1000   250 32   0.282 synergistic
E. coli (AE-23) 1000 500   1000 32   1.064 indifferent
OX11 with AMP Acinetobacter sp. (EH-8) 1000   32 62.5   4 0.187 synergistic
K. pneumoniae (KP) 1000   1000 31.25   1 0.032 synergistic
K. georgiana (SH-14) 32   500 8   32 0.314 synergistic
C. werkmanii (SH-52) 32   8 16   2 0.750 indifferent
E. coli (AE-44) 1000   500 500   16 0.532 indifferent
E. coli (AE-23) 1000   500 500   32 0.564 indifferent
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a

Fractional inhibitory concentration index.

2.7. HSA Binding Studies

The interaction between HSA and OX11 was studied using UV–vis, steady state fluorescence, synchronous, 3D fluorescence, and CD spectroscopy spectroscopic techniques. These results provide accurate insights regarding the binding of OX11 with HSA.32 The fluorescence spectra of HSA in the absence and presence of OX11 were noted, as shown in Figure 11. The maximum fluorescence emission was observed at 353 nm, which is a characteristic peak of the Trp residue in HSA. The fluorescence intensity was found to be decreasing with the addition of a higher concentration of OX11, and the process is referred as the quenching process (Figures 12 and 13). The quenching constant was calculated using a Stern–Volmer equation (eq 4) at different temperatures, and the values are listed in Table 6. From Table 6, the increasing value of Ksv with increasing temperature suggests the involvement of dynamic quenching. The values of the different thermodynamic parameters, such as enthalpy change (H), entropy change (S), and Gibbs free energy change (G), are also determined using eqs 6 and 7, and the values are listed in Table 6. The positive sign of ΔS and ΔH suggests the involvement of the hydrophobic interaction prevailing between the HSA and OX11 according to the Ross and Subramanian theory.33 Further, the binding constant (Kb) was calculated using a Van’t Hoff plot (Figure 14), and the values are summarized in Table 6. The value of Kb showed an increase with temperature, which suggests that, at higher temperature, the complex formed between HSA and OX11 is strong. Also, the value of ΔG was found to be negative, which showed that its interaction process is spontaneous in nature.34 In addition, the synchronous spectra were also recorded to determine the involvement of the residue in the quenching process. Figures 15 and 16 show the synchronous fluorescence spectra at Δλ = 60 and 15 nm, which clarify the involvement of Trp and Tyr, respectively.35 The synchronous results shown in Figures 15 and 16 suggest that, with the increasing concentration of OX11, the quenching of Trp was much stronger than the quenching of Tyr, which depicts that the quenching of HSA is mainly because of the involvement of the Trp residue. Also, the three-dimensional fluorescence spectra of the HSA and HSA-OX11 complex were recorded, as shown in Figure 17. The spectra revealed three distinct peaks. Peak 1 corresponds to Raleigh scattering, whereas peak 2 corresponds to the presence of tyrosine and tryptophan residue, and peak 3 is the characteristic peak of the polypeptide backbone structure. From Figure 17, it was observed that OX11 quenched the fluorescence intensity of peaks 2 and 3. The relative fluorescence intensity of peaks 2 and 3 of HSA alone dropped from 132.78 to 118.22 and 153.05 to 97.71, respectively, whereas in the presence of OX11, peaks 2 and 3 decreased from 65.07 to 1 and 107.03 to 90, respectively (Table 7). The decrease in stock shift suggests the binding between HSA and OX11, which was also established by our steady state fluorescence results. The decrease in fluorescence intensity of both peaks is evident of a conformational change occurring in the HSA molecule in the presence of OX11.36

Figure 11.

Figure 11

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Fluorescence emission spectra of HSA (5 μM) in the absence and presence of varied concentrations of OX11 at 298 K and pH 7.4.

Figure 12.

Figure 12

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Stern–Volmer plot for the quenching of HSA by OX11 at 298, 303, and 308 K and pH 7.4.

Figure 13.

Figure 13

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Double log plot for the quenching of HSA by OX11 at 298, 303, and 308 K and pH 7.4.

Table 6. Binding Parameters and Thermodynamic Parameters of HSA-OX11 Using Fluorescence Spectroscopy.

temp (K) Ksv (L/mol) Kb (L/mol) n R2 ΔH (kJ/mol) ΔS (kJ mol–1 K–1) ΔG (kJ/mol)
298 162.08 11.55 0.62 0.996 36.86 144.16 –6.09
303 234.98 15.79 0.66 0.996 –6.81
308 309.54 18.70 0.69 0.997 –7.53
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Figure 14.

Figure 14

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Vant’t Hoff plot for the quenching of HSA by OX11 at 298 K and pH 7.4.

Figure 15.

Figure 15

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Synchronous fluorescence spectra of HSA (5 μM) in the absence and presence of varied concentrations of OX11 at Δλ = 60 nm at 298 K and 7.4 pH.

Figure 16.

Figure 16

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Synchronous fluorescence spectra of HSA (5 μM) in the absence and presence of varied concentrations of OX11 at Δλ = 15 nm at 298 K and pH 7.4.

Figure 17.

Figure 17

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Three-dimensional fluorescence spectra of the (a) HSA (5 μM) and (b) HSA-OX11 complex.

Table 7. Parameters of the 3D Fluorescence of the HSA and HSA-OX11 Complex.

  HSA
 HSA-OX11
  peak 2 peak 3 peak 2 peak 3
peak position 280/345.07 230/337.03 280/279.06 230/320
relative intensity 132.78 153.05 118.22 97.71
Δλ (nm) 65.07 107.03 1 90
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In addition, the complexation was further confirmed by UV–vis spectroscopy. The UV–vis spectra of pure HSA are shown in Figure 18, where a strong band at 278 nm provides the information about the buried aromatic amino acids (Trp, Tyr, and Phe). Figure 18 shows the continuous decrease in the maximum absorbance on the addition of increasing concentration of OX11, which signifies the complex formation between HSA and OX11. Further, absorption spectra were used to calculate the value of the binding constant using the double reciprocal plot (as shown in Figure 19) using eq 3. The value of the binding constant was in the same order and was in good agreement with the result obtained from fluorescence spectroscopy.37 Additionally, a significant change in the value of the α-helical content was observed from CD spectra (shown in Figure 20). At lower concentration, the value of the α-helical content increased from 58.8 to 60.3% as compared to the native protein, whereas it decreased from 60.3 to almost 58.3% (toward native protein), which suggests the significant change in the secondary structure of HSA in the presence of OX11. A lower concentration of OX11 stabilized the secondary structure (movement to a more folded structure), whereas unfolding (folded to unfolded state) of HSA at a higher concentration of OX11 was seen.38 The present study could play an important role in pharmacological applications of drug-protein complexation.

Figure 18.

Figure 18

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UV–vis spectra of HSA (5 μM) in the absence and presence of varied concentrations of OX11 at 298 K and pH 7.4.

Figure 19.

Figure 19

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Double reciprocal plot for the UV–vis spectra of HSA (5 μM) in the absence and presence of OX11 at 298 K and pH 7.4.

Figure 20.

Figure 20

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Far UV CD of HSA with OX11 at 298 K and pH 7.4.

3. Discussion

Bacterial infections remain a risk to public health despite the availability of antibiotics due to the rising level of the multidrug resistant bacteria.39,40 Antibiotic resistance is one of the top 10 threats, according to the World Health Organization (WHO).41 The Center for Disease Control and Prevention (CDC) predicted that a “post-antibiotic era” will begin soon.42 Therefore, it is obvious for medical science researchers to find new ways to overcome the scarcity of effective antibacterial agents with better efficacy. In this intensive study, to develop a better antibacterial agent, we first synthesized a diverse series of oxadiazole-sulfonamide-based compounds (OX1–OX27), and through the preliminary screening, two compounds OX7 and OX11 were picked as selective inhibitors of S. pneumoniae, P. aeruginosa, and E. coli bacterial strains. The bactericidal action against the selected bacterial strains revealed through various antibacterial studies conducted on these two test compounds clearly suggests that the OX11 compound can be taken as the lead compound. Drugs that show synergy are always considered better than drugs that do not because by counteracting biological compensation, sparing doses on each chemical, or utilizing context-specific multitarget processes, synergistic combinations of two or more pharmaceuticals can prevent toxicity and other side effects associated with high doses of single treatments.43−45 We examined the antibacterial efficacy of compound OX11 against 28 multidrug resistant strains (MDR) strains. Although our lead compound OX11 is found to be indifferent toward S. pneumoniae-sensitive bacterial strain in combination with CIP, indeed, it shows synergy with CIP when tested against E. coli cells. Further, when tested in combination with AMP, the interaction pattern of OX11 was found synergistic with the tested sensitive bacterial strains. Interestingly, OX11 also showed synergy with AMP and SMX against 3 out of 28 MDR bacterial strains. The findings of the cell viability tests suggested that OX11 could be used as a promising antibacterial agent against specific bacterial strains because it is non-toxic to mammalian cells but inhibits bacterial cells selectively. Moreover, the viability of G. mellonella larvae was not affected in an in vivo toxicity assay using OX11 up to a dose of 125 μg/mL, showing its nontoxic behavior toward the larvae. However, the hemocyte density of the larvae shows some variation, which is indicative of a stress response in larvae. Single cells (planktonic mode) and biofilms are two alternative “lifestyles” that bacteria can adopt. As the estimated 80% of all bacterial infections are biofilm-related,46,47 we examined the effect of OX7 and OX11 on biofilm formation. We observed that both the test compounds emerge as a significant disruptor of biofilm formation, damaging its formation by up to 90%. Unfortunately, the molecular mechanism of biofilm disruption could not be elucidated. Our findings through confocal laser microscopy clearly show cell lysis due to the presence of OX11, confirming its significant antibacterial property, which indicates its potential as a better pharmacophore that may form the basis of further study. Binding of plasma protein plays an important role in drug disposition and its efficacy.48 Human serum albumin (HSA) binds a diverse set of drugs, especially neutral and negatively charged hydrophobic compounds.49 On binding with HSA, OX11 was found to stabilize the secondary structure, forming a spontaneous and stable complex even at higher temperature.

4. Conclusions

In summary, a series of oxadiazole-sulfonamide-based compounds bearing various substitutions were synthesized and screened as an effective antibacterial agent against a group of Gram-positive as well as Gram-negative strains. OX7 and OX11 emerged out as significant antibacterial compounds against sensitive strains like S. pneumoniae, P.aeruginosa, and E. coli strains, and OX11 is moderately effective against MDR K. georgiana and C. werkmanii strains. Compound OX11 exhibited a synergistic effect with AMP against the Acinetobacter isolate in addition to K. pneumoniae and K. georgiana. Compound OX11 with SMX showed a synergistic effect against C. werkmanii, E. coli, K. pneumoniae, and K. georgiana strains. Further research revealed that growth kinetic analyses validated the bacteriocidal effect of the test compounds. The CLSM study indicated that OX11 can be further explored as a better pharmacophore. The TEM study revealed that OX11 exhibited considerable cell wall destruction and membrane rupture in E. coli bacterial cells, resulting in cell death. Furthermore, these compounds were found to be effective anti-biofilm agents in E. coli and to be non-cytotoxic in the HEK293 cell line at concentrations of up to 250 μM. OX11, on the other hand, showed modest change in hemocyte density, indicating a stress response, and was found to be safe to G. mellonella larvae up to a concentration of 125 μg/mL. Our research suggests that the compound OX11 would be further optimized to generate a more effective and safer antibacterial agent.

5. Materials and Methods

5.1. Chemistry

All the oxadiazole-based compounds bearing various substitutions (OX1–OX27) were synthesized and recently reported by our research group.50 The purity of all the compounds was confirmed by ultraperformance liquid chromatography (UPLC) before any biological studies (spectra given in the Supporting Information). The fresh stock solution of all the compounds was made to be 5 mg/mL for each compound and was dissolved in 1 mL of the DMSO (molecular biology grade) solvent.

5.2. Antibacterial Activity

5.2.1. Culture Preparation and Maintenance

Five bacterial strains viz.S. pneumoniae (MTCC 655), E. faecalis (MTCC 439), P. aeruginosa (ATCC 2453), S. typhimurium (MTCC 3224), and E. coli (ATCC 25922) were streaked on nutrient agar plates and kept in an incubator overnight at 37 °C. A pure and single colony of each isolate was picked and inoculated into the nutrient broth and cultured in an incubator shaker overnight (Orbitech).

5.2.2. Minimum Inhibitory Concentration (MIC) Determination

To evaluate the MIC of the test compounds, the standard protocol recommended by the National Committee for Clinical Laboratory Standards (NCCLS) was followed. The test compounds (OX1–OX27) were studied for their antibacterial activity against two Gram-positive strains (S. pneumoniae and E. faecalis) and three Gram-negative strains (S. typhimurium, P. aeruginosa, and E. coli) using the conventional broth dilution method. The MIC values were calculated and compared with CIP and AMP as the reference antibiotics. The stock solution (5 mg/mL) of the standard drug (AMP and CIP) and synthesized compounds (OX1–OX27) was prepared in DMSO (molecular biology grade). To acquire the requisite concentrations of 500, 250, 125, 62.50, 31.25, 15.62, 7.81, 3.90, 1.95, 0.97, 0.48, and 0.24 μg/mL, the progressive serial broth dilution method was used. A positive and a negative control were also taken in the experiment. The cultures were incubated at 37 °C for 24 h and then compared with blank in terms of turbidity caused by the microbial growth.

5.2.3. Disk Diffusion Assay

The disk diffusion assay was performed using aforesaid strains for the selected compounds OX7 and OX11, which may have antimicrobial potential as they showed a low MIC value among tested compounds against the bacterial isolates.51 The bacteria were cultivated overnight at 37 °C after being inoculated in a liquid broth medium. Approximately, 105 cells/mL was taken from that liquid broth medium and placed into Petri plates (Tarsons) after being inoculated into a molten nutritional agar medium. After solidification, sterilized 4 mm diameter disks of Whatman paper were placed on solid agar at an appropriate distance. Different concentrations of compounds, i.e., half MIC, MIC, and double MIC, were placed on the disks. For the positive and negative control, the standard drug and DMSO were applied onto the disks, respectively, while one disk was left blank for comparison. It was then kept in the incubator for 24 h at 37 °C. After 24 h, the diameter of the zone of inhibition (ZOI) was accurately measured in millimeters, and the ZOI values of the positive and negative controls were compared.

5.2.4. Growth Kinetics Assay

The Gram-positive and Gram-negative strains of S. pneumoniae and E. coli cells were revived by sub-culture on the nutrient agar plate, respectively. An inoculum was then transferred into the liquid nutrient broth and before use; the cells were grown for 24 h at 37 °C to get a fresh culture. The growth kinetic assay was performed according to Saigal et al.5 A graph was plotted between the O.D. and time duration (in h), and the effect of OX7 and OX11 on the growth of test organisms was determined.

5.2.5. Time Kill Curve Study

Time kill curves were examined to check the bacteriostatic/bactericidal effect of the compounds OX7 and OX11. S. pneumoniae and E. coli strains were used in this study. A control was also used to monitor the extent of full growth. The fresh culture (∼2 × 106 cells) of test organisms was inoculated in freshly prepared media. These cells were then treated to the test chemicals at MIC and 4MIC doses. For the time kill curve study, the protocol given by Saigal et al. was followed.5

5.2.6. Synergistic Antibacterial Activity

The synergistic activity of the test compounds OX7 and OX11 with standard drugs CIP and MP was determined using the microdilution checkerboard method.52 CIP and AMP were serially diluted in columns from 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.062 and 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, and 0.078 μg/mL, respectively, while test compounds were diluted in rows from 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg/mL in a 96-microwell plate to obtain large numbers of combinations. The plates were inoculated with 100 μL of the freshly prepared culture of S. pneumoniae or E. coli cells. After incubation, the combinatorial MIC value was evaluated at which no visible growth appeared. Using the equation (eq 1) given below, the synergy of compounds in terms of FICI (fractional inhibitory concentration index) was calculated

5.2.6. 1

5.3. Assessment of Biofilm Disruption

5.3.1. XTT Assay

To observe the effect of compounds OX7 and OX11 on the metabolic activity of biofilm formation in the tested bacterial strains, the XTT (2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide) assay was conducted on E. coli and S. pneumoniae strains, with slight modifications as the previously reported method.5 Briefly, 100 μL of sterile nutrient broth was poured in the 96-well microtiter plate, inoculated with desired bacterial strains, and incubated at 37 °C for 24 h to allow biofilm formation. After incubation, the medium was discarded gently followed by gentle washing with PBS. Then, the fresh medium containing various concentrations of test compounds (64–0.5 μg/mL) was poured gently into each well and further incubated for 24 h at 37 °C. After incubation, the medium was evacuated and the non-adherent cells were cleaned using PBS. In each well, 50 μL of XTT salt solution (HiMedia) and the plates were incubated in the dark for 90 min at 37 °C. Bacterial dehydrogenase converts XTT tetrazolium salt to XTT formazan, which causes a colorimetric change (turns orange) and has been related to cell survival. These colorimetric variations were determined by taking their optical densities at 490 nm on a spectrophotometer.53 The % inhibition data was further interpreted from dose–response curves.

5.3.2. Visualization of Biofilm Disruption by Scanning Electron Microscopic (SEM) Analysis

The ability of the selected compounds OX7 and OX11 to disrupt biofilm formation was visualized by SEM analysis using E. coli cells by using a previouly described protocol.5 Test compounds were added in the plate and again incubated for the next 24 h at 37 °C. Samples were again washed with PBS and dried to examine under scanning electron microscopy.54

5.4. In Vitro (Hemolytic and MTT Assay) and In Vivo (G. mellonella Larvae) Toxicity Studies of Test Compounds

5.4.1. Hemolytic Assay

The hemolytic activity of the compounds OX7 and OX11 was studied using human red blood cells (hRBCs).55 The procedures used to perform the hemolytic study were similar to those reported earlier.56

% hemolysis was calculated from the following equation (eq 2).

5.4.1. 2

5.4.2. MTT Assay

To confirm the cytotoxic effect of selected compounds, the MTT assay was also performed following the encouraging results of the hemolytic assay. A typical MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used.57 Cell line human embryonic kidney (HEK293) cells are widely used in cell biological research because of their consistent growth and inclination for transfection.58−60 HEK293 cells were maintained and cultured in Dulbecco’s modified Eagle’s medium (DMEM) media enriched with 10% heat inactivated fetal bovine serum (FBS) and 1% penicillin and streptomycin solution in a humidified incubator (5% CO2) in T-25 flasks at 37 °C. Approximately 2 × 104 cells (150 μL) were seeded per well in a 96-well plate and incubated for 24 h before treatment. After a day, cells were treated with increasing concentrations (0–250 μM) of each test compound in 200 μL as the final volume at 37 °C for 48 h in a CO2 incubator. Further assesment of the cytotoxicity was dertermine as mentioned by Uddin et al.61 The control cells were treated with cell culture media only, and to nullify the effect of respective DMSO concentration, the cells were correspondingly treated with DMSO and subtracted from the respective compound-treated well readings. The percent of viable cells was determined and shown as a function of compound concentration.62

5.4.3. In Vivo Toxicity Evaluation of Lead Compound OX11 on G. mellonella Larvae

In vivo activity of OX11 was assessed in G. mellonella larvae, as previously described.63 Larvae were inoculated with 20 μL of OX11 solutions (7, 31, 62, or 125 μg/mL) and incubated in the dark for 24 h at 30 °C. The effect of the compound on hemocyte density was assessed as described.63

5.5. TEM (Transmission Electron Microscopy) Analysis

The morphology of E. coli cells, both treated (with OX11) and untreated, was examined by TEM analysis according to the standard protocol.64 First of all, the mid-log phase E. coli cells were harvested and then treated to MIC concentation of OX11. Afterward, washing of cells was done three times with PBS solution and fixed in 2.5% glutaraldehyde in phosphate/magnesium buffer overnight. By using 0.1 M sodium phosphate buffer (pH 6.0), the cells were washed twice for 15 min and cells were fixed in 2% osmium tetroxide for 2 h. After 2 h, cells were again washed twice in distilled water for 15 min and then en bloc stained for 30 min with 1% aqueous uranyl acetate. Cells were dehydrated in 95 and 100% ethanol after two more washing. The cells were then treated to propylene oxide for 2–10 min before being infiltrated for 1 h in a 1:1 propylene/epoxy embedding medium (Epon) combination and then overnight in fresh Epon. The ultrathin sections were cut after polymerization for 48 h at 60 °C using a microtome (Leica EM UC6) and transferred to a copper grid. After staining with a saturated solution of uranyl acetate in 50% alcohol, the samples were stained with lead citrate. These samples were washed three times in Milli-Q (MQ) water and dried by touching gently with Whatman filter paper. These thin sections were then examined with a FEI Tecnai G2 at 200 KV.

5.6. Confocal Laser Scanning Microscopic (CLSM) Analysis

The lead compound OX11 was analyzed through CLSM to determine its effect on E. coli cells. The bacterial cells were stained using DAPI (4′,6-diamidino-2-phenylindole), which is a fluorescent dye that binds tightly to the DNA’s A-T rich region and emits blue fluorescence at 461 nm as emission spectra, upon excitation at 358 nm. The log-phase cells of E. coli were harvested by centrifugation for 15 min and washed twice with PBS buffer solution and futher examined as mentioned previously.5

5.7. Effect of the Lead Compound (OX11) on Resistant Bacterial Strains

Based on the above pharmacological investigations, OX11 was found to be the promising antibacterial agent among all the tested compounds. The MIC of OX11 was determined against 19 different environmental isolates of E. coli, two strains of K. pneumoniae, and one strain each of Acinetobacter sp., K. georgiana, and C. werkmanii.(30,31) These environmental isolates were taken from the River Yamuna Delhi stretch (India) and drain effluent water of the Ghazipur Slaughter House (India). Some of these environmental isolates are multidrug resistant (MDR) phenotypes. Details of their resistance pattern against different antibiotics are given in Table 8. The studies on the synergistic effect (with AMP and sulfamethoxazole) were performed using a previously mentioned protocol.51 Because of the structural similarities with OX11, we included the sulfamethoxazole (SMX) antibiotic in our study on resistant strains for comparison.

Table 8. Phenotype and Resistance Pattern against Different Antibiotics of Environmental Isolates30,31a.

bacterial isolate code strain resistant phenotype
AE-9 E. coli AMP, AT, CTN, CXM, CZ, ETP, CIP, RIF MDR
AE-27 E. coli AMP, A/S, AT, CTN, CXM, CZ, ETP, IPM CIP, LE, OF, RIF MDR
AE-44 E. coli AMP, A/S, AT, CTN, CXM, CZ, ETP, CIP, LE, OF, RIF MDR
AE-32 E. coli AMP, A/S, AT, CTN, CXM, CZ, ETP, CIP, LE, OF, PB,TOB, TR MDR
AE-17 E. coli AMP, AT, CTN, CXM, CZ, ETP, CIP, RIF MDR
AE-23 E. coli AMP, CTN, CX CXM, CZ, ETP, RIF MDR
AE-42 E. coli AMP, A/S, AT, CTN, CXM, CZ, ETP, CIP, LE, RIF,TOB, TR MDR
AE-21 E. coli AMP, A/S, CXM non-MDR
AE-31 E. coli AMP, A/S, AT, CTN, CXM, CZ, ETP, IPM, CX, CIP, LE, OF, RIF, TE, AK, TOB MDR
EC-36 E. coli AMP, CTN non-MDR
EC-2 E. coli AMP, CTN, CXM, CZ, RIF MDR
EC-3 E. coli AMP, CTN, CXM, CZ, RIF MDR
EC-6 E. coli AMP, A/S, CTN, CXM, CZ, LE, CIP, OF, RIF, TE, TR MDR
EC-25 E. coli AMP, CTX non-MDR
OB-18 E. coli AMP, AT, CZ, ETP, RIF, TR MDR
OB-6 E. coli AMP, AT, CZ, ETP, RIF, TR MDR
AE-2 E. coli AMP, A/S, AT, CTN, CXM, CZ, ETP, CIP MDR
OE-11 E. coli CTX, AMP non-MDR
EE-2 E. coli CIP, LE, OF, RIF, AMP, CTN, CXM, CZ MDR
EH-8 Acinetobacter sp. AT, CTN, CX, CXM, CZ, ETP, CIP, PB, AK MDR
KP K. pneumoniae AT, CTN, CXM, CZ, ETP, IPM, RIF, TE MDR
EA-13 K. pneumoniae CIP, OF, RIF, TR, AMP MDR
SH-14 K. georgiana CAZ, CTX, CTR non-MDR
SH-52 C. werkmanii CL, PB, TR, RIF, AT, ETP, CZ, CX, CAZ, CTX MDR
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a

AMP: ampicillin, A/S: ampicillin/sulbactam, AT: aztreonam, CTN: cefotetan, CX: cefoxitin, CTX: cefotaxime, CXM: cefuroxime, CZ: cefazolin, ETP: ertapenem, IPM: imipenem, CIP: ciprofloxacin, LE: levofloxacin, OF: ofloxacin, PB: plymixin B, RIF: rifampicin, TE: tetracyclin, AK: amikacin, TOB: tobramycin, TR: trimethoprim.

5.8. Human Serum Albumin (HSA) Binding Study

UV–vis, fluorescence, synchronous, and CD spectroscopic techniques were employed to study the binding between HSA and OX11. The UV–vis spectra of HSA (5 μM) in the presence and absence of various concentrations of OX11 (0.1–6.8 μM) were recorded using an Analytik Jena Specord-250 spectrophotometer (USA) using a 1.0 cm cell at 298 K. The binding constant (Ka) was also analyzed using the double reciprocal plot between 1/A0 – Avs 1/[OX11] by eq 3.36

5.8. 3

Further, to confirm the quenching mechanism and the binding forces involved in complexation, fluorescence spectroscopy was employed. The fluorescence spectra of HSA (5 μM) in the presence and absence of different concentrations of OX11 (0.1–6.8 μM) were recorded on a Cary Eclipse spectrofluorometer (Varian, USA) equipped with a 150 W xenon lamp at 298, 303, and 308 K using a quartz cuvette (1.0 cm) at 280 nm as the excitation wavelength. Temperature was maintained in the assays using a constant temperature cell holder coupled to a constant temperature water circulator (Varian, USA). In addition, synchronous spectra were obtained using the same spectrofluorometer. The difference between the excitation and emission (Δλ = λem – λex) wavelength was kept constant. The Δλ at 15 nm or 60 nm showed by synchronous fluorescence spectra gave characteristic information of tyrosine (Tyr) residues or tryptophan (Trp) residues with the excitation and emission slit widths at 5 nm. The quenching mechanism was determined according to the Stern–Volmer equation (eqs 4 and 5).65

5.8. 4
5.8. 5

where F0 and F represent the protein’s relative fluorescence intensity in the presence and absence of the quencher OX11. The Stern–Volmer quenching constant is Ksv, and the molar concentration of OX11 is [Q]. The Ksv values were calculated using the slope of the HSA-OX11 system’s Stern–Volmer plot. Also, the binding parameters such as the binding constant (Kb) and the number of binding sites (n) are calculated using eq 3 and thermodynamic parameters like enthalpy change (ΔH), entropy change (ΔS), and Gibbs energy change (ΔG) were also determined by carefully analyzing the fluorescence data according to the thermodynamic equations (eqs 6 and 7).66

5.8. 6
5.8. 7

Also, three-dimensional fluorescence was noted on the same instrument. The excitation wavelength was set at 200 nm, and emission was recorded between 200 and 400 with an interval of 10 nm. The CD spectra were recorded on a JASCO J-815 spectropolarimeter at 298 K with a cell holder (thermostatically controlled) attached to a Neslab RTE-110 water bath with an accuracy of ±0.1 K. The CD instrument was calibrated with D6 10-camphorsulfonic acid. Each spectrum was recorded using a quartz cuvette of 0.1 cm path length at 298 K. The spectra were recorded in the far UV range from 200 to 250 nm at a scan rate of 50 nm/min, and each spectrum is the average of three scans with a full-scale sensitivity of 10mdeg. The concentration of HSA used was 5 μM. The α-helical contents of free and combined protein were calculated from mean residue ellipticity (MRE) values at 208 nm by using eqs 8 and 9.67

5.8. 8
5.8. 9

Acknowledgments

M.A. gratefully acknowledges the financial support in the form of the Core Research Grant from the Science & Engineering Research Board (SERB), Government of India (file no. CRG/2018/003967). A.U. is a recipient of the Senior Research Fellowship from ICMR (Fellowship/48/2019-ECD-II). Authors also acknowledge SAIF, AIIMS, New Delhi, for providing scanning and transmission electron microscopic facilities.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03379.

  • Supplementary data includes spectral data of the test compounds OX7 and OX11 and images of agar plates showing the zone of inhibition (PDF)

Author Contributions

○ A.A. and P.H. are equal contributors. M.A. designed the project and optimized the experiments. P.H. synthesized the compounds. A.A., M.I., A.U., and A.K. screened and performed all the biological experiments. R.M. and K.K. performed in vivo toxicological studies using G. mellonella. J.S. and R.P. performed HSA binding studies. M.A., A.A., M.I., A.U., and A.K. interpreted the data and wrote the manuscript. M.A., A.A., M.M., and M.C.J. edited the final manuscript. M.A. provided funding acquisition and aided administrative processing. All the authors approved and reviewed the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ao1c03379_si_001.pdf (1MB, pdf)

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