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Binding mode of dihydroquinazolinones with lysozyme and its antifungal activity against Aspergillus species
Binding mode of dihydroquinazolinones with lysozyme and its antifungal activity against Aspergillus species K. Hemalatha, G. Madhumitha, Lokesh Ravi, V. Gopiesh Khanna, Naif Abdullah Al-Dhabi, Mariadhas Valan Arasu PII: DOI: Reference:
S1011-1344(16)30183-X doi: 10.1016/j.jphotobiol.2016.05.005 JPB 10373
To appear in: Received date: Accepted date:
18 March 2016 3 May 2016
Please cite this article as: K. Hemalatha, G. Madhumitha, Lokesh Ravi, V. Gopiesh Khanna, Naif Abdullah Al-Dhabi, Mariadhas Valan Arasu, Binding mode of dihydroquinazolinones with lysozyme and its antifungal activity against Aspergillus species, (2016), doi: 10.1016/j.jphotobiol.2016.05.005
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ACCEPTED MANUSCRIPT
Binding mode of dihydroquinazolinones with lysozyme and its antifungal activity against Aspergillus species
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K. Hemalathaa, G. Madhumithaa*, Lokesh Ravib, V. Gopiesh Khannab, Naif Abdullah AlDhabic, Mariadhas Valan Arasuc a Chemistry of Heterocycles & Natural Product Research Laboratory, Department of Chemistry, School of Advanced Sciences, VIT University, Vellore 632 014, Tamil Nadu, India. b Division of Bio-medical Sciences, School of Biosciences and Technology, VIT University, Vellore 632 014, Tamil Nadu, India. c Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King Saud University, P. O. Box 2455, Riyadh, 11451, Kingdom of Saudi Arabia.
Aspergillosis is one of the infectious fungal diseases affecting mainly the
immunocompromised patients.
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Abstract:
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Corresponding author. Tel.: 04162202336; fax: 04162245544/5766. E-mail address: [email protected]; [email protected]
The scarcity of the antifungal targets has identified the
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importance of N-myristoyl transferase (NMT) in the regulation of fungal pathway.
The
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dihydroquinazolinone molecules were designed on the basis of fragments responsible for binding with the target enzyme. The aryl halide, 1(a-g), aryl boronic acid and potassium carbonate were heated together in water and dioxane mixture to yield new C-C bond formation in dihydroquinazolinone. The bis(triphenylphosphine)palladium(II) dichloride was used as catalyst for the C-C bond formation. The synthesized series were screened for their in vitro antifungal activity against Aspergillus niger and Aspergillus fumigatus. The binding interactions of the active compound with lysozyme were explored using multiple spectroscopic studies. Molecular docking study of dihydroquinazolinones with the enzyme revealed the information regarding various binding forces involved in the interaction. Keywords: Aspergillosis, N-myristoyl transferase, dihydroquinazolinones, antifungal activity, lysozyme, spectroscopy. 1. Introduction: Aspergillosis is an infection/allergic response caused by Aspergillus species. It is a type of fungus whose spores are omnipresent in air, water and soil. It does not cause illness in the 1
ACCEPTED MANUSCRIPT normal patient whereas it causes life threatening infections in the immunocompromised patient [1]. The patients with hematological malignancies [2], advanced HIV infection [3] and those who have undergone solid organ transplantation are more susceptible to aspergillus infection [4].
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The principle organ of infection is lungs and it produces syndromes such as allergic bronchopulmonary aspergillosis (ABPA), aspergilloma and invasive aspergillosis (IA) [5]. IA
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was found to be the main reason for the infectious pneumonic mortality in an
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immunocompromised patient. Aspergillus genus possesses more than 180 different species. Aspergillus fumigatus was recovered from the clinical isolates of IA patients and it accounts for
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the majority of pathological conditions. Aspergillus niger is a less prevalent disease causing organism but inhalation of a lot of spores can cause serious lung disorder. It can also cause
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fungal infections in the ear which leads to temporary pain and hearing loss. If left untreated, it leads to damage in the inner parts of the ear [6]. The treatment for aspergillosis includes various azoles, polyene antibiotics, and echinocandins [7]. Azoles are advantageous over the other drug
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categories because of its availability as oral formulations. But azoles are displaying resistance
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towards the Aspergillus species. The urge for the development of novel antifungal agent and the pathway through which it acts is always in progress [8].
Heterocycles and nanoparticles
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scaffolds possessing antimicrobial activity targeting various pathways has been reviewed [9,10]. The N-myristoyl transferase (NMT) was identified as a novel mode of action for the antifungal therapy and it is an enzyme catalyzing the N-myristoylation reaction. It is a reaction in which Nterminal part of glycine in the cellular proteins were transferred with myristoyl-CoA group. These proteins are vital for the growth and function of the fungi [11] (Fig. 1). The role of NMT as the potential target in the aspergillosis treatment has been reported [12]. From the literature, it was found that the NMT inhibitors with linkers containing ether oxygen atom and sulfonamide group were essential for the formation of hydrogen bonding between the amino acid residues [13-15] (Fig. 2). Therefore, the quinazolinones containing these moieties (ether oxygen and sulfonamide group) were designed and synthesized. The antifungal activity of these compounds was compared with that of the quinazolinone containing other functional moieties. Quinazolinones [16-18] were proven with various pharmacological actions [19] and its importance as an antifungal agent was also reported [20, 21].
Heterocycles are bioactive
compounds [22, 23] and various reaction conditions [24, 25] are utilized in the formation of the nucleus. Even though they possess promising biological activities it fails because of the poor 2
ACCEPTED MANUSCRIPT physicochemical properties. The ADME (absorption, distribution, metabolism, and excretion) properties of the bioactive compounds are strongly influenced by its binding properties with the protein. Lysozyme is an important enzyme which is present in almost all the body fluids [26]. It
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is an antimicrobial peptide and it is an essential component of the innate immune system. It causes bacterial cell lysis by hydrolyzing the β-linkage of the bacterial peptidoglycan layer. The
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antifungal activity of lysozyme has also been reported [27]. It belongs to the category of low
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molecular weight proteins (LMWPs) with a molecular weight of 14 kDa. It is used as a carrier for many drugs resulted in target oriented treatment [28, 29].
The investigation on the
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interaction of lysozyme with various drugs has already been reported [30-32]. In this work, the antifungal activities of the synthesized series were tested against the Aspergillus species.
various spectroscopic methodologies.
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2.1. Chemicals and instruments
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2. Materials and methods
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Further, the interaction of dihydroquinazolinones with lysozyme has been studied by using
The reagents used in the synthesis were of analytical grade and are used without any
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further processing techniques.
The synthesized compounds were purified by column
chromatographic separations using silica gel of mesh size 60-120. NMR spectra were recorded on Bruker Avance 400 MHz spectrometer using TMS as an internal standard. The melting points were measured in Elchem Microprocessor based DT apparatus and was corrected using standard benzoic acid. The exact molecular weight was predicted using the ESI-MS Thermo Fleet instrument. Chicken egg white lysozyme from sigma Aldrich was stored at 4 ºC and was used without further purification. Sodium phosphate buffer of pH 7.4 was prepared using double distilled water. The absorption spectra were recorded on a Hitachi U-2800 double beam UV/Vis spectrophotometer. The instrument was equipped with 1 cm quartz cell and slit width was set at 1.5 nm.
Fluorescence spectra were recorded from Hitachi F-7000 fluorescence
spectrofluorometer.
Circular dichroism (CD) spectrum was recorded on JASCO J-715
chiroptical spectrometer (JASCO corp.,) and the bandwidth was set at 1 nm at a scanning speed of 500 nm/min. 2.2. Synthesis 3
ACCEPTED MANUSCRIPT The reaction mixture containing aryl halide, 1(a-g) (1 Equiv), aryl boronic acid (1.1 Equiv) and potassium carbonate (1.5 Equiv) was taken in a sealed tube containing 1:3 ratio of water:1,4-dioxane. The nitrogen gas was purged into the sealed tube for 15 minutes. The
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catalyst bis(triphenylphosphine)palladium(II) dichloride of 0.1 equivalent was added to the reaction mixture and stirred at 80 ⁰C for 1-2 h. The completion of the reaction was confirmed by
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thin layer chromatography. The reaction mixture was filtered through the celite and the collected
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filtrate was extracted with chloroform. The chloroform layer was washed several times with water.
The residual moisture was removed by drying over anhydrous sodium sulfate and
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concentrated under reduced pressure. The obtained crude product was purified by column chromatography using petroleum ether and ethyl acetate as a solvent system (Scheme 1).
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2.2.1. Tert-butyl 4- (4- (2, 2, 3-trimethyl -4- oxo-1, 2, 3, 4- tetrahydroquinazolin-6- l) phenyl) piperazine-1-carboxylate (2a)
Off-white solid; yield: 92%; m.p.: 238-240 C. FT-IR (KBr disc) (νmax/cm-1): 1691
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(C=O), 2974 (methylene C-H stretch), 3304 (NH). 1H NMR (400 MHz, CDCl3): δ (ppm), 1.48
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(s, 9H), 1.54 (s, 6H), 3.07 (s, 3H), 3.16 (d, J=4.4 Hz, 4H), 3.58 (t, J=4.8 Hz, 4H), 4.22 (bs, 1H), 13
C
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6.65 (d, J=8 Hz, 1H), 6.95 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.4 Hz, 3H), 8.13 (d, J=2 Hz, 1H).
NMR (100 MHz, CDCl3): 2 X 21.5, 22.1, 3 X 23.2, 2 X 44.1, 2 X 66.0, 74.6, 109.7, 110.7, 2 X 111.5, 121.0, 3 X 121.8, 126.1, 126.4, 127.0, 138.2, 144.7, 149.5, 158.2. HRMS m/z calculated C26H34N4O3 (M+) 450.2631; found 450.2360. 2.2.2. 6- (4- (benzyloxy) phenyl) - 2, 2, 3- trimethyl -2, 3 -dihydroquinazolin- 4(1H)- one (2b) Off-white solid; yield: 88%; m.p.: 230-232 C. FT-IR (KBr disc) (νmax/cm-1): 1624 1
(C=O), 2970 (methylene C-H stretch), 3296 (NH).
H NMR (400 MHz, DMSO-d6): δ (ppm),
1.46 (s, 6H), 2.95 (s, 3H), 5.13 (s, 2H), 6.75 (d, J= 8 Hz, 1H), 6.83 (bs, 1H), 7.06 (d, J= 8.8 Hz, 2H), 7.34-7.55 (m, 8H), 7.84 (d, J= 2 Hz, 1H).
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C NMR (100 MHz, DMSO- d6): 2 X 25.8,
26.6, 69.2, 70.8, 114.3, 114.8, 2 X 115.2, 124.7, 2 X 126.6, 2 X 127.6, 2 X 127.7, 128.4, 128.6, 131.0, 132.6, 137.1, 145.0, 157.1, 162.3. HRMS m/z calculated C24H24N2O2 (M+) 372.1838; found 372.1835. 2.2.3. 6- (4- (benzyloxy) phenyl) -3- ethyl -1H- spiro [cyclopentane -1, 2- quinazolin] -4(3H)one (2c)
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ACCEPTED MANUSCRIPT Off-white solid; yield: 85%; m.p.: 196-198 C. FT-IR (KBr disc) (νmax/cm-1): 1620 (C=O), 2954 (methylene C-H stretch), 3452 (NH).
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H NMR (400 MHz, DMSO-d6): δ (ppm),
1.13 (t, J= 6.8 Hz, 3H), 1.66-1.94 (m, 8H), 3.38-3.43 (m, 2H), 5.14 (s, 2H), 6.82 (d, J= 8.3 Hz,
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1H), 6.89 (bs, 1H), 7.06 (d, J= 8.6 Hz, 2H), 7.34-7.54 (m, 8H), 7.82 (d, J= 1.8 Hz, 1H).
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NMR (100 MHz, DMSO- d6): 15.4, 2 X 22.5, 36.1, 2 X 36.5, 69.2, 81.1, 115.1, 2 X 115.2,
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115.4, 124.7, 2 X 126.7, 2 X 127.6, 127.7, 2 X 128.4, 128.8, 130.8, 132.7, 137.1, 144.9, 157.2,
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162.6. HRMS m/z calculated C27H28N2O2 (M+) 412.2151; found 412.2150. 2.2.4. 6- (4- (benzyloxy) phenyl) -3- methyl -1H- spiro [cyclopentane-1, 2-quinazolin] -4(3H)-
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one (2d)
Off-white solid; yield: 84%; m.p.: 220-222 C. FT-IR (KBr disc) (νmax/cm-1): 1616 1
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(C=O), 2931 (methylene C-H stretch), 3471 (NH).
H NMR (400 MHz, DMSO- d6): δ (ppm),
1.63-2.08 (m, 8H), 2.95 (s, 3H), 5.13 (s, 2H), 6.82 (d, J= 8.3 Hz, 1H), 6.91 (bs, 1H), 7.06 (d, J= 13
C NMR (100
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8.6 Hz, 2H), 7.34-7.42 (m, 3H), 7.46-7.55 (m, 5H), 7.83 (d, J= 1.8 Hz, 1H).
MHz, DMSO- d6): 2 X 23.0, 27.3, 2 X 35.8, 69.2, 81.1, 115.1, 2 X 115.2, 2 X 124.7, 2 X 126.7,
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2 X 127.6, 2 X127.7, 2 X 128.4, 128.8, 130.9, 132.6, 137.1, 157.2, 163.0. HRMS m/z calculated
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C26H26N2O2 (M+) 398.1994; found 398.1992. 2.2.5. 6- (4- (benzyloxy) phenyl) -3- ethyl -2, 2-dimethyl -2, 3- dihydroquinazolin -4(1H)- one (2e)
Off-white solid; yield: 88%; m.p.: 258-260 C. FT-IR (KBr disc) (νmax/cm-1): 1614 (C=O), 2970 (methylene C-H stretch), 3294 (NH).
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H NMR (400 MHz, DMSO- d6): δ (ppm),
1.13 (t, J= 6.9 Hz, 3H), 1.49 (s, 6H), 3.44-3.48 (m, 2H), 5.14 (s, 2H), 6.73 (d, J= 8.4 Hz, 1H), 6.80 (bs, 1H), 7.06 (d, J= 9.2 Hz, 2H), 7.34-7.54 (m, 8H), 7.83 (d, J= 2 Hz, 1H).
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C NMR (100
MHz, DMSO- d6): 15.4, 2 X 26.8, 35.8, 69.2, 71.2, 114.5, 114.7, 2 X 115.2, 124.6, 2 X 126.6, 2 X 127.6, 127.7, 2 X 128.4, 128.6, 131.0, 132.7, 137.1, 144.9, 157.1, 162.0.
HRMS m/z
calculated C25H26N2O2 (M+) 386.1994; found 386.1992. 2.2.6. N- (3- (2, 2, 3- trimethyl -4- oxo- 1, 2, 3, 4- tetrahydroquinazolin -6 -l) phenyl) methane sulfonamide (2f) Off-white solid; yield: 90%; m.p.: 220-222 C. FT-IR (KBr disc) (νmax/cm-1): 1629 (C=O), 3070 (methylene C-H stretch), 3365 (NH). 5
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H NMR (400 MHz, DMSO- d6): δ (ppm),
ACCEPTED MANUSCRIPT 1.47 (s, 6H), 2.95 (s, 3H), 3.02 (s, 3H), 6.78 (d, J=8.4 Hz, 1H), 6.95 (bs, 1H), 7.14-7.16 (m, 1H), 7.31-7.42 (m, 3H), 7.54-7.57 (m, 1H), 7.87 (d, J=2.2 Hz, 1H), 9.76 (s, 1H).
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C NMR (100
MHz, DMSO- d6): 2 X 25.9, 26.6, 39.1, 70.9, 114.2, 114.8, 116.8, 117.6, 121.1, 125.3, 128.1,
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129.8, 131.3, 138.9, 141, 145.8, 162.2. HRMS m/z calculated C18H21N3O3S (M+) 359.1304;
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found 359.1300.
methane sulfonamide (2g)
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2.2.7. N- (3- (3- ethyl- 2, 2-dimethyl -4- oxo-1, 2, 3, 4- tetrahydroquinazolin -6- yl) phenyl) Off-white solid; yield: 87%; m.p.: 168-170 C. FT-IR (KBr disc) (νmax/cm-1): 1629 1
H NMR (400 MHz, DMSO- d6): δ (ppm),
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(C=O), 2972 (methylene C-H stretch), 3371 (NH).
1.14 (t, J=6.9 Hz, 3H), 1.50 (s, 6H), 3.02 (s, 3H), 3.44-3.49 (m, 2H), 6.77 (d, J=8.4 Hz, 1H),
9.76 (s, 1H).
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6.93 (bs, 1H), 7.13-7.16 (m, 1H), 7.31-7.42 (m, 3H), 7.54-7.56 (m, 1H), 7.87 (d, J=2.2 Hz, 1H), C NMR (100 MHz, DMSO- d6): 15.3, 2 X 26.9, 35.8, 39.1, 71.3, 114.4, 114.7,
116.8, 117.6, 121.7, 125.2, 128.1, 129.8, 131.3, 138.9, 141, 145.7, 161.8. HRMS m/z calculated
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C19H23N3O3S (M+) 373.1460; found 373.1458. 2.2.8. 3- methyl -6- (4- nitrophenyl) -1H- spiro [cyclopentane- 1, 2- quinazolin] -4(3H)- one (2h)
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Off-white solid; yield: 89%; m.p: 160-162 C. FT-IR (KBr disc) (νmax/cm-1): 1625 (C=O), 2953 (methylene C-H stretch), 3296 (NH).
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H NMR (400 MHz, DMSO- d6): δ (ppm),
1.67-2.10 (m, 8H), 2.96 (s, 3H), 6.90 (d, J= 8.8 Hz, 1H), 7.30 (bs, 1H), 7.74-7.77 (m, 1H), 7.88 (t, J= 7.2 Hz, 2H), 8.05 (d, J= 2.4 Hz, 1H), 8.26 (t, J= 2 Hz, 2H).
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C NMR (100 MHz,
DMSO- d6): 2 X 23.0, 27.4, 2 X 36.0, 81.1, 114.9, 115.3, 2 X 124.1, 126.0, 126.2, 2 X 126.7, 131.7, 145.5, 146.3, 146.8, 162.5. HRMS m/z calculated C19H19N3O3 (M+) 337.1426; found 337.1422. 2.2.9. 3- ethyl- 2, 2- dimethyl -6- (4- nitrophenyl) -2, 3- dihydroquinazolin -4(1H)- one (2i) Off-white solid; yield: 85%; m.p.: 254-256 C. FT-IR (KBr disc) (νmax/cm-1): 1616 (C=O), 2926 (methylene C-H stretch), 3290 (NH).
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H NMR (400 MHz, DMSO- d6): δ (ppm),
1.14 (t, J=6.9 Hz, 3H), 1.52 (s, 6H), 3.45-3.50 (m, 2H), 6.81 (d, J=8.4 Hz, 1H), 7.19 (bs, 1H), 7.74-7.76 (m, 1H), 7.87-7.89 (m, 2H), 8.04 (d, J=2.2 Hz, 1H), 8.24 -8.26 (m, 2H).
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C NMR
(100 MHz, DMSO- d6): 15.3, 2 X 27, 35.9, 71.3, 114.3, 114.9, 2 X 124.1, 3 X 125.8, 126.1,
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2.3. Anti-fungal activity [33] 2.3.1. Determination of zone of inhibition (ZOI)
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Clinical isolates of Aspergillus niger and Aspergillus fumigatus were grown in Sabouraud dextrose broth (SDB) till adequate turbidity is obtained. The broth was then used as inoculum
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for lawn culture in Kirby Bauer’s well diffusion method to check for anti-fungal activity. Sabouraud dextrose agar (SDA) was used for the well diffusion method. The individual test
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compounds were dissolved in DMSO and diluted to obtain a final volume of 200 µl. The final concentration of 10 μg/ml for all the test compounds was used for this study. The 8 mm wells
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were punched on the SDB and total of 200 μl of the test compound solution was added to each well. Fluconazole and nystatin were used as standard antibiotics. The zone of inhibition was
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observed after 48-72 h.
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2.3.2. Determination of minimum inhibitory concentration (MIC) Minimum inhibitory concentration was performed on a 96-well plate equally dispensed
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with fresh SDB. The test compounds were serially diluted in the individual column to obtain a range of concentrations. The uniform volume of freshly prepared fungal broth was added to all the wells and incubated at 28 ˚C for 24 hours and was recorded from 96-well plate reader. The culture becomes turbid because of the growth of the fungi in the tube containing low inhibitory concentration. However, the culture remains clear in the tube containing above the level of inhibitory concentration. The standard drugs used for the comparative study was Nystatin and fluconazole. 2.4. Lysozyme interaction studies The wavelength range for UV-Vis absorption spectra were scanned from 190-310 nm. Fluorescence emission spectra were obtained by titrating lysozyme (5 μM) with increasing concentrations (0-10 μM) of the ligand at 298 K. The excitation wavelength was set at 280 nm. The slit widths of excitation and emission wavelength were set at 5 nm. The scan speed was set at 2400 nm/min.
The three-dimensional fluorescence spectra were performed using the
excitation wavelength scan range from 200-400 nm and emission wavelength scan range from 7
ACCEPTED MANUSCRIPT 200-800 nm. The excitation and emission slit width was fixed at 5 nm. The sampling interval was fixed at 5 nm and 10 nm for excitation and emission wavelength respectively. The CD spectra of lysozyme in the presence (2 μM) and absence of ligand (2 μM) were recorded
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from 190-260 nm.
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2.5. Molecular docking
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The interaction between the synthesized dihydroquinazolinones 2(a-i) and Aspergillus fumigatus NMT was found out by molecular modeling using Auto Dock 4.0. The synthesized The crystal
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molecules were constructed and energy minimized using Chem3D Pro 12.0.
structure of A. fumigatus NMT (PDB code: 4CAW) and hen egg-white lysozyme (PDB code:
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6LYZ) [34] were obtained from Brookhaven protein data bank. The preparation of the protein was performed by adding essential hydrogen atoms and Kollman charges. Similarly, ligands were prepared by incorporating non-polar hydrogen atoms and Gasteiger charges. A grid box of
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size 90 Å x 90 Å x 90 Å (NMT) and 80 Å x 80 Å x 80 Å (lysozyme) were created using Auto
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Grid. The Lamarckian Genetic Algorithm was employed in Auto Dock tool. The detailed information on the docked conformation of the best fit was retrieved from the Accelrys discovery
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studio and LigPlot software. 3. Results and discussion 3.1. Antifungal activity
The antifungal activity of the newly synthesized compounds against Aspergillus species was evaluated by the well diffusion method. Among the compound series, compound 2b and 2c are effective against both the species of Aspergillus.
The sulfonamide-containing
dihydroquinazolinones (2f and 2g) exhibited significant action against A. fumigatus when compared with the A. niger. Interestingly, other derivatives also displayed strong inhibitory potential against the fungal growth.
The antifungal activity of the compounds almost
comparable to the standard drug nystatin [Table 1]. The widely used antifungal standard drug fluconazole was ineffective against Aspergillus [34]. Overall, A. fumigatus was found to be more sensitive than A. niger against the tested compounds. 3.2. Lysozyme interaction studies 8
ACCEPTED MANUSCRIPT 3.2.1. UV-Vis absorption spectra The UV-Vis absorption spectrum is a preliminary method of analysis to explore the structural change of the protein. The changes in the absorption spectra of the protein in the
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presence and absence of ligand (2b) are displayed in the Fig. 3. The strong absorption peak at 190-200 nm reflects the conformation of the peptide backbone. The aromatic amino acid such as
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tyrosine and tryptophan exhibits weak band at 280 nm. The addition of ligand to the lysozyme
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solution displays an increase in the intensity of the peak. This change in the absorption spectra indicates the conformational change of the peptide backbone due to complexation with the
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3.2.2. Fluorescence quenching studies [36]
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ligand.
Fluorescence quenching study has been used to gain information regarding the interaction of the ligand with the protein, quenching mechanism, binding constant and the number of The regular decrease in the fluorescence intensity of the protein with the
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binding sites.
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subsequent addition of ligand indicates its interaction with the lysozyme (Fig. 4). Further confirmation of the quenching mechanism can be obtained from the Stern-Volmer equation.
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F0/F= 1+Kqτ0[Q]= 1+Ksv[Q]
Where F0 and F are the fluorescence intensities in the absence and presence of quencher respectively, Kq is the quenching rate constant of the biomacromolecule, τ0 is the fluorescence lifetime in the absence of quencher, [Q] is the concentration of the quencher and Ksv is the SternVolmer quenching constant. The value of slope obtained from the Stern-Volmer plot (Fig. 5) was found to be 4.431 x 104 L mol-1. The resulted value of Kq (4.431 x 1012 L mol-1 S-1) was greater than 2 x 1010 L mol-1 S-1. This standard value is the maximum scatter collision quenching constant of various quenchers with the biopolymers. Since the obtained value of Kq was greater than the standard value, the quenching mechanism was confirmed to be static quenching process. The static quenching process is expected due to the formation of ground state complex between the quencher and the fluorophore. The double-logarithmic equation is represented as: log [F0-F/F] = log Kb + n log [Q] Where Kb is the binding constant and n is the number of binding site between the protein and the ligand. The slope value (0.99) of the double-logarithmic plot (Fig. 6) is approximately equal to 1 which indicates that one binding site is present between the lysozyme and the ligand. 9
ACCEPTED MANUSCRIPT 3.2.3. Circular dichroism study The circular dichroism measurement was carried out to ascertain the changes in the secondary structure of lysozyme after interacting with the ligand. The presence of two negative
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bands in the far-UV region (190-250 nm) is the characteristics of the native lysozyme. The absorption in this region is mainly because of the peptide bond. The negative band at 208 nm
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(intense band) and 222 nm (weak and broadband) represents the π-π* transition of α-helix and
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n-π* transition of both α-helix and random coil respectively (Fig. 7). The change in the α-helical
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content of lysozyme from 38.9% to 26.7% was observed after interacting with the ligand.
3.2.4. Three-dimensional fluorescence spectra [37]
Three-dimensional spectra provide detailed information regarding the conformational
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change of the protein. It contains four peaks of which ‘peak a’ and ‘peak b’ are called Rayleigh scattering peak (λex=λem) and second-order scattering peak (λex=2λem) (Fig. 8 and Fig. 9). The
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interaction of the ligand with lysozyme can only be predicted through the spectral changes in
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‘peak 1’ and ‘peak 2’. The ‘peak 1’ represents the spectral characteristics of tyrosine and tryptophan residues (λex=280 nm, λem=375 nm). The changes in the fluorescence intensity
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(112.6-104.1) were observed after interaction with the ligand. The ‘peak 2’ reveals information regarding the peptide backbone of the protein and its fluorescence intensity was also found to be decreased (82.4 to 77.67). The quantum yield of the native protein was decreased leading to decreased fluorescence intensity.
Through these results, the conformational change of the
protein can be ascertained along with the supportive information from circular dichroism. 3.3. Molecular docking
3.3.1. Docking interactions with NMT The
molecular
docking
was
employed
to
identify
the
exact
location
of
dihydroquinazolinones in the active site of NMT using Autodock software. The compounds containing C-O bond in the side chain of the 6th position phenyl ring (2a, 2b, 2c, 2d, and 2e) may be responsible for the less binding energies (Table 2). The binding energy of the compounds was also justified through the various non-covalent interactions (Fig. 10). The hydrogen bonding interaction was formed between the carbonyl group of dihydroquinazolinones and the amino acid residues such as Tyr393, Asn434, Gly251, and Tyr374. In addition, to this hydrogen bonding 10
ACCEPTED MANUSCRIPT was also formed between the –NH group of dihydroquinazolinones and C=O group of Asn213 amino acid residue. The involvement of various other hydrophobic interactions such as π-alkyl, π-π stacking, alkyl-alkyl and π- were also formed within the active site of the enzyme. The
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lone pair of electrons on the sulfur atom of sulphonamide containing compounds (2f and 2g) was
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involved in the interaction with π-electrons of the aromatic amino acid residues. 3.3.2. Docking interactions with lysozyme
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Lysozyme contains 129 amino acid residues among which six of them are tryptophan residues. The fluorescence of lysozyme is mainly due to the Trp62, Trp63, and Trp108 residues
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which lie mainly in the substrate binding site [38]. The quenching of fluorescence is because of the interaction of the ligand with these three amino acids. All the compounds are docked inside
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the binding pocket containing Trp108 residue (Fig. 11). The π-electrons of both Trp108 and 6th position side chain phenyl ring interacted with each other through π- π- stacking mechanism. In addition to this interaction, Trp62 and Trp63 are also responsible for the fluorescence
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quenching in the compound, 2a. This is because of the π-alkyl interaction between the π-
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electrons of tryptophan residues and the t-butyloxycarbonyl group of the compound.
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hydrogen bond is formed between the hydrogen atom of the Trp63 residue and the oxygen atom in the C-O bond containing compounds (2b, 2c, 2d and 2e). Therefore, hydrogen bonding and hydrophobic type of interactions are responsible for the stability of the ligand-lysozyme system. 4. Conclusion
A series of Suzuki Miyaura coupled products of dihydroquinazolinones were synthesized and characterized by IR, 1H-NMR,
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C-NMR and HRMS. The antifungal activities of the
compounds against the Aspergillus species were evaluated. The compounds containing C-O bond in the side chain (2a, 2b, 2c, 2d, and 2e) exhibited superior antifungal activity. The compounds are comparatively effective towards A. fumigatus than A. niger.
Further, the
interaction of the compound with lysozyme protein was evaluated and confirmed by means of various spectroscopic studies.
The quenching of fluorescence was predicted to be static
quenching mechanism. The molecular modeling studies provided additional information for analyzing the results obtained from the other experimentations. In conclusion, an effective agent for the treatment of patients infected with Aspergillus species was explored.
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ACCEPTED MANUSCRIPT Acknowledgment Author Dr. G. Madhumitha thank to the research grants provided by DST-FTYS (No.
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SB/FT/CS-113/2013), Government of India, New Delhi. The author’s acknowledge the seed fund provided by VIT University Management for carrying out the research. The authors are
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thankful for the NMR facilities provided by DST-FIST to School of Advanced Sciences, VIT University, Vellore. The acknowledgement was also extended to the support provided by VIT-
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SIF for GC-MS and UV-analysis. Prof. Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu extend their sincere appreciation to the Deanship of Scientific Research at King Saud University
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for its funding this Prolific Research Group (PRG-1437-28).
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ACCEPTED MANUSCRIPT [38] S.R. Feroz, Y, J. Teoh, S.B. Mohamad, S.L. Hong, S.N.A. Malek, S. Tayyab, Interaction of flavokawain B with lysozyme: A photophysical and molecular simulation
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study, J. Lumin. 160 (2015) 101-109.
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ACCEPTED MANUSCRIPT Table captions Table 1. Antifungal activity of the compounds 2(a-i) against Aspergillus species
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Table 2. The binding energies of the compounds 2(a-i) with NMT and lysozyme enzyme Figure captions
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Fig. 1. Mechanism of action NMT inhibitors
Fig. 2. Ether linkage and sulfonamide group of NMT inhibitors involved in hydrogen bonding
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Fig. 3. Absorption spectra of lysozyme (1 μM) in the presence and absence of the ligand (2b) (1 μM).
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Fig. 4. Emission spectra of lysozyme (5 μM) at various concentrations of the ligand (2b). The concentration of the compound (2b) are [a] to [k]=0-10 μM
concentrations of the compound (2b)
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Fig. 5. Stern-Volmer plot for the fluorescence quenching of lysozyme with various
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Fig. 6. Double-logarithmic plot for the fluorescence quenching of lysozyme with various concentrations of the compound (2b)
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Fig. 7. CD spectra of lysozyme (1 μM) in the absence and presence of the ligand (2b) (1 μM)
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Fig. 8. Three-dimensional fluorescence spectra (B’1) and contour map (B’2) of lysozyme (1 μM) in the absence of ligand
Fig. 9. Three-dimensional fluorescence spectra (A’1) and contour map (A’2) of lysozyme (1 μM) in the presence of ligand (2b) (1 μM) Fig. 10. Docking interactions of the compound (2b) with the NMT enzyme (PDB code: 4CAW). The various non-covalent interactions between the ligand and the enzyme are represented as dashed lines Fig. 11. The binding mode between the compound (2b) and lysozyme (PDB code: 6LYZ). The representation of hydrogen bonding residues along with various hydrophobic interactions is given
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ACCEPTED MANUSCRIPT Table 1 Compound
Aspergillus niger
Aspergillus fumigatus
MIC (µg/ml)
2a
14
6.2
2b
17
3.1
2c
16
3.1
2d
13
6.2
2e
15
9.5
2f
14
6.2
2g
15
2h
11
2i
18
Nystatin
18
Fluconazole
Nil
MIC (µg/ml) 6.2
18
3.1
3.1
13
6.2
14
3.1
6.2
15
3.1
6.2
11
6.2
6.2
12
6.2
3.1
18
3.1
Nil
Nil
Nil
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Binding energy (ΔGBE) in kcal/mol Lysozyme (PDB code: 6LYZ)
2a
-9.09
-8.07
2b
-9.13
2c
-8.89
2d
-9.54
2e
-9.65
2f
-8.24
2g
-8.37
2h
-8.78
2i
-7.22
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NMT (PDB code: 4CAW)
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-8.69
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-9.09 -8.92 -8.55 -9.37 -9.33 -8.20 -7.15
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Fig. 11
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Scheme 1. Synthesis of 6-substituted 2,3-dihydroquinazolinones via Suzuki Miyaura coupling
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Graphical abstract
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Highlights
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1. Synthesis of 2,3-dihydroquinazolinones via Suzuki Miyaura coupling reaction. 2. Screening the antifungal activity of the compounds against Aspergillus species. 3. Interaction with lysozyme was determined by various spectroscopic studies.
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S1011-1344(16)30183-X doi: 10.1016/j.jphotobiol.2016.05.005 JPB 10373
To appear in: Received date: Accepted date:
18 March 2016 3 May 2016
Please cite this article as: K. Hemalatha, G. Madhumitha, Lokesh Ravi, V. Gopiesh Khanna, Naif Abdullah Al-Dhabi, Mariadhas Valan Arasu, Binding mode of dihydroquinazolinones with lysozyme and its antifungal activity against Aspergillus species, (2016), doi: 10.1016/j.jphotobiol.2016.05.005
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Binding mode of dihydroquinazolinones with lysozyme and its antifungal activity against Aspergillus species
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K. Hemalathaa, G. Madhumithaa*, Lokesh Ravib, V. Gopiesh Khannab, Naif Abdullah AlDhabic, Mariadhas Valan Arasuc a Chemistry of Heterocycles & Natural Product Research Laboratory, Department of Chemistry, School of Advanced Sciences, VIT University, Vellore 632 014, Tamil Nadu, India. b Division of Bio-medical Sciences, School of Biosciences and Technology, VIT University, Vellore 632 014, Tamil Nadu, India. c Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King Saud University, P. O. Box 2455, Riyadh, 11451, Kingdom of Saudi Arabia.
Aspergillosis is one of the infectious fungal diseases affecting mainly the
immunocompromised patients.
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Abstract:
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Corresponding author. Tel.: 04162202336; fax: 04162245544/5766. E-mail address: [email protected]; [email protected]
The scarcity of the antifungal targets has identified the
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importance of N-myristoyl transferase (NMT) in the regulation of fungal pathway.
The
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dihydroquinazolinone molecules were designed on the basis of fragments responsible for binding with the target enzyme. The aryl halide, 1(a-g), aryl boronic acid and potassium carbonate were heated together in water and dioxane mixture to yield new C-C bond formation in dihydroquinazolinone. The bis(triphenylphosphine)palladium(II) dichloride was used as catalyst for the C-C bond formation. The synthesized series were screened for their in vitro antifungal activity against Aspergillus niger and Aspergillus fumigatus. The binding interactions of the active compound with lysozyme were explored using multiple spectroscopic studies. Molecular docking study of dihydroquinazolinones with the enzyme revealed the information regarding various binding forces involved in the interaction. Keywords: Aspergillosis, N-myristoyl transferase, dihydroquinazolinones, antifungal activity, lysozyme, spectroscopy. 1. Introduction: Aspergillosis is an infection/allergic response caused by Aspergillus species. It is a type of fungus whose spores are omnipresent in air, water and soil. It does not cause illness in the 1
ACCEPTED MANUSCRIPT normal patient whereas it causes life threatening infections in the immunocompromised patient [1]. The patients with hematological malignancies [2], advanced HIV infection [3] and those who have undergone solid organ transplantation are more susceptible to aspergillus infection [4].
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The principle organ of infection is lungs and it produces syndromes such as allergic bronchopulmonary aspergillosis (ABPA), aspergilloma and invasive aspergillosis (IA) [5]. IA
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was found to be the main reason for the infectious pneumonic mortality in an
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immunocompromised patient. Aspergillus genus possesses more than 180 different species. Aspergillus fumigatus was recovered from the clinical isolates of IA patients and it accounts for
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the majority of pathological conditions. Aspergillus niger is a less prevalent disease causing organism but inhalation of a lot of spores can cause serious lung disorder. It can also cause
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fungal infections in the ear which leads to temporary pain and hearing loss. If left untreated, it leads to damage in the inner parts of the ear [6]. The treatment for aspergillosis includes various azoles, polyene antibiotics, and echinocandins [7]. Azoles are advantageous over the other drug
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categories because of its availability as oral formulations. But azoles are displaying resistance
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towards the Aspergillus species. The urge for the development of novel antifungal agent and the pathway through which it acts is always in progress [8].
Heterocycles and nanoparticles
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scaffolds possessing antimicrobial activity targeting various pathways has been reviewed [9,10]. The N-myristoyl transferase (NMT) was identified as a novel mode of action for the antifungal therapy and it is an enzyme catalyzing the N-myristoylation reaction. It is a reaction in which Nterminal part of glycine in the cellular proteins were transferred with myristoyl-CoA group. These proteins are vital for the growth and function of the fungi [11] (Fig. 1). The role of NMT as the potential target in the aspergillosis treatment has been reported [12]. From the literature, it was found that the NMT inhibitors with linkers containing ether oxygen atom and sulfonamide group were essential for the formation of hydrogen bonding between the amino acid residues [13-15] (Fig. 2). Therefore, the quinazolinones containing these moieties (ether oxygen and sulfonamide group) were designed and synthesized. The antifungal activity of these compounds was compared with that of the quinazolinone containing other functional moieties. Quinazolinones [16-18] were proven with various pharmacological actions [19] and its importance as an antifungal agent was also reported [20, 21].
Heterocycles are bioactive
compounds [22, 23] and various reaction conditions [24, 25] are utilized in the formation of the nucleus. Even though they possess promising biological activities it fails because of the poor 2
ACCEPTED MANUSCRIPT physicochemical properties. The ADME (absorption, distribution, metabolism, and excretion) properties of the bioactive compounds are strongly influenced by its binding properties with the protein. Lysozyme is an important enzyme which is present in almost all the body fluids [26]. It
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is an antimicrobial peptide and it is an essential component of the innate immune system. It causes bacterial cell lysis by hydrolyzing the β-linkage of the bacterial peptidoglycan layer. The
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antifungal activity of lysozyme has also been reported [27]. It belongs to the category of low
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molecular weight proteins (LMWPs) with a molecular weight of 14 kDa. It is used as a carrier for many drugs resulted in target oriented treatment [28, 29].
The investigation on the
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interaction of lysozyme with various drugs has already been reported [30-32]. In this work, the antifungal activities of the synthesized series were tested against the Aspergillus species.
various spectroscopic methodologies.
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2.1. Chemicals and instruments
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2. Materials and methods
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Further, the interaction of dihydroquinazolinones with lysozyme has been studied by using
The reagents used in the synthesis were of analytical grade and are used without any
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further processing techniques.
The synthesized compounds were purified by column
chromatographic separations using silica gel of mesh size 60-120. NMR spectra were recorded on Bruker Avance 400 MHz spectrometer using TMS as an internal standard. The melting points were measured in Elchem Microprocessor based DT apparatus and was corrected using standard benzoic acid. The exact molecular weight was predicted using the ESI-MS Thermo Fleet instrument. Chicken egg white lysozyme from sigma Aldrich was stored at 4 ºC and was used without further purification. Sodium phosphate buffer of pH 7.4 was prepared using double distilled water. The absorption spectra were recorded on a Hitachi U-2800 double beam UV/Vis spectrophotometer. The instrument was equipped with 1 cm quartz cell and slit width was set at 1.5 nm.
Fluorescence spectra were recorded from Hitachi F-7000 fluorescence
spectrofluorometer.
Circular dichroism (CD) spectrum was recorded on JASCO J-715
chiroptical spectrometer (JASCO corp.,) and the bandwidth was set at 1 nm at a scanning speed of 500 nm/min. 2.2. Synthesis 3
ACCEPTED MANUSCRIPT The reaction mixture containing aryl halide, 1(a-g) (1 Equiv), aryl boronic acid (1.1 Equiv) and potassium carbonate (1.5 Equiv) was taken in a sealed tube containing 1:3 ratio of water:1,4-dioxane. The nitrogen gas was purged into the sealed tube for 15 minutes. The
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catalyst bis(triphenylphosphine)palladium(II) dichloride of 0.1 equivalent was added to the reaction mixture and stirred at 80 ⁰C for 1-2 h. The completion of the reaction was confirmed by
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thin layer chromatography. The reaction mixture was filtered through the celite and the collected
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filtrate was extracted with chloroform. The chloroform layer was washed several times with water.
The residual moisture was removed by drying over anhydrous sodium sulfate and
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concentrated under reduced pressure. The obtained crude product was purified by column chromatography using petroleum ether and ethyl acetate as a solvent system (Scheme 1).
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2.2.1. Tert-butyl 4- (4- (2, 2, 3-trimethyl -4- oxo-1, 2, 3, 4- tetrahydroquinazolin-6- l) phenyl) piperazine-1-carboxylate (2a)
Off-white solid; yield: 92%; m.p.: 238-240 C. FT-IR (KBr disc) (νmax/cm-1): 1691
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(C=O), 2974 (methylene C-H stretch), 3304 (NH). 1H NMR (400 MHz, CDCl3): δ (ppm), 1.48
TE
(s, 9H), 1.54 (s, 6H), 3.07 (s, 3H), 3.16 (d, J=4.4 Hz, 4H), 3.58 (t, J=4.8 Hz, 4H), 4.22 (bs, 1H), 13
C
AC CE P
6.65 (d, J=8 Hz, 1H), 6.95 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.4 Hz, 3H), 8.13 (d, J=2 Hz, 1H).
NMR (100 MHz, CDCl3): 2 X 21.5, 22.1, 3 X 23.2, 2 X 44.1, 2 X 66.0, 74.6, 109.7, 110.7, 2 X 111.5, 121.0, 3 X 121.8, 126.1, 126.4, 127.0, 138.2, 144.7, 149.5, 158.2. HRMS m/z calculated C26H34N4O3 (M+) 450.2631; found 450.2360. 2.2.2. 6- (4- (benzyloxy) phenyl) - 2, 2, 3- trimethyl -2, 3 -dihydroquinazolin- 4(1H)- one (2b) Off-white solid; yield: 88%; m.p.: 230-232 C. FT-IR (KBr disc) (νmax/cm-1): 1624 1
(C=O), 2970 (methylene C-H stretch), 3296 (NH).
H NMR (400 MHz, DMSO-d6): δ (ppm),
1.46 (s, 6H), 2.95 (s, 3H), 5.13 (s, 2H), 6.75 (d, J= 8 Hz, 1H), 6.83 (bs, 1H), 7.06 (d, J= 8.8 Hz, 2H), 7.34-7.55 (m, 8H), 7.84 (d, J= 2 Hz, 1H).
13
C NMR (100 MHz, DMSO- d6): 2 X 25.8,
26.6, 69.2, 70.8, 114.3, 114.8, 2 X 115.2, 124.7, 2 X 126.6, 2 X 127.6, 2 X 127.7, 128.4, 128.6, 131.0, 132.6, 137.1, 145.0, 157.1, 162.3. HRMS m/z calculated C24H24N2O2 (M+) 372.1838; found 372.1835. 2.2.3. 6- (4- (benzyloxy) phenyl) -3- ethyl -1H- spiro [cyclopentane -1, 2- quinazolin] -4(3H)one (2c)
4
ACCEPTED MANUSCRIPT Off-white solid; yield: 85%; m.p.: 196-198 C. FT-IR (KBr disc) (νmax/cm-1): 1620 (C=O), 2954 (methylene C-H stretch), 3452 (NH).
1
H NMR (400 MHz, DMSO-d6): δ (ppm),
1.13 (t, J= 6.8 Hz, 3H), 1.66-1.94 (m, 8H), 3.38-3.43 (m, 2H), 5.14 (s, 2H), 6.82 (d, J= 8.3 Hz,
PT
1H), 6.89 (bs, 1H), 7.06 (d, J= 8.6 Hz, 2H), 7.34-7.54 (m, 8H), 7.82 (d, J= 1.8 Hz, 1H).
13
C
NMR (100 MHz, DMSO- d6): 15.4, 2 X 22.5, 36.1, 2 X 36.5, 69.2, 81.1, 115.1, 2 X 115.2,
RI
115.4, 124.7, 2 X 126.7, 2 X 127.6, 127.7, 2 X 128.4, 128.8, 130.8, 132.7, 137.1, 144.9, 157.2,
SC
162.6. HRMS m/z calculated C27H28N2O2 (M+) 412.2151; found 412.2150. 2.2.4. 6- (4- (benzyloxy) phenyl) -3- methyl -1H- spiro [cyclopentane-1, 2-quinazolin] -4(3H)-
NU
one (2d)
Off-white solid; yield: 84%; m.p.: 220-222 C. FT-IR (KBr disc) (νmax/cm-1): 1616 1
MA
(C=O), 2931 (methylene C-H stretch), 3471 (NH).
H NMR (400 MHz, DMSO- d6): δ (ppm),
1.63-2.08 (m, 8H), 2.95 (s, 3H), 5.13 (s, 2H), 6.82 (d, J= 8.3 Hz, 1H), 6.91 (bs, 1H), 7.06 (d, J= 13
C NMR (100
D
8.6 Hz, 2H), 7.34-7.42 (m, 3H), 7.46-7.55 (m, 5H), 7.83 (d, J= 1.8 Hz, 1H).
MHz, DMSO- d6): 2 X 23.0, 27.3, 2 X 35.8, 69.2, 81.1, 115.1, 2 X 115.2, 2 X 124.7, 2 X 126.7,
TE
2 X 127.6, 2 X127.7, 2 X 128.4, 128.8, 130.9, 132.6, 137.1, 157.2, 163.0. HRMS m/z calculated
AC CE P
C26H26N2O2 (M+) 398.1994; found 398.1992. 2.2.5. 6- (4- (benzyloxy) phenyl) -3- ethyl -2, 2-dimethyl -2, 3- dihydroquinazolin -4(1H)- one (2e)
Off-white solid; yield: 88%; m.p.: 258-260 C. FT-IR (KBr disc) (νmax/cm-1): 1614 (C=O), 2970 (methylene C-H stretch), 3294 (NH).
1
H NMR (400 MHz, DMSO- d6): δ (ppm),
1.13 (t, J= 6.9 Hz, 3H), 1.49 (s, 6H), 3.44-3.48 (m, 2H), 5.14 (s, 2H), 6.73 (d, J= 8.4 Hz, 1H), 6.80 (bs, 1H), 7.06 (d, J= 9.2 Hz, 2H), 7.34-7.54 (m, 8H), 7.83 (d, J= 2 Hz, 1H).
13
C NMR (100
MHz, DMSO- d6): 15.4, 2 X 26.8, 35.8, 69.2, 71.2, 114.5, 114.7, 2 X 115.2, 124.6, 2 X 126.6, 2 X 127.6, 127.7, 2 X 128.4, 128.6, 131.0, 132.7, 137.1, 144.9, 157.1, 162.0.
HRMS m/z
calculated C25H26N2O2 (M+) 386.1994; found 386.1992. 2.2.6. N- (3- (2, 2, 3- trimethyl -4- oxo- 1, 2, 3, 4- tetrahydroquinazolin -6 -l) phenyl) methane sulfonamide (2f) Off-white solid; yield: 90%; m.p.: 220-222 C. FT-IR (KBr disc) (νmax/cm-1): 1629 (C=O), 3070 (methylene C-H stretch), 3365 (NH). 5
1
H NMR (400 MHz, DMSO- d6): δ (ppm),
ACCEPTED MANUSCRIPT 1.47 (s, 6H), 2.95 (s, 3H), 3.02 (s, 3H), 6.78 (d, J=8.4 Hz, 1H), 6.95 (bs, 1H), 7.14-7.16 (m, 1H), 7.31-7.42 (m, 3H), 7.54-7.57 (m, 1H), 7.87 (d, J=2.2 Hz, 1H), 9.76 (s, 1H).
13
C NMR (100
MHz, DMSO- d6): 2 X 25.9, 26.6, 39.1, 70.9, 114.2, 114.8, 116.8, 117.6, 121.1, 125.3, 128.1,
PT
129.8, 131.3, 138.9, 141, 145.8, 162.2. HRMS m/z calculated C18H21N3O3S (M+) 359.1304;
RI
found 359.1300.
methane sulfonamide (2g)
SC
2.2.7. N- (3- (3- ethyl- 2, 2-dimethyl -4- oxo-1, 2, 3, 4- tetrahydroquinazolin -6- yl) phenyl) Off-white solid; yield: 87%; m.p.: 168-170 C. FT-IR (KBr disc) (νmax/cm-1): 1629 1
H NMR (400 MHz, DMSO- d6): δ (ppm),
NU
(C=O), 2972 (methylene C-H stretch), 3371 (NH).
1.14 (t, J=6.9 Hz, 3H), 1.50 (s, 6H), 3.02 (s, 3H), 3.44-3.49 (m, 2H), 6.77 (d, J=8.4 Hz, 1H),
9.76 (s, 1H).
13
MA
6.93 (bs, 1H), 7.13-7.16 (m, 1H), 7.31-7.42 (m, 3H), 7.54-7.56 (m, 1H), 7.87 (d, J=2.2 Hz, 1H), C NMR (100 MHz, DMSO- d6): 15.3, 2 X 26.9, 35.8, 39.1, 71.3, 114.4, 114.7,
116.8, 117.6, 121.7, 125.2, 128.1, 129.8, 131.3, 138.9, 141, 145.7, 161.8. HRMS m/z calculated
TE
D
C19H23N3O3S (M+) 373.1460; found 373.1458. 2.2.8. 3- methyl -6- (4- nitrophenyl) -1H- spiro [cyclopentane- 1, 2- quinazolin] -4(3H)- one (2h)
AC CE P
Off-white solid; yield: 89%; m.p: 160-162 C. FT-IR (KBr disc) (νmax/cm-1): 1625 (C=O), 2953 (methylene C-H stretch), 3296 (NH).
1
H NMR (400 MHz, DMSO- d6): δ (ppm),
1.67-2.10 (m, 8H), 2.96 (s, 3H), 6.90 (d, J= 8.8 Hz, 1H), 7.30 (bs, 1H), 7.74-7.77 (m, 1H), 7.88 (t, J= 7.2 Hz, 2H), 8.05 (d, J= 2.4 Hz, 1H), 8.26 (t, J= 2 Hz, 2H).
13
C NMR (100 MHz,
DMSO- d6): 2 X 23.0, 27.4, 2 X 36.0, 81.1, 114.9, 115.3, 2 X 124.1, 126.0, 126.2, 2 X 126.7, 131.7, 145.5, 146.3, 146.8, 162.5. HRMS m/z calculated C19H19N3O3 (M+) 337.1426; found 337.1422. 2.2.9. 3- ethyl- 2, 2- dimethyl -6- (4- nitrophenyl) -2, 3- dihydroquinazolin -4(1H)- one (2i) Off-white solid; yield: 85%; m.p.: 254-256 C. FT-IR (KBr disc) (νmax/cm-1): 1616 (C=O), 2926 (methylene C-H stretch), 3290 (NH).
1
H NMR (400 MHz, DMSO- d6): δ (ppm),
1.14 (t, J=6.9 Hz, 3H), 1.52 (s, 6H), 3.45-3.50 (m, 2H), 6.81 (d, J=8.4 Hz, 1H), 7.19 (bs, 1H), 7.74-7.76 (m, 1H), 7.87-7.89 (m, 2H), 8.04 (d, J=2.2 Hz, 1H), 8.24 -8.26 (m, 2H).
13
C NMR
(100 MHz, DMSO- d6): 15.3, 2 X 27, 35.9, 71.3, 114.3, 114.9, 2 X 124.1, 3 X 125.8, 126.1,
6
ACCEPTED MANUSCRIPT 131.8, 145.5, 146.3, 146.7, 161.6. HRMS m/z calculated C18H19N3O3 (M+) 325.1426; found 325.1425.
PT
2.3. Anti-fungal activity [33] 2.3.1. Determination of zone of inhibition (ZOI)
RI
Clinical isolates of Aspergillus niger and Aspergillus fumigatus were grown in Sabouraud dextrose broth (SDB) till adequate turbidity is obtained. The broth was then used as inoculum
SC
for lawn culture in Kirby Bauer’s well diffusion method to check for anti-fungal activity. Sabouraud dextrose agar (SDA) was used for the well diffusion method. The individual test
NU
compounds were dissolved in DMSO and diluted to obtain a final volume of 200 µl. The final concentration of 10 μg/ml for all the test compounds was used for this study. The 8 mm wells
MA
were punched on the SDB and total of 200 μl of the test compound solution was added to each well. Fluconazole and nystatin were used as standard antibiotics. The zone of inhibition was
D
observed after 48-72 h.
TE
2.3.2. Determination of minimum inhibitory concentration (MIC) Minimum inhibitory concentration was performed on a 96-well plate equally dispensed
AC CE P
with fresh SDB. The test compounds were serially diluted in the individual column to obtain a range of concentrations. The uniform volume of freshly prepared fungal broth was added to all the wells and incubated at 28 ˚C for 24 hours and was recorded from 96-well plate reader. The culture becomes turbid because of the growth of the fungi in the tube containing low inhibitory concentration. However, the culture remains clear in the tube containing above the level of inhibitory concentration. The standard drugs used for the comparative study was Nystatin and fluconazole. 2.4. Lysozyme interaction studies The wavelength range for UV-Vis absorption spectra were scanned from 190-310 nm. Fluorescence emission spectra were obtained by titrating lysozyme (5 μM) with increasing concentrations (0-10 μM) of the ligand at 298 K. The excitation wavelength was set at 280 nm. The slit widths of excitation and emission wavelength were set at 5 nm. The scan speed was set at 2400 nm/min.
The three-dimensional fluorescence spectra were performed using the
excitation wavelength scan range from 200-400 nm and emission wavelength scan range from 7
ACCEPTED MANUSCRIPT 200-800 nm. The excitation and emission slit width was fixed at 5 nm. The sampling interval was fixed at 5 nm and 10 nm for excitation and emission wavelength respectively. The CD spectra of lysozyme in the presence (2 μM) and absence of ligand (2 μM) were recorded
PT
from 190-260 nm.
RI
2.5. Molecular docking
SC
The interaction between the synthesized dihydroquinazolinones 2(a-i) and Aspergillus fumigatus NMT was found out by molecular modeling using Auto Dock 4.0. The synthesized The crystal
NU
molecules were constructed and energy minimized using Chem3D Pro 12.0.
structure of A. fumigatus NMT (PDB code: 4CAW) and hen egg-white lysozyme (PDB code:
MA
6LYZ) [34] were obtained from Brookhaven protein data bank. The preparation of the protein was performed by adding essential hydrogen atoms and Kollman charges. Similarly, ligands were prepared by incorporating non-polar hydrogen atoms and Gasteiger charges. A grid box of
D
size 90 Å x 90 Å x 90 Å (NMT) and 80 Å x 80 Å x 80 Å (lysozyme) were created using Auto
TE
Grid. The Lamarckian Genetic Algorithm was employed in Auto Dock tool. The detailed information on the docked conformation of the best fit was retrieved from the Accelrys discovery
AC CE P
studio and LigPlot software. 3. Results and discussion 3.1. Antifungal activity
The antifungal activity of the newly synthesized compounds against Aspergillus species was evaluated by the well diffusion method. Among the compound series, compound 2b and 2c are effective against both the species of Aspergillus.
The sulfonamide-containing
dihydroquinazolinones (2f and 2g) exhibited significant action against A. fumigatus when compared with the A. niger. Interestingly, other derivatives also displayed strong inhibitory potential against the fungal growth.
The antifungal activity of the compounds almost
comparable to the standard drug nystatin [Table 1]. The widely used antifungal standard drug fluconazole was ineffective against Aspergillus [34]. Overall, A. fumigatus was found to be more sensitive than A. niger against the tested compounds. 3.2. Lysozyme interaction studies 8
ACCEPTED MANUSCRIPT 3.2.1. UV-Vis absorption spectra The UV-Vis absorption spectrum is a preliminary method of analysis to explore the structural change of the protein. The changes in the absorption spectra of the protein in the
PT
presence and absence of ligand (2b) are displayed in the Fig. 3. The strong absorption peak at 190-200 nm reflects the conformation of the peptide backbone. The aromatic amino acid such as
RI
tyrosine and tryptophan exhibits weak band at 280 nm. The addition of ligand to the lysozyme
SC
solution displays an increase in the intensity of the peak. This change in the absorption spectra indicates the conformational change of the peptide backbone due to complexation with the
MA
3.2.2. Fluorescence quenching studies [36]
NU
ligand.
Fluorescence quenching study has been used to gain information regarding the interaction of the ligand with the protein, quenching mechanism, binding constant and the number of The regular decrease in the fluorescence intensity of the protein with the
D
binding sites.
TE
subsequent addition of ligand indicates its interaction with the lysozyme (Fig. 4). Further confirmation of the quenching mechanism can be obtained from the Stern-Volmer equation.
AC CE P
F0/F= 1+Kqτ0[Q]= 1+Ksv[Q]
Where F0 and F are the fluorescence intensities in the absence and presence of quencher respectively, Kq is the quenching rate constant of the biomacromolecule, τ0 is the fluorescence lifetime in the absence of quencher, [Q] is the concentration of the quencher and Ksv is the SternVolmer quenching constant. The value of slope obtained from the Stern-Volmer plot (Fig. 5) was found to be 4.431 x 104 L mol-1. The resulted value of Kq (4.431 x 1012 L mol-1 S-1) was greater than 2 x 1010 L mol-1 S-1. This standard value is the maximum scatter collision quenching constant of various quenchers with the biopolymers. Since the obtained value of Kq was greater than the standard value, the quenching mechanism was confirmed to be static quenching process. The static quenching process is expected due to the formation of ground state complex between the quencher and the fluorophore. The double-logarithmic equation is represented as: log [F0-F/F] = log Kb + n log [Q] Where Kb is the binding constant and n is the number of binding site between the protein and the ligand. The slope value (0.99) of the double-logarithmic plot (Fig. 6) is approximately equal to 1 which indicates that one binding site is present between the lysozyme and the ligand. 9
ACCEPTED MANUSCRIPT 3.2.3. Circular dichroism study The circular dichroism measurement was carried out to ascertain the changes in the secondary structure of lysozyme after interacting with the ligand. The presence of two negative
PT
bands in the far-UV region (190-250 nm) is the characteristics of the native lysozyme. The absorption in this region is mainly because of the peptide bond. The negative band at 208 nm
RI
(intense band) and 222 nm (weak and broadband) represents the π-π* transition of α-helix and
SC
n-π* transition of both α-helix and random coil respectively (Fig. 7). The change in the α-helical
NU
content of lysozyme from 38.9% to 26.7% was observed after interacting with the ligand.
3.2.4. Three-dimensional fluorescence spectra [37]
Three-dimensional spectra provide detailed information regarding the conformational
MA
change of the protein. It contains four peaks of which ‘peak a’ and ‘peak b’ are called Rayleigh scattering peak (λex=λem) and second-order scattering peak (λex=2λem) (Fig. 8 and Fig. 9). The
D
interaction of the ligand with lysozyme can only be predicted through the spectral changes in
TE
‘peak 1’ and ‘peak 2’. The ‘peak 1’ represents the spectral characteristics of tyrosine and tryptophan residues (λex=280 nm, λem=375 nm). The changes in the fluorescence intensity
AC CE P
(112.6-104.1) were observed after interaction with the ligand. The ‘peak 2’ reveals information regarding the peptide backbone of the protein and its fluorescence intensity was also found to be decreased (82.4 to 77.67). The quantum yield of the native protein was decreased leading to decreased fluorescence intensity.
Through these results, the conformational change of the
protein can be ascertained along with the supportive information from circular dichroism. 3.3. Molecular docking
3.3.1. Docking interactions with NMT The
molecular
docking
was
employed
to
identify
the
exact
location
of
dihydroquinazolinones in the active site of NMT using Autodock software. The compounds containing C-O bond in the side chain of the 6th position phenyl ring (2a, 2b, 2c, 2d, and 2e) may be responsible for the less binding energies (Table 2). The binding energy of the compounds was also justified through the various non-covalent interactions (Fig. 10). The hydrogen bonding interaction was formed between the carbonyl group of dihydroquinazolinones and the amino acid residues such as Tyr393, Asn434, Gly251, and Tyr374. In addition, to this hydrogen bonding 10
ACCEPTED MANUSCRIPT was also formed between the –NH group of dihydroquinazolinones and C=O group of Asn213 amino acid residue. The involvement of various other hydrophobic interactions such as π-alkyl, π-π stacking, alkyl-alkyl and π- were also formed within the active site of the enzyme. The
PT
lone pair of electrons on the sulfur atom of sulphonamide containing compounds (2f and 2g) was
RI
involved in the interaction with π-electrons of the aromatic amino acid residues. 3.3.2. Docking interactions with lysozyme
SC
Lysozyme contains 129 amino acid residues among which six of them are tryptophan residues. The fluorescence of lysozyme is mainly due to the Trp62, Trp63, and Trp108 residues
NU
which lie mainly in the substrate binding site [38]. The quenching of fluorescence is because of the interaction of the ligand with these three amino acids. All the compounds are docked inside
MA
the binding pocket containing Trp108 residue (Fig. 11). The π-electrons of both Trp108 and 6th position side chain phenyl ring interacted with each other through π- π- stacking mechanism. In addition to this interaction, Trp62 and Trp63 are also responsible for the fluorescence
D
quenching in the compound, 2a. This is because of the π-alkyl interaction between the π-
TE
electrons of tryptophan residues and the t-butyloxycarbonyl group of the compound.
The
AC CE P
hydrogen bond is formed between the hydrogen atom of the Trp63 residue and the oxygen atom in the C-O bond containing compounds (2b, 2c, 2d and 2e). Therefore, hydrogen bonding and hydrophobic type of interactions are responsible for the stability of the ligand-lysozyme system. 4. Conclusion
A series of Suzuki Miyaura coupled products of dihydroquinazolinones were synthesized and characterized by IR, 1H-NMR,
13
C-NMR and HRMS. The antifungal activities of the
compounds against the Aspergillus species were evaluated. The compounds containing C-O bond in the side chain (2a, 2b, 2c, 2d, and 2e) exhibited superior antifungal activity. The compounds are comparatively effective towards A. fumigatus than A. niger.
Further, the
interaction of the compound with lysozyme protein was evaluated and confirmed by means of various spectroscopic studies.
The quenching of fluorescence was predicted to be static
quenching mechanism. The molecular modeling studies provided additional information for analyzing the results obtained from the other experimentations. In conclusion, an effective agent for the treatment of patients infected with Aspergillus species was explored.
11
ACCEPTED MANUSCRIPT Acknowledgment Author Dr. G. Madhumitha thank to the research grants provided by DST-FTYS (No.
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SB/FT/CS-113/2013), Government of India, New Delhi. The author’s acknowledge the seed fund provided by VIT University Management for carrying out the research. The authors are
RI
thankful for the NMR facilities provided by DST-FIST to School of Advanced Sciences, VIT University, Vellore. The acknowledgement was also extended to the support provided by VIT-
SC
SIF for GC-MS and UV-analysis. Prof. Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu extend their sincere appreciation to the Deanship of Scientific Research at King Saud University
NU
for its funding this Prolific Research Group (PRG-1437-28).
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ACCEPTED MANUSCRIPT [38] S.R. Feroz, Y, J. Teoh, S.B. Mohamad, S.L. Hong, S.N.A. Malek, S. Tayyab, Interaction of flavokawain B with lysozyme: A photophysical and molecular simulation
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study, J. Lumin. 160 (2015) 101-109.
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ACCEPTED MANUSCRIPT Table captions Table 1. Antifungal activity of the compounds 2(a-i) against Aspergillus species
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Table 2. The binding energies of the compounds 2(a-i) with NMT and lysozyme enzyme Figure captions
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Fig. 1. Mechanism of action NMT inhibitors
Fig. 2. Ether linkage and sulfonamide group of NMT inhibitors involved in hydrogen bonding
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Fig. 3. Absorption spectra of lysozyme (1 μM) in the presence and absence of the ligand (2b) (1 μM).
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Fig. 4. Emission spectra of lysozyme (5 μM) at various concentrations of the ligand (2b). The concentration of the compound (2b) are [a] to [k]=0-10 μM
concentrations of the compound (2b)
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Fig. 5. Stern-Volmer plot for the fluorescence quenching of lysozyme with various
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Fig. 6. Double-logarithmic plot for the fluorescence quenching of lysozyme with various concentrations of the compound (2b)
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Fig. 7. CD spectra of lysozyme (1 μM) in the absence and presence of the ligand (2b) (1 μM)
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Fig. 8. Three-dimensional fluorescence spectra (B’1) and contour map (B’2) of lysozyme (1 μM) in the absence of ligand
Fig. 9. Three-dimensional fluorescence spectra (A’1) and contour map (A’2) of lysozyme (1 μM) in the presence of ligand (2b) (1 μM) Fig. 10. Docking interactions of the compound (2b) with the NMT enzyme (PDB code: 4CAW). The various non-covalent interactions between the ligand and the enzyme are represented as dashed lines Fig. 11. The binding mode between the compound (2b) and lysozyme (PDB code: 6LYZ). The representation of hydrogen bonding residues along with various hydrophobic interactions is given
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Aspergillus niger
Aspergillus fumigatus
MIC (µg/ml)
2a
14
6.2
2b
17
3.1
2c
16
3.1
2d
13
6.2
2e
15
9.5
2f
14
6.2
2g
15
2h
11
2i
18
Nystatin
18
Fluconazole
Nil
MIC (µg/ml) 6.2
18
3.1
3.1
13
6.2
14
3.1
6.2
15
3.1
6.2
11
6.2
6.2
12
6.2
3.1
18
3.1
Nil
Nil
Nil
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Binding energy (ΔGBE) in kcal/mol Lysozyme (PDB code: 6LYZ)
2a
-9.09
-8.07
2b
-9.13
2c
-8.89
2d
-9.54
2e
-9.65
2f
-8.24
2g
-8.37
2h
-8.78
2i
-7.22
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NMT (PDB code: 4CAW)
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-8.69
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-9.09 -8.92 -8.55 -9.37 -9.33 -8.20 -7.15
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Scheme 1. Synthesis of 6-substituted 2,3-dihydroquinazolinones via Suzuki Miyaura coupling
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Graphical abstract
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Highlights
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1. Synthesis of 2,3-dihydroquinazolinones via Suzuki Miyaura coupling reaction. 2. Screening the antifungal activity of the compounds against Aspergillus species. 3. Interaction with lysozyme was determined by various spectroscopic studies.
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