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A study on catalytic and non-catalytic sites of H5N1 and H1N1 neuraminidase as the target for chalcone inhibitors

A study on catalytic and non-catalytic sites of H5N1 and H1N1 neuraminidase as the target for... The H1N1 pandemic in 2009 and the H5N1 outbreak in 2005 have shocked the world as millions of people were infected and hundreds of thousands died due to the infections by the influenza virus. Oseltamivir, the most common drug to block the viral life cycle by inhibiting neuraminidase (NA) enzyme, has been less effective in some resistant cases due to the virus mutation. Presently, the binding of 10 chalcone derivatives towards H5N1 and H1N1 NAs in the non-catalytic and catalytic sites was studied using molecular docking. The in silico study was also conducted for its drug-like likeness such as Lipinski Rule, mutagenicity, toxicity and pharmacokinetic profiles. The result demonstrates that two chalcones (1c and 2b) have the potential for future NA inhibitor development. Compound 1c inhibits H5N1 NA and H1N1 NA with IC of 27.63 µM and 28.11 µM, respectively, whereas compound 2b inhibits NAs with IC of 50 50 87.54 µM and 73.17 µM for H5N1 and H1N1, respectively. The in silico drug-like likeness prediction reveals that 1c is 62% better than 2b (58%) in meeting the criteria. The results suggested that 1c and 2b have potencies to be devel- oped as non-competitive inhibitors of neuraminidase for the future development of anti-influenza drugs. Keywords: Neuraminidase, Non-catalytic site, Chalcone, Influenza, H5N1, H1N1 Introduction Fortunately, there are no reported human cases of this The novel Severe Acute Respiratory Syndrome (SARS) H5N1 avian flu, although the transmission of the disease coronavirus, SARS-CoV-2, which was first discovered in to humans had been reported since the first infection to December 2019 in Wuhan, China, infected approximately humans recorded in 2003 [4]. 64,000 people with about 1400 deaths announced at that Since the H5N1 outbreak in 2005, the world has been time, mainly in China [1]. At almost the same time, the prepared for the impending influenza pandemic, and in country was also reportedly dealing with an outbreak of 2009, H1N1 strains emerged and threatened humanity the deadly H5N1 avian influenza in Hunan, an area that with its spread in more than 70 countries [5]. However, borders Hubei, the province where the new coronavirus the severity of the 2009 H1N1 pandemic was modest emerged, according to the South China Morning Post [2]. compared to the H5N1 outbreak. H5N1 strain is con- As of February 1, 2020, local authorities had culled 17,828 sidered particularly dangerous because of its human poultries after the H5N1 outbreak, according to China’s fatality rate of over 50% to date and because of the risk Ministry of Agriculture and Rural Affairs statement [3]. that the virus may develop the ability to pass efficiently between humans. As of October 2020, the World Health Organization (WHO) reported a total of 861 confirmed *Correspondence: mhariono@usd.ac.id human cases which resulted in the deaths of 455 peo- Faculty of Pharmacy, Sanata Dharma University, Campus III, Paingan, ple since 2003 [6]. Thus, these instances in history war - Maguwoharjo, Depok, Sleman 55282, Yogyakarta, Indonesia rant an enhanced pandemic preparedness, especially Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Hariyono et al. Appl Biol Chem (2021) 64:69 Page 2 of 17 in this COVID-19 season. Although there have been no strong as the known drugs, it may overcome the drug i.e. detected complications across the population in either H274Y for the N1 protein [22]. seasonal or pandemic H1N1 influenza, the elderly patient In one of the more recent research, safflomin A (SA) has a higher risk for hospitalization and mortality during was reported to show inhibition against NAs from H1N1 the seasonal H1N1 influenza [7]. In particular, patients and H3N2 influenza viruses. The antiviral assay using having specific chronic conditions such as heart disor - MDCK cells which was infected by H1N1 and H3N2 der, lung disease, hyperglycemia, renal failure, rheumat- influenza virus exhibited the synergistic effect of SA with ics, dementia, and thromboembolism are at high risk of oseltamivir in viral cell proliferation. The kinetics study influenza complications [8, 9]. of SA demonstrated a non-competitive inhibition against The influenza virus is known to be rapidly mutated in N1 and N2 NA. Furthermore, a molecular docking study which oral anti-influenza, oseltamivir (Tamiflu), has been predicted that SA interacted with N1 and N2 NA at the less potent to the H1N1 infection [10]. Therefore, a study non-catalytic site. These results suggested that SA, which on finding a new anti-influenza agent should be continu - has a chalcone backbone, may serve as a potential thera- ously encouraged. Neuraminidase is the most common peutic option to the currently available anti-influenza targeted protein in the therapy of influenza, although agents to overcome the drug resistance [23]. The struc - hemaglutinin and M2 ion channels were also studied ture of NSC89853, SA, oseltamivir and zanamivir are [11]. NA has an important role in cleaving new virions presented in Fig. 1. from the infected cell [12]. Once it is blocked, the new In this present study, we examined 10 chalcone deriva- virus could not spread over other organs [13]. Reverting tives for their predicted binding affinities into the non- to the problem of virus resistance, a new chemical that catalytic site of H1N1 and H5N1 NA using a molecular has not been recognized by the virus is highly desired docking study. The non-catalytic location is at the back of [14]. the NA’s catalytic site within loop 150. Molecular dock- A series of chalcone compounds have been reported to ing of chalcones into the NA catalytic site was also con- demonstrate anti-neuraminidase activity [15] together ducted to compare their predicted binding affinities with with their quantitative-structure activity relationships the ones in the non-catalytic site. Further in silico stud- (QSARs) study. The QSAR model suggested that a func - ies to predict the drug-like likeness were also carried out tional group having electronic characters would increase employing Lipinski Rule, pharmacokinetic profile and the activity of the chalcone. In contrast, the steric effect toxicity prediction. The study was then followed by test - would decrease its activity against H1N1 NA. Other ing for their biological activities against H5N1 and H1N1 chalcones having anti-NA activities were isolated from NA using in vitro MUNANA assay. In addition, the safety Glycyrrhiza uralensis [16], Erythrina addisoniae [17], index was also calculated by studying the cytotoxicity Glycyrrhiza inflate [18], Polygala karensium [18], and effect of the most active compound toward a normal cell. Angelica keiskei [19] further supporting the claim of chal- Two chalcones demonstrated potential activity as neu- cones as potential NA inhibitors. raminidase of H5N1 and H1N1 inhibitors, respectively, However, upon inspection of the chemical structure, with low toxicities to the normal cell lines and predicted chalcone might not be a favorable scaffold for NA inhibi - to have considerably good drug-like structures. tors which usually belong to the shikimic acid structure. The shikimic acid inhibitors such as oseltamivir and zan - Results amivir bind to the catalytic site of NA, which is a small Molecular docking pocket surrounded by basic sub-pocket, acidic sub- The ten chalcone derivatives have been predicted for pocket as well as hydrophobic sub-pocket alongside its their molecular interactions using docking study as conserved amino acid residues [20]. Chalcone is a phe- presented in Table  1. The ΔG ranged at −  6.12 to bind nyl ring extended by an α,β-unsaturated carbonyl with − 7.97 kcal/mol and − 6.34 to − 8.31 kcal/mol at H1N1 various alkyl or aryl moieties next to the carbonyl group, NA and H5N1 NA non-catalytic sites, respectively. These making it too bulky to fit into the NA catalytic site [21]. results describe that chalcone has a potency from a low A chalcone compound, namely 2-amino-5-[3-[4-[(E)-4- to moderate micromolar activity (K 32.71 to 1.44 µM at chloro-3-oxobut-1-enyl]anilino]propyl]-4-methyl-1H-py- H1N1 NA; and K 22.58 to 0.82 µM at H5N1 NA) to bind rimidin-6-one (NSC89853) is reported to show H5N1 at this non-catalytic site and disrupt the enzyme activity. NA inhibition from the screening of 20 compounds from The superimposition of ten chalcone derivatives in the NCI database. This inhibition may be due to the bind - non-catalytic site of H1N1 and H5N1 NAs is illustrated ing to the protein with an alternative mode, that is dif- in Fig. 2. ferent from the known drugs (oseltamivir or zanamivir). To compare the binding mode of the ligands to the Although the potency of such a compound was not as common site, a molecular docking study was also Har iyono et al. Appl Biol Chem (2021) 64:69 Page 3 of 17 Fig. 1 The structure of a NSC89853, and b SA, which are suggested to be non-catalytic site inhibitors of H5N1 and H1N1, respectively, whereas c oseltamivir, and d zanamivir are known NA inhibitors targetting the catalytic sites Table 1 The docking results of 10 chalcone derivatives in the non-catalytic site of H1N1 and H5N1 NA Compounds H1N1 NA H5N1 NA ΔG ResiduesPredicted K ΔG ResiduesPredicted K bind i bind i (kcal/mol) (kcal/mol) 1a − 6.48 ARG118, ARG156, ARG430 17.68 μM − 7.12 ARG118, ARG156, ARG430 6.02 μM 2a − 6.92 SER196, VAL203, LYS207 8.50 μM − 8.31 LEU134, ARG156, ARG430 815.58 nM 3a − 6.68 SER196, ARG207 12.66 μM − 8.18 VAL116, ARG118, LEU134, THR135, 1.00 μM ARG156, ARG430 1b − 6.12 PHE174, SER196 32.71 μM − 6.34 ARG118, ARG430, THR439 22.58 μM 2b − 6.18 LYS150, VAL177 29.34 μM − 7.32 VAL116, GLN136, GLY147, VAL149, 4.33 μM HIS155 3b − 7.15 VAL116, ALA138, HIS144, ILE149 5.74 μM − 7.58 VAL116, ARG118, VAL149, ASP151, 2.80 μM THR439 4b − 7.97 LYS150, VAL177, SER196, ASP199, 1.44 μM − 7.89 ARG118, ARG156, ARG430, PRO431 1.65 μM VAL205 1c − 6.72 HIS144, ILE149, THR438 11.82 μM − 6.53 VAL116, ILE117, ARG118, LEU134, 18.30 μM ARG430 2c − 6.02 LYS143, HIS144 38.83 μM − 7.55 ARG118, HIS144, VAL149, ASP151, 2.95 μM HIS155, THR439 3c − 6.27 HIS144, TYR155 25.18 μM − 7.94 VAL116, ARG118, LEU134, ARG156, 1.52 μM ARG430, PRO431 conducted into the H1N1 and H5N1 NA catalytic sites. individual NA which are overlapped with its initial pose The parameterization of molecular docking was con - is presented in Additional file 1: Figure S1. trolled by re-docking oseltamivir-triazole into the H1N1 The control docking pose closely interacts with the NA and oseltamivir into the H5N1 NA catalytic site. This conserved amino acid residues such as ARG118, ASP151, resulted in RMSD 1.02  Å of the most populated cluster TRP179, ARG293, ARG368 and TYR402 via hydrogen bondings. The parameter of that control docking is then (85%) with ΔG − 9.35 kcal/mol at H1N1 NA, whereas bind used to dock 10 chalcone derivatives into both H1N1 the RMSD of oseltamivir at H5N1 NA active site was and H5N1 NA active site binding pockets. The docking 1.9  Å of the most populated cluster (74%) with the low- results of 10 chalcone derivatives are presented in Table 2 est energy (ΔG − 7.52 kcal/mol). This defines that the bind represented by the ΔG parameter is accepted for further docking of chalcone , interacting residues and the bind compounds. The control docking pose of ligands to their predicted K . The docking into H1N1 results in ΔG i bind Hariyono et al. Appl Biol Chem (2021) 64:69 Page 4 of 17 Fig. 2 The superimposition of 10 chalcone derivatives (yellow sticks) in the non-catalytic site of a H1N1 and b H5N1 NA. The NA is presented in the surface model and the ligands are in the stick models, docked in the behind of the catalytic site (yellow circles) Table 2 The docking results of 10 chalcone derivatives in the catalytic site of H1N1 and H5N1 NA Compounds H1N1 NA H5N1 NA ΔG ResiduesPredicted K ΔG (kcal/mol) ResiduesPredicted K bind i bind i (kcal/mol) 1a − 6.37 ARG152, ARG156, ARG225, GLU277, 21.55 μM − 5.69 GLU119, ARG152, ARG156, ILE222, 67.9 μM GLU278 SER246 2a − 7.71 ARG152, ARG225, THR226, GLU278 2.22 μM − 6.66 ARG118, ARG152, ILE222, GLY244, 13.07 μM SER246, ARG292, ARG371 3a − 7.39 GLU119, LYS150, ASP151, ASP152, 3.86 μM − 6.07 ARG118, ARG152, ARG224 THR225, 35.7 μM ARG225, GLU277, GLU278 GLU277, TYR347, ARG371, TYR406 1b − 7.36 ARG118, ARG152, TRP179, GLU277, 4.03 μM − 6.26 ARG152, ARG156, ILE222, SER246, 25.86 μM GLU278, ARG293 TYR406 2b − 8.10 ARG118, ARG152, TRP179, GLU277, 1.15 μM − 6.52 ARG118, ASP151, ARG156, ARG292, 16.63 μM ARG293, ASN295 TYR347, ARG371 3b − 8.34 ARG118, ARG152, ARG156, TRP179, 0.772 μM − 6.41 ARG118, ASP151, ARG156, ARG224, 20.05 μM GLU278, ARG293, ASN295 GLU276, ARG277, ARG371 4b − 7.13 ARG118, LEU134, LYS150, ASP151, 5.91 μM − 6.88 ARG118, ASP151, ARG156, GLU277, 9.04 μM ARG152, ARG293, ASN344 ARG292, TYR347, ARG371 1c − 6.57 GLU119, ARG152, ARG156, GLU277, 15.34 μM − 5.05 ARG118, ASP151, ARG152, TRP178, 200.37 μM GLU278 ARG224, TYR347, ARG371, ARG430, PRO431 2c − 7.49 GLU119, LYS150, ASP151, ARG152, 3.25 μM − 6.86 ARG118, GLU119, LEU134, ASP151, 9.41 μM TRP179, ARG293, ASN344, ARG368, ARG152, ARG156, ARG224, TYR402 GLU227, TYR347, ARG371, ILE427, ARG430, PRO431 3c − 7.75 ARG118, GLU119, LYS150, ASP151, 2.09 μM − 6.93 GLU119, ARG152, ARG156, ILE222, 8.32 μM ARG152, TRP179, ARG293, ASN344, ARG224, GLY244 ARG368, TYR402 of docked chalcones into the H1N1 catalytic site are − 6.37 to − 8.34 kcal/mol, whereas it is ΔG − 5.05 to bind likely lower than that of H1N1 at the non-catalytic one. −  6.93  kcal/mol into H5N1 for the overlapped-docked In contrast, the chalcones docked into the H5N1 have pose which is presented in Fig. 3. The calculated energies Har iyono et al. Appl Biol Chem (2021) 64:69 Page 5 of 17 Fig. 3 The superimposition of 10 chalcone derivatives (yellow sticks) in the active site of a H1N1 NA (blue) and b H5N1 NA (red) in the ribbon model lower free energy of binding in the non-catalytic than in via hydrogen bond with its receptor [24]. The rotat - the catalytic sites. However, except for compounds 1a, able bonds may influence their stability during ADME 2a and 3a, its long alkyl chain escaped from the catalytic (absorption, distribution, metabolism, and excretion) and site, indicating their non-fit binding into both H1N1 and the receptor binding. Thus, the less flexible the chain in H5N1 NA’s catalytic sites. Table  2 tabulated the docking the molecule, the more stable the drug is to perform its results of the 10 compounds in the active site of H1N1, activity [25]. The Polar Surface Area (PSA) is related to H5N1 NA, and their superposition as illustrated in Fig. 3. the permeability of drugs across the cell membrane in which the higher the PSA, the poorer the cell permeabil- Lipinski rule ity (oral bioavailability) is [25]. According to the Lipinski Rule, an ideal drug should have In general, the results show that all chalcones meet the a molecular weight (MW) which is less than 500  Da, < 5 MW, the number of HBD, HBA and the rotatable bonds log P, ≤ 5 Hydrogen Bond Donor (HBD), ≤ 10 Hydrogen requirements. However, compounds 2b and 3b slightly Bond Acceptor (HBA), ≤ 10 rotatable bonds, and ≤ 140 Å deviate on the log P limit, whereas compounds 2c, 3c and surface area [24, 25]. The potency of the compound to be 4b do not fulfill the limit of the surface area requirement. drug is commonly expressed by I C , which is affected Table  3 presents the chalcones with their Lipinski Rule by its MW. The more potent drug should have a higher profiles. MW to minimize its dose in performing a pharmacologi- cal activity. Nevertheless, the MW should not be higher Mutagenicity and toxicity profiles than 500 considering the drug absorption via the intesti- The in silico toxicity predictions have become a routine nal membrane [24]. before processing compounds to be drug candidates. In The log P is the concentration of the compound in this study, we also predicted the mutagenicity and toxic- n-octanol divided by its concentration in water. There - ity profiles of the 10 chalcones. Generally, a compound’s fore, it reflects the balance of the compound’s solubility mutagenic properties are usually confirmed using the in water during oral dissolution steps with its oral bioa- AMES test [26, 27]. The human Maximum Tolerated vailability in the blood system [24, 25]. The ideal log P < 5 Dose (hMTD) is acceptable when the predicted toxic indicates that the compounds are well soluble in the body dose threshold in humans is more than 0.477. The potas - fluid as well as absorbed through the gastrointestinal cell sium channels that mediate the cardiac repolarization membrane, which then be transferred into the blood vas- in humans are represented by the values of hERG I and cular. On the other hand, the number of HBD or HBA is II. Hence, inhibiting this kind of protein would cause a associated with polarity to interact with water during the long QT syndrome development that might lead to a dissolution process as well as their molecular interaction fatal arrhythmia. The in  vivo toxicity is often expressed Hariyono et al. Appl Biol Chem (2021) 64:69 Page 6 of 17 Table 3 The drug-like likeness profile of 10 chalcones was studied as NA inhibitors Ligands MW log P HBD HBA Rotatable bonds Surface area 1a 242.249 3.4274 1 2 3 104.015 1b 284.330 4.5090 0 2 5 123.429 1c 312.365 4.0857 1 4 7 135.536 2a 269.256 3.1965 1 4 4 114.502 2b 310.368 5.0432 0 2 5 135.153 2c 390.435 4.8845 1 5 8 169.342 3a 268.268 2.9865 2 3 4 115.170 3b 318.347 5.5141 0 2 5 139.391 3c 360.409 4.8759 1 4 7 157.864 4b 337.375 4.8123 0 4 6 145.641 by LD value which can be defined as the required dose be interpreted as they are able to cause any observable given to cause 50% death of a group of rats in evaluating a chronic toxicity at the lower amount and/or shorter time compound acute toxicity represented by ORAT (log L D of exposure. Several compounds (2c, 3a, 3b and 3c) dem- value) prediction. onstrate T. Pyriformis toxicity potency, whereas, based LOAEL (lowest-observed-adverse-effect level) associ - on the minnow toxicity prediction, compounds 1a, 2a, 3a ates with the compound’s lowest concentration which and 4b are deemed to be safe. Table 4 presents the AMES causes an adverse effect in human physiology. This is test results of the chalcones for mutagenicity prediction indicated by the alteration of morphology, function, along with other toxicity profiles. growth, or development. The safety of a compound is higher as the LOAEL value increases. The liver injury Pharmacokinetic profiles commonly reflects the drug hepatotoxic properties, Table 5 demonstrates that all chalcones are well absorbed whereas the potential dermal adverse effects are deter - through the human intestinal with near values to 100% mined by skin sensitization properties. The value of toxic into the blood system. In general, the water solubility of endpoints, however, is measured by  T. pyriformis  and all chalcones are most likely poor as their log S value are minnow toxicity [28]. lower than − 4, except for 3a which has a log S value of Nowadays, the drugs’ safety to the environment is a − 2.601; indicating that it may dissolve readily in the dis- concern. Low environmental damage is demonstrated by solution step. Furthermore, oral absorption prediction the values of T. pyriformis and minnow toxicity which are using Caco2 cell model [32] with the required value is to be respectively higher than 0.5 and − 0.3. Based on the higher than 0.90. Therefore, except for 2c and 4b, chal - AMES test, two compounds i.e. 2a and 4b are predicted cones show good human gastrointestinal absorption. not to be toxic, while 4b might have the possibility to be The skin permeability of chalcones is most likely suit - hepatotoxic. No chalcones inhibit the hERG I protein, able for the transdermal route because the values are but 1c, 2b, 2c and 3c inhibit hERG II. Most of the chal- approximately −  2.5. A protein transport namely P-gly- cones have a low maximum dose which is weakly toler- coprotein (P-gp) is crucial during the pharmacokinet- ated in humans, except for 2a, 2b and 4b. In addition, all ics steps, however, this could have either advantages or chalcones exhibit no skin sensitization. The oral rat acute disadvantages in therapeutic effect [33]. A compound is toxicity LD can be classified as very toxic (≤ 5  mg/kg), supposed to not inhibit P-gp, either P-gp I or P-gp II. In toxic or moderately toxic (> 5 to < 500  mg/kg), harmful the ideal situation, it should not be acting as P-gp sub- or slightly toxic (> 500 to < 2000  mg/kg), and non-toxic strate either. According to the prediction, compounds (> 2000  mg/kg) [29]. All compounds are predicted to be 1c and 2b are predicted to inhibit the P-gp I activity, potentially less toxic with L D ranging from 194.01 to whereas compounds 2c, 3c, and 4b inhibited both P-gp 399  mg/kg. Oral rat LOAEL value shows that the inges- I and P-gp II. In contrast, 2a acts like the substrate for tion chronic toxicity is relative to the limit of concen- P-gp, accordingly. tration and length of exposure, however, LOAEL does One of the distribution parameters is defined by the not solely represent drug safety accurately [30, 31]. This number of VDss or volume of distribution at a steady prediction showed that compounds 2a, 2c, 3a and 4b state, which  is directly proportional with the amount have lower values than the other compounds, which can of drug distributed into tissue; a higher V   indicates a D Har iyono et al. Appl Biol Chem (2021) 64:69 Page 7 of 17 Table 4 The AMES test result of the chalcones for mutagenicity prediction along with other toxicity profiles Ligands AMES toxicity hMTD hERG I hERG II ORAT (log LD ) ORCT (log Hepatotoxicity Skin T. Pyriformis toxicity Minnow toxicity inhibitor inhibitor LOAEL) Sensitisation 1a No 0.646 No No 2.228 2.203 No No 1.380 0.262 1b No 0.830 No No 2.324 2.321 No No 1.115 − 1.141 1c No 0.611 No Yes 2.318 2.044 No No 1.207 − 0.820 2a Yes 0.039 No No 2.485 1.267 No No 1.231 − 0.229 2b No 0.429 No Yes 2.360 2.117 No No 1.664 − 0.595 2c No 0.521 No Yes 2.577 1.164 No No 0.353 − 1.641 3a No 0.638 No No 2.233 1.715 No No 0.286 0.520 3b No 0.669 No Yes 2.519 2.330 No No 0.421 − 1.293 3c No 0.502 No Yes 2.524 2.125 No No 0.419 − 2.152 4b Yes 0.039 No No 2.601 1.231 Yes No 1.207 − 0.232 Hariyono et al. Appl Biol Chem (2021) 64:69 Page 8 of 17 Table 5 The absorption profiles of chalcones as predicted by the software Ligands Water solubility Caco2 Intestinal Skin permeability P-glycoprotein P-glycoprotein I P-glycoprotein permeability absorption substrate inhibitor II inhibitor (human) 1a − 4.140 1.426 93.706 − 2.315 No No No 1b − 5.655 1.423 96.342 − 2.434 No No No 1c − 4.798 1.570 93.360 − 2.483 No Yes No 2a − 4.312 0.904 91.606 − 2.627 Yes No No 2b − 5.636 1.636 95.062 − 2.327 No Yes No 2c − 6.726 0.556 93.240 − 2.699 No Yes Yes 3a − 2.601 0.949 100.000 − 2.683 No No No 3b − 6.714 1.684 93.229 − 2.677 No No Yes 3c − 6.342 1.078 95.102 − 2.678 No Yes Yes 4b − 5.522 0.343 93.495 − 2.659 Yes Yes Yes greater amount of tissue distribution. As a rule, VDss subfamilies, which play significant roles in drug metabo - should be ≥ − 0.15. Among the 10 chalcones, three com- lism [35]. CYP2D6 and CYP3A4 are found in the brain pounds (2c, 3a, and 3c) do not meet these criteria. The and intestines, respectively, and are most likely respon- fraction unbound (fu) for all chalcones are predicted to sible to metabolize the drug in their surrounding areas. be ≤ 0.15, indicating that more fractions of drug molecule Furthermore, CYP3A4 also affects oral bioavailability by are bound to the plasma protein. Besides VDss, the chal- first-pass metabolism. A compound that binds strongly cones were also predicted for their ability to cross the to these CYPs could be acting as the substrate or inhibi- brain membrane which is important as the compounds tor leading to the lower / higher bioavailability as well may affect the Central Nervous System (CNS) [34]. Val - as activity of other drugs. This could be potential for the ues < − 1 indicate poor Blood–Brain Barrier (BBB) per- drug causing clinical drug-drug interactions, leading to meability and < − 3 for CNS permeability. In other words, adverse reactions or therapeutic failures. In this predic- the compound is poorly distributed to the brain and una- tion, neither chalcones act as the substrate nor inhibi- ble to penetrate CNS. From the results, all the 10 com- tor for CYP2D6. However, except for 3a, all compounds pounds should be carefully managed as there is potential act as CYP3A4 substrate, whereas 2c and 3c act as this for these chalcones to enter the CNS especially for 1a, enzyme’s inhibitor. Furthermore, chalcones are likely to 1b, 2b and 3b which can also penetrate BBB. The distri - inhibit the CYP1A2, CYP2C19, and CYP2C9 presented bution profiles of the chalcones as predicted by software in Table  7, which should be of concern when the com- are presented in Table 6. pound is consumed with other drugs. Metabolisms are also an important indicator of good Total clearance describes the compound’s rate while drug-like properties. CYP1A2, CYP2C9, CYP2C19, being removed from the body [36]. One of the main renal CYP2D6, CYP3A4 are among the cytochrome P450 uptake transporters to remove the drug from the blood Table 6 The distribution profile of chalcones as predicted by the software Ligands VDss (human) Fraction unbound (fu) BBB permeability CNS permeability (human) 1a 0.015 0.058 0.012 − 1.641 1b 0.106 0.000 0.425 − 1.298 1c 0.087 0.000 − 0.233 − 2.219 2a 0.041 0.000 − 0.328 − 2.123 2b 0.527 0.000 0.398 − 1.465 2c − 0.476 0.017 − 0.257 − 2.157 3a − 2.366 0.000 − 0.200 − 2.253 3b − 0.083 0.035 0.479 − 1.054 3c − 0.227 0.024 − 0.258 − 2.053 4b 0.638 0.000 − 0.341 − 1.930 Har iyono et al. Appl Biol Chem (2021) 64:69 Page 9 of 17 Table 7 The interaction between chalcones with a diverse CYP subfamily Ligands CYP2D6 CYP3A4 CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 substrate substrate inhibitor inhibitor inhibitor inhibitor inhibitor 1a No Yes Yes Yes No No No 1b No Yes Yes Yes Yes No No 1c No Yes Yes Yes Yes No No 2a No Yes Yes Yes Yes No No 2b No Yes Yes Yes Yes No No 2c No Yes Yes Yes Yes No Yes 3a No No No No No No No 3b No Yes Yes Yes Yes No No 3c No Yes Yes Yes Yes No Yes 4b No Yes Yes Yes Yes No No is the OCT2 transporter. It plays a pivotal role in the Neuraminidase assay removal and renal clearance of mostly cationic drugs as The chalcone derivatives have been examined for their well as endogenous compounds [37]. Inhibition of OCT2 inhibitions toward the activity of H1N1 and H5N1 influ - (such as by cimetidine) elevates OCT2-dependent renal enza neuraminidase. The first screening using 100  µg/ clearance drugs, hence altering pharmacokinetics and ml of the sample’s concentration showed that five com - pharmacodynamics profiles. Compound 3a is predicted pounds exhibited 0–10% inhibition toward H1N1 NA, to be the fastest compound excreted from the body due while only one compound showed activity in the same to its highest total clearance. In contrast, compound 2b range against H5N1. Furthermore, three compounds is the slowest compound to be removed from the body exhibited 10–50% inhibition toward H1N1, while five due to its lowest total clearance. All chalcones have low compounds demonstrated inhibition against H5N1 at the total clearance (log Cl < 0.763), yet, it is generally desir- same inhibition percentage. In the H5N1 NA inhibition able to develop a drug for oral administration without a assay, two compounds (1b and 2c) were inactive. Inter- high dosage regimen [38]. One chalcone, 2b, is predicted estingly, two compounds (1c and 2b) showed more than to act as renal OCT2 substrate that might lead to unde- 50% inhibition to both H5N1 and H1N1. Therefore, they sirable side effects. Figure  4 illustrates the total clearance were selected to be further studied on their IC as well of all chalcones that reflects their rate to be eliminated as CC . Table 8 presents the assay results of ten chalcone from the body system. compounds compared to vanillin as the positive control. Fig. 4 The histogram of total renal clearance profiles of the chalcones with its OCT2-dependence clearance, green block = OC T2-independent and yellow block = OC T2-dependent Hariyono et al. Appl Biol Chem (2021) 64:69 Page 10 of 17 Table 8 The assay result of ten chalcone compounds with their R side chains as depicted in Fig. 5 (100 µg/mL) against H5N1 and H1N1 NA compared to vanillin as the positive control Compounds R R R R % Inhibition ± SE 1 2 3 4 H1N1 NA H5N1 NA 1a H OH H F 21 ± 7 23 ± 11 2a H OH H NO 16 ± 2 21 ± 2 3a H OH H COOH 19 ± 2 27 ± 0 1b H O-isopropyl H F 5 ± 8 0 ± 3 2b H O-cyclopentyl H F 69 ± 2 70 ± 1 3b H O-benzyl H F 2 ± 4 18 ± 3 4b H O-cyclopentyl H NO 5 ± 5 15 ± 3 1c OCH OH H O-butyl 83 ± 1 82 ± 3 2c OCH OH OCH O-benzyl 1 ± 4 − 2 ± 16 3 3 3c H OH OCH O-benzyl 2 ± 4 7 ± 8 Vanillin – – – – 86 ± 2 84 ± 4 Cytotoxicity assay R R 2 4 In the cytotoxicity assay results (Fig. 7), 1c showed a high concentration to inhibit the Vero cell proliferation with CC 968.16 µM. The safety index of this compound cal - culated with the safety index (SI) values was 34.44 and R R 1 3 35.69 for H1N1 and H5N1 NA, respectively. In addition, 2b showed CC 757.18  µM with the SI values = 8.65 and 10.35 for H1N1 and H5N1, respectively. The graph Fig. 5 The chalcone backbone scaffold of 10 chalcone derivatives plotting log concentration of 1c and 2b vs % cell viability against Vero cell in the cytotoxicity studies is presented in Fig. 7a. Furthermore, the cell imaging in Fig. 7b exhib- The IC of 1c was then estimated as 28.11  µM and ited the cell proliferation survival, when there was no 27.63  µM for H1N1 and H5N1, respectively. Compound treatment to the cell, which was indicated by the forma- 2b also demonstrated similar % inhibition toward H1N1 tion of formazan crystals upon MTT reaction. Formazan and H5N1 NA with its I C 87.54  µM and 73.17  µM, crystal indicates the ability of the cell to express NADPH- respectively. This is quite interesting because the com - dependent oxidoreductase to reduce the MTT, which pound showed similar IC when it was applied to both leads to cell proliferation. In contrast, the formazan crys- H5N1 and H1N1 NA. Figure  6 presents the drug-dose tal formation will be reduced along with the treatment of depending curve of 1c and 2b in inhibiting the H1N1 and 1c and 2b on the Vero cell leading to their cytotoxicity H5N1 NA. properties (Fig. 7a). Fig. 6 The drug–dose depending curve of 1c and 2b in inhibiting H1N1 and H5N1 NA Har iyono et al. Appl Biol Chem (2021) 64:69 Page 11 of 17 Fig. 7 The cytotoxicity profiles of 1c and 2b plotting a log concentration of vs % Vero cell viability of 1c and 2b, whereas b is the Vero cell imaging without any compound exposure (negative control), and c with 1c treatment to the cells. The orange arrows indicate the formazan crystal formation which is higher in 1c absence than its presence, accordingly a particularly striking feature in the catalytic domain Discussion referred to as the ‘150 loop’ [39, 45]. The shikimic acid The 2009 H1N1 NA is one of several 2009 pandemic scaffold has been classically patterned for NA inhibitors influenza virus mutants in which the I223 changed to due to its capability to form a stable chair conformation R223 (I223R). This mutation results in shrinkage of the transition state of the non-aromatic six-member ring enzyme’s active site affecting the binding of NA inhibi - with the rather flat oxonium cation, and thereby arrange tor [39]. Another mutant is H274Y (H1N1), which was the position of important residues to interact with the also reported to be resistant to oseltamivir as confirmed shikimic binding groups [45]. Taking this into consid- in about 20% isolates from humans in Europe [40, 41]. eration, some aromatic compounds have been devised NA’s active site is composed of eight functional resi- to mimic this transition state leading to their inhibition dues (ARG118, ASP151, ARG152, ARG224, GLU276, against NA [46, 47]. ARG292, ARG371 and TYR406) and surrounded by In the present study, two chalcones (1c and 2b) have 11 framework residues (GLU119, ARG156, TRP178, been observed to have the best in  vitro results com- SER179, ASP198, ILE222, GLU227, HIS274, GLU277, pared to the other 8 chalcones. Figures 8 and 9 illustrate ASN294 and GLU425) [42]. the molecular interactions of 1c and 2b, respectively, Structurally, chalcone consists of one aromatic ring while binding into the H1N1 (non- and catalytic sites) extended by a predominantly trans-configuration of α,β- and H5N1 (non- and catalytic sites). During the bind- unsaturated carbonyl. The relatively short carbon–carbon ing into the non-catalytic site of H1N1 NA, 1c interacts distance between α,β-unsaturated alongside the relatively via H-bond with only THR438. The other interactions long C–C distances is 1.326 Å and 1.46 Å, which is con- are vdW interactions with ASN146 and HIS144; amide sistent with a localized double bond in the enone unit in pi-stacked with HIS144 and ILE149; and pi-alkyl inter- this structure [43]. The chain next to the carbonyl group actions with VAL116, ALA138 and SER145. In contrast, is usually prolonged by either alkyl or aryl moieties with the binding to the catalytic site of H1N1 NA shows that various functional groups being attached. The conjugated 1c interacts with ASP151, ARG293, ASN344, ARG368, double bond could make this type of compound less and TYR402 via H-bonds, whereas pi-alkyl and alkyl- flexible. However, the C=O bond can present as either alkyl interactions are also observed with LYS150 and S-cis or S-trans conformation with respect to the vinylic LEU134, respectively. In addition, pi-anion interaction double bond due to the free rotation along with the sin- is observed with GLU119. Compound 1c interacts with gle bond between C-carbonylic and Cα. This, therefore, the non-catalytic site of H5N1 NA via H-bonds with could increase the conformation stability of such com- VAL116, ARG118, and ARG430; amide pi-stacked with pounds during a dynamic environment [44]. ILE117; and pi-alkyl with VAL116, ARG118, ARG430 The combination of in silico and in  vitro studies has and PRO431. Furthermore, in its catalytic site, the suggested a new binding mode for chalcone compounds interactions are observed via H-bonds with ARG152, into N1 NA. The active site of NA is decorated by a rel - ARG371, TYR347, and ARG430; alkyl interactions with atively small pocket surrounded by some sub-pockets TRP178, ARG224, and PRO431; pi-cation as well as determining the binding region of such enzyme with Hariyono et al. Appl Biol Chem (2021) 64:69 Page 12 of 17 Fig. 8 The molecular interactions of 1c with a non-catalytic H1N1, b catalytic H1N1, c non-catalytic H5N1, and d catalytic H5N1 NA sites. The green, pale green, purple, magenta, pink, cyan, and orange represent H-bond, vdW, pi-sigma, amide pi-stacked, pi-alkyl, pi-halogen, and pi-cation/anion, respectively pi-cation interactions are observed with ARG118 and The possibility of chalcone to interact with the active ASP151, respectively. site of NA is only in one aromatic ring augmented by the The second best compound (2b) also interacts via binding group such as OH or OCH . The α,β-unsaturated H-bond with the non-catalytic site of H1N1 NA with chain most likely protruded outside of the active pocket. LYS150; pi-alkyl with PRO154, VAL177, PRO198 and Although the ΔG of chalcone in the active site is con- bind VAL205. In contrast, the interactions with the cata- sidered acceptable, it is inconsistent with the in  vitro lytic site via H-bonds are absent. However, the remain- results. Compound 1c has relatively higher ΔG in both bind ing interactions of 2b are observed with ARG118 and NAs’ active sites than other compounds with poor inhibi- ARG152 via pi-alkyl interactions; GLU277, ARG293 tion against NAs. However, the in  vitro results showed and ASN295 via pi-halogen interactions; with TRP179 that 1c is the most active compared to others; indicating via pi-lone pair electron interaction. The non-catalytic a poor correlation between in vitro and in silico studies. site of H5N1 NA shows interactions with chalcones In contrast, although 2c is inactive against NAs in  vitro, via H-bonds with GLY147 and pi-alkyl with VAL116, the ΔG in both NA’s active sites is considered fair. bind ALA138, VAL149. In addition, amide pi-stacked with Notably, compounds with –NO group (2a and 4b) HIS155 is also observed. In the H5N1 NA catalytic show the lowest binding energy in either non-catalytic site, chalcones make interactions via H-bonds with or catalytic sites for both H1N1 and H5N1 NAs. How- ARG118, ARG156 and ARG371. Furthermore, there ever, the in  vitro results demonstrated a low percentage are also amide pi-stacked interaction with TYR347, of inhibition toward both NAs. Nitro group is the strong- as well as pi-cation/anion with ASP151 and ARG292, est electron-withdrawing, which reactively interacts respectively. with atoms having an electron-donating group to form Har iyono et al. Appl Biol Chem (2021) 64:69 Page 13 of 17 Fig. 9 The molecular interactions of 2b with a non-catalytic H1N1, b catalytic H1N1, c non-catalytic H5N1, and d catalytic H5N1 NA sites. The green, pale green, purple, magenta, pink, cyan, yellowish-green and orange represent H-bond, vdW, pi-sigma, amide pi-stacked, pi-alkyl, pi-halogen, pi-lone pair electrons, and pi-cation/anion, respectively H-bond as well as electronic interactions [48]. This false- association and dissociation rates using Surface Plasmon positive results could be due to the stability of the –NO Resonance (SPR). In this method, the protein is immo- group, which is easily reduced by the acidic pH turn- bilised onto a biosensor surface while the drug ligand is ing into an amine group (nitro reduction) [49], which is continuously flowing across the biosensor surface, where measured in pH 6.5 in this MUNANA system. This con - it binds to the immobilised receptor. The binding is meas - version could make the 2b and 4b lose their interactions ured by the kinetic association and dissociation rates (k / as suggested by the docking study, leading to their low k ) for several different ligand concentrations [50]. inhibition against NA. The inconsistent results of 2b and The second method is ITC, which measures the heat 3b are not fully understood as well. Therefore, we sug - transfer during binding that enables accurate determi- gest that a molecular dynamics simulation is performed nation of binding constants (K ), reaction stoichiom- to elucidate this phenomenon in future studies. On the etry (n), enthalpy (∆H) and entropy (ΔS). This provides a other hand, there are inconsistent results between dock- complete thermodynamic profile of the molecular inter - ing and in vitro results when the chalcones were docked action. This deeper understanding of structure–function into either non-catalytic and catalytic sites of H5N1. relationships enables more confident decision making in Therefore, it is recommended to study the kinetics of NA hit selection and lead optimization [51]. inhibition for the two most active compounds (1c and In this present study, an array of  in silico  predictions 2b) to confirm whether the mode of inhibition is com - has been performed on the chalcones including Lipin- petitive or non-competitive. ski Rule, mutagenicity, toxicity and pharmacokinetic The kinetic assay can be carried out using meth - profiles. The chalcones have diverse functional groups ods such as Biacore or Isothermal Titration Calorim- conferring the distinction of their drug-like properties, etry (ITC). Biacore measures the real-time binding mutagenicity, toxicity, and pharmacokinetics, which will Hariyono et al. Appl Biol Chem (2021) 64:69 Page 14 of 17 contribute to their overall therapeutic effects. Given that compounds have a good safety index augmenting their the two compounds (1c and 2b) are active in the in vitro potency as the potential H5N1 and H1N1 NA inhibitors. study, we are motivated to understand the possibility of these compounds to be considered as lead candidates Materials and methods for optimization. They have good Lipinski Rule profiles Chemistry by meeting the requirements of MW, log P, number of The chalcone compounds (Fig.  10) were synthesized HBD-HBA, number of rotatable bonds and the surface using the established method in our laboratory [21, 52, area. Both compounds are also not responsive against 53]. We scale up the production of the compounds and the AMES test describing their non-mutagenic potency. confirm their purity using a thin-layer chromatographic Unfortunately, 2b is predicted to have a low tolerance method and melting point test with the data of published in the maximum dose. In addition, both compounds are compounds as the reference. responsive toward hERG II as inhibitors and having an acute toxic dose of around 200 mg/kg, but irresponsive to hERG I as an inhibitor, not toxic in chronic ingestion, and Molecular docking neither hepatotoxic nor skin sensitized. In environmen- The proteins used are the resistant I223R NA mutant of tal damage, both are non-toxic to T. pyriformis as well as H1N1 2009 pandemic influenza virus (PDB ID 4B7M) minnow species. [39] and the wild type H5N1 NA (PDB ID 2HU0) [54]. These two compounds are insoluble in water. Thus, The protein structure was processed using AutoDock - consideration should be given for a suitable delivery sys- Tools 1.5.6 (www. autod ock. scrip ps. edu) with the ligand tem. They have good in Caco2 permeability, intestinal separated from the protein. The grid for docking to the human absorption and skin permeability; reflecting their non-catalytic site of H1N1 NA and H5N1 NA was set good absorption profile either in oral or topical admin - to 94 × 72 × 96 and 80 × 80 × 80 with its spacing set istration. Both compounds have potencies to inhibit the to 0.375  Å. The center of mass of the ligand was set to protein carrier during absorption but neither of them acts x = 26.852, y = −  32.014, z = −  1.019 for H1N1 NA, and as the protein substrate. In the distribution profiles, both x = − 7.07, y = 28.242, z = 107.729 for H5N1 NA. Genetic compounds bind tightly in the plasma protein, which Algorithm was chosen for docking calculation in Auto- could reduce the therapeutic dose. Furthermore, the Dock 4.2.3. The searching parameters were set to the BBB and CNS permeability should be taken into account. default values (Population size = 150, maximum number Interaction with other drugs should be evaluated as of evals = 2,500,000, maximum of generations = 27,000, well, as these two compounds are predicted to act as the maximum number of top individuals that automatically CYP3A4 substrate, CYP1A2, CYP2C19, and CYP2C9 survives = 1). The number of GA run was set to 100. inhibitors. In contrast, they are irresponsive toward Docking parameters such as random number genera- CYP2D6 and CYP3A4. The excretion of 1c is faster tion, energy parameters, and step size were also set to the than 2b since the total clearance of 1c is higher than 2b default values. The results were analyzed by checking the in addition to the potential of this compound as Renal RMSD values, ligand–protein interactions, free energy OCT2’s substrate. Overall, 1c meets the requirements of of binding (FEB) as well as the number of conformations drug-like properties at approximately 62%, whereas 2b that exist in a population cluster [55]. For the subse- is at 58%. These percentages are above 50%, therefore, quent molecular docking, the chalcone structure deriva- these two compounds should be given the opportunity to tives were sketched and energetically optimized using be optimized and developed further as drug candidates. Hyperchem Professional version 8.0 (www. hyper. com) This conclusion is supported by the in  vitro cytotoxic - with MM + force field and Polak-Ribiere (Conjugate Gra - ity study against normal cell lines confirming that both dient). The visualization of ligand–protein interaction Fig. 10 The structure of 10 chalcone derivatives (1a–4b) with different set of functional groups Har iyono et al. Appl Biol Chem (2021) 64:69 Page 15 of 17 Neuraminidase assay was conducted using Biovia Discovery Studio 2016 The H1N1 NA assay followed the general procedure of (www. accel rys. com). Fluorometric Neuraminidase Assay [57]. The H1N1 NA The docking of compounds to the catalytic site of NA (A/California/04/2009) and H5N1 NA (A/Anhui/1/2005) was carried out by using PDB 2HU0 (H5N1 NA in com- enzymes were purchased from Sinobio. The fixed con - plex with oseltamivir) [54] as well as PDB 6HP0 (H1N1 centrations of H1N1 NA (0.3  u/mL) and MUNANA NA in complex with oseltamivir triazole) [56] with (100  mM) were optimized employing the previously the same default protocol being used in PDB 4B7M, described method. Vanillin was used as the positive con- except for the number of points and its center of mass. trol inhibitors [58], and the H1N1 neuraminidase assay For 6HP0, the number of points is 40 × 40 × 40 with was prepared by mixing an assay buffer, tested samples the center of mass (x = 43.641; y = 0.53; z = 20.263). For (at concentrations 100  μg/mL in 1% of DMSO-Buffer), 2HU0, the number of points is 40 × 40 × 40 with the and a constant 0.3 unit/mL of neuraminidase which were center of mass (x = 1.763; y = 19.33; z = 108.34). pre-incubated at 37 °C for 30 min with 200 rpm. After the addition of 100  μM substrates, the reaction assays were Lipinski rule of five incubated at 37 °C for 60 min with 200 rpm. To stop the The Lipinski Rule profiles were individually predicted by reaction, 100  μl of glycine stop solution was added. The inputting SMILES string, which is automatically done by assays were carried out in triplicate. The assay proto - the server. The value of molecular weight (MW), log P, col for H5N1 inhibition assay is similar to that of H1N1 the number of hydrogen bond donors (HBD), the num- but the concentrations being used were 0.15  u/mL, and ber of hydrogen bond acceptors (HBA), the number of 50  µM for the enzyme and substrate, respectively. A rotatable bonds and the surface area were observed and series of concentrations were prepared for those demon- then tabulated. strating > 50% of enzyme inhibition to calculate the IC . The fluorescence intensity of NANA was measured by Modulus Microplate Reader with a UV optical kit at λ Mutagenicity and toxicity studies 340/440 nm. The drug-dose dependent curve and its sta - Using the same protocol as in “Lipinski rule of five” sec - tistical analysis (95% confident interval) were generated tion, the mutagenic potency of the ligands was repre- using GraphPad Prism 5.0 (https:// graph pad- prism. softw sented by the AMES test results. Other parameters such are. infor mer. com/5. 0/). as maximum tolerated dose (human) (hMTD), hERG I inhibitor, hERG II inhibitor, oral rat acute toxicity (log LD ), oral rat chronic toxicity (log LOAEL), hepatotoxic- Cytotoxicity assay ity, skin sensitization, T. pyriformis toxicity, and minnow The cytotoxicity of each compound on Vero cells was toxicity represented the toxicity properties of the ligands. determined using MTT assay. Vero cell is a non-tum- origenic cell from the kidney tissue of African green monkeys [59]. Cells will proliferate by expressing the Pharmacokinetics study NADPH-dependent oxidoreductase in mitochondria Using the same protocol in 4.3, the ADME (absorp- that reduces the MTT reagent into the reduction state tion, distribution, metabolism, and excretion) profiles of formazan crystal [60]. Cells (1 × 10 /well) were seeded of the ligands were predicted. Subsequently, the absorp- in 96-well flat-bottomed plates and incubated with each tion is influenced by water solubility, Caco2 perme - sample at various concentrations for 24  h. Compound ability, skin permeability, P-glycoprotein substrate, solutions were prepared in the following concentrations: P-glycoprotein I inhibitor, and P-glycoprotein II inhibi- 10, 20, 40, 80, 160, 320, 640 and 1280  µg/mL. 30  μL of tor instead of human gastrointestinal absorption. The MTT solution (5 mg/mL in PBS) was added to each well distribution is represented by VDss (human), fraction and the plate was incubated at 37  °C for another 4  h. unbound (human), blood–brain barrier (BBB) perme- Then, the medium was discarded and 150  µl of DMSO ability, and central nervous system (CNS) permeability. was added to dissolve the formazan crystals. The absorb - The metabolism is represented by the CYP2D6 substrate, ance of each sample was read at 595  nm using a micro- CYP3A4 substrate, CYP1A2 inhibitor, CYP2C19 inhibi- plate reader. Results were expressed as a percentage of tor, CYP2C9 inhibitor, CYP2D6 inhibitor, and CYP3A4 cell viability with respect to untreated control cells (as inhibitor. Lastly, the excretion is represented by the total 100%) [61]. The drug-dose dependent curve and its sta - clearance and renal OCT2 substrate. The Lipinski Rule, tistical analysis (95% confident interval) were generated mutagenicity, toxicity and pharmacokinetic prediction using GraphPad Prism 5.0 (https:// graph pad- prism. softw were carried out using pkCSM online tool (http:// biosig. are. infor mer. com/5. 0/). unime lb. edu. au/ pkcsm/ predi ction) [28]. Hariyono et al. Appl Biol Chem (2021) 64:69 Page 16 of 17 9. 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A study on catalytic and non-catalytic sites of H5N1 and H1N1 neuraminidase as the target for chalcone inhibitors

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Abstract

The H1N1 pandemic in 2009 and the H5N1 outbreak in 2005 have shocked the world as millions of people were infected and hundreds of thousands died due to the infections by the influenza virus. Oseltamivir, the most common drug to block the viral life cycle by inhibiting neuraminidase (NA) enzyme, has been less effective in some resistant cases due to the virus mutation. Presently, the binding of 10 chalcone derivatives towards H5N1 and H1N1 NAs in the non-catalytic and catalytic sites was studied using molecular docking. The in silico study was also conducted for its drug-like likeness such as Lipinski Rule, mutagenicity, toxicity and pharmacokinetic profiles. The result demonstrates that two chalcones (1c and 2b) have the potential for future NA inhibitor development. Compound 1c inhibits H5N1 NA and H1N1 NA with IC of 27.63 µM and 28.11 µM, respectively, whereas compound 2b inhibits NAs with IC of 50 50 87.54 µM and 73.17 µM for H5N1 and H1N1, respectively. The in silico drug-like likeness prediction reveals that 1c is 62% better than 2b (58%) in meeting the criteria. The results suggested that 1c and 2b have potencies to be devel- oped as non-competitive inhibitors of neuraminidase for the future development of anti-influenza drugs. Keywords: Neuraminidase, Non-catalytic site, Chalcone, Influenza, H5N1, H1N1 Introduction Fortunately, there are no reported human cases of this The novel Severe Acute Respiratory Syndrome (SARS) H5N1 avian flu, although the transmission of the disease coronavirus, SARS-CoV-2, which was first discovered in to humans had been reported since the first infection to December 2019 in Wuhan, China, infected approximately humans recorded in 2003 [4]. 64,000 people with about 1400 deaths announced at that Since the H5N1 outbreak in 2005, the world has been time, mainly in China [1]. At almost the same time, the prepared for the impending influenza pandemic, and in country was also reportedly dealing with an outbreak of 2009, H1N1 strains emerged and threatened humanity the deadly H5N1 avian influenza in Hunan, an area that with its spread in more than 70 countries [5]. However, borders Hubei, the province where the new coronavirus the severity of the 2009 H1N1 pandemic was modest emerged, according to the South China Morning Post [2]. compared to the H5N1 outbreak. H5N1 strain is con- As of February 1, 2020, local authorities had culled 17,828 sidered particularly dangerous because of its human poultries after the H5N1 outbreak, according to China’s fatality rate of over 50% to date and because of the risk Ministry of Agriculture and Rural Affairs statement [3]. that the virus may develop the ability to pass efficiently between humans. As of October 2020, the World Health Organization (WHO) reported a total of 861 confirmed *Correspondence: mhariono@usd.ac.id human cases which resulted in the deaths of 455 peo- Faculty of Pharmacy, Sanata Dharma University, Campus III, Paingan, ple since 2003 [6]. Thus, these instances in history war - Maguwoharjo, Depok, Sleman 55282, Yogyakarta, Indonesia rant an enhanced pandemic preparedness, especially Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Hariyono et al. Appl Biol Chem (2021) 64:69 Page 2 of 17 in this COVID-19 season. Although there have been no strong as the known drugs, it may overcome the drug i.e. detected complications across the population in either H274Y for the N1 protein [22]. seasonal or pandemic H1N1 influenza, the elderly patient In one of the more recent research, safflomin A (SA) has a higher risk for hospitalization and mortality during was reported to show inhibition against NAs from H1N1 the seasonal H1N1 influenza [7]. In particular, patients and H3N2 influenza viruses. The antiviral assay using having specific chronic conditions such as heart disor - MDCK cells which was infected by H1N1 and H3N2 der, lung disease, hyperglycemia, renal failure, rheumat- influenza virus exhibited the synergistic effect of SA with ics, dementia, and thromboembolism are at high risk of oseltamivir in viral cell proliferation. The kinetics study influenza complications [8, 9]. of SA demonstrated a non-competitive inhibition against The influenza virus is known to be rapidly mutated in N1 and N2 NA. Furthermore, a molecular docking study which oral anti-influenza, oseltamivir (Tamiflu), has been predicted that SA interacted with N1 and N2 NA at the less potent to the H1N1 infection [10]. Therefore, a study non-catalytic site. These results suggested that SA, which on finding a new anti-influenza agent should be continu - has a chalcone backbone, may serve as a potential thera- ously encouraged. Neuraminidase is the most common peutic option to the currently available anti-influenza targeted protein in the therapy of influenza, although agents to overcome the drug resistance [23]. The struc - hemaglutinin and M2 ion channels were also studied ture of NSC89853, SA, oseltamivir and zanamivir are [11]. NA has an important role in cleaving new virions presented in Fig. 1. from the infected cell [12]. Once it is blocked, the new In this present study, we examined 10 chalcone deriva- virus could not spread over other organs [13]. Reverting tives for their predicted binding affinities into the non- to the problem of virus resistance, a new chemical that catalytic site of H1N1 and H5N1 NA using a molecular has not been recognized by the virus is highly desired docking study. The non-catalytic location is at the back of [14]. the NA’s catalytic site within loop 150. Molecular dock- A series of chalcone compounds have been reported to ing of chalcones into the NA catalytic site was also con- demonstrate anti-neuraminidase activity [15] together ducted to compare their predicted binding affinities with with their quantitative-structure activity relationships the ones in the non-catalytic site. Further in silico stud- (QSARs) study. The QSAR model suggested that a func - ies to predict the drug-like likeness were also carried out tional group having electronic characters would increase employing Lipinski Rule, pharmacokinetic profile and the activity of the chalcone. In contrast, the steric effect toxicity prediction. The study was then followed by test - would decrease its activity against H1N1 NA. Other ing for their biological activities against H5N1 and H1N1 chalcones having anti-NA activities were isolated from NA using in vitro MUNANA assay. In addition, the safety Glycyrrhiza uralensis [16], Erythrina addisoniae [17], index was also calculated by studying the cytotoxicity Glycyrrhiza inflate [18], Polygala karensium [18], and effect of the most active compound toward a normal cell. Angelica keiskei [19] further supporting the claim of chal- Two chalcones demonstrated potential activity as neu- cones as potential NA inhibitors. raminidase of H5N1 and H1N1 inhibitors, respectively, However, upon inspection of the chemical structure, with low toxicities to the normal cell lines and predicted chalcone might not be a favorable scaffold for NA inhibi - to have considerably good drug-like structures. tors which usually belong to the shikimic acid structure. The shikimic acid inhibitors such as oseltamivir and zan - Results amivir bind to the catalytic site of NA, which is a small Molecular docking pocket surrounded by basic sub-pocket, acidic sub- The ten chalcone derivatives have been predicted for pocket as well as hydrophobic sub-pocket alongside its their molecular interactions using docking study as conserved amino acid residues [20]. Chalcone is a phe- presented in Table  1. The ΔG ranged at −  6.12 to bind nyl ring extended by an α,β-unsaturated carbonyl with − 7.97 kcal/mol and − 6.34 to − 8.31 kcal/mol at H1N1 various alkyl or aryl moieties next to the carbonyl group, NA and H5N1 NA non-catalytic sites, respectively. These making it too bulky to fit into the NA catalytic site [21]. results describe that chalcone has a potency from a low A chalcone compound, namely 2-amino-5-[3-[4-[(E)-4- to moderate micromolar activity (K 32.71 to 1.44 µM at chloro-3-oxobut-1-enyl]anilino]propyl]-4-methyl-1H-py- H1N1 NA; and K 22.58 to 0.82 µM at H5N1 NA) to bind rimidin-6-one (NSC89853) is reported to show H5N1 at this non-catalytic site and disrupt the enzyme activity. NA inhibition from the screening of 20 compounds from The superimposition of ten chalcone derivatives in the NCI database. This inhibition may be due to the bind - non-catalytic site of H1N1 and H5N1 NAs is illustrated ing to the protein with an alternative mode, that is dif- in Fig. 2. ferent from the known drugs (oseltamivir or zanamivir). To compare the binding mode of the ligands to the Although the potency of such a compound was not as common site, a molecular docking study was also Har iyono et al. Appl Biol Chem (2021) 64:69 Page 3 of 17 Fig. 1 The structure of a NSC89853, and b SA, which are suggested to be non-catalytic site inhibitors of H5N1 and H1N1, respectively, whereas c oseltamivir, and d zanamivir are known NA inhibitors targetting the catalytic sites Table 1 The docking results of 10 chalcone derivatives in the non-catalytic site of H1N1 and H5N1 NA Compounds H1N1 NA H5N1 NA ΔG ResiduesPredicted K ΔG ResiduesPredicted K bind i bind i (kcal/mol) (kcal/mol) 1a − 6.48 ARG118, ARG156, ARG430 17.68 μM − 7.12 ARG118, ARG156, ARG430 6.02 μM 2a − 6.92 SER196, VAL203, LYS207 8.50 μM − 8.31 LEU134, ARG156, ARG430 815.58 nM 3a − 6.68 SER196, ARG207 12.66 μM − 8.18 VAL116, ARG118, LEU134, THR135, 1.00 μM ARG156, ARG430 1b − 6.12 PHE174, SER196 32.71 μM − 6.34 ARG118, ARG430, THR439 22.58 μM 2b − 6.18 LYS150, VAL177 29.34 μM − 7.32 VAL116, GLN136, GLY147, VAL149, 4.33 μM HIS155 3b − 7.15 VAL116, ALA138, HIS144, ILE149 5.74 μM − 7.58 VAL116, ARG118, VAL149, ASP151, 2.80 μM THR439 4b − 7.97 LYS150, VAL177, SER196, ASP199, 1.44 μM − 7.89 ARG118, ARG156, ARG430, PRO431 1.65 μM VAL205 1c − 6.72 HIS144, ILE149, THR438 11.82 μM − 6.53 VAL116, ILE117, ARG118, LEU134, 18.30 μM ARG430 2c − 6.02 LYS143, HIS144 38.83 μM − 7.55 ARG118, HIS144, VAL149, ASP151, 2.95 μM HIS155, THR439 3c − 6.27 HIS144, TYR155 25.18 μM − 7.94 VAL116, ARG118, LEU134, ARG156, 1.52 μM ARG430, PRO431 conducted into the H1N1 and H5N1 NA catalytic sites. individual NA which are overlapped with its initial pose The parameterization of molecular docking was con - is presented in Additional file 1: Figure S1. trolled by re-docking oseltamivir-triazole into the H1N1 The control docking pose closely interacts with the NA and oseltamivir into the H5N1 NA catalytic site. This conserved amino acid residues such as ARG118, ASP151, resulted in RMSD 1.02  Å of the most populated cluster TRP179, ARG293, ARG368 and TYR402 via hydrogen bondings. The parameter of that control docking is then (85%) with ΔG − 9.35 kcal/mol at H1N1 NA, whereas bind used to dock 10 chalcone derivatives into both H1N1 the RMSD of oseltamivir at H5N1 NA active site was and H5N1 NA active site binding pockets. The docking 1.9  Å of the most populated cluster (74%) with the low- results of 10 chalcone derivatives are presented in Table 2 est energy (ΔG − 7.52 kcal/mol). This defines that the bind represented by the ΔG parameter is accepted for further docking of chalcone , interacting residues and the bind compounds. The control docking pose of ligands to their predicted K . The docking into H1N1 results in ΔG i bind Hariyono et al. Appl Biol Chem (2021) 64:69 Page 4 of 17 Fig. 2 The superimposition of 10 chalcone derivatives (yellow sticks) in the non-catalytic site of a H1N1 and b H5N1 NA. The NA is presented in the surface model and the ligands are in the stick models, docked in the behind of the catalytic site (yellow circles) Table 2 The docking results of 10 chalcone derivatives in the catalytic site of H1N1 and H5N1 NA Compounds H1N1 NA H5N1 NA ΔG ResiduesPredicted K ΔG (kcal/mol) ResiduesPredicted K bind i bind i (kcal/mol) 1a − 6.37 ARG152, ARG156, ARG225, GLU277, 21.55 μM − 5.69 GLU119, ARG152, ARG156, ILE222, 67.9 μM GLU278 SER246 2a − 7.71 ARG152, ARG225, THR226, GLU278 2.22 μM − 6.66 ARG118, ARG152, ILE222, GLY244, 13.07 μM SER246, ARG292, ARG371 3a − 7.39 GLU119, LYS150, ASP151, ASP152, 3.86 μM − 6.07 ARG118, ARG152, ARG224 THR225, 35.7 μM ARG225, GLU277, GLU278 GLU277, TYR347, ARG371, TYR406 1b − 7.36 ARG118, ARG152, TRP179, GLU277, 4.03 μM − 6.26 ARG152, ARG156, ILE222, SER246, 25.86 μM GLU278, ARG293 TYR406 2b − 8.10 ARG118, ARG152, TRP179, GLU277, 1.15 μM − 6.52 ARG118, ASP151, ARG156, ARG292, 16.63 μM ARG293, ASN295 TYR347, ARG371 3b − 8.34 ARG118, ARG152, ARG156, TRP179, 0.772 μM − 6.41 ARG118, ASP151, ARG156, ARG224, 20.05 μM GLU278, ARG293, ASN295 GLU276, ARG277, ARG371 4b − 7.13 ARG118, LEU134, LYS150, ASP151, 5.91 μM − 6.88 ARG118, ASP151, ARG156, GLU277, 9.04 μM ARG152, ARG293, ASN344 ARG292, TYR347, ARG371 1c − 6.57 GLU119, ARG152, ARG156, GLU277, 15.34 μM − 5.05 ARG118, ASP151, ARG152, TRP178, 200.37 μM GLU278 ARG224, TYR347, ARG371, ARG430, PRO431 2c − 7.49 GLU119, LYS150, ASP151, ARG152, 3.25 μM − 6.86 ARG118, GLU119, LEU134, ASP151, 9.41 μM TRP179, ARG293, ASN344, ARG368, ARG152, ARG156, ARG224, TYR402 GLU227, TYR347, ARG371, ILE427, ARG430, PRO431 3c − 7.75 ARG118, GLU119, LYS150, ASP151, 2.09 μM − 6.93 GLU119, ARG152, ARG156, ILE222, 8.32 μM ARG152, TRP179, ARG293, ASN344, ARG224, GLY244 ARG368, TYR402 of docked chalcones into the H1N1 catalytic site are − 6.37 to − 8.34 kcal/mol, whereas it is ΔG − 5.05 to bind likely lower than that of H1N1 at the non-catalytic one. −  6.93  kcal/mol into H5N1 for the overlapped-docked In contrast, the chalcones docked into the H5N1 have pose which is presented in Fig. 3. The calculated energies Har iyono et al. Appl Biol Chem (2021) 64:69 Page 5 of 17 Fig. 3 The superimposition of 10 chalcone derivatives (yellow sticks) in the active site of a H1N1 NA (blue) and b H5N1 NA (red) in the ribbon model lower free energy of binding in the non-catalytic than in via hydrogen bond with its receptor [24]. The rotat - the catalytic sites. However, except for compounds 1a, able bonds may influence their stability during ADME 2a and 3a, its long alkyl chain escaped from the catalytic (absorption, distribution, metabolism, and excretion) and site, indicating their non-fit binding into both H1N1 and the receptor binding. Thus, the less flexible the chain in H5N1 NA’s catalytic sites. Table  2 tabulated the docking the molecule, the more stable the drug is to perform its results of the 10 compounds in the active site of H1N1, activity [25]. The Polar Surface Area (PSA) is related to H5N1 NA, and their superposition as illustrated in Fig. 3. the permeability of drugs across the cell membrane in which the higher the PSA, the poorer the cell permeabil- Lipinski rule ity (oral bioavailability) is [25]. According to the Lipinski Rule, an ideal drug should have In general, the results show that all chalcones meet the a molecular weight (MW) which is less than 500  Da, < 5 MW, the number of HBD, HBA and the rotatable bonds log P, ≤ 5 Hydrogen Bond Donor (HBD), ≤ 10 Hydrogen requirements. However, compounds 2b and 3b slightly Bond Acceptor (HBA), ≤ 10 rotatable bonds, and ≤ 140 Å deviate on the log P limit, whereas compounds 2c, 3c and surface area [24, 25]. The potency of the compound to be 4b do not fulfill the limit of the surface area requirement. drug is commonly expressed by I C , which is affected Table  3 presents the chalcones with their Lipinski Rule by its MW. The more potent drug should have a higher profiles. MW to minimize its dose in performing a pharmacologi- cal activity. Nevertheless, the MW should not be higher Mutagenicity and toxicity profiles than 500 considering the drug absorption via the intesti- The in silico toxicity predictions have become a routine nal membrane [24]. before processing compounds to be drug candidates. In The log P is the concentration of the compound in this study, we also predicted the mutagenicity and toxic- n-octanol divided by its concentration in water. There - ity profiles of the 10 chalcones. Generally, a compound’s fore, it reflects the balance of the compound’s solubility mutagenic properties are usually confirmed using the in water during oral dissolution steps with its oral bioa- AMES test [26, 27]. The human Maximum Tolerated vailability in the blood system [24, 25]. The ideal log P < 5 Dose (hMTD) is acceptable when the predicted toxic indicates that the compounds are well soluble in the body dose threshold in humans is more than 0.477. The potas - fluid as well as absorbed through the gastrointestinal cell sium channels that mediate the cardiac repolarization membrane, which then be transferred into the blood vas- in humans are represented by the values of hERG I and cular. On the other hand, the number of HBD or HBA is II. Hence, inhibiting this kind of protein would cause a associated with polarity to interact with water during the long QT syndrome development that might lead to a dissolution process as well as their molecular interaction fatal arrhythmia. The in  vivo toxicity is often expressed Hariyono et al. Appl Biol Chem (2021) 64:69 Page 6 of 17 Table 3 The drug-like likeness profile of 10 chalcones was studied as NA inhibitors Ligands MW log P HBD HBA Rotatable bonds Surface area 1a 242.249 3.4274 1 2 3 104.015 1b 284.330 4.5090 0 2 5 123.429 1c 312.365 4.0857 1 4 7 135.536 2a 269.256 3.1965 1 4 4 114.502 2b 310.368 5.0432 0 2 5 135.153 2c 390.435 4.8845 1 5 8 169.342 3a 268.268 2.9865 2 3 4 115.170 3b 318.347 5.5141 0 2 5 139.391 3c 360.409 4.8759 1 4 7 157.864 4b 337.375 4.8123 0 4 6 145.641 by LD value which can be defined as the required dose be interpreted as they are able to cause any observable given to cause 50% death of a group of rats in evaluating a chronic toxicity at the lower amount and/or shorter time compound acute toxicity represented by ORAT (log L D of exposure. Several compounds (2c, 3a, 3b and 3c) dem- value) prediction. onstrate T. Pyriformis toxicity potency, whereas, based LOAEL (lowest-observed-adverse-effect level) associ - on the minnow toxicity prediction, compounds 1a, 2a, 3a ates with the compound’s lowest concentration which and 4b are deemed to be safe. Table 4 presents the AMES causes an adverse effect in human physiology. This is test results of the chalcones for mutagenicity prediction indicated by the alteration of morphology, function, along with other toxicity profiles. growth, or development. The safety of a compound is higher as the LOAEL value increases. The liver injury Pharmacokinetic profiles commonly reflects the drug hepatotoxic properties, Table 5 demonstrates that all chalcones are well absorbed whereas the potential dermal adverse effects are deter - through the human intestinal with near values to 100% mined by skin sensitization properties. The value of toxic into the blood system. In general, the water solubility of endpoints, however, is measured by  T. pyriformis  and all chalcones are most likely poor as their log S value are minnow toxicity [28]. lower than − 4, except for 3a which has a log S value of Nowadays, the drugs’ safety to the environment is a − 2.601; indicating that it may dissolve readily in the dis- concern. Low environmental damage is demonstrated by solution step. Furthermore, oral absorption prediction the values of T. pyriformis and minnow toxicity which are using Caco2 cell model [32] with the required value is to be respectively higher than 0.5 and − 0.3. Based on the higher than 0.90. Therefore, except for 2c and 4b, chal - AMES test, two compounds i.e. 2a and 4b are predicted cones show good human gastrointestinal absorption. not to be toxic, while 4b might have the possibility to be The skin permeability of chalcones is most likely suit - hepatotoxic. No chalcones inhibit the hERG I protein, able for the transdermal route because the values are but 1c, 2b, 2c and 3c inhibit hERG II. Most of the chal- approximately −  2.5. A protein transport namely P-gly- cones have a low maximum dose which is weakly toler- coprotein (P-gp) is crucial during the pharmacokinet- ated in humans, except for 2a, 2b and 4b. In addition, all ics steps, however, this could have either advantages or chalcones exhibit no skin sensitization. The oral rat acute disadvantages in therapeutic effect [33]. A compound is toxicity LD can be classified as very toxic (≤ 5  mg/kg), supposed to not inhibit P-gp, either P-gp I or P-gp II. In toxic or moderately toxic (> 5 to < 500  mg/kg), harmful the ideal situation, it should not be acting as P-gp sub- or slightly toxic (> 500 to < 2000  mg/kg), and non-toxic strate either. According to the prediction, compounds (> 2000  mg/kg) [29]. All compounds are predicted to be 1c and 2b are predicted to inhibit the P-gp I activity, potentially less toxic with L D ranging from 194.01 to whereas compounds 2c, 3c, and 4b inhibited both P-gp 399  mg/kg. Oral rat LOAEL value shows that the inges- I and P-gp II. In contrast, 2a acts like the substrate for tion chronic toxicity is relative to the limit of concen- P-gp, accordingly. tration and length of exposure, however, LOAEL does One of the distribution parameters is defined by the not solely represent drug safety accurately [30, 31]. This number of VDss or volume of distribution at a steady prediction showed that compounds 2a, 2c, 3a and 4b state, which  is directly proportional with the amount have lower values than the other compounds, which can of drug distributed into tissue; a higher V   indicates a D Har iyono et al. Appl Biol Chem (2021) 64:69 Page 7 of 17 Table 4 The AMES test result of the chalcones for mutagenicity prediction along with other toxicity profiles Ligands AMES toxicity hMTD hERG I hERG II ORAT (log LD ) ORCT (log Hepatotoxicity Skin T. Pyriformis toxicity Minnow toxicity inhibitor inhibitor LOAEL) Sensitisation 1a No 0.646 No No 2.228 2.203 No No 1.380 0.262 1b No 0.830 No No 2.324 2.321 No No 1.115 − 1.141 1c No 0.611 No Yes 2.318 2.044 No No 1.207 − 0.820 2a Yes 0.039 No No 2.485 1.267 No No 1.231 − 0.229 2b No 0.429 No Yes 2.360 2.117 No No 1.664 − 0.595 2c No 0.521 No Yes 2.577 1.164 No No 0.353 − 1.641 3a No 0.638 No No 2.233 1.715 No No 0.286 0.520 3b No 0.669 No Yes 2.519 2.330 No No 0.421 − 1.293 3c No 0.502 No Yes 2.524 2.125 No No 0.419 − 2.152 4b Yes 0.039 No No 2.601 1.231 Yes No 1.207 − 0.232 Hariyono et al. Appl Biol Chem (2021) 64:69 Page 8 of 17 Table 5 The absorption profiles of chalcones as predicted by the software Ligands Water solubility Caco2 Intestinal Skin permeability P-glycoprotein P-glycoprotein I P-glycoprotein permeability absorption substrate inhibitor II inhibitor (human) 1a − 4.140 1.426 93.706 − 2.315 No No No 1b − 5.655 1.423 96.342 − 2.434 No No No 1c − 4.798 1.570 93.360 − 2.483 No Yes No 2a − 4.312 0.904 91.606 − 2.627 Yes No No 2b − 5.636 1.636 95.062 − 2.327 No Yes No 2c − 6.726 0.556 93.240 − 2.699 No Yes Yes 3a − 2.601 0.949 100.000 − 2.683 No No No 3b − 6.714 1.684 93.229 − 2.677 No No Yes 3c − 6.342 1.078 95.102 − 2.678 No Yes Yes 4b − 5.522 0.343 93.495 − 2.659 Yes Yes Yes greater amount of tissue distribution. As a rule, VDss subfamilies, which play significant roles in drug metabo - should be ≥ − 0.15. Among the 10 chalcones, three com- lism [35]. CYP2D6 and CYP3A4 are found in the brain pounds (2c, 3a, and 3c) do not meet these criteria. The and intestines, respectively, and are most likely respon- fraction unbound (fu) for all chalcones are predicted to sible to metabolize the drug in their surrounding areas. be ≤ 0.15, indicating that more fractions of drug molecule Furthermore, CYP3A4 also affects oral bioavailability by are bound to the plasma protein. Besides VDss, the chal- first-pass metabolism. A compound that binds strongly cones were also predicted for their ability to cross the to these CYPs could be acting as the substrate or inhibi- brain membrane which is important as the compounds tor leading to the lower / higher bioavailability as well may affect the Central Nervous System (CNS) [34]. Val - as activity of other drugs. This could be potential for the ues < − 1 indicate poor Blood–Brain Barrier (BBB) per- drug causing clinical drug-drug interactions, leading to meability and < − 3 for CNS permeability. In other words, adverse reactions or therapeutic failures. In this predic- the compound is poorly distributed to the brain and una- tion, neither chalcones act as the substrate nor inhibi- ble to penetrate CNS. From the results, all the 10 com- tor for CYP2D6. However, except for 3a, all compounds pounds should be carefully managed as there is potential act as CYP3A4 substrate, whereas 2c and 3c act as this for these chalcones to enter the CNS especially for 1a, enzyme’s inhibitor. Furthermore, chalcones are likely to 1b, 2b and 3b which can also penetrate BBB. The distri - inhibit the CYP1A2, CYP2C19, and CYP2C9 presented bution profiles of the chalcones as predicted by software in Table  7, which should be of concern when the com- are presented in Table 6. pound is consumed with other drugs. Metabolisms are also an important indicator of good Total clearance describes the compound’s rate while drug-like properties. CYP1A2, CYP2C9, CYP2C19, being removed from the body [36]. One of the main renal CYP2D6, CYP3A4 are among the cytochrome P450 uptake transporters to remove the drug from the blood Table 6 The distribution profile of chalcones as predicted by the software Ligands VDss (human) Fraction unbound (fu) BBB permeability CNS permeability (human) 1a 0.015 0.058 0.012 − 1.641 1b 0.106 0.000 0.425 − 1.298 1c 0.087 0.000 − 0.233 − 2.219 2a 0.041 0.000 − 0.328 − 2.123 2b 0.527 0.000 0.398 − 1.465 2c − 0.476 0.017 − 0.257 − 2.157 3a − 2.366 0.000 − 0.200 − 2.253 3b − 0.083 0.035 0.479 − 1.054 3c − 0.227 0.024 − 0.258 − 2.053 4b 0.638 0.000 − 0.341 − 1.930 Har iyono et al. Appl Biol Chem (2021) 64:69 Page 9 of 17 Table 7 The interaction between chalcones with a diverse CYP subfamily Ligands CYP2D6 CYP3A4 CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 substrate substrate inhibitor inhibitor inhibitor inhibitor inhibitor 1a No Yes Yes Yes No No No 1b No Yes Yes Yes Yes No No 1c No Yes Yes Yes Yes No No 2a No Yes Yes Yes Yes No No 2b No Yes Yes Yes Yes No No 2c No Yes Yes Yes Yes No Yes 3a No No No No No No No 3b No Yes Yes Yes Yes No No 3c No Yes Yes Yes Yes No Yes 4b No Yes Yes Yes Yes No No is the OCT2 transporter. It plays a pivotal role in the Neuraminidase assay removal and renal clearance of mostly cationic drugs as The chalcone derivatives have been examined for their well as endogenous compounds [37]. Inhibition of OCT2 inhibitions toward the activity of H1N1 and H5N1 influ - (such as by cimetidine) elevates OCT2-dependent renal enza neuraminidase. The first screening using 100  µg/ clearance drugs, hence altering pharmacokinetics and ml of the sample’s concentration showed that five com - pharmacodynamics profiles. Compound 3a is predicted pounds exhibited 0–10% inhibition toward H1N1 NA, to be the fastest compound excreted from the body due while only one compound showed activity in the same to its highest total clearance. In contrast, compound 2b range against H5N1. Furthermore, three compounds is the slowest compound to be removed from the body exhibited 10–50% inhibition toward H1N1, while five due to its lowest total clearance. All chalcones have low compounds demonstrated inhibition against H5N1 at the total clearance (log Cl < 0.763), yet, it is generally desir- same inhibition percentage. In the H5N1 NA inhibition able to develop a drug for oral administration without a assay, two compounds (1b and 2c) were inactive. Inter- high dosage regimen [38]. One chalcone, 2b, is predicted estingly, two compounds (1c and 2b) showed more than to act as renal OCT2 substrate that might lead to unde- 50% inhibition to both H5N1 and H1N1. Therefore, they sirable side effects. Figure  4 illustrates the total clearance were selected to be further studied on their IC as well of all chalcones that reflects their rate to be eliminated as CC . Table 8 presents the assay results of ten chalcone from the body system. compounds compared to vanillin as the positive control. Fig. 4 The histogram of total renal clearance profiles of the chalcones with its OCT2-dependence clearance, green block = OC T2-independent and yellow block = OC T2-dependent Hariyono et al. Appl Biol Chem (2021) 64:69 Page 10 of 17 Table 8 The assay result of ten chalcone compounds with their R side chains as depicted in Fig. 5 (100 µg/mL) against H5N1 and H1N1 NA compared to vanillin as the positive control Compounds R R R R % Inhibition ± SE 1 2 3 4 H1N1 NA H5N1 NA 1a H OH H F 21 ± 7 23 ± 11 2a H OH H NO 16 ± 2 21 ± 2 3a H OH H COOH 19 ± 2 27 ± 0 1b H O-isopropyl H F 5 ± 8 0 ± 3 2b H O-cyclopentyl H F 69 ± 2 70 ± 1 3b H O-benzyl H F 2 ± 4 18 ± 3 4b H O-cyclopentyl H NO 5 ± 5 15 ± 3 1c OCH OH H O-butyl 83 ± 1 82 ± 3 2c OCH OH OCH O-benzyl 1 ± 4 − 2 ± 16 3 3 3c H OH OCH O-benzyl 2 ± 4 7 ± 8 Vanillin – – – – 86 ± 2 84 ± 4 Cytotoxicity assay R R 2 4 In the cytotoxicity assay results (Fig. 7), 1c showed a high concentration to inhibit the Vero cell proliferation with CC 968.16 µM. The safety index of this compound cal - culated with the safety index (SI) values was 34.44 and R R 1 3 35.69 for H1N1 and H5N1 NA, respectively. In addition, 2b showed CC 757.18  µM with the SI values = 8.65 and 10.35 for H1N1 and H5N1, respectively. The graph Fig. 5 The chalcone backbone scaffold of 10 chalcone derivatives plotting log concentration of 1c and 2b vs % cell viability against Vero cell in the cytotoxicity studies is presented in Fig. 7a. Furthermore, the cell imaging in Fig. 7b exhib- The IC of 1c was then estimated as 28.11  µM and ited the cell proliferation survival, when there was no 27.63  µM for H1N1 and H5N1, respectively. Compound treatment to the cell, which was indicated by the forma- 2b also demonstrated similar % inhibition toward H1N1 tion of formazan crystals upon MTT reaction. Formazan and H5N1 NA with its I C 87.54  µM and 73.17  µM, crystal indicates the ability of the cell to express NADPH- respectively. This is quite interesting because the com - dependent oxidoreductase to reduce the MTT, which pound showed similar IC when it was applied to both leads to cell proliferation. In contrast, the formazan crys- H5N1 and H1N1 NA. Figure  6 presents the drug-dose tal formation will be reduced along with the treatment of depending curve of 1c and 2b in inhibiting the H1N1 and 1c and 2b on the Vero cell leading to their cytotoxicity H5N1 NA. properties (Fig. 7a). Fig. 6 The drug–dose depending curve of 1c and 2b in inhibiting H1N1 and H5N1 NA Har iyono et al. Appl Biol Chem (2021) 64:69 Page 11 of 17 Fig. 7 The cytotoxicity profiles of 1c and 2b plotting a log concentration of vs % Vero cell viability of 1c and 2b, whereas b is the Vero cell imaging without any compound exposure (negative control), and c with 1c treatment to the cells. The orange arrows indicate the formazan crystal formation which is higher in 1c absence than its presence, accordingly a particularly striking feature in the catalytic domain Discussion referred to as the ‘150 loop’ [39, 45]. The shikimic acid The 2009 H1N1 NA is one of several 2009 pandemic scaffold has been classically patterned for NA inhibitors influenza virus mutants in which the I223 changed to due to its capability to form a stable chair conformation R223 (I223R). This mutation results in shrinkage of the transition state of the non-aromatic six-member ring enzyme’s active site affecting the binding of NA inhibi - with the rather flat oxonium cation, and thereby arrange tor [39]. Another mutant is H274Y (H1N1), which was the position of important residues to interact with the also reported to be resistant to oseltamivir as confirmed shikimic binding groups [45]. Taking this into consid- in about 20% isolates from humans in Europe [40, 41]. eration, some aromatic compounds have been devised NA’s active site is composed of eight functional resi- to mimic this transition state leading to their inhibition dues (ARG118, ASP151, ARG152, ARG224, GLU276, against NA [46, 47]. ARG292, ARG371 and TYR406) and surrounded by In the present study, two chalcones (1c and 2b) have 11 framework residues (GLU119, ARG156, TRP178, been observed to have the best in  vitro results com- SER179, ASP198, ILE222, GLU227, HIS274, GLU277, pared to the other 8 chalcones. Figures 8 and 9 illustrate ASN294 and GLU425) [42]. the molecular interactions of 1c and 2b, respectively, Structurally, chalcone consists of one aromatic ring while binding into the H1N1 (non- and catalytic sites) extended by a predominantly trans-configuration of α,β- and H5N1 (non- and catalytic sites). During the bind- unsaturated carbonyl. The relatively short carbon–carbon ing into the non-catalytic site of H1N1 NA, 1c interacts distance between α,β-unsaturated alongside the relatively via H-bond with only THR438. The other interactions long C–C distances is 1.326 Å and 1.46 Å, which is con- are vdW interactions with ASN146 and HIS144; amide sistent with a localized double bond in the enone unit in pi-stacked with HIS144 and ILE149; and pi-alkyl inter- this structure [43]. The chain next to the carbonyl group actions with VAL116, ALA138 and SER145. In contrast, is usually prolonged by either alkyl or aryl moieties with the binding to the catalytic site of H1N1 NA shows that various functional groups being attached. The conjugated 1c interacts with ASP151, ARG293, ASN344, ARG368, double bond could make this type of compound less and TYR402 via H-bonds, whereas pi-alkyl and alkyl- flexible. However, the C=O bond can present as either alkyl interactions are also observed with LYS150 and S-cis or S-trans conformation with respect to the vinylic LEU134, respectively. In addition, pi-anion interaction double bond due to the free rotation along with the sin- is observed with GLU119. Compound 1c interacts with gle bond between C-carbonylic and Cα. This, therefore, the non-catalytic site of H5N1 NA via H-bonds with could increase the conformation stability of such com- VAL116, ARG118, and ARG430; amide pi-stacked with pounds during a dynamic environment [44]. ILE117; and pi-alkyl with VAL116, ARG118, ARG430 The combination of in silico and in  vitro studies has and PRO431. Furthermore, in its catalytic site, the suggested a new binding mode for chalcone compounds interactions are observed via H-bonds with ARG152, into N1 NA. The active site of NA is decorated by a rel - ARG371, TYR347, and ARG430; alkyl interactions with atively small pocket surrounded by some sub-pockets TRP178, ARG224, and PRO431; pi-cation as well as determining the binding region of such enzyme with Hariyono et al. Appl Biol Chem (2021) 64:69 Page 12 of 17 Fig. 8 The molecular interactions of 1c with a non-catalytic H1N1, b catalytic H1N1, c non-catalytic H5N1, and d catalytic H5N1 NA sites. The green, pale green, purple, magenta, pink, cyan, and orange represent H-bond, vdW, pi-sigma, amide pi-stacked, pi-alkyl, pi-halogen, and pi-cation/anion, respectively pi-cation interactions are observed with ARG118 and The possibility of chalcone to interact with the active ASP151, respectively. site of NA is only in one aromatic ring augmented by the The second best compound (2b) also interacts via binding group such as OH or OCH . The α,β-unsaturated H-bond with the non-catalytic site of H1N1 NA with chain most likely protruded outside of the active pocket. LYS150; pi-alkyl with PRO154, VAL177, PRO198 and Although the ΔG of chalcone in the active site is con- bind VAL205. In contrast, the interactions with the cata- sidered acceptable, it is inconsistent with the in  vitro lytic site via H-bonds are absent. However, the remain- results. Compound 1c has relatively higher ΔG in both bind ing interactions of 2b are observed with ARG118 and NAs’ active sites than other compounds with poor inhibi- ARG152 via pi-alkyl interactions; GLU277, ARG293 tion against NAs. However, the in  vitro results showed and ASN295 via pi-halogen interactions; with TRP179 that 1c is the most active compared to others; indicating via pi-lone pair electron interaction. The non-catalytic a poor correlation between in vitro and in silico studies. site of H5N1 NA shows interactions with chalcones In contrast, although 2c is inactive against NAs in  vitro, via H-bonds with GLY147 and pi-alkyl with VAL116, the ΔG in both NA’s active sites is considered fair. bind ALA138, VAL149. In addition, amide pi-stacked with Notably, compounds with –NO group (2a and 4b) HIS155 is also observed. In the H5N1 NA catalytic show the lowest binding energy in either non-catalytic site, chalcones make interactions via H-bonds with or catalytic sites for both H1N1 and H5N1 NAs. How- ARG118, ARG156 and ARG371. Furthermore, there ever, the in  vitro results demonstrated a low percentage are also amide pi-stacked interaction with TYR347, of inhibition toward both NAs. Nitro group is the strong- as well as pi-cation/anion with ASP151 and ARG292, est electron-withdrawing, which reactively interacts respectively. with atoms having an electron-donating group to form Har iyono et al. Appl Biol Chem (2021) 64:69 Page 13 of 17 Fig. 9 The molecular interactions of 2b with a non-catalytic H1N1, b catalytic H1N1, c non-catalytic H5N1, and d catalytic H5N1 NA sites. The green, pale green, purple, magenta, pink, cyan, yellowish-green and orange represent H-bond, vdW, pi-sigma, amide pi-stacked, pi-alkyl, pi-halogen, pi-lone pair electrons, and pi-cation/anion, respectively H-bond as well as electronic interactions [48]. This false- association and dissociation rates using Surface Plasmon positive results could be due to the stability of the –NO Resonance (SPR). In this method, the protein is immo- group, which is easily reduced by the acidic pH turn- bilised onto a biosensor surface while the drug ligand is ing into an amine group (nitro reduction) [49], which is continuously flowing across the biosensor surface, where measured in pH 6.5 in this MUNANA system. This con - it binds to the immobilised receptor. The binding is meas - version could make the 2b and 4b lose their interactions ured by the kinetic association and dissociation rates (k / as suggested by the docking study, leading to their low k ) for several different ligand concentrations [50]. inhibition against NA. The inconsistent results of 2b and The second method is ITC, which measures the heat 3b are not fully understood as well. Therefore, we sug - transfer during binding that enables accurate determi- gest that a molecular dynamics simulation is performed nation of binding constants (K ), reaction stoichiom- to elucidate this phenomenon in future studies. On the etry (n), enthalpy (∆H) and entropy (ΔS). This provides a other hand, there are inconsistent results between dock- complete thermodynamic profile of the molecular inter - ing and in vitro results when the chalcones were docked action. This deeper understanding of structure–function into either non-catalytic and catalytic sites of H5N1. relationships enables more confident decision making in Therefore, it is recommended to study the kinetics of NA hit selection and lead optimization [51]. inhibition for the two most active compounds (1c and In this present study, an array of  in silico  predictions 2b) to confirm whether the mode of inhibition is com - has been performed on the chalcones including Lipin- petitive or non-competitive. ski Rule, mutagenicity, toxicity and pharmacokinetic The kinetic assay can be carried out using meth - profiles. The chalcones have diverse functional groups ods such as Biacore or Isothermal Titration Calorim- conferring the distinction of their drug-like properties, etry (ITC). Biacore measures the real-time binding mutagenicity, toxicity, and pharmacokinetics, which will Hariyono et al. Appl Biol Chem (2021) 64:69 Page 14 of 17 contribute to their overall therapeutic effects. Given that compounds have a good safety index augmenting their the two compounds (1c and 2b) are active in the in vitro potency as the potential H5N1 and H1N1 NA inhibitors. study, we are motivated to understand the possibility of these compounds to be considered as lead candidates Materials and methods for optimization. They have good Lipinski Rule profiles Chemistry by meeting the requirements of MW, log P, number of The chalcone compounds (Fig.  10) were synthesized HBD-HBA, number of rotatable bonds and the surface using the established method in our laboratory [21, 52, area. Both compounds are also not responsive against 53]. We scale up the production of the compounds and the AMES test describing their non-mutagenic potency. confirm their purity using a thin-layer chromatographic Unfortunately, 2b is predicted to have a low tolerance method and melting point test with the data of published in the maximum dose. In addition, both compounds are compounds as the reference. responsive toward hERG II as inhibitors and having an acute toxic dose of around 200 mg/kg, but irresponsive to hERG I as an inhibitor, not toxic in chronic ingestion, and Molecular docking neither hepatotoxic nor skin sensitized. In environmen- The proteins used are the resistant I223R NA mutant of tal damage, both are non-toxic to T. pyriformis as well as H1N1 2009 pandemic influenza virus (PDB ID 4B7M) minnow species. [39] and the wild type H5N1 NA (PDB ID 2HU0) [54]. These two compounds are insoluble in water. Thus, The protein structure was processed using AutoDock - consideration should be given for a suitable delivery sys- Tools 1.5.6 (www. autod ock. scrip ps. edu) with the ligand tem. They have good in Caco2 permeability, intestinal separated from the protein. The grid for docking to the human absorption and skin permeability; reflecting their non-catalytic site of H1N1 NA and H5N1 NA was set good absorption profile either in oral or topical admin - to 94 × 72 × 96 and 80 × 80 × 80 with its spacing set istration. Both compounds have potencies to inhibit the to 0.375  Å. The center of mass of the ligand was set to protein carrier during absorption but neither of them acts x = 26.852, y = −  32.014, z = −  1.019 for H1N1 NA, and as the protein substrate. In the distribution profiles, both x = − 7.07, y = 28.242, z = 107.729 for H5N1 NA. Genetic compounds bind tightly in the plasma protein, which Algorithm was chosen for docking calculation in Auto- could reduce the therapeutic dose. Furthermore, the Dock 4.2.3. The searching parameters were set to the BBB and CNS permeability should be taken into account. default values (Population size = 150, maximum number Interaction with other drugs should be evaluated as of evals = 2,500,000, maximum of generations = 27,000, well, as these two compounds are predicted to act as the maximum number of top individuals that automatically CYP3A4 substrate, CYP1A2, CYP2C19, and CYP2C9 survives = 1). The number of GA run was set to 100. inhibitors. In contrast, they are irresponsive toward Docking parameters such as random number genera- CYP2D6 and CYP3A4. The excretion of 1c is faster tion, energy parameters, and step size were also set to the than 2b since the total clearance of 1c is higher than 2b default values. The results were analyzed by checking the in addition to the potential of this compound as Renal RMSD values, ligand–protein interactions, free energy OCT2’s substrate. Overall, 1c meets the requirements of of binding (FEB) as well as the number of conformations drug-like properties at approximately 62%, whereas 2b that exist in a population cluster [55]. For the subse- is at 58%. These percentages are above 50%, therefore, quent molecular docking, the chalcone structure deriva- these two compounds should be given the opportunity to tives were sketched and energetically optimized using be optimized and developed further as drug candidates. Hyperchem Professional version 8.0 (www. hyper. com) This conclusion is supported by the in  vitro cytotoxic - with MM + force field and Polak-Ribiere (Conjugate Gra - ity study against normal cell lines confirming that both dient). The visualization of ligand–protein interaction Fig. 10 The structure of 10 chalcone derivatives (1a–4b) with different set of functional groups Har iyono et al. Appl Biol Chem (2021) 64:69 Page 15 of 17 Neuraminidase assay was conducted using Biovia Discovery Studio 2016 The H1N1 NA assay followed the general procedure of (www. accel rys. com). Fluorometric Neuraminidase Assay [57]. The H1N1 NA The docking of compounds to the catalytic site of NA (A/California/04/2009) and H5N1 NA (A/Anhui/1/2005) was carried out by using PDB 2HU0 (H5N1 NA in com- enzymes were purchased from Sinobio. The fixed con - plex with oseltamivir) [54] as well as PDB 6HP0 (H1N1 centrations of H1N1 NA (0.3  u/mL) and MUNANA NA in complex with oseltamivir triazole) [56] with (100  mM) were optimized employing the previously the same default protocol being used in PDB 4B7M, described method. Vanillin was used as the positive con- except for the number of points and its center of mass. trol inhibitors [58], and the H1N1 neuraminidase assay For 6HP0, the number of points is 40 × 40 × 40 with was prepared by mixing an assay buffer, tested samples the center of mass (x = 43.641; y = 0.53; z = 20.263). For (at concentrations 100  μg/mL in 1% of DMSO-Buffer), 2HU0, the number of points is 40 × 40 × 40 with the and a constant 0.3 unit/mL of neuraminidase which were center of mass (x = 1.763; y = 19.33; z = 108.34). pre-incubated at 37 °C for 30 min with 200 rpm. After the addition of 100  μM substrates, the reaction assays were Lipinski rule of five incubated at 37 °C for 60 min with 200 rpm. To stop the The Lipinski Rule profiles were individually predicted by reaction, 100  μl of glycine stop solution was added. The inputting SMILES string, which is automatically done by assays were carried out in triplicate. The assay proto - the server. The value of molecular weight (MW), log P, col for H5N1 inhibition assay is similar to that of H1N1 the number of hydrogen bond donors (HBD), the num- but the concentrations being used were 0.15  u/mL, and ber of hydrogen bond acceptors (HBA), the number of 50  µM for the enzyme and substrate, respectively. A rotatable bonds and the surface area were observed and series of concentrations were prepared for those demon- then tabulated. strating > 50% of enzyme inhibition to calculate the IC . The fluorescence intensity of NANA was measured by Modulus Microplate Reader with a UV optical kit at λ Mutagenicity and toxicity studies 340/440 nm. The drug-dose dependent curve and its sta - Using the same protocol as in “Lipinski rule of five” sec - tistical analysis (95% confident interval) were generated tion, the mutagenic potency of the ligands was repre- using GraphPad Prism 5.0 (https:// graph pad- prism. softw sented by the AMES test results. Other parameters such are. infor mer. com/5. 0/). as maximum tolerated dose (human) (hMTD), hERG I inhibitor, hERG II inhibitor, oral rat acute toxicity (log LD ), oral rat chronic toxicity (log LOAEL), hepatotoxic- Cytotoxicity assay ity, skin sensitization, T. pyriformis toxicity, and minnow The cytotoxicity of each compound on Vero cells was toxicity represented the toxicity properties of the ligands. determined using MTT assay. Vero cell is a non-tum- origenic cell from the kidney tissue of African green monkeys [59]. Cells will proliferate by expressing the Pharmacokinetics study NADPH-dependent oxidoreductase in mitochondria Using the same protocol in 4.3, the ADME (absorp- that reduces the MTT reagent into the reduction state tion, distribution, metabolism, and excretion) profiles of formazan crystal [60]. Cells (1 × 10 /well) were seeded of the ligands were predicted. Subsequently, the absorp- in 96-well flat-bottomed plates and incubated with each tion is influenced by water solubility, Caco2 perme - sample at various concentrations for 24  h. Compound ability, skin permeability, P-glycoprotein substrate, solutions were prepared in the following concentrations: P-glycoprotein I inhibitor, and P-glycoprotein II inhibi- 10, 20, 40, 80, 160, 320, 640 and 1280  µg/mL. 30  μL of tor instead of human gastrointestinal absorption. The MTT solution (5 mg/mL in PBS) was added to each well distribution is represented by VDss (human), fraction and the plate was incubated at 37  °C for another 4  h. unbound (human), blood–brain barrier (BBB) perme- Then, the medium was discarded and 150  µl of DMSO ability, and central nervous system (CNS) permeability. was added to dissolve the formazan crystals. The absorb - The metabolism is represented by the CYP2D6 substrate, ance of each sample was read at 595  nm using a micro- CYP3A4 substrate, CYP1A2 inhibitor, CYP2C19 inhibi- plate reader. Results were expressed as a percentage of tor, CYP2C9 inhibitor, CYP2D6 inhibitor, and CYP3A4 cell viability with respect to untreated control cells (as inhibitor. Lastly, the excretion is represented by the total 100%) [61]. The drug-dose dependent curve and its sta - clearance and renal OCT2 substrate. The Lipinski Rule, tistical analysis (95% confident interval) were generated mutagenicity, toxicity and pharmacokinetic prediction using GraphPad Prism 5.0 (https:// graph pad- prism. softw were carried out using pkCSM online tool (http:// biosig. are. infor mer. com/5. 0/). unime lb. edu. au/ pkcsm/ predi ction) [28]. Hariyono et al. Appl Biol Chem (2021) 64:69 Page 16 of 17 9. 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Journal

Applied Biological ChemistrySpringer Journals

Published: Dec 1, 2021

Keywords: Neuraminidase; Non-catalytic site; Chalcone; Influenza; H5N1; H1N1

References