Chemoinformatic Design and Profiling of Derivatives of Dasabuvir, Efavirenz, and Tipranavir as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase and Methyltransferase

Zika virus (ZIKV) infection is one of the mosquito-borne flaviviruses of human importance with more than 2 million suspected cases and more than 1 million people infected in about 30 countries. There are reported inhibitors of the zika virus replication machinery, but no approved effective antiviral therapy including vaccines directed against the virus for treatment or prevention is currently available. The study investigated the chemoinformatic design and profiling of derivatives of dasabuvir, efavirenz, and tipranavir as potential inhibitors of the zika virus RNA-dependent RNA polymerase (RdRP) and/or methyltransferase (MTase). The three-dimensional (3D) coordinates of dasabuvir, efavirenz, and tipranavir were obtained from the PubChem database, and their respective derivatives were designed with DataWarrior-5.2.1 using an evolutionary algorithm. Derivatives that were not mutagenic, tumorigenic, or irritant were selected; docked into RdRP and MTase; and further subjected to absorption, distribution, metabolism, excretion, and toxicity (ADMET) evaluation with Swiss-ADME and pkCSM web tools. Some of the designed compounds are Lipinski’s rule-of-five compliant, with good synthetic accessibilities. Compounds 20d, 21d, 22d, and 1e are nontoxic with the only limitation of CYP1A2, CYP2C19, and/or CYP2C9 inhibition. Replacements of −CH3 and −NH– in the methanesulfonamide moiety of dasabuvir with −OH and −CH2– or −CH2CH2–, respectively, improved the safety/toxicity profile. Hepatotoxicity in 5d, 4d, and 18d is likely due to −NH– in their methanesulfonamide/sulfamic acid moieties. These compounds are potent inhibitors of N-7 and 2′-methylation activities of ZIKV methyltransferase and/or RNA synthesis through interactions with amino acid residues in the priming loop/“N-pocket” in the virus RdRP. Synthesis of these compounds and wet laboratory validation against ZIKV are recommended.


■ INTRODUCTION
Zika virus (ZIKV) is an arbovirus of the Flaviviridae family and flavivirus genus, primarily transmitted by the bite of infected Aedes spp. mosquitoes. 1 ZIKV infection is one of the mosquitoborne flaviviruses of human importance 2 first identified in 1947 in Uganda, 3 and more than 2 million suspected cases have been reported 4 with more than 1 million people infected in about 30 countries. 5 The virus has resonated with global concern that the World Health Organization (WHO) on February 1, 2016 declared it "a public health emergency of international concern". 6 Also, a potential pandemic spread of nonvector-borne ZIKV transmission has been reported elsewhere. 7 Again, the zika virus can cause several neurological complications, including microcephaly in newborns and Guillain−Barre syndrome in adults. 8 There are conspiracy theories, controversies, and other pseudoscientific claims about ZIKV. A 2019 report showed belief in genetically modified mosquitoes as the cause of ZIKV, 9,10 and the first genetically modified mosquitoes were released in 2021. 11 The controversies, especially the controversial tissue research, could hamper essential research on ZIKV, like probing the link between the virus and birth defects 12 including drug discovery. There are reported inhibitors of the zika virus replication/ protein synthesis machinery including amodiaquine, hydroxychloroquine, dasabuvir, efavirenz, and tipranavir, 2,13−17 but no approved effective antiviral therapy (drugs or vaccines) directed against the virus for treatment (or prevention) is currently available. 2,18,19 Therefore, the discovery of novel antivirals against ZIKV is a research priority. To address this urgent medical need, we designed and profiled derivatives of dasabuvir, efavirenz, and tipranavir as potential inhibitors of the zika virus NS5 protein (RNA-dependent RNA polymerase (RdRP) and methyltransferase (MTase)). This is because the three approved drugs (dasabuvir, efavirenz, and tipranavir) have been reported to inhibit the replication of the zika virus. 2 Again, they have shown some unfavorable therapeutic outcomes. For example, dasabuvir and tipranavir have been associated with mutations in hepatitis C NS5B polymerase and human immunodeficiency virus (HIV) protease, respectively, leading to resistance. 20−22 Increased damage indexes and frequencies have been observed in the brain of mice treated with efavirenz, 23 and WHO guidelines do not recommend efavirenz use during the first trimester of pregnancy due to concern for teratogenicity. 24 Therefore, there is a need to discover/identify more potent and nontoxic/safer derivatives of the parent compounds.
The NS5 protein is the largest and most conserved flaviviral protein with an RNA-dependent RNA polymerase (RdRP) domain at its C-terminal end, which is vital for viral replication. 25, 26 Zika virus NS5 protein also has an N-terminal methyltransferase (MTase) domain that plays a critical role in viral RNA genome capping 13,26 and interacts with the NS5polymerase domain during RNA synthesis. 5 S-Adenosylmethionine (SAM) MTase substrate binding sites have been implicated in zika virus methyltransferase inhibition with sinefungin as its analogue. 27 Specifically, the MTase domain of the the zika virus NS5 protein catalyzes methylation of the RNA 5′-cap structure to generate N7-methyl-guanosine and 2′-O-methyl adenosine (GpppA-RNA → m 7 GpppAm-RNA) using SAM as a methyl group donor. The NS5 protein has been validated as a promising antiviral drug target. 5,26 Taken together, inhibition of both RNA-dependent RNA polymerase and methyltransferase activities of the zika virus will halt viral replication and appears a promising drug target option. Therefore, the study is aimed at chemoinformatic design and profiling of derivatives of dasabuvir, efavirenz, and tipranavir as potential inhibitors of the zika virus RNA-dependent RNA polymerase and/or methyltransferase. There are several computational techniques for drug discovery against diseases like the zika virus. They include, but are not limited to, virtual ligand screening, molecular docking, and molecular dynamics simulations including free energy and steered molecular dynamics simulations. 28,29 ■ METHODS Description of Data Sets. The data sets used for this study are dasabuvir (PubChem identifier: CID 56640146), efavirenz (PubChem identifier: CID 64139), and tipranavir (PubChem identifier: CID 54682461) and were obtained from the PubChem database. 30 The drugs have been reported as inhibitors of zika virus replication at micromolar concentrations 2 with selective toxicities in Vero, UKF-NB-4, and HBCA cells. 2 Again, dasabuvir, efavirenz, and tipranavir have been reported to have some unfavorable therapeutic outcomes as mentioned earlier.
In Silico Design of the Hypothetical Compounds. The three-dimensional (3D) coordinates of dasabuvir (PubChem identifier: CID 56640146), efavirenz (PubChem identifier: CID 64139), and tipranavir (PubChem identifier: CID 54682461) were obtained from the PubChem database 30 in the Structure Data File (SDF) format and used for the design of their respective derivatives. Briefly, approved druglike derivatives of dasabuvir, efavirenz, and tipranavir (Supporting Information 1) were designed with DataWarrior-5.2.1 31 using an evolutionary algorithm, SkelSphere descriptor, automatic number of generations, fitness criteria of structural (dis)similarity, 128 compounds per generation, and eight (8) compounds survival per generation. The number of heavy atoms, calculated logP (cLog P), and other pharmacokinetic/ toxicity (e.g., mutagenic, tumorigenic, irritant) properties were calculated. The designed compounds that were not mutagenic, tumorigenic, and irritants (Supporting Information 1) were identified after extracting the calculated parameters, including their Simplified Molecular Input Line Entry System (SMILES) with a shell script (Supporting Information 2), and used for further studies.
Predocking Preparations of Designed Compounds and Target Proteins. The designed compounds were saved in the SDF format in the DataWarrior-5.2.1 platform. The files were separated into their compounds using Open Babel. 32 Designed compounds that were not mutagenic, tumorigenic, and irritant (Supporting Information 1) were obtained and prepared for docking simulations using shell script (Code S2 in Supporting Information 2). The script utilized obminimize 32 and General Amber Force Field (GAFF), which have parameters for most organic and pharmaceutical molecules. 33 The compounds were subjected to 2500 steps of geometry optimization using the steepest descent algorithm before generating the PDBQT (Protein Data Bank, Partial Charge (Q), and Atom Type (T)) file format for docking simulation studies. Zika virus NS5-RNA-dependent RNA polymerase (PDB ID: 5U04) and NS5-methyltransferase (PDB ID: 5MRK) were used for the study. Their 3D atomic coordinates were obtained from Protein Data Bank 34 and prepared for molecular docking simulations as previously reported, which involved the deletion of duplicate chains, heteromolecules, water molecules, and the addition of polar hydrogen. 2 Validation of Docking Protocols. To substantiate our evidence, the experimental crystal complex of NS5-methyltransferase with sinefungin was reproduced in silico as previously reported 2 using AutoDockVina. 35 The sinefungin obtained in a complex with NS5-methyltransferase from the protein data bank was removed, optimized, and redocked with NS5-methyltransferase using the grid box sizes (13 × 18 × 15) and centers (18.216 × 7.699 × 4.793), xyz coordinates, at 1.0 Å grid space and 8 exhaustiveness, as reported in a previous study. 2 Also, blind docking and redocking in the case of RNAdependent RNA polymerase with sizes (12 × 16 × 16) and centers (25.036 × 68.817 × 103.577) at 1.0 Å grid space and 8 exhaustiveness were performed, as previously reported. 2 Molecular Docking Simulations. The prepared designed compounds that were not mutagenic, tumorigenic, and irritant were docked into the zika virus NS5 protein (RNA-dependent RNA polymerase and methyltransferase) using AutoDockVina 35 after validation of molecular docking protocols or blind docking and redocking simulations. Specifically, the compounds were docked into the priming loop/allosteric pocket ("N-pocket") site 16,36 in RNA-dependent RNA polymerase and substrate (S-adenosylmethionine) binding site in methyltransferase with docking parameters mentioned in the Validation of Docking Protocols section. Additionally, dasabuvir, efavirenz, and tipranavir, which were previously reported as inhibitors of flavivirus/zika virus replication in cell culture, 2 were also docked into the zika virus NS5 protein as the control/standard. The simulations were performed locally on a Linux platform using a configuration file and script from a previous study 2 containing information on the grid box centers and sizes of prepared receptors (RNA-dependent RNA polymerase and methyltransferase) and the designed compounds. The binding energies (BEs) (kcal/mol) (n = 4) from the molecular docking simulations were calculated and expressed as mean ± standard deviation. Designed compounds with better binding energies for RNA-dependent RNA polymerase and/or methyltransferase than their respective parent compounds (dasabuvir, efavirenz, and tipranavir) were identified, and their binding poses were visualized with Maestro, version 12.8, Schrodinger, LLC, New York, NY.
Calculation of Bioactivities. The AutoDockVina docking score corresponds to the predicted binding energy (BE). 35 Inhibition constants (K i ) of the designed compounds with better BEs were obtained using eq 1 as reported elsewhere, 37 where R is the gas constant (1.987 × 10 −3 kcal/(K mol)) and T (298.15 K) is the absolute temperature of the protein− ligand complex. All ligand efficiency-related parameters 38 were derived from the binding energy and thus calculated using eqs 2−5 as reported elsewhere. 37  (5) where LELP means ligand efficiency-dependent lipophilicity, FQ means fit quality, and HA means heavy atoms.
In Silico Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) Predictions of the Designed Compounds. The designed compounds with better BEs than their respective parent compounds (dasabuvir, efavirenz, and tipranavir) were further subjected to absorption, distribution, metabolism, excretion, and toxicity (ADMET) and drug-likeness evaluation. The ADMET and drug-likeness predictions were performed with Swiss-ADME and pkCSM web tools. 39,40 ■ RESULTS AND DISCUSSION Designed Compounds. A total of 597, 582, and 609 approved druglike derivatives of dasabuvir, efavirenz, and tipranavir, respectively, were designed with DataWarrior-5.2.1. It is important to identify potentially mutagenic, tumorigenic, and irritant compounds and exclude them from the drug discovery compound library. Therefore, 77, 29, and 436 designed derivatives of dasabuvir, efavirenz, and tipranavir, representing 12.91, 4.98, and 71.59% of their respective designed derivatives, respectively, were identified as nonmutagenic, nontumorigenic, and nonirritant (Supporting Information 1). The summary of mutagenic, tumorigenic, and irritant assessments of all designed derivatives can be found in Figure S1 in Supporting Information 3. This study showed that dasabuvir is a low-mutagenic and highly tumorigenic agent but not an irritant (Supporting Information 1). This observation may be due to the amide group in the sulfonamide moiety of dasabuvir since amine/amide drugs/ compounds and their nitrosation products are mutagenic. 41,42 The previous reports have shown that treatment of hepatitis C with dasabuvir is associated with mutations including S556G and C316Y mutations in the viral NS5B polymerase, leading to resistance. 20,21 Increased damage indexes and frequencies have been observed in the brain of mice treated with efavirenz, 23 and findings from this study showed that efavirenz is a highly tumorigenic agent but not mutagenic nor irritant (Supporting Information 1). The tumorigenic effect of efavirenz may be responsible for adverse effects (e.g., headache, dizziness, insomnia, impaired concentration, agitation, amnesia, fatigue, and hallucinations) of efavirenz on the central nervous system. 24 Also, the World Health Organization (WHO) guidelines does not recommend efavirenz use during the first trimester of pregnancy due to the concern for teratogenicity. 24 This study showed that tipranavir was not mutagenic, tumorigenic, and irritant (Supporting Information 1). However, experimentally resolved structures of wild-type and L76V mutant of the HIV protease in a complex with tipranavir have been reported. 22 All of the nonmutagenic, nontumorigenic, and nonirritant derivatives were selected as libraries 28 and used for molecular docking simulations.
Molecular Docking and Bioactivity Results of the Designed Compounds. The results of the molecular docking simulation binding energies/affinities (kcal/mol) of the parent compounds and their designed derivatives that were not mutagenic, tumorigenic, and irritant derivatives (Supporting Information 1) with the zika virus RNA-dependent RNA polymerase and methyltransferase are presented in Figure 1a,c   and Tables S1−S4 in Supporting Information 3 and Supporting Information 1. The binding energy score is an indication of how strongly the compounds interact with drug targets. The lower the numeric value, the better the interaction. This study identified derivatives of dasabuvir 15d, efavirenz 3e, and tipranavir 21t, with binding energies for the zika virus RNA-dependent RNA polymerase only (41, 2, and 18, respectively), methyltransferase only (2, 2, and 50, respectively), and both RNA-dependent RNA polymerase and methyltransferase (14, 0, and 20, respectively) equivalent to or better than their respective parent compounds (dasabuvir, 15d; efavirenz, 3e; and tipranavir, 21t), as can be seen in Supporting Information 1. This is possibly due to the changes in the structures of the parent compounds. For example, some  of the derivatives are due to the isosteric replacement of carbon with a nitrogen atom, and nitrogen-containing compounds have a reported exceptional broad-spectrum biological activity against viruses. 43 The names and structures of some of the designed derivatives of dasabuvir 15d, efavirenz 3e, and tipranavir 21t, including their parent compounds (1d to 19d, 1t to 30t, 1e to 5e) were determined by their respective SMILES (Supporting Information 1) using Chem-Draw-12.0 and are presented in Tables 1, 4 This study identified some derivatives of the said parent compounds as potential inhibitors of the virus NS5 protein, and a previous report had validated N- [4-[6-tert-butyl-5methoxy-8-(6-methoxy-2-oxo-1H-pyridin-3-yl)-3 quinolyl] phenyl] methanesulfonamide (PubChem identifier: CID 49835560), a derivative of dasabuvir, against hepatitis C virus NS5B polymerase 44 but not zika virus. Again, novel guanosine derivatives against the zika virus polymerase have been reported. 49 The inhibition constant value in the micromolar range, fit quality (FQ) of ≥0.8, and the range of LELP between −10 and 10 have been reported for acceptable lead/druglike compounds. 37,50 The majority of the designed derivatives (1d to 14d, 16d to 22d, 1t to 20t, 22t to 26t, 1e, 2e, 3e, and 4e) have better K i , FQ, and LELP values when compared with their respective parent compounds (Tables S6  and S5 in Supporting Information 3). This suggests that they may have better antiviral activities against the virus than their respective parent compounds. Also, they have good pharmacokinetic parameters, drug-likeness, and toxicity profiles (Tables 1−10), including synthetic accessibilities.
Molecular Interactions. Molecular interactions of two representatives of derivatives of dasabuvir (3d and 5d) with better binding energies and K i for both zika virus methyltransferase and RNA-dependent RNA polymerase (Figure 1a,b) than dasabuvir, 15d, are presented in Figure 2. It is important to mention that some derivatives of dasabuvir (16d and 17d) showed better binding energies and K i (Tables S1 and S5 in Supporting Information 3) than 3d and 5d for RNA-dependent RNA polymerase but not for methyltransferase. Figure 3 shows the molecular interactions of 16d and 17d with RNA-dependent RNA polymerase. Also, molecular interactions of two representatives of derivatives of tipranavir (3t and 10t) with better binding energies and K i for both zika virus methyltransferase and RNA-dependent RNA polymerase (Figure 1c,d) than tipranavir, 21t, are presented in Figure 5. Again, some derivatives of tipranavir (e.g., 22t to 26t) showed better binding energies (−10.00 ± 0.00 to −10.25 ± 0.006 kcal/mol) and K i (0.030192974 ± 0.002938 to 0.045899983 ± 0.000000 μM) (Tables S2 and S6 in Supporting Information 3) than 3t and 10t for methyltransferase but not for RNAdependent RNA polymerase. Docking results showed that derivatives of dasabuvir (3d, 5d, 16d, and 17d) and tipranavir (3t, 10t, 22t to 26t) complexes with methyltransferase and RNA-dependent RNA polymerase are stabilized by a significant number of noncovalent interactions (Figures 2−5). Specifically, there are four intermolecular hydrogen bonds between four amino acid residues (Ser56, Gly85, Gly86, and Asp131) in the methyltransferase and two derivatives of dasabuvir (3d, 5d) compared with three (Ser56, Gly85, and Gly86) between methyltransferase and dasabuvir (Figure 2a−c). Also, the study observed a hydrogen bond between Gly148 in the methyl-transferase with tipranavir derivatives (3t, 10t, 22t to 26t) (Figures 4 and 5a−c). Additionally, other amino acid residues in the methyltransferase with direct contacts (hydrogen bond, π−π stacking, and/or π−cation) with 3t, 10t, 22t to 26t are Lys105, Gly106, Glu111, Phe133, Asp146, and/or Lys182 (Figures 4, 5a−c, and S2 in Supporting Information 3). Similar observations have been made for a reference inhibitor (2-(4- 47 Again, a previous study has reported intermolecular interactions between S-adenosylmethionine (SAM) with Lys105, Ile147, Val132, Asp131, Gly106, Glu111, Thr104, Ser56, Asp146, Gly86, and Trp87 in zika virus methyltransferase. 51 Also, interactions between Gly148, Cys82, Arg84, Lys105, Val132, and Gly77 in zika virus MTase with in vitro validated inhibitors of the virus MTase have been reported. 47 This suggests that the designed derivatives of dasabuvir and tipranavir bind to the SAM binding site and are possible inhibitors of zika virus methyltransferase. The possible inhibition of N-7 and 2′ methylation activities of the virus MTase by the designed compounds due to the mentioned hydrogen bonds implies virus death through host immune response. This is because methylation at the N-7 position is essential for viral replication and methylation at the 2′-O position helps RNA evade host immune response, 52 and mutating Gly148 to Ala148 abolishes the N-7, and 2′-O methylation capacity of dengue virus MTase activity. 47 This may be the case for the zika virus MTase. Again, Arg160, and Arg163 are important for N-7 methylation, while Phe133 is important for 2′-O methylation. 47 This study observed intermolecular interactions between Phe133, Arg160, and Arg163 in the methyltransferase with the designed compounds (Figures 2 and 4). Other compounds like theaflavin, a natural compound, have been reported to directly bind to Asp146 of zika virus NS5 MTase to inhibit replication. 48 Asp146 in zika virus is part of the Lys61− Asp146−Lys182−Glu218 tetrad motif that is important for both N-7 and 2′O methylation, 51 and the designed compounds (3d, 5d, 16d, 17d, 3t, 10t, 22t to 26t) showed interaction with Asp146 (Figures 2a−c, 4, 5a−c, and S2 in Supporting Information 3). This implies that the designed derivatives of dasabuvir and tipranavir are possible inhibitors of methylation at the N-7 position, which is essential for viral replication and methylation at the 2′-O position and helps the virus evade the host immune response. 52 However, there is a need for in vitro validations of the designed compounds against zika virus methyltransferase activity.
In the case of interactions of derivatives of dasabuvir and tipranavir with the virus RNA-dependent RNA polymerase, this study observed hydrogen-bonding or π−cation interaction between Arg731 and dasabuvir and its derivatives (3d, 5d, 16d, 17d) (Figures 2d−f and 3). Other amino acid residues in the RNA-dependent RNA polymerase that have direct contacts (hydrogen bonding, π−π stacking, and/or π−cation) with dasabuvir and its derivatives (3d, 5d, 16d, 17d) including tipranavir and its derivatives (3t and 10t) are Tyr609, Arg612, Asp665, Cys711, Ser712, Arg739, Arg794, Trp797, and/or Ser798 (Figures 2d−f, 3, and 5d−f). Intermolecular hydrogen bonds have been reported between the sulfonamide moiety of nonnucleoside inhibitors and Arg739, Trp797, Arg731, and Thr796 residues in the allosteric pocket ("N-pocket") of the zika virus RNA-dependent RNA polymerase. 16 Also, similar interactions have been reported for novel guanosine derivatives and nonnucleoside inhibitors like 5-(5-fluorothiophen-2-yl)-2hydroxy-4-methoxy-N-((3-(trifluoromethyl)phenyl)sulfonyl) benzamide against the zika virus polymerase. 16,49 This largely validates the designed compounds as potential inhibitors of the zika virus RNA-dependent RNA polymerase. It has been reported elsewhere that fidaxomicin inhibits zika virus replication and prevents the virus-infected mice from death. 36 Also, strong binding between fidaxomicin and zika virus RNA-dependent RNA polymerase has implicated Thr795 and Ser798 to be located in the priming loop (residues 787− 809), an important stacking platform for initiation of nucleotide triphosphate entry into de novo RNA synthesis. 36 This suggests that the designed compounds have the potential for in vitro and in vivo activities against the zika virus since they showed intermolecular hydrogen bond(s) with amino acid residues from the priming loop region. However, there is a need for laboratory confirmations of the activities of the designed compounds against the zika virus RNA-dependent RNA polymerase. A previous report showed that tipranavir contains stereocentres at both C-3α and C-6 and a robust resistance profile to HIV. Tipranavir binding with few hydrogen bonds creates increased flexibility to adjust to amino acid changes in the HIV protease active site, which is largely responsible for its resistance profile. 53 This may explain why this study observed fewer hydrogen bonds in the interactions of the designed derivatives of tipranavir with RNA-dependent RNA polymerase and methyltransferase (Figures 4 and 5) when compared with those of dasabuvir (Figures 2 and 3). Generally, some of the designed derivatives of dasabuvir and tipranavir showed better/improved binding energies/inhibition constants than their respective parent compounds (dasabuvir and tipranavir). However, derivatives of efavirenz (1e, 2e, 4e, and 4e) did not show appreciable improvement in binding energies and inhibition constants (Table 7). Interestingly, atoms/groups or substituents in these designed derivatives with better/improved binding energies and inhibition constants that make them different from their parent compounds were not directly involved in intermolecular    (Figures 3a and 4). Quinoline nitrogen (N) and oxygen (O) atoms in carboxylic functional groups of 16d and 24t are involved in interactions with RNA-dependent RNA polymerase and methyltransferase, respectively, when compared with dasabuvir and tipranavir (Figures 2c and 5c). It is possible that

Results of In Silico ADMET Predictions of the Designed Compounds.
The predicted drug-likeness, pharmacokinetics, and ADMET parameters of the designed compounds with better binding energies than dasabuvir or tipranavir are presented in Tables 1−6, 9, and 10 and Supporting Information 1. Tables 1, 3, 9, and 10 illustrate the parameters suggested by Lipinski drug-likeness and oral bioavailability, gastrointestinal adsorption (GIA), pan-assay interference compounds (PAINS) alert, synthetic accessibility, and bioavailability score. Tables 2 and 4−6 present some selected toxicity profiles of the designed compounds with better binding energies than dasabuvir or tipranavir. Determination of the drug-likeness of any potential drug candidate is paramount in the drug discovery/development process. The drug-likeness properties for potential drug candidates proposed by Lipinski 40,54 and implemented in the Swiss-ADME web tool were utilized. It could be observed from Tables 1, 3, 9, and 10 that the designed compounds with better binding energies/ inhibition constants than dasabuvir or tipranavir did not violate more than one of the rules proposed by Lipinski. This suggests good drug-likeness and oral bioavailability 54 of the compounds. Other information in Tables 1, 3, 9, and 10 includes glycoprotein (P-gp) substrate, gastrointestinal adsorption (GIA), pan-assay interference compounds (PAINS) alert, bioavailability score, and synthetic accessibility. It is clear from Tables 1, 9, and 10 that the derivatives of dasabuvir are all not substrates for permeability glycoprotein (P-gp), the most important member of ATP-binding cassette (ABC) transporters for increasing active efflux through biological membranes, 40 except 6d and 7d. Then, all of the derivatives of tipranavir (Tables 3 and 9) are substrates of P-gp. Again, derivatives of efavirenz (2e, 4e, and 5e) are P-gp substrates just like efavirenz (Tables 9 and 10). Previous reports have shown that P-gp inhibitors can overcome multidrug resistance and poor bioavailability problems of therapeutic P-gp substrates. 55,56 The designed derivatives of dasabuvir and tipranavir showed low gastrointestinal absorption (GIA) like their parent compounds, except 23d, 24d, and 6d, while all derivatives of efavirenz showed high GIA like their parent compound (efavirenz) (Tables 9 and 10). The high GIA observed in the derivatives of dasabuvir was due to methylene (−CH 2 −) ( Figure S3 in Supporting Information 3) with increased cLog P compared with that of dasabuvir (Tables S10 and S11). This is possible because the methyl group has been associated with increased lipophilicity, log P, of compounds, 57 which can lead to their solubility in biomembranes. This has been implicated in the passage of compounds from the gastrointestinal tract to target tissues and transport through the bloodstream. 57 However, 23d, 24d, and 6d were positive for Ames toxicity, hERG II inhibition, and hepatotoxicity ( Figure  S3 in Supporting Information 3). The designed compounds were predicted to possess no PAINS alert (Tables 1, 3 , 9, and 10). This suggests that the designed compounds are not frequent hitters or promiscuous compounds and do not contain substructures that will show potent responses in assays irrespective of the protein target. 40 Prediction of synthetic accessibility (ease of synthesis) of bioactive compounds possessing drug-likeness is paramount for the selection of the most promising molecules for syntheses, biological assays, or other experiments. 40,58 A good number of the designed compounds, especially 6d (3.26) and 3t (5.17), demonstrated ease of synthesis due to  Figure 6. Hepatotoxic structural alerts for derivatives of dasabuvir. Compounds 4d, 5d, and 18d are noninhibitors of the human ether-ago-go-related gene (hERG) but showed hepatotoxicity. Also, dasabuvir (15d) is the parent compound that showed both hERG II inhibition and hepatotoxicity. Figure 7. Structural determinants of noninhibition of hERG and no hepatotoxicity in derivatives of dasabuvir (19d, 20d, 21, and 22d) compared with dasabuvir (15d) in Figure 6, which showed both hERG II inhibition and hepatotoxicity.
their lower synthetic accessibility score (Tables 1, 3, 9, and 10) compared with dasabuvir (3.46) and tipranavir (5.29), respectively. This is because the smaller the value, the easier the synthesis of a compound. 58 Synthetic accessibility of designed derivatives of 1,2,4-oxadiazole has been reported elsewhere. 45 The bioavailability scores of the designed compounds are similar to those of their parent compounds and within the probability of 55% or more, except 5d, 9t, 18d, and 24t, which showed 11% 58 each (Tables 1, 3, 9, and 10). Similar observations have been made elsewhere. 45 Prediction of interactions of molecules/compounds with cytochromes P450 (CYP) during drug discovery/development is very important. This is because cytochrome P450 and its isoenzymes play a key role in drug elimination through metabolic biotransformation. 40 The propensity with which the designed compounds caused drug interactions through inhibition of CYPs, and the isoforms affected, 40 was evaluated, and it shows that they are inhibitors of at least one CYP450 isoform, except 1d, 5d, and 18d, which are not inhibitors of any of the isoforms (Tables 1, 3, 9, and 10). However, the bioavailability score of 5d and 18d is 0.11 each, while that of 1d is 0.55 compared with 0.55 of dasabuvir. Previous reports revealed that many therapeutic compounds are a substrate of one of the five major cytochrome P450 isoforms (CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4). 59,60 Therefore, inhibition of one or more of the isoforms could lead to pharmacokinetics-related drug−drug interactions, 61−63 which may result in toxic or other unwanted adverse effects. 64 This suggests that 1d, 5d, and 18d, which are not inhibitors of any of the isoforms, will not lead to pharmacokinetics-related drug−drug interactions and toxic or other unwanted adverse effects. However, the isoforms not inhibited by the designed derivatives may selectively process the compounds for elimination through metabolic biotransformation such that inhibition by a specific inhibitor will lead to an increase in the exposure to the substrate. 61 Also, CYP and P-gp can process small molecules synergistically to improve the protection of Table 9. Drug-Likeness and Pharmacokinetic Parameters of Derivatives of Dasabuvir, Efavirenz, and Tipranavir That Are Noninhibitors of the Human Ether-a-go-go-Related Gene (hERG)  It is also possible that pkCSM did not classify as a hepatotoxic agent due to certain substructural elements, but this is unlikely because pkCSM relies on learning patterns between chemical composition, similarity, pharmacokinetics, and safety properties for predictive models capable of identifying implicit patterns consistent and valid for unseen data. 39 Again, the generation of toxic/reactive metabolites is a possibility for liver failure due to efavirenz.
tissues and organisms. 65 Again, 5d and 18d showed lower bioavailability scores (Tables 1 and 9). The toxicity of the designed compounds with binding energies equal to or better than their respective parent compounds (dasabuvir, efavirenz, and tipranavir) was further assessed using the pkCSM web tool, and the results are presented in Tables 2 and 4−6 and Supporting Information 1. The results showed that six of the designed derivatives of dasabuvir (6d, 13d, 17d, 19d, 23d, and 24d) showed Ames toxicity, whereas derivatives of efavirenz and tipranavir did not (Tables 2 and 4−6). The Ames test is a short-term bacterial reverse mutation assay detecting a large number of compounds that can induce genetic damage and frameshift mutations. 66 Toxicity is a leading cause of attrition at all stages of drug development, 66 and the identification of compounds capable of inducing mutations has become an important procedure in safety assessment 67 during drug discovery/development. This suggests that the designed derivatives without Ames toxicity are relatively safe than those that show Ames toxicity. No skin sensitization was observed in the designed derivatives of dasabuvir, efavirenz, and tipranavir (Tables 2 and4−6). This suggests that the compounds will not cause allergic reactions such as rash, blisters, or itch when in contact with the skin. 68 Few of the designed compounds (12) with better binding energies are noninhibitors of human ether-a-go-go-related genes I and II (hERG I and hERG II) (Tables 5 and 6). The rest of the designed compounds are inhibitors of hERG II but not hERG I (Tables 2 and 4−6). Activation of hERG channels mediates cardiac action potential repolarization. 69 Loss-offunction mutations in hERG and its inhibition can lead to inherited and acquired long QT syndrome (LQTS), respectively, 69−71 which can in turn lead to fatal cardiac arrhythmia 70 and sudden death. 69 However, small molecules that increase/activate hERG have found application in the treatment of LQTS. 69,71 This suggests that the identified noninhibitors of hERG I and hERG II are less cardiovascular toxic, making them safer than noninhibitors of only hERG I. Similar observations have been made for derivatives of 1,2,4oxadiazole. 45 Also, the predicted toxicities of designed derivatives of dasabuvir, efavirenz, and tipranavir that are noninhibitors of the human ether-a-go-go-related gene (hERG) and/or noninducers of hepatotoxicity, binding energies, bioactivities, drug-likeness, and pharmacokinetic parameters are presented in Tables 5−10. They showed no Ames toxicity, except 19d, and no skin sensitization ( Table 7).
The predicted toxicity of designed derivatives of dasabuvir, efavirenz, and tipranavir that are noninhibitors of the human ether-a-go-go-related gene (hERG) and noninducers of hepatotoxicity is presented in Table 6. The rest of the designed compounds showed hepatoxicity just like their parent compounds, except efavirenz 3e. Reports have shown that drug-induced hepatotoxicity is the main cause of drug nonapproval and/or withdrawal. 72,73 Therefore, the identification of non-hepatotoxic compounds early in drug discovery will offer drugs devoid of hepatotoxicity. This is important because drug-induced liver injury may lead to acute hepatitis and/or acute liver failures. 72,74,75 Interestingly, only five compounds (19d, 20d, 21d, 22d, 1e) showed no hepatotoxicity (Table 6). They also showed no Ames toxicity, except 19d, no skin sensitization, hERG inhibition, and better K i for RdRP (Table 8) but not MTase. Above all, they did not violate more than one Lipinski rule and showed no PAINS alerts (Table 10). Based on the toxicity profile of the designed compounds, it may be reasonable to classify 20d, 21d, 22d, and 1e as nontoxic and those in Tables 5, 7, and 9 (5d, 4d, 18d, 24t, 5e, 4e, and 2e) as moderately toxic (i.e., only hepatotoxic) than the rest of the compounds.
Structural Alerts for hERG II Inhibition and Hepatotoxicity. Molecular structures of the designed compounds that showed no hERG inhibition and/or hepatotoxicity (Table  10) including high GIA were visualized to identify structural alerts/features associated with the observed hERG II inhibition, high GIA, and/or hepatotoxicity (Figures 6−8 and S3 in Supporting Information 3). Such structural alerts are indicative of probable hERG II inhibition, high GIA, or hepatotoxicity. However, it is also possible that certain substitutions can block the resulting toxicity, absorption, or inhibition. 76 It can be observed from Figure 6 that compounds 4d, 5d, and 18d contain quinoline or isoquinoline moieties. Liver tumors have been observed in rats and mice exposed to quinoline. 77,78 This may be the case for compounds 4d, 5d, and 18d. However, this is unlikely since dasabuvir, which also showed hepatotoxicity, does not have a quinoline or isoquinoline moiety ( Figure 6). Structural alerts for hepatotoxicity due to para-aminophenylsulphonamides such as sulfadimethoxine, sulfaphenazole, and sulfamethoxazole have been reported elsewhere. 76 Since −NH− in the methanesulfonamide/sulfamic acid moieties (red color in Figure 6) and attached to the quinoline/isoquinoline moieties (green color in Figure 6) is common in the para-aminophenylsulphonamides and compounds 4d, 5d, 15d, and 18d, it may be responsible for the observed hepatotoxicity. Moreover, its absence in 19d to 22d (Figure 7) supports this view. Interestingly, sulfonamides/nitromethanesulfonamides have been linked to cases of hepatic injury and acute liver failure. 76,79−81 It is unlikely that −NH− in the pyrimidine moiety is responsible for hepatotoxicity because it is also present in compounds 19d to 22d, which did not show hepatotoxicity. Ingestion/overdose of other drugs like acetaminophen (a drug containing −NH−) has been associated with hepatotoxicity 82 through their conversion to a toxic metabolite, N-acetyl-p-benzoquinone imine. 83 Again, high acute toxicity has been reported for some piperine amide analogues containing either −NH or −N−. 84 Therefore, the observed hepatotoxicity of 4d, 5d, 15d, and 18d is likely due to the presence of −NH− in the methanesulfonamide/sulfamic acid moieties. Introduction of hydrophilic and/or polar hydroxyl groups (−NH, −N−, and/or −OH) in dasabuvir results in compounds 4d, 5d, and 18d with no hERG inhibition and containing quinoline or isoquinoline instead of the naphthalene moiety ( Figure 6). Similar findings have been reported elsewhere. 85 These modifications made 4d, 5d, and 18d have cLog P values of 3.38, 2.85, and 2.73, respectively, compared with 3.8 for dasabuvir (Table 6). It is widely admitted that compounds that have higher log P and pK a tend to be at higher risk of hERG inhibition, 85 and this may be the case for dasabuvir. Quinoline-based kinase inhibitors with reduced hERG activity have been reported elsewhere. 86 Again, drugs with a quinoline structure, particularly with high molecular polarity, can exert a significant potential hERG inhibitory activity. 87 This may be the case for 4d, 5d, 18d, 19d, 20d, 21d, and 22d since they are more polar than dasabuvir. It is important to note that some hERG inhibition cannot be explained by the decrease in lipophilicity or basicity but by subtle changes in the compound that can lead to the flexibility of groups like methylene. 85 The introduction of two fluorine (F) atoms and replacement of piperidine with piperazine leading to methylene (−CH 2 −) flexibility have been used to overcome hERG inhibition. 85 Indeed, 19d, 20d, 21d, and 22d were a result of replacements of −NH− with −CH 2 −, −CH 2 CH 2 − or −CH 2 CH 2 CH 2 −, and −CH 3 with −OH including replacement of the hydrogen atom (H) with −CH 3 in the case of 21d. A previous study showed that modification of aryl-1H-imidazole compounds with the gem-dimethyl moiety between hydroxyl and amide groups has resulted in compounds with reduced hERG channel interactions. 85 A closer look at Figures 4 and 5c shows that 24t was derived from tipranavir, 21t, by replacement of a hydrogen atom (H) with −CH 2 COOH. This caused a decrease in cLog P from 6.06 to 5.27 (Table 9), making 24t more polar with the added benefit of being a noninhibitor of hERG II (Table 5). However, this modification caused a decrease in the 24t bioavailability score, which is 0.11 compared with 0.56 of tipranavir. Also, the bioavailability score of 5d and 18d is 0.11 each compared with 0.55 of dasabuvir. The modifications in 5d, 18d, and 1d did not lead to inhibition of any of the CYP450 isoforms. This suggests that they will not lead to pharmacokinetics-related drug−drug interactions, 61−63 which may result in toxic or other unwanted adverse effects. 64 However, they showed a smaller bioavailability score than   (Tables 9 and  10) when compared with 3.8 of efavirenz. These changes were due to the replacement of the oxygen (O) atom with S, Cl with F, and H with N. Compound 1e had higher cLog P because the S atom is less electronegative than the O atom. This implies hERG inhibition. Fluorine incorporation in 1,4-substituted piperidines during optimization showed improved hERG channel selectivity, 85 while that of nitrogen atom on 7,9dihydro-8H-purin-8-ones led to its triazolopyridine analogue, which keeps its activity toward DNA-dependent protein kinase with reduced hERG potency. 85 This may be the case for the derivatives during interactions with zika virus RNA-dependent RNA polymerase and methyltransferase in vivo. Hepatotoxicity of compounds 2e and 4e may be similar to that of adenosine-based reverse transcriptase inhibitors such as tenofovir, vidarabine, and clofarabine 76 due to the substituted nitrogen atom(s). Efavirenz can be considered a safer drug for the liver. 88 However, there are reported cases of acute liver failure among patients on efavirenz-based antiretroviral therapy due to efavirenz, 89 but our findings did not mark efavirenz as a hepatotoxic agent.

■ CONCLUSIONS
Some of the designed compounds follow Lipinski's rule-of-five, with no mutagenic, tumorigenic, or irritant properties. Again, the compounds did not show skin sensitization and PAINS alert while possessing good synthetic accessibilities. Compounds 20b, 21b, and 22b are noninhibitors of hERG while possessing no hepatotoxicity and Ames toxicity. Based on the toxicity profile of the designed compounds, 20d, 21d, 22d, and 1e are classified as nontoxic with the only limitation of CYP1A2, CYP2C19, and/or CYP2C9 inhibition, which can be managed with careful administration to avoid pharmacokinetics-related drug−drug interactions and toxicity or other unwanted adverse effects when developed and brought to the clinic. Replacement of −CH 3 and −NH− in the methanesulfonamide moiety of dasabuvir with −OH and −CH 2 − or −CH 2 CH 2 − respectively improved the safety/toxicity profiles of 20d, 21d, and 22d. Hepatotoxicity observed in some of the derivatives of dasabuvir (5d, 4d, 18d) is likely due to the presence of −NH− in their methanesulfonamide or sulfamic acid moieties. The designed compounds are potent inhibitors of N-7 and 2′ methylation activities of zika virus MTase, which can lead to virus death and/or the ability to not evade host immune response. Also, they are potent inhibitors of the virus RNA synthesis through interactions with amino acid residues in the priming loop and/or "N-pocket" in the virus RNAdependent RNA polymerase. Synthesis and wet laboratory validations of the designed compounds against the zika virus are recommended.
Designed derivatives of dasabuvir, efavirenz, and tipranavir; their binding energies and bioactivities; predicted ADME properties evaluated with Swiss-ADME; and toxicity and other parameters evaluated with pkCSM (Supporting Information 1) (XLSX) Shell scripts used for extraction of calculated parameters and preparation of designed derivatives for docking simulations ( Supporting Information 2)