key: cord-0721034-3twbyjj9
authors: Chatterjee, Satyaki; Kumar, Neeraj; Sehrawat, Hitesh; Yadav, Nisha; Mishra, Vivek
title: Click Triazole as a Linker for Drug Repurposing Against SARs-CoV-2: A Greener Approach in Race to Find COVID-19 Therapeutic
date: 2021-02-10
journal: nan
DOI: 10.1016/j.crgsc.2021.100064
sha: 814539818e4533ad5c4777528f338705f369f0e9
doc_id: 721034
cord_uid: 3twbyjj9

WHO holding the hands of the scientific commune and trying to repurpose the drugs against the SARS-CoV-2. The robust scientific data has illustrated the probable mechanistic path of SARS-CoV-2 entry and action in damaging the cells. Which further has demonstrated Hydroxychloroquine (HCQ; antimalarial drug) as promising drug therapeutic; apart from certain setbacks to be an excellent agent in treating COVID-19. In the present study, we have explored the derivatives of HCQ, conjugated with bioactive agents by the virtue of sustainably modified clicked triazole approach as potential Mpro enzyme inhibitors. In results, we found the chloroquinetrithaizone has strong binding affinity for the Mpro enzyme of SARS CoV-2. We also found the stable binding of CQ-TrOne conjugate with Mpro by MD simulation studies through RMSD, RMSF and Rg calculations. Moreover, in conjunction with critical reaction coordinate outcomes, binding MMGB/PB energy profile depicted the efficient binding affinity towards Mpro. Also, DFT analyses illustrated the stability of the repurposed drug under study. These significant outcomes have shown high potency of compounds and can be further assessed through in vitro and in vivo assays to develop the effective drug against COVID-19.

study. These significant outcomes have shown high potency of compounds and can be further assessed through in vitro and in vivo assays to develop the effective drug against COVID-19.

Since December 2019, a race has begun to bring a conclusion to the chapter entitled "COVID-19 A PANDEMIC". The year 2020, a mirror couple year, is now 11 months old with an unforgettable & undesirable memory with cases of a novel coronavirus from Wuhan to Worli; pushing the global death toll beyond imagination. SARS CoV-2 has emerged highly infectious with infection of over 25.5 million people worldwide in the large number of countries and caused the lethal results leading to the death. The WHO has shown critical importance on the use of existing FDA approved drugs as an anti-covid agent rather than developing agents from scratch. The biggest question that subsequently became a challenge at the clinical level was ruling out when to use the drug candidate. The idea to start now and move rapidly became a pioneer in coping up with the pandemic constructing SARS-CoV-2 [1, 2] as with passing time different strains of Human coronaviruses (HCoVs) have started coming up. 2019n-CoV is a part of the Betacoronaviruses genus similar to the Severe Acute Respiratory Syndrome Human coronavirus (SARS-HCoVs) and the Middle-East Respiratory Syndrome Human coronavirus (MERS-HCoVs) attempted to treat by Ebola antiviral Remdesivir, which failed for two major reasons; primarily it works only if given at an early stage and on the other side, the drug is expensive, whereas anti-HIV agent Lopinavir and ritonavir were completely inactive [3] .

Chloroquine (CQ) and Hydrochloroquine (HCQ) showed some hope as being a basic unit; it dropped the pH in endosomes restricting the entry of viruses changing the glycosylation of ACE2 receptor and spike protein [4, 5] , although the entry mechanism for novel SARs-CoV-2, structured with a Spike, Nucleocapsid, Matrix, and Envelope, and non-structural proteins such as J o u r n a l P r e -p r o o f RNA dependent RNA polymerase which is a crucial enzyme in the life cycle of RNA viruses, is still not clear (Fig. 1) . i) The viral RNA copy inhibited by the action of both CQ and HCQ, and ii) the action of CQ and HCQ, blocked the transport of novel coronaviruses from early endosomes (EEs) to endolysosomes (ELs), which is a key step in the release of viral genomes and iii) the cytokines were high in the plasma of infected patients making the importance of cytokines with the disease and hydrochloroquine can significantly reduce the production of cytokines as well, making chloroquine and hydrochloroquine an imperative precursor in treatment [6] which is even supported by molecular modelling study from Fantini et al. [7] In 2010, the role of Zn 2+ in inhibiting RNA polymerase in coronavirus and arterivirus was established by in-vitro study [8] and amazingly Chloroquine appeared as an astounding zinc ionophore [9] . However, in the end, HCQ failed at its safe dosage amount; which is around 6-6.5 mg/kg per day and is not up to the mark. Repurposing of 'existing' drugs to treat coronavirus is an attractive way of designing drugs from risk-free agents, with promising lower overall improvement costs and petite growth J o u r n a l P r e -p r o o f timelines [10] . In challenging biology-oriented synthesis, KB Sharpless defined Click Chemistry [11] as a stronghold owing to its greener nature (modest reaction conditions, readily and easily available starting materials and reagents, use of no solvent, a benign solvent (such as water), or one that is easily removed, simple product isolation). This powerful toolbox facilitates selective C-Hetero-C bond formation in environment friendly and cost-viable route [12] [13] [14] [15] , this nonchromatographic high yielding method repurposes drugs by creating a click link between two different bioactive agents. We have made attempt to elucidate the binding affinity of HCQ modified with click ready azide conjugated with existing pharmacophores with a triazole ring.

The triazole moiety formed here is not only acting as a linker but it may act as a basic unit (pKa = 9.3), elevating the pH level in eukaryote cells inhibiting the entry of spiked virus as well as the clicked triazole link has also shown medicinal & biological applications [16] [17] [18] , further the purpose is to explore the potency of click chemistry in drug development for COVID-19 treatment. The novelty of this work included the application of click chemistry and its impact for enhancement of drug binding efficacy and potency to combat the Mpro. Significantly, these results will also be applicable to other strain of virus, since we have employed the widely accepted target model which has high similarity with multiple variants of target Mpro. Viral RNA replication to yield functional viral protein is a decisive regulatory step and Mpro is the main protease enzyme. Depending on this fact, researchers tried to investigate the action of compounds on this particular protease, inhibiting the replication of the virus. The reported computer-aided assay of HCQ, CQ [19] , and Artemisinin analogs [20] and subsequent inhibition of protease enzyme Mpro in terms of binding energy motivated us to use this technique.

Molecular dynamics simulation, a tool to analyze the physical movement of atoms and molecules, elucidate the free energy change of the drug upon binding with the target protein J o u r n a l P r e -p r o o f through the root mean square deviation (RMSD), the heat-map of decomposing, and other relevant parameters [21, 22] . In addition, molecular docking lends a hand in recognizing the mechanistic interaction of the drug with the target receptor [23, 24] . Mechanistically, molecular modeling studies are widely employed and proved to be very efficient to understand the background and preliminary efficacy of drugs out of large datasets [25] [26] [27] . To investigate the mode of binding action and binding of drugs with their specific target receptors. Hence, herein details advanced compoutational assays are employed to study the clicked analogs to acquire the highly competent drug against Mpro of SARS-CoV-2 for further corroboration with investigational lab assay.

To perform the molecular binding analyses of proposed drugs, the main protease enzyme (Mpro) of SARS CoV-2 was used, it has main regulatory role in coronavirus infection, progression, and further regulation The crystal structure of SARS-CoV-2 main protease was retrieved from the Protein Data Bank (http://www.rcsb.org/) [28] and the structure was energy minimized to avail the confined and stabilized structure. The 3D crystal structure was also assessed for its physicochemical and stereochemical properties using the Ramachandran plot and other physicochemical servers [29, 30] . The receptor files were prepared using the Whatif prepdock module for molecular modeling analyses.

J o u r n a l P r e -p r o o f

The chloroquine-clicked drug analogs were designed drawn using the Chemdrawn software.

Designed conjugates ligands were optimized and energy minimized for molecular docking and molecular dynamics simulations analyses. The drug files (ligands) were assessed and corrected with Ligprep module and further chilarties were optimized.

To evaluate the binding affinity of proposed drugs with the Mpro enzyme, first molecular docking analyses were conducted. We have employed the HEX 8.0 molecular docking software, which works grounded on a fast Fourier algorithm [29, 31] . The shape and dynamics and 3D FFT mode route was opted and further the steps through interface energy with steric shape coordination and the electrostatic potential were investigated. Docking program was set to avail the top 25 drug binding poses with Mpro, which were further analyzed through the drug-protein interaction profilers. Importantly, to strengthen our results we have also used another molecular docking software Swissdock and molecular contacts, involved non-covalent bonds and binding domains were analyzed using the Ligplot tool.

The molecular dynamics simulation using the Amber16 program with a build force field (ff14SB and gaff force) and to evaluate the binding mechanism of chloroquine-clicked analogs with the Mpro enzyme. Amber-16 program was used for MD simulation in a set parameter of 1 nanosecond. In explicit water mode, using the steepest descent step method internal approach simulation was initiated [32] . The all-atoms MD simulation in explicit solvent water system was run for one nanosecond. The binding trajectory was minimized and optimized through the Where, ∆E MM is denoted as the variation of the MM energy in the gas phase, which includes ∆E internal , electrostatic energy (∆E elec ) and van der Waals energy (∆E vdw ), solvation free energy (∆G solv ) which is the sum of electrostatic solvation energy, ∆G GB is the polar influence, designed by solvent accessible surface area (SASA) and the non-electrostatic solvation ∆G SA is subsidized by non-polar part.

J o u r n a l P r e -p r o o f Furthermore, pharmacological assessment of lead drugs, along with known drugs was performed. Pharmacokinetic and pharmacodynamics properties for Chloroquine modified CQclicked analogs were studied using SwissADME, pkCSM, ACD/I-Lab, and Molinspiration servers. Many pharmacological parameters including the Lipinski rule of five, drug-likeliness, and ADMET (Absorption, metabolism, excretion, and toxicity) were examined.

The crystal structure of SARS-CoV-2 main protease (PDB ID 6LU7) was retrieved from

Protein Data Bank (http://www.rcsb.org/). The coronavirus main protease (Mpro) was used as a potential target for repurposing therapeutics as it plays essential roles in the induction and life cycle progression of coronavirus. The 3D crystal structure of Mpro has been reported to premeditate through the X-ray diffraction technique in the expression system Escherichia coli.

from Bat SARS-like coronavirus. Prior to perform, the molecular modelling studies, the 3D structure of Mpro was examined through the Ramachandran plot along with its 3D structure local quality estimations. The structural sequence plot assessment depicted the >90.6% residues lie in the favorable region, 9.1% residues lie in the optimal region and only 0.4% residues lie in the outlier region of the Ramachandran plot. Moreover, high structural eminence with superior homology showed the pragmatic in local quality assessment of indigenous structures of Mpro.

Also, the Verify3D server affirmed the optimal quality of structure by >94% of Residues possessing the 3D/1D profiles of native structures. These outcomes gave the refined drug target Mpro for coronavirus for molecular modeling studies. Importantly, redocking results by SwissDock analysis of CQ-TrOne supported the obtained data with a high docking score (Table 1) . From chloroquine to chloroquine-N 3 the docking score dropped by 38.21 kJ/mol defining the role of the substituent in the binding activity. It describes the role of click triazole in enhanced binding affinity towards the Mpro and possible role in COVID-19 treatment. Interestingly, the redocking outcome was apposite and the efficacy of CQ-TrOne was again optimal by molecular binding ∆G energy score of -9.66 kcal/mol, also better J o u r n a l P r e -p r o o f than other repurposed candidates (Fig. 3) . With these unambiguous results, it can be concluded that CQ-TrOne has a high binding potential with the target Mpro and can be considered as valuable therapeutics for COVID-19. interaction at 290A-GLU-5.03Å (Fig. 4) . These molecular contact analyses revealed the strong binding of lead drug conjugate with catalytic domain-2 and domain-3, results were taken further to study through MD simulation to evaluate the binding statistics. (Fig. 5A) . In addition, the receptor-ligand duo was gaged through RMSD vs. density histogram plots, which showed receptor Mpro enzyme and the drug candidate CQ-TrOne were normally distributed around 1.4 and 2.5 Å (Fig. 5E, 5F ). The RMSD values to the MD simulation run suggested the stabilized interaction and ideal for further detailed analysis.

Further, to examine every residue dynamics of the target receptor, a RMSF plot was studied. It depicted the affirmative and indigenous fluctuation of the sequence with a peak around 150 residues and in a range from 280 to 305 residues. The fluctuations in the above-stated statement can be reasonably expected due to the proximal binding of the drug candidate with the target protein, it also explains that it is binding in coils and beta sheets of the Mpro enzyme, which has been espied molecular binding exploration (Fig. 5C) . With the aim of realizing the compactness and structural activity of macromolecule Mpro, the radii of gyration (Rg) of resulting trajectories were calculated as Rg varies depending upon the folding state of the protein complex. The data indicated fluctuation in an assortment of 21.7−22.2 Å, and this marginal flux showed the stability of the Mpro to CQ-TrOne while binding (Fig. 5B) . As Q of trajectories defines the transition states for all proteins subjected to conformational changes with a free energy barrier, these native contacts were also considered for analysis. The assimilated domino effect describes that the native contacts were preferred by the coarse-grained theoretical models. And, Q value above 96% illustrated the conformation dynamics of the macromolecule system along with the energetics of the bound ligand (Fig. 5D ). On the foundation of these substantial outcomes through RMSD, RMSF, Rg, and Q values recommended the stability of the Mpro enzyme−CQ-TrOne complex, that CQ-TrOne can be a potential drug candidate against SARS-CoV-2.

Principal component analysis (PCA) was executed to get insight into the conformational inconsistencies within the experimental structures and nature of clusters. Conformation frames were divided into two clusters in red and black colors. The rotational calculation of the residues for three different conformations PC1, PC2, and PC3 were obtained through the normal mode molecular dynamics, which signposted that the PCA results were optimal. The conformational analysis revealed that the PC1 cluster has the maximum variability of 31.49% in terms of internal gesticulations of proteins of the Mpro enzyme conformation with the binding of CQ-TrOne, while PC2 and PC3 showed 19.63% & 6.73% the remaining variability of all the atomic motions through the key components respectively (Fig. 7) .

We observed that the conformations changed from the black cluster to the red cluster and supplementary recuperated from the first cluster blue to white to red during the molecular dynamics time. Additionally, the two different conformations from the two clusters were allied and superimposed, shown in the plot (Fig. 8) . To add on, a dynamical residue cross-correlation J o u r n a l P r e -p r o o f map was computed, which designated a high pairwise correlation at binding residues 280-305, close to binding groove-3 of the Mpro enzyme (Fig. 7D) . (Table 3 ). In addition to pharmacodynamics studies, the toxicity profile (side effects) of CQ-TrOne were measured (Table 3) . Toxicity profiling depicted, CQ-TrOne is of non-carcinogenic in nature by Ames test. The acute toxicity lethal dosage value (LD50) was calculated to be 2.626 mol/kg, which indicated the concentration that may lead to 50% death of animals in the study. The oral rat chronic toxicity score was studied to 1.295 log mg/kg BW/day and no side effects with T. Pyriformis and no skin sensitivity to the humans.

Also, apart from CQ-TrOne the pharmacological analyses of chloroquine, ribavirin, and favipiravir drugs were performed, shown in the table. Pharmacology parameters calculation and comparative evaluation showed that CQ-TrOne has high druggability, drug-likeness, and potency for repurposing therapeutics inconsideration to chloroquine, ribavirin, and favipiravir drugs.

J o u r n a l P r e -p r o o f

For repurposing the drugs, we chose non-chromatographic and regioselective click reaction.

CQ-based click products are well known in the field of bio for their aforementioned unique properties. We followed Yu et al. reported pathway [35] accompanied by Subhashini et al. [36] and proposed the synthesis of CQ-TrOne. Diabetic drug A and propargyl bromide B were treated in a mild aprotic alkaline medium to generate C by nucleophilic substitution reaction. Then, the prepared CQ-N 3 and alkyne linked thiazolidine-2,4-dione C were treated under Cu(II) medium to generate the CQ-clicked repurposed drug. 

The authors declare no conflict of interest.

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☐The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SC and NK contributed equally J o u r n a l P r e -p r o o f