Establishing an Analogue Based In Silico Pipeline in the Pursuit of Novel Inhibitory Scaffolds against the SARS Coronavirus 2 Papain-Like Protease

The ongoing coronavirus pandemic has been a burden on the worldwide population, with mass fatalities and devastating socioeconomic consequences. It has particularly drawn attention to the lack of approved small-molecule drugs to inhibit SARS coronaviruses. Importantly, lessons learned from the SARS outbreak of 2002–2004, caused by severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), can be applied to current drug discovery ventures. SARS-CoV-1 and SARS-CoV-2 both possess two cysteine proteases, the main protease (M^pro) and the papain-like protease (PL^pro), which play a significant role in facilitating viral replication, and are important drug targets. The non-covalent inhibitor, GRL-0617, which was found to inhibit replication of SARS-CoV-1, and more recently SARS-CoV-2, is the only PL^pro inhibitor co-crystallised with the recently solved SARS-CoV-2 PL^pro crystal structure. Therefore, the GRL-0617 structural template and pharmacophore features are instrumental in the design and development of more potent PL^pro inhibitors. In this work, we conducted scaffold hopping using GRL-0617 as a reference to screen over 339,000 ligands in the chemical space using the ChemDiv, MayBridge, and Enamine screening libraries. Twenty-four distinct scaffolds with structural and electrostatic similarity to GRL-0617 were obtained. These proceeded to molecular docking against PL^pro using the AutoDock tools. Of two compounds that showed the most favourable predicted binding affinities to the target site, as well as comparable protein-ligand interactions to GRL-0617, one was chosen for further analogue-based work. Twenty-seven analogues of this compound were further docked against the PL^pro, which resulted in two additional hits with promising docking profiles. Our in silico pipeline consisted of an integrative four-step approach: (1) ligand-based virtual screening (scaffold-hopping), (2) molecular docking, (3) an analogue search, and, (4) evaluation of scaffold drug-likeness, to identify promising scaffolds and eliminate those with undesirable properties. Overall, we present four novel, and lipophilic, scaffolds obtained from an exhaustive search of diverse and uncharted regions of chemical space, which may be further explored in vitro through structure-activity relationship (SAR) studies in the search for more potent inhibitors. Furthermore, these scaffolds were predicted to have fewer off-target interactions than GRL-0617. Lastly, to our knowledge, this work contains the largest ligand-based virtual screen performed against GRL-0617.     

Design,Microwave-Assisted Synthesis and In Silico Prediction Study of Novel Isoxazole Linked Pyranopyrimidinone Conjugates as New Targets for Searching Potential Anti-SARS-CoV-2 Agents

A series of novel naphthopyrano[2,3-d]pyrimidin-11(12H)-one containing isoxazole nucleus 4 was synthesized under microwave irradiation and classical conditions in moderate to excellent yields upon 1,3-dipolar cycloaddition reaction using various arylnitrile oxides under copper(I) catalyst. A one-pot, three-component reaction, N-propargylation and Dimroth rearrangement were used as the key steps for the preparation of the dipolarophiles3. The structures of the synthesized compounds were established by ¹H NMR, ¹³C NMR and HRMS-ES means. The present study aims to also predict the theoretical assembly of the COVID-19 protease (SARS-CoV-2 M^pro) and to discover in advance whether this protein can be targeted by the compounds 4a–1 and thus be synthesized. The docking scores of these compounds were compared to those of the co-crystallized native ligand inhibitor (N3) which was used as a reference standard. The results showed that all the synthesized compounds (4a–l) gave interesting binding scores compared to those of N3 inhibitor. It was found that compounds 4a, 4e and 4i achieved greatly similar binding scores and modes of interaction than N3, indicating promising affinity towards SARS-CoV-2 M^pro. On the other hand, the derivatives 4k, 4h and 4j showed binding energy scores (−8.9, −8.5 and −8.4 kcal/mol, respectively) higher than the M^pro N3 inhibitor (−7.0 kcal/mol), revealing, in their turn, a strong interaction with the target protease, although their interactions were not entirely comparable to that of the reference N3.     

A new asymmetric synthesis of (+)-12b-epidevinylantirhine

We report a new asymmetric synthesis of the indole alkaloid derivative (+)-12b-epidevinylantirhine through stereoselective cyclization of a tethered indole nucleus onto an N-acyliminium ion intermediate, generated from a readily available non-racemic bicyclic lactam building block, and subsequent template modification through a highly diastereoselective conjugate addition protocol. In addition, we present the first X-ray crystal structure of this indole target.     

Stereoselective photochemical 1,3-dioxolane addition to 5-alkoxymethyl-2(5H)-furanone: synthesis of bis-tetrahydrofuranyl ligand for HIV protease inhibitor UIC-94017 (TMC-114)

A convenient synthesis of (3R,3aS,6aR)-3-hydroxyhexahydrofuro[2,3-b]furan, a high-affinity nonpeptidal ligand for HIV protease inhibitor UIC-94017, is described. This inhibitor is undergoing advanced clinical trials. The synthesis utilizes a novel stereoselective photochemical 1,3-dioxolane addition to 5(S)-benzyloxymethyl-2(5H)-furanone as the key step. The requisite furanone derivative was prepared in high enantiomeric excess by an immobilized lipase-catalyzed selective acylation of (+/-)-1-(benzyloxy)-3-buten-2-ol and a ring-closing olefin metathesis with Grubbs' catalyst. Optically active bis-THF was converted to protease inhibitor 2 (UIC-94017).     

Towards Eumelanin@Zeolite Hybrids: Pore‐Size‐Controlled 5,6‐Dihydroxyindole Polymerization

5,6‐Dihydroxyindole ( 1 ) and its N‐methyl derivative ( 2 ), key eumelanin building blocks, were inserted into zeolite L by sublimation at 175 °C for 5 days. At a 10 mg/300 mg indole/zeolite ratio, the resulting hybrids displayed a stable deep red coloration. CP/MAS ¹³C NMR and UV/Vis spectroscopy of the red species suggested the generation and accommodation of quinonoid biindole derivative(s) within the void space of the acidic zeolite channels. Removal of the zeolite matrix by treatment with HF gave a stable species that could be separated by HPLC and characterized by mass spectrometry as an oxygenated biindole derivative (or a mixture of isomers), suggesting addition of water to the original dimer and subsequent re‐oxidation. The characterization was corroborated by optimized molecular geometries and simulated UV spectra with density functional calculations. Loading 1 or 2 into the larger pores of SBA‐15 type mesoporous silica resulted in black eumelanin‐type polymers, confirming channel size dependence over the polymerization process.     

Comparison of clinically approved molecules on SARS-CoV-2 drug target proteins: a molecular docking study

The new type of coronavirus, SARS-CoV-2 has affected more than 22.6 million people worldwide. Since the first day the virus was spotted in Wuhan, China, numerous drug design studies have been conducted all over the globe. Most of these studies target the receptor-binding domain of spike protein of SARS-CoV-2, which is known to bind to the human ACE2 receptor and SARS-CoV-2 main protease, vital for the virus’ replication. However, there might be a third target, human furin protease, which cleaves the virus’ S1-S2 domains playing an active role in its entry into the host cell. In this study, we docked five clinically used drug molecules, favipiravir, hydroxychloroquine, remdesivir, lopinavir, and ritonavir onto three target proteins, the receptor-binding domain of SARS-CoV-2 spike protein, SARS-CoV-2 main protease, and human furin protease. Results of molecular docking simulations revealed that human furin protease might be targeted by COVID-19. Remdesivir, a nucleic acid derivative, strongly bound to the active site of this protease, suggesting that this molecule can be used as a template for designing novel furin protease inhibitors to fight against the disease. Protein-drug interactions revealed in this study at the molecular level, can pave the way for better drug design for each specific target.     

Purification and Biochemical Characterization of a New Protease Inhibitor from Conyza dioscoridis with Antimicrobial,Antifungal and Cytotoxic Effects

The main objective of the current study was the extraction, purification, and biochemical characterization of a protein protease inhibitor from Conyza dioscoridis. Antimicrobial potential and cytotoxic effects were also examined. The protease inhibitor was extracted in 0.1 M phosphate buffer (pH 6–7). Then, the protease inhibitor, named PDInhibitor, was purified using ammonium sulfate precipitation followed by filtration through a Sephadex G-50 column and had an apparent molecular weight of 25 kDa. The N-terminal sequence of PDInhibitor showed a high level of identity with those of the Kunitz family. PDInhibitor was found to be active at pH values ranging from 5.0 to 11.0, with maximal activity at pH 9.0. It was also fully active at 50 °C and maintained 90% of its stability at over 55 °C. The thermostability of the PDInhibitor was clearly enhanced by CaCl₂ and sorbitol, whereas the presence of Ca²⁺ and Zn²⁺ ions, Sodium taurodeoxycholate (NaTDC), Sodium dodecyl sulfate (SDS), Dithiothreitol (DTT), and β-ME dramatically improved the inhibitory activity. A remarkable affinity of the protease inhibitor with available important therapeutic proteases (elastase and trypsin) was observed. PDInhibitor also acted as a potent inhibitor of commercial proteases from Aspergillus oryzae and of Proteinase K. The inhibitor displayed potent antimicrobial activity against gram+ and gram- bacteria and against fungal strains. Interestingly, PDInhibitor affected several human cancer cell lines, namely HCT-116, MDA-MB-231, and Lovo. Thus, it can be considered a potentially powerful therapeutic agent.     

Synthesis and Biochemical Evaluation of 8H-Indeno[1,2-d]thiazole Derivatives as Novel SARS-CoV-2 3CL Protease Inhibitors

The COVID-19 pandemic caused by SARS-CoV-2 is a global burden on human health and economy. The 3-Chymotrypsin-like cysteine protease (3CL^pro) becomes an attractive target for SARS-CoV-2 due to its important role in viral replication. We synthesized a series of 8H-indeno[1,2-d]thiazole derivatives and evaluated their biochemical activities against SARS-CoV-2 3CL^pro. Among them, the representative compound 7a displayed inhibitory activity with an IC₅₀ of 1.28 ± 0.17 μM against SARS-CoV-2 3CL^pro. Molecular docking of 7a against 3CL^pro was performed and the binding mode was rationalized. These preliminary results provide a unique prototype for the development of novel inhibitors against SARS-CoV-2 3CL^pro.     

Excited State Photoreaction between the Indole Side Chain of Tryptophan and Halocompounds Generates New Fluorophores and Unique Modifications

Photoreaction of indole containing compounds with chloroform and other trichlorocompounds generates products with redshifted fluorescence. In proteins, this reaction can be used for the fluorescent detection of proteins. Little characterization of products generated through the photochemical reaction of indoles with halocompounds has been done, yet is fundamental for the development of other fluorophores, protein labeling agents, and bioactive indole derivatives. Here, we have characterized which isomers form in the photoreaction between tryptophan and chloroform using ¹H‐NMR of tryptophan and methylated derivatives to reveal that the two major products that are formed result from modification at the 4‐ and 6‐carbon positions of the indole ring. Reaction at position 6 generates 6‐formyl tryptophan and the reaction at position 4 generates an imine because the formyl derivative that is initially formed reacts further with the tryptophan amine group. The spectroscopic properties and product molecular weights of photoproducts formed from photoreaction of tryptophan with other trihalo and monohalocompounds are also determined. The indole ring of tryptophan can be modified with various additions from halocompounds, including the addition of labels to the indole ring via methylene groups. This opens possibilities for generating novel tryptophan based fluorophores and protein labeling strategies using this photochemistry.     

A fluorescence turn-on method for real-time monitoring of protease activity based on the electron transfer between a fluorophore labeled oligonucleotide and cytochrome c

A new continuous fluorescence turn-on assay for protease activity and inhibitor screening has been developed. A fluorophore labeled single stranded DNA (FAM-DNA) and cytochrome c (cyt c) were employed. The fluorescence of the FAM-DNA was efficiently quenched when binding to cyt c, through the electron transfer between the FAM fluorophore and the heme cofactor of cyt c. In the presence of a protease, such as trypsin, cyt c was digested into small peptide fragments. The FAM-DNA was released, which resulted in the recovery of the FAM fluorescence. The rate of the cyt c digestion could be reduced via the addition of an inhibitor. As a result, reduced degree of the fluorescence recovery was obtained. The limit of detection of our assay is 1 nM trypsin and the IC₅₀ values are 3.23 μg mL⁻¹ and 0.303 μg mL⁻¹ for the inhibitor from egg white and the inhibitor from soybean, respectively. Our method could be used for the sensing of protease activity for various biochemical applications, and for the screening of protease inhibitors as drugs for the treatment of various related diseases.     

Photo-switchable molecular devices based on metal-ionic recognition

A novel calix[4]arene derivative, bearing two spirobenzopyran moieties in the lower rim, can recognize lanthanide ions. Alternating irradiation with ultraviolet and visible light controls the ligand-to-metal charge transfer (LMCT) and energy transfer of the host-Eu³⁺ complexes. Thus, fluorescence of Eu³⁺ can be switched on and off through light. The system may be applied as molecular logic switches.     

A convenient route to prepare isodicyclopentadiene—precursor of 1,5-dihydro-pentalene

A new route to prepare tricyclo[5.2.1.0^2,6]deca-2,4-diene (isodicyclopentadiene) was developed. This new route passes through a brominated (5-bromotricyclo[5.2.1.0^2,6]dec-3-ene) derivative obtained from tricycle[5.2.1.0^2,6]dec-3-ene (8,9-dihydrodicyclopentadiene) and NBS with a good yield. The complete assignment of protons and carbons on nuclear magnetic resonance spectra was done for dicyclopentadiene and the chemically transformed compounds by 2D NMR techniques.     

Anti-corrosion and anti-microbial evaluation of novel water-soluble bis azo pyrazole derivative for carbon steel pipelines in petroleum industries by experimental and theoretical studies

In this research, we investigated the synthesis of a novel water-soluble bis azo pyrazolin-5-one (ABP) which was synthesized efficiently via the regioselective reaction of hydrazine with coumarin hydrazone (CMH). Also, we evaluate their anti-corrosion and anti-bacterial behavior. The inhibition efficiency of ABP in an acidic medium (1.0 M HCl) was evaluated using various electrochemical and surface morphology measurements. The novel bis pyrazole-based azo dye ABP (16 × 10^?6 M) demonstrated a higher protection capacity (93.3 %). Tafel curves revealed that ABP was a mixed-type inhibitor. The adsorption of ABP on the C-steel (CS) surface is proven by the alteration in (R_ct and C_dl) impedance characteristics and obeyed the Langmuir isotherm model. SEM/EDX, AFM, and XPS surface examinations confirmed the enhancement of an adsorbed film protects the CS surface from acid corrosion at the appropriate dose. Furthermore, theoretical calculations using DFT and MC simulations were performed to identify the active sites on ABP molecules in charge of the adsorption and surface protection of the CS. The adsorption of bis pyrazole-based azo dye on the metal surface explained the protection mechanism. Moreover, the ABP screened for its antimicrobial activity against sulfate-reducing bacteria (SRB), and the calculated inhibition efficiency was 100 %. The current work presents significant results in manufacturing and producing novel water-soluble bis pyrazole-based azo dye derivative with high anti-corrosion and anti-microbial efficiency.     

Synthesis,crystal structure,and density functional theory study of a new compound 4-(2-chlorobenzyl)-1-(5-fluoro-2-hydroxy-3-(thiomorpholinomethyl)phenyl [1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one

In this paper, a novel SHP244 derivative 4-(2-chlorobenzyl)-1- (5-fluoro-2-hydroxy-3- [thiomorph-olinomethyl] phenyl)- [1,2,4]triazolo [4,3-a] quinazolin-5(4H)-one was synthesized through five steps. The single crystals were grown in a suitable solvent system (dichloromethane and methanol). Its structure was confirmed by ¹H NMR, ¹³C NMR spectroscopy, ESI-MS, m/z: 534.12[M-H] (MS), FT-IR, and X-ray single crystal diffraction. The crystal structure of the title compound was optimized by density functional theory (DFT) calculation. The crystal structure after X-ray single crystal diffraction was compared with the structure optimized by DFT calculation, and the result shows that the two structures are consistent. In order to explore certain physical and chemical properties, the frontier molecular orbital and molecular electrostatic potential of the title compound were analyzed. In addition, the docking of the title compound to the target protein was studied to understand the docking effect of the compound with the target protein.     

Mechanism of inhibition of SARS-CoV-2 Mpro by N3 peptidyl Michael acceptor explained by QM/MM simulations and design of new derivatives with tunable chemical reactivity

The SARS-CoV-2 main protease (M^pro) is essential for replication of the virus responsible for the COVID-19 pandemic, and one of the main targets for drug design. Here, we simulate the inhibition process of SARS-CoV-2 M^pro with a known Michael acceptor (peptidyl) inhibitor, N3. The free energy landscape for the mechanism of the formation of the covalent enzyme-inhibitor product is computed with QM/MM molecular dynamics methods. The simulations show a two-step mechanism, and give structures and calculated barriers in good agreement with experiment. Using these results and information from our previous investigation on the proteolysis reaction of SARS-CoV-2 M^pro, we design two new, synthetically accessible N3-analogues as potential inhibitors, in which the recognition and warhead motifs are modified. QM/MM modelling of the mechanism of inhibition of M^pro by these novel compounds indicates that both may be promising candidates as drug leads against COVID-19, one as an irreversible inhibitor and one as a potential reversible inhibitor.

QM/MM simulations identify the mechanism of reaction of N3, a covalent peptidyl inhibitor of SARS-CoV-2 main protease. Modelling of two novel proposed compounds, B1 and B2, suggests that reversibility of covalent inhibition could be tailored.     

In Silico Screening of Semi-Synthesized Compounds as Potential Inhibitors for SARS-CoV-2 Papain-like Protease: Pharmacophoric Features,Molecular Docking,ADMET, Toxicity and DFT Studies

Papain-like protease is an essential enzyme in the proteolytic processing required for the replication of SARS-CoV-2. Accordingly, such an enzyme is an important target for the development of anti-SARS-CoV-2 agents which may reduce the mortality associated with outbreaks of SARS-CoV-2. A set of 69 semi-synthesized molecules that exhibited the structural features of SARS-CoV-2 papain-like protease inhibitors (PLPI) were docked against the coronavirus papain-like protease (PLpro) enzyme (PDB ID: (4OW0). Docking studies showed that derivatives 34 and 58 were better than the co-crystallized ligand while derivatives 17, 28, 31, 40, 41, 43, 47, 54, and 65 exhibited good binding modes and binding free energies. The pharmacokinetic profiling study was conducted according to the four principles of the Lipinski rules and excluded derivative 31. Furthermore, ADMET and toxicity studies showed that derivatives 28, 34, and 47 have the potential to be drugs and have been demonstrated as safe when assessed via seven toxicity models. Finally, comparing the molecular orbital energies and the molecular electrostatic potential maps of 28, 34, and 47 against the co-crystallized ligand in a DFT study indicated that 28 is the most promising candidate to interact with the target receptor (PLpro).     

Isolation and structure of trichotomine and trichotomine G1

The new pigments, trichotomine (1) and trichotomine G₁ (12), which have a novel carbon skelton, have been isolated from the fruits of the plant Clerodendron trichotomum Thunb. On the basis of physical and chemical evidence, the structure of trichotomine (1)^1,2 was elucidated as an oxidative dimeric compound of 3 - oxo - 2,3 - 5,6 - tetrahydro - 11 H indolizino[8,7-b]indole 5-carboxylic acid and its stereostructure containing the absolute configuration was further determined by means of an x-ray crystallographic analysis.³ The structure of trichotomine G₁ (12), a N-β-D-glucopyranosyl trichotomine, is also based on spectral and chemical evidence. A N-β-D-glucopyranosyl indole derivative such as (12) is the first naturally occurring example.⁴     

A confocal study of mechanism of 5-hydroxytryptamino induced intracellular calcium dynamics in cultured rat stomach fundus smooth muscle cells with a new Ca2+ indicator STDln-AM

A new fluorescent Ca²⁺ indicator STDln-AM for detecting [Ca²⁺], transients in cultured smooth muscle cells is presented. By making a comparison, the difference between STDln and fluo-3 is discussed in detail. Using the new Ca²⁺ indicator, the mechanism of 5-hydroxytryptamino (5-HT) induced intracellular calcium dynamics in stomach fundus smooth muscle cells (SFSMC) of rats is investigated. It is shown that in contrast with fluo-3, STDln is uniformly distributed in the cytosolic compartment but excluded from the nucleus, when it is transfected into cells. This feature makes it a real cytosol Ca²⁺ indicator and can reflect changes of cytosol [Ca²⁺] more accurately than that of fluo-3. In addition, STDln responds to the [Ca²⁺], transients more sensitive and faster than fluo-3. The results also show that, the L-type Ca²⁺ channel inhibitor Mn9202 and the PLC inhibitor Compound 48/80 can significantly inhibit the [Ca²⁺], elevation induced by 5-HT, while the PKC inhibitor D-Sphingosine can enhance the effect of 5-HT. The results suggest that 5-HT acts by the way of 5-HT₂ receptors on SFSMC, then through 5-HT₂ receptors coupled IP₃/Ca²⁺ and GC/PKC double signal transduction pathways to make Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ influx possibly through L-type calcium channels.     

2-methylindole analogs as cholinesterases and glutathione S-transferase inhibitors: Synthesis,biological evaluation,molecular docking,and pharmacokinetic studies

In this study, we aimed to (i) synthesize new 2-methylindole analogs containing various amino structures, pyrrolidine, piperidine, morpholine, and substituted phenyl groups through structural and molecular modifications, (ii) evaluate the pharmaceutical potential of 2-methylindole analogs via assessing enzyme inhibitory activity against glutathione S-transferase (GST), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE), (iii) predict ADMET and pharmacokinetic properties of the synthesized 2-methylindole analogs, (iv) reveal the possible interactions between the synthesized 2-methylindole analogs with GST, AChE, and BChE enzymes using several molecular docking software. In vitro enzyme inhibition assays showed that the synthesized indole analogs exhibited moderate to good inhibitory activities against GST, AChE, and BChE enzymes. Briefly, the inhibitory activities of the analogs 4b and 4i against AChE, 4a and 4b against BChE, and analogs 1 and 4i against GST were detected to be higher or close to the standard inhibitor compounds. The analog 4b was detected to have the best inhibitory activity against both AChE and BChE enzymes with the lowest IC₅₀ values as 0.648 µM for AChE and 0.745 µM for BChE. The analyses of enzyme inhibition relationship with the synthesized analogs could help to design new analogs for the inhibitors of cholinergic and glutathione pathways based on the indole derivatives.