Broad-Spectrum Extracellular Antiviral Properties of Cucurbit[n]urils.

Viruses are microscopic pathogens capable of causing disease and are responsible for a range of human mortalities and morbidities worldwide. They can be rendered harmless or destroyed with a range of antiviral chemical compounds. Cucurbit[n]urils (CB[n]s) are a family of macrocycle chemical compounds existing as a range of homologues; due to their structure, they can bind to biological materials, acting as supramolecular "hosts" to "guests", such as amino acids. Due to the increasing need for a nontoxic antiviral compound, we investigated whether cucurbit[n]urils could act in an antiviral manner. We have found that certain cucurbit[n]uril homologues do indeed have an antiviral effect against a range of viruses, including herpes simplex virus 2 (HSV-2), respiratory syncytial virus (RSV) and SARS-CoV-2. In particular, we demonstrate that CB[7] is the active homologue of CB[n], having an antiviral effect against enveloped and nonenveloped species. High levels of efficacy were observed with 5 min contact times across different viruses. We also demonstrate that CB[7] acts with an extracellular virucidal mode of action via host-guest supramolecular interactions between viral surface proteins and the CB[n] cavity, rather than via cell internalization or a virustatic mechanism. This finding demonstrates that CB[7] acts as a supramolecular virucidal antiviral (a mechanism distinct from other current extracellular antivirals), demonstrating the potential of supramolecular interactions for future antiviral disinfectants.

Design, Synthesis, and Application of Chiral Bicyclic Imidazole Catalysts.

ConspectusAsymmetric organocatalysis has been considered to be an efficient and reliable strategy for the stereoselective preparation of optically active chemicals. In particular, chiral tertiary amines as Lewis base organocatalysts bearing core structures including quinuclidine, dimethylaminopyridine (DMAP), N-methylimidazole (NMI), amidine, etc. have provided new and powerful tools for various chemical transformations. However, due to the limitations in structural complexity, synthetic difficulty, low catalytic efficiency, and high cost, the industrial application of such catalysts is still far from being widely adopted. Therefore, the development of new chiral tertiary amine catalysts with higher activity and selectivity is greatly desired.In order to address the contradiction between activity and selectivity caused by the ortho group, a bicyclic imidazole structure bearing a relatively large bond angle ∠θ was designed as the skeleton of our new catalysts. 6,7-Dihydro-5H-pyrrolo[1,2-a]imidazole (abbreviated as DPI) and 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine (abbreviated as TIP) are two of the utilized skeletons. In addition to obtaining satisfactory catalytic activity, excellent enantioselectivity would also be expected because the stereocontrol R group is neither far nor close to the catalytic active site (sp2-N atom) and is adjustable. Based on this skeleton, a family of chiral bicyclic imidazole catalysts were easily prepared and successfully applied in several enantioselective reactions for the synthesis of a variety of valuable chiral compounds.6,7-Dihydro-5H-pyrrolo[1,2-a]imidazole (abbreviated as DPI) is the predominantly utilized skeleton. First, HO-DPI, the key intermediate of the designed chiral bicyclic imidazole catalysts, could be efficiently synthesized from imidazole and acrolein, then separated by kinetic resolution or optical resolution. Second, Alkoxy-DPI, the alkyloxy-substituted chiral bicyclic imidazole catalysts, were synthesized by a one-step alkylation from HO-DPI. This type of catalyst has been successfully applied in asymmetric Steglich rearrangement (C-acylation rearrangement of O-acylated azlactones), asymmetric phosphorylation of lactams, and a sequential four-step acylation reaction. Third, Acyloxy-DPI, the acyloxy-substituted chiral bicyclic imidazole catalysts, were synthesized with a one-step acetylative kinetic resolution from racemic HO-DPI or acylation from enantiopure HO-DPI. The catalyst AcO-DPI has been successfully applied in enantioselective Black rearrangement and in direct enantioselective C-acylation of 3-substituted benzofuran-2(3H)-ones and 2-oxindoles. Fourth, Alkyl-DPI was synthesized via a two-step reaction from racemic HO-DPI and separated easily by resolution. The catalyst Cy-DPI has been successfully applied in dynamic kinetic resolution of 3-hydroxyphthalides through enantioselective O-acylation. Cy-PDPI was synthesized through a Cu-catalyzed amidation from Cy-DPI and successfully applied in the kinetic resolution of secondary alcohols with good to excellent enantioselectivities. Finally, the carbamate type chiral bicyclic imidazole catalysts, Carbamate-DPI, were readily synthesized from HO-DPI, and the catalyst Ad-DPI bearing a bulky adamantyl group was successfully applied in the synthesis of the anti-COVID-19 drug remdesivir via asymmetric phosphorylation. Alongside our initial work, this Account also introduces four elegant studies by other groups concerning asymmetric phosphorylation utilizing chiral bicyclic imidazole catalysts.In summary, this Account focuses on the chiral bicyclic imidazole catalysts developed in our group and provides an overview on their design, synthesis, and application that will serve as inspiration for the exploration of new organocatalysts and related reactions.

Synthesis, In Silico Prediction and In Vitro Evaluation of Antimicrobial Activity, DFT Calculation and Theoretical Investigation of Novel Xanthines and Uracil Containing Imidazolone Derivatives.

Novel xanthine and imidazolone derivatives were synthesized based on oxazolone derivatives 2a-c as a key intermediate. The corresponding xanthine 3-5 and imidazolone derivatives 6-13 were obtained via reaction of oxazolone derivative 2a-c with 5,6-diaminouracils 1a-e under various conditions. Xanthine compounds 3-5 were obtained by cyclocondensation of 5,6-diaminouracils 1a-c with different oxazolones in glacial acetic acid. Moreover, 5,6-diaminouracils 1a-e were reacted with oxazolones 2a-c in presence of drops of acetic acid under fused condition yielding the imidazolone derivatives 6-13. Furthermore, Schiff base of compounds 14-16 were obtained by condensing 5,6-diaminouracils 1a,b,e with 4-dimethylaminobenzaldehyde in acetic acid. The structural identity of the resulting compounds was resolved by IR, 1H-, 13C-NMR and Mass spectral analyses. The novel synthesized compounds were screened for their antifungal and antibacterial activities. Compounds 3, 6, 13 and 16 displayed the highest activity against Escherichia coli as revealed from the IC50 values (1.8-1.9 µg/mL). The compound 16 displayed a significant antifungal activity against Candia albicans (0.82 µg/mL), Aspergillus flavus (1.2 µg/mL) comparing to authentic antibiotics. From the TEM microgram, the compounds 3, 12, 13 and 16 exhibited a strong deformation to the cellular entities, by interfering with the cell membrane components, causing cytosol leakage, cellular shrinkage and irregularity to the cell shape. In addition, docking study for the most promising antimicrobial tested compounds depicted high binding affinity against acyl carrier protein domain from a fungal type I polyketide synthase (ACP), and Baumannii penicillin- binding protein (PBP). Moreover, compound 12 showed high drug- likeness, and excellent pharmacokinetics, which needs to be in focus for further antimicrobial drug development. The most promising antimicrobial compounds underwent theoretical investigation using DFT calculation.

Mechanism of Ligand Recognition by Human ACE2 Receptor.

Angiotensin converting enzyme 2 (ACE2) plays a key role in renin-angiotensin system regulation and amino acid homeostasis. Human ACE2 acts as the receptor for severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2. ACE2 is also widely expressed in epithelial cells of the lungs, heart, kidney, and pancreas. It is considered an important drug target for treating SARS-CoV-2 as well as pulmonary diseases, heart failure, hypertension, renal diseases, and diabetes. Despite the critical importance, the mechanism of ligand binding to the human ACE2 receptor remains unknown. Here, we have addressed this challenge through all-atom simulations using a novel ligand Gaussian accelerated molecular dynamics (LiGaMD) method. Microsecond time scale LiGaMD simulations have unprecedentedly captured multiple times of spontaneous binding and unbinding of a potent inhibitor MLN-4760 in the ACE2 receptor. With ligand far away in the unbound state, the ACE2 receptor samples distinct Open, Partially Open, Closed, and Fully Closed conformations. Upon ligand binding to the active site, conformational ensemble of the ACE2 receptor is biased toward the Closed state as observed in the X-ray experimental structure. The LiGaMD simulations thus suggest a conformational selection mechanism for ligand recognition by the highly flexible ACE2 receptor, which is expected to facilitate rational drug design targeting human ACE2 against coronaviruses and other related human diseases.

Characterization of the Modulatory Effect of Hydroxychloroquine on ACE2 Activity: New Insights in relation to COVID-19.

Chloroquine (CQ) and hydroxychloroquine (HCQ) have shown the ability to inhibit in vitro viral replications of coronaviridae viruses such as SARS-CoV and SARS-CoV-2. However, clinical trial outcomes have been disparate, suggesting that CQ and HCQ antiviral mechanisms are not fully understood. Based on three-dimensional structural similarities between HCQ and the known ACE2 specific inhibitor MLN-4760, we compared their modulation on ACE2 activity. Here we describe, for the first time, in a cell-free in vitro system that HCQ directly and dose-dependently inhibits the activity of recombinant human ACE2, with a potency similar to the MLN-4760. Further analysis suggests that HCQ binds to a noncompetitive site other than the one occupied by MLN-4760. We also determined that the viral spike glycoprotein segment that comprises the RBD segment has no effect on ACE2 activity but unexpectedly was able to partially reverse the inhibition induced by HCQ but not that by MLN-4760. In summary, here we demonstrate the direct inhibitory action of HCQ over the activity of the enzyme ACE2. Then, by determining the activity of ACE2, we reveal that the interaction with the spike protein of SARS-CoV-2 leads to structural changes that at least partially displace the interaction of the said enzyme with HCQ. These results may help to explain why the effectiveness of HCQ in clinical trials has been so variable. Additionally, this knowledge could be used for to develop techniques for the detection of SARS-CoV-2.

Repurposing of FDA-approved drugs against active site and potential allosteric drug-binding sites of COVID-19 main protease.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still has serious negative effects on health, social life, and economics. Recently, vaccines from various companies have been urgently approved to control SARS-CoV-2 infections. However, any specific antiviral drug has not been confirmed so far for regular treatment. An important target is the main protease (Mpro ), which plays a major role in replication of the virus. In this study, Gaussian and residue network models are employed to reveal two distinct potential allosteric sites on Mpro that can be evaluated as drug targets besides the active site. Then, Food and Drug Administration (FDA)-approved drugs are docked to three distinct sites with flexible docking using AutoDock Vina to identify potential drug candidates. Fourteen best molecule hits for the active site of Mpro are determined. Six of these also exhibit high docking scores for the potential allosteric regions. Full-atom molecular dynamics simulations with MM-GBSA method indicate that compounds docked to active and potential allosteric sites form stable interactions with high binding free energy (∆Gbind ) values. ∆Gbind values reach -52.06 kcal/mol for the active site, -51.08 kcal/mol for the potential allosteric site 1, and - 42.93 kcal/mol for the potential allosteric site 2. Energy decomposition calculations per residue elucidate key binding residues stabilizing the ligands that can further serve to design pharmacophores. This systematic and efficient computational analysis successfully determines ivermectine, diosmin, and selinexor currently subjected to clinical trials, and further proposes bromocriptine, elbasvir as Mpro inhibitor candidates to be evaluated against SARS-CoV-2 infections.

Computational study of the therapeutic potentials of a new series of imidazole derivatives against SARS-CoV-2.

Owing to the urgent need for therapeutic interventions against the SARS-coronavirus 2 (SARS-CoV-2) pandemic, we employed an in silico approach to evaluate the SARS-CoV-2 inhibitory potential of newly synthesized imidazoles. The inhibitory potentials of the compounds against SARS-CoV-2 drug targets - main protease (Mpro), spike protein (Spro) and RNA-dependent RNA polymerase (RdRp) were investigated through molecular docking analysis. The binding free energy of the protein-ligand complexes were estimated, pharmacophore models were generated and the absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of the compounds were determined. The compounds displayed various levels of binding affinities for the SARS-CoV-2 drug targets. Bisimidazole C2 scored highest against all the targets, with its aromatic rings including the two imidazole groups contributing to the binding. Among the phenyl-substituted 1H-imidazoles, C9 scored highest against all targets. C11 scored highest against Spro and C12 against Mpro and RdRp among the thiophene-imidazoles. The compounds interacted with HIS 41 - CYS 145 and GLU 288 - ASP 289 - GLU 290 of Mpro, ASN 501 of Spro receptor binding motif and some active site amino acids of RdRp. These novel imidazole compounds could be further developed as drug candidates against SARS-CoV-2 following lead optimization and experimental studies.

High-throughput screening identifies established drugs as SARS-CoV-2 PLpro inhibitors.

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (Mpro), PLpro is responsible for processing the viral replicase polyprotein into functional units. Therefore, it is an attractive target for antiviral drug development. Here we discovered four compounds, YM155, cryptotanshinone, tanshinone I and GRL0617 that inhibit SARS-CoV-2 PLpro with IC50 values ranging from 1.39 to 5.63 µmol/L. These compounds also exhibit strong antiviral activities in cell-based assays. YM155, an anticancer drug candidate in clinical trials, has the most potent antiviral activity with an EC50 value of 170 nmol/L. In addition, we have determined the crystal structures of this enzyme and its complex with YM155, revealing a unique binding mode. YM155 simultaneously targets three "hot" spots on PLpro, including the substrate-binding pocket, the interferon stimulating gene product 15 (ISG15) binding site and zinc finger motif. Our results demonstrate the efficacy of this screening and repurposing strategy, which has led to the discovery of new drug leads with clinical potential for COVID-19 treatments.

Direct Potentiometric Study of Cationic and Nonionic Surfactants in Disinfectants and Personal Care Products by New Surfactant Sensor Based on 1,3-Dihexadecyl-1H-benzo[d]imidazol-3-ium.

A novel, simple, low-cost, and user-friendly potentiometric surfactant sensor based on the new 1,3-dihexadecyl-1H-benzo[d]imidazol-3-ium-tetraphenylborate (DHBI-TPB) ion-pair for the detection of cationic surfactants in personal care products and disinfectants is presented here. The new cationic surfactant DHBI-Br was successfully synthesized and characterized by nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectrometry, liquid chromatography-mass spectrometry (LC-MS) and elemental analysis and was further employed for DHBI-TPB ion-pair preparation. The sensor gave excellent response characteristics for CTAB, CPC and Hyamine with a Nernstian slope (57.1 to 59.1 mV/decade) whereas the lowest limit of detection (LOD) value was measured for CTAB (0.3 × 10-6 M). The sensor exhibited a fast dynamic response to dodecyl sulfate (DDS) and TPB. High sensor performances stayed intact regardless of the employment of inorganic and organic cations and in a broad pH range (2-11). Titration of cationic and etoxylated (EO)-nonionic surfactant (NSs) (in Ba2+) mixtures with TPB revealed the first inflexion point for a cationic surfactant and the second for an EO-nonionic surfactant. The increased concentration of EO-nonionic surfactants and the number of EO groups had a negative influence on titration curves and signal change. The sensor was successfully applied for the quantification of technical-grade cationic surfactants and in 12 personal care products and disinfectants. The results showed good agreement with the measurements obtained by a commercial surfactant sensor and by a two-phase titration. A good recovery for the standard addition method (98-102%) was observed.

Interactive Molecular Dynamics in Virtual Reality Is an Effective Tool for Flexible Substrate and Inhibitor Docking to the SARS-CoV-2 Main Protease.

The main protease (Mpro) of the SARS-CoV-2 virus is one focus of drug development efforts for COVID-19. Here, we show that interactive molecular dynamics in virtual reality (iMD-VR) is a useful and effective tool for creating Mpro complexes. We make these tools and models freely available. iMD-VR provides an immersive environment in which users can interact with MD simulations and so build protein complexes in a physically rigorous and flexible way. Recently, we have demonstrated that iMD-VR is an effective method for interactive, flexible docking of small molecule drugs into their protein targets (Deeks et al. PLoS One 2020, 15, e0228461). Here, we apply this approach to both an Mpro inhibitor and an oligopeptide substrate, using experimentally determined crystal structures. For the oligopeptide, we test against a crystallographic structure of the original SARS Mpro. Docking with iMD-VR gives models in agreement with experimentally observed (crystal) structures. The docked structures are also tested in MD simulations and found to be stable. Different protocols for iMD-VR docking are explored, e.g., with and without restraints on protein backbone, and we provide recommendations for its use. We find that it is important for the user to focus on forming binding interactions, such as hydrogen bonds, and not to rely on using simple metrics (such as RMSD), in order to create realistic, stable complexes. We also test the use of apo (uncomplexed) crystal structures for docking and find that they can give good results. This is because of the flexibility and dynamic response allowed by the physically rigorous, atomically detailed simulation approach of iMD-VR. We make our models (and interactive simulations) freely available. The software framework that we use, Narupa, is open source, and uses commodity VR hardware, so these tools are readily accessible to the wider research community working on Mpro (and other COVID-19 targets). These should be widely useful in drug development, in education applications, e.g., on viral enzyme structure and function, and in scientific communication more generally.

Repurposing of renin inhibitors as SARS-COV-2 main protease inhibitors: A computational study.

The COVID-19 pandemic has urged for the repurposing of existing drugs for rapid management and treatment. Renin inhibitors down regulation of ACE2, which is an essential receptor for SARS-CoV-2 infection that is responsible for COVID-19, in addition to their ability to act as protease inhibitors were encouraging aspects for their investigation as possible inhibitors of main protease of SARS-CoV-2 via computational studies. A Pharmacophore model was generated using the newly released SARS-COV-2 main protease inhibitors. Virtual screening was performed on renin inhibitors, and Drug likeness filter identified remikiren and 0IU as hits. Molecular docking for both compounds showed that the orally active renin inhibitor remikiren (Ro 42-5892) of Hoffmann-La Roche exhibited good molecular interaction with Cys145 and His41 in the catalytic site of SARS-CoV-2 main protease. Molecular dynamics simulation suggested that the drug is stable in the active site of the enzyme.

Assessment of effective imidazole derivatives against SARS-CoV-2 main protease through computational approach.

Because of the fast increase in deaths due to Corona Viral Infection in majority region in the world, the detection of drugs potent of this infection is a major need. With this idea, docking study was executed on eighteen imidazole derivatives based on 7-chloro-4-aminoquinoline against novel Coronavirus (SARS-CoV-2). In this study, we carried out a docking study of these molecules in the active site of SARS-CoV-2 main protease. The result indicate that Molecules N° 3, 7 and 14 have more binding energy with SARS-CoV-2 main protease recently crystallized (pdb code 6LU7) in comparison with the other imidazole derivatives and the two drug; Chloroquine and hydroxychloroquine. Because of the best energy of interaction, these three molecules could have the most potential antiviral treatment of COVID-19 than the other studied compounds. The structures with best affinity in the binding site of the protease have more than 3 cycles and electronegative atoms in the structure. This may increase the binding affinity of these molecules because of formation of π-bonds, halogen interactions and/or Hydrogen bond interactions between compounds and the enzyme. So, compounds with more cycles and electronegative atoms could have a potent inhibition of SARS-CoV-2 main protease.

Catalytic Asymmetric Synthesis of the anti-COVID-19 Drug Remdesivir.

The catalytic asymmetric synthesis of the anti-COVID-19 drug Remdesivir has been realized by the coupling of the P-racemic phosphoryl chloride with protected nucleoside GS441524. The chiral bicyclic imidazole catalyst used is crucial for the dynamic kinetic asymmetric transformation (DyKAT) to proceed smoothly with high reactivity and excellent stereoselectivity (96 % conv., 22:1 SP :RP ). Mechanistic studies showed that this DyKAT is a first-order visual kinetic reaction dependent on the catalyst concentration. The unique chiral bicyclic imidazole skeleton and carbamate substituent of the catalyst are both required for the racemization process, involving the phosphoryl chloride, and subsequent stereodiscriminating step. A 10 gram scale reaction was also conducted with comparably excellent results, showing its potential for industrial application.

Solid-Phase Microextraction Fiber in Face Mask for in Vivo Sampling and Direct Mass Spectrometry Analysis of Exhaled Breath Aerosol.

Molecular analysis of exhaled breath aerosol (EBA) with simple procedures represents a key step in clinical and point-of-care applications. Due to the crucial health role, a face mask now is a safety device that helps protect the wearer from breathing in hazardous particles such as bacteria and viruses in the air; thus exhaled breath is also blocked to congregate in the small space inside of the face mask. Therefore, direct sampling and analysis of trace constituents in EBA using a face mask can rapidly provide useful insights into human physiologic and pathological information. Herein, we introduce a simple approach to collect and analyze human EBA by combining a face mask with solid-phase microextraction (SPME) fiber. SPME fiber was inserted into a face mask to form SPME-in-mask that covered nose and mouth for in vivo sampling of EBA, and SPME fiber was then coupled with direct analysis in real-time mass spectrometry (DART-MS) to directly analyze the molecular compositions of EBA under ambient conditions. The applicability of SPME-in-mask was demonstrated by direct analysis of drugs and metabolites in oral and nasal EBA. The unique features of SPME-in-mask were also discussed. Our results showed that this method is enabled to analyze volatile and nonvolatile analytes in EBA and is expected to have a significant impact on human EBA analysis in clinical applications. We also hope this method will inspire biomarker screening of some respiratory diseases that usually required wearing of a face mask in daily life.

QSAR Modeling of SARS-CoV Mpro Inhibitors Identifies Sufugolix, Cenicriviroc, Proglumetacin, and other Drugs as Candidates for Repurposing against SARS-CoV-2.

The main protease (Mpro) of the SARS-CoV-2 has been proposed as one of the major drug targets for COVID-19. We have identified the experimental data on the inhibitory activity of compounds tested against the closely related (96 % sequence identity, 100 % active site conservation) Mpro of SARS-CoV. We developed QSAR models of these inhibitors and employed these models for virtual screening of all drugs in the DrugBank database. Similarity searching and molecular docking were explored in parallel, but docking failed to correctly discriminate between experimentally active and inactive compounds, so it was not relied upon for prospective virtual screening. Forty-two compounds were identified by our models as consensus computational hits. Subsequent to our computational studies, NCATS reported the results of experimental screening of their drug collection in SARS-CoV-2 cytopathic effect assay (https://opendata.ncats.nih.gov/covid19/). Coincidentally, NCATS tested 11 of our 42 hits, and three of them, cenicriviroc (AC50 of 8.9 µM), proglumetacin (tested twice independently, with AC50 of 8.9 µM and 12.5 µM), and sufugolix (AC50 12.6 µM), were shown to be active. These observations support the value of our modeling approaches and models for guiding the experimental investigations of putative anti-COVID-19 drug candidates. All data and models used in this study are publicly available via Supplementary Materials, GitHub (https://github.com/alvesvm/sars-cov-mpro), and Chembench web portal (https://chembench.mml.unc.edu/).