Cannabinoids Block Cellular Entry of SARS-CoV-2 and the Emerging Variants
Advertisement

Caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the COVID-19 pandemic includes at least 272 million cases worldwide, 5.3 million deaths, and over 600 000 new cases daily as of December 2021. (1) Vaccines have been developed, but due to their limited availability and the rate of virus mutation, SARS-CoV-2 infections are likely to continue for many years. As the pandemic continues, several SARS-CoV-2 variants have emerged that are circulating globally, including the variant B.1.1.7 (alpha, first detected in the United Kingdom), variant B.1.351 (beta, first detected in South Africa), and variant B.1.617.2 (delta, first detected in India). (2) These variants of concern are reported to have the capacity to escape humoral immunity elicited by natural infection or the current vaccinations. Moreover, the variants are associated with increases in infections and hospitalizations, suggesting a competitive fitness advantage over the original strain. (3)

A member of the Coronaviridae family, SARS-CoV-2 is an enveloped, nonsegmented, positive sense RNA virus that is characterized by crown-like spikes on the outer surface. (4,5) SARS-CoV-2 contains RNA strands 29.9 kb long (6) that encode the four main structural proteins, spike, envelope, membrane, and nucleocapsid, 16 nonstructural proteins, and several accessory proteins. (7) Any step of the SARS-CoV-2 virus infection and replication cycle is a potential target for antiviral intervention including cell entry, genome replication, viral maturation, or viral release. However, binding of the viral spike protein of SARS-CoV-2 to the human cell surface receptor angiotensin converting enzyme-2 (ACE2) is a critical step during the infection of human cells. Therefore, cell entry inhibitors could be used to prevent SARS-CoV-2 infection as well as to shorten the course of COVID-19 infections by preventing virus particles from infecting human cells.

A transmembrane protein with a molecular mass of ∼150 kDa, the spike protein forms homotrimers protruding from the SARS-CoV-2 surface. Subunits of the SARS-CoV-2 spike protein trimer consist of an S1 subunit that binds to ACE2 of the host cell to initiate infection, an S2 subunit that mediates virus fusion with host cells, and a transmembrane domain (Figure 1A). The infection of host cells by SARS-CoV-2 begins with the attachment of the receptor-binding domain (RBD) of the S1 protein, (8) which has been identified as residues 331 to 524, (9) to the host cell receptor ACE2. An enzyme on the outer cell membrane of host cells, ACE2 is expressed abundantly on human endothelial cells in the lungs, arteries, heart, kidney, and intestines. (10) TMPRSS2 protease on the host cell membrane activates the spike protein by cleaving it at S1/S2 and S2 sites, (11) leading to conformational changes that allow the virus to fuse with the host membrane and enter the cytoplasm. The S1 subunit is primarily responsible for the determination of the host virus range and cellular tropism. (12)

Figure 1

Figure 1. Affinity selection–mass spectrometric (AS-MS) discovery of natural ligands to the SARS-CoV-2 spike protein. (A) The spike protein of SARS-CoV-2 consists of trimers of a protein containing an S1 subunit, an S2 subunit, and a transmembrane domain. The S1 subunit binds to human ACE2 to initiate cell entry. Recombinant S1 containing a His-tag was immobilized on magnetic microbeads for affinity selection of ligands. (B) AS-MS was used to isolate and identify natural ligands to the spike protein S1 subunit. A magnetic probe retained the microbeads containing the S1 subunit and bound ligands, while unbound compounds were washed away. Ligands were released using organic solvent and then analyzed using UHPLC-MS. (C) During AS-MS, the SBP-1 peptide bound to immobilized S1 (equivalent to 0.17 μM) (positive control) but not to immobilized denatured S1 (negative control). (D) MagMASS was used for the affinity selection and identification of cannabinoid acids (0.10 μM each in this confirmatory chromatogram) as ligands from hemp extracts. Negative controls using denatured S1 showed no significant binding of cannabinoid acids.

Ligands with high affinity to the receptor binding domain on the S1 protein have the potential to function as entry inhibitors and prevent infection of human cells by SARS-CoV-2. (13) For example, small peptides derived from the heptad repeat regions of SARS-CoV-1 spike S2 subunit have been shown to inhibit SARS-CoV infection by the interference of fusion with target cells. (14,15) The approach of utilizing compounds that block virus–receptor interaction has also been useful for other viruses, including HIV-1 and hepatitis C virus. (16,17)

Advertisement

Natural products are the most successful source of drugs and drug leads in the history of pharmacology. (18,19) Although combinatorial chemistry currently receives more emphasis for lead discovery by the pharmaceutical industry, (20) nature continues to be a source of unique chemical structural diversity for new drug discovery. (21) Approximately two-thirds of new small-molecule drugs since 1981 have been natural products, derivatives of natural products, natural product pharmacophores, or mimics of natural products. (18,22) Less than 10% of the world’s biodiversity has been evaluated for potential biological activity, so that many more useful natural lead compounds await discovery. (18) As an example of a natural product with anti-SARS-CoV-2 activity, panduratin from the medicinal plant Boesenbergia rotunda was reported recently to be active against SARS-CoV-2 at both pre-entry and postinfection phases. (23)

Although bioassay-guided fractionation is widely used for natural products drug discovery, affinity selection–mass spectrometry (AS-MS) provides a more efficient alternative. (24) AS-MS involves incubating a therapeutically important receptor like the SARS-CoV-2 spike protein with a mixture of possible ligands such as a botanical extract. The ligand–receptor complexes are separated from nonbinding molecules using one of several methods such as ultrafiltration, (25) size exclusion, (26) or magnetic microbeads, (27) and then ultra-high-pressure liquid chromatography–mass spectrometry (UHPLC-MS) is used to characterize the affinity-extracted ligands. In this investigation, we used the AS-MS approach of magnetic microbead affinity selection screening (MagMASS). (28,29)

Hemp (Cannabis sativa L., Cannabaceae) is used for fiber, food, and animal feed, and various hemp extracts and compounds have become popular additions to cosmetics, body lotions, dietary supplements, and food. Over 170 secondary metabolites including some unique compounds are produced by hemp (30,31) including flavonoids, diterpenes, triterpenes, lignans, and cannabinoids. Orally bioavailable, (32) there are at least 70 cannabinoids including cannabidiols, Δ9-tetrahydrocannabinols, Δ8-tetrahydrocannabinols, cannabigerols, cannabinols, cannabichromenes, and cannabitriols, and in 2018, the U.S. FDA approved a cannabidiol isolate (Epidiolex), for the treatment of seizures associated with certain types of epileptic seizures. (33)

Using MagMASS to screen hemp extracts for ligands to the SARS-CoV-2 spike protein, several cannabinoid ligands were identified and ranked by affinity to the spike protein. Two cannabinoids with the highest affinities for the spike protein, cannabidiolic acid (CBDA) and cannabigerolic acid (CBGA), were confirmed to block infection of human epithelial cells by a pseudovirus expressing the spike protein. More importantly, both CBDA and CBGA block infection of the original live SARS-CoV-2 virus and variants of concern, including the B.1.1.7 and B.1.351.


Discovery of Hemp Ligands against SARS-CoV-2

To discover natural ligands to the SARS-CoV-2 spike protein, a MagMASS (27) assay was developed using the spike protein S1 subunit immobilized on magnetic microbeads (Figure 1). To confirm that the immobilized S1 subunit retained selectivity for the human cell surface protein ACE2, magnetic microbeads containing the S1 subunit were incubated with SBP-1, which is a peptide containing the amino acid sequence of human ACE2 to which the SARS-CoV-2 virus spike protein recognition site binds (amino acid residues 331 to 524). After processing using MagMASS (Figure 1B), SBP-1 showed selective binding to the immobilized spike protein S1 subunit (positive control), whereas SBP-1 did not bind to the denatured S1 subunit that had been immobilized in an identical manner (Figure 1C).

During screening of botanical extracts using MagMASS, extracts of hemp (C. sativa L.) produced several hits (Figure 1D). Based on dereplication of the hits and follow-up assays using cannabinoid standards, the spike protein ligands with the highest affinities were identified as CBGA, tetrahydrocannabinolic acid (THCA-A), and CBDA (Figures 2

4, Supporting Information). The cannabinoids Δ9-tetrahydrocannabinol, Δ8-tetrahydrocannabinol, cannabichromene, cannabigerol, cannabinol, and cannabidiol showed only weak or no binding based on competitive binding MagMASS assays (34) using equimolar mixtures (Table 1).

Table 1. MagMASS Ranking of Hemp Cannabinoids for Binding to the SARS-CoV-2 Spike Proteina

Figure 2

Figure 2. Computational based modeling of the binding of cannabinoid acids to the SARS-CoV-2 spike protein S1 C-terminal domain using AutoDock Vina. The active site residues of the S1 subunit are shown in yellow. (A) CBGA (pink) is predicted to bind to the anallosteric site (−6.6 kcal/mol free energy of binding). (B) Although less favorable (−6.2 kcal/mol), CBGA (magenta) can also bind to the orthosteric site on the S1 C-terminal domain. (C) THCA-A (cyan) and (D) CBDA (teal) are predicted to bind at the orthosteric site with free energies of binding of −6.5 kcal/mol and −6.3 kcal/mol, respectively.

Figure 3

Figure 3. CBD compounds block viral entry of SARS-CoV-2 through spike binding. Neutralization of spike protein pseudotyped lentivirus and multiple variants of live SARS-CoV-2 virus by cannabinoids CBDA and CBGA. (A) Representative images of high-resolution microscopy of SARS-CoV-2 (WA1/2020)-infected Vero E6 cells treated with 25 μg/mL CBDA, CBGA, or vehicle (control). Cells were stained with anti-ds-RNA (red) antibody to visualize replication sites formed during infection. DAPI (blue) was used to stain nuclei. (B) Infection of ACE2 293T cells with SARS-CoV-2 spike pseudotyped lentivirus in the presence of CBDA or CBGA. Percent neutralization was determined by quantification of total GFP signal resulting from successful pseudovirus infection, normalized to vehicle control (n = 3). (C) Table of IC50 values for pseudovirus experiments. (D and E) Live-virus infection of Vero E6 cells with SARS-CoV-2 variants (WA1/2020, B.1.1.7, and B.1.351) in the presence of CBDA (D) or CBGA (E). Percent neutralization was normalized to vehicle control wells (n = 3). (F) Table of IC50 values for live-virus experiments shown in D and E. IC50 values were determined by fitting data to a three-parameter model for pseudotype infection (C) and live-infection (F) experiments.

Figure 4

Figure 4. Orthosteric site residues of the spike S1 receptor binding domain. The residues in magenta are mutated in the B.1.351 variant (K417N, E484 K, N501Y). The B.1.1.7 variant mutation occurs at N501Y.

Dissociation Constants and Ligand Docking

The Kd values for the binding of CBGA and CBDA to the SARS-CoV-2 spike protein S1 subunit were determined using equilibrium dialysis. The optimum time for full equilibration of CBGA was 5 h, while that of CBDA was 4 h. The Kd values for CBGA and CBDA were 19.8 ± 2.7 and 5.6 ± 2.2 μM, respectively. Because THCA-A is a controlled substance, insufficient quantities were available for determination of binding affinity or antiviral activity.

The binding interactions of CBDA, THCA-A, and CBGA with the SARS-CoV-2 spike protein S1 C-terminal domain were modeled using AutoDock Vina (Figure 2). In agreement with the MagMASS rank ordering of ligands (Table 1), the free energy of binding was greatest for CBGA (−6.6 kcal/mol) followed by THCA-A (−6.5 kcal/mol) and CBDA (−6.3 kcal/mol). The optimum binding mode for CBGA was at an allosteric site with a binding pocket dominated by hydrophobic residues within a van der Waals distance of 4 Å, namely, F374, L368, F342, W436, A344, and L441 (Figure 2A). The hydrophobic isoprenyl group of CBGA interacted with the hydrophobic residues L368, F342, F374, and W436, while the pentyl group interacted with A344, L441, and the amide of T345. The carboxylic acid group formed a hydrogen bond with the D343 amide side chain, while the hydroxy groups at positions 1 and 5 formed hydrogen bonds with the side chains of D343 and R509, respectively. Although less favorable, CBGA was also predicted to bind orthosterically to the spike protein S1 C-terminal domain with −6.2 kcal/mol free energy of binding (Figure 2B).

Unlike CBGA, CBDA and THCA-A were predicted to bind preferentially within the orthosteric site of the spike protein S1 subunit. The key interactions for CBDA include hydrogen bonding between the carboxylic acid group and the R403 side chain and hydrophobic interactions between the CBDA aromatic ring and the Y495 side chain (Figure 2C). Additional hydrophobic contributions were made by Y505, G496, and Y453. The hydroxy group at position 5 of CBDA formed a hydrogen bond with the amide group of G496. THCA-A was predicted to bind at the surface of the orthosteric site in a hydrophobic region consisting of Y495, F497, Y505, and G496 (Figure 2D). Hydrogen bond interactions could form between the carboxylic acid and D501 as well as between the hydroxy group at position 1 and the carbonyl group of Y505.

Inhibition of SARS-CoV-2 Cell Entry

To determine if CBDA or CBGA could prevent infection by blocking SARS-CoV-2 cell entry, pseudovirus and live SARS-CoV-2 virus cell infection assays were carried out. We incubated the live SARS-CoV-2 virus with 25 μg/mL of either CBDA, CBGA, or vehicle control (DMSO) and then infected Vero E6 cells. At 24 h postinfection, cells were stained with anti-double-stranded RNA (dsRNA) antibody known to bind specifically to viral RNA. We found an absence of SARS-CoV-2 viral RNA in cells treated with either cannabinoid (Figure 3A). To quantify the level of inhibition, we produced spike protein pseudotyped lentiviral particles with a GFP reporter gene, and HEK 293T cells overexpressing ACE-2 were infected for 48 h with these lentiviral particles following treatment with varying concentrations of CBDA, CBGA, or vehicle control. The number of infected cells was quantified by fluorescence microscopy, and the concentration that reduced pseudovirus infections by half (IC50) was 7.7 μg/mL for CBDA and 8.4 μg/mL for CBGA (Figure 3B,C). The cytotoxicity of these compounds was insignificant at concentrations below 50 μg/mL for Caco2, 293T-ACE2, and Vero cell lines (Figure 4, Supporting Information).

To validate the virus neutralizing capabilities of CBDA and CBGA, we next performed focus forming assays using authentic SARS-CoV-2 virus (Isolate USA-WA1/2020). We utilized Vero E6 cells for these experiments due to their high susceptibility to the virus and common use in SARS-CoV-2 live-virus studies. Focus forming assays were performed using serial dilutions of CBDA or CBGA that were incubated with infectious SARS-CoV-2 for 1 h prior to infection. As in the pseudovirus neutralization assay, CBDA and CBGA prevented SARS-CoV-2 entry into Vero E6 cells with IC50 values of 24 and 37 μg/mL (Figure 3D–F), respectively.

Emerging variants of concern (VOC), including B.1.1.7 and B.1.351, have been shown to resist neutralization by antibodies generated against earlier lineages of SARS-CoV-2. To assess whether blockage of cell entry by CBDA and CBGA is variant dependent, we performed additional focus forming assays using the live SARS-CoV-2 variants B.1.1.7, containing the N501Y spike protein mutation, and B.1.351, containing the K417N, E484 K, and N501Y spike mutations. Like WA1/2020 infections, CBDA and CBGA both blocked B.1.1.7 infection with IC50 values of 11 and 26 μg/mL, respectively. B.1.351 was neutralized as well by both compounds with IC50 values of 19 and 37 μg/mL, respectively (Figure 3D–F), indicating no substantial loss of activity against these VOCs.

Originally invented for high-throughput screening of pools of combinatorial libraries, (35) the selectivity, sensitivity, and speed of AS-MS approaches like MagMASS are also ideal for screening natural products mixtures such as botanical extracts. (36) Compared with conventional high-throughput screening utilizing fluorescence or absorbance readouts (such as FRET or fluorescence polarization), AS-MS offers advantages such as compatibility with any type of ligand mixture, matrix, and assay buffer and no requirement for fluorescent tags. Uniquely, AS-MS does not suffer from interference from samples containing fluorophores or chromophores, which are common in natural products. One of the newer AS-MS techniques, (27,37) MagMASS offers advantages compared with the other AS-MS approaches that use ultrafiltration or size exclusion such as ease of automation and faster separation of receptor–ligand complexes from unbound compounds, which minimizes ligand loss due to premature dissociation from the receptor and maximizes sensitivity. (24) Therefore, MagMASS is an ideal platform for the discovery of natural ligands of the SARS-CoV-2 spike protein.

Recently, the crystal structure of the C-terminal domain of the SARS-CoV-2 spike protein in complex with the human ACE2 receptor was solved. The key interactions involve residues along the spike protein C-terminal domain interface that contribute to a network of hydrogen bonding and salt-bridge interactions with the ACE2 receptor. The residues on the spike protein involved in the binding to ACE2 include A475, N487, E484, Y453, K417, G446, Y449, G496, Q498, T500, G502, Y489, and F486. The interaction of the virus with the ACE2 receptor is mainly contributed by the polar interactions resulting from hydrophilic residues on the surface of the spike protein C-terminal domain. (38)

In the AutoDock Vina docking program, the ligand docking in the active site is based on algorithms that take into consideration the steric, hydrophobic bonding, and hydrogen bonding interactions between the ligand and active site residues. The best predicted binding conformation should have the lowest free energy of binding (kcal/mol). CBGA gave the lowest free energy of binding (−6.7 kcal/mol) to an allosteric site, with a root-mean-square deviation of 24.3 from the orthosteric site. On the other hand, THCA-A and CBDA had slightly higher energies of binding at −6.5 and −6.3 kcal/mol, respectively. Overall, the MagMASS data show that CBGA binds to the spike protein S1 subunit strongly in cannabinoid mixtures, suggesting that it binds allosterically and does not compete for binding with orthosteric cannabinoid ligands.

Variants of the SARS-CoV-2 virus such as B.1.1.7 and B.1.351 include amino acids in the spike protein S1 subunit that interact with the ACE2 receptor. (39) For example, the N501Y mutation was identified by bioinformatics analysis of data derived by metagenomics sequencing of samples obtained from a patient with persistent SARS-CoV-2 infection. Other highly infectious variants identified that include mutation of the active site residues include N501T, K417, and E484 K (Figure 4). (40) With the rapid mutations occurring, a novel inhibitor capable of binding to an orthosteric site would be of great interest in the intervention of SARS-CoV-2 variants characterized by active site mutations.

Our infection inhibition assay results clearly indicate that CBDA and CBGA are both able to block cell entry by SARS-CoV-2. The concentrations needed to block infection by 50% of viruses is high but might be clinically achievable. For example, CBDA administered orally to human volunteers at 0.063 mg/kg showed greater bioavailability than CBD and produced maximum plasma concentrations of 0.21 μM. (32) In beagle dogs, oral administration of CBDA at 1 mg/kg was well tolerated, was 2-fold more bioavailable than CBD, and produced serum levels up to 1.42 μM. (41) Although no data on the bioavailability of CBGA are yet available, the data for CBDA suggest that μM plasma and serum concentrations for CBGA should also be possible.

Previous reports have indicated that one possible mechanism for inhibition of SARS-CoV-2 by decarboxylated cannabidiol (CBD) is activation of innate immune mechanisms. (42) However, our live-virus data indicate that inhibition by CBDA and CBGA occurs at the point of cell entry. These mechanisms are not mutually exclusive, and it remains possible that multiple cannabinoids in complex mixtures from plant extracts could act independently to inhibit SARS-CoV-2, potentially leading to enhanced effectiveness when compared to individual compounds.

One of the primary concerns in the ongoing pandemic is the spread of viral variants, of which there are many, with some of the most concerning and widespread being B.1.1.7 and B.1.351. These variants are well known for evading antibodies against early lineage SARS-CoV-2, which is particularly concerning due to the fact that current vaccination strategies rely on the early lineage spike RBD as an antigen. Our data show minimal impact of the variant lineages on the effectiveness of CBDA and CBGA, a trend that will hopefully extends to other existing and future variants. Because we believe that the primary binding site for CBGA is allosteric, there may even be reduced evolutionary pressure for SARS-CoV-2 to mutate their binding sites compared to the orthosteric binding sites typically favored by neutralizing antibodies. With widespread use of cannabinoids, resistant variants could still arise, but the combination of vaccination and CBDA/CBGA treatment should create a more challenging environment with which SARS-CoV-2 must contend, reducing the likelihood of escape.


General Experimental Procedures

Mass spectrometric analyses were carried out using a Shimadzu (Kyoto, Japan) Nexera UHPLC system interfaced with an LCMS-9030 Q-ToF hybrid high-resolution mass spectrometer or an LCMS-8050 triple quadrupole mass spectrometer.

Plant Material

Extracts of hemp and isolates of specific cannabinoids were obtained from the Global Hemp Innovation Center (Oregon State University, Corvallis, OR, USA). Plant taxonomy was confirmed by Jay S. Noller of the Global Hemp Innovation Center. Certified cannabinoid standards were purchased from Cayman Chemical (Ann Arbor, MI, USA).

Affinity Selection–Mass Spectrometry

Recombinant SARS-CoV-2 spike protein (RayBiotech; Peachtree Corners, GA, USA) (∼72 kDa) containing an N-terminal His-tag was immobilized on Ni2+-nitrilotriacetic acid-derivatized magnetic microbeads (EmerTher; Parsippany, NJ, USA) for use in the affinity selection–mass spectrometry approach MagMASS. As a negative control, denatured spike protein was immobilized on identical magnetic microbeads. The spike protein for the negative control incubations was denatured by incubating in a 95 °C water bath for 15 min. Positive control incubations used SBP-1 (RayBiotech), a 23 amino acid peptide with the sequence of IEEQAKTFLDKFNHEAEDLFYQS, which is identical to the ACE2 α1 helix sequence recognized by the SARS-CoV-2 spike protein. SBP-1 (33 nM) was incubated for 60 min with magnetic microbeads containing 50 pmol of immobilized active or denatured S1 protein in 300 μL of binding buffer. After washing twice with 500 μL of 30 mM ammonium acetate to remove unbound ligand while the beads were retained by a magnetic field, ligand was released from the beads using 90% methanol in water (200 μL) and analyzed using UHPLC-LC/MS. SBP-1 was measured using positive ion electrospray with selected reaction monitoring on a triple quadrupole mass spectrometer at unit resolution. For SBP-1, the selected reaction monitoring transitions were m/z 701.6 ([M + 4H]4+) to m/z 136.2 (quantifier) and m/z 701.6 ([M + 4H]4+) and m/z 120.2 (qualifier) at collision energies of −30 and −45 V, respectively, with a dwell time of 25 ms per transition.

Extracts (10 μg), mixtures of cannabinoid standards (0.10 μM each), or cannabinoid standards (0.10 μM) were incubated with 50 pmol of immobilized SARS-CoV-2 spike protein and screened using MagMASS as described above. The released ligand was analyzed using UHPLC-LC/MS with reversed phase UHPLC separation on a Waters (Milford, MA, USA) Acquity UPLC BEH C18 column (1.7 μm, 130 Å, 2.1 mm × 50 mm) with a 5 min linear gradient from 20% to 80% acetonitrile in 0.1% aqueous formic acid at a flow rate of 0.3 mL/min for the analysis of SBP-1 peptide. Cannabinoid separations were similar except that a 100 mm Waters Acquity UPLC BEH C18 column was used with a 1 min gradient from 50% to 75% acetonitrile followed by an 11 min gradient to 80% acetonitrile. The column was equilibrated to initial conditions for 1 min between analyses.

Ligands eluting from the column were detected using positive ion or negative ion electrospray mass spectrometry on the Q-ToF mass spectrometer at a resolving power of 30 000. The electrospray temperature was 300 °C, and voltages of 4.5 and −3.5 kV were used for positive or negative ion mode, respectively. The nitrogen gas flow rates for the electrospray ion source were 10 L/min for drying, 10 L/min for heating, and 3 L/min for nebulization. Data-dependent product ion tandem mass spectrometry was used such that mass spectra and product ion tandem mass spectra were acquired every 100 ms over the scan range of m/z 100–1200 and m/z 70–1200, respectively. For product ion MS/MS, the collision energy was 35 V with an energy spread of 17 V.

Following affinity selection, the UHPLC-MS chromatograms of each sample and corresponding negative control were compared using the metabolomics software Online XCMS (Scripps Research, La Jolla, CA, USA) to identify peaks enriched due to specific binding to the S1 protein. (43) High-resolution mass spectra for each enriched peak were processed using Shimadzu LabSolutions V5.2 software. Natural product ligands for which structures have been reported in the literature were identified by comparison with authentic standards based on their elemental compositions determined using high-resolution accurate mass measurements, tandem mass spectra, and UHPLC retention times.

Equilibrium Dissociation Constants

The affinity constants for the binding of active compounds to the spike protein S1 subunit were determined by rapid equilibrium dialysis. First, the optimum time for full equilibration of the RED device obtained from ThermoFisher (Waltham, MA, USA) was determined with each of the compounds by adding 1 μM spike protein in buffer (300 μL) to the protein chamber and adding blank phosphate-buffered saline, pH 7.2 (500 μL), to the buffer chamber. CBGA or CBDA was spiked into the protein chamber at a final concentration of 2.5 μM. During incubation at 37 °C on an orbital shaker at 200 rpm, aliquots (30 μL) were sampled from the sample and buffer chambers at 0.5, 1, 2, 3, 4, 5, 6, and 7 h, mixed with equal volumes of buffer, 300 μL of ice-cold 90% aqueous acetonitrile containing 0.1% formic acid, and 500 ng/mL d4-daidzein (internal standard), vortex mixed, and incubated on ice for 1 h. After centrifugation at 18000g for 30 min, the supernatant was removed and ligand concentration was measured using UHPLC-MS/MS with selected reaction monitoring MS/MS and negative ion electrospray on a triple quadrupole mass spectrometer. For the measurement of CBGA, the selected reaction monitoring transitions were m/z 359 to m/z 341 (quantifier) and m/z 359 to m/z 315 (qualifier). For the measurement of CBDA, the selected reaction monitoring transitions were m/z 357 to m/z 339 (quantifier) and m/z 357 to m/z 245 (qualifier).

Next, the equilibrium dissociation constants of CBGA and CBDA were determined by incubating the spike protein S1 subunit with different concentrations of the ligands ranging from 0.05 to 500 μM in triplicate. After 5 h for CBGA or 4 h for CBDA, the concentrations of each ligand in the sample and buffer chambers were measured using UHPLC-MS/MS with a triple quadruple mass spectrometer as described above. Data analysis and fitting was carried out using Microsoft Excel (Seattle, WA, USA) and KaleidaGraph v4.1 (Reading, PA, USA).

Ligand Docking

The computational aided modeling of cannabinoids was carried out using AutoDock Vina (Scripps Research Institute, La Jolla, CA, USA). (44) The coordinates of the crystal structure of SARS-CoV-2 spike protein C-terminal domain were downloaded from the Protein Data Bank (PDB, ID number 6LZG). (45) The ChemDraw structures of the ligands were converted to .pdb files using Pymol. The protein data were loaded into the AutoDock Vina program, the search space was defined around the known orthosteric site, and the file was converted to .pdbqt. Similarly, the ligands were individually loaded and converted to .pdbqt files.

Pseudotyped Lentivirus Production

Pseudovirus was prepared as previously described. (46) 293T cells, seeded 1 day ahead with 2 million cells in 6 cm TC-treated dishes, were transfected with lentivirus packaging plasmids, SARS-CoV-2 S plasmid, and lzGreen reporter plasmid. (47) After transfection, cells were incubated at 37 °C for 60 h. Viral media were filtered with a 0.45 μm syringe filter and snap frozen in liquid nitrogen before storing at −80 °C. Virus stocks were titrated on 293T-ACE2 cells treated with 50 μL of 5 μg/mL Polybrene (Sigma-Aldrich, St. Louis, MO, USA). Titer was determined by fluorescence microscopy using a BZ-X700 all-in-one fluorescent microscope (Keyence, Itasca, IL, USA).

SARS-CoV-2 Virus Propagation

SARS-CoV-2 isolates USA/CA_CDC_5574/2020 [lineage B.1.1.7] (NR-54011), hCoV-19/South Africa/KRISP-K005325/2020 [lineage B.1.351] (NR-54009), and USA-WA1/2020 1 [lineage A] (NR-52281) were obtained through BEI Resources, diluted 1:10, and added onto 70% confluent Vero E6 cells. The cells were incubated for 1 h at 37 °C with rocking every 15 min. Additional media were added according to the manufacturer’s recommended culture volume, and the cells were incubated for 72 h in a tissue culture incubator. Supernatant was centrifuged at 3000g for 5 min before aliquoting and freezing at −80 °C.

Pseudovirus Neutralization Assay

Pseudovirus neutralization was performed as previously described. (46) Briefly, 293T-ACE2 cells were seeded at 10 000 cells per well on tissue culture treated, poly lysine treated 96-well plates. Cells were grown overnight at 37 °C. LzGreen SARS-COV-2 S pseudotyped lentivirus was combined with 2-fold serial dilutions of CBDA and CBGA in DMSO or vehicle control. The virus–drug mixture was incubated at 37 °C for 1 h, after which virus was added to 293T-ACE2 treated with 5 μg/mL Polybrene. Cells were incubated with neutralized virus for 44 h, then fixed with 4% formaldehyde for 1 h at room temperature, incubated with DAPI for 10 min at room temperature, and imaged with a BZ-X700 all-in-one fluorescent microscope (Keyence, Itasca, IL, USA). Total areas of DAPI and GFP fluorescent signal were calculated using included microscope software (Keyence). To account for variability in cell count, green fluorescent signal was normalized to DAPI signal. For conditions with fewer DAPI foci, the modal value of DAPI signal for each set of replicates was used for normalization across that condition. Otherwise DMSO control values were used in normalization to manage DAPI inconsistency across replicates. IC50 values were calculated with combined replicate data in python using a three-parameter logistic model and plotted with the matplotlib data visualization library.

Focus Forming Assay for Live SARS-CoV-2

Focus forming assays were performed as previously described. (46,48) In brief, 96-well plates with subconfluent Vero E6 cells were infected with 50–100 virus titer per well of the original SARS-CoV-2 strain (WA-1/2020) or the variants (B.1.1.7 or B.1.351) in buffer containing CBDA or CBGA ranging from 100 to 0.625 μg/mL. DMSO was used as a vehicle control. The virus and drug mixtures were incubated for 1 h at 37 °C prior to addition to cells. The mixture was incubated with cells for 1 h at 37 °C before addition of overlay media (Opti-MEM, 2% FBS, 2% methylcellulose). Infection was allowed to proceed for 48 h; then plates were fixed for 1 h in 4% formaldehyde in phosphate-buffered saline (PBS). Cells were permeabilized (PBS, 0.1% saponin, 0.1% bovine serum albumin) for 30 min. Anti-SARS-CoV-2 spike protein alpaca immune serum was diluted 1:5000 in permeabilization buffer and incubated on plates overnight at 4 °C. Plates were washed three times with PBS with 0.1% Tween-20 (wash buffer) and incubated with antillama-HRP at 1:20 000 for 1 h at room temperature. Following three more washes in wash buffer, plates were developed with TrueBlue (Seracare) for 30 min before being imaged (CTL immunospot) and counted (Viridot). (49) Three separate dilution series were prepared for each experiment, each of which was used to prepare three technical replicates. IC50 values were calculated with combined replicate data in python using a three-parameter logistic model and plotted with the matplotlib data visualization library.

Immunofluorescence

Vero E6 cells were seeded on 96-well glass-bottom optical plates coated with poly lysine solution; 20 000 cells were seeded per well. Cells were infected with SARS-CoV-2 as described above. At 24 h postinfection, cells were fixed with 4% paraformaldehyde in PBS for 1 h. The 96-well plates with SARS-CoV-2-infected Vero E6 cells were permeabilized with 2% bovine serum albumin and 0.1% Triton-X-100 in PBS. Transfected cells were incubated for 2 h at room temperature with a mouse anti-dsRNA antibody (Millipore Sigma) to stain SARS-CoV-2 replication sites in infected cells. Anti-mouse IgG AF555 conjugated secondary antibodies were added at 1:500 dilution for 1 h at RT (Invitrogen, Carlsbad, CA, USA). Confocal imaging was performed with a Zeiss LSM 980 using a 63× Plan-Achromatic 1.4 NA oil immersion objective. Images were processed with Zeiss Zen Blue software. Maximum intensity z-projections were prepared in Fiji.


The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.1c00946.

  • Tandem mass spectra of affinity selected CBDA, CBGA, and THCA-A and the corresponding standards; cytotoxicity of CBDA in mammalian cell lines (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.


    • Ruth N. MuchiriLinus
      Pauling Institute, Department of Pharmaceutical Sciences, College
      of Pharmacy, Oregon State University, 2900 SW Campus Way, Corvallis, Oregon 97331, United States

    • Timothy A. BatesMolecular
      Microbiology & Immunology, Oregon Health
      & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, United States

    • Jules B. WeinsteinMolecular
      Microbiology & Immunology, Oregon Health
      & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, United States

    • Hans C. LeierMolecular
      Microbiology & Immunology, Oregon Health
      & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, United States

    • Scotland FarleyMolecular
      Microbiology & Immunology, Oregon Health
      & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, United States

    • Fikadu G. TafesseMolecular
      Microbiology & Immunology, Oregon Health
      & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, United States

  • The authors declare no competing financial interest.


The authors thank Shimadzu Scientific Instruments for mass spectrometry support, the Global Hemp Innovation Center for supplying hemp extracts, and the EmerTher company for providing the Ni-NTA magnetic microbeads used in this investigation

This article references 49 other publications.

  1. 3

    Walensky, R. P.; Walke, H. T.; Fauci, A. S. JAMA 2021, 325, 10371038,  DOI: 10.1001/jama.2021.2294

    [Crossref], [PubMed], [CAS], Google Scholar

    3

    SARS-CoV-2 variants of concern in the United States-challenges and opportunities

    Walensky, Rochelle P.; Walke, Henry T.; Fauci, Anthony S.

    JAMA, the Journal of the American Medical Association
    (2021),
    325
    (11),
    1037-1038CODEN:
    JAMAAP;
    ISSN:1538-3598.

    (American Medical Association)

    As of Feb. 3, 2021, 468 000 sequences of SARS-CoV-2 from COVID-19 cases glob- ally have been uploaded into publicly available data- bases, including more than 93 000 from individuals in the US. SARS-CoV-2, like other RNA viruses, constantly changes through mutation, with new variants occurring over time. Among the numerous SARS-CoV-2 variants that have been detected, only a very small pro- portion are of public health concern because they are more transmissible, cause more severe illness, or can elude the immune response that develops following infection and possibly from vaccination. The B.1.1.7 lineage (known as 20I/501Y.V1 or variant of concern [VOC] 202012/01) was first detected in the UK in Dec. 2020 with likely emergence during the preceding Sept.; this variant has now been identified in at least 80 countries. Modeling data have illustrated how a more conta- gious variant, such as B.1.1.7, has the potential to exac- erbate the trajectory of the US pandemic and to reverse the present downward trend in new infections and further delay control of the pandemic.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXntlWru70%253D&md5=0ee1b97c474060440f82b9bc9051990b

  2. 4

    Tahir
    ul Qamar, M.
    ; Alqahtani, S. M.; Alamri, M. A.; Chen, L. L. J. Pharm. Anal. 2020, 10, 313319,  DOI: 10.1016/j.jpha.2020.03.009

    [Crossref], [PubMed], [CAS], Google Scholar

    4

    Structural basis of SARS-CoV-2 3CL(pro) and anti-COVID-19 drug discovery from medicinal plants

    Tahir Ul Qamar Muhammad; Chen Ling-Ling; Tahir Ul Qamar Muhammad; Chen Ling-Ling; Alqahtani Safar M; Alamri Mubarak A

    Journal of pharmaceutical analysis
    (2020),
    10
    (4),
    313-319
    ISSN:.

    The recent pandemic of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has raised global health concerns. The viral 3-chymotrypsin-like cysteine protease (3CL(pro)) enzyme controls coronavirus replication and is essential for its life cycle. 3CL(pro) is a proven drug discovery target in the case of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Recent studies revealed that the genome sequence of SARS-CoV-2 is very similar to that of SARS-CoV. Therefore, herein, we analysed the 3CL(pro) sequence, constructed its 3D homology model, and screened it against a medicinal plant library containing 32,297 potential anti-viral phytochemicals/traditional Chinese medicinal compounds. Our analyses revealed that the top nine hits might serve as potential anti- SARS-CoV-2 lead molecules for further optimisation and drug development process to combat COVID-19.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38zlvVCksQ%253D%253D&md5=c4f1e4ccbce32c8a68c720d44c27062d

  3. 5

    Huang, C.; Wang, Y.; Li, X.; Ren, L.; Zhao, J.; Hu, Y.; Zhang, L.; Fan, G.; Xu, J.; Gu, X.; Cheng, Z.; Yu, T.; Xia, J.; Wei, Y.; Wu, W.; Xie, X.; Yin, W.; Li, H.; Liu, M.; Xiao, Y.; Gao, H.; Guo, L.; Xie, J.; Wang, G.; Jiang, R.; Gao, Z.; Jin, Q.; Wang, J.; Cao, B. Lancet 2020, 395, 497506,  DOI: 10.1016/S0140-6736(20)30183-5

    [Crossref], [PubMed], [CAS], Google Scholar

    5

    Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

    Huang, Chaolin; Wang, Yeming; Li, Xingwang; Ren, Lili; Zhao, Jianping; Hu, Yi; Zhang, Li; Fan, Guohui; Xu, Jiuyang; Gu, Xiaoying; Cheng, Zhenshun; Yu, Ting; Xia, Jiaan; Wei, Yuan; Wu, Wenjuan; Xie, Xuelei; Yin, Wen; Li, Hui; Liu, Min; Xiao, Yan; Gao, Hong; Guo, Li; Xie, Jungang; Wang, Guangfa; Jiang, Rongmeng; Gao, Zhancheng; Jin, Qi; Wang, Jianwei; Cao, Bin

    Lancet
    (2020),
    395
    (10223),
    497-506CODEN:
    LANCAO;
    ISSN:0140-6736.

    (Elsevier Ltd.)

    A recent cluster of pneumonia cases in Wuhan, China, was caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-nCoV). We report the epidemiol., clin., lab., and radiol. characteristics and treatment and clin. outcomes of these patients. All patients with suspected 2019-nCoV were admitted to a designated hospital in Wuhan. We prospectively collected and analyzed data on patients with lab.-confirmed 2019-nCoV infection by real-time RT-PCR and next-generation sequencing. Data were obtained with standardised data collection forms shared by the International Severe Acute Respiratory and Emerging Infection Consortium from electronic medical records. Researchers also directly communicated with patients or their families to ascertain epidemiol. and symptom data. Outcomes were also compared between patients who had been admitted to the intensive care unit (ICU) and those who had not. By Jan 2, 2020, 41 admitted hospital patients had been identified as having lab.-confirmed 2019-nCoV infection. Most of the infected patients were men (30 [73%] of 41); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). Median age was 49·0 years (IQR 41·0-58·0). 27 (66%) of 41 patients had been exposed to Huanan seafood market. One family cluster was found. Common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum prodn. (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38). Dyspnoea developed in 22 (55%) of 40 patients (median time from illness onset to dyspnoea 8·0 days [IQR 5·0-13·0]). 26 (63%) Of 41 patients had lymphopenia. All 41 patients had pneumonia with abnormal findings on chest CT. Complications included acute respiratory distress syndrome (12 [29%]), RNAemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). 13 (32%) patients were admitted to an ICU and six (15%) died. Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα. The 2019-nCoV infection caused clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus and was assocd. with ICU admission and high mortality. Major gaps in our knowledge of the origin, epidemiol., duration of human transmission, and clin. spectrum of disease need fulfilment by future studies. Ministry of Science and Technol., Chinese Academy of Medical Sciences, National Natural Science Foundation of China, and Beijing Municipal Science and Technol. Commission.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1Kqu7c%253D&md5=b279b965c1054d99e60f673859c03b49

  4. 6

    Wu, F.; Zhao, S.; Yu, B.; Chen, Y.-M.; Wang, W.; Song, Z.-G.; Hu, Y.; Tao, Z.-W.; Tian, J.-H.; Pei, Y.-Y.; Yuan, M.-L.; Zhang, Y.-L.; Dai, F.-H.; Liu, Y.; Wang, Q.-M.; Zheng, J.-J.; Xu, L.; Holmes, E. C.; Zhang, Y.-Z. Nature 2020, 579, 265269,  DOI: 10.1038/s41586-020-2008-3

    [Crossref], [PubMed], [CAS], Google Scholar

    6

    A new coronavirus associated with human respiratory disease in China

    Wu, Fan; Zhao, Su; Yu, Bin; Chen, Yan-Mei; Wang, Wen; Song, Zhi-Gang; Hu, Yi; Tao, Zhao-Wu; Tian, Jun-Hua; Pei, Yuan-Yuan; Yuan, Ming-Li; Zhang, Yu-Ling; Dai, Fa-Hui; Liu, Yi; Wang, Qi-Min; Zheng, Jiao-Jiao; Xu, Lin; Holmes, Edward C.; Zhang, Yong-Zhen

    Nature (London, United Kingdom)
    (2020),
    579
    (7798),
    265-269CODEN:
    NATUAS;
    ISSN:0028-0836.

    (Nature Research)

    Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health. Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 Jan. 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 Dec. 2019. Epidemiol. investigations have suggested that the outbreak was assocd. with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 Dec. 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here ‘WH-Human 1’ coronavirus (and has also been referred to as ‘2019-nCoV’). Phylogenetic anal. of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China. This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXksFKlsLc%253D&md5=0163a684829e880a0c3347e19f0ce52a

  5. 7

    Jiang, S.; Hillyer, C.; Du, L. Trends Immunol. 2020, 41, 355359,  DOI: 10.1016/j.it.2020.03.007

    [Crossref], [PubMed], [CAS], Google Scholar

    7

    Neutralizing Antibodies against SARS-CoV-2 and Other Human Coronaviruses

    Jiang, Shibo; Hillyer, Christopher; Du, Lanying

    Trends in Immunology
    (2020),
    41
    (5),
    355-359CODEN:
    TIRMAE;
    ISSN:1471-4906.

    (Elsevier Ltd.)

    A review. Coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome (SARS)-CoV-2 (also known as 2019-nCoV) is threatening global public health, social stability, and economic development. To meet this challenge, this article discusses advances in the research and development of neutralizing antibodies (nAbs) for the prevention and treatment of infection by SARS-CoV-2 and other human CoVs.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlslKmurw%253D&md5=c6f3920a1d0b8ad71fe5dcaebcd4db30

  6. 8

    Rabi, F. A.; Al Zoubi, M. S.; Kasasbeh, G. A.; Salameh, D. M.; Al-Nasser, A. D. Pathogens 2020, 9, 231,  DOI: 10.3390/pathogens9030231

    [Crossref], [CAS], Google Scholar

    8

    SARS-CoV-2 and coronavirus disease 2019: what we know so far

    Rabi, Firas A.; Al Zoubi, Mazhar S.; Kasasbeh, Ghena A.; Salameh, Dunia M.; Al-Nasser, Amjad D.

    Pathogens
    (2020),
    9
    (3),
    231CODEN:
    PATHCD;
    ISSN:2076-0817.

    (MDPI AG)

    In Dec. 2019, a cluster of fatal pneumonia cases presented in Wuhan, China. They were caused by a previously unknown coronavirus. All patients had been assocd. with the Wuhan Wholefood market, where seafood and live animals are sold. The virus spread rapidly and public health authorities in China initiated a containment effort. However, by that time, travelers had carried the virus to many countries, sparking memories of the previous coronavirus epidemics, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), and causing widespread media attention and panic. Based on clin. criteria and available serol. and mol. information, the new disease was called coronavirus disease of 2019 (COVID-19), and the novel coronavirus was called SARS Coronavirus-2 (SARS-CoV-2), emphasizing its close relationship to the 2002 SARS virus (SARS-CoV). The scientific community raced to uncover the origin of the virus, understand the pathogenesis of the disease, develop treatment options, define the risk factors, and work on vaccine development. Here we present a summary of current knowledge regarding the novel coronavirus and the disease it causes.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisFGgtb%252FE&md5=e54c637f6b70a2d6bbee4ca56ac6f89d

  7. 9

    Tai, W.; He, L.; Zhang, X.; Pu, J.; Voronin, D.; Jiang, S.; Zhou, Y.; Du, L. Cell. Mol. Immunol. 2020, 17, 613620,  DOI: 10.1038/s41423-020-0400-4

    [Crossref], [PubMed], [CAS], Google Scholar

    9

    Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine

    Tai, Wanbo; He, Lei; Zhang, Xiujuan; Pu, Jing; Voronin, Denis; Jiang, Shibo; Zhou, Yusen; Du, Lanying

    Cellular & Molecular Immunology
    (2020),
    17
    (6),
    613-620CODEN:
    CMIEAO;
    ISSN:1672-7681.

    (Nature Research)

    The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a serious threat to global public health, calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV). The CoV spike (S) protein plays the most important roles in viral attachment, fusion and entry, and serves as a target for development of antibodies, entry inhibitors and vaccines. Here, we identified the receptor-binding domain (RBD) in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2 (ACE2) receptors. SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and, hence, attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells, thus inhibiting their infection to host cells. SARS-CoV RBD-specific antibodies could cross-react with SARS-CoV-2 RBD protein, and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2, suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlt1Chsrw%253D&md5=87bc49d070c84e78b01230518aaa465a

  8. 10

    Turner, A. J. In Protective Arm of the Renin Angiotensin System (RAS); Unger, T.; Steckelings, U. M.; dos Santos, R. A. S., Eds.; Elsevier: New York, 2015; pp 185189. DOI: 10.1016/B978-0-12-801364-9.00025-0

  9. 11

    Hoffmann, M.; Kleine-Weber, H.; Schroeder, S.; Krüger, N.; Herrler, T.; Erichsen, S.; Schiergens, T. S.; Herrler, G.; Wu, N.-H.; Nitsche, A.; Müller, M. A.; Drosten, C.; Pöhlmann, S. Cell. 2020, 181, 271280,  DOI: 10.1016/j.cell.2020.02.052

    [Crossref], [PubMed], [CAS], Google Scholar

    11

    SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

    Hoffmann, Markus; Kleine-Weber, Hannah; Schroeder, Simon; Krueger, Nadine; Herrler, Tanja; Erichsen, Sandra; Schiergens, Tobias S.; Herrler, Georg; Wu, Nai-Huei; Nitsche, Andreas; Mueller, Marcel A.; Drosten, Christian; Poehlmann, Stefan

    Cell (Cambridge, MA, United States)
    (2020),
    181
    (2),
    271-280.e8CODEN:
    CELLB5;
    ISSN:0092-8674.

    (Cell Press)

    The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clin. use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXktl2qtb8%253D&md5=60aea5c939a2d4df034a91d6198fb3ef

  10. 12

    Guo, Y.-R.; Cao, Q.-D.; Hong, Z.-S.; Tan, Y.-Y.; Chen, S.-D.; Jin, H.-J.; Tan, K.-S.; Wang, D.-Y.; Yan, Y. Military Med. Res. 2020, 7, 11,  DOI: 10.1186/s40779-020-00240-0

    [Crossref], [PubMed], [CAS], Google Scholar

    12

    The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak – an update on the status

    Guo, Yan-Rong; Cao, Qing-Dong; Hong, Zhong-Si; Tan, Yuan-Yang; Chen, Shou-Deng; Jin, Hong-Jun; Tan, Kai-Sen; Wang, De-Yun; Yan, Yan

    Military Medical Research
    (2020),
    7
    (1),
    11CODEN:
    MMRICN;
    ISSN:2054-9369.

    (BioMed Central Ltd.)

    A review. An acute respiratory disease, caused by a novel coronavirus (SARS-CoV-2, previously known as 2019-nCoV), the coronavirus disease 2019 (COVID-19) has spread throughout China and received worldwide attention. On 30 Jan. 2020, World Health Organization (WHO) officially declared the COVID-19 epidemic as a public health emergency of international concern. The emergence of SARS-CoV-2, since the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, marked the 3rd introduction of a highly pathogenic and large-scale epidemic coronavirus into the human population in the 21st century. As of 1 March 2020, a total of 87,137 confirmed cases globally, 79,968 confirmed in China and 7169 outside of China, with 2977 deaths (3.4%) had been reported by WHO. Meanwhile, several independent research groups have identified that SARS-CoV-2 belongs to β-coronavirus, with highly identical genome to bat coronavirus, pointing to bat as the natural host. The novel coronavirus uses the same receptor, angiotensin-converting enzyme 2 (ACE2) as that for SARS-CoV, and mainly spreads through the respiratory tract. Importantly, increasingly evidence showed sustained human-to-human transmission, along with many exported cases across the globe. The clin. symptoms of COVID-19 patients include fever, cough, fatigue, and a small population of patients appeared gastrointestinal infection symptoms. The elderly and people with underlying diseases are susceptible to infection and prone to serious outcomes, which may be assocd. with acute respiratory distress syndrome (ARDS) and cytokine storm. Currently, there are few specific antiviral strategies, but several potent candidates of antivirals and repurposed drugs are under urgent investigation. We summarized the latest research progress of the epidemiol., pathogenesis, and clin. characteristics of COVID-19, and discussed the current treatment and scientific advancements to combat the epidemic novel coronavirus.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXns1aht78%253D&md5=040aa4a4b4c385793ea2e49c536299f3

  11. 13

    Du, L.; He, Y.; Zhou, Y.; Liu, S.; Zheng, B.-J.; Jiang, S. Nat. Rev. Microbiol. 2009, 7, 226236,  DOI: 10.1038/nrmicro2090

    [Crossref], [PubMed], [CAS], Google Scholar

    13

    The spike protein of SARS-CoV – a target for vaccine and therapeutic development

    Du, Lanying; He, Yuxian; Zhou, Yusen; Liu, Shuwen; Zheng, Bo-Jian; Jiang, Shibo

    Nature Reviews Microbiology
    (2009),
    7
    (3),
    226-236CODEN:
    NRMACK;
    ISSN:1740-1526.

    (Nature Publishing Group)

    A review. Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease caused by a novel coronavirus, SARS-coronavirus (SARS-CoV). The SARS-CoV spike (S) protein is composed of two subunits; the S1 subunit contains a receptor-binding domain that engages with the host cell receptor angiotensin-converting enzyme 2 and the S2 subunit mediates fusion between the viral and host cell membranes. The S protein plays key parts in the induction of neutralizing-antibody and T-cell responses, as well as protective immunity, during infection with SARS-CoV. In this Review, we highlight recent advances in the development of vaccines and therapeutics based on the S protein.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFeqs7Y%253D&md5=610a46a8fed820bf5d0076e7c7d03c13

  12. 14

    Sainz, B., Jr; Mossel, E. C.; Gallaher, W. R.; Wimley, W. C.; Peters, C. J.; Wilson, R. B.; Garry, R. F. Virus Res. 2006, 120, 146155,  DOI: 10.1016/j.virusres.2006.03.001

    [Crossref], [PubMed], [CAS], Google Scholar

    14

    Inhibition of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infectivity by peptides analogous to the viral spike protein

    Sainz, Bruno; Mossel, Eric C.; Gallaher, William R.; Wimley, William C.; Peters, C. J.; Wilson, Russell B.; Garry, Robert F.

    Virus Research
    (2006),
    120
    (1-2),
    146-155CODEN:
    VIREDF;
    ISSN:0168-1702.

    (Elsevier B.V.)

    Severe acute respiratory syndrome-assocd. coronavirus (SARS-Co-V) is the cause of an atypical pneumonia that affected Asia, North America and Europe in 2002-2003. The viral spike (S) glycoprotein is responsible for mediating receptor binding and membrane fusion. Recent studies have proposed that the carboxyl terminal portion (S2 subunit) of the S protein is a class I viral fusion protein. The Wimley and White interfacial hydrophobicity scale was used to identify regions within the Co-V S2 subunit that may preferentially assoc. with lipid membranes with the premise that peptides analogous to these regions may function as inhibitors of viral infectivity. Five regions of high interfacial hydrophobicity spanning the length of the S2 subunit of SARS-Co-V and murine hepatitis virus (MHV) were identified. Peptides analogous to regions of the N-terminus or the pretransmembrane domain of the S2 subunit inhibited SARS-Co-V plaque formation by 40-70% at concns. of 15-30 μM. Interestingly, peptides analogous to the SARS-Co-V or MHV loop region inhibited viral plaque formation by >80% at similar concns. The obsd. effects were dose-dependent (IC50 values of 2-4 μM) and not a result of peptide-mediated cell cytotoxicity. The antiviral activity of the Co-V peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xms1egtbY%253D&md5=75ac62c39705ba25f468dab2f8912ae3

  13. 15

    Yuan, K.; Yi, L.; Chen, J.; Qu, X.; Qing, T.; Rao, X.; Jiang, P.; Hu, J.; Xiong, Z.; Nie, Y.; Shi, X.; Wang, W.; Ling, C.; Yin, X.; Fan, K.; Lai, L.; Ding, M.; Deng, H. Biochem. Biophys. Res. Commun. 2004, 319, 746752,  DOI: 10.1016/j.bbrc.2004.05.046

    [Crossref], [PubMed], [CAS], Google Scholar

    15

    Suppression of SARS-CoV entry by peptides corresponding to heptad regions on spike glycoprotein

    Yuan, Kehu; Yi, Ling; Chen, Jian; Qu, Xiuxia; Qing, Tingting; Rao, Xi; Jiang, Pengfei; Hu, Jianhe; Xiong, Zikai; Nie, Yuchun; Shi, Xuanling; Wang, Wei; Chen, Ling; Yin, Xiaolei; Fan, Keqiang; Lai, Luhua; Ding, Mingxiao; Deng, Hongkui

    Biochemical and Biophysical Research Communications
    (2004),
    319
    (3),
    746-752CODEN:
    BBRCA9;
    ISSN:0006-291X.

    (Elsevier Science)

    Heptad repeat regions (HR1 and HR2) are highly conserved sequences located in the glycoproteins of enveloped viruses. They form a six-helix bundle structure and are important in the process of virus fusion. Peptides derived from the HR regions of some viruses have been shown to inhibit the entry of these viruses. SARS-CoV was also predicted to have HR1 and HR2 regions in the S2 protein. Based on this prediction, we designed 25 peptides and screened them using a HIV-luc/SARS pseudotyped virus assay. Two peptides, HR1-1 and HR2-18, were identified as potential inhibitors, with EC50 values of 0.14 and 1.19 μM, resp. The inhibitory effects of these peptides were validated by the wild-type SARS-CoV assay. HR1-1 and HR2-18 can serve as functional probes for dissecting the fusion mechanism of SARS-CoV and also provide the potential of further identifying potent inhibitors for SARS-CoV entry.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXkslWnu7o%253D&md5=9764a88c9fc49e1e3bfaaefa5c681f87

  14. 16

    De
    Clercq, E.
    J. Clin. Virol. 2001, 22, 7389,  DOI: 10.1016/S1386-6532(01)00167-6

    [Crossref], [PubMed], [CAS], Google Scholar

    16

    Antiviral drugs: current state of the art

    De Clercq, E.

    Journal of Clinical Virology
    (2001),
    22
    (1),
    73-89CODEN:
    JCVIFB;
    ISSN:1386-6532.

    (Elsevier Science Ireland Ltd.)

    A review. The chemotherapy of virus infections has definitely come of age. There are now 15 antiviral agents that have been formally licensed for the treatment of human immunodeficiency virus infections (zidovudine, didanosine, zalcitabine, stavudine, Lamivudine, Abacavir, Nevirapine, Delavirdine, Efavirenz, Saquinavir, Ritonavir, Indinavir, Nelfinavir, Amprenavir, Lopinavir) and several others, such as Tenofovir Disoproxil, Emtricitabine, Capravirine, Emivirine, T-20 (Pentafuside), and AMD3100 (bicyclam), are under clin. development. Lamivudine has been approved, and several other compds. (such as Adefovir Dipivoxil, Emtricitabine, and Entecavir) are under clin. development, for the treatment of hepatitis B virus infections. Among the anti-herpesvirus agents, Aciclovir, Valaciclovir, Penciclovir, Famciclovir, Idoxuridine, Trifluridine, and Brivudin are used in the treatment of herpes simplex virus and varicella-zoster virus infections, and Ganciclovir, Foscarnet, Cidofovir, Fomivirsen, and Maribavir (the latter in the developmental stage) are used in the treatment of cytomegalovirus infections. Following amantadine and Rimantadine, the neuraminidase inhibitors, Zanamivir and Oseltamivir, have now become available for the therapy and prophylaxis of influenza virus infections, and so is Ribavirin for the treatment of respiratory syncytial virus infections and the combination of Ribavirin with interferon-α for the treatment of hepatitis C virus infections.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXksVGqur0%253D&md5=c6a22c42d6d86c0c7241472997eb19f1

  15. 17

    VanCompernolle, S. E.; Wiznycia, A. V.; Rush, J. R.; Dhanasekaran, M.; Baures, P. W.; Todd, S. C. Virology 2003, 314, 371380,  DOI: 10.1016/S0042-6822(03)00406-9

    [Crossref], [PubMed], [CAS], Google Scholar

    17

    Small molecule inhibition of hepatitis C virus E2 binding to CD81

    VanCompernolle, Scott E.; Wiznycia, Alexander V.; Rush, Jeremy R.; Dhanasekaran, Muthu; Baures, Paul W.; Todd, Scott C.

    Virology
    (2003),
    314
    (1),
    371-380CODEN:
    VIRLAX;
    ISSN:0042-6822.

    (Elsevier Science)

    The hepatitis C virus (HCV) is a causal agent of chronic liver infection, cirrhosis, and hepatocellular carcinoma infecting more than 170 million people. CD81 is a receptor for HCV envelope glycoprotein E2. Although the binding of HCV-E2 with CD81 is well documented the role of this interaction in the viral life cycle remains unclear. Host specificity and mutagenesis studies suggest that the helix D region of CD81 mediates binding to HCV-E2. Structural anal. of CD81 has enabled the synthesis of small mols. designed to mimic the space and hydrophobic features of the solvent-exposed face on helix D. Utilizing a novel bis-imidazole scaffold a series of over 100 compds. has been synthesized. Seven related, imidazole-based compds. were identified that inhibit binding of HCV-E2 to CD81. The inhibitory compds. have no short-term effect on cellular expression of CD81 or other tetraspanins, do not disrupt CD81 assocns. with other cell surface proteins, and bind reversibly to HCV-E2. These results provide an important proof of concept that CD81-based mimics can disrupt binding of HCV-E2 to CD81.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXnsVWqsb4%253D&md5=9c3dab64e81daeb818c1d433f5fef65c

  16. 18

    Cragg, G. M.; Newman, D. J. Pure Appl. Chem. 2005, 77, 724,  DOI: 10.1351/pac200577010007

    [Crossref], [CAS], Google Scholar

    18

    Biodiversity: a continuing source of novel drug leads

    Cragg, Gordon M.; Newman, David J.

    Pure and Applied Chemistry
    (2005),
    77
    (1),
    7-24CODEN:
    PACHAS;
    ISSN:0033-4545.

    (International Union of Pure and Applied Chemistry)

    A review. Nature has been a source of medicinal agents for thousands of years and continues to be an abundant source of novel chemotypes and pharmacophores. With only 5 to 15% of the approx. 250 000 species of higher plants systematically investigated, and the potential of the marine environment barely tapped, these areas will remain a rich source of novel bioactive compds. Less than 1% of bacterial and 5% of fungal species are currently known, and the potential of novel microbial sources, particularly those found in extreme environments, seems unbounded. To these natural sources can be added the potential to investigate the rational design of novel structure types within certain classes of microbial metabolites through genetic engineering. It is apparent that Nature can provide the novel chem. scaffolds for elaboration by combinatorial approaches (chem. and biochem.), thus leading to agents that have been optimized on the basis of their pharmacol. activities. The proven natural product drug discovery track record, coupled with the continuing threat to biodiversity through the destruction of terrestrial and marine ecosystems and the current low no. of new chem. entities in pharmaceutical industry pipelines, provides a compelling argument in favor of expanded multidisciplinary and international collaboration in the exploration of Nature as a source of novel leads for the development of drugs and other valuable bioactive agents.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXisVWmsrk%253D&md5=e50b8c45b05a0abe0c272e710449e26d

  17. 19

    Dias, D. A.; Urban, S.; Roessner, U. Metabolites 2012, 2, 303336,  DOI: 10.3390/metabo2020303

    [Crossref], [PubMed], [CAS], Google Scholar

    19

    A historical overview of natural products in drug discovery

    Dias, Daniel A.; Urban, Sylvia; Roessner, Ute

    Metabolites
    (2012),
    2
    (2),
    303-336CODEN:
    METALU;
    ISSN:2218-1989.

    (MDPI AG)

    A review. Historically, natural products have been used since ancient times and in folklore for the treatment of many diseases and illnesses. Classical natural product chem. methodologies enabled a vast array of bioactive secondary metabolites from terrestrial and marine sources to be discovered. Many of these natural products have gone on to become current drug candidates. This brief review aims to highlight historically significant bioactive marine and terrestrial natural products, their use in folklore and dereplication techniques to rapidly facilitate their discovery. Furthermore a discussion of how natural product chem. has resulted in the identification of many drug candidates; the application of advanced hyphenated spectroscopic techniques to aid in their discovery, the future of natural product chem. and finally adopting metabolomic profiling and dereplication approaches for the comprehensive study of natural product exts. will be discussed.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xnt1SltLs%253D&md5=d02ea918e56b6cc7de17053adaf47650

  18. 20

    von
    Nussbaum, F.
    ; Brands, M.; Hinzen, B.; Weigand, S.; Habich, D. Angew.Chem. Int. Ed. 2006, 45, 50725129,  DOI: 10.1002/anie.200600350

    [Crossref], [CAS], Google Scholar

    20

    Antibacterial natural products in medicinal chemistry – exodus or revival?

    von Nussbaum, Franz; Brands, Michael; Hinzen, Berthold; Weigand, Stefan; Haebich, Dieter

    Angewandte Chemie, International Edition
    (2006),
    45
    (31),
    5072-5129CODEN:
    ACIEF5;
    ISSN:1433-7851.

    (Wiley-VCH Verlag GmbH & Co. KGaA)

    A review. To create a drug, nature’s blueprints often have to be improved through semi-synthesis or total synthesis (chem. post-evolution). Selected contributions from industrial and academic groups highlight the arduous but rewarding path from natural products to drugs. Principle modification types for natural products to drugs. Principle modification types for natural products are discussed herein, such as decoration, substitution, and degrdn. The biol., chem., and socioeconomic environments of antibacterial research are dealt with in context. Natural products, many from soil organisms, have provided the majority of lead structures for marketed anti-infectives. Surprisingly, numerous “old” classes of antibacterial natural products have never been intensively explored by medical chemists. Nevertheless, research on antibacterial natural products is flagging. Apparently, the “old fashioned” natural products no longer fit into modern drug discovery. The handling of natural products is cumbersome, requiring nonstandardized workflows and extended timelines. Revisiting natural products with modern chem. and target-finding tools from biol. (reversed genomics) is one option for their revival.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xot12guro%253D&md5=4521f405a693b7090394a5df52f44b05

  19. 21

    Mishra, B. B.; Tiwari, V. K. Eur. J. Med. Chem. 2011, 46, 47694807,  DOI: 10.1016/j.ejmech.2011.07.057

    [Crossref], [PubMed], [CAS], Google Scholar

    21

    Natural products: An evolving role in future drug discovery

    Mishra, Bhuwan B.; Tiwari, Vinod K.

    European Journal of Medicinal Chemistry
    (2011),
    46
    (10),
    4769-4807CODEN:
    EJMCA5;
    ISSN:0223-5234.

    (Elsevier Masson SAS)

    A review. The therapeutic areas of infectious diseases and oncol. have benefited from abundant scaffold diversity in natural products, able to interact with many specific targets within the cell and indeed for many years have been source or inspiration for the majority of FDA approved drugs. The present review describes natural products (NPs), semi-synthetic NPs and NP-derived compds. that have undergone clin. evaluation or registration from 2005 to 2010 by disease area i.e. infectious (bacterial, fungal, parasitic and viral), immunol., cardiovascular, neurol., inflammatory and related diseases and oncol.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1amt7zP&md5=62a6656e02e672ec35a35d30dd9c4397

  20. 22

    Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2016, 79, 629661,  DOI: 10.1021/acs.jnatprod.5b01055

    [ACS Full Text ACS Full Text], [CAS], Google Scholar

    22

    Natural Products as Sources of New Drugs from 1981 to 2014

    Newman, David J.; Cragg, Gordon M.

    Journal of Natural Products
    (2016),
    79
    (3),
    629-661CODEN:
    JNPRDF;
    ISSN:0163-3864.

    (American Chemical Society-American Society of Pharmacognosy)

    This contribution is a completely updated and expanded version of the four prior analogous reviews that were published in this journal in 1997, 2003, 2007, and 2012. In the case of all approved therapeutic agents, the time frame has been extended to cover the 34 years from Jan. 1, 1981, to Dec. 31, 2014, for all diseases worldwide, and from 1950 (earliest so far identified) to Dec. 2014 for all approved antitumor drugs worldwide. As mentioned in the 2012 review, we have continued to utilize our secondary subdivision of a “natural product mimic”, or “NM”, to join the original primary divisions and the designation “natural product botanical”, or “NB”, to cover those botanical “defined mixts.” now recognized as drug entities by the U.S. FDA (and similar organizations). From the data presented in this review, the utilization of natural products and/or their novel structures, in order to discover and develop the final drug entity, is still alive and well. For example, in the area of cancer, over the time frame from around the 1940s to the end of 2014, of the 175 small mols. approved, 131, or 75%, are other than “S” (synthetic), with 85, or 49%, actually being either natural products or directly derived therefrom. In other areas, the influence of natural product structures is quite marked, with, as expected from prior information, the anti-infective area being dependent on natural products and their structures. We wish to draw the attention of readers to the rapidly evolving recognition that a significant no. of natural product drugs/leads are actually produced by microbes and/or microbial interactions with the “host from whence it was isolated”, and therefore it is considered that this area of natural product research should be expanded significantly.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xit1Kqu7k%253D&md5=c9f2a44ab6b66331b7ef6ca64029328a

  21. 23

    Kanjanasirirat, P.; Suksatu, A.; Manopwisedjaroen, S.; Munyoo, B.; Tuchinda, P.; Jearawuttanakul, K.; Seemakhan, S.; Charoensutthivarakul, S.; Wongtrakoongate, P.; Rangkasenee, N.; Pitiporn, S.; Waranuch, N.; Chabang, N.; Khemawoot, P.; Sa-Ngiamsuntorn, K.; Pewkliang, Y.; Thongsri, P.; Chutipongtanate, S.; Hongeng, S.; Borwornpinyo, S.; Thitithanyanont, A. Sci. Rep. 2020, 10, 19963,  DOI: 10.1038/s41598-020-77003-3

    [Crossref], [PubMed], [CAS], Google Scholar

    23

    High-content screening of Thai medicinal plants reveals Boesenbergia rotunda extract and its component Panduratin A as anti-SARS-CoV-2 agents

    Kanjanasirirat, Phongthon; Suksatu, Ampa; Manopwisedjaroen, Suwimon; Munyoo, Bamroong; Tuchinda, Patoomratana; Jearawuttanakul, Kedchin; Seemakhan, Sawinee; Charoensutthivarakul, Sitthivut; Wongtrakoongate, Patompon; Rangkasenee, Noppawan; Pitiporn, Supaporn; Waranuch, Neti; Chabang, Napason; Khemawoot, Phisit; Sa-ngiamsuntorn, Khanit; Pewkliang, Yongyut; Thongsri, Piyanoot; Chutipongtanate, Somchai; Hongeng, Suradej; Borwornpinyo, Suparerk; Thitithanyanont, Arunee

    Scientific Reports
    (2020),
    10
    (1),
    19963CODEN:
    SRCEC3;
    ISSN:2045-2322.

    (Nature Research)

    Abstr.: Since Dec. 2019, the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused severe pneumonia, a disease named COVID-19, that became pandemic and created an acute threat to public health. The effective therapeutics are in urgent need. Here, we developed a high-content screening for the antiviral candidates using fluorescence-based SARS-CoV-2 nucleoprotein detection in Vero E6 cells coupled with plaque redn. assay. Among 122 Thai natural products, we found that Boesenbergia rotunda ext. and its phytochem. compd., panduratin A, exhibited the potent anti-SARS-CoV-2 activity. Treatment with B. rotunda ext. and panduratin A after viral infection drastically suppressed SARS-CoV-2 infectivity in Vero E6 cells with IC50 of 3.62μg/mL (CC50 = 28.06μg/mL) and 0.81μΜ (CC50 = 14.71μM), resp. Also, the treatment of panduratin A at the pre-entry phase inhibited SARS-CoV-2 infection with IC50 of 5.30μM (CC50 = 43.47μM). Our study demonstrated, for the first time, that panduratin A exerts the inhibitory effect against SARS-CoV-2 infection at both pre-entry and post-infection phases. Apart from Vero E6 cells, treatment with this compd. was able to suppress viral infectivity in human airway epithelial cells. This result confirmed the potential of panduratin A as the anti-SARS-CoV-2 agent in the major target cells in human. Since B. rotunda is a culinary herb generally grown in China and Southeast Asia, its ext. and the purified panduratin A may serve as the promising candidates for therapeutic purposes with economic advantage during COVID-19 situation.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitl2nsLvO&md5=85efcfa2ea697cdb22d7300e2e1ac37f

  22. 24

    Muchiri, R. N.; van Breemen, R. B. J. Mass Spectrom. 2021, 56, e4647,  DOI: 10.1002/jms.4647

    [Crossref], [PubMed], [CAS], Google Scholar

    24

    Affinity selection-mass spectrometry for the discovery of pharmacologically active compounds from combinatorial libraries and natural products

    Muchiri, Ruth N.; van Breemen, Richard B.

    Journal of Mass Spectrometry
    (2021),
    56
    (5),
    e4647CODEN:
    JMSPFJ;
    ISSN:1076-5174.

    (John Wiley & Sons Ltd.)

    A review. Invented to address the high-throughput screening (HTS) demands of combinatorial chem., affinity selection-mass spectrometry (AS-MS) utilizes binding interactions between ligands and receptors to isolate pharmacol. active compds. from mixts. of small mols. and then relies on the selectivity, sensitivity, and speed of mass spectrometry to identify them. No radiolabels, fluorophores, or chromophores are required. Although many variations of AS-MS have been devised, three approaches have emerged as the most flexible, productive, and popular, and they differ primarily in how ligand-receptor complexes are sepd. from nonbinding compds. in the mixt. These are pulsed ultrafiltration (PUF) AS-MS, size exclusion chromatog. (SEC) AS-MS, and magnetic microbead affinity selection screening (MagMASS). PUF and SEC AS-MS are soln.-phase screening approaches, and MagMASS uses receptors immobilized on magnetic microbeads. Because pools of compds. are screened using AS-MS, each contg. hundreds to thousands of potential ligands, hundreds of thousands of compds. can be screened per day. AS-MS is also compatible with complex mixts. of chem. diverse natural products in exts. of botanicals and fungi and microbial cultures, which often contain fluorophores and chromophores that can interfere with convention HTS. Unlike conventional HTS, AS-MS may be used to discover ligands binding to allosteric as well as orthosteric receptor sites, and AS-MS has been useful for discovering ligands to targets that are not easily incorporated into conventional HTS such as membrane-bound receptors.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvVymt7%252FN&md5=fa2b0774631da4dff58d761f277cefc1

  23. 25

    van
    Breemen, R. B.
    ; Huang, C. R.; Nikolic, D.; Woodbury, C. P.; Zhao, Y. Z.; Venton, D. L. Anal. Chem. 1997, 69, 21592164,  DOI: 10.1021/ac970132j

    [ACS Full Text ACS Full Text], [CAS], Google Scholar

    25

    Pulsed Ultrafiltration Mass Spectrometry: A New Method for Screening Combinatorial Libraries

    van Breemen, Richard B.; Huang, Chao-Ran; Nikolic, Dejan; Woodbury, Charles P.; Zhao, Yong-Zhong; Venton, Duane L.

    Analytical Chemistry
    (1997),
    69
    (11),
    2159-2164CODEN:
    ANCHAM;
    ISSN:0003-2700.

    (American Chemical Society)

    In response to the need for rapid screening of combinatorial libraries to identify new lead compds. during drug discovery, we have developed an online combination of ultrafiltration and electrospray mass spectrometry, called pulsed ultrafiltration mass spectrometry, which facilitates the identification of soln.-phase ligands in library mixts. that bind to soln.-phase receptors. After ligands contained in a library mixt. were bound to a macromol. receptor, e.g., human serum albumin or calf intestine adenosine deaminase, the ligand-receptor complexes were purified by ultrafiltration and then dissocd. using methanol to elute the ligands into the electrospray mass spectrometer for detection. Ligands with dissocn. consts. in the micromolar to nanomolar range were successfully bound, released, and detected using this method, including warfarin, salicylate, furosemide, and thyroxine binding to human serum albumin, and erythro-9-(2-hydroxy-3-nonyl)adenine binding to calf intestine adenosine deaminase. Repetitive bind-and-release expts. demonstrated that the receptor could be reused. Thus, pulsed ultrafiltration mass spectrometry was shown to provide a simple and powerful new method for the screening of combinatorial libraries in support of new drug discovery.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXivVertb8%253D&md5=2fbae2fe277ad721bba9477c12eef120

  24. 26

    Kaur, S.; McGuire, L.; Tang, D.; Dollinger, G.; Huebner, V. J. Protein Chem. 1997, 16, 505511,  DOI: 10.1023/A:1026369729393

    [Crossref], [PubMed], [CAS], Google Scholar

    26

    Affinity selection and mass spectrometry-based strategies to identify lead compounds in combinatorial libraries

    Kaur, Surinder; McGuire, Lisa; Tang, Dazhi; Dollinger, Gavin; Huebner, Verena

    Journal of Protein Chemistry
    (1997),
    16
    (5),
    505-511CODEN:
    JPCHD2;
    ISSN:0277-8033.

    (Plenum)

    The screening of diverse libraries of small mols. created by combinatorial synthetic methods is a recent development which has the potential to accelerate the identification of lead compds. in drug discovery. We developed a direct and rapid method to identify lead compds. in libraries involving affinity selection and mass spectrometry. In our strategy, the receptor or target mol. of interest is used to isolate the active components from the library phys., followed by direct structural identification of the active compds. bound to the target mol. by mass spectrometry. In a drug design strategy, structurally diverse libraries can be used for the initial identification of lead compds. Once lead compds. have been identified, libraries contg. compds. chem. similar to the lead compd. can be generated and used to optimize the binding characteristics. These strategies have also been adopted for more detailed studies of protein-ligand interactions.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXks1aqt7Y%253D&md5=5a83b161e45e515d18c85bf6687f086b

  25. 27

    Choi, Y.; van Breemen, R. B. Combin. Chem. High Throughput Screen. 2008, 11, 16,  DOI: 10.2174/138620708783398340

    [Crossref], [PubMed], [CAS], Google Scholar

    27

    Development of a screening assay for ligands to the estrogen receptor based on magnetic microparticles and LC-MS

    Choi, Yongsoo; van Breemen, Richard B.

    Combinatorial Chemistry & High Throughput Screening
    (2008),
    11
    (1),
    1-6CODEN:
    CCHSFU;
    ISSN:1386-2073.

    (Bentham Science Publishers Ltd.)

    A high throughput screening assay for the identification of ligands to pharmacol. significant receptors was developed based on magnetic particles contg. immobilized receptors followed by liq. chromatog.-mass spectrometry (LC-MS). This assay is suitable for the screening of complex mixts. such as botanical exts. For proof-of-principle, estrogen receptor-α (ER-α) and ER-β were immobilized on magnetic particles functionalized with aldehyde or carboxylic acid groups. Alternatively, biotinylated ER was immobilized onto streptavidin-derivitized magnetic particles. The ER that was immobilized using the streptavidin-biotin chem. showed higher activity than that immobilized on aldehyde or carboxylic acid functionalized magnetic particles. Immobilized ER was incubated with exts. of Trifolium pratense (red clover) or Humulus lupulus (hops). As a control for non-specific binding, each botanical ext. was incubated with magnetic particles contg. no ER. After magnetic sepn. of the particles contg. bound ligands from the unbound components in the ext., the particles were washed, ligands were released using methanol, and then the ligands were identified using LC-MS. The estrogens genistein and daidzein were identified in the red clover ext., and the estrogen 8-prenylnaringenin was identified in the hop ext. These screening results are consistent with those obtained using previous screening approaches.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXitVSgs78%253D&md5=50e9fbfab98b56836fe724e3f4024c09

  26. 28

    Rush, M. D.; Walker, E. M.; Prehna, G.; Burton, T.; van Breemen, R. B. J. Am. Soc. Mass Spectrom. 2017, 28, 479448,  DOI: 10.1007/s13361-016-1564-0

    [ACS Full Text ACS Full Text], [CAS], Google Scholar

    28

    Development of a Magnetic Microbead Affinity Selection Screen (MagMASS) Using Mass Spectrometry for Ligands to the Retinoid X Receptor-α

    Rush, Michael D.; Walker, Elisabeth M.; Prehna, Gerd; Burton, Tristesse; van Breemen, Richard B.

    Journal of the American Society for Mass Spectrometry
    (2017),
    28
    (3),
    479-485CODEN:
    JAMSEF;
    ISSN:1044-0305.

    (Springer)

    To overcome limiting factors in mass spectrometry-based screening methods such as automation while still facilitating the screening of complex mixts. such as botanical exts., magnetic microbead affinity selection screening (MagMASS) was developed. The screening process involves immobilization of a target protein on a magnetic microbead using a variety of possible chemistries, incubation with mixts. of mols. contg. possible ligands, a washing step that removes nonbound compds. while a magnetic field retains the beads in the microtiter well, and an org. solvent release step followed by LC-MS anal. Using retinoid X receptor-α (RXRα) as an example, which is a nuclear receptor and target for anti-inflammation therapy as well as cancer treatment and prevention, a MagMASS assay was developed and compared with an existing screening assay, pulsed ultrafiltration (PUF)-MS. Optimization of MagMASS involved evaluation of multiple protein constructs and several magnetic bead immobilization chemistries. The full-length RXRα construct immobilized with amylose beads provided optimum results. Addnl. enhancements of MagMASS were the application of 96-well plates to enable automation, use of UHPLC instead of HPLC for faster MS analyses, and application of metabolomics software for faster, automated data anal. Performance of MagMASS was demonstrated using mixts. of synthetic compds. and known ligands spiked into botanical exts.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVylt7zK&md5=3559923c001633605cd534903e08a653

  27. 29

    van
    Breemen, R. B.
    Curr. Trends Mass Spectrom. 2020, 18, 1825

    [CAS], Google Scholar

    29

    Affinity selection-mass spectrometry: defining the bioactive compounds in complex mixtures of natural products and combinatorial libraries

    van Breemen, Richard

    Current Trends in Mass Spectrometry
    (2020),
    18
    (1),
    18-25CODEN:
    CTMSAX
    ISSN:.

    (MultiMedia Pharma Sciences, LLC)

    Drug discovery from combinatorial libraries typically utilizes high-throughput screening of discreet compds., and the discovery of natural products with pharmacol. mechanisms of action relies on bioassay-guided fractionation. Both processes can be expedited through the application of affinity selection-mass spectrometry (AS-MS). AS-MS includes a family of MS-based affinity screening methods, including pulsed ultrafiltration (PUF)-AS-MS, size exclusion chromatog. AS-MS, and magnetic microbead affinity selection screening (MagMASS). All AS-MS approaches begin by incubating a pharmacol. important receptor with a mixt. of possible ligands, sepg. the ligand-receptor complexes from non-binding mols. (the approaches differ in this sepn. step), and then using LC-MS to characterize the affinity-extd. ligands. The speed, selectivity, and sensitivity of mass spectrometry and ultrahigh-pressure liq. chromatog. (UHPLC)-compatible MS ionization techniques, like electrospray and atm. pressure chem. ionization, make AS-MS ideal for characterizing ligands. Recent applications of AS-MS include allosteric as well as orthosteric ligand discovery, and finding ligands to membrane-bound proteins and RNA targets.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1CgtLzM&md5=8bbbd21bf76d9f5ea4ae51741cfae3fd

  28. 30

    Citti, C.; Linciano, P.; Panseri, S.; Vezzalini, F.; Forni, F.; Vandelli, M. A.; Cannazza, G. Front. Plant Sci. 2019, 10, 120,  DOI: 10.3389/fpls.2019.00120

    [Crossref], [PubMed], [CAS], Google Scholar

    30

    Cannabinoid Profiling of Hemp Seed Oil by Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

    Citti Cinzia; Linciano Pasquale; Vezzalini Francesca; Forni Flavio; Vandelli Maria Angela; Cannazza Giuseppe; Citti Cinzia; Cannazza Giuseppe; Panseri Sara

    Frontiers in plant science
    (2019),
    10
    (),
    120
    ISSN:1664-462X.

    Hemp seed oil is well known for its nutraceutical, cosmetic and pharmaceutical properties due to a perfectly balanced content of omega 3 and omega 6 polyunsaturated fatty acids. Its importance for human health is reflected by the success on the market of organic goods in recent years. However, it is of utmost importance to consider that its healthy properties are strictly related to its chemical composition, which varies depending not only on the manufacturing method, but also on the hemp variety employed. In the present work, we analyzed the chemical profile of ten commercially available organic hemp seed oils. Their cannabinoid profile was evaluated by a liquid chromatography method coupled to high-resolution mass spectrometry. Besides tetrahydrocannabinol and cannabidiol, other 30 cannabinoids were identified for the first time in hemp seed oil. The results obtained were processed according to an untargeted metabolomics approach. The multivariate statistical analysis showed highly significant differences in the chemical composition and, in particular, in the cannabinoid content of the hemp oils under investigation.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cfosFCjuw%253D%253D&md5=d84097c06eebd8798e0d84c30e4cc374

  29. 31

    Hazekamp, A.; Fischedick, J. T.; Díez, M. L.; Lubbe, A.; Ruhaak, R. L. In Comprehensive Natural Products II; Mander, L.; Lui, H.-W.; Eds.; Elsevier: Oxford, UK, 2010; pp 10331084.

  30. 32

    Pellesi, L.; Licata, M.; Verri, P.; Vandelli, D.; Palazzoli, F.; Marchesi, F.; Cainazzo, M. M.; Pini, L. A.; Guerzoni, S. Eur. J. Clin. Pharmacol. 2018, 74, 14271436,  DOI: 10.1007/s00228-018-2516-3

    [Crossref], [PubMed], [CAS], Google Scholar

    32

    Pharmacokinetics and tolerability of oral cannabis preparations in patients with medication overuse headache (MOH)-a pilot study

    Pellesi, Lanfranco; Licata, Manuela; Verri, Patrizia; Vandelli, Daniele; Palazzoli, Federica; Marchesi, Filippo; Cainazzo, Maria Michela; Pini, Luigi Alberto; Guerzoni, Simona

    European Journal of Clinical Pharmacology
    (2018),
    74
    (11),
    1427-1436CODEN:
    EJCPAS;
    ISSN:0031-6970.

    (Springer)

    Purpose: The recent release of a medical cannabis strain has given a new impulse for the study of cannabis in Italy. The National Health Service advises to consume medical cannabis by vaporizing, in decoction or oil form. This is the first study that explores the pharmacokinetics and tolerability of a single oral dose of cannabis as decoction (200 mL) or in olive oil (1 mL), as a first step to improve the prescriptive recommendations. Methods: This is a single-center, open-label, two-period crossover study designed to assess the pharmacokinetics and tolerability of oral cannabis administered to 13 patients with medication overuse headache (MOH). A liq. chromatog. tandem-mass spectrometry (LC-MS/MS) method was conducted for the quantification of THC, CBD, 11-OH-THC, THC-COOH, THC-COOH-glucuronide, THCA-A, and CBDA. Blood pressure, heart rate, and a short list of symptoms by numerical rating scale (NRS) were assessed. Results: Decoctions of cannabis showed high variability in cannabinoids content, compared to cannabis oil. For both prepns., THCA-A and CBDA were the most widely absorbed cannabinoids, while THC and CBD were less absorbed. The most important differences concern the bioavailability of THC, higher in oil (AUC0-24 7.44, 95% CI 5.19, 9.68) than in decoction (AUC0-24 3.34, 95% CI 2.07, 4.60), and the bioavailability of CBDA. No serious adverse events were reported. Conclusions: Cannabis decoction and cannabis oil showed different pharmacokinetic properties, as well as distinct consequences on patients. This study was performed in a limited no. of patients; future studies should be performed to investigate the clin. efficacy in larger populations.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht12qu7rI&md5=ce0b1bfcb797eb45df5f0447000d9a5e

  31. 34

    Sun, Y.; Gu, C.; Liu, X.; Liang, W.; Yao, P.; Bolton, J. L.; van Breemen, R. B. J. Am. Soc. Mass Spectrom. 2005, 16, 271279,  DOI: 10.1016/j.jasms.2004.11.002

    [ACS Full Text ACS Full Text], [CAS], Google Scholar

    34

    Ultrafiltration tandem mass spectrometry of estrogens for characterization of structure and affinity for human estrogen receptors

    Sun, Yongkai; Gu, Chungang; Liu, Xuemei; Liang, Wenzhong; Yao, Ping; Bolton, Judy L.; van Breemen, Richard B.

    Journal of the American Society for Mass Spectrometry
    (2005),
    16
    (2),
    271-279CODEN:
    JAMSEF;
    ISSN:1044-0305.

    (Elsevier Inc.)

    Although hormone replacement therapy (HRT) is used by post-menopausal women for the relief of menopausal symptoms and the potential redn. of osteoporosis, HRT also increases their risk of Alzheimer’s disease, stroke, breast cancer, and endometrial cancer. Since the majority of these effects are assocd. primarily with estrogen binding to only one of the estrogen receptors (ER), new assays are needed that can more efficiently evaluate ER-binding and identify ligands selective for ER-α and ER-β. HPLC-tandem mass spectrometry (LC-MS-MS) was combined with ultrafiltration as a new method to investigate the relative binding of compds. to the ERs and to evaluate the structures of these estrogens. Mixts. of estradiol and six equine estrogens, including equilin, equilenin, 8,9-dehydroestrone, and their 17β-hydroxyl derivs., were assayed simultaneously to det. their relative binding to human ER-α and ER-β. Estrogens contg. a 17β-OH group were found to have higher relative affinities for the estrogen receptors than their ketone analogs. In addn., 17β-EN showed selectivity for binding to ER-β over ER-α. The results were compared to the IC50 values obtained by using a conventional radiolabeled estradiol competitive binding assay. Finally, the utility of neg. ion electrospray tandem mass spectrometry for the unambiguous identification of these estrogen isomers was investigated. Several characteristic recyclization pathways during tandem mass spectrometry were identified, which might be useful for distinguishing related estrogens.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1Cqtbc%253D&md5=7a6f2771791223985049a0b6bfe0a8cd

  32. 35

    Zhao, Y. Z.; van Breemen, R. B.; Nikolic, D.; Huang, C. R.; Woodbury, C. P.; Schilling, A.; Venton, D. L. J. Med. Chem. 1997, 40, 40064012,  DOI: 10.1021/jm960729b

    [ACS Full Text ACS Full Text], [CAS], Google Scholar

    35

    Screening Solution-Phase Combinatorial Libraries Using Pulsed Ultrafiltration/Electrospray Mass Spectrometry

    Zhao, Yong-Zhong; van Breemen, Richard B.; Nikolic, Dejan; Huang, Chao-Ran; Woodbury, Charles P.; Schilling, Alexander; Venton, Duane L.

    Journal of Medicinal Chemistry
    (1997),
    40
    (25),
    4006-4012CODEN:
    JMCMAR;
    ISSN:0022-2623.

    (American Chemical Society)

    A method is described whereby a family of homologues is synthesized in a one-pot reaction, without isolation or purifn., and the reaction mixt. is screened using a competitive binding assay based on pulsed ultrafiltration/electrospray mass spectrometry (PUF/ESMS) to tentatively identify those derivs. having the highest affinity for a target receptor. As a model system to test this approach, a synthetic scheme designed to prep. a series of analogs of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), as diastereomeric mixts., was carried out. Pulsed ultrafiltration screening of the crude reaction mixt. against controls without protein detected protonated mols. corresponding to EHNA-type derivs. and three of its linear, alkyl homologues but did not show protonated mols. for an iso-Bu or benzylic EHNA deriv., suggesting the latter was inactive. To verify this conclusion, we prepd. E/THNA, the linear homologues, and the benzylic deriv. (each as a diastereomeric mixt.) and bioassayed them for their adenosine deaminase inhibition index ([I]/[S]0.5). The bioassay results for the individually synthesized analogs were in good agreement with that predicted by the obsd. relative ion enhancement in the PUF expts. Thus, the PUF protocol might be used as a general method to quickly provide direction to the chemist in search of drug candidates.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXnt1OgtLY%253D&md5=8f783c80c74421d1214a5831730df855

  33. 36

    Liu, J.; Burdette, J. E.; Xu, H.; Gu, C.; van Breemen, R. B.; Bhat, K. P.; Booth, N.; Constantinou, A. I.; Pezzuto, J. M.; Fong, H. H.; Farnsworth, N. R.; Bolton, J. L. J. Agric. Food Chem. 2001, 49, 24722479,  DOI: 10.1021/jf0014157

    [ACS Full Text ACS Full Text], [CAS], Google Scholar

    36

    Evaluation of Estrogenic Activity of Plant Extracts for the Potential Treatment of Menopausal Symptoms

    Liu, Jianghua; Burdette, Joanna E.; Xu, Haiyan; Gu, Chungang; van Breemen, Richard B.; Bhat, Krishna P. L.; Booth, Nancy; Constantinou, Andreas I.; Pezzuto, John M.; Fong, Harry H. S.; Farnsworth, Norman R.; Bolton, Judy L.

    Journal of Agricultural and Food Chemistry
    (2001),
    49
    (5),
    2472-2479CODEN:
    JAFCAU;
    ISSN:0021-8561.

    (American Chemical Society)

    Eight botanical prepns. that are commonly used for the treatment of menopausal symptoms were tested for estrogenic activity. Methanol exts. of red clover (Trifolium pratense L.), chasteberry (Vitex agnus-castus L.), and hops (Humulus lupulus L.) showed significant competitive binding to estrogen receptors α (ERα) and β (ERβ). With cultured Ishikawa (endometrial) cells, red clover and hops exhibited estrogenic activity as indicated by induction of alk. phosphatase (AP) activity and up-regulation of progesterone receptor (PR) mRNA. Chasteberry also stimulated PR expression, but no induction of AP activity was obsd. In S30 breast cancer cells, pS2 (presenelin-2), another estrogen-inducible gene, was up-regulated in the presence of red clover, hops, and chasteberry. Interestingly, exts. of Asian ginseng (Panax ginseng C.A. Meyer) and North American ginseng (Panax quinquefolius L.) induced pS2 mRNA expression in S30 cells, but no significant ER binding affinity, AP induction, or PR expression was noted in Ishikawa cells. Dong quai [Angelica sinensis (Oliv.) Diels] and licorice (Glycyrrhiza glabra L.) showed only weak ER binding and PR and pS2 mRNA induction. Black cohosh [Cimicifuga racemosa (L.) Nutt.] showed no activity in any of the above in vitro assays. Bioassay-guided isolation utilizing ER competitive binding as a monitor and screening using ultrafiltration LC-MS revealed that genistein was the most active component of red clover. Consistent with this observation, genistein was the most effective of four red clover isoflavones tested in the above in vitro assays. Therefore, estrogenic components of plant exts. can be identified using assays for estrogenic activity along with screening and identification of the active components using ultrafiltration LC-MS. These data suggest a potential use for some dietary supplements, ingested by human beings, in the treatment of menopausal symptoms.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXitFentLs%253D&md5=5433658c43caff5f53645c5c638efe94

  34. 37

    Rush, M. D.; Walker, E. M.; Burton, T.; van Breemen, R. B. J. Nat. Prod. 2016, 79, 28982902,  DOI: 10.1021/acs.jnatprod.6b00693

    [ACS Full Text ACS Full Text], [CAS], Google Scholar

    37

    Magnetic Microbead Affinity Selection Screening (MagMASS) of Botanical Extracts for Inhibitors of 15-Lipoxygenase

    Rush, Michael D.; Walker, Elisabeth M.; Burton, Tristesse; van Breemen, Richard B.

    Journal of Natural Products
    (2016),
    79
    (11),
    2898-2902CODEN:
    JNPRDF;
    ISSN:0163-3864.

    (American Chemical Society-American Society of Pharmacognosy)

    To expedite the identification of active natural products in complex mixts. such as botanical exts., a Magnetic Microbead Affinity Selection Screening (MagMASS) procedure was developed. This technique utilizes target proteins immobilized on magnetic beads for rapid bioaffinity isolation of ligands from complex mixts. A MagMASS method was developed and validated for 15-lipoxygenase. As a proof of concept, several North American prairie plants used medicinally by Native Americans were extd. with MeOH and screened. A hit from an ext. of Proserpinaca palustris, also known as mermaid weed, was flagged for further characterization using high-resoln. tandem mass spectrometry, dereplication, and identification using XCMS online. Through the application of high-resoln. product ion tandem mass spectrometry, comparison with natural product databases and confirmation using stds., the hit was identified as quercitrin, which is a known inhibitor of 15-lipoxygenase. The overall workflow of MagMASS is faster and more amendable to automation than alternative methods designed for screening botanical exts. or complex mixts. of combinatorial libraries.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslGqt7nE&md5=274bfaf440ae494133c764360dc6e937

  35. 38

    Wang, Q.; Zhang, Y.; Wu, L.; Niu, S.; Song, C.; Zhang, Z.; Lu, G.; Qiao, C.; Hu, Y.; Yuen, K. Y.; Wang, Q.; Zhou, H.; Yan, J.; Qi, J. Cell 2020, 181, 894904,  DOI: 10.1016/j.cell.2020.03.045

    [Crossref], [PubMed], [CAS], Google Scholar

    38

    Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2

    Wang, Qihui; Zhang, Yanfang; Wu, Lili; Niu, Sheng; Song, Chunli; Zhang, Zengyuan; Lu, Guangwen; Qiao, Chengpeng; Hu, Yu; Yuen, Kwok-Yung; Wang, Qisheng; Zhou, Huan; Yan, Jinghua; Qi, Jianxun

    Cell (Cambridge, MA, United States)
    (2020),
    181
    (4),
    894-904.e9CODEN:
    CELLB5;
    ISSN:0092-8674.

    (Cell Press)

    The recent emergence of a novel coronavirus (SARS-CoV-2) in China has caused significant public health concerns. Recently, ACE2 was reported as an entry receptor for SARS-CoV-2. In this study, we present the crystal structure of the C-terminal domain of SARS-CoV-2 (SARS-CoV-2-CTD) spike (S) protein in complex with human ACE2 (hACE2), which reveals a hACE2-binding mode similar overall to that obsd. for SARS-CoV. However, at. details at the binding interface demonstrate that key residue substitutions in SARS-CoV-2-CTD slightly strengthen the interaction and lead to higher affinity for receptor binding than SARS-RBD. Addnl., a panel of murine monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) against SARS-CoV-S1/receptor-binding domain (RBD) were unable to interact with the SARS-CoV-2 S protein, indicating notable differences in antigenicity between SARS-CoV and SARS-CoV-2. These findings shed light on the viral pathogenesis and provide important structural information regarding development of therapeutic countermeasures against the emerging virus.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmvFGrur4%253D&md5=188108e44f104860d4a2b83707ce7230

  36. 39

    Yi, C.; Sun, X.; Ye, J.; Ding, L.; Liu, M.; Yang, Z.; Lu, X.; Zhang, Y.; Ma, L.; Gu, W.; Qu, A.; Xu, J.; Shi, Z.; Ling, Z.; Sun, B. Cell Mol. Immunol. 2020, 17, 621630,  DOI: 10.1038/s41423-020-0458-z

    [Crossref], [PubMed], [CAS], Google Scholar

    39

    Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies

    Yi, Chunyan; Sun, Xiaoyu; Ye, Jing; Ding, Longfei; Liu, Meiqin; Yang, Zhuo; Lu, Xiao; Zhang, Yaguang; Ma, Liyang; Gu, Wangpeng; Qu, Aidong; Xu, Jianqing; Shi, Zhengli; Ling, Zhiyang; Sun, Bing

    Cellular & Molecular Immunology
    (2020),
    17
    (6),
    621-630CODEN:
    CMIEAO;
    ISSN:1672-7681.

    (Nature Research)

    Abstr.: Coronavirus disease 2019 (COVID-19), caused by the novel human coronavirus SARS-CoV-2, is currently a major threat to public health worldwide. The viral spike protein binds the host receptor angiotensin-converting enzyme 2 (ACE2) via the receptor-binding domain (RBD), and thus is believed to be a major target to block viral entry. Both SARS-CoV-2 and SARS-CoV share this mechanism. Here we functionally analyzed the key amino acid residues located within receptor binding motif of RBD that may interact with human ACE2 and available neutralizing antibodies. The in vivo expts. showed that immunization with either the SARS-CoV RBD or SARS-CoV-2 RBD was able to induce strong clade-specific neutralizing antibodies in mice; however, the cross-neutralizing activity was much weaker, indicating that there are distinct antigenic features in the RBDs of the two viruses. This finding was confirmed with the available neutralizing monoclonal antibodies against SARS-CoV or SARS-CoV-2. It is worth noting that a newly developed SARS-CoV-2 human antibody, HA001, was able to neutralize SARS-CoV-2, but failed to recognize SARS-CoV. Moreover, the potential epitope residues of HA001 were identified as A475 and F486 in the SARS-CoV-2 RBD, representing new binding sites for neutralizing antibodies. Overall, our study has revealed the presence of different key epitopes between SARS-CoV and SARS-CoV-2, which indicates the necessity to develop new prophylactic vaccine and antibody drugs for specific control of the COVID-19 pandemic although the available agents obtained from the SARS-CoV study are unneglectable.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXpsVOitLc%253D&md5=6386997d8670e5b9e863dc423839b07c

  37. 40

    Tegally, H.; Wilkinson, E.; Giovanetti, M.; Iranzadeh, A.; Fonseca, V.; Giandhari, J.; Doolabh, D.; Pillay, S.; San, E. J.; Msomi, N.; Mlisana, K.; von Gottberg, A.; Walaza, S.; Allam, M.; Ismail, A.; Mohale, T.; Glass, A. J.; Engelbrecht, S.; Van Zyl, G.; Preiser, W.; Petruccione, F.; Sigal, A.; Hardie, D.; Marais, G.; Hsiao, N. Y.; Korsman, S.; Davies, M. A.; Tyers, L.; Mudau, I.; York, D.; Maslo, C.; Goedhals, D.; Abrahams, S.; Laguda-Akingba, O.; Alisoltani-Dehkordi, A.; Godzik, A.; Wibmer, C. K.; Sewell, B. T.; Lourenço, J.; Alcantara, L. C. J.; Kosakovsky Pond, S. L.; Weaver, S.; Martin, D.; Lessells, R. J.; Bhiman, J. N.; Williamson, C.; de Oliveira, T. Nature 2021, 592, 438443,  DOI: 10.1038/s41586-021-03402-9

    [Crossref], [PubMed], [CAS], Google Scholar

    40

    Detection of a SARS-CoV-2 variant of concern in South Africa

    Tegally, Houriiyah; Wilkinson, Eduan; Giovanetti, Marta; Iranzadeh, Arash; Fonseca, Vagner; Giandhari, Jennifer; Doolabh, Deelan; Pillay, Sureshnee; San, Emmanuel James; Msomi, Nokukhanya; Mlisana, Koleka; von Gottberg, Anne; Walaza, Sibongile; Allam, Mushal; Ismail, Arshad; Mohale, Thabo; Glass, Allison J.; Engelbrecht, Susan; Van Zyl, Gert; Preiser, Wolfgang; Petruccione, Francesco; Sigal, Alex; Hardie, Diana; Marais, Gert; Hsiao, Nei-yuan; Korsman, Stephen; Davies, Mary-Ann; Tyers, Lynn; Mudau, Innocent; York, Denis; Maslo, Caroline; Goedhals, Dominique; Abrahams, Shareef; Laguda-Akingba, Oluwakemi; Alisoltani-Dehkordi, Arghavan; Godzik, Adam; Wibmer, Constantinos Kurt; Sewell, Bryan Trevor; Lourenco, Jose; Alcantara, Luiz Carlos Junior; Kosakovsky Pond, Sergei L.; Weaver, Steven; Martin, Darren; Lessells, Richard J.; Bhiman, Jinal N.; Williamson, Carolyn; de Oliveira, Tulio

    Nature (London, United Kingdom)
    (2021),
    592
    (7854),
    438-443CODEN:
    NATUAS;
    ISSN:0028-0836.

    (Nature Portfolio)

    Continued uncontrolled transmission of SARS-CoV-2 in many parts of the world is creating conditions for substantial evolutionary changes to the virus. We describe a newly arisen lineage of SARS-CoV-2 (designated 501Y.V2; also known as B.1.351 or 20H) that is defined by 8 mutations in the spike protein, including 3 substitutions (K417N, E484K, and N501Y) at residues in its receptor-binding domain that may have functional importance. This lineage was identified in South Africa after the 1st wave of the epidemic in a severely affected metropolitan area (Nelson Mandela Bay) that is located on the coast of the Eastern Cape province. This lineage spread rapidly, and became dominant in Eastern Cape, Western Cape, and KwaZulu-Natal provinces within weeks. Although the full import of the mutations is yet to be detd., the genomic data, which show rapid expansion and displacement of other lineages in several regions, suggest that this lineage is assocd. with a selection advantage that most plausibly results from increased transmissibility or immune escape.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXnslyju7Y%253D&md5=5c506e6dfc5abc7ed82669c3214a286b

  38. 41

    Wakshlag, J. J.; Schwark, W. S.; Deabold, K. A.; Talsma, B. N.; Cital, S.; Lyubimov, A.; Iqbal, A.; Zakharov, A. Front. Vet. Sci. 2020, 7, 505,  DOI: 10.3389/fvets.2020.00505

    [Crossref], [PubMed], [CAS], Google Scholar

    41

    Pharmacokinetics of Cannabidiol, Cannabidiolic Acid, Δ9-Tetrahydrocannabinol, Tetrahydrocannabinolic Acid and Related Metabolites in Canine Serum After Dosing With Three Oral Forms of Hemp Extract

    Wakshlag Joseph J; Schwark Wayne S; Deabold Kelly A; Talsma Bryce N; Cital Stephen; Lyubimov Alex; Iqbal Asif; Zakharov Alexander

    Frontiers in veterinary science
    (2020),
    7
    (),
    505
    ISSN:2297-1769.

    Cannabidiol (CBD)-rich hemp extract use is increasing in veterinary medicine with little examination of serum cannabinoids. Many products contain small amounts of Δ9-tetrahydrocannabinol (THC), and precursor carboxylic acid forms of CBD and THC known as cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA). Examination of the pharmacokinetics of CBD, CBDA, THC, and THCA on three oral forms of CBD-rich hemp extract that contained near equal amounts of CBD and CBDA, and minor amounts (<0.3% by weight) of THC and THCA in dogs was performed. In addition, we assess the metabolized psychoactive component of THC, 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and CBD metabolites 7-hydroxycannabidiol (7-OH-CBD) and 7-nor-7-carboxycannabidiol (7-COOH-CBD) to better understand the pharmacokinetic differences between three formulations regarding THC and CBD, and their metabolism. Six purpose-bred female beagles were utilized for study purposes, each having an initial 7-point, 24-h pharmacokinetic study performed using a dose of 2 mg/kg body weight of CBD/CBDA (~1 mg/kg CBD and ~1 mg/kg CBDA). Dogs were then dosed every 12 h for 2 weeks and had further serum analyses at weeks 1 and 2, 6 h after the morning dose to assess serum cannabinoids. Serum was analyzed for each cannabinoid or cannabinoid metabolite using liquid chromatography and tandem mass spectroscopy (LC-MS/MS). Regardless of the form provided (1, 2, or 3) the 24-h pharmacokinetics for CBD, CBDA, and THCA were similar, with only Form 2 generating enough data above the lower limit of quantitation to assess pharmacokinetics of THC. CBDA and THCA concentrations were 2- to 3-fold higher than CBD and THC concentrations, respectively. The 1- and 2-week steady-state concentrations were not significantly different between the two oils or the soft chew forms. CBDA concentrations were statistically higher with Form 2 than the other forms, showing superior absorption/retention of CBDA. Furthermore, Form 1 showed less THCA retention than either the soft chew Form 3 or Form 2 at weeks 1 and 2. THC was below the quantitation limit of the assay for nearly all samples. Overall, these findings suggest CBDA and THCA are absorbed or eliminated differently than CBD or THC, respectively, and that a partial lecithin base provides superior absorption and/or retention of CBDA and THCA.

    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&c