Affinity selection-mass spectrometry in the discovery of anti-SARS-CoV-2 compounds.

Small molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which is the cause of the COVID-19 pandemic. To expedite the discovery of lead compounds for development, assays have been developed based on affinity selection-mass spectrometry (AS-MS), which enables the rapid screening of mixtures such as combinatorial libraries and extracts of botanicals or other sources of natural products. AS-MS assays have been used to find ligands to the SARS-CoV-2 spike protein for inhibition of cell entry as well as to the 3-chymotrypsin-like cysteine protease (3CLpro) and the RNA-dependent RNA polymerase complex constituent Nsp9, which are targets for inhibition of viral replication. The AS-MS approach of magnetic microbead affinity selection screening has been used to discover high-affinity peptide ligands to the spike protein as well as the hemp cannabinoids cannabidiolic acid and cannabigerolic acid, which can prevent cell infection by SARS-CoV-2. Another AS-MS method, native mass spectrometry, has been used to discover that the flavonoids baicalein, scutellarein, and ganhuangenin, can inhibit the SARS-CoV-2 protease 3CLpro. Native mass spectrometry has also been used to find an ent-kaurane natural product, oridonin, that can bind to the viral protein Nsp9 and interfere with RNA replication. These natural lead compounds are under investigation for the development of therapeutic agents to prevent or treat SARS-CoV-2 infection.

Pharmacokinetics and Early Tumor Response to Conventional Transarterial Chemoembolization with Sorafenib and Doxorubicin in a VX2 Rabbit Tumor Model.

PURPOSE: To investigate the pharmacokinetics (PK) and early effects of conventional transarterial chemoembolization (TACE) using sorafenib and doxorubicin on tumor necrosis, hypoxia markers, and angiogenesis in a rabbit VX2 liver tumor model. MATERIALS AND METHODS: VX2 tumor-laden New Zealand White rabbits (N = 16) were divided into 2 groups: 1 group was treated with hepatic arterial administration of ethiodized oil and doxorubicin emulsion (DOX-TACE), and the other group was treated with ethiodized oil, sorafenib, and doxorubicin emulsion (SORA-DOX-TACE). Animals were killed within 3 days of the procedure. Levels of sorafenib and doxorubicin were measured in blood, tumor, and adjacent liver using mass spectrometry. Tumor necrosis was determined by histopathological examination. Intratumoral hypoxia-inducible factor (HIF) 1α, vascular endothelial growth factor (VEGF), and microvessel density (MVD) were determined by immunohistochemistry. RESULTS: The median intratumoral concentration of sorafenib in the SORA-DOX-TACE group was 17.7 µg/mL (interquartile range [IQR], 7.42-33.5 µg/mL), and its maximal plasma concentration (Cmax) was 0.164 µg/mL (IQR, 0.0798-0.528 µg/mL). The intratumoral concentration and Cmax of doxorubicin were similar between the groups: 4.08 µg/mL (IQR, 3.18-4.79 µg/mL) and 0.677 µg/mL (IQR, 0.315-1.23 µg/mL), respectively, in the DOX-TACE group and 1.68 µg/mL (IQR, 0.795-4.08 µg/mL) and 0.298 µg/mL (IQR, 0.241-0.64 µg/mL), respectively, in the SORA-DOX-TACE group. HIF-1α expression was increased in the SORA-DOX-TACE group than in the DOX-TACE group. Tumor volume, tumor necrosis, VEGF expression, and MVD were similar between the 2 groups. CONCLUSIONS: The addition of sorafenib to DOX-TACE delivered to VX2 liver tumors resulted in high intratumoral and low systemic concentrations of sorafenib without altering the PK of doxorubicin.

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

As a complement to vaccines, small-molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, which cause COVID-19. Affinity selection-mass spectrometry was used for the discovery of botanical ligands to the SARS-CoV-2 spike protein. Cannabinoid acids from hemp (Cannabis sativa) were found to be allosteric as well as orthosteric ligands with micromolar affinity for the spike protein. In follow-up virus neutralization assays, cannabigerolic acid and cannabidiolic acid prevented infection of human epithelial cells by a pseudovirus expressing the SARS-CoV-2 spike protein and prevented entry of live SARS-CoV-2 into cells. Importantly, cannabigerolic acid and cannabidiolic acid were equally effective against the SARS-CoV-2 alpha variant B.1.1.7 and the beta variant B.1.351. Orally bioavailable and with a long history of safe human use, these cannabinoids, isolated or in hemp extracts, have the potential to prevent as well as treat infection by SARS-CoV-2.

Drug discovery from natural products using affinity selection-mass spectrometry.

As a starting point for drug discovery, affinity selection-mass spectrometry (AS-MS) is ideal for the discovery of lead compounds from chemically diverse sources such as botanical, fungal and microbial extracts. Based on binding interactions between macromolecular receptors and ligands of low molecular mass, AS-MS enables the rapid isolation of pharmacologically active small molecules from complex mixtures for mass spectrometric characterization and identification. Unlike conventional high-throughput screening, AS-MS requires no radiolabels, no UV or fluorescent chromophores, and is compatible with all classes of receptors, enzymes, incubation buffers, cofactors, and ligands. The most successful types of AS-MS include pulsed ultrafiltration (PUF) AS-MS, size exclusion chromatography (SEC) AS-MS, and magnetic microbead affinity selection screening (MagMASS), which differ in their approaches for separating the ligand-receptor complexes from the non-binding compounds in mixtures. After affinity isolation, the ligand(s) from the mixture are characterized using high resolution UHPLC-MS and tandem mass spectrometry. Based on these elemental composition and structural data, the identities of the lead compounds are determined by searching on-line databases for known natural products and by comparison with standards. The structures of novel natural products are determined using a combination of spectroscopic techniques including two-dimensional NMR and MS.

Analysis of lanthionine ketimine ethyl ester in mouse serum, whole blood and tissues using ultrahigh-pressure liquid chromatography/tandem mass spectrometry.

RATIONALE: Preclinical studies in the search for treatments for several neurodegenerative diseases have identified lanthionine ketimine (LK) and its monoethyl ester derivative (LKE) as potential candidates. An ultrahigh-pressure liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) assay was developed to evaluate bioavailability by measuring these compounds in mouse serum, whole blood and brain tissue. METHODS: Following administration of LKE to mice for 3 days in chow at 300 ppm, the animals were sacrificed, and LKE was extracted from serum, whole blood and brain tissues through protein precipitation using cold methanol. To enhance chromatographic separation and electrospray ionization, LK was methylated using diazomethane. Separations were carried out using C18 reversed-phase UHPLC, and quantitative measurements were obtained using on-line triple-quadruple mass spectrometry with positive ion electrospray ionization, collision-induced dissociation and selected reaction monitoring. Tolbutamide was used as internal standard. RESULTS: LKE showed good recovery ranging from 77-90% in serum and 82-88% in brain tissue. An eight-point standard curve ranging from 0.005 to 4.6 µM was linear (R2 0.998). The average LKE detected in mouse serum was 277.42 nM, while the concentration in whole blood was 38 nM. Neither LK nor LKE was detected in brain tissues. CONCLUSIONS: A rapid quantitative method to measure LKE in mouse serum, whole blood and brain tissues using UHPLC/MS/MS was developed and validated following FDA guidelines. This method is suitable for bioavailability and pharmacokinetic studies.

Paclitaxel Biosynthesis: Adenylation and Thiolation Domains of an NRPS TycA PheAT Module Produce Various Arylisoserine CoA Thioesters.

Structure-activity relationship studies show that the phenylisoserinyl moiety of paclitaxel (Taxol) is largely necessary for the effective anticancer activity. Several paclitaxel analogues with a variant isoserinyl side chain have improved pharmaceutical properties versus those of the parent drug. To produce the isoserinyl CoAs as intermediates needed for enzyme catalysis on a semibiosynthetic pathway to paclitaxel analogues, we repurposed the adenylation and thiolation domains (Phe-AT) of a nonribosomal peptide synthetase (TycA) so that they would function as a CoA ligase. Twenty-eight isoserine analogue racemates were synthesized by an established procedure based on the Staudinger [2+2] cycloaddition reaction. Phe-AT converted 16 substituted phenylisoserines, one ß-(heteroaryl)isoserine, and one ß-(cyclohexyl)isoserine to their corresponding isoserinyl CoAs. We imagine that these CoA thioesters can likely serve as linchpin biosynthetic acyl donors transferred by a 13-O-acyltransferase to a paclitaxel precursor baccatin III to make drug analogues with better efficacy. It was also interesting to find that an active site mutant [Phe-AT (W227S)] turned over 2-pyridylisoserine and the sterically demanding p-methoxyphenylisoserine substrates to their CoA thioesters, while Phe-AT did not. This mutant is promising for further development to make 3-fluoro-2-pyridylisoserinyl CoA, a biosynthetic precursor of the oral pharmaceutical tesetaxel used for gastric cancers.

Chemical Standardization of Milk Thistle (Silybum marianum L.) Extract Using UHPLC-MS/MS and the Method of Standard Addition.

Extracts prepared from the seeds of the medicinal plant milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)] are widely used as dietary supplements due to anti-inflammatory, antitumor, and hepatoprotective effects. Called silymarin, the main components of lipophilic extracts of milk thistle seeds are flavonoids and flavonolignans including silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, taxifolin, and 2,3-dehydrosilybins. The aim of this study was to develop a method based on UHPLC-MS/MS for the chemical authentication and standardization of milk thistle silymarin. Validation included the method of standard addition to account for the lack of a blank matrix. Potential matrix effects were investigated by analyzing silymarin standards dissolved only in the initial UHPLC mobile phase. Measurements of six flavonolignans and taxifolin in the milk thistle extract using UHPLC-MS/MS with standard addition or external standard calibration produced similar results for all analytes except silydianin and 2,3-dehydrosilybin B, which showed significant peak enhancement during negative ion electrospray due to botanical matrix effects. The UHPLC-MS/MS-based method of standard addition requires <10 min per injection and is suitable for the standardization of silymarin from milk thistle in support of preclinical and clinical studies of safety and efficacy.

Cannabinoid residuals in tissues of lambs fed spent hemp biomass and consumer's exposure assessment.

Spent hemp biomass (SHB) contains trace amounts of cannabinoids, including Δ9-tetrahydrocannabinol (Δ9-THC), that may accumulate in the tissues of animals consuming SHB. We measured cannabinoid residues in the liver, adipose tissue, and muscle of finishing lambs fed either 10% or 20% SHB for 8 weeks, or 4 weeks followed by 4 weeks SHB withdrawal. We detected multiple cannabinoids in the liver at a similar proportion to the SHB. However, CBD and Δ9-THC were enriched >20-fold in the adipose and muscle, compared to their proportion in SHB. The highest concentration of Δ9-THC was detected in adipose tissue and was 7.4-times higher than in muscle. Most cannabinoids were undetectable in tissues after 4 weeks of clearance. The consumers' exposure assessment on Δ9-THC revealed tissue levels of total THC (THCA+Δ9-THC) that exceed the acute reference dose of 1 µg/kg BW across population groups. When consuming meat from the lambs fed 10% and 20% SHB, the maximum total THC exposure was 2.03 and 7.32 µg/kg BW, respectively, equal to or below the Lowest Observed Adverse Effect Level of 36 µg/kg BW, the No Observed Adverse Effect Level of 12 µg/kg BW or a tolerable dose intake of 7 µg/kg BW.

Short-term treatment with cholestyramine increases long-acting anticoagulant rodenticide clearance from rabbits without affecting plasma vitamin K1 levels or blood coagulation.

Administration of high-dose vitamin K1 (VK1) overcomes coagulopathy and bleeding elicited by acute poisoning with long-acting anticoagulant rodenticides (LAARs). However, long-term (months) treatment is required due to long LAAR biological half-lives that may lead to poor compliance and recurrent coagulopathy. The half-lives of LAARs are extended by slow metabolism, and similar to warfarin, are thought to undergo enterohepatic recirculation. We now show that treatment with the bile acid sequestrant cholestyramine (CSA) administered concomitantly with VK1 decreases plasma LAAR levels and increases LAAR fecal excretion. Daily CSA treatment for 14 days did not reduce plasma VK1 levels, or increase prothrombin time. Collectively, these data show that CSA accelerates LAAR clearance from rabbits without adverse effects on VK1 anticoagulation, and could provide an additional therapeutic option for treatment of LAAR poisoning.

Advances in Magnetic Microbead Affinity Selection Screening: Discovery of Natural Ligands to the SARS-CoV-2 Spike Protein.

Affinity selection-mass spectrometry, which includes magnetic microbead affinity selection-screening (MagMASS), is ideal for the discovery of ligands in complex mixtures that bind to pharmacological targets. Therapeutic agents are needed to prevent or treat COVID-19, which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection of human cells by SARS-CoV-2 involves binding of the virus spike protein subunit 1 (S1) to the human cell receptor angiotensin converting enzyme-2 (ACE2). Like antibodies, small molecules have the potential to block the interaction of the viral S1 protein with human ACE2 and prevent SARS-CoV-2 infection. Therefore, a MagMASS assay was developed for the discovery of ligands to the S1 protein. Unlike previous MagMASS approaches, this new assay used robotics for 5-fold enhancement of throughput and sensitivity. The assay was validated using the SBP-1 peptide, which is identical to the ACE2 amino acid sequence recognized by the S1 protein, and then applied to the discovery of natural ligands from botanical extracts. Small molecule ligands to the S1 protein were discovered in extracts of the licorice species, Glycyrrhiza inflata. In particular, the licorice ligand licochalcone A was identified through dereplication and comparison with standards using HPLC with high-resolution tandem mass spectrometry.

Conventional versus drug-eluting embolic transarterial chemoembolization with doxorubicin: comparative drug delivery, pharmacokinetics, and treatment response in a rabbit VX2 tumor model.

The purpose of this study was to compare intra-tumoral drug delivery, pharmacokinetics, and treatment response after doxorubicin (DOX) conventional (c-) versus drug-eluting embolic (DEE-) transarterial chemoembolization (TACE) in a rabbit VX2 liver tumor model. Twenty-four rabbits with solitary liver tumors underwent c-TACE (n = 12) (1:2 water-in-oil emulsion, 0.6 mL volume, 2 mg DOX) or DEE-TACE (n = 12) (130,000 70-150 µm 2 mg DOX-loaded microspheres). Systemic, intra-tumoral, and liver DOX levels were measured using mass spectrometry up to 7-day post-procedure. Intra-tumoral DOX distribution was quantified using fluorescence imaging. Percent tumor necrosis was quantified by a pathologist blinded to treatment group. Lobar TACE was successfully performed in all cases. Peak concentration (CMAX, µg/mL) for plasma, tumor tissue, and liver were 0.666, 4.232, and 0.270 for c-TACE versus 0.103, 8.988, and 0.610 for DEE-TACE. Area under the concentration versus time curve (AUC, µg/mL ∗ min) for plasma, tumor tissue, and liver were 18.3, 27,078.8, and 1339.1 for c-TACE versus 16.4, 26,204.8, and 1969.6 for DEE-TACE. A single dose of intra-tumoral DOX maintained cytotoxic levels through 7-day post-procedure for both TACE varieties, with a half-life of 1.8 (c-TACE) and 0.8 (DEE-TACE) days. Tumor-to-normal liver DOX ratio was high (c-TACE, 20.2; DEE-TACE, 13.3). c-TACE achieved significantly higher DOX coverage of tumor vs. DEE-TACE (10.8% vs. 2.3%; P = 0.003). Percent tumor necrosis was similar (39% vs. 37%; P = 0.806). In conclusion, in a rabbit VX2 liver tumor model, both c-TACE and DEE-TACE achieved tumoricidal intra-tumoral DOX levels and high tumor-to-normal liver drug ratios, though c-TACE resulted in significantly greater tumor coverage.

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

Invented to address the high-throughput screening (HTS) demands of combinatorial chemistry, affinity selection-mass spectrometry (AS-MS) utilizes binding interactions between ligands and receptors to isolate pharmacologically active compounds from mixtures of small molecules 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 separated from nonbinding compounds in the mixture. These are pulsed ultrafiltration (PUF) AS-MS, size exclusion chromatography (SEC) AS-MS, and magnetic microbead affinity selection screening (MagMASS). PUF and SEC AS-MS are solution-phase screening approaches, and MagMASS uses receptors immobilized on magnetic microbeads. Because pools of compounds are screened using AS-MS, each containing hundreds to thousands of potential ligands, hundreds of thousands of compounds can be screened per day. AS-MS is also compatible with complex mixtures of chemically diverse natural products in extracts 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.

Single Laboratory Validation of UHPLC-MS/MS Assays for Six Milk Thistle Flavonolignans in Human Serum.

BACKGROUND: Extracts of milk thistle, Silybum marianum (L.) Gaertn., are used as dietary supplements for their hepatoprotective, anti-inflammatory, and anti-tumor activities. OBJECTIVE: An assay based on UHPLC-MS/MS was developed and validated for the quantitative analysis of six major milk thistle flavonolignans extracted from human serum. METHODS: Ethyl acetate containing 0.1% formic acid was used to extract flavonolignans from human serum. A 10-min UHPLC-MS/MS method using selected reaction ion monitoring was developed for measuring extracts for silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin. RESULTS: The quantitative method was validated with respect to selectivity, specificity, accuracy, linearity, precision, LOD, and LLOQ. Extraction efficiency for the quality control standards at LLOQ, low, medium, and high concentrations ranged between 81% and 109%, and the calibration curves were linear (R2 > 0.997) for all flavonolignans. The method precision was determined using coefficients of variation, which were <15%. The method accuracy was assessed using percent relative error which was <15%. CONCLUSIONS: The UHPLC-MS/MS assay is fast, precise, sensitive, selective, accurate, and useful for the analysis of milk thistle flavonolignans in human serum. HIGHLIGHTS: The UHPLC-MS/MS assay is suitable for rapid quantitative analysis of milk thistle flavonolignans in human serum.

Single-Laboratory Validation of UHPLC-MS/MS Assays for Red Clover Isoflavones in Human Serum and Dietary Supplements.

BACKGROUND: Extracts of red clover (Trifolium pratense L.) containing estrogenic and pro-estrogenic isoflavones are used in dietary supplements primarily for the management of menopausal symptoms in women. OBJECTIVE: A UHPLC-MS/MS assay was developed and validated for the quantitative analysis of the six major red clover isoflavones in dietary supplements and in human serum in support of clinical trials. METHODS: Enzymatic deconjugation of isoflavone glucuronides and sulfate conjugates in human serum specimens was carried out followed by protein precipitation. Isoflavones in red clover dietary supplements were acid hydrolyzed to release aglycons from glycosides. UHPLC separations (< 4 min) were combined with MS/MS using collision-induced dissociation, selective reaction monitoring and deuterated internal standards to measure biochanin A, formononetin, daidzein, genistein, irilone, and prunetin. RESULTS: The method was validated with respect to selectivity, specificity, accuracy, linearity, precision, LOD, and LOQ. The calibration curves for all analytes were linear (R2 > 0.998). The mean recovery for low-, medium- and high-quality control standards ranged between 80% and 108%. The precision of the method was assessed using coefficients of variation, which were <15%. CONCLUSIONS: The UHPLC-MS/MS method is fast, precise, sensitive, selective, accurate, and applicable to the quantitative analysis of red clover isoflavones in different matrices. HIGHLIGHTS: This validated UHPLC-MS/MS assay is applicable to the rapid quantitative analysis of red clover isoflavones in human serum and in dietary supplements.

Taxol biosynthesis: tyrocidine synthetase A catalyzes the production of phenylisoserinyl CoA and other amino phenylpropanoyl thioesters.

In Taxus plants the biosynthesis of the pharmaceutical paclitaxel includes the transfer of ß-amino phenylpropanoyls from coenzyme A to the diterpenoid baccatin III by an acyl CoA-dependent acyltransferase. Several enzymes on the pathway are known, yet a few remain unidentified, including the putative ligase that biosynthesizes key ß-amino phenylpropanoyl CoAs. The multienzyme, nonribosomal peptide synthetase that produces tyrocidines contains a tridomain starter module tyrocidine synthetase A that normally activates (S)-α-Phe to an adenylate anhydride in the adenylation domain. The Phe moiety is then thioesterified by the pendent pantetheine of the adjacent thiolation domain. Herein, the adenylation domain was found to function as a CoA ligase, making α-, ß-phenylalanyl, and phenylisoserinyl CoA. The latter two are substrates of a phenylpropanoyltransferase on the biosynthetic pathway of the antimitotic paclitaxel.