Structure-based identification of a G protein-biased allosteric modulator of cannabinoid receptor CB1.

Cannabis sativa is known for its therapeutic benefit in various diseases including pain relief by targeting cannabinoid receptors. The primary component of cannabis, Δ9-tetrahydrocannabinol (THC), and other agonists engage the orthosteric site of CB1, activating both Gi and ß-arrestin signaling pathways. The activation of diverse pathways could result in on-target side effects and cannabis addiction, which may hinder therapeutic potential. A significant challenge in pharmacology is the design of a ligand that can modulate specific signaling of CB1. By leveraging insights from the structure-function selectivity relationship (SFSR), we have identified Gi signaling-biased agonist-allosteric modulators (ago-BAMs). Further, two cryoelectron microscopy (cryo-EM) structures reveal the binding mode of ago-BAM at the extrahelical allosteric site of CB1. Combining mutagenesis and pharmacological studies, we elucidated the detailed mechanism of ago-BAM-mediated biased signaling. Notably, ago-BAM CB-05 demonstrated analgesic efficacy with fewer side effects, minimal drug toxicity and no cannabis addiction in mouse pain models. In summary, our finding not only suggests that ago-BAMs of CB1 provide a potential nonopioid strategy for pain management but also sheds light on BAM identification for GPCRs.

Discovery and structure-activity relationship studies of novel α-ketoamide derivatives targeting the SARS-CoV-2 main protease.

The SARS-CoV-2 main protease (Mpro, also named 3CLpro) is a promising antiviral target against COVID-19 due to its functional importance in viral replication and transcription. Herein, we report the discovery of a series of α-ketoamide derivatives as a new class of SARS-CoV-2 Mpro inhibitors. Structure-activity relationship (SAR) of these compounds was analyzed, which led to the identification of a potent Mpro inhibitor (27h) with an IC50 value of 10.9 nM. The crystal structure of Mpro in complex with 27h revealed that α-ketoamide warhead covalently bound to Cys145s of the protease. In an in vitro antiviral assay, 27h showed excellent activity with an EC50 value of 43.6 nM, comparable to the positive control, Nirmatrelvir. This compound displayed high target specificity for Mpro against human proteases and low toxicity. It also possesses favorable pharmacokinetic properties. Overall, compound 27h could be a promising lead compound for drug discovery targeting SARS-CoV-2 Mpro and deserves further in-depth studies.

A new generation Mpro inhibitor with potent activity against SARS-CoV-2 Omicron variants.

Emerging SARS-CoV-2 variants, particularly the Omicron variant and its sublineages, continually threaten the global public health. Small molecule antivirals are an effective treatment strategy to fight against the virus. However, the first-generation antivirals either show limited clinical efficacy and/or have some defects in pharmacokinetic (PK) properties. Moreover, with increased use of these drugs across the globe, they face great pressure of drug resistance. We herein present the discovery and characterization of a new generation antiviral drug candidate (SY110), which is a potent and selective inhibitor of SARS-CoV-2 main protease (Mpro). This compound displayed potent in vitro antiviral activity against not only the predominant SARS-CoV-2 Omicron sublineage BA.5, but also other highly pathogenic human coronaviruses including SARS-CoV-1 and MERS-CoV. In the Omicron-infected K18-hACE2 mouse model, oral treatment with SY110 significantly lowered the viral burdens in lung and alleviated the virus-induced pathology. Importantly, SY110 possesses favorable PK properties with high oral drug exposure and oral bioavailability, and also an outstanding safety profile. Furthermore, SY110 exhibited sensitivity to several drug-resistance Mpro mutations. Collectively, this investigation provides a promising new drug candidate against Omicron and other variants of SARS-CoV-2.

Discovery of 3-phenyl-1,2,4-oxadiazole derivatives as a new class of SARS-CoV-2 main protease inhibitors.

The ongoing COVID-19 pandemic has led to massive infections and deaths and caused tremendous grief among the people. Although vaccines have played an important role in fighting COVID-19, the situation that the protective effect of current vaccines significantly decreases against mutated strains reminds us of the pressing need for developing effective antiviral therapeutics. The main protease (Mpro) is a key enzyme for SARS-CoV-2 viral replication and transcription and an attractive target for drug development. In this research, we report a new series of Mpro inhibitors containing 3-phenyl-1,2,4-oxadiazole. Structure-activity relationship (SAR) studies led to the discovery of the most active compound, 16d, which showed an IC50 value of 5.27 ± 0.26 µM. Collectively, we obtained a new small molecular inhibitor targeting SARS-CoV-2 Mpro, which contains a new scaffold. This compound could be taken as a lead compound for subsequent drug discovery against SARS-CoV-2.

Discovery of 2-vinyl-10H-phenothiazine derivatives as a class of ferroptosis inhibitors with minimal human Ether-a-go-go related gene (hERG) activity for the treatment of DOX-induced cardiomyopathy.

Ferroptosis was an iron-dependent, nonapoptotic form of regulated cell death. In our previous study, we discovered a potent ferroptosis inhibitor with phenothiazine scaffold (1), but subsequent investigation showed that this compound had potent hERG binding affinity. Herein, we report the discovery of a series of 2-vinyl-10H-phenothiazine derivatives as new class of ferroptosis inhibitors. Structure-activity relationship (SAR) analyses led to the identification of compound 7j, which exhibited significantly reduced hERG inhibition (IC50 > 30 µM) while maintaining high ferroptosis inhibitory activity (EC50 = 0.001 µM on the erastin-induced HT1080 cell ferroptosis model). Further studies confirmed 7j acted as a ROS scavenger and could relieve DOX-induced cardiomyopathy. 7j also displayed favorable pharmacokinetic properties and exhibited no obvious toxicity in vivo and vitro. Overall, this study provides a promising lead compound for drug discovery targeting ferroptosis.

Discovery of a potent, selective and cell active inhibitor of m6A demethylase ALKBH5.

AlkB homolog 5 (ALKBH5) is an RNA m6A demethylase involved in the regulation of genes transcription, translation and metabolism and has been considered as a promising therapeutic target for various human diseases, especially cancers. However, there is still a lack of potent and selective ALKBH5 inhibitors. Herein, we report a new class of ALKBH5 inhibitors containing the 1-aryl-1H-pyrazole scaffold, which were obtained through fluorescence polarization-based screening, structural optimization and structure-activity relationship analysis. Among these compounds, 20m was the most potent one, which showed an IC50 value of 0.021 µM in fluorescence polarization assay. Compound 20m exhibited high selectivity towards ALKBH5 versus FTO as well as other AlkB subfamily members, indicating good selectivity for ALKBH5. Cellular thermal shift assay (CETSA) analysis showed that 20m could efficiently stabilize ALKBH5 in HepG2 cells. Dot blot assay demonstrated that 20m could increase m6A level in intact cells. Collectively, 20m is a potent, selective and cell active ALKBH5 inhibitor and could be used as a versatile chemical probe to explore the biological function of ALKBH5.

Real-time fluorometric monitoring of monophenolase activity using a matrix-matched calibration curve.

Tyrosinase is the key enzyme for the metabolism of tyrosine and inherently comprises both monophenolase activity and diphenolase activity. A real-time fluorometric assay method was established to exclusively monitor the monophenolase activity by eliminating interference from diphenolase reactions through a combination of borate and hydroxylamine. Synthetic matrices comprised of tyrosine and DOPA (L-3,4-dihydroxyphenylalanine) preincubated with tyrosinase with the consistent sum concentration of 70 µM to mimic the monophenolase reaction mixture in borate buffer according to law of mass conservation. A matrix-matched calibration curve for determination of tyrosine was established using the synthetic matrices as standard sample to eliminate spectral interference from DOPA. The limit of detection (LOD) for tyrosine was 0.61 µM. The time course for consumption of tyrosine was established to measure the initial velocity through real-time reading out the tyrosine fluorescence intensity of the reaction mixture in a cuvette in situ. The assay worked in the monophenolase activity range from 0.2839 to 1.7308 U mL-1 with LOD of 0.0851 U mL-1. The proposal sensing system successfully afforded a prospective potential for application in enzyme kinetics and screening of inhibitor. Graphical abstract.

Spectrofluorometric method for the determination of ascorbic acid in pharmaceutical preparation using l-tyrosine as fluorescence probe.

Ascorbic acid is a vital nutrient and antioxidant that is commonly used as an additive in commercial products. Quantitation of ascorbic acid is highly desired in the medical, food, and cosmetic industries. A spectrofluorometric assay for sensitive determination of ascorbic acid was developed using l-tyrosine as a fluorescent probe. The native fluorescence intensity of tyrosine was quenched using ascorbic acid. The linear range was 0.03-30.00 µM, and the limit of detection was 0.01 µM. The method exhibited excellent precision, accuracy, specificity, and robustness. Components of pharmaceutical preparations that are commonly found with ascorbic acid did not interfere with detection. The procedure was successfully employed for determination of ascorbic acid content in pharmaceutical tablets, injections, and nutrient supplements with satisfactory results. A Stern-Volmer plot and fluorescence lifetime revealed that quenching was attributed to the inner filter effect and static quenching. Isothermal titration calorimetry confirmed the formation of a complex between tyrosine and ascorbic acid, with a binding constant of 1.68 × 103 M-1 and reaction stoichiometry of 0.94. Thermodynamic parameters suggested spontaneous complexation via hydrophobic interactions as the dominant binding force. This method is promising for the simple and rapid determination of ascorbic in the pharmaceutical industry.

Continuous Fluorometric Method for Determining the Monophenolase Activity of Tyrosinase on L-Tyrosine, through Quenching L-DOPA Fluorescence by Borate.

Tyrosinase is the key enzyme in melanin biosynthesis and inherently involves both monophenolase activity and diphenolase activity. A continuous fluorometric assay method was developed for the first time to directly monitor the real monophenolase activity without the interference of diphenolase reactions through exclusively quenching the native fluorescence of DOPA by borate. Complexation with borate at pH 8.0 allowed for selective quantitation of tyrosine in a binary mixture of tyrosine and DOPA at 335 nm. The time course for consumption of tyrosine was established to measure the initial velocity by recording the tyrosine fluorescence intensity at discrete intervals. The assay worked in the monophenolase activity range from 0.13 to 2.01 U mL-1 with the limit of detection (LOD) of 0.10 U mL-1. The assay method exhibited a promising prospect in application in kinetics of monophenolase and high throughput screening for monophenolase inhibitors.

Integrated study of the mechanism of tyrosinase inhibition by baicalein using kinetic, multispectroscopic and computational simulation analyses.

Controlling the activity of tyrosinase, which plays an important role in melanin synthesis, is essential endeavor in the field of medicine, food, cosmetics, and agriculture. Baicalein is a widely available inhibitor of tyrosinase. The mechanism of this inhibition was studied through an integrated approach coupling enzyme kinetics, multiple types of spectroscopy and computational simulation. Baicalein conspicuously inhibited the diphenolase activity with an IC50 value of 0.11 mM. The inhibtion kinetics indicated that baicalein was a mix-type tight binding inhibitor with a Ki of 0.17 mM and an α value of 0.56. UV absorption spectra showed that a complex formed. Baicalein quenched the fluorescence via both static and dynamic quenching though a single binding site with a binding constant of 278,000 M-1. Thermodynamic parameters suggested spontaneous binding through hydrogen bonding and van der Waals forces. The binding distance was estimated at 3.41 nm. Phase diagram analysis suggested baicalein induced at least two structurally distinct intermediates. Synchronous fluorescence and three-dimensional fluorescence spectra showed blue shift of the maximum emission wavelength. The content of α-helix decreased from 32.67% to 29.00% according to circular dichroism spectra. It was revealed that baicalein induced conformational changes in tyrosinase and enhanced the hydrophobicity of the microenvironment around Trp residues. Docking simulations predicted that baicalein mainly bound tyrosinase via its Met280 residue. This research will provide reliable basis concerning the inhibitory mechanism of baicalein against tyrosianse, and therefore eventually contribute to development of anti-tyrosinase agents.

The effect of 7,8,4´-trihydroxyflavone on tyrosinase activity and conformation: Spectroscopy and docking studies.

Tyrosinase is a ubiquitous enzyme that plays an essential role in the production of melanin. Effective inhibitors of tyrosinase have extensive applications in the medical, cosmetic and food industries. In this study, a combination of enzyme kinetics, ultraviolet (UV)-visible absorption, fluorescence spectroscopic techniques and a computational simulation method was used to characterize the inhibitory mechanism of 7,8,4´-trihydroxyflavone on tyrosinase. 7,8,4´-Trihydroxyflavone was found to strongly inhibit the oxidation of l-DOPA by tyrosinase with an IC50 value of 10.31 ± 0.41 µM. The inhibitory mechanism was determined to be reversible and non-competitive with a Ki of 9.50 ± 0.40 µM. The UV absorption spectra showed that 7,8,4´-trihydroxyflavone could chelate with copper ions and form a complex with tyrosinase. The intrinsic fluorescence of tyrosinase was quenched by 7,8,4´-trihydroxyflavone through a static quenching mechanism. 7,8,4´-Trihydroxyflavone was found to occupy a single binding site with a binding constant of 7.50 ± 1.20 × 104  M-1 at 298 K. The conformation of tyrosinase changed, and the microenvironment became more hydrophilic after 7,8,4´-trihydroxyflavone binding. Thermodynamics parameters indicated that the binding was a spontaneous process and involved hydrogen bonds and van der Waals forces. The binding distance was evaluated to be 4.54 ± 0.05 nm. Docking simulation analysis further authenticated that 7,8,4´-trihydroxyflavone could form hydrogen bonds with the residues His244 and Met280 within the tyrosinase active site. Our results will contribute to further understanding of the inhibitory mechanisms of 7,8,4´-trihydroxyflavone against tyrosinase and will facilitate future screening for tyrosinase inhibitors.