The main protease of SARS‐CoV‐2 (M pro ), the causative agent of COVID‐19, constitutes a significant drug target. A new fluorogenic substrate was kinetically compared to an internally quenched fluorescent peptide and shown to be ideally suitable for high throughput screening with recombinantly expressed M pro . Two classes of protease inhibitors, azanitriles and pyridyl esters, were identified, optimized and subjected to in‐depth biochemical characterization. Tailored peptides equipped with the unique azanitrile warhead exhibited concomitant inhibition of M pro and cathepsin L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards M pro inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (k inac /K i =37 500 m −1 s −1 , K i =24.0 n m ) and pyridyl ester 17 (k inac /K i =29 100 m −1 s −1 , K i =10.0 n m ), promising drug candidates for further development have been discovered.
Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A 1 , A 2A , A 2B , and A 3 , of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A 1 AR of rat and mouse), CGS-21680 (for A 2A AR of rat), and Cl-IB-MECA (for A 3 AR of all three species). The functionally selective partial A 2B agonist BAY60-6583 was found to additionally bind to A 1 and A 3 AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A 1 ), preladenant (A 2A ), and PSB-603 (A 2B ) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A 3 AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.
The four adenosine receptors (ARs), A, A, A, and A, constitute a subfamily of G protein-coupled receptors (GPCRs) with exceptional foundations for structure-based ligand design. The vast amount of mutagenesis data, accumulated in the literature since the 1990s, has been recently supplemented with structural information, currently consisting of several inactive and active structures of the A and inactive conformations of the A ARs. We provide the first integrated view of the pharmacological, biochemical, and structural data available for this receptor family, by mapping onto the relevant crystal structures all site-directed mutagenesis data, curated and deposited at the GPCR database (available through http://www.gpcrdb.org). This analysis provides novel insights into ligand binding, allosteric modulation, and signaling of the AR family.
The adenosine receptor (AR) subtypes A2A and A2B are rhodopsin-like Gs protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A2A-A2BAR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A2AAR when co-expressed with the A2BAR (A2B ≥ A2A) in recombinant as well as in native cells. In the presence of A2BARs, A2A-selective ligands lost high affinity binding to A2AARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A2AAR ligands as drugs as they will fail to modulate the receptor in an A2A-A2B heteromer context. Accordingly, A2A-A2BAR heteromers represent novel pharmacological targets.
8-Amido-chromen-4-one-2-carboxylic acid derivatives were identified as novel agonists at the G protein-coupled orphan receptor GPR35. They were characterized by a β-arrestin recruitment assay and optimized to obtain agonists with nanomolar potency for the human GPR35. The compounds were found to exhibit high selectivity versus the related GPR55. The most potent agonists were 6-bromo-8-(4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (85, EC50 12.1 nM) and 6-bromo-8-(2-chloro-4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (90, EC50 11.1 nM), both of which were >1700-fold selective versus GPR55. Most compounds were considerably less potent at rat and mouse than at human GPR35. 6-Bromo-8-(2-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (87) was the only derivative that activated GPR35 of all three species at similar, low micromolar concentration. Compounds 85 and 90 are the most potent agonists at the human GPR35 known to date and might thus serve as powerful pharmacological tools to further elucidate the receptor's (patho)physiological role and its potential as a future drug target.
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