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.

The G protein-coupled receptor GPR84, which is activated by (hydroxy)fatty acids, is highly expressed on immune cells. Recently, 3,3'-diindolylmethane was identified as a heterocyclic, nonlipid-like GPR84 agonist. We synthesized a broad range of diindolylmethane derivatives by condensation of indoles with formaldehyde in water under microwave irradiation. The products were evaluated at the human GPR84 in cAMP and β-arrestin assays. Structure-activity relationships (SARs) were steep. 3,3'-Diindolylmethanes bearing small lipophilic residues at the 5- and/or 7-position of the indole rings displayed the highest activity in cAMP assays, the most potent agonists being di(5-fluoro-1H-indole-3-yl)methane (38, PSB-15160, EC 80.0 nM) and di(5,7-difluoro-1H-indole-3-yl)methane (57, PSB-16671, EC 41.3 nM). In β-arrestin assays, SARs were different, indicating biased agonism. The new compounds were selective versus related fatty acid receptors and the arylhydrocarbon receptor. Selected compounds were further investigated and found to display an ago-allosteric mechanism of action and increased stability in comparison to the lead structure.

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.

GPR84, a Gi protein-coupled receptor that is activated by medium-chain (hydroxy)fatty acids, appears to play an important role in inflammation, immunity, and cancer. Recently, 6-octylaminouracil (4) has been reported to act as an agonist at GPR84. Here, we describe the synthesis of 69 derivatives and analogs of 4, 66 of which represent new compounds. They were evaluated in (a) cyclic adenosine monophosphate accumulation and (b) β-arrestin assays in human GPR84-expressing cells. Potent nonbiased as well as G protein-biased agonists were developed, e.g., 6-hexylamino-2,4(1H,3H)-pyrimidinedione (20, PSB-1584, EC50 5.0 nM (a), 3.2 nM (b), bias factor: 0) and 6-((p-chloro- and p-bromo-phenylethyl)amino)-2,4(1H,3H)-pyrimidinedione (47, PSB-16434, EC50 7.1 nM (a), 520 nM (b), bias factor: 1.9 = 79-fold Gi pathway-selective; 48, PSB-17365, EC50 2.5 nM (a), 100 nM (b), bias factor 1.3 = 20-fold selective), which were selective versus other free fatty acid-activated receptors. Compounds 20 and 48 were found to be metabolically stable upon incubation with human liver microsomes. A pharmacophore model was created on the basis of structurally diverse lipidlike GPR84 agonists.

The adenosine A(2B) receptor is of considerable interest as a new drug target for the treatment of asthma, inflammatory diseases, pain, and cancer. In the present study we investigated the role of the cysteine residues in the extracellular loop 2 (ECL2) of the receptor, which is particularly cysteine-rich, by a combination of mutagenesis, molecular modeling, chemical and pharmacological experiments. Pretreatment of CHO cells recombinantly expressing the human A(2B) receptor with dithiothreitol led to a 74-fold increase in the EC(50) value of the agonist NECA in cyclic AMP accumulation. In the C78(3.25)S and the C171(45.50)S mutant high-affinity binding of the A(2B) antagonist radioligand [(3)H]PSB-603 was abolished and agonists were virtually inactive in cAMP assays. This indicates that the C3.25-C45.50 disulfide bond, which is highly conserved in GPCRs, is also important for binding and function of A(2B) receptors. In contrast, the C166(45.45)S and the C167(45.46)S mutant as well as the C166(45.45)S-C167(45.46)S double mutant behaved like the wild-type receptor, while in the C154(45.33)S mutant significant, although more subtle effects on cAMP accumulation were observed - decrease (BAY60-6583) or increase (NECA) - depending on the structure of the investigated agonist. In contrast to the X-ray structure of the closely related A(2A) receptor, which showed four disulfide bonds, the present data indicate that in the A(2B) receptor only the C3.25-C45.50 disulfide bond is essential for ligand binding and receptor activation. Thus, the cysteine residues in the ECL2 of the A(2B) receptor not involved in stabilization of the receptor structure may have other functions.