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 adenosine A 2A receptor (A 2A AR) is an important new (potential) drug target in immuno-oncology, and for neurodegenerative diseases. Preladenant and its derivatives belong to the most potent A 2A AR antagonists displaying exceptional selectivity. While crystal structures of the human A 2A AR have been solved, mostly using the A 2A -StaR2 protein that bears 9 point mutations, co-crystallization with Preladenant derivatives has so far been elusive. We developed a new A 2A AR construct harboring a single point mutation (S91 3.39 K) which renders it extremely thermostable. This allowed the co-crystallization of two novel Preladenant derivatives, the polyethylene glycolconjugated (PEGylated) PSB-2113, and the fluorophore-labeled PSB-2115. The obtained crystal structures (2.25 Å and 2.6 Å resolution) provide explanations for the high potency and selectivity of Preladenant derivatives. They represent the first crystal structures of a GPCR in complex with PEG-and fluorophore-conjugated ligands. The applied strategy is predicted to be applicable to further class A GPCRs.

Die Hauptprotease von SARS-CoV-2 (M pro ), dem Auslçser von COVID-19, ist ein wichtiges Arzneistoff-Target. Ein neues fluorogenes Substrat, das kinetischmit einem intern gequenchten fluoreszierenden Peptid verglichen wurde, erwies sich als ideal geeignet füre in Hochdurchsatz-Screening mit rekombinant exprimierter M pro .Z wei Klassen von Protease-Inhibitoren, Azanitrile und Pyridylester,w urden identifiziert, optimiert und biochemisch charakterisiert. Maßgeschneiderte Peptide mit einer reaktiven Azanitril-Kopfgruppe zeigten eine duale Inhibition von M pro und Cathepsin L, einer Protease, welche die virale Zellinvasion befçrdert. Zur Optimierung der Pyridylindolester wurde ein Positions-Scanning durchgeführt. Unser fokussierter Ansatz zur Entwicklung von M pro -Inhibitoren erwies sich dem virtuellen Screening als überlegen. Mit den beiden irreversiblen Inhibitoren Azanitril 8 (k inac /K i = 37 500 m À1 s À1 ,K i = 24.0 nm)u nd Pyridylester 17 (k inac /K i = 29 100 m À1 s À1 ,K i = 10.0 nm)w urden vielversprechende Kandidaten fürdie zukünftige Arzneistoffentwicklung entdeckt.

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Blockade of the adenosine A2B receptor (A2BAR) represents a potential novel strategy for the immunotherapy of cancer. In the present study, we designed, synthesized, and characterized irreversible A2BAR antagonists based on an 8-p-sulfophenylxanthine scaffold. Irreversible binding was confirmed in radioligand binding and bioluminescence resonance energy transfer(BRET)-based Gα15 protein activation assays by performing ligand wash-out and kinetic experiments. p-(1-Propylxanthin-8-yl)benzene sulfonyl fluoride (6a, PSB-21500) was the most potent and selective irreversible A2BAR antagonist of the present series with an apparent Ki value of 10.6 nM at the human A2BAR and >38-fold selectivity versus the other AR subtypes. The corresponding 3-cyclopropyl-substituted xanthine derivative 6c (PSB-21502) was similarly potent, but was non-selective versus A1- and A2AARs. Attachment of a reactive sulfonyl fluoride group to an elongated xanthine 8-substituent (12, Ki 7.37 nM) resulted in a potent, selective, reversibly binding antagonist. Based on previous docking studies, the lysine residue K2697.32 was proposed to react with the covalent antagonists. However, the mutant K269L behaved similarly to the wildtype A2BAR, indicating that 6a and related irreversible A2BAR antagonists do not interact with K2697.32. The new irreversible A2BAR antagonists will be useful tools and have the potential to be further developed as therapeutic drugs.