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 complex nature of multifactorial diseases, such as Morbus Alzheimer, has produced a strong need to design multitarget-directed ligands to address the involved complementary pathways. We performed a purposive structural modification of a tetratarget small-molecule, that is contilisant, and generated a combinatorial library of 28 substituted chromen-4-ones. The compounds comprise a basic moiety which is linker-connected to the 6-position of the heterocyclic chromenone core. The syntheses were accomplished by Mitsunobu- or Williamson-type ether formations. The resulting library members were evaluated at a panel of seven human enzymes, all of which being involved in the pathophysiology of neurodegeneration. A concomitant inhibition of human acetylcholinesterase and human monoamine oxidase B, with IC50 values of 5.58 and 7.20 μM, respectively, was achieved with the dual-target 6-(4-(piperidin-1-yl)butoxy)-4H-chromen-4-one (7).

The cysteine protease cathepsin K is a target for the treatment of diseases associated with high bone turnover. Cathepsin K is mainly expressed in osteoclasts and responsible for the destruction of the proteinaceous components of the bone matrix. We designed various fluorescent activity-based probes (ABPs) and their precursors that bind to and inactivate cathepsin K. ABP 25 exhibited extraordinary potency (k inac/K i = 35,300 M–1s–1) and selectivity for human cathepsin K. Crystal structures of cathepsin K in complex with ABP 25 and its nonfluorescent precursor 21 were determined to characterize the binding mode of this new type of acrylamide-based Michael acceptor with the particular orientation of the dibenzylamine moiety to the primed subsite region. The cyanine-5 containing probe 25 allowed for sensitive detection of cathepsin K, selective visualization in complex proteomes, and live cell imaging of a human osteosarcoma cell line, underlining its applicability in a pathophysiological environment.

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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