Development of an active-site titrant for SARS-CoV-2 main protease as an indispensable tool for evaluating enzyme kinetics.

A titrant for the SARS-CoV-2 main protease (M) was developed that enables, for the first time, the exact determination of the concentration of the enzymatically active M by active-site titration. The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme-titrant complex. Four fluorogenic substrates of M, including a prototypical, internally quenched Dabcyl-EDANS peptide, were compared in terms of solubility under typical assay conditions.

Expanding the Structural Diversity at the Phenylene Core of Ligands for the von Hippel-Lindau E3 Ubiquitin Ligase: Development of Highly Potent Hypoxia-Inducible Factor-1α Stabilizers.

Hypoxia-inducible factor-1α (HIF-1α) constitutes the principal mediator of cellular adaptation to hypoxia in humans. The HIF-1α protein level and activity are tightly regulated by the ubiquitin E3 ligase von Hippel-Lindau (VHL). Here, we performed a structure-guided and bioactivity-driven design of new VHL inhibitors.

Combinatorial assembly, traceless generation and in situ evaluation of inhibitors for therapeutically relevant serine proteases.

A combinatorial method was devised and applied for the design and identification of substrate-analogue inhibitors of therapeutically relevant serine proteases, such as thrombin and factor Xa. We conceptualized imino acid derived diketomorpholines as generally applicable key intermediates prepared through solid-phase synthesis and prone to be cleaved with primary amines in a traceless fashion. The approach led to a compound library whose members were prepared under bioassay-compatible conditions and directly subjected to the in situ evaluation, allowing a fast prediction of hit compounds.

Diketomorpholines: Synthetic Accessibility and Utilization.

Diketomorpholines (DKMs; morpholine-2,5-diones) possess a six-membered ring with a lactone and lactam moiety and belong to the family of cyclodepsipeptides. In this review, the synthetic accessibility of DKMs is summarized and their utilization, in particular, for ring-opening polymerization reactions, is highlighted. The occurrence of the DKM scaffold in natural products encompasses small monocyclic compounds but also complex, polycyclic representatives with a fused DKM ring.

Targeting the Main Protease of SARS-CoV-2: From the Establishment of High Throughput Screening to the Design of Tailored Inhibitors.

The main protease of SARS-CoV-2 (M ), 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 . Two classes of protease inhibitors, azanitriles and pyridyl esters, were identified, optimized and subjected to in-depth biochemical characterization.

Systematic exploration of different E3 ubiquitin ligases: an approach towards potent and selective CDK6 degraders.

Cyclin-dependent kinase 6 (CDK6) is an important regulator of the cell cycle. Together with CDK4, it phosphorylates and inactivates retinoblastoma (Rb) protein. In tumour cells, CDK6 is frequently upregulated and CDK4/6 kinase inhibitors like palbociclib possess high activity in breast cancer and other malignancies.

PROTAC-mediated crosstalk between E3 ligases.

Small-molecule heterobifunctional degraders can effectively control protein levels and are useful research tools. We assembled proteolysis targeting chimeras (PROTACs) from a cereblon (CRBN) and a von-Hippel-Lindau (VHL) ligase ligand and demonstrated a PROTAC-induced heterodimerization of the two E3 ligases leading to unidirectional and efficient degradation of CRBN.