A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility

We have developed a simple protocol for amino acid type selective isotope labeling in insect cells. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. Similar protein amounts as with full media are obtained, without compromising on isotope incorporation ratios, which are consistently ≥ 90% for all amino acids tested in single and dual amino acid labeling experiments. This enables NMR studies of eukaryotic proteins and their interactions even for proteins with low expression levels. We show applications with human kinases, where protein-ligand interactions are characterized by 2D [15N,1H]- and [13C,1H]-HSQC spectra

Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate–rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3′ untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.ISSN:0027-8424ISSN:1091-649

Design of potent and selective covalent inhibitors of Bruton’s Tyrosine Kinase targeting an inactive conformation

Bruton’s tyrosine kinase (BTK) is a member of the TEC kinase family and is selectively expressed in a subset of immune cells. It is a key regulator of antigen receptor signaling in B cells and of Fc receptor signaling in mast cells and macrophages. A BTK inhibitor will likely have a positive impact on autoimmune diseases which are caused by autoreactive B cells and immune-complex driven inflammation. We report the design, optimization, and characterization of potent and selective covalent BTK inhibitors. Starting from the selective reversible inhibitor 3 binding to an inactive conformation of BTK, we designed covalent irreversible compounds by attaching an electrophilic warhead to reach Cys481. The first prototype 4 covalently modified BTK and showed an excellent kinase selectivity including several Cys-containing kinases, validating the design concept. In addition, this compound blocked FcγR-mediated hypersensitivity in vivo. Optimization of whole blood potency and metabolic stability resulted in compounds such as 8, which maintained the excellent kinase selectivity and showed improved BTK occupancy in vivo

Discovery of Potent, Orally Bioavailable, Tricyclic NLRP3 Inhibitors.

NLRP3 is a molecular sensor recognizing a wide range of danger signals. Its activation leads to the assembly of an inflammasome that allows for activation of caspase-1 and subsequent maturation of IL-1β and IL-18, as well as cleavage of Gasdermin-d and pyroptotic cell death. The NLRP3 inflammasome has been implicated in a plethora of diseases including gout, type 2 diabetes, atherosclerosis, Alzheimer's disease, and cancer. In this publication, we describe the discovery of a novel, tricyclic, NLRP3-binding scaffold by high-throughput screening. The hit (1) could be optimized into an advanced compound NP3-562 demonstrating excellent potency in human whole blood and full inhibition of IL-1β release in a mouse acute peritonitis model at 30 mg/kg po dose. An X-ray structure of NP3-562 bound to the NLRP3 NACHT domain revealed a unique binding mode as compared to the known sulfonylurea-based inhibitors. In addition, NP3-562 shows also a good overall development profile

The Crystal structure of the cancer osaka thyroid kinase reveals an unexpected kinase domain fold

Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune disorders such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Cancer Osaka Thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a Serine-Threonine (ST) kinase and is a key regulator in the production of proinflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective and potent inhibitors for the treatment of autoimmune disorders. The production of monomeric, recombinant COT kinase has proven to be very difficult and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the X-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this publication reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The glycine-rich loop contains a 15 amino acid insert that forms a β-hairpin structure and partially covers the ATP-binding site cleft, which – together with the structurally versatile active site – significantly impacts the design of potent, low-molecular weight COT inhibitors

Gift from nature : cyclomarin s kills Mycobacteria and malaria parasites by distinct modes of action

Malaria continues to be one of the most devastating human diseases despite many efforts to limit its spread by prevention of infection or by pharmaceutical treatment of patients. We have conducted a screen for antiplasmodial compounds by using a natural product library. Here we report on cyclomarin A as a potent growth inhibitor of Plasmodium falciparum and the identification of its molecular target, diadenosine triphosphate hydrolase (PfAp3Aase), by chemical proteomics. Using a biochemical assay, we could show that cyclomarin A is a specific inhibitor of the plasmodial enzyme but not of the closest human homologue hFHIT. Co-crystallisation experiments demonstrate a unique binding mode of the inhibitor. One molecule of cyclomarin A binds a dimeric PfAp3Aase and prevents the formation of the enzyme⋅substrate complex. These results validate PfAp3Aase as a new drug target for the treatment of malaria. We have previously elucidated the structurally unrelated regulatory subunit ClpC1 of the ClpP protease as the molecular target of cyclomarin A in Mycobacterium tuberculosis. Thus, cyclomarin A is a rare example of a natural product with two distinct and specific modes of action

Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton’s Tyrosine Kinase

Bruton’s tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncology indications based on their suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064 (25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in Phase 2 clinical studies for chronic spontaneous urticaria and Sjoegren’s Syndrome

PAX8 and MECOM are interaction partners driving Ovarian Carcinogenesis

The transcription factor PAX8 is critical for the development of the thyroid and urogenital system. Comprehensive genomic screens furthermore indicate an additional oncogenic role for PAX8 in renal and ovarian carcinogenesis. While a plethora of PAX8-regulated genes in different contexts have been proposed, we still lack a mechanistic understanding of how PAX8 engages molecular complexes to drive disease-relevant oncogenic transcriptional programs. Here we show that protein isoforms originating from the MECOM locus form a complex with PAX8. This includes PRDM3 (also called MDS1-EVI1) for which we map its interaction with PAX8 in vitro and in vivo. We show that PAX8 binds a large number of genomic sites and forms transcriptional hubs. At a subset of these, PAX8 together with PRDM3 regulate a specific gene expression module involved in adhesion and extracellular matrix. This gene module correlates with PAX8 and MECOM expression in large scale profiling of cell lines, PTXs and clinical cases and stratifies gynecological cancer cases with worse prognosis. PRDM3 is amplified in Ovarian Cancers and we show that the MECOM locus and PAX8 sustain in-vivo ovarian cancer growth, further supporting that the identified function of the MECOM locus underlies PAX8-driven oncogenic functions in ovarian cancer