Identification and Characterization of Novel Receptor-Interacting Serine/Threonine-Protein Kinase 2 Inhibitors Using Structural Similarity Analysis.

Receptor-interacting protein kinase 2 (RIP2 or RICK, herein referred to as RIPK2) is linked to the pathogen pathway that activates nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) and autophagic activation. Using molecular modeling (docking) and chemoinformatics analyses, we used the RIPK2/ponatinib crystal structure and searched in chemical databases for small molecules exerting binding interactions similar to those exerted by ponatinib. The identified RIPK2 inhibitors potently inhibited the proliferation of cancer cells by > 70% and also inhibited NFκB activity. More importantly, in vivo inhibition of intestinal and lung inflammation rodent models suggests effectiveness to resolve inflammation with low toxicity to the animals. Thus, our identified RIPK2 inhibitor may offer possible therapeutic control of inflammation in diseases such as inflammatory bowel disease, asthma, cystic fibrosis, primary sclerosing cholangitis, and pancreatitis.

Stereoselective Deuteration in Aspartate, Asparagine, Lysine, and Methionine Amino Acid Residues Using Fumarate as a Carbon Source for Escherichia coli in D2O.

Perdeuteration with selective 1H,13C-enrichment of methyl groups has enabled solution NMR studies of large (>30 kDa) protein systems. However, we propose that for all non-methyl positions, only magnetization originating from 1H-12C groups is sufficiently long-lived, and it can be transferred via through-space NOEs to slowly relaxing 1H-15N or 1H-13C methyl groups to achieve multidimensional solution NMR. We demonstrate stereoselective 1H,12C-labeling by adding relatively inexpensive unlabeled carbon sources to Escherichia coli growth media in D2O. Using our model system, a mutant WW domain from human Pin1, we compare deuteration patterns in 19 amino acids (all except cysteine). Protein grown using glucose as the sole carbon source had high levels of protonation in aromatic rings and the Hß positions of serine and tryptophan. In contrast, using our FROMP media (fumarate, rhamnose, oxalate, malonate, pyruvate), stereoselective protonation of Hß2 with deuteration at Hα and Hß3 was achieved in Asp, Asn, Lys, and Met residues. In solution NMR, stereospecific chemical shift assignments for Hß are typically obtained in conjunction with χ1 dihedral angle determinations using 3-bond J-coupling (3JN-Hß, 3JCO-Hß, 3JHα-Hß) experiments. However, due to motional averaging, the assumption of a pure rotameric state can yield incorrect χ1 dihedral angles with incorrect stereospecific assignments. This was the case for three residues in the Pin1 WW domain (Lys28, Met30, and Asn44). Thus, stereoselective 1H,12C-labeling will be useful not only for NMR studies of large protein systems, but also for determining side chain rotamers and dynamics in any protein system.