Two tryptophan residues were incorporated on one face of a β-hairpin peptide to form an aromatic pocket that interacts with a lysine or N-methylated lysine via cation-π interactions. The two tryptophan residues were found to pack against the lysine side chain forming an aromatic pocket similar to those observed in trimethylated lysine receptor proteins. Thermal analysis of methylated lysine variant hairpin peptides revealed an increase in thermal stability as the degree of methylation was increased resulting in the most thermally stable β-hairpin reported to date.

Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization of this 3-acylaminoindazole scaffold furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.The human protein Ser/Thr kinases Adaptor protein 2-Associated Kinase 1 (AAK1) and BMP-2-Inducible Kinase (BMP2K/BIKE) play critical roles in mediating endocytosis and other key signaling pathways. Both are broadly expressed and are members of the NAK family of human kinases, which also includes Cyclin G-Associated Kinase (GAK) and Myristoylated and Palmitoylated Serine/Threonine Kinase 1 (MPSK1/STK16). The family shares little homology outside of their kinase domains. 1 AAK1 and BMP2K are the most closely related, with overall sequence identity of 50% and kinase domain sequence identity of 74%. 2 A key function of AAK1 is regulation of receptor-mediated endocytosis via binding directly to clathrin and phosphorylating the medium subunit of AP2 (adaptor protein 2), which stimulates binding to cargo proteins. [3][4][5] AAK1 also modulates the Notch pathway, partially through its phosphorylation of Numb. 6, 7 BMP2K plays a role in osteoblast differentiation, is a clathrin-coated vesicle-associated protein, and, like AAK1, also associates with Numb. 8, 9 Due to their many functions, AAK1 and BMP2K have been implicated as potential drug targets for diverse conditions. AAK1 has been linked to diseases affecting the brain such as schizophrenia, Parkinson's disease and amyotrophic lateral sclerosis as well as implicated as a potential anti-viral target for the treatment of Hepatitis C. 5, 10, 11 BMP2K has been associated with myopia and evaluated as a potential treatment for HIV. 12, 13 A dual AAK1/BMP2K small molecule inhibitor was recently reported as a novel therapeutic to treat neuropathic pain. 14 X-ray crystal structures for the kinase domains of all NAK family members have been solved and reported. 2, 15, 16 Published and novel high-resolution crystal structures of AAK1 and BMP2K reveal target-specific structural features that have enabled our design of specific chemical probes and allowed further

Phosphorylated amino acids were incorporated into a designed β-hairpin peptide to study the effect on β-hairpin structure when the phosphate group is positioned to interact with a tryptophan residue on the neighboring strand. The three commonly phosphorylated residues in biological systems, serine, threonine, and tyrosine, were studied in same β-hairpin system. It was found that phosporylation destabilizes the hairpin structure by approximately 1.0 kcal/mol regardless of the type of phosphorylated residue. In contrast, destabilization due to glutamic acid was about 0.3 kcal/mol. Double mutant cycles and pH studies are consistent with a repulsive interaction as the source of destabilization. These findings demonstrate a novel mechanism by which phosphorylation may influence protein structure and function.

Thiazine formation during the conjugation of N‐terminal cysteine peptides to maleimides is an underreported side reaction in the peptide literature. When the conjugation was performed at neutral and basic pH, we observed the thiazine isomer as a significant by‐product. Nuclear magnetic resonance (NMR) spectroscopy confirmed the structure of the six‐membered thiazine and ultra‐high performance liquid chromatography (UHPLC) combined with tandem mass spectrometry (MS/MS) allowed for facile, unambiguous detection due to a unique thiazine mass fragment. Furthermore, substitution of various amino acids adjacent to the N‐terminal cysteine in a tripeptide model system resulted in different rates of thiazine formation, albeit within the same order of magnitude. We also determined that varying the N‐substitution of the maleimide affects the thiazine conversion rate. Altogether, our findings suggest that thiazine rearrangement for N‐terminal cysteine‐maleimide adducts is a general side reaction that is applicable to other peptide or protein systems. Performing the conjugation reaction under acidic conditions or avoiding the use of an N‐terminal cysteine with a free amino group prevents the formation of the thiazine impurity.

Protein post-translational modifications (PTMs) are used in nature as a means of turning on or off a myriad of biological events. Methylation of lysine and phosphorylation of serine are important PTMs in the histone code found to modulate chromatin packing, which in turn affects gene expression. The design of peptides that fold into secondary structures can help to further our understanding of complex protein interactions. Here we report the design of the Trpswitch peptide sequence that folds into a moderately stable beta-hairpin structure in aqueous solution and show that the stability of the structure can be tuned by incorporation of dimethyllysine or phosphoserine. Dimethylated Trpswitch results in an increase in beta-hairpin stability, while phosphorylated Trpswitch is unstructured at neutral pH. When both modifications are incorporated into Trpswitch, a less stable beta-hairpin structure is observed. This system provides a model to demonstrate how multiple PTMs may work in concert or against each other to influence structure.