Dynamic combinatorial chemistry was utilized to identify a novel small molecule receptor, A2D, for asymmetric dimethyl arginine (aRMe2), which is a post-translational modification (PTM) in proteins. It is known to play a role in a number of diseases, including spinal muscular atrophy, leukemia, lymphoma, and breast cancer. The receptor exhibits 2.5-7.5-fold selectivity over the isomeric symmetric dimethyl arginine, depending on the surrounding sequence, with binding affinities in the low micromolar range. The affinity and selectivity of A2D for the different methylated states of Arg parallels that of proteins that bind to these PTMs. Characterization of the receptor-PTM complex indicates that cation-π interactions provide the main driving force for binding, loosely mimicking the binding mode found in the recognition of dimethyl arginine by native protein receptors.
A network of reader proteins and enzymes precisely controls gene transcription through the dynamic addition, removal, and recognition of post-translational modifications (PTMs) of histone tails. Histone PTMs work in concert with this network to regulate gene transcription through the histone code, and the dysregulation of PTM maintenance is linked to a large number of diseases, including many types of cancer. A wealth of research aims to elucidate the functions of this code, but our understanding of the effects of PTMs, specifically the methylation of lysine (Lys) and arginine (Arg), is lacking. The development of new tools to study PTMs relies on a sophisticated understanding of the mechanisms that drive protein and small molecule recognition in water. In this review, we outline the physical organic concepts that drive the molecular recognition of Lys and Arg methylation by reader proteins and draw comparisons to the binding mechanisms of small molecule receptors for methylated Lys and Arg that have been developed recently.
Dynamic combinatorial chemistry was used to generate a set of receptors for peptides containing methylated lysine (KMen, n = 0-3) and study the contribution of electrostatic effects and pocket depth to binding affinity and selectivity. We found that changing the location of a carboxylate resulted in an increase in preference for KMe2, presumably based on ability to form a salt bridge with KMe2. The number of charged groups on either the receptor or peptide guest systematically varied the binding affinities to all guests by approximately 1-1.5 kcal mol(-1), with little influence on selectivity. Lastly, formation of a deeper pocket led to both increased affinity and selectivity for KMe3 over the lower methylation states. From these studies, we identified that the tightest binder was a receptor with greater net charge, with a Kd of 0.2 μM, and the receptor with the highest selectivity was the one with the deepest pocket, providing 14-fold selectivity between KMe3 and KMe2 and a Kd for KMe3 of 0.3 μM. This work provides key insights into approaches to improve binding affinity and selectivity in water, while also demonstrating the versatility of dynamic combinatorial chemistry for rapidly exploring the impact of subtle changes in receptor functionality on molecular recognition in water.
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