Drug Design, Development and Therapy (Apr 2023)
Synthesis of Amino Acids Bearing Halodifluoromethyl Moieties and Their Application to p53-Derived Peptides Binding to Mdm2/Mdm4
Abstract
Sebastian Vaas,1 Markus O Zimmermann,1 Theresa Klett,1 Frank M Boeckler1,2 1Department of Pharmacy and Biochemistry, Eberhard Karls Universität Tübingen, Laboratory for Molecular Design and Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences, Tübingen, 72076, Germany; 2Institute for Bioinformatics and Medical Informatics (IBMI), Eberhard Karls Universität Tübingen, Tübingen, 72076, GermanyCorrespondence: Frank M Boeckler, Molecular Design and Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8 (Haus B), Tübingen, D-72076, Germany, Tel +49 7071 29 74567, Fax +49 7071 29 5637, Email [email protected]: Therapeutic peptides are a significant class of drugs in the treatment of a wide range of diseases. To enhance their properties, such as stability or binding affinity, they are usually chemically modified. This includes, among other techniques, cyclization of the peptide chain by bridging, modifications to the backbone, and incorporation of unnatural amino acids. One approach previously established, is the use of halogenated aromatic amino acids. In principle, they are thereby enabled to form halogen bonds (XB). In this study, we focus on the -R-CF2X moiety (R = O, NHCO; X = Cl, Br) as an uncommon halogen bond donor. These groups enable more spatial variability in protein–protein interactions. The chosen approach via Fmoc-protected building blocks allows for the incorporation of these modified amino acids in peptides using solid-phase peptide synthesis.Results and Discussion: Using a competitive fluorescence polarization assay to monitor binding to Mdm4, we demonstrate that a p53-derived peptide with Lys24Nle(ϵNHCOCF2X) exhibits an improved inhibition constant Ki compared to the unmodified peptide. Decreasing Ki values observed with the increasing XB capacity of the halogen atoms (F ≪ Cl < Br) indicates the formation of a halogen bond. By reducing the side chain length of Nle(ϵNHCOCF2X) to Abu(γNHCOCF2X) as control experiments and through quantum mechanical calculations, we suggest that the observed affinity enhancement is related to halogen bond-induced intramolecular stabilization of the α-helical binding mode of the peptide or a direct interaction with His54 in human Mdm4.Graphical Abstract: Keywords: halogen bonding, solid-phase peptide synthesis, unnatural amino acids, fluorescence polarization assay, oncological target, MdmX
