Structural waters in the S1 binding pocket of β-trypsin are critical for the stabilization of the complex of β-trypsin with its inhibitor bovine pancreatic trypsin inhibitor (BPTI). The inhibitor strength of BPTI can be modulated by replacing the critical lysine residue at the P1 position by non-natural amino acids. We study BPTI variants in which the critical Lys15 in BPTI has been replaced by α-aminobutyric acid (Abu) and its fluorinated derivatives monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly), and trifluoroethylglycine (TfeGly). We investigate the hypothesis that additional water molecules in the binding pocket can form specific noncovalent interactions with the fluorinated side chains and thereby act as an extension of the inhibitors. We report potentials of mean force (PMF) of the unbinding process for all four complexes and enzyme activity inhibition assays. Additionally, we report the protein crystal structure of the Lys15MfeGly–BPTI−β-trypsin complex (pdb: 7PH1). Both experimental and computational data show a stepwise increase in inhibitor strength with increasing fluorination of the Abu side chain. The PMF additionally shows a minimum for the encounter complex and an intermediate state just before the bound state. In the bound state, the computational analysis of the structure and dynamics of the water molecules in the S1 pocket shows a highly dynamic network of water molecules that does not indicate a rigidification or stabilizing trend in regard to energetic properties that could explain the increase in inhibitor strength. The analysis of the energy and the entropy of the water molecules in the S1 binding pocket using grid inhomogeneous solvation theory confirms this result. Overall, fluorination systematically changes the binding affinity, but the effect cannot be explained by a persistent water network in the binding pocket. Other effects, such as the hydrophobicity of fluorinated amino acids and the stability of the encounter complex as well as the additional minimum in the potential of mean force in the bound state, likely influence the affinity more directly.
Target druggability assessment is an integral part of the early target characterization and selection process in pharmaceutical industry. Here, we investigate a set of five different serine proteases from the blood coagulation cascade. The aim of this study is twofold. Firstly, leveraging the wealth of available in‐house high‐throughput screening (HTS) data, we analyze HTS hit rates and discuss their predictive value for the development of small molecule (SMOL) candidates. Purely structure‐activity relationship (SAR) based druggability ratings are compared with computational protein‐structure based druggability assessments. Secondly, we evaluate the impact of using conformational ensembles from molecular dynamics (MD) simulations instead of single static crystal structures as basis for computational druggability assessments. Based on this study, we recommend incorporating molecular dynamics routinely into the early target characterization process, especially if only a single X‐ray structure is available.
Phosphotyrosine residues are essential functional switches in health and disease. Thus, phosphotyrosine biomimetics are crucial for the development of chemical tools and drug molecules. We report here the discovery and investigation of pentafluorophosphato amino acids as novel phosphotyrosine biomimetics. A mild acidic pentafluorination protocol was developed and two PF 5 -amino acids were prepared and employed in peptide synthesis. Their structures, reactivities, and fluorine-specific interactions were studied by NMR and IR spectroscopy, X-ray diffraction, and in bioactivity assays. The mono-anionic PF 5 motif displayed an amphiphilic character binding to hydrophobic surfaces, to water molecules, and to protein-binding sites, exploiting charge and HÀ F-bonding interactions. The novel motifs bind 25-to 30-fold stronger to the phosphotyrosine binding site of the protein tyrosine phosphatase PTP1B than the best current biomimetics, as rationalized by computational methods, including molecular dynamics simulations.
Discovery of protein‐binding fragments for precisely defined binding sites is an unmet challenge to date. Herein, formylglycine is investigated as a molecular probe for the sensitive detection of fragments binding to a spatially defined protein site . Formylglycine peptide 3 was derived from a phosphotyrosine‐containing peptide substrate of protein tyrosine phosphatase PTP1B by replacing the phosphorylated amino acid with the reactive electrophile. Fragment ligation with formylglycine occurred in situ in aqueous physiological buffer. Structures and kinetics were validated by NMR spectroscopy. Screening and hit validation revealed fluorinated and non‐fluorinated hit fragments being able to replace the native phosphotyrosine residue. The formylglycine probe identified low‐affinity fragments with high spatial resolution as substantiated by molecular modelling. The best fragment hit, 4‐amino‐phenyl‐acetic acid, was converted into a cellularly active, nanomolar inhibitor of the protein tyrosine phosphatase SHP2.
Phosphotyrosinreste sind essenzielle funktionale Schalter in Gesundheit und Krankheit. Daher sind Phosphotyrosin‐Biomimetika entscheidend für die Entwicklung von chemischen Werkzeugen und Wirkstoffmolekülen. Hier beschreiben wir die Entdeckung und Untersuchung von Pentafluorphosphato‐Aminosäuren als neuartige Phosphotyrosinmimetika. Ein mildes Pentafluorierungsprotokoll wurde entwickelt, zwei PF5‐Aminosäuren hergestellt und für die Peptidsynthese verwendet. Ihre Strukturen, Reaktivitäten und fluorspezifischen Wechselwirkungen wurden mittels NMR‐ und IR‐Spektroskopie, Röntgenstreuung und biologischen Assays untersucht. Das monoanionische PF5‐Motiv weist einen amphiphilen Charakter auf und bindet an hydrophobe Oberflächen, Wassermoleküle und Proteinbindungstaschen mittels Ladungs‐ und H−F‐Brückenwechselwirkungen. Die neuen Motive binden 25‐ bis 30‐fach stärker an die Phosphotyrosinbindungsstelle der Proteintyrosin‐Phosphatase PTP1B als das derzeit beste Biomimetikum, wie durch computergestützte Methoden wie Moleküldynamiksimulationen rationalisiert werden konnte.
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