Letter to John Hodsdon from Stephen W. Laughton, August 18, 1862

https://digitalmaine.com/adj_gen_corr_town_appleton/1006/thumbnail.jp

Conversion of a non-selective adenosine receptor antagonist into A3-selective high affinity fluorescent probes using peptide-based linkers

Advances in fluorescence-based imaging technologies have helped propel the study of real-time biological readouts and analysis across many different areas. In particular the use of fluorescent ligands as chemical tools to study proteins such as G protein-coupled receptors (GPCRs) has received ongoing interest. Methods to improve the efficient chemical synthesis of fluorescent ligands remain of paramount importance to ensure this area of bioanalysis continues to advance. Here we report conversion of the non-selective GPCR adenosine receptor antagonist Xanthine Amine Congener into higher affinity and more receptor subtype-selective fluorescent antagonists. This was achieved through insertion and optimisation of a dipeptide linker between the adenosine receptor pharmacophore and the fluorophore. Fluorescent probe 27 containing BODIPY 630/650 (pKD = 9.12 ± 0.05 [hA3AR]), and BODIPY FL-containing 28 (pKD = 7.96 ± 0.09 [hA3AR]) demonstrated clear, displaceable membrane binding using fluorescent confocal microscopy. From in silico analysis of the docked ligand-receptor complexes of 27, we suggest regions of molecular interaction that could account for the observed selectivity of these peptide-linker based fluorescent conjugates. This general approach of converting a non-selective ligand to a selective biological tool could be applied to other ligands of interest

Letter to John Hodsdon from Stephen W. Laughton, August 18, 1862

https://digitalmaine.com/adj_gen_corr_town_appleton/1006/thumbnail.jp

Molecular Dynamics Simulations of the Adenosine A2a Receptor: Structural Stability, Sampling, and Convergence

Molecular dynamics (MD) simulations of membrane-embedded G-protein coupled receptors (GPCRs) have rapidly gained popularity among the molecular simulation community in recent years, a trend which has an obvious link to the tremendous pharmaceutical importance of this group of receptors and the increasing availability of crystal structures. In view of the widespread use of this technique, it is of fundamental importance to ensure the reliability and robustness of the methodologies so they yield valid results and enable sufficiently accurate predictions to be made. In this work, 200 ns simulations of the A2a adenosine receptor (A2a AR) have been produced and evaluated in the light of these requirements. The conformational dynamics of the target protein, as obtained from replicate simulations in both the presence and absence of an inverse agonist ligand (ZM241385), have been investigated and compared using principal component analysis (PCA). Results show that, on this time scale, convergence of the replicates is not readily evident and dependent on the types of the protein motions considered. Thus rates of inter- as opposed to intrahelical relaxation and sampling can be different. When studied individually, we find that helices III and IV have noticeably greater stability than helices I, II, V, VI, and VII in the apo form. The addition of the inverse agonist ligand greatly improves the stability of all helices

Molecular Dynamics Simulations of the Adenosine A2a Receptor in POPC and POPE Lipid Bilayers: Effects of Membrane on Protein Behavior

Analysis of 300 ns (ns) molecular dynamics (MD) simulations of an adenosine A2a receptor (A2a AR) model, conducted in triplicate, in 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) bilayers reveals significantly different protein dynamical behavior. Principal component analysis (PCA) shows that the dissimilarities stem from interhelical rather than intrahelical motions. The difference in the hydrophobic thicknesses of these simulated lipid bilayers is potentially a significant reason for the observed difference in results. The distinct lipid headgroups might also lead to different molecular interactions and hence different protein loop motions. Overall, the A2a AR shows higher mobility and flexibility in POPC as compared to POPE

Irreversible growth plate fusions in children with medulloblastoma treated with a targeted hedgehog pathway inhibitor

The permanent defects in bone growth observed in preclinical studies of hedgehog (Hh) pathway inhibitors were not substantiated in early phase clinical studies of vismodegib in children. Consequently, vismodegib advanced into pediatric trials for malignancies suspected of being driven by aberrant activation of the Hh pathway. In one multicenter phase II trial, vismodegib was added to the therapy regimen for newly diagnosed Hh pathway activated medulloblastoma. Herein, we report on 3 children (2 on trial and one off trial) treated with vismodegib who developed widespread growth plate fusions that persist long after cessation of therapy. Currently, all 3 patients exhibit profound short stature and disproportionate growth, and 2 subsequently developed precocious puberty. Notably, the growth plate fusions only developed after a prolonged exposure to the drug (> 140 days). These findings resulted in a major trial amendment to restrict the agent to skeletally mature patients as well as a product label warning and update. Moreover, these findings alter the risk-benefit ratio of Hh inhibitors and underscore the importance of careful study of targeted agents in children