title: Toward a biorelevant structure of protein kinase C bound modulators: design, synthesis, and evaluation of labeled bryostatin analogues for analysis with rotational echo double resonance NMR spectroscopy.
abstract: Protein kinase C (PKC) modulators are currently of great importance in preclinical and clinical studies directed at cancer, immunotherapy, HIV eradication, and Alzheimer's disease. However, the bound conformation of PKC modulators in a membrane environment is not known. Rotational echo double resonance (REDOR) NMR spectroscopy could uniquely address this challenge. However, REDOR NMR requires strategically labeled, high affinity ligands to determine interlabel distances from which the conformation of the bound ligand in the PKC-ligand complex could be identified. Here we report the first computer-guided design and syntheses of three bryostatin analogues strategically labeled for REDOR NMR analysis. Extensive computer analyses of energetically accessible analogue conformations suggested preferred labeling sites for the identification of the PKC-bound conformers. Significantly, three labeled analogues were synthesized, and, as required for REDOR analysis, all proved highly potent with PKC affinities (∼1 nM) on par with bryostatin. These potent and strategically labeled bryostatin analogues are new structural leads and provide the necessary starting point for projected efforts to determine the PKC-bound conformation of such analogues in a membrane environment, as needed to design new PKC modulators and understand PKC-ligand-membrane structure and dynamics.
authors.affiliation: †Department of Chemistry and ‡Department of Chemical and Systems Biology, Stanford University, Stanford, California 94305, United States.
authors.forename: Brian A
authors.initials: BA
authors.lastname: Loy
authors.affiliation: †Department of Chemistry and ‡Department of Chemical and Systems Biology, Stanford University, Stanford, California 94305, United States.
authors.forename: Adam B
authors.initials: AB
authors.lastname: Lesser
authors.affiliation: †Department of Chemistry and ‡Department of Chemical and Systems Biology, Stanford University, Stanford, California 94305, United States.
authors.forename: Daryl
authors.initials: D
authors.lastname: Staveness
authors.affiliation: †Department of Chemistry and ‡Department of Chemical and Systems Biology, Stanford University, Stanford, California 94305, United States.
authors.forename: Kelvin L
authors.initials: KL
authors.lastname: Billingsley
authors.affiliation: †Department of Chemistry and ‡Department of Chemical and Systems Biology, Stanford University, Stanford, California 94305, United States.
authors.forename: Lynette
authors.initials: L
authors.lastname: Cegelski
authors.affiliation: †Department of Chemistry and ‡Department of Chemical and Systems Biology, Stanford University, Stanford, California 94305, United States.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Wender
mesh_terms: D054713:Bryostatins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D060326:Chemistry Techniques, Synthetic; D015195:Drug Design; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012399:Rotation
chemical_list: D054713:Bryostatins; D008024:Ligands; C046785:bryostatin 1; D011493:Protein Kinase C