Steven M. Pitzenberger

High Fatty Acid Content in Rabbit Serum Is Responsible for the Differentiation of Osteoblasts Into Adipocyte‐like Cells

Osteoblasts and adipocytes originate from common mesenchymal precursors. With aging, there is a decrease in osteoprogenitor cells that parallels an increase of adipocytes in bone marrow. We observed that rabbit serum (RS) induces adipocyte‐like differentiation in human osteosarcoma SaOS‐2/B10 and MG‐63 cell lines, in rat ROS17/2.8 cells, and in mouse calvaria‐derived osteoblastic MB1.8 cells, as evidenced by the accumulation of Oil Red O positive lipid vesicles and the decrease in alkaline phosphatase expression. Both SaOS‐2/B10 and MG‐63 cells, but not ROS17/2.8 nor MB1.8 cells, express significant levels of PPARγ mRNA, a member of the peroxisome proliferator activated receptor (PPAR) family that has been implicated in the control of adipocyte differentiation. However, both ROS17/2.8 and MG‐63 cells express significant levels of the adipocyte selective marker, aP2 fatty acid binding mRNA, which can be further increased by RS. These cell types express PPARδ/NUC‐1 but not PPARα, indicating that cells that do not express either PPARγ or PPARα are capable of differentiating into adipocyte‐like cells. Transfection experiments in COS cells showed that compared with fetal bovine serum (FBS), RS is rich in agents that stimulate PPAR‐dependent transcription. The stimulatory activity was ethyl acetate extractable and was 35‐fold more abundant in RS than in FBS. Purification and analysis revealed that the major components of this extract are free fatty acids. Furthermore, the same fatty acids, a mixture of palmitic, oleic, and linoleic acids, activate the PPARs and induce adipocyte‐like differentiation of both ROS17/2.8 and SaOS‐2/B10 cells. These findings suggest that fatty acids or their metabolites can initiate the switch from osteoblasts to adipocyte‐like cells.

Characterization of a solid state reaction product from a lyophilized formulation of a cyclic heptapeptide. A novel example of an excipient-induced oxidation

AbstractPurpose. To elucidate the structure of a degradation product arising from a lyophilized formulation of a cyclic heptapeptide, and to provide a mechanism to account for its formation.nMethods. Preparative HPLC was used to isolate the degradate in quantities sufficient for structural studies. A structure assignment was made on the basis of the compounds spectroscopic properties (UV, MS, NMR) and the results of amino acid analysis.nResults. The degradate was identified as a benzaldehyde derivative arising from the oxidative deamination of an aminomethyl phenylalanine moiety. The extent of formation of this product is influenced by the amount of mannitol used as an excipient in the formulation. A mechanism is proposed whereby reducing sugar impurities in mannitol act as an oxidizing agent via the intermediacy of Schiff base adducts which subsequently undergo tautomerization and hydrolysis.nConclusions. Reducing sugar impurities in mannitol are responsible for the oxidative degradation of the peptide via a mechanism that involves Schiff base intermediates. This mechanism may be a potential route of degradation of other arylmethyl amines in mannitol-based formulations.

Identification of fatty acid methyl ester as naturally occurring transcriptional regulators of the members of the peroxisome proliferator-activated receptor family.

The nuclear hormone receptors NUC-1 (PPARξ) and PPARα are members of the peroxisome proliferator-activated receptor (PPAR) family. The members of this receptor family are activated by agents that stimulate peroxisome proliferation, free fatty acids, prostaglandin J2 metabolites, and agents considered for the therapy of insulin-independent diabetes mellitus. To identify putative physiological agents that activate NUC-1, we tested the ability of acetone extracts of various rat tissues to activate the transcription of an MMTV-luciferase reporter gene, via a GR/NUC-1 hybrid receptor. GR/NUC-1 contains the ligand binding region of the NUC-1 receptor and the DNA binding domain of the glucocorticoid receptor. Using this assay, we found stimulatory activity in the pancreas, which upon purification and characterization was identified as methylpalmitate, known to be enriched in pancreatic lipids. In addition, we determined that ethyl esters of palmitic and oleic acids are also potent activators of this receptor. thus, fatty acid ester formation may control the cellular concentrations of fatty acids, and acyl-ester formation may play a role in the control of metabolic pathways and the activation of the PPAR.

Design and Synthesis of 2,3,5-Substituted Imidazolidin-4-one Inhibitors of BACE-1

Alzheimer’s disease is a slowly progressing neurodegenerative condition that is increasing in prevalence because of the ageing population and is a significant healthcare burden. Although the pathophysiology of the disease has not been completely elucidated, abnormal production and/or clearance of a small peptide called Ab has been implicated from genetic and other studies. The Ab peptide arises from proteolytic processing of the APP protein, first by b-secretase followed by g-secretase. Based on these observations, b-secretase (BACE-1) has been identified as a promising drug target for disease-modifying therapy and has attracted significant attention from the medicinal chemistry community. BACE-1 is an aspartyl protease with a site of action inside the CNS and thus represents a challenging target. Hydroxyethylamine (HEA) transition state isosteres are well-known inhibitory motifs for aspartyl proteases, and prior work from these laboratories identified 1 (Figure 1) as a potent inhibitor of BACE-1. Whereas this compound has good enzyme potency (IC50=11 nm) and cell activity (sAPPb_NF= 29 nm), molecular weight is still high (578 Daltons) and the compound is a P-glycoprotein transporter (PGP) substrate with poor brain penetration. To improve brain-penetration properties, truncation of the structure and removal of hydrogen bond donors and acceptors was deemed necessary. One approach is to further optimize the P3-P1 portion and eliminate the hydroxyethyl amine. The work described in this report instead focuses on prime side modifications with the goal of improving potency sufficiently to allow removal of the P2P3 portion of the inhibitor. Closer examination of the X-ray crystal structure of 1 in the BACE-1 active site shows that the basic amine makes hydrogen-bond contacts to both Asp228 and the Gly34 carbonyl (Figure 2), and SAR studies have shown that primary and secondary amines are preferred over tertiary amines in this posi-

Three-dimensional structure of echistatin and dynamics of the active site

SummaryThe snake venom protein echistatin contains the cell recognition sequence Arg-Gly-Asp and is a potent inhibitor of platelet aggregation. The three-dimensional structure of echistatin and the dynamics of the active RGD site are presented. A set of structures was determined using the Distance Geometry method and subsequently refined by Molecular Dynamics and energy minimization. Disulfide pairings are suggested, based on violations of experimental constraints. The structures satisfy 230 interresidue distance constraints, derived from nuclear Overhauser effect measurements, five hydrogen-bonding constraints, and 21 torsional constraints from vicinal spin-spin coupling constants. The segment from Gly5 to Cys20 and from Asp30 to Asn42 has a well-defined conformation and the Arg-Gly-Asp sequence, which adopts a turn-like structure, is located at the apex of a nine-residue loop connecting the two strands of a distorted β-sheet. The mobility of the Arg-Gly-Asp site has been quantitatively characterized by 15N relaxation measurements. The overall correlation time of echistatin was determined from fluorescence measurements, and was used in a model-free analysis to determine internal motional parameters. The active site has order parameters of 0.3–0.5, i.e., among the smallest values ever observed at the active site of a protein. Correlation of the flexible region of the protein as characterized by relaxation experiments and the NMR solution structures was made by calculating generalized order parameters from the ensemble of three-dimensional structures. The motion of the RGD site detected experimentally is more extensive than a simple RGD loop ‘wagging’ motional model, suggested by an examination of superposed solution structures.