Insulin receptor, AMPK and CPT-2: Crystallization experiments and structural characterization of three proteins relevant to diabetes mellitus

Impaired insulin signaling due to peripheral insulin resistance and failure of pancreatic insulin secretion is a key factor for the onset and manifestation of type 2 diabetes mellitus (T2D). Insulin receptor (IR) agonists are being developed to restore insulin signaling via direct activation of the cytoplasmic insulin receptor tyrosine kinase activity. Two classes of such compounds have been developed by the pharmaceutical companies Merck & Co., Inc., and Telik, Inc., but in patents and literature there is no data as to how these compounds activate the IR kinase activity. Attempts to solve the crystal structures of dimeric human IR kinase domains and complexes of these with IR agonists within this thesis failed. However, biophysical characterization of the full-length intracellular part of the IR containing the kinase domain demonstrated an intrinsic ability to dimerize independently of the enzyme-substrate type of interaction seen in IR autophosphorylation. This dimer formation was found to be enhanced in the presence of a distinct IR agonist. Crystals of various constructs and isoforms of the key antidiabetic target AMP-activated protein kinase (AMPK) were obtained. Despite substantial optimization trials these crystals were not sufficient for solving the structure of AMPK. However, the crystal structure of carnitine palmitoyltransferase 2 (CPT-2), a protein downstream of the metabolite signaling initiated by AMPK, was solved. CPT-1 and -2 facilitate the import of long-chain fatty acids into mitochondria. Modulation of the catalytic activity of the CPT system is currently under investigation for the development of novel drugs against diabetes mellitus. The crystal structure of the full-length mitochondrial membrane protein CPT-2 was solved at a resolution of 1.6 Å. The structure of CPT-2 in complex with the generic CPT-inhibitor ST1326 [(R)-N-tetradecylcarbamoyl-aminocarnitine], a substrate analog mimicking palmitoylcarnitine and currently in clinical trials for T2D treatment, was solved at 2.5 Å resolution. These structures of CPT-2 provide insight into the function of residues involved in substrate binding and determination of substrate specificity, thereby facilitating the rational design of novel antidiabetic drugs. A sequence insertion uniquely found in CPT-2 was identified that mediates membrane localization. Mapping of mutations described for CPT-2 deficiency, a hereditary disorder of lipid metabolism, implies effects on substrate recognition and structural integrity of CPT-2

Cathepsin S inhibition combines control of systemic and peripheral pathomechanisms of autoimmune tissue injury

Cathepsin(Cat)-S processing of the invariant chain-MHC-II complex inside antigen presenting cells is a central pathomechanism of autoimmune-diseases. Additionally, Cat-S is released by activated-myeloid cells and was recently described to activate protease-activated-receptor-(PAR)-2 in extracellular compartments. We hypothesized that Cat-S blockade targets both mechanisms and elicits synergistic therapeutic effects on autoimmune tissue injury. MRL-(Fas)lpr mice with spontaneous autoimmune tissue injury were treated with different doses of Cat-S inhibitor RO5459072, mycophenolate mofetil or vehicle. Further, female MRL-(Fas) lpr mice were injected with recombinant Cat-S with/without concomitant Cat-S or PAR-2 blockade. Cat-S blockade dose-dependently reversed aberrant systemic autoimmunity, e.g. plasma cytokines, activation of myeloid cells and hypergammaglobulinemia. Especially IgG autoantibody production was suppressed. Of note (MHC-II-independent) IgM were unaffected by Cat-S blockade while they were suppressed by MMF. Cat-S blockade dose-dependently suppressed immune-complex glomerulonephritis together with a profound and early effect on proteinuria, which was not shared by MMF. In fact, intravenous Cat-S injection induced severe glomerular endothelial injury and albuminuria, which was entirely prevented by Cat-S or PAR-2 blockade. In-vitro studies confirm that Cat-S induces endothelial activation and injury via PAR-2. Therapeutic Cat-S blockade suppresses systemic and peripheral pathomechanisms of autoimmune tissue injury, hence, Cat-S is a promising therapeutic target in lupus nephritis

X-ray structure of glutathione S-transferase from Schistosoma japonicum in a new crystal form reveals flexibility of the substrate-binding site

The crystal structure of the 26 kDa glutathione S-transferase from S. japonicum (SjGST) was determined at 3 Å resolution in the new space group P212121. The structure of orthorhombic SjGST reveals unique features of the ligand-binding site and dimer interface when compared with previously reported structures

Potent and Selective BACE-1 Peptide Inhibitors Lower Brain Aβ Levels Mediated by Brain Shuttle Transport

Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance