Design of (ω-N-(O-acyl)hydroxy amid) aminodicarboxylic acid pyrrolidides as potent inhibitors of proline-specific peptidases

AbstractA novel class of competitive, acylating inhibitors for the proline-specific peptidases: dipeptidyl peptidase IV, dipeptidyl peptidase II and prolyl endopeptidase, has been developed. The inhibitor molecules combine the efficacy of aminoacyl pyrrolidides and the potential transacylating capability of diacyl hydroxyl amines. The N-terminal deblocked inhibitors are potent reversible inhibitors of porcine kidney dipeptidyl peptidase IV, human placenta dipeptidyl peptidase II exhibiting K1 values in the μM range. Boc-protected (ω-N-hydroxy acyl amid) aminodiacarboxylic acid pyrrolidides inhibit substrate hydrolysis by prolyl endopeptidases from different sources competitively reaching K, values of 30 nM to 60 μM. Additionally, α-N-BOC-(ω-N-hydroxy acetyl) glutaminyl pyrrolidide modifies human placenta prolyl endopeptidase in a time-dependent reaction

Methodology

© The Author(s) 2019. A detailed overview of the methodologies used to develop the 2.0 °C and 1.5 °C scenario presented in this book. Starting with the overall modelling approach, the interaction of seven different models is explained which are used to calculate and developed detailed scenarios for greenhouse gas emission and energy pathways to stay within a 2.0 °C and 1.5 °C global warming limit. The following models are presented: For the non-energy GHG emission pathways, the Generalized Equal Quantile Walk (GQW)method, the land-based sequestration design method and the Carbon cycle and climate (MAGICC) model. For the energy pathways, a renewable energy resources assessment for space constrained environments ([R]E-SPACE, the transport scenario model (TRAEM), the Energy System Model (EM) and the power system model [R]E 24/7. The methodologies of an employment analysis model, and a metal resource assessment tool are outlined. These models have been used to examine the analysis of the energy scenario results

Distinct glutaminyl cyclase expression in Edinger–Westphal nucleus, locus coeruleus and nucleus basalis Meynert contributes to pGlu-Aβ pathology in Alzheimer’s disease

Glutaminyl cyclase (QC) was discovered recently as the enzyme catalyzing the pyroglutamate (pGlu or pE) modification of N-terminally truncated Alzheimer’s disease (AD) Aβ peptides in vivo. This modification confers resistance to proteolysis, rapid aggregation and neurotoxicity and can be prevented by QC inhibitors in vitro and in vivo, as shown in transgenic animal models. However, in mouse brain QC is only expressed by a relatively low proportion of neurons in most neocortical and hippocampal subregions. Here, we demonstrate that QC is highly abundant in subcortical brain nuclei severely affected in AD. In particular, QC is expressed by virtually all urocortin-1-positive, but not by cholinergic neurons of the Edinger–Westphal nucleus, by noradrenergic locus coeruleus and by cholinergic nucleus basalis magnocellularis neurons in mouse brain. In human brain, QC is expressed by both, urocortin-1 and cholinergic Edinger–Westphal neurons and by locus coeruleus and nucleus basalis Meynert neurons. In brains from AD patients, these neuronal populations displayed intraneuronal pE-Aβ immunoreactivity and morphological signs of degeneration as well as extracellular pE-Aβ deposits. Adjacent AD brain structures lacking QC expression and brains from control subjects were devoid of such aggregates. This is the first demonstration of QC expression and pE-Aβ formation in subcortical brain regions affected in AD. Our results may explain the high vulnerability of defined subcortical neuronal populations and their central target areas in AD as a consequence of QC expression and pE-Aβ formation

First insight into structure-activity relationships of selective meprin beta inhibitors

The astacin proteases meprin a and beta are emerging drug targets for treatment of disorders such as kidney failure, fibrosis or inflammatory bowel disease. However, there are only few inhibitors of both proteases reported to date. Starting from NNGH as lead structure, a detailed elaboration of the structure-activity relationship of meprin beta inhibitors was performed, leading to compounds with activities in the lower nanomolar range. Considering the preference of meprin 0 for acidic residues in the P1' position, the compounds were optimized. Acidic modifications induced potent inhibition and >100-fold selectivity over other structurally related metalloproteases such as MMP-2 or ADAM10

Purification of recombinant Aβ(1-42) and pGlu-Aβ(3-42) using preparative SDS-PAGE

Recombinant expression and purification of amyloid peptides represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. However, the isolation of the recombinant peptides is hampered by inefficient separation from contaminants such as the fusion protein required for efficient expression in E. coli. Here, we present a new approach for the isolation of highly purified Aβ(1-42) and pGlu-Aβ(3-42), which is based on a separation using preparative SDS-PAGE. The method relies on the purification of the Aβ fusion protein by affinity chromatography followed by preparative SDS-PAGE under reducing conditions and subsequent removal of detergents by precipitation. The application of preparative SDS-PAGE represents the key step to isolate highly pure recombinant Aβ, which has been applied for characterization of aggregation and toxicity. Thereby, the yield of the purification strategy was >60%. To the best of our knowledge, this is the first description of an electrophoresis-based method for purification of a recombinant Aβ peptide. Therefore, the method might be of interest for isolation of other amyloid peptides, which are critical for conventional purification strategies due to their aggregation propensity