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Numerous studies suggest that the majority of amyloid- β (A β ) peptides deposited in Alzheimer's disease (AD) are truncated and post-translationally modified at the N terminus. Among these modified species, pyroglutamyl-A β (pE-A β , including N3pE-A β 40/42 and N11pE-A β 40/42) has been identified as particularly neurotoxic. The N-terminal modification renders the peptide hydrophobic, accelerates formation of oligomers, and reduces degradation by peptidases, leading ultimately to the accumulation of the peptide and progression of AD. It has been shown that the formation of pyroglutamyl residues is catalyzed by glutaminyl cyclase (QC). Here, we present data about the pharmacological in vitro and in vivo efficacy of the QC inhibitor (S)-1-(1H-benzo[d]imidazol-5-yl)-5-(4-propoxyphenyl)imidazolidin-2-one (PQ912), the first-in-class compound that is in clinical development. PQ912 inhibits human, rat, and mouse QC activity, with K i values ranging between 20 and 65 nM. Chronic oral treatment of hAPP SL xhQC double-transgenic mice with approximately 200 mg/kg/day via chow shows a significant reduction of pE-A β levels and concomitant improvement of spatial learning in a Morris water maze test paradigm. This dose results in a brain and cerebrospinal fluid concentration of PQ912 which relates to a QC target occupancy of about 60%. Thus, we conclude that >50% inhibition of QC activity in the brain leads to robust treatment effects. Secondary pharmacology experiments in mice indicate a fairly large potency difference for A β cyclization compared with cyclization of physiologic substrates, suggesting a robust therapeutic window in humans. This information constitutes an important translational guidance for predicting the therapeutic dose range in clinical studies with PQ912., (Copyright © 2017 by The Author(s).)

Rheumatoid arthritis is characterised by synovial inflammation and proliferation of fibroblast-like synoviocytes. The induction of apoptosis has long been proposed as a target for proliferative autoimmune diseases, and has further been shown to act as a successful treatment of experimental models of arthritis, such as collagen-induced arthritis. Here we examined the effects of specific oral small-molecule inhibitors of the transcription regulating cyclin-dependent kinase 9 on the development and progression of collagen-induced arthritis. DBA/1 mice were immunised with bovine collagen type II and treated orally with specific CDK9 inhibitors. The effects of CDK9 inhibition on RNA levels and protein expression, apoptosis induction, caspase activation and lymphocyte phenotype were further analysed. Mice showed a significant delay in disease onset and a reduction in disease severity following treatment with CDK9 inhibitors. Inhibiting CDK9 activity in peripheral blood mononuclear cells resulted in the loss of Mcl-1 expression at both the protein and RNA levels, along with a subsequent increase in apoptosis. CDK9 specific inhibitors may be a potential alternative treatment not only of cancer, but also for autoimmune- and inflammatory diseases. Taken together, these results show that transient inhibition of CDK9 induces apoptosis in leukocyte subsets and modulates the immune response.

Inflammation is an integral part of non-alcoholic fatty liver disease (NAFLD), the most prevalent form of hepatic pathology found in the general population. In this context, recently we have examined the potential role of Glutaminyl Cyclases (QC and isoQC), and their inhibitors, in the maturation of chemokines, for example, monocyte chemoattractant protein 1 (MCP-1, CCL2), to generate their bioactive conformation. Catalysis by isoQC leads to the formation of an N-terminal pyroglutamate residue protecting CCL2 against degradation by aminopeptidases. This is of importance because truncated forms possess a reduced potential to attract immune cells. Since liver inflammation is characterized by the up-regulation of different chemokine pathways, and within this CCL2 is known to be a prominent example, we hypothesised that application of QC/isoQC inhibitors may alleviate liver inflammation by destabilizing CCL2. Therefore, we investigated the role of QC/isoQC inhibition, in comparison with the angiotensin receptor blocker Telmisartan, during development of pathology in a mouse model of non-alcoholic fatty liver disease. Application of a QC/isoQC inhibitor led to a significant reduction in circulating alanine aminotransferase and NAFLD activity score accompanied by an inhibitory effect on hepatocyte ballooning. Further analysis revealed a specific reduction of inflammation by decreasing the number of F4/80-positive macrophages, which is in agreement with the proposed CCL2-related mechanism of action of QC/isoQC inhibitors. Finally, QC/isoQC inhibitor application attenuated liver fibrosis as characterized by reduced collagen deposition in the liver parenchyma. Thus in conclusion, QC/isoQC inhibitors are a promising novel class of anti-non-alcoholic steatohepatitis drugs which have a comparable disease-modifying effect to that of Telmisartan, which is probably mediated via specific interference with a comparable monocyte/macrophage infiltration that occurs under inflammatory conditions., (© 2013 The Authors. International Journal of Experimental Pathology © 2013 International Journal of Experimental Pathology.)

Background: Septic arthritis is a severe and rapidly debilitating disease mainly caused by Staphylococcus aureus. Here, we assess the antiarthritic efficiency of glutaminyl cyclase (QC) inhibitors., Methods: Mice were inoculated with an arthritogenic amount of S. aureus intravenously or by local administration into the knee joint. Animals were treated with QC inhibitors (PBD155 and PQ529) via chow during the experiment. QC and isoQC knockout mice were also analyzed for arthritis symptoms after local administration of bacteria., Results: Both QC inhibitors significantly delayed the onset of clinical signs of arthritis, and inhibitors significantly decreased weight loss in treated animals. Following intraarticular injection of S. aureus, PBD155-treated mice had lower levels of synovitis and bone erosion, as well as less myeloperoxidase in synovial tissue. Fluorescence-activated cell sorter analysis revealed that PBD155 treatment affected the expression pattern of adhesion molecules, preventing the upregulation of cells expressing CD11b/CD18., Conclusion: The compounds investigated here represent a novel class of small molecular antiarthritic inhibitors. In our studies, they exerted strong antiinflammatory actions, and therefore they might be suited for disease-modifying treatment of infectious arthritis.

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate-modified amyloid peptides deposited in neurodegenerative disorders such as Alzheimer's disease. Inhibitors of QC are currently in development as potential therapeutics. The crystal structures of the potent inhibitor PBD150 bound to human and murine QC (hQC, mQC) have been described recently. The binding modes of a dimethoxyphenyl moiety of the inhibitor are significantly different between the structures, which contrasts with a similar K(i) value. We show the conformation of PBD150 prone to disturbance by protein-protein interactions within the crystals. Semi-empirical calculations of the enzyme-inhibitor interaction within the crystal suggest significant differences in the dissociation constants between the binding modes. To probe for interactions in solution, a site-directed mutagenesis on hQC was performed. The replacement of F325 and I303 by alanine or asparagine resulted in a 800-fold lower activity of the inhibitor, whereas the exchange of S323 by alanine or valine led to a 20-fold higher activity of PBD150. The results provide an example of deciphering the interaction mode between a target enzyme and lead substance in solution, if co-crystallization does not mirror such interactions properly. Thus, the study might provide implications for rapid screening of binding modes also for other drug targets., (© 2012 John Wiley & Sons A/S.)

The inhibition of human glutaminyl cyclase (hQC) has come into focus as a new potential approach for the treatment of Alzheimer's disease. The hallmark of this principle is the prevention of the formation of Abeta(3,11(pE)-40,42), as these Abeta-species were shown to be of elevated neurotoxicity and likely to act as a seeding core leading to an accelerated formation of Abeta-oligomers and fibrils. Starting from 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea, bioisosteric replacements led to the development of new classes of inhibitors. The optimization of the metal-binding group was achieved by homology modeling and afforded a first insight into the probable binding mode of the inhibitors in the hQC active site. The efficacy assessment of the hQC inhibitors was performed in cell culture, directly monitoring the inhibition of Abeta(3,11(pE)-40,42) formation.

Because of their abundance, resistance to proteolysis, rapid aggregation and neurotoxicity, N-terminally truncated and, in particular, pyroglutamate (pE)-modified Abeta peptides have been suggested as being important in the initiation of pathological cascades resulting in the development of Alzheimer's disease. We found that the N-terminal pE-formation is catalyzed by glutaminyl cyclase in vivo. Glutaminyl cyclase expression was upregulated in the cortices of individuals with Alzheimer's disease and correlated with the appearance of pE-modified Abeta. Oral application of a glutaminyl cyclase inhibitor resulted in reduced Abeta(3(pE)-42) burden in two different transgenic mouse models of Alzheimer's disease and in a new Drosophila model. Treatment of mice was accompanied by reductions in Abeta(x-40/42), diminished plaque formation and gliosis and improved performance in context memory and spatial learning tests. These observations are consistent with the hypothesis that Abeta(3(pE)-42) acts as a seed for Abeta aggregation by self-aggregation and co-aggregation with Abeta(1-40/42). Therefore, Abeta(3(pE)-40/42) peptides seem to represent Abeta forms with exceptional potency for disturbing neuronal function. The reduction of brain pE-Abeta by inhibition of glutaminyl cyclase offers a new therapeutic option for the treatment of Alzheimer's disease and provides implications for other amyloidoses, such as familial Danish dementia.

A new inhibitor, H-Ala-Ile-pyrrolidin-2-yl boronic acid, was developed as an inhibitor against prolyl tripeptidyl aminopeptidase with a K(i) value of 88.1 nM. The structure of the prolyl tripeptidyl aminopeptidase complexed with the inhibitor (enzyme-inhibitor complex) was determined at 2.2 A resolution. The inhibitor was bound to the active site through a covalent bond between Ser603 and the boron atom of the inhibitor. This structure should closely mimic the structure of the reaction intermediate between the enzyme and substrate. We previously proposed that two glutamate residues, Glu205 and Glu636, are involved in the recognition of substrates. In order to clarify the function of these glutamate residues in substrate recognition, three mutant enzymes, E205A, E205Q, and E636A were generated by site-directed mutagenesis. The E205A mutant was expressed as an inclusion body. The E205Q mutant was expressed in soluble form, but no activity was detected. Here, the structures of the E636A mutant and its complex with the inhibitor were determined. The inhibitor was located at almost the same position as in the wild-type enzyme-inhibitor complex. The amino group of the inhibitor interacted with Glu205 and the main-chain carbonyl group of Gln203. In addition, a water molecule in the place of Glu636 of the wild-type enzyme interacted with the amino group of the inhibitor. This water molecule was located near the position of Glu636 in the wild-type and formed a hydrogen bond with Gln203. The k(cat)/K(M) values of the E636A mutant toward the two substrates used were smaller than those of the wild-type by two orders of magnitude. The K(i) value of our inhibitor for the E636A mutant was 48.8 microM, which was 554-fold higher than that against the wild-type enzyme. Consequently, it was concluded that Glu205 and Glu636 are significant residues for the N-terminal recognition of a substrate.