Inhibiting the immunoproteasome's ß5i catalytic activity affects human peripheral blood-derived immune cell viability.

Small molecule inhibitors selectively targeting the immunoproteasome subunit ß5i are currently being developed for the treatment of autoimmune disorders. However, patients carrying loss-of-function mutations in the gene encoding ß5i (Psmb8) suffer from the proteasome-associated autoinflammatory syndromes (PRAAS) emphasizing the need to study pharmacological inhibition of immunoproteasome function in human cells. Here, we characterized the immunomodulatory potential of the selective ß5i inhibitor ONX 0914 and Bortezomib, a pan-proteasome inhibitor, in human peripheral blood mononuclear cells (PBMCs). Both compounds efficiently blocked pro-inflammatory cytokine secretion in human whole blood and PBMC cultures stimulated with toll-like receptor (TLR) agonists. Furthermore, the compounds inhibited T cell cytokine production induced by recall antigen CMVpp65 or by polyclonal stimulation. The viability of PBMCs, however, was rapidly decreased in the presence of ONX 0914 and Bortezomib demonstrated by decreased residual cytosolic ATP and increased Annexin V surface binding. Interestingly, HLA-DR + monocytes were rapidly depleted from the cultures in the presence of ONX 0914 as a ß5i-selective inhibitor and Bortezomib. In conclusion, the anti-inflammatory potential of ß5i-selective inhibitors is correlating with a cytotoxicity increase in human PBMC subsets ex vivo. Our results provide important insights into the anti-inflammatory mechanism of action of ß5i-inhibitors which currently hold the promise as a novel therapy for autoinflammatory diseases.

Fragment-Based Discovery of Novel Potent Sepiapterin Reductase Inhibitors.

Genome-wide-association studies in chronic low back pain patients identified sepiapterin reductase as a high interest target for developing new analgesics. Here we used 19F NMR fragment screening for the discovery of novel, ligand-efficient SPR inhibitors. We report the crystal structures of six chemically diverse inhibitors complexed with SPR, identifying relevant interactions and binding modes in the sepiapterin pocket. Exploration of our initial fragment screening hit led to double-digit nanomolar inhibitors of SPR with excellent ligand efficiency.

The design of potent and selective inhibitors of DPP-4: optimization of ADME properties by amide replacements.

For a series of beta-homophenylalanine based inhibitors of dipeptidyl peptidase IV ADME properties were improved by the incorporation of amide replacements. These efforts led to a novel series of potent and selective inhibitors of DPP-4 that exhibit an attractive pharmacokinetic profile and show excellent efficacy in an animal model of diabetes.

From lead to preclinical candidate: optimization of beta-homophenylalanine based inhibitors of dipeptidyl peptidase IV.

A series of highly potent and selective inhibitors of DPP-4 was optimized for ADMET properties. The effort resulted in the discovery of inhibitor 1g, that exhibits excellent efficacy in an oral glucose tolerance test and an attractive pharmacokinetic profile.

Discovery of beta-homophenylalanine based pyrrolidin-2-ylmethyl amides and sulfonamides as highly potent and selective inhibitors of dipeptidyl peptidase IV.

Modifications of DPP-4 inhibitor 5, that was discovered by structure based design, are described and structure-activity relationships discussed. With analogue 7k one of the most potent non-covalent inhibitors of DPP-4 reported to date (IC(50)=0.38nM) was discovered. X-ray structure of inhibitor 7k bound to DPP-4 revealed a hydrogen bonding interaction with Q553. First successful efforts in balancing overall properties, as demonstrated by improved metabolic stability, highlight the potential of this series.

Orally available selective melanocortin-4 receptor antagonists stimulate food intake and reduce cancer-induced cachexia in mice.

BACKGROUND: Cachexia is among the most debilitating and life-threatening aspects of cancer. It represents a metabolic syndrome affecting essential functional circuits involved in the regulation of homeostasis, and includes anorexia, fat and muscle tissue wasting. The anorexigenic peptide alpha-MSH is believed to be crucially involved in the normal and pathologic regulation of food intake. It was speculated that blockade of its central physiological target, the melanocortin (MC)-4 receptor, might provide a promising anti-cachexia treatment strategy. This idea is supported by the fact that in animal studies, agouti-related protein (AgRP), the endogenous inverse agonist at the MC-4 receptor, was found to affect two hallmark features of cachexia, i.e. to increase food intake and to reduce energy expenditure. METHODOLOGY/PRINCIPAL FINDINGS: SNT207707 and SNT209858 are two recently discovered, non peptidic, chemically unrelated, orally active MC-4 receptor antagonists penetrating the blood brain barrier. Both compounds were found to distinctly increase food intake in healthy mice. Moreover, in mice subcutaneously implanted with C26 adenocarcinoma cells, repeated oral administration (starting the day after tumor implantation) of each of the two compounds almost completely prevented tumor induced weight loss, and diminished loss of lean body mass and fat mass. CONCLUSIONS/SIGNIFICANCE: In contrast to the previously reported peptidic and small molecule MC-4 antagonists, the compounds described here work by the oral administration route. Orally active compounds might offer a considerable advantage for the treatment of cachexia patients.

The reversed binding of beta-phenethylamine inhibitors of DPP-IV: X-ray structures and properties of novel fragment and elaborated inhibitors.

The co-crystal structure of beta-phenethylamine fragment inhibitor 5 bound to DPP-IV revealed that the phenyl ring occupied the proline pocket of the enzyme. This finding provided the basis for a general hypothesis of a reverse binding mode for beta-phenethylamine-based DPP-IV inhibitors. Novel inhibitor design concepts that obviate substrate-like structure-activity relationships (SAR) were thereby enabled, and novel, potent inhibitors were discovered.

In silico fragment-based discovery of DPP-IV S1 pocket binders.

Dipeptidyl peptidase IV is a clinically validated target for type-2 diabetes and belongs to a family of peptidases with a quite unique post-proline cleavage specificity. Known inhibitors contain a limited number of molecular anchors occupying the small prototypical S1 pocket. A virtual screening approach for such S1-binding fragments was carried out using FlexX docking to evaluate its potential to confirm known and find novel compounds. Several low molecular weight inhibitors exhibiting activities in the micromolar range could be identified as starting points for structure-based design.

A novel biosynthetic pathway providing precursors for fatty acid biosynthesis and secondary metabolite formation in myxobacteria.

Short chain carboxylic acids are well known as the precursors of fatty acid and polyketide biosynthesis. Iso-fatty acids, which are important for the control of membrane fluidity, are formed from branched chain starter units (isovaleryl-CoA and isobutyryl-CoA), which in turn are derived from the degradation of leucine and valine, respectively. Branched chain carboxylic acids are also employed as starter molecules for the biosynthesis of secondary metabolites, e.g. the therapeutically important anthelmintic agent avermectin or the electron transport inhibitor myxothiazol. During our studies on myxothiazol biosynthesis in the myxobacterium, Stigmatella aurantiaca, we detected a novel biosynthetic route to isovaleric acid. After cloning and inactivation of the branched chain keto acid dehydrogenase complex, which is responsible for the degradation of branched chain amino acids, the strain is still able to produce iso-fatty acids and myxothiazol. Incorporation studies employing deuterated leucine show that it can only serve as precursor in the wild type strain but not in the esg mutant. Feeding experiments using (13)C-labeled precursors show that isovalerate is efficiently made from acetate, giving rise to a labeling pattern in myxothiazol that provides evidence for a novel branch of the mevalonate pathway involving the intermediate 3,3-dimethylacryloyl-CoA. 3,3-Dimethylacrylic acid was synthesized in deuterated form and fed to the esg mutant, resulting in strong incorporation into myxothiazol and iso-fatty acids. Similar experiments employing Myxococcus xanthus revealed that the discovered biosynthetic route described is present in other myxobacteria as well.