Discovery of selective fragment-sized immunoproteasome inhibitors.

Proteasomes contribute to maintaining protein homeostasis and their inhibition is beneficial in certain types of cancer and in autoimmune diseases. However, the inhibition of the proteasomes in healthy cells leads to unwanted side-effects and significant effort has been made to identify inhibitors specific for the immunoproteasome, especially to treat diseases which manifest increased levels and activity of this proteasome isoform. Here, we report our efforts to discover fragment-sized inhibitors of the human immunoproteasome. The screening of an in-house library of structurally diverse fragments resulted in the identification of benzo[d]oxazole-2(3H)-thiones, benzo[d]thiazole-2(3H)-thiones, benzo[d]imidazole-2(3H)-thiones, and 1-methylbenzo[d]imidazole-2(3H)-thiones (with a general term benzoXazole-2(3H)-thiones) as inhibitors of the chymotrypsin-like (ß5i) subunit of the immunoproteasome. A subsequent structure-activity relationship study provided us with an insight regarding growing vectors. Binding to the ß5i subunit was shown and selectivity against the ß5 subunit of the constitutive proteasome was determined. Thorough characterization of these compounds suggested that they inhibit the immunoproteasome by forming a disulfide bond with the Cys48 available specifically in the ß5i active site. To obtain fragments with biologically more tractable covalent interactions, we performed a warhead scan, which yielded benzoXazole-2-carbonitriles as promising starting points for the development of selective immunoproteasome inhibitors with non-peptidic scaffolds.

Discovery of Immunoproteasome Inhibitors Using Large-Scale Covalent Virtual Screening.

Large-scale virtual screening of boronic acid derivatives was performed to identify nonpeptidic covalent inhibitors of the ß5i subunit of the immunoproteasome. A hierarchical virtual screening cascade including noncovalent and covalent docking steps was applied to a virtual library of over 104,000 compounds. Then, 32 virtual hits were selected, out of which five were experimentally confirmed. Biophysical and biochemical tests showed micromolar binding affinity and time-dependent inhibitory potency for two compounds. These results validate the computational protocol that allows the screening of large compound collections. One of the lead-like boronic acid derivatives identified as a covalent immunoproteasome inhibitor is a suitable starting point for chemical optimization.

Drug discovery strategies and the preclinical development of D-amino-acid oxidase inhibitors as antipsychotic therapies.

INTRODUCTION: D-amino-acid oxidase (DAAO) degrades D-serine, a co-agonist of the NMDA receptor whose dysfunction is involved in the positive, negative, and cognitive symptoms of schizophrenia. The inhibition of DAAO appears to be a viable strategy to increase D-serine level and to have therapeutic potential in schizophrenia. Areas covered: This review describes the efforts to develop DAAO inhibitors and to optimize their in vitro and in vivo effects in preclinical settings. The structural evolution of DAAO inhibitors is presented from simple carboxylic acid derivatives via small, planar compounds with carboxylic acid mimetics to extended compounds whose binding is possible owing to DAAO flexibility. Inhibitory potency and pharmacokinetic properties are discussed in the context of compounds' ability to increase D-serine level and to show efficacy in animal models of schizophrenia. Expert opinion: The accumulated knowledge on the structural requirements of DAAO inhibitors and on their in vitro and in vivo effects provides appropriate basis to develop inhibitors with optimized potency, selectivity and pharmacokinetic profile including blood-brain penetration. In addition, the validation of DAAO inhibition therapy in alleviating the symptoms of schizophrenia requires further studies on the efficacy of DAAO inhibitors in behavioral assays of animals and on the species differences in D-serine metabolism.

Discovery of d-amino acid oxidase inhibitors based on virtual screening against the lid-open enzyme conformation.

d-Amino acid oxidase (DAAO) inhibitors are typically small polar compounds with often suboptimal pharmacokinetic properties. Features of the native binding site limit the operational freedom of further medicinal chemistry efforts. We therefore initiated a structure based virtual screening campaign based on the X-ray structures of DAAO complexes where larger ligands shifted the loop (lid opening) covering the native binding site. The virtual screening of our in-house collection followed by the in vitro test of the best ranked compounds led to the identification of a new scaffold with micromolar IC50. Subsequent SAR explorations enabled us to identify submicromolar inhibitors. Docking studies supported by in vitro activity measurements suggest that compounds bind to the active site with a salt-bridge characteristic to DAAO inhibitor binding. In addition, displacement of and interaction with the loop covering the active site contributes significantly to the activity of the most potent compounds.

Structure-based Virtual Screening Approaches in Kinase-directed Drug Discovery.

Protein kinases are one of the most targeted protein families in current drug discovery pipelines. They are implicated in many oncological, inflammatory, CNS-related and other clinical indications. Virtual screening is a computational technique with a diverse set of available tools that has been shown many times to provide novel starting points for kinase-directed drug discovery. This review starts with a concise overview of the function, structural features and inhibitory mechanisms of protein kinases. In addition to briefly reviewing the practical aspects of structure-based virtual screenings, we discuss several case studies to illustrate the state of the art in the virtual screening for type I, type II, allosteric (type III-V) and covalent (type VI) kinase inhibitors. With this review, we strive to provide a summary of the latest advances in the structure-based discovery of novel kinase inhibitors, as well as a practical tool to anyone who wishes to embark on such an endeavor.

Ensemble docking-based virtual screening yields novel spirocyclic JAK1 inhibitors.

Small molecule inhibition of Janus kinases (JAKs) has been demonstrated as a viable strategy for the treatment of various inflammatory conditions and continues to emerge in cancer-related indications. In this study, a large supplier database was screened to identify novel chemistry starting points for JAK1. The docking-based screening was followed up by testing ten hit compounds experimentally, out of which five have displayed single-digit micromolar and submicromolar IC50 values on JAK1. Thus, the study was concluded with the discovery of five novel JAK inhibitors from a tiny screening deck with a remarkable hitrate of 50%. The results have highlighted spirocyclic pyrrolopyrimidines with submicromolar JAK1 IC50 values and a preference for JAK1 over JAK2 as potential starting points in developing a novel class of JAK1 inhibitors.

Discovery of Subtype Selective Janus Kinase (JAK) Inhibitors by Structure-Based Virtual Screening.

Janus kinase inhibitors represent a promising opportunity for the pharmaceutical intervention of various inflammatory and oncological indications. Subtype selective inhibition of these enzymes, however, is still a very challenging goal. In this study, a novel, customized virtual screening protocol was developed with the intention of providing an efficient tool for the discovery of subtype selective JAK2 inhibitors. The screening protocol involves protein ensemble-based docking calculations combined with an Interaction Fingerprint (IFP) based scoring scheme for estimating ligand affinities and selectivities, respectively. The methodology was validated in retrospective studies and was applied prospectively to screen a large database of commercially available compounds. Six compounds were identified and confirmed in vitro, with an indazole-based hit exhibiting promising selectivity for JAK2 vs JAK1. Having demonstrated that the described methodology is capable of identifying subtype selective chemical starting points with a favorable hit rate (11%), we believe that the presented screening concept can be useful for other kinase targets with challenging selectivity profiles.