Characterization of a small molecule inhibitor of plasminogen activator inhibitor type 1 that accelerates the transition into the latent conformation.

A novel class of small molecule inhibitors for plasminogen activator inhibitor type 1 (PAI-1), represented by AZ3976, was identified in a high throughput screening campaign. AZ3976 displayed an IC(50) value of 26 µm in an enzymatic chromogenic assay. In a plasma clot lysis assay, the compound was active with an IC(50) of 16 µm. Surprisingly, AZ3976 did not bind to active PAI-1 but bound to latent PAI-1 with a K(D) of 0.29 µm at 35 °C and a binding stoichiometry of 0.94, as measured by isothermal calorimetry. Reversible binding was confirmed by surface plasmon resonance direct binding experiments. The x-ray structure of AZ3976 in complex with latent PAI-1 was determined at 2.4 Å resolution. The inhibitor was bound in the flexible joint region with the entrance to the cavity located between α-helix D and ß-strand 2A. A set of surface plasmon resonance experiments revealed that AZ3976 inhibited PAI-1 by enhancing the latency transition of active PAI-1. Because AZ3976 only had measurable affinity for latent PAI-1, we propose that its mechanism of inhibition is based on binding to a small fraction in equilibrium with active PAI-1, a latent-like prelatent form, from which latent PAI-1 is then generated more rapidly. This mode of action, with induced accelerated latency transition of active PAI-1 may, together with supporting x-ray data, provide improved opportunities for small molecule drug design in the hunt for therapeutically useful PAI-1 inhibitors.

AZD0284, a Potent, Selective, and Orally Bioavailable Inverse Agonist of Retinoic Acid Receptor-Related Orphan Receptor C2.

Inverse agonists of the nuclear receptor RORC2 have been widely pursued as a potential treatment for a variety of autoimmune diseases. We have discovered a novel series of isoindoline-based inverse agonists of the nuclear receptor RORC2, derived from our recently disclosed RORC2 inverse agonist 2. Extensive structure-activity relationship (SAR) studies resulted in AZD0284 (20), which combined potent inhibition of IL-17A secretion from primary human TH17 cells with excellent metabolic stability and good PK in preclinical species. In two preclinical in vivo studies, compound 20 reduced thymocyte numbers in mice and showed dose-dependent reduction of IL-17A containing γδ-T cells and of IL-17A and IL-22 RNA in the imiquimod induced inflammation model. Based on these data and a favorable safety profile, 20 was progressed to phase 1 clinical studies.

Discovery of retinoic acid receptor agonists as proliferators of cardiac progenitor cells through a phenotypic screening approach.

Identification of small molecules with the potential to selectively proliferate cardiac progenitor cells (CPCs) will aid our understanding of the signaling pathways and mechanisms involved and could ultimately provide tools for regenerative therapies for the treatment of post-MI cardiac dysfunction. We have used an in vitro human induced pluripotent stem cell-derived CPC model to screen a 10,000-compound library containing molecules representing different target classes and compounds reported to modulate the phenotype of stem or primary cells. The primary readout of this phenotypic screen was proliferation as measured by nuclear count. We identified retinoic acid receptor (RAR) agonists as potent proliferators of CPCs. The CPCs retained their progenitor phenotype following proliferation and the identified RAR agonists did not proliferate human cardiac fibroblasts, the major cell type in the heart. In addition, the RAR agonists were able to proliferate an independent source of CPCs, HuES6. The RAR agonists had a time-of-differentiation-dependent effect on the HuES6-derived CPCs. At 4 days of differentiation, treatment with retinoic acid induced differentiation of the CPCs to atrial cells. However, after 5 days of differentiation treatment with RAR agonists led to an inhibition of terminal differentiation to cardiomyocytes and enhanced the proliferation of the cells. RAR agonists, at least transiently, enhance the proliferation of human CPCs, at the expense of terminal cardiac differentiation. How this mechanism translates in vivo to activate endogenous CPCs and whether enhancing proliferation of these rare progenitor cells is sufficient to enhance cardiac repair remains to be investigated.

Discovery of Potent and Orally Bioavailable Inverse Agonists of the Retinoic Acid Receptor-Related Orphan Receptor C2.

The further optimization of a recently disclosed series of inverse agonists of the nuclear receptor RORC2 is described. Investigations into the left-hand side of compound 1, guided by X-ray crystal structures, led to the substitution of the 4-aryl-thiophenyl residue with the hexafluoro-2-phenyl-propan-2-ol moiety. This change resulted in to compound 28, which combined improved drug-like properties with good cell potency and a significantly lower dose, using an early dose to man prediction. Target engagement in vivo was demonstrated in the thymus of mice by a reduction in the number of double positive T cells after oral dosing.

Potent and Orally Bioavailable Inverse Agonists of RORγt Resulting from Structure-Based Design.

Retinoic acid receptor related orphan receptor γt (RORγt), has been identified as the master regulator of TH17-cell function and development, making it an attractive target for the treatment of autoimmune diseases by a small-molecule approach. Herein, we describe our investigations on a series of 4-aryl-thienyl acetamides, which were guided by insights from X-ray cocrystal structures. Efforts in targeting the cofactor-recruitment site from the 4-aryl group on the thiophene led to a series of potent binders with nanomolar activity in a primary human-TH17-cell assay. The observation of a DMSO molecule binding in a subpocket outside the LBD inspired the introduction of an acetamide into the benzylic position of these compounds. Hereby, a hydrogen-bond interaction of the introduced acetamide oxygen with the backbone amide of Glu379 was established. This greatly enhanced the cellular activity of previously weakly cell-active compounds. The best compounds combined potent inhibition of IL-17 release with favorable PK in rodents, with compound 32 representing a promising starting point for future investigations.

Development of an ELISA for High-Throughput Screening of Inhibitors of Cdk5-Mediated PPARγ Phosphorylation.

The peroxisome proliferator-activated receptor gamma (PPARγ) is the target for the thiazolidinedione class of potent insulin-sensitizing drugs, which includes rosiglitazone and pioglitazone. However, their usage has been restricted due to severe side effects. Recent data have shown that specifically inhibiting the cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation of PPARγ at Ser273 may lead to novel insulin sensitizers with fewer side effects. Here we describe a novel enzyme-linked immunosorbent assay (ELISA) in the 384-well format, which enables screening for PPARγ ligands that inhibit phosphorylation at Ser273 by Cdk5. The assay is robust with a Z-factor > 0.6, demonstrating its suitability for high-throughput screening. We demonstrate the suitability of this assay for profiling of published PPARγ ligands and identification of novel compounds that prevent the Cdk5-mediated phosphorylation of PPARγ at Ser273 in a 622 compound pilot study. Our assay enables the discovery and development of novel therapeutic agents for use in type-2 diabetes. Furthermore, our results in combination with structural analysis of reported PPARγ ligand binding domain X-ray structures give a molecular rationale for the Cdk5-mediated phosphorylation of PPARγ at Ser273.

Benzoxazepines Achieve Potent Suppression of IL-17 Release in Human T-Helper 17 (TH 17) Cells through an Induced-Fit Binding Mode to the Nuclear Receptor RORγ.

RORγt, an isoform of the retinoic acid-related orphan receptor gamma (RORc, RORγ), has been identified as the master regulator of T-helper 17 (TH 17) cell function and development, making it an attractive target for the treatment of autoimmune diseases. Validation for this target comes from antibodies targeting interleukin-17 (IL-17), the signature cytokine produced by TH 17 cells, which have shown impressive results in clinical trials. Through focused screening of our compound collection, we identified a series of N-sulfonylated benzoxazepines, which displayed micromolar affinity for the RORγ ligand-binding domain (LBD) in a radioligand binding assay. Optimization of these initial hits resulted in potent binders, which dose-dependently decreased the ability of the RORγ-LBD to interact with a peptide derived from steroid receptor coactivator 1, and inhibited the release of IL-17 secretion from isolated and cultured human TH 17 cells with nanomolar potency. A cocrystal structure of inverse agonist 15 (2-chloro-6-fluoro-N-(4-{[3-(trifluoromethyl)phenyl]sulfonyl}-2,3,4,5-tetrahydro-1,4-benzoxazepin-7-yl)benzamide) bound to the RORγ-LBD illustrated that both hydrophobic interactions, leading to an induced fit around the substituted benzamide moiety of 15, as well as a hydrogen bond from the amide NH to His479 seemed to be important for the mechanism of action. This structure is compared with the structure of agonist 25 (N-(2-fluorophenyl)-4-[(4-fluorophenyl)sulfonyl]-2,3,4,5-tetrahydro-1,4-benzoxazepin-6-amine ) and structures of other known RORγ modulators.

A serendipitously identified novel small molecule procoagulant compound giving rise to a high-throughput screening assay based on human plasma.

INTRODUCTION: Oral treatment is lacking for haemophilia, the rare bleeding disorders, and some severe forms of von Willebrand's disease. We have serendipitously identified a small molecule procoagulant compound (AZ10047130). This publication describes some characteristics of AZ10047130 and a systematic search for novel hits using a, human plasma-based, high-throughput screening (HTS) assay. MATERIAL AND METHODS: Coagulation, thrombin generation, chromogenic assays and surface plasmon resonance (SPR) experiments were used to characterise AZ10047130. A 1536-well formatted human plasma coagulation assay for HTS was developed. RESULTS: In the plasma clot assay (re-calcified plasma with low tissue factor) AZ10047130 shortened time to coagulation with an EC50 value of 3.9 µM (assay concentration). AZ10047130 was similarly effective in immunodepleted human and haemophilia A plasmas. SPR and chromogenic substrate experiments indicated that AZ10047130 binds to the heparin binding site of several coagulation factors. The HTS screened in excess of one million compounds. It generated some hits belonging to the same pharmacophore as AZ10047130 but also some entirely novel hits. CONCLUSION: These novel small molecule procoagulant compounds may serve as templates for discovery of oral procoagulant drugs.

Evaluation of human liver slices and reporter gene assays as systems for predicting the cytochrome p450 induction potential of drugs in vivo in humans.

PURPOSE: The aim of the study was to investigate the feasibility of predicting human in vivo cytochrome P450 (CYP) induction properties of drugs using in vitro methods. METHODS: The CYP induction potential of compounds was tested in human liver slices and in reporter gene assays for the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR). RESULTS: In human liver slices, CYP activities decreased dramatically over the experimental period, whereas mRNA levels could reliably be used to investigate CYP1A, 2C9, and 3A4 induction. However, the interindividual variations and demanding experimentation limit the use of liver slices in screening programs. Reporter gene assays are robust and reliable assays, amenable to high throughput screening. Several compounds activated AhR. The relevance of this activation, however, needs to be further investigated since there are no clear reports on drugs inducing CYP1A in vivo. The results from the PXR assay could be used to correctly classify compounds with known CYP3A induction properties when relating in vivo AUCtot to PXR EC50 values. CONCLUSIONS: Liver slices are a valuable model to study the regulation of a larger number of enzymes by single compounds. The PXR reporter gene assay could be used as a reliable screening method to predict CYP3A induction in vivo.

Identification of the human ApoAV gene as a novel RORalpha target gene.

Retinoic acid receptor-related orphan receptor-alpha (RORalpha) (NR1F1) is an orphan nuclear receptor with a potential role in metabolism. Previous studies have shown that RORalpha regulates transcription of the murine Apolipoprotein AI gene and human Apolipoprotein CIII genes. In the present study, we present evidence that RORalpha also induces transcription of the human Apolipoprotein AV gene, a recently identified apolipoprotein associated with triglyceride levels. Adenovirus-mediated overexpression of RORalpha increased the endogenous expression of ApoAV in HepG2 cells and RORalpha also enhanced the activity of an ApoAV promoter construct in transiently transfected HepG2 cells. Deletion and mutation studies identified three AGGTCA motifs in the ApoAV promoter that mediate RORalpha transactivation, one of which overlaps with a previously identified binding site for PPARalpha. Together, these results suggest a novel mechanism whereby RORalpha modulates lipid metabolism and implies RORalpha as a potential target for the treatment of dyslipidemia and atherosclerosis.