Discovery of a novel, highly potent and orally bioavailable pyrrolidinone indole series of irreversible Myeloperoxidase (MPO) inhibitors.

Myeloperoxidase (MPO) is a heme-containing peroxidase from phagocytic cells, which plays an important role in the innate immune response. The primary anti-microbial function of MPO is achieved by catalyzing the oxidation of halides by hydrogen peroxide (H2O2). Upon activation of phagocytes, MPO activity is detectable in both phagosomes and extracellularly, where it can remain or transcytose into interstitial compartments. Activated MPO leads to oxidative stress and tissue damage in many inflammatory states, including cardiovascular disease. Starting from a low molecular weight (LMW) high throughput screening (HTS) hit, here we report the discovery of a novel pyrrolidinone indole (IN-4) as a highly potent MPO inhibitor. This compound displays similar in vitro potency across peroxidation, plasma and NETosis assays. In a dilution/dialysis study, <5% of the original MPO activity was detected post-incubation of MPO with IN-4, suggesting irreversible enzyme inhibition. A fast MPO inactivation rate (kinact/Ki) and low partition ratio (k3/k4) make IN-4 kinetic properties attractive for an MPO inhibitor. This compound also displays significant selectivity over the closely related thyroid peroxidase (TPO), and is selective for extracellular MPO over intracellular (neutrophil) MPO. Moreover, IN-4 shows good exposure, low clearance and high oral bioavailability in mice, rats and dogs. The high in vitro MPO activity and high oral exposure observed with IN-4 result in a dose-dependent inhibition of MPO activity in three mouse models of inflammation. In conclusion, IN-4 is a novel, potent, mechanism-based and selective MPO inhibitor, which may be used as superior therapeutic agent to treat multiple inflammatory conditions, including cardiovascular disease.

Discovery and Targeting of the Signaling Controls of PNPLA3 to Effectively Reduce Transcription, Expression, and Function in Pre-Clinical NAFLD/NASH Settings.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are emerging worldwide epidemics, projected to become the leading cause of liver transplants. The strongest genetic risk factor for NAFLD/NASH susceptibility and progression is a single-nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 gene (PNPLA3), rs738409, encoding the missense mutation I148M. This aminoacidic substitution interferes with the normal remodeling of lipid droplets in hepatocytes. It is also thought to play a key role in promoting liver fibrosis by inhibiting the release of retinol from hepatic stellate cells. Reducing PNPLA3 levels in individuals homozygous for 148M may be an effective treatment for the entire spectrum of NAFLD, based on gene dosage analysis in the human population, as well as the protective effect of another naturally occurring SNP (rs2294918) in PNPLA3 which, when co-inherited, reduces PNPLA3 mRNA levels to 50% and counteracts disease risk. By screening a clinical compound library targeting specific signaling pathways active in primary human hepatocytes, we identified momelotinib, a drug evaluated in clinical trials to treat myelofibrosis, as a potent down-regulator of PNPLA3 expression, across all genotypes. We found that momelotinib treatment yielded >80% reduction in PNPLA3 mRNA in human primary hepatocytes and stellate cells, as well as in vivo via acute and chronic treatment of WT mice. Using a human multilineage 3D spheroid model of NASH homozygous for the PNPLA3 mutant protein, we additionally show that it decreases PNPLA3 mRNA as well as intracellular lipid content. Furthermore, we show that the effects on PNPLA3 coincide with changes in chromatin accessibility within regulatory regions of the PNPLA3 locus, consistent with inhibition occurring at the level of transcription. In addition to its primary reported targets, the JAK kinases, momelotinib inhibits several non-JAK kinases, including ACVR1. Using a combination of targeted siRNA knockdowns and signaling pathway perturbations, we show that momelotinib reduces the expression of the PNPLA3 gene largely through the inhibition of BMP signaling rather than the JAK/STAT pathway. Overall, our work identified momelotinib as a potential NASH therapeutic and uncovered previously unrecognized connections between signaling pathways and PNPLA3. These pathways may be exploited by drug modalities to "tune down" the level of gene expression, and therefore offer a potential therapeutic benefit to a high at-risk subset of NAFLD/NASH patients.

Discovery of a novel indole pharmacophore for the irreversible inhibition of myeloperoxidase (MPO).

Myeloperoxidase (MPO) activity and subsequent generation of hypochlorous acid has been associated with the killing of host-invading microorganisms (e.g. bacteria, viruses, and fungi). However, during oxidative stress, high MPO activity can damage host tissue and is linked to several chronic inflammatory conditions. Herein, we describe the development of a novel biaryl, indole-pyrazole series of irreversible mechanism-based inhibitors of MPO. Derived from an indole-containing high-throughput screen hit, optimization efforts resulted in potent and selective 6-substituted indoles with good oral bioavailability and in vivo activity.

A Systematic Approach to Identify Candidate Transcription Factors that Control Cell Identity.

Hundreds of transcription factors (TFs) are expressed in each cell type, but cell identity can be induced through the activity of just a small number of core TFs. Systematic identification of these core TFs for a wide variety of cell types is currently lacking and would establish a foundation for understanding the transcriptional control of cell identity in development, disease, and cell-based therapy. Here, we describe a computational approach that generates an atlas of candidate core TFs for a broad spectrum of human cells. The potential impact of the atlas was demonstrated via cellular reprogramming efforts where candidate core TFs proved capable of converting human fibroblasts to retinal pigment epithelial-like cells. These results suggest that candidate core TFs from the atlas will prove a useful starting point for studying transcriptional control of cell identity and reprogramming in many human cell types.

Characterization of twenty-five ovarian tumour cell lines that phenocopy primary tumours.

Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the 25 new ovarian tumour cell lines described here retain the genomic landscape, histopathology and molecular features of the original tumours. Furthermore, the molecular profile and drug response of these cell lines correlate with distinct groups of primary tumours with different outcomes. Thus, tumour cell lines derived using this methodology represent a significantly improved platform to study human tumour pathophysiology and response to therapy.

Induction of FoxP3+CD4+CD25+ regulatory T cells by a bone marrow population distinct from plasmacytoid-DC.

Facilitating cells (FC) are bone marrow-derived cells that facilitate allogeneic hematopoietic stem cell (SC) engraftment and induce transplantation tolerance without causing graft vs. host disease. Although there is evidence for FC directing the development of FoxP3+CD4+CD25+ regulatory T cells, the specific FC subsets that control regulatory T cell development have not been defined. The current study investigates the role of FC-CD3epsilon+ and FC-CD3epsilon- subpopulations in the development of FoxP3+CD4+CD25+ regulatory T cells. Here, we demonstrate that the induction of FoxP3+CD4+CD25+ regulatory T cells in coculture is mediated by not only the FC-CD3epsilon- subset but also the FC-CD3epsilon+ subset, which is distinct from plasmacytoid precursor dendritic cells (p-preDC). The identification of cell populations distinct from p-preDC that efficiently induce the generation of FoxP3+CD4+CD25+ regulatory T cells may prove useful for future therapeutic applications for the induction of tolerance following allogeneic SC transplantation.

Induction of FoxP3+CD4+25+ regulatory T cells following hemopoietic stem cell transplantation: role of bone marrow-derived facilitating cells.

The establishment of donor cell lineages following allogeneic bone marrow transplantation is frequently associated with the development of graft-vs-host disease (GVHD). The identification of cell populations that are capable of supporting allogeneic stem cell (SC) engraftment and the induction of tolerance without inducing GVHD could expand the use of this therapy. CD8(+)TCR(-) facilitating cells (FC) have been shown to promote allogeneic SC engraftment with resulting transplantation tolerance across complete MHC barriers without inducing GVHD. Although donor reconstitution in SC plus FC recipients is associated with the induction of regulatory T cell-associated factors, it is not known whether an induction of regulatory T cells and subsequent tolerance is a direct effect of the FC. The current study demonstrates that 1) SC plus FC transplantation results in the induction of donor CD4(+)25(+) regulatory T cells and that FC are present in the spleen of recipients before the induction of these cells, 2) activation of FC with CpG-oligodeoxynucleotide promotes CD4(+)25(-) T cell differentiation into CD4(+)25(+) regulatory T cells in vitro, as demonstrated by cytokine and forkhead/winged helix transcription factor (FoxP3) gene and protein expression, and 3) direct contact between FC and CD4(+)25(-) T cells is required for FoxP3(+)CD4(+)25(+) regulatory T cell induction and is dependent on CD86 expression on FC. This is the first report to demonstrate a mechanism for FC in the induction of regulatory T cells following allogeneic SC plus FC transplantation. The transplantation of donor FC may provide an alternative approach to permit clinical SC engraftment and induction of transplantation tolerance in the future.

Mutations in the Arabidopsis phosphoinositide phosphatase gene SAC9 lead to overaccumulation of PtdIns(4,5)P2 and constitutive expression of the stress-response pathway.

Phosphoinositides (PIs) are signaling molecules that regulate cellular events including vesicle targeting and interactions between membrane and cytoskeleton. Phosphatidylinositol (PtdIns)(4,5)P(2) is one of the best characterized PIs; studies in which PtdIns(4,5)P(2) localization or concentration is altered lead to defects in the actin cytoskeleton and exocytosis. PtdIns(4,5)P(2) and its derivative Ins(1,4,5)P(3) accumulate in salt, cold, and osmotically stressed plants. PtdIns(4,5)P(2) signaling is terminated through the action of inositol polyphosphate phosphatases and PI phosphatases including supressor of actin mutation (SAC) domain phosphatases. In some cases, these phosphatases also act on Ins(1,4,5)P(3). We have characterized the Arabidopsis (Arabidopsis thaliana) sac9 mutants. The SAC9 protein is different from other SAC domain proteins in several ways including the presence of a WW protein interaction domain within the SAC domain. The rice (Oryza sativa) and Arabidopsis SAC9 protein sequences are similar, but no apparent homologs are found in nonplant genomes. High-performance liquid chromatography studies show that unstressed sac9 mutants accumulate elevated levels of PtdIns(4,5)P(2) and Ins(1,4,5)P(3) as compared to wild-type plants. The sac9 mutants have characteristics of a constitutive stress response, including dwarfism, closed stomata, and anthocyanin accumulation, and they overexpress stress-induced genes and overaccumulate reactive-oxygen species. These results suggest that the SAC9 phosphatase is involved in modulating phosphoinsitide signals during the stress response.