Discovery of azaspirocyclic 1H-3,4,5-Trisubstitued pyrazoles as novel G2019S-LRRK2 selective kinase inhibitors.

Mutations in the Leucine Rich Repeat Protein Kinase 2 gene (LRRK2) are genetic predispositions for Parkinson's Disease, of which the G2019S (GS) missense mutation is the most common. GS-LRRK2 has a hyperactive kinase, and although numerous drug discovery programs have targeted the LRRK2 kinase, few have reached clinical trials. We recently reported on the discovery of a novel LRRK2 kinase inhibitor chemotype, 1H-pyrazole biaryl sulfonamides. Although both potent and selective GS-LRRK2 inhibitors, 1H-pyrazole biaryl sulfonamides are incapable of crossing the blood-brain barrier. Retaining the core 1H-pyrazole and focusing our efforts on a phenylsulfonamide bioisosteric replacement, we report the discovery and preliminary development of azaspirocyclic 1H-3,4,5-trisubstituted pyrazoles as potent and selective (>2000-fold) GS-LRRK2 kinase inhibitors capable of entering rodent brain. The compounds disclosed here present an excellent starting point for the development of more brain penetrant compounds.

Discovery of 1H-Pyrazole Biaryl Sulfonamides as Novel G2019S-LRRK2 Kinase Inhibitors.

G2019S (GS) is the most prevalent mutation in the leucine rich repeat protein kinase 2 gene (LRRK2), a genetic predisposition that is common for Parkinson's disease, as well as for some forms of cancer, and is a shared risk allele for Crohn's disease. GS-LRRK2 has a hyperactive kinase, and although numerous drug discovery programs have targeted LRRK2 kinase, few have reached clinical development. We report the discovery and preliminary development of an entirely novel structural class of potent and selective GS-LRRK2 kinase inhibitors: biaryl-1H-pyrazoles.

Discovery of G2019S-Selective Leucine Rich Repeat Protein Kinase 2 inhibitors with in vivo efficacy.

Mutations in the Leucine Rich Repeat Protein Kinase 2 gene (LRRK2) are the most common genetic causes of Parkinson's Disease (PD). The G2019S mutation is the most common inherited LRRK2 mutation, occurs in the kinase domain, and results in increased kinase activity. We report the discovery and development of compound 38, an indazole-based, G2019S-selective (>2000-fold vs. WT) LRRK2 inhibitor capable of entering rodent brain (Kp = 0.5) and selectively inhibiting G2019S-LRRK2. The compounds disclosed herein present a starting point for further development of brain penetrant G2019S selective inhibitors that hopefully reduce lung phenotype side-effects and pave the way to providing a precision medicine for people with PD who carry the G2019S mutation.

Expression of P450c17 in the human fetal nervous system.

P450c17 catalyzes steroid 17α-hydroxylase and 17,20 lyase activities. P450c17 is expressed in human fetal and postnatal adrenals and gonads and in the developing mouse nervous system, but little is known about its expression in the human nervous system. We obtained portions of 9-, 10-, and 11-wk gestation human fetuses and delineated the pattern of expression of P450c17 in their peripheral nervous systems by immunocytochemistry using the P450c17 antiserum previously used to characterize P450c17 in the mouse brain. P450c17 was readily detected in the dorsal root ganglia (DRG) and spinal cord. Neural structures were identified with antisera to the cytoskeletal protein neural cell adhesion molecule; DRG were identified with antisera to the neuronal transcription factor BRN3A and neurotrophin receptor tropomyosin-receptor-kinase B. The identification of P450c17 was confirmed using commercial antisera directed against different domains of P450c17 and by using antisera immunodepleted with authentic human P450c17. We also found expression of the P450 cholesterol side-chain cleavage enzyme (P450scc) in the spinal cord and DRG. Expression of P450scc is limited to cell bodies; unlike P450c17, we never detected P450scc in fiber tracts. Catalysis by P450c17 requires electron donation from P450 oxidoreductase (POR). Dual-label immunohistochemistry detected P450c17 and POR colocalized in DRG bundles, but some fibers containing P450c17 lacked POR. These data suggest that neurosteroids synthesized via these two enzymes may act in the developing human nervous system. The expression of P450c17 in structures lacking POR means that P450c17 may not be steroidogenic in those locations, suggesting that P450c17 may have additional functions that do not require POR.

Oligogenesis and oligodendrocyte progenitor maturation vary in different brain regions and partially correlate with local angiogenesis after ischemic stroke.

Oligogenesis plays an important role in functional recovery after ischemic stroke. We tested the hypothesis that oligogenesis and the maturation of oligodendrocyte progenitor cells (OPCs) vary in different brain regions using a rat transient middle cerebral artery occlusion (tMCAO) model. Compared to Day 1, olig2(+) OPCs and oligodendrocytes (OLGs) increased in the peri-infarct basal ganglia (BG) 7 (44%) and 14 (61%) days after 2 hours of MCAO; OPCs (PDGFRα(+)) and OLGs (CC1(+)) increased in this region 14 days after tMCAO by 139% and 126%, respectively. Although the olig2(+) cells and OLGs did not increase significantly in the peri-infarct cortex (CTX), the OPCs increased in this region by 95% at Day 14 vs. Day 1 after tMCAO. The numbers of OPCs and OLGs remained low after an initial reduction at Day 1 in the peri-infarct corpus callosum (CC). Correlation analyses showed that the numbers of olig2(+) cells (r=0.73, P=0.03) and OLGs (r=0.74, P=0.02) correlated with local vessel density; however, the number of OPCs did not correlate with vessel density (r=0.43, P=0.24). Our data show that oligogenesis and the maturation of OPCs differ in various brain regions and the difference in regional angiogenic response is one of the potential reasons.

Estrogen receptor beta-selective agonists stimulate calcium oscillations in human and mouse embryonic stem cell-derived neurons.

Estrogens are used extensively to treat hot flashes in menopausal women. Some of the beneficial effects of estrogens in hormone therapy on the brain might be due to nongenomic effects in neurons such as the rapid stimulation of calcium oscillations. Most studies have examined the nongenomic effects of estrogen receptors (ER) in primary neurons or brain slices from the rodent brain. However, these cells can not be maintained continuously in culture because neurons are post-mitotic. Neurons derived from embryonic stem cells could be a potential continuous, cell-based model to study nongenomic actions of estrogens in neurons if they are responsive to estrogens after differentiation. In this study ER-subtype specific estrogens were used to examine the role of ERalpha and ERbeta on calcium oscillations in neurons derived from human (hES) and mouse embryonic stem cells. Unlike the undifferentiated hES cells the differentiated cells expressed neuronal markers, ERbeta, but not ERalpha. The non-selective ER agonist 17beta-estradiol (E(2)) rapidly increased [Ca2+]i oscillations and synchronizations within a few minutes. No change in calcium oscillations was observed with the selective ERalpha agonist 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT). In contrast, the selective ERbeta agonists, 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), MF101, and 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3 benzoxazol-5-ol (ERB-041; WAY-202041) stimulated calcium oscillations similar to E(2). The ERbeta agonists also increased calcium oscillations and phosphorylated PKC, AKT and ERK1/2 in neurons derived from mouse ES cells, which was inhibited by nifedipine demonstrating that ERbeta activates L-type voltage gated calcium channels to regulate neuronal activity. Our results demonstrate that ERbeta signaling regulates nongenomic pathways in neurons derived from ES cells, and suggest that these cells might be useful to study the nongenomic mechanisms of estrogenic compounds.

Regulation of P450c17 expression in the early embryo depends on GATA factors.

The enzyme P450c17 is required for glucocorticoid, sex steroid, and some neurosteroid biosynthesis. Defective human P450c17 causes sexual infantilism and 46,XY sex reversal but is compatible with life, whereas ablation of the corresponding mouse gene causes embryonic lethality at around E7. Normal mouse embryos express P450c17 protein and activity in the embryonic endoderm at E7. Adult adrenal and gonadal steroidogenesis requires steroidogenic factor-1 (SF-1), but SF-1 is not expressed in the early mouse embryo. We show that P450c17 is expressed in differentiated mouse parietal and visceral endoderm lineages, in cultured mouse F9 embryonic carcinoma stem cells, in mouse embryonic stem cells, and in cultured mouse P19 stem cells. Bases -110 to -55 (which contain an SF-1 site and two potential GATA sites) of the rat cyp17 gene confer promoter activity in F9 cells. Overexpression of SF-1 has no effect, whereas overexpression of GATA4 in F9 cells increases transcription from -110/-55 fused to a reporter and increases endogenous P450c17 mRNA. Chromatin immunoprecipitation assays show that GATA4 binds to -215/+55 of mouse cyp17. Stimulating F9 cells with retinoic acid and cAMP differentiates them into visceral and parietal endoderm. Commensurate with cell differentiation, quantitative PCR showed increased GATA4 and GATA6 mRNAs, temporally followed by increased P450c17 mRNA. Small interfering RNA inhibition of GATA4 or GATA6 in undifferentiated or differentiated F9 cells diminished endogenous cyp17 expression. Thus, P450c17 is expressed in mouse embryonic stem cells, its expression increases upon differentiation to an early embryonic endoderm lineage, and GATA4/6 are responsible for activation of P450c17 gene expression at this early stage of embryonic development.

Endogenous and synthetic neurosteroids in treatment of Niemann-Pick Type C disease.

The functions for neurosteroids during development and in response to nervous system injury are beginning to be identified. We focused on a mouse model in which we believed neurosteroid production would be altered, and which had a neurodegenerative phenotype. Niemann-Pick Type-C (NP-C) is an autosomal recessive neurodegenerative disease caused by mutations in NPC1 (95%) or NPC2 (5%), resulting in lysosomal accumulation of unesterified cholesterol and glycolipids. The NIH mouse model of NP-C has a mutation in the NPC1 gene, and exhibits several pathological features of the most severe NP-C patients. How lysosomal storage and trafficking defects lead to neurodegeneration is unknown. We found that these mice had normal neurosteroidogenic enzyme activity during development, but lost this activity in the early neonatal period, prior to onset of neurological symptoms. Neurons that expressed P450scc, 3beta HSD, as well as those that expressed 3alpha HSD and 5alpha reductase were lost in adult NP-C brains, resulting in diminished concentrations of allopregnanolone. We treated NP-C mice with allopregnanolone and found that a single dose in the neonatal period resulted in a doubling of life span, substantial delay in onset of neurological symptoms, survival of cerebellar Purkinje and granule cell neurons, and reduction in cholesterol and ganglioside accumulation. The mechanism by which allopregnanolone elicited these effects is unknown. Our in vitro studies showed that Purkinje cell survival promoted by allopregnanolone was lost by treatment with bicuculline, suggesting GABA(A) receptors may play a role. We treated NP-C mice with a synthetic GABA(A) neurosteroid, ganaxolone (3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one). Ganaxolone treatment of NP-C mice produced beneficial neurological effects, but these effects were not as robust as those obtained using allopregnanolone. Thus, allopregnanolone may elicit its effects through GABA(A) receptors and through other mechanisms. Additional studies also suggest that allopregnanolone may elicit its effects through pregnane-X-receptors (PXR). Our data suggest that mouse models of neurodegeneration may be beneficial in establishing both physiologic and pharmacologic actions of neurosteroids. These animal models further establish the wide range of functions of these compounds, which may ultimately be useful for treatment of human diseases.

Inactivation of Numb and Numblike in embryonic dorsal forebrain impairs neurogenesis and disrupts cortical morphogenesis.

Numb and Numblike, conserved homologs of Drosophila Numb, have been implicated in cortical neurogenesis; however, analysis of their involvement in later stages of cortical development has been hampered by early lethality of double mutants in previous studies. Using Emx1(IREScre) to induce more restricted inactivation of Numb in the dorsal forebrain of numblike null mice beginning at E9.5, we have generated viable double mutants that displayed striking brain defects. It was thus possible to examine neurogenesis during the later peak phase (E12.5-E16.5). Loss of Numb and Numblike in dorsal forebrain resulted in neural progenitor hyperproliferation, delayed cell cycle exit, impaired neuronal differentiation, and concomitant defects in cortical morphogenesis. These findings reveal novel and essential function of Numb and Numblike during the peak period of cortical neurogenesis. Further, these double mutant mice provide an unprecedented viable animal model for severe brain malformations due to defects in neural progenitor cells.

Transcriptional regulation of cortical neuron migration by POU domain factors.

The identification of pathways mediated by the kinase Cdk5 and the ligand reelin has provided a conceptual framework for exploring the molecular mechanisms underlying proper lamination of the developing mammalian cerebral cortex. In this report, we identify a component of the regulation of Cdk5-mediated cortical lamination by genetic analysis of the roles of the class III POU domain transcription factors, Brn-1 and Brn-2, expressed during the development of the forebrain and coexpressed in most layer II-V cortical neurons. Brn-1 and Brn-2 appear to critically control the initiation of radial migration, redundantly regulating the cell-autonomous expression of the p35 and p39 regulatory subunits of Cdk5 in migrating cortical neurons, with Brn-1(-/-)/Brn-2(-/-) mice exhibiting cortical inversion.