Process Development and Scale-Up of a Novel Atypical DAT Inhibitor (S)-CE-123.

Large-scale syntheses of small molecules and kilo laboratories are crucial steps in drug development, especially in advanced stages. (S)-5-((Benzhydrylsulfinyl)methyl)thiazole, (S)-CE-123, a potent, selective, and novel atypical DAT inhibitor, has undergone iterative testing as part of the preclinical evaluation step. This required the process transfer, scale-up, and synthesis of a 1 kg preclinical batch. The Kagan protocol for asymmetric sulfide to sulfoxide oxidation was successfully applied within a four-step synthetic process for the successful upscaling of (S)-CE-123. During the scale-up of the last step, several changes were made to the original synthetic procedure, as with every increase in batch size, new problems had to be overcome. These include, among others, the workup optimization of the last step, the simplification of chromatographic purification, elution modification to improve the purity of the product and saving of workup time. Two washing steps were added to the original procedure to enhance both the yield and the enantiomeric excess value of the final product. The modifications introduced allowed access to a 1 kg (S)-CE-123 batch with a purity >99% and an enantiomeric excess value of 95%.

Chirality Matters: Fine-Tuning of Novel Monoamine Reuptake Inhibitors Selectivity through Manipulation of Stereochemistry.

The high structural similarity, especially in transmembrane regions, of dopamine, norepinephrine, and serotonin transporters, as well as the lack of all crystal structures of human isoforms, make the specific targeting of individual transporters rather challenging. Ligand design itself is also rather limited, as many chemists, fully aware of the synthetic and analytical challenges, tend to modify lead compounds in a way that reduces the number of chiral centers and hence limits the potential chemical space of synthetic ligands. We have previously shown that increasing molecular complexity by introducing additional chiral centers ultimately leads to more selective and potent dopamine reuptake inhibitors. Herein, we significantly extend our structure-activity relationship of dopamine transporter-selective ligands and further demonstrate how stereoisomers of defined absolute configuration may fine-tune and direct the activity towards distinct targets. From the pool of active compounds, using the examples of stereoisomers 7h and 8h, we further showcase how in vitro activity significantly differs in in vivo drug efficacy experiments, calling for proper validation of individual stereoisomers in animal studies. Furthermore, by generating a large library of compounds with defined absolute configurations, we lay the groundwork for computational chemists to further optimize and rationally design specific monoamine transporter reuptake inhibitors.

Low-Affinity/High-Selectivity Dopamine Transport Inhibition Sufficient to Rescue Cognitive Functions in the Aging Rat.

The worldwide increase in cognitive decline, both in aging and with psychiatric disorders, warrants a search for pharmacological treatment. Although dopaminergic treatment approaches represent a major step forward, current dopamine transporter (DAT) inhibitors are not sufficiently specific as they also target other transporters and receptors, thus showing unwanted side effects. Herein, we describe an enantiomerically pure, highly specific DAT inhibitor, S-CE-123, synthetized in our laboratory. Following binding studies to DAT, NET and SERT, GPCR and kinome screening, pharmacokinetics and a basic neurotoxic screen, S-CE-123 was tested for its potential to enhance and/or rescue cognitive functions in young and in aged rats in the non-invasive reward-motivated paradigm of a hole-board test for spatial learning. In addition, an open field study with young rats was carried out. We demonstrated that S-CE-123 is a low-affinity but highly selective dopamine reuptake inhibitor with good bioavailability. S-CE-123 did not induce hyperlocomotion or anxiogenic or stereotypic behaviour in young rats. Our compound improved the performance of aged but not young rats in a reward-motivated task. The well-described impairment of the dopaminergic system in aging may underlie the age-specific effect. We propose S-CE-123 as a possible candidate for developing a tentative therapeutic strategy for age-related cognitive decline and cognitive dysfunction in psychiatric disorders.

The Novel Analogue of Modafinil CE-158 Protects Social Memory against Interference and Triggers the Release of Dopamine in the Nucleus Accumbens of Mice.

Previous studies have shown that atypical dopamine-transporter-inhibitors such as modafinil and its analogues modify behavioral and cognitive functions in rodents. Here, we tested potential promnestic effects of the novel, more dopamine-transporter selective modafinil analogue CE-158 in the social discrimination memory task in male mice. Systemic administration of CE-158 1 h before the social learning event prevented the impairment of social-recognition memory following retroactive interference 3 h after the learning session of a juvenile conspecific. This effect was dose-dependent, as mice treated with 10 mg/kg, but not with 1 mg/kg CE-158, were able to discriminate between the novel and familiar conspecific despite the presentation of an interference stimulus, both 3 h and 6 h post learning. However, when 10 mg/kg of the drug was administered after learning, CE-158 failed to prevent social memory from interference. Paralleling these behavioral effects, the systemic administration of 10 mg/kg CE-158 caused a rapid and sustained elevation of extracellular dopamine in the nucleus accumbens, a brain area where dopaminergic signaling plays a key role in learning and memory function, of freely moving mice, while 1 mg/kg was not sufficient for altering dopamine levels. Taken together, our findings suggest promnestic effects of the novel dopamine-transporter-inhibitor CE-158 in a social recognition memory test that may be in part mediated via increased dopamine-neurotransmission in the nucleus accumbens. Thus, selective-dopamine-transporter-inhibitors such as CE-158 may represent interesting drug candidates for the treatment of memory complaints observed in humans with cognitive impairments and dementia.

Reinstatement of synaptic plasticity in the aging brain through specific dopamine transporter inhibition.

Aging-related neurological deficits negatively impact mental health, productivity, and social interactions leading to a pronounced socioeconomic burden. Since declining brain dopamine signaling during aging is associated with the onset of neurological impairments, we produced a selective dopamine transporter (DAT) inhibitor to restore endogenous dopamine levels and improve cognitive function. We describe the synthesis and pharmacological profile of (S,S)-CE-158, a highly specific DAT inhibitor, which increases dopamine levels in brain regions associated with cognition. We find both a potentiation of neurotransmission and coincident restoration of dendritic spines in the dorsal hippocampus, indicative of reinstatement of dopamine-induced synaptic plasticity in aging rodents. Treatment with (S,S)-CE-158 significantly improved behavioral flexibility in scopolamine-compromised animals and increased the number of spontaneously active prefrontal cortical neurons, both in young and aging rodents. In addition, (S,S)-CE-158 restored learning and memory recall in aging rats comparable to their young performance in a hippocampus-dependent hole board test. In sum, we present a well-tolerated, highly selective DAT inhibitor that normalizes the age-related decline in cognitive function at a synaptic level through increased dopamine signaling.

Structure-Activity Relationships of Novel Thiazole-Based Modafinil Analogues Acting at Monoamine Transporters.

Atypical dopamine reuptake inhibitors, such as modafinil, are used for the treatment of sleeping disorders and investigated as potential therapeutics against cocaine addiction and for cognitive enhancement. Our continuous effort to find modafinil analogues with higher inhibitory activity on and selectivity toward the dopamine transporter (DAT) has previously led to the promising thiazole-containing derivatives CE-103, CE-111, CE-123, and CE-125. Here, we describe the synthesis and activity of a series of compounds based on these scaffolds, which resulted in several new selective DAT inhibitors and gave valuable insights into the structure-activity relationships. Introduction of the second chiral center and subsequent chiral separations provided all four stereoisomers, whereby the S-configuration on both generally exerted the highest activity and selectivity on DAT. The representative compound of this series was further characterized by in silico, in vitro, and in vivo studies that have demonstrated both safety and efficacy profile of this compound class.

Discovery of Inhibitor of Wnt Production 2 (IWP-2) and Related Compounds As Selective ATP-Competitive Inhibitors of Casein Kinase 1 (CK1) δ/ε.

Inhibitors of Wnt production (IWPs) are known antagonists of the Wnt pathway, targeting the membrane-bound O-acyltransferase porcupine (Porcn) and thus preventing a crucial Wnt ligand palmitoylation. Since IWPs show structural similarities to benzimidazole-based CK1 inhibitors, we hypothesized that IWPs could also inhibit CK1 isoforms. Molecular modeling revealed a plausible binding mode of IWP-2 in the ATP binding pocket of CK1δ which was confirmed by X-ray analysis. In vitro kinase assays demonstrated IWPs to be ATP-competitive inhibitors of wtCK1δ. IWPs also strongly inhibited the gatekeeper mutant M82FCK1δ. When profiled in a panel of 320 kinases, IWP-2 specifically inhibited CK1δ. IWP-2 and IWP-4 also inhibited the viability of various cancer cell lines. By a medicinal chemistry approach, we developed improved IWP-derived CK1 inhibitors. Our results suggest that the effects of IWPs are not limited to Porcn, but also might influence CK1δ/ε-related pathways.

A daily single dose of a novel modafinil analogue CE-123 improves memory acquisition and memory retrieval.

Dopamine reuptake inhibitors have been shown to improve cognitive parameters in various tasks and animal models. We recently reported a series of modafinil analogues, of which the most promising, 5-((benzhydrylsulfinyl)methyl) thiazole (CE-123), was selected for further development. The present study aims to characterize pharmacological properties of CE-123 and to investigate the potential to enhance memory performance in a rat model. In vitro transporter assays were performed in cells expressing human transporters. CE-123 blocked uptake of [3H] dopamine (IC50 = 4.606 µM) while effects on serotonin (SERT) and the norepinephrine transporter (NET) were negligible. Blood-brain barrier and pharmacokinetic studies showed that the compound reached the brain and lower elimination than R-modafinil. The Pro-cognitive effect was evaluated in a spatial hole-board task in male Sprague-Dawley rats and CE-123 enhances memory acquisition and memory retrieval, represented by significantly increased reference memory indices and shortened latency. Since DAT blockers can be considered as indirect dopamine receptor agonists, western blotting was used to quantify protein levels of dopamine receptors D1R, D2R and D5R and DAT in the synaptosomal fraction of hippocampal subregions CA1, CA3 and dentate gyrus (DG). CE-123 administration in rats increased total DAT levels and D1R protein levels were significantly increased in CA1 and CA3 in treated/trained groups. The increase of D5R was observed in DG only. Dopamine receptors, particularly D1R, seem to play a role in mediating CE-123-induced memory enhancement. Dopamine reuptake inhibition by CE-123 may represent a novel and improved stimulant therapeutic for impairments of cognitive functions.

Heterocyclic Analogues of Modafinil as Novel, Atypical Dopamine Transporter Inhibitors.

Modafinil is a wake promoting compound with high potential for cognitive enhancement. It is targeting the dopamine transporter (DAT) with moderate selectivity, thereby leading to reuptake inhibition and increased dopamine levels in the synaptic cleft. A series of modafinil analogues have been reported so far, but more target-specific analogues remain to be discovered. It was the aim of this study to synthesize and characterize such analogues and, indeed, a series of compounds were showing higher activities on the DAT and a higher selectivity toward DAT versus serotonin and norepinephrine transporters than modafinil. This was achieved by substituting the amide moiety by five- and six-membered aromatic heterocycles. In vitro studies indicated binding to the cocaine pocket on DAT, although molecular dynamics revealed binding different from that of cocaine. Moreover, no release of dopamine was observed, ruling out amphetamine-like effects. The absence of neurotoxicity of a representative analogue may encourage further preclinical studies of the above-mentioned compounds.

A novel heterocyclic compound improves working memory in the radial arm maze and modulates the dopamine receptor D1R in frontal cortex of the Sprague-Dawley rat.

A series of compounds have been shown to enhance cognitive function via the dopaminergic system and indeed the search for more active and less toxic compounds is continuing. It was therefore the aim of the study to synthetise and test a novel heterocyclic compound for cognitive enhancement in a paradigm for working memory. Specific and effective dopamine re-uptake inhibition DAT (IC50=4,1±0,8µM) made us test this compound in a radial arm maze (RAM) in the rat. CE-125 (4-((benzhydrylsulfinyl)methyl)-2-cyclopropylthiazole), was tested for dopamine (DAT), serotonin and norepinephrine re-uptake inhibition by a well-established system. The working memory index (WMI) was evaluated in male Sprague Dawley rats that were intraperitoneally injected with CE-125 (1 or 10mg/kg body weight). In order to evaluate basic neurotoxicity, the open field, elevated plus maze, rota rod studies and the forced swim test were carried out. Frontal cortex was taken at the last day of the RAM test and dopamine receptors D1R and D2R, DAT and phosphorylated DAT protein levels were determined. On the 10th day both doses were increasing the WMI as compared to the vehicle-treated group. In both, trained and treated groups, D1R levels were significantly reduced while D2R levels were unchanged. DAT levels were comparable between all groups while phosphorylated DAT levels were increased in the trained group treated with 1mg/kg body weight. CE-125 as a probably non-neurotoxic compound and specific reuptake inhibitor was shown to increase performance (WMI) and modulation of the dopaminergic system is proposed as a possible mechanism of action.

Inhibitors of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) exert a strong anti-herpesviral activity.

Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of (val)ganciclovir therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy of antiviral treatment is based on the exploitation of cell-directed signaling, e. g. pathways with a known relevance for carcinogenesis and tumor drug development. Here we describe a principle for putative antiviral drugs based on targeting dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). DYRKs constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell proliferation and differentiation. Members of the DYRK family are capable of phosphorylating a number of substrate proteins, including regulators of the cell cycle, e.g. DYRK1B can induce cell cycle arrest, a critical step for the regulation of HCMV replication. Here we provide first evidence for a critical role of DYRKs during viral replication and the high antiviral potential of DYRK inhibitors (SC84227, SC97202 and SC97208, Harmine and AZ-191). Using established replication assays for laboratory and clinically relevant strains of HCMV, concentration-dependent profiles of inhibition were obtained. Mean inhibitory concentrations (EC50) of 0.98 ± 0.08 µM/SC84227, 0.60 ± 0.02 µM/SC97202, 6.26 ± 1.64 µM/SC97208, 0.71 ± 0.019 µM/Harmine and 0.63 ± 0.23 µM/AZ-191 were determined with HCMV strain AD169-GFP for the infection of primary human fibroblasts. A first analysis of the mode of antiviral action suggested a block of viral replication at the early-late stage of HCMV gene expression. Moreover, rhesus macaque cytomegalovirus (RhCMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV-1) showed a similarly high sensitivity to these compounds. Thus, we conclude that DYRK signaling represents a promising target pathway for the development of novel anti-herpesviral strategies.

A novel heterocyclic compound targeting the dopamine transporter improves performance in the radial arm maze and modulates dopamine receptors D1-D3.

A series of compounds targeting the dopamine transporter (DAT) haS been shown to improve memory performance most probably by re-uptake inhibition. Although specific DAT inhibitors are available, there is limited information about specificity, mechanism and in particular the effect on dopamine receptors. It was therefore the aim of the study to test the DAT inhibitor 4-(diphenyl-methanesulfinylmethyl)-2-methyl-thiazole (code: CE-111), synthetized in our laboratory for the specificity to target DAT, for the effects upon spatial memory and for induced dopamine receptor modulation. Re-uptake inhibition was tested for DAT (IC50=3.2µM), serotonin transporter, SERT (IC50=272291µM) and noradrenaline transporter, NET (IC50=174µM). Spatial memory was studied in the radial arm maze (RAM) in male Sprague-Dawley rats that were intraperitoneally injected with CE-111 (1 or 10mg/kg body weight). Performance in the RAM was improved using 1 and 10mg/kg body weight of CE-111. Training and treatment effects on presynaptic, postsynaptic and extrasynaptic D1 and D2- receptors and dopamine receptor containing complexes as well as on activated DAT were observed. CE-111 was crossing the blood-brain barrier comparable to modafinil and was identified as effective to improve memory performance in the RAM. Dopamine re-uptake inhibition along with modulations in dopamine receptors are proposed as potential underlying mechanisms.

A Novel Heterocyclic Compound CE-104 Enhances Spatial Working Memory in the Radial Arm Maze in Rats and Modulates the Dopaminergic System.

Various psychostimulants targeting monoamine neurotransmitter transporters (MATs) have been shown to rescue cognition in patients with neurological disorders and improve cognitive abilities in healthy subjects at low doses. Here, we examined the effects upon cognition of a chemically synthesized novel MAT inhibiting compound 2-(benzhydrylsulfinylmethyl)-4-methylthiazole (named as CE-104). The efficacy of CE-104 in blocking MAT [dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter] was determined using in vitro neurotransmitter uptake assay. The effect of the drug at low doses (1 and 10 mg/kg) on spatial memory was studied in male rats in the radial arm maze (RAM). Furthermore, the dopamine receptor and transporter complex levels of frontal cortex (FC) tissue of trained and untrained animals treated either with the drug or vehicle were quantified on blue native PAGE (BN-PAGE). The drug inhibited dopamine (IC50: 27.88 µM) and norepinephrine uptake (IC50: 160.40 µM), but had a negligible effect on SERT. In the RAM, both drug-dose groups improved spatial working memory during the performance phase of RAM as compared to vehicle. BN-PAGE Western blot quantification of dopamine receptor and transporter complexes revealed that D1, D2, D3, and DAT complexes were modulated due to training and by drug effects. The drug's ability to block DAT and its influence on DAT and receptor complex levels in the FC is proposed as a possible mechanism for the observed learning and memory enhancement in the RAM.

DYRK1B as therapeutic target in Hedgehog/GLI-dependent cancer cells with Smoothened inhibitor resistance.

A wide range of human malignancies displays aberrant activation of Hedgehog (HH)/GLI signaling, including cancers of the skin, brain, gastrointestinal tract and hematopoietic system. Targeting oncogenic HH/GLI signaling with small molecule inhibitors of the essential pathway effector Smoothened (SMO) has shown remarkable therapeutic effects in patients with advanced and metastatic basal cell carcinoma. However, acquired and de novo resistance to SMO inhibitors poses severe limitations to the use of SMO antagonists and urgently calls for the identification of novel targets and compounds.Here we report on the identification of the Dual-Specificity-Tyrosine-Phosphorylation-Regulated Kinase 1B (DYRK1B) as critical positive regulator of HH/GLI signaling downstream of SMO. Genetic and chemical inhibition of DYRK1B in human and mouse cancer cells resulted in marked repression of HH signaling and GLI1 expression, respectively. Importantly, DYRK1B inhibition profoundly impaired GLI1 expression in both SMO-inhibitor sensitive and resistant settings. We further introduce a novel small molecule DYRK1B inhibitor, DYRKi, with suitable pharmacologic properties to impair SMO-dependent and SMO-independent oncogenic GLI activity. The results support the use of DYRK1B antagonists for the treatment of HH/GLI-associated cancers where SMO inhibitors fail to demonstrate therapeutic efficacy.

A heterocyclic compound CE-103 inhibits dopamine reuptake and modulates dopamine transporter and dopamine D1-D3 containing receptor complexes.

A series of compounds have been reported to enhance memory via the DA system and herein a heterocyclic compound was tested for working memory (WM) enhancement. 2-((benzhydrylsulfinyl)methyl)thiazole (CE-103) was synthesized in a six-step synthesis. Binding of CE-103 to the dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters and dopamine reuptake inhibition was tested as well as blood brain permeation and a screen for GPCR targets. 60 male Sprague Dawley rats were divided into six groups: CE-103 treated 1-10 mg/kg body weight, trained (TDI) and yoked (YDI) and vehicle treated, trained (TVI) and yoked (YVI) rats. Daily single intraperitoneal injections for a period of 10 days were administered and rats were tested in a radial arm maze (RAM). Hippocampi were taken 6 h following the last day of training and complexes containing the unphosphorylated or phosphorylated dopamine transporter (DAT) and complexes containing the D1-3 dopamine receptor subunits were determined. CE-103 was binding to the DAT but insignificantly to SERT or NET and dopamine reuptake was blocked specifically (IC50 = 14.73 µM). From day eight the compound was decreasing WM errors in the RAM significantly at both doses tested as compared to the vehicle controls. In the trained CE-103-treated group levels of the complex containing the phosphorylated dopamine transporter (pDAT) as well as D1R were decreased while levels of complexes containing D2R and D3R were significantly increased. CE-103 was shown to enhance spatial WM and DA reuptake inhibition with subsequent modulation of D1-3 receptors is proposed as a possible mechanism of action.

The effect of modafinil on the rat dopamine transporter and dopamine receptors D1-D3 paralleling cognitive enhancement in the radial arm maze.

A series of drugs have been reported to increase memory performance modulating the dopaminergic system and herein modafinil was tested for its working memory (WM) enhancing properties. Reuptake inhibition of dopamine, serotonin (SERT) and norepinephrine (NET) by modafinil was tested. Sixty male Sprague-Dawley rats were divided into six groups (modafinil-treated 1-5-10 mg/kg body weight, trained and untrained and vehicle treated trained and untrained rats; daily injected intraperitoneally for a period of 10 days) and tested in a radial arm maze (RAM), a paradigm for testing spatial WM. Hippocampi were taken 6 h following the last day of training and complexes containing the unphosphorylated or phosphorylated dopamine transporter (DAT-CC and pDAT-CC) and complexes containing the D1-3 dopamine receptor subunits (D1-D3-CC) were determined. Modafinil was binding to the DAT but insignificantly to SERT or NET and dopamine reuptake was blocked specifically (IC50 = 11.11 µM; SERT 1547 µM; NET 182 µM). From day 8 (day 9 for 1 mg/kg body weight) modafinil was decreasing WM errors (WMEs) in the RAM significantly and remarkably at all doses tested as compared to the vehicle controls. WMEs were linked to the D2R-CC and the pDAT-CC. pDAT and D1-D3-CC levels were modulated significantly and modafinil was shown to enhance spatial WM in the rat in a well-documented paradigm at all the three doses and dopamine reuptake inhibition with subsequent modulation of D1-3-CC is proposed as a possible mechanism of action.

Effects of altered expression and activity levels of CK1δ and ɛ on tumor growth and survival of colorectal cancer patients.

Colorectal cancer (CRC) is the fourth leading cause of cancer related death worldwide due to high apoptotic resistance and metastatic potential. Because mutations as well as deregulation of CK1 isoforms contribute to tumor development and tumor progression, CK1 has become an interesting drug target. In this study we show that CK1 isoforms are differently expressed in colon tumor cell lines and that growth of these cell lines can be inhibited by CK1-specific inhibitors. Furthermore, expression of CK1δ and ɛ is changed in colorectal tumors compared to normal bowel epithelium, and high CK1ɛ expression levels significantly correlate with prolonged patients' survival. In addition to changes in CK1δ and ɛ expression, mutations within exon 3 of CK1δ were detected in colorectal tumors. These mutations influence ATP binding resulting in changes in kinetic parameters of CK1δ. Overexpression of these mutants in HT29 cells alters their ability to grow anchorage independently. Consistent with these results, these CK1δ mutants lead to differences in proliferation rate and tumor size in xenografts due to changes in gene expression, especially in genes involved in regulation of cell proliferation, cell cycle, and apoptosis. In summary, our results provide evidence that changes in the expression levels of CK1 isoforms in colorectal tumors correlate with patients' survival. Furthermore, CK1 mutants affect growth and proliferation of tumor cells and induce tumor growth in xenografts, leading to the assumption that CK1 isoforms provide interesting targets for the development of novel effective therapeutic concepts to treat colorectal cancer.

A new small molecule for treating inflammation and chorioretinal neovascularization in relapsing-remitting and chronic experimental autoimmune uveitis.

PURPOSE: We investigated the effect of PP-001, a new small molecule inhibitor of dihydro-orotate dehydrogenase in two experimental rat experimental autoimmune uveitis (EAU) models: a spontaneously relapsing-remitting model and a monophasic/chronic disease model that results in late chorioretinal neovascularization. Both of the diseases are induced by immunization with autoantigen peptides. METHODS: Prevention was tested using daily oral applications of PP-001 after immunization with the retinal S-antigen peptide PDSAg (for induction of monophasic uveitis and neovascularization) or the interphotoreceptor retinoid-binding protein peptide R14 (for induction of spontaneously relapsing-remitting EAU). Treatment to inhibit relapses and neovascularization was tested using PP-001 daily after the first attack of R14-induced or after onset of PDSAg-induced EAU. Uveitis was graded clinically and histologically. The effect of PP-001 on cytokine secretion and proliferation was evaluated using rat T-cell lines. RESULTS: Preventive feeding of PP-001 abrogated both types of EAU. Starting treatment after the resolution of the first attack led to a significant reduction of the number and intensity of relapses in R14-induced EAU. PP-001-treatment initiated after onset or after peak of PDSAg-induced EAU significantly reduced neovascularization (as determined by histology). Proliferation of antigen-specific T-cell lines and secretion of IFN-γ, IL-17, IL-10, IP-10, and VEGF were efficiently suppressed by PP-001. CONCLUSIONS: We investigated a new dihydroorotate dehydrogenase inhibitor as treatment for primary and recurrent disease in relapsing-remitting and chronic rat models of experimental autoimmune uveitis. The small molecule compound PP-001 suppressed proliferation and cytokine secretion of autoreactive T cells (i.e., IFN-g, IL-17, and VEGF) and chorioretinal neovascularization in chronic EAU.

Difluoro-dioxolo-benzoimidazol-benzamides as potent inhibitors of CK1δ and ε with nanomolar inhibitory activity on cancer cell proliferation.

Deregulation of CK1 (casein kinase 1) activity can be involved in the development of several pathological disorders and diseases such as cancer. Therefore, research interest in identifying potent CK1-specific inhibitors is still increasing. A previously published potent and selective benzimidazole-derived CK1δ/ε-specific inhibitor compound with significant effects on several tumor cell lines was further modified to difluoro-dioxolo-benzoimidazole derivatives displaying remarkable inhibitory effects and increased intracellular availability. In the present study, we identified two heterocyclic molecules as new CK1-specific inhibitor compounds with favorable physicochemical properties and notable selectivity in a kinome-wide screen. Being compared to other CK1 isoforms, these compounds exhibited advanced isoform selectivity toward CK1δ. Moreover, newly designed compounds showed increased growth inhibitory activity in a panel of different tumor cell lines as determined by analyses of cell viability and cell cycle distribution. In summary, presented lead optimization resulted in new highly selective CK1δ-specific small molecule inhibitors with increased biological activity.

A novel DRAK inhibitor, SC82510, promotes axon branching of adult sensory neurons in vitro.

Recently, a new potent protein kinase inhibitor, SC82510, was identified acting on DRAK2 and stimulating axon outgrowth at low concentrations. DRAK is the Drosophila homologue of death-associated protein kinase that phosphorylates myosin-II regulatory light chain in a similar fashion as ROCK, the downstream target of RhoA mediating axon outgrowth inhibition. While higher concentrations of this novel compound exhibited toxic effects, significant promotion of process outgrowth of PC12 cells and of adult primary neurons was observed at 1 nM which could be further enhanced by addition of a neuronal growth factor (FGF-2). Unlike the effects of ROCK inhibitors on axon outgrowth that stimulate both, elongation and branching, SC82510 primarily promoted axon branching, whereas axon elongation was not increased in this cell culture model of peripheral axon regeneration.