A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis.

Diarrhoeal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite Cryptosporidium to be a leading cause of paediatric diarrhoea, with particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor an effective treatment. Here we establish a drug discovery process built on scalable phenotypic assays and mouse models that take advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity, we identify pyrazolopyridines as inhibitors of Cryptosporidium parvum and Cryptosporidium hominis. Oral treatment with the pyrazolopyridine KDU731 results in a potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhoea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest that the Cryptosporidium lipid kinase PI(4)K (phosphatidylinositol-4-OH kinase) is a target for pyrazolopyridines and that KDU731 warrants further preclinical evaluation as a drug candidate for the treatment of cryptosporidiosis.

Indolyl-naphthyl-maleimides as potent and selective inhibitors of protein kinase C-α/ß.

The indolyl-naphthyl maleimide 7 is a potent inhibitor of the classical PKC isotypes α,ß and shows excellent selectivity over the novel PKC isotypes δ,ε,η,θ and other kinases belonging to the AGC family. The SAR around 7 as well as the physico-chemical characteristics of selected derivatives and their activity in T and B cell activation and proliferation assays are discussed.

Cyanotriazoles are selective topoisomerase II poisons that rapidly cure trypanosome infections.

Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo-electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.

The PKC inhibitor AEB071 may be a therapeutic option for psoriasis.

PKC isoforms tau, alpha, and beta play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40+ dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.

2,6-Naphthyridines as potent and selective inhibitors of the novel protein kinase C isozymes.

The present study describes a novel series of ATP-competitive PKC inhibitors based on the 2,6-naphthyridine template. Example compounds potently inhibit the novel Protein Kinase C (PKC) isotypes δ, ε, η, θ (in particular PKCε/η, and display a 10-100-fold selectivity over the classical PKC isotypes. The prototype compound 11 was found to inhibit PKCθ-dependent pathways in vitro and in vivo. In vitro, a-CD3/a-CD28-induced lymphocyte proliferation could be effectively blocked in 10% rat whole blood. In mice, 11 dose-dependently inhibited Staphylococcus aureus enterotoxin B-triggered IL-2 serum levels after oral dosing.

Effects of the novel protein kinase C inhibitor AEB071 (Sotrastaurin) on rat cardiac allograft survival using single agent treatment or combination therapy with cyclosporine, everolimus or FTY720.

NVP-AEB071 (AEB, sotrastaurin), an oral inhibitor of protein kinase C (PKC), effectively blocks T-cell activation. The immunosuppressive effects of oral AEB were demonstrated in a rat local graft versus host (GvH) reaction and rat cardiac transplantation models. T-cell activation was suppressed by 95% in blood from AEB-treated rats, with a positive correlation between T-cell inhibition and AEB blood concentration. In GvH studies, AEB inhibited lymph node swelling dose-dependently (3-30 mg/kg). BN and DA cardiac allografts were acutely rejected within 6-10 days post-transplantation in untreated LEW rats. AEB at 10 and 30 mg/kg b.i.d. prolonged BN graft survival to a mean survival time of 15 and >28 days, and DA grafts to 6.5 and 17.5 days, respectively. In the DA to LEW model, combining a nonefficacious dose of AEB (10 mg/kg b.i.d.) with a nonefficacious dose of cyclosporine, everolimus or FTY720 led to prolonged median survival times (26 days, >68 days and >68 days, respectively). Pharmacokinetic monitoring excluded drug-drug interactions, suggesting synergy. In conclusion, these studies are the first to demonstrate that AEB prolongs rat heart allograft survival safely as monotherapy and in combination with nonefficacious doses of cyclosporine, everolimus or FTY720. Thus, AEB may have the potential to offer an alternative to calcineurin inhibitor-based therapies.

Structure-Based and Property-Driven Optimization of N-Aryl Imidazoles toward Potent and Selective Oral RORγt Inhibitors.

Retinoic acid receptor-related orphan receptor gamma-t (RORγt) is considered to be the master transcription factor for the development of Th17 cells that produce proinflammatory cytokines such as IL-17A. Overproportionate Th17 cell abundance is associated with the pathogenesis of many inflammatory conditions including psoriasis. In a high-throughput fluorescence resonance energy transfer (FRET) screen, we identified compound 1 as a hit with promising lipophilic efficiency (LipE). Using structure-based drug design based on a number of X-ray cocrystal structures, we morphed this hit class into potent imidazoles, exemplified by compound 3. To improve the poor absorption, distribution, metabolism, and excretion (ADME) properties of neutral imidazoles, we extended our ligands with carboxylic acid substituents toward a polar, water-rich area of the protein. This highly lipophilicity-efficient modification ultimately led to the discovery of compound 14, a potent and selective inhibitor of RORγt with good ADME properties and excellent in vivo pharmacokinetics. This compound showed good efficacy in an in vivo delayed-type hypersensitivity pharmacology model in rats.

Optimizing a Weakly Binding Fragment into a Potent RORγt Inverse Agonist with Efficacy in an in Vivo Inflammation Model.

The transcription factor RORγt is an attractive drug-target due to its role in the differentiation of IL-17 producing Th17 cells that play a critical role in the etiopathology of several autoimmune diseases. Identification of starting points for RORγt inverse agonists with good properties has been a challenge. We report the identification of a fragment hit and its conversion into a potent inverse agonist through fragment optimization, growing and merging efforts. Further analysis of the binding mode revealed that inverse agonism was achieved by an unusual mechanism. In contrast to other reported inverse agonists, there is no direct interaction or displacement of helix 12 observed in the crystal structure. Nevertheless, compound 9 proved to be efficacious in a delayed-type hypersensitivity (DTH) inflammation model in rats.

Synthesis and Biological Evaluation of New Triazolo- and Imidazolopyridine RORγt Inverse Agonists.

Retinoic-acid-related orphan receptor γt (RORγt) is a key transcription factor implicated in the production of pro-inflammatory Th17 cytokines, which drive a number of autoimmune diseases. Despite diverse chemical series having been reported, combining high potency with a good physicochemical profile has been a very challenging task in the RORγt inhibitor field. Based on available chemical structures and incorporating in-house knowledge, a new series of triazolo- and imidazopyridine RORγt inverse agonists was designed. In addition, replacement of the terminal cyclopentylamide metabolic soft spot by five-membered heterocycles was investigated. From our efforts, we identified an optimal 6,7,8-substituted imidazo[1,2-a]pyridine core system and a 5-tert-butyl-1,2,4-oxadiazole as cyclopentylamide replacement leading to compounds 10 ((S)-N-(8-((4-(cyclopentanecarbonyl)-3-methylpiperazin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-5-carboxamide) and 33 ((S)-N-(8-((4-(5-(tert-butyl)-1,2,4-oxadiazol-3-yl)-3-methylpiperazin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-5-carboxamide). Both derivatives showed good pharmacological potencies in biochemical and cell-based assays combined with excellent physicochemical properties, including low to medium plasma protein binding across species. Finally, 10 and 33 were shown to be active in a rodent pharmacokinetic/pharmacodynamic (PK/PD) model after oral gavage at 15 mg kg-1 , lowering IL-17 cytokine production in ex vivo antigen recall assays.

Retinoic acid receptor alpha and retinoid X receptor specific agonists reduce renal injury in established chronic glomerulonephritis of the rat.

Retinoids, derivatives of vitamin A, inhibit mesangial cell proliferation, glomerular inflammation, and extracellular matrix deposition in acute anti-Thy1.1 glomerulonephritis (Thy-GN) of the rat. We examined a model, chronic mesangioproliferative Thy-GN (MoAb 1-22-3), which is more akin to human disease. Treatment started on day 23 when Thy-GN had already been established. Nonnephritic control and Thy-GN rats were treated orally for 67 days with vehicle or with two doses of either the retinoic acid receptor alpha-specific agonist AGN 195183 (RARalpha agonist) or the retinoid X receptor specific agonist AGN 194204 (RXR agonist). Doses of either the RARalpha or the RXR agonist significantly reduced albuminuria and normalized blood pressure during the course of treatment. The glomerulosclerosis index, glomerular cell and interstitial cell counts, and area of the interstitial space were significantly lower in nephritic rats treated with the RARalpha agonist or RXR agonist than with vehicle. The RARalpha and RXR agonist significantly reduced the infiltration of the glomerulus by macrophages. The increase in glomerular TGFbeta1 and prepro-ET(1) gene expression in vehicle-treated nephritic rats was significantly attenuated by RARalpha or RXR agonists. Glomerular expression of RXRalpha and RARalpha receptor mRNA was significantly greater in vehicle-treated nephritic rats than in nonnephritic controls. Treatment with RARalpha or RXR agonists tended to normalize retinoid-receptor gene expression. Our data indicate that both RARalpha agonists and RXR agonists reduce renal damage in rats with established chronic glomerulonephritis. Receptor-specific retinoids may provide a novel therapeutic approach for the treatment of chronic glomerulonephritis.

Identification of a novel series of orexin receptor antagonists with a distinct effect on sleep architecture for the treatment of insomnia.

Dual orexin receptor (OXR) antagonists (DORAs) such as almorexant, 1 (SB-649868), or suvorexant have shown promise for the treatment of insomnias and sleep disorders in several recent clinical trials in volunteers and primary insomnia patients. The relative contribution of antagonism of OX1R and OX2R for sleep induction is still a matter of debate. We therefore initiated a drug discovery project with the aim of creating both OX2R selective antagonists and DORAs. Here we report that the OX2R selective antagonist 26 induced sleep in mice primarily by increasing NREM sleep, whereas the DORA suvorexant induced sleep largely by increasing REM sleep. Thus, OX2R selective antagonists may also be beneficial for the treatment of insomnia.

PKC theta cooperates with PKC alpha in alloimmune responses of T cells in vivo.

The physiological roles of PKC alpha and PKC theta were defined in T cell immune functions downstream of the antigen receptor. To investigate the hypothesis that both PKC isotypes may have overlapping functions, we generated mice lacking both genes. We find that PKC alpha(-/-)/theta(-/-) animals have additive T cell response defects in comparison to animals carrying single mutations in these genes. Our studies demonstrate that the activities of PKC alpha and PKC theta converge to regulate both IL-2 cytokine responses and T cell intrinsic alloreactivity in vivo. Mechanistically, this PKC alpha/theta crosstalk primarily affects the NFAT transactivation pathway in T lymphocytes, as observed by decreased phosphorylation of Ser-9 on GSK3 beta, reduced nuclear translocation and DNA binding of NFAT in isolated PKC alpha(-/-)/theta(-/-) CD3(+) T cells. This additive defect proved to be of physiological relevance, because PKC alpha(-/-)/theta(-/-) mice demonstrated significantly prolonged allograft survival in heart transplantation experiments, whereas both PKC alpha(-/-) and PKC theta(-/-) mice showed only minimal graft prolongation when compared to wild type controls. While PKC theta appears to be the rate-limiting PKC isotype mediating T lymphocyte activation, we here provide genetic evidence that PKC alpha and PKC theta have overlapping functions in alloimmunoreactivity in vivo and both PKC theta and PKC alpha isotypes must be targeted to prevent organ allograft rejection.

Structure of human cyclophilin A in complex with the novel immunosuppressant sanglifehrin A at 1.6 A resolution.

Sanglifehrin A (SFA) is a novel immunosuppressant isolated from Streptomyces sp. that binds strongly to the human immunophilin cyclophilin A (CypA). SFA exerts its immunosuppressive activity through a mode of action different from that of all other known immunophilin-binding substances, namely cyclosporine A (CsA), FK506, and rapamycin. We have determined the crystal structure of human CypA in complex with SFA at 1.6 A resolution. The high resolution of the structure revealed the absolute configuration at all 17 chiral centers of SFA as well as the details of the CypA/SFA interactions. In particular, it was shown that the 22-membered macrocycle of SFA is deeply embedded in the same binding site as CsA and forms six direct hydrogen bonds with CypA. The effector domain of SFA, on the other hand, has a chemical and three-dimensional structure very different from CsA, already strongly suggesting different immunosuppressive mechanisms. Furthermore, two CypA.SFA complexes form a dimer in the crystal as well as in solution as shown by light scattering and size exclusion chromatography experiments. This observation raises the possibility that the dimer of CypA.SFA complexes is the molecular species mediating the immunosuppressive effect.

Nuclear (receptor) power: retinoids in rat mesangioproliferative disease.

Cytokine-driven proliferation and inflammation play important roles in the response of the kidney to injury and precede the development of glomerulosclerosis. There is great interest in agents which may interfere with such proliferation and inflammation. Therefore, a rat model of mesangioproliferative glomerulonephritis was studied and the effects of all-trans retinoid acid (RA) and isotretinoin, powerful anti-proliferative and anti-inflammatory substances, on glomerular damage and cell proliferation were examined. The use of retinoids was also warranted because of their known (suppressive) effects on genes involved in the pathogenesis of renal damage. RA prevented the blood pressure increase evoked by anti-Thy1.1 nephritis. Treatment with either RA or isotretinoin reduced the albumin excretion rate by 70%. Periodic acid-Schiff (PAS) stains revealed significantly fewer glomerular cells in nephritic rats treated with retinoids. Similarly, the number of mitoses and of cells which stained positively for proliferating cell nuclear antigen was significantly less in nephritic glomeruli treated with retinoids compared with the vehicle-treated group. Glomerular expression of platelet-derived growth factor B-chain was significantly reduced in the presence of retinoids. It was concluded that retinoids limit glomerular proliferation, glomerular lesions and albuminuria in an established model of renal damage. These findings point to retinoids as potential novel modulators of glomerular injury.

Retinoid agonist isotretinoin ameliorates obstructive renal injury.

PURPOSE: Interstitial fibrosis is a major cause of end stage renal failure. Retinoids, which are involved in tissue repair and fibrosis, inhibit inflammatory and proliferative pathways. Therefore, we studied the dose dependent effects of the retinoid receptor agonist isotretinoin 13-cis retinoic acid in the unilateral ureteral obstruction model (UUO). MATERIALS AND METHODS: Sham operated control rats were compared with UUO rats treated with vehicle (UUO-Veh), or low (5 mg/kg body weight (UUO-LD) or high (25 mg/kg) (UUO-HD) dose isotretinoin. Kidneys were evaluated using reverse transcriptase-polymerase chain reaction and immunohistology 7 days after UUO. Renal injury and fibrosis were quantified by immunostaining and expression measurements of the genes involved in renal fibrosis. RESULTS: In UUO-Veh kidneys the interstitial area was expanded 5-fold but only 3-fold in UUO-HD and 3.5-fold in UUO-LD rats. Interstitial cell counts were 3-fold higher in UUO-Veh rats but significantly less in UUO-HD or UUO-LD animals. Tubular and interstitial cell proliferation was significantly higher in UUO-Veh rats compared with sham operated control plus vehicle animals but less so in UUO-LD and UUO-HD rats. In UUO-Veh rats interstitial infiltration by monocytes/macrophages was higher compared with unobstructed controls. It was significantly less after isotretinoin treatment. In UUO-Veh rats mRNA for procollagen I, and transforming growth factor-beta1 and II receptor was significantly increased. It was significantly less after treatment with isotretinoin. Fibronectin and collagen I immunostaining was also decreased by isotretinoin. CONCLUSIONS: Since isotretinoin limits proliferation, inflammation and fibrosis after UUO, retinoids should be further investigated as potentially promising therapeutic agents for renal disease.

Retinoid receptor-specific agonists alleviate experimental glomerulonephritis.

Retinoids are potent antiproliferative and anti-inflammatory compounds. We previously demonstrated that the natural pan-agonists all-trans retinoic acid (RA) and 13-cis RA efficiently preserve renal structure and function in rat mesangioproliferative glomerulonephritis. We examine effects of synthetic retinoid receptor-specific agonists 1) to identify common and receptor subtype-specific pathways in this model and 2) to characterize effects of retinoids on the renal endothelin (ET) system. Vehicle-injected control rats were compared with rats treated with daily subcutaneous injections of agonists specific for retinoid A (Ro-137410) and retinoid X (Ro-257386) receptors and the complex anti-activator protein-1 active retinoid BMS-453 7 days after induction of anti-Thy1.1 nephritis (n = 7-9/group). The different retinoids lowered glomerular ET-1 and ET type A and B receptor gene expression in control and nephritic rats with comparable efficacy. Reduction of glomerular c-Fos and GATA-2 mRNA expression levels suggests downregulation of transcription factors required for ET expression. The different retinoids were similar in their action on the glomerular capillary occlusion score, number of total glomerular cells, and glomerular infiltrating macrophage count. They differed in their ability to normalize blood pressure (Ro-257386 > BMS-453 > arotinoid), albuminuria (BMS-453 > Ro-257386 > arotinoid), and creatinine clearance (arotinoid > BMS-453 > Ro-257386). No signs of toxicity were observed. We conclude that all retinoid agonists with different subtype specificity are highly efficient in reducing renal damage and proliferation of mesangial cells. Retinoid X and A receptor-specific pathways are apparently involved in the antiproliferative, anti-inflammatory, and anti-ET action. Further studies are indicated to define the potential use of retinoid agonists in inflammatory renal disease.