Discovery of IRAK4 Inhibitors BAY1834845 (Zabedosertib) and BAY1830839.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in innate inflammatory processes. Here, we describe the discovery of two clinical candidate IRAK4 inhibitors, BAY1834845 (zabedosertib) and BAY1830839, starting from a high-throughput screening hit derived from Bayer's compound library. By exploiting binding site features distinct to IRAK4 using an in-house docking model, liabilities of the original hit could surprisingly be overcome to confer both candidates with a unique combination of good potency and selectivity. Favorable DMPK profiles and activity in animal inflammation models led to the selection of these two compounds for clinical development in patients.

Discovery of BAY-390, a Selective CNS Penetrant Chemical Probe as Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonist.

Transient receptor potential ankyrin 1 (TRPA1) is a voltage-dependent, ligand-gated ion channel, and activation thereof is linked to a variety of painful conditions. Preclinical studies have demonstrated the role of TRPA1 receptors in a broad range of animal models of acute, inflammatory, and neuropathic pain. In addition, a clinical study using the TRPA1 antagonist GRC-17536 (Glenmark Pharmaceuticals) demonstrated efficacy in a subgroup of patients with painful diabetic neuropathy. Consequently, there is an increasing interest in TRPA1 inhibitors as potential analgesics. Herein, we report the identification of a fragment-like hit from a high-throughput screening (HTS) campaign and subsequent optimization to provide a novel and brain-penetrant TRPA1 inhibitor (compound 18, BAY-390), which is now being made available to the research community as an open-source in vivo probe.

Discovery and Characterization of BAY 1214784, an Orally Available Spiroindoline Derivative Acting as a Potent and Selective Antagonist of the Human Gonadotropin-Releasing Hormone Receptor as Proven in a First-In-Human Study in Postmenopausal Women.

The growth of uterine fibroids is sex hormone-dependent and commonly associated with highly incapacitating symptoms. Most treatment options consist of the control of these hormonal effects, ultimately blocking proliferative estrogen signaling (i.e., oral contraceptives/antagonization of human gonadotropin-releasing hormone receptor [hGnRH-R] activity). Full hGnRH-R blockade, however, results in menopausal symptoms and affects bone mineralization, thus limiting treatment duration or demanding estrogen add-back approaches. To overcome such issues, we aimed to identify novel, small-molecule hGnRH-R antagonists. This led to the discovery of compound BAY 1214784, an orally available, potent, and selective hGnRH-R antagonist. Altering the geminal dimethylindoline core of the initial hit compound to a spiroindoline system significantly improved GnRH-R antagonist potencies across several species, mandatory for a successful compound optimization in vivo. In a first-in-human study in postmenopausal women, once daily treatment with BAY 1214784 effectively lowered plasma luteinizing hormone levels by up to 49%, at the same time being associated with low pharmacokinetic variability and good tolerability.

Author Correction: Amine oxidase 3 is a novel pro-inflammatory marker of oxidative stress in peritoneal endometriosis lesions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Amine oxidase 3 is a novel pro-inflammatory marker of oxidative stress in peritoneal endometriosis lesions.

Endometriosis is a common gynaecological disease of women in reproductive age, and is thought to arise from retrograde menstruation and implantation of endometrial tissue, mostly into the peritoneal cavity. The condition is characterized by a chronic, unresolved inflammatory process thereby contributing to pain as cardinal symptom in endometriosis. Elevated reactive oxygen species (ROS) and oxidative stress have been postulated as factors in endometriosis pathogenesis. We here set out for a systematic study to identify novel mechanisms and pathways relating to oxidative stress in ectopic peritoneal lesions. Using combined proteomic and transcriptomic approaches, we identified novel targets including upregulated pro-oxidative enzymes, such as amine oxidase 3/vascular adhesion protein 1 (AOC3/VAP1) as well as downregulated protective factors, in particular alkenal reductase PTGR1 and methionine sulfoxide reductase. Consistent with an altered ROS landscape, we observed hemoglobin / iron overload, ROS production and lipid peroxidation in ectopic lesions. ROS-derived 4-hydroxy-2-nonenal induced interleukin IL-8 release from monocytes. Notably, AOC3 inhibitors provoked analgesic effects in inflammatory pain models in vivo, suggesting potential translational applicability.

Discovery and Characterization of the Potent and Selective P2X4 Inhibitor N-[4-(3-Chlorophenoxy)-3-sulfamoylphenyl]-2-phenylacetamide (BAY-1797) and Structure-Guided Amelioration of Its CYP3A4 Induction Profile.

The P2X4 receptor is a ligand-gated ion channel that is expressed on a variety of cell types, especially those involved in inflammatory and immune processes. High-throughput screening led to a new class of P2X4 inhibitors with substantial CYP 3A4 induction in human hepatocytes. A structure-guided optimization with respect to decreased pregnane X receptor (PXR) binding was started. It was found that the introduction of larger and more polar substituents on the ether linker led to less PXR binding while maintaining the P2X4 inhibitory potency. This translated into significantly reduced CYP 3A4 induction for compounds 71 and 73. Unfortunately, the in vivo pharmacokinetic (PK) profiles of these compounds were insufficient for the desired profile in humans. However, BAY-1797 (10) was identified and characterized as a potent and selective P2X4 antagonist. This compound is suitable for in vivo studies in rodents, and the anti-inflammatory and anti-nociceptive effects of BAY-1797 were demonstrated in a mouse complete Freund's adjuvant (CFA) inflammatory pain model.

Discovery and optimization of pyridyl-cycloalkyl-carboxylic acids as inhibitors of microsomal prostaglandin E synthase-1 for the treatment of endometriosis.

Here we report on novel and potent pyridyl-cycloalkyl-carboxylic acid inhibitors of microsomal prostaglandin E synthase-1 (PTGES). PTGES produces, as part of the prostaglandin pathway, prostaglandin E2 which is a well-known driver for pain and inflammation. This fact together with the observed upregulation of PTGES during inflammation suggests that blockade of the enzyme might provide a beneficial treatment option for inflammation related conditions such as endometriosis. Compound 5a, a close analogue of the screening hit, potently inhibited PTGES in vitro, displayed excellent PK properties in vitro and in vivo and demonstrated efficacy in a CFA-induced pain model in mice and in a rat dyspareunia endometriosis model and was therefore selected for further studies.

Identification of a Benzimidazolecarboxylic Acid Derivative (BAY 1316957) as a Potent and Selective Human Prostaglandin E2 Receptor Subtype 4 (hEP4-R) Antagonist for the Treatment of Endometriosis.

The presence and growth of endometrial tissue outside the uterine cavity in endometriosis patients are primarily driven by hormone-dependent and inflammatory processes-the latter being frequently associated with severe, acute, and chronic pelvic pain. The EP4 subtype of prostaglandin E2 (PGE2) receptors (EP4-R) is a particularly promising anti-inflammatory and antinociceptive target as both this receptor subtype and the pathways forming PGE2 are highly expressed in endometriotic lesions. High-throughput screening resulted in the identification of benzimidazole derivatives as novel hEP4-R antagonists. Careful structure-activity relationship investigation guided by rational design identified a methyl substitution adjacent to the carboxylic acid as an appropriate means to accomplish favorable pharmacokinetic properties by reduction of glucuronidation. Further optimization led to the identification of benzimidazolecarboxylic acid BAY 1316957, a highly potent, specific, and selective hEP4-R antagonist with excellent drug metabolism and pharmacokinetics properties. Notably, treatment with BAY 1316957 can be expected to lead to prominent and rapid pain relief and significant improvement of the patient's quality of life.

An approach towards enhancement of a screening library: The Next Generation Library Initiative (NGLI) at Bayer - against all odds?

Pharmaceutical companies often refer to 'screening their library' when performing high-throughput screening (HTS) on a corporate compound collection to identify lead structures for small-molecule drug discovery programs. Characteristics of such a library, including the size, chemical space covered, and physicochemical properties, often determine the success of a screening campaign. Therefore, strategies to maintain and enhance the overall quality of screening collections are crucial to stay competitive and to cope with the 'novelty erosion' that is observed gradually. The Next Generation Library Initiative (NGLI), the enhancement of Bayer's HTS collection by 500000 newly designed compounds within 5 years, is addressing exactly this challenge. Here, we describe this collaborative project, which involves all internal medicinal chemists in a crowd-sourcing approach, as well as selected external partners, to reach this ambitious goal.

Role of vitamin D derivatives in intestinal tissue of patients with inflammatory bowel diseases.

BACKGROUND AND AIM: The adhesion molecule expression and matrix metalloproteinases (MMPs) are proposed to be major factors for intestinal injury mediated by T cells in (IBD) and are up-regulated in intestinal mucosa of IBD patients. To investigate the effect of vitamin D derivatives on adhesion molecules and MMPs in colonic biopsies of IBD patients. METHODS: Biopsies from inflamed and non-inflamed tract of terminal ileum and colon and PBMC from the same IBD patients were cultured with or without vitamin D derivatives. MMP activity and adhesion molecule levels were determined. RESULTS: 1,25(OH)2D3 and ZK 191784 significantly decrease ICAM-1 protein levels in the biopsies obtained only from the inflamed region of intestine of UC patients, while MAdCAM-1 levels decrease in the presence of 1,25(OH)2D3 in the non-inflamed region, and, in the presence of ZK, in the inflamed one. In CD patients 1,25(OH)2D3 and ZK decrease ICAM-1 and MAdCAM-1 in the biopsies obtained from the non-inflamed and inflamed regions, with the exception of ICAM-1 in the inflamed region in the presence of 1,25(OH)2D3. The expression of MMP-9, MMP-2, and MMP-3 decreases in the presence of vitamin D derivatives in UC and CD with the exception of 1,25(OH)2D3 that does not affect the levels of MMP-9 and MMP-2 in CD. Vitamin D derivatives always affect MMP-9, MMP-2 and ICAM-1 in PBMC of UC and CD patients. CONCLUSIONS: Based on the increased expression of ICAM-1, MAdCAM-1 and MMP-2,-9,-3 in IBD, our study suggests that vitamin D derivatives may be effective in the management of these diseases.

The vitamin D analog ZK191784 normalizes decreased bone matrix mineralization in mice lacking the calcium channel TRPV5.

Mice lacking the renal epithelial Ca(2+) channel TRPV5 (TRPV5(-/-)) display impaired renal Ca(2+) reabsorption, hypercalciuria, and intestinal Ca(2+) hyperabsorption, due to secondary hypervitaminosis D. Using these mice, we previously demonstrated that ZK191784 acts as an intestine-specific 1,25(OH)(2) D(3) antagonist without affecting serum calcium levels. On the other hand, it acted as an agonist in the kidney and the effects of ZK191784 on bone were ambiguous. The present study was undertaken to further evaluate the effect of the vitamin D receptor antagonist on murine bone in mice lacking TRPV5. Eight-week-old female Trpv5(+/+) and Trpv5(-/-) mice were treated for 4 weeks with or without 50 µg/kg/day ZK191784. Quantitative backscattered electron imaging showed that the reduced bone matrix mineralization found in femoral bones of Trpv5(-/-) mice was partially but significantly restored upon ZK191784 treatment, just as we observed for trabecular bone thickness. This supports the significance of 1,25(OH)(2) D(3) and optimal control of Ca(2+) homeostasis for bone formation and matrix mineralization. Restoration also took place at the bone gene expression level, where 1α-hydroxylase (Cyp27b1) mRNA in femurs from ZK-treated Trpv5(-/-) mice was upregulated compared to control levels. The downregulated 24-hydroxylase (Cyp24a1) gene expression in femoral bone indicated local vitamin D resistance in the mice treated with ZK191784. Phosphate homeostasis was unaffected between the groups as shown by unaltered serum PO(4)(3-) and fibroblast growth factor (FGF) 23 as well as Fgf23 mRNA expression in bone. In conclusion, circulating 1,25(OH)(2) D(3) is important for optimal control of Ca(2+) homeostasis but also for controlled bone formation and matrix mineralization.

Vitamin D metabolism and effects on pluripotency genes and cell differentiation in testicular germ cell tumors in vitro and in vivo.

Testicular germ cell tumors (TGCTs) are classified as either seminomas or nonseminomas. Both tumors originate from carcinoma in situ (CIS) cells, which are derived from transformed fetal gonocytes. CIS, seminoma, and the undifferentiated embryonal carcinoma (EC) retain an embryonic phenotype and express pluripotency factors (NANOG/OCT4). Vitamin D (VD) is metabolized in the testes, and here, we examined VD metabolism in TGCT differentiation and pluripotency regulation. We established that the VD receptor (VDR) and VD-metabolizing enzymes are expressed in human fetal germ cells, CIS, and invasive TGCTs. VD metabolism diminished markedly during the malignant transformation from CIS to EC but was reestablished in differentiated components of nonseminomas, distinguished by coexpression of mesodermal markers and loss of OCT4. Subsequent in vitro studies confirmed that 1,25(OH)(2)D(3) (active VD) downregulated NANOG and OCT4 through genomic VDR activation in EC-derived NTera2 cells and, to a lesser extent, in seminoma-derived TCam-2 cells, and up-regulated brachyury, SNAI1, osteocalcin, osteopontin, and fibroblast growth factor 23. To test for a possible therapeutic effect in vivo, NTera2 cells were xenografted into nude mice and treated with 1,25(OH)(2)D(3), which induced down-regulation of pluripotency factors but caused no significant reduction of tumor growth. During NTera2 tumor formation, down-regulation of VDR was observed, resulting in limited responsiveness to cholecalciferol and 1,25(OH)(2)D(3) treatment in vivo. These novel findings show that VD metabolism is involved in the mesodermal transition during differentiation of cancer cells with embryonic stem cell characteristics, which points to a function for VD during early embryonic development and possibly in the pathogenesis of TGCTs.

Vitamin D receptor activation improves allergen-triggered eczema in mice.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease that has increased in prevalence over the last several decades in industrialized countries. AD is a multifactorial, heterogenous disease with a variety of defects in the immune system, in antimicrobial defense mechanisms and epidermal barrier integrity, which collectively contribute to the risk and severity of AD development. Vitamin D receptor (VDR) signaling has been shown to be important not only in the immune system but also in the skin and in particular keratinocytes to regulate skin homeostasis and epidermal barrier function. However, this work aimed to analyze the role and clinical efficiency of VDR activation by a VDR agonist without calcium-mobilizing activity in a mouse model of allergen-triggered eczema. We show that the systemic administration of the low-calcemic VDR agonist significantly improved the allergen-triggered eczema. Thereby, forkhead box P3 (Foxp3)-expressing regulatory T cells, revealed to have a role in AD, were selectively increased in the skin of VDR agonist-treated mice. Moreover, our results demonstrate a marked induction of skin barrier gene and antimicrobial peptide gene expression in skin lesions of VDR agonist-treated mice. Thus, our study provides evidence that systemic VDR agonist treatment may improve allergen-triggered eczema in vivo.

The novel vitamin D analog ZK191784 inhibits prostate cancer cell invasion.

BACKGROUND: Low serum levels of 1,25(OH)(2)D(3) (1,25D), have been associated with aggressive biologic behavior of prostate cancer (PCa). In the present study, we examined the effects of 1,25D and its novel, low-calcemic analog ZK191784 (ZK) on matrix metalloproteinases (MMPs), as well as on intercellular adhesion molecule-1 (ICAM-1) protein levels in human PCa cell lines LNCaP and DU-145. MATERIALS AND METHODS: Cells were incubated with either vehicle (control), 1,25D or ZK. MMP-2 and MMP-9 activity was determined by gelatin zymography, while ICAM-1 levels were assessed by Western blot analysis and immunocytochemistry. RESULTS: Compared to the controls, 1,25D and ZK caused a marked dose-dependent decrease in the gelatinolytic activity of the MMPs under study, particularly when ZK was used. Likewise, ICAM-1 was down-regulated in the cells incubated with 1,25D or ZK. CONCLUSION: Vitamin D analogs appear to be involved in the regulation of extracellular MMP activity and membrane adhesion molecule expression. Further studies, both in vitro and in vivo, are needed to define their role as potential therapeutic tools.

Vitamin D is required for IFN-gamma-mediated antimicrobial activity of human macrophages.

Control of tuberculosis worldwide depends on our understanding of human immune mechanisms, which combat the infection. Acquired T cell responses are critical for host defense against microbial pathogens, yet the mechanisms by which they act in humans remain unclear. We report that T cells, by the release of interferon-γ (IFN-γ), induce autophagy, phagosomal maturation, the production of antimicrobial peptides such as cathelicidin, and antimicrobial activity against Mycobacterium tuberculosis in human macrophages via a vitamin D-dependent pathway. IFN-γ induced the antimicrobial pathway in human macrophages cultured in vitamin D-sufficient sera, but not in sera from African-Americans that have lower amounts of vitamin D and who are more susceptible to tuberculosis. In vitro supplementation of vitamin D-deficient serum with 25-hydroxyvitamin D3 restored IFN-γ-induced antimicrobial peptide expression, autophagy, phagosome-lysosome fusion, and antimicrobial activity. These results suggest a mechanism in which vitamin D is required for acquired immunity to overcome the ability of intracellular pathogens to evade macrophage-mediated antimicrobial responses. The present findings underscore the importance of adequate amounts of vitamin D in all human populations for sustaining both innate and acquired immunity against infection.

Vitamin D(3) down-regulates proinflammatory cytokine response to Mycobacterium tuberculosis through pattern recognition receptors while inducing protective cathelicidin production.

A well-known association between vitamin D(3) and infection with Mycobacterium tuberculosis has previously been reported, but little is known regarding the underlying mechanisms. We have investigated how 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] affects the proinflammatory cytokine production induced by M. tuberculosis. Furthermore, we explored whether 1,25(OH)(2)D(3) influence the production of the protective antimycobacterial peptide cathelicidin. Upon in vitro stimulation with M. tuberculosis, 1,25(OH)(2)D(3) induced a dose-dependent down-regulation of IL-6, TNFα and IFNγ, while increasing the production of IL-10 in culture supernatant as well as cathelicidin mRNA expression. This effect on cytokine response was not due to modulation of T-helper cell differentiation, as T-bet, GATA3, Foxp3 and ROR-γt mRNA expression remained unaffected. Similarly, 1,25(OH)(2)D(3) did not affect suppressor of cytokine signaling (SOCS)1 and SOCS3 mRNA expression. The mechanism whereby 1,25(OH)(2)D(3) inhibited the proinflammatory cytokine response was through reduced expression of the pattern recognition receptors (PRR) - TLR2, TLR4, Dectin-1 and mannose receptor, whose mRNA and protein expression were both reduced. The suppression of PRRs could be restored by a VDR antagonist. Upon M. tuberculosis stimulation, 1,25(OH)(2)D(3) modulates the balance in cytokine production towards an anti-inflammatory profile by repression of TLR2, TLR4, Dectin-1 and mannose receptor expression, while increasing cathelicidin production. These two effects may have beneficial consequences, by reducing the collateral tissue damage induced by proinflammatory cytokines, while the antibacterial effects of cathelicidin are enhanced.

Rendezvous in chemical space? Comparing the small molecule compound libraries of Bayer and Schering.

Here, we compare the entire compound collections of Bayer HealthCare and Schering AG with respect to structural identities, similarities and physico-chemical properties. We discuss possible consequences stemming from unexpected findings in light of new collaborative models in pharmaceutical research.

Calcitriol down-regulates human ether a go-go 1 potassium channel expression in cervical cancer cells.

BACKGROUND/AIM: Human ether à-go-go-1 (EAG1) potassium channels are promising anticancer targets. Calcitriol has antitumoural properties. This study investigated EAG1 regulation by calcitriol in normal and cancer cells. MATERIALS AND METHODS: Cancer cell lines from cervix, prostate, mammary gland, and normal placenta trophoblasts were cultured. Calcitriol was determined by HPLC. Gene and protein expression were assessed by real-time RT-PCR and western blot analysis, respectively. Calcitriol-synthesising enzyme CYP27B1 or vitamin D receptor (VDR), were transfected in cervical cancer SiHa cells. Cell proliferation was assayed with XTT. RESULTS: Calcitriol decreased EAG1 mRNA in all cell types, and EAG1 protein and proliferation in SiHa cells. VDR antagonist ZK-159222 prevented the calcitriol effect on EAG1 mRNA. CYP27B1-transfected cells produced more calcitriol and less EAG1 mRNA. EAG1 mRNA was more potently inhibited by calcitriol in VDR-transfected cells. CONCLUSION: EAG1 is a calcitriol target in normal and cancer cells and calcitriol is a potential therapy for cervical cancer.

Down-regulation of adhesion molecules and matrix metalloproteinases by ZK 156979 in inflammatory bowel diseases.

Intracellular adhesion molecules and matrix metalloproteinases (MMPs) are up-regulated in intestinal mucosa of patients with inflammatory bowel diseases (IBD), i.e. ulcerative colitis (UC) or Crohn's disease (CD). Our aim was to verify whether the vitamin D analogue ZK 156979 (ZK) down-regulates adhesion molecules, and decreases MMPs production by PBMC of IBD patients. ICAM-1 and LFA-1 levels increase, when PBMC were incubated with PHA or LPS or TNF-alpha, and decrease when these substances were used in combination with ZK. MMPs activity increases incubating the cells with PHA or LPS or TNF-alpha. MMP-9 decreases when ZK was used in association, while MMP-2 decreases only when ZK was used in combination with anti-TNF-alpha. Our results suggest that the down-regulation of ICAM-1 and LFA-1 on PBMC and the inhibition of MMP-9 activity by ZK could provide a potential role of this low calcemic vitamin D derivative in future strategies in IBD therapy.