Safety and Dose-Response of Vidofludimus Calcium in Relapsing Multiple Sclerosis: Extended Results of a Placebo-Controlled Phase 2 Trial.

BACKGROUND AND OBJECTIVES: Vidofludimus calcium suppressed MRI disease activity compared with placebo in patients with relapsing-remitting multiple sclerosis (RRMS) in the first cohort of the phase 2 EMPhASIS study. Because 30 mg and 45 mg showed comparable activity on multiple end points, the study enrolled an additional low-dose cohort to further investigate a dose-response relationship. METHODS: In a randomized, placebo-controlled, phase 2 trial, patients with RRMS, aged 18-55 years, and with ≥2 relapses in the last 2 years or ≥1 relapse in the last year, and ≥1 gadolinium-enhancing brain lesion in the last 6 months. Patients were randomly assigned (1:1:1) vidofludimus calcium (30 or 45 mg) or placebo in cohort 1 and vidofludimus calcium (10 mg) or placebo (4:1) in cohort 2 for 24 weeks. The primary end point was the cumulative number of combined unique active (CUA) lesions at week 24. Secondary end points were clinical outcomes and safety. RESULTS: Across cohorts 1 and 2, 268 patients were randomized to placebo (n = 81), 10 mg (n = 47) vidofludimus calcium, 30 mg (n = 71) vidofludimus calcium, or 45 mg (n = 69) vidofludimus calcium. The mean cumulative CUA lesions over 24 weeks was 5.8 (95% CI 4.1-8.2) for placebo, 5.9 (95% CI 3.9-9.0) for 10 mg treatment group, 1.4 (95% CI 0.9-2.1) for 30 mg treatment group, and 1.7 (95% CI 1.1-2.5) for 45 mg treatment group. Serum neurofilament light chain decreased in a dose-dependent manner. The number of patients with confirmed disability worsening after 24 weeks was 3 (3.7%) patients receiving placebo and 3 (1.6%) patients receiving any dose of vidofludimus calcium. Treatment-emergent adverse events occurred in 35 (43%) placebo patients compared with 11 (23%) and 71 (37%) patients in the 10 mg or any dose of vidofludimus calcium groups, respectively. The incidence of liver enzyme elevations and infections were similar between placebo and any dose of vidofludimus calcium. No new safety signals were observed. DISCUSSION: Compared with placebo, vidofludimus calcium suppressed the development of new brain lesions with daily doses of 30 mg and 45 mg, but not 10 mg, establishing the lowest efficacious dose is 30 mg. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that among adults with active RRMS and ≥1 Gd+ brain lesion in the past 6 months, the cumulative number of active lesions decreased with vidofludimus calcium. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov (NCT03846219) and EudraCT (2018-001896-19).

Validation of nuclear receptor RORγ isoform 1 as a novel host-directed antiviral target based on the modulation of cholesterol levels.

Currently, the clinically approved repertoire of antiviral drugs predominantly comprises direct-acting antivirals (DAAs). However, the use of DAAs is frequently limited by adverse effects, restriction to individual virus species, or the induction of viral drug resistance. These issues will likely be resolved by the introduction of host-directed antivirals (HDAs) targeting cellular proteins crucial for viral replication. However, experiences with the development of antiviral HDAs and clinical applications are still in their infancy. With the present study, we explored the human nuclear receptor and transcription factor RORγ isoform 1 (RORγ1), a member of the retinoic acid receptor-related orphan receptor (ROR) family, as a putative target of antiviral HDAs. To this end, cell culture models were used to investigate major viral human pathogens, i.e. the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human cytomegalovirus (HCMV), varicella zoster virus (VZV) and human immunodeficiency virus 1 (HIV-1). Our results demonstrated (i) an antiviral activity of the clinically relevant RORγ modulators cedirogant and others, (ii) that isoform RORγ1 acts as the responsible determinant and drug target in the analyzed cell culture-based models, (iii) a selectivity of the antiviral effect for RORγ1 over related receptors RORα and RORß, (iv) a late-phase inhibition exerted by cedirogant in HCMV replication and (v) a mechanistic link to the cellular cholesterol biosynthesis. Combined, the data highlight this novel RORγ-specific antiviral targeting concept and the developmental potential of RORγ-directed small molecules.

Safety, Tolerability, and Pharmacokinetics of IMU-935, a Novel Inverse Agonist of Retinoic Acid Receptor-Related Orphan Nuclear Receptor γt: Results From a Double-Blind, Placebo-Controlled, First-in-Human Phase 1 Study.

Retinoic acid receptor-related orphan nuclear receptor (ROR)γt regulates the transcription of interleukin-17 and other cytokines implicated in inflammatory and autoimmune diseases. We assessed the safety, tolerability, and pharmacokinetics (PK) of IMU-935, an inverse agonist of RORγt, in a first-in-human phase 1 study. This was a double-blind, placebo-controlled trial that randomly assigned healthy subjects single ascending doses (25-400 mg) or multiple ascending doses (150 mg once or twice daily for 14 days) of IMU-935 or placebo. Dose escalation was determined by the safety, tolerability, and PK. Twenty-four and 70 subjects received placebo or IMU-935, respectively. Of the 70 subjects who received IMU-935, 59 received a single dose and 11 received multiple doses. Treatment-emergent adverse events (TEAEs) occurred in 21 subjects (88%) and 58 (83%) given any dose of placebo or IMU-935, respectively. Treatment-related TEAEs occurred in 6 (30%) and 25 (42%) subjects given a single dose of placebo and IMU-935, respectively. All treatment-related TEAEs were mild except for 2 moderate TEAEs and 1 moderate TEAE in the IMU-935 group and placebo group, respectively. No treatment-related discontinuations or serious adverse events occurred. The PK of IMU-935 were dose proportional with a half-life of ≈24 hours. In conclusion, IMU-935 was safe with no dose-limiting toxicities and had a PK profile that supports once-daily dosing.

Mechanistic Impact of Different Ligand Scaffolds on FXR Modulation Suggests Avenues to Selective Modulators.

The bile-acid sensing nuclear farnesoid X receptor (FXR) is an attractive target for the treatment of hepatic and metabolic diseases, but application of this chemotherapeutic concept remains limited due to adverse effects of FXR activation observed in clinical trials. To elucidate the mechanistic basis of FXR activation at the molecular level, we have systematically studied FXR co-regulator interactions and dimerization in response to seven chemically diverse FXR ligands. Different molecular effects on FXR activation mediated by different scaffolds were evident and aligned with characteristic structural changes within the ligand binding domain of FXR. A partial FXR agonist acted mainly through co-repressor displacement from FXR and caused an FXR-regulated gene expression pattern markedly differing from FXR agonist effects. These results suggest selective modulation of FXR dimerization and co-regulator interactions for different ligands, offering a potential avenue for the design of gene- or tissue-selective FXR modulators.

Safety and Efficacy of Vidofludimus Calcium in Patients Hospitalized with COVID-19: A Double-Blind, Randomized, Placebo-Controlled, Phase 2 Trial.

INTRODUCTION: Vidofludimus calcium has shown anti-inflammatory effects in clinical trials of autoimmune diseases and recently demonstrated antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We performed a double-blind, randomized, placebo-controlled, phase 2 trial to evaluate the safety and efficacy of vidofludimus calcium in patients hospitalized for coronavirus disease 2019 (COVID-19) in Europe and the USA. METHODS: Patients aged 18 years or older who positive for COVID-19 were randomized (1:1) to receive placebo or 45 mg vidofludimus calcium for 14 days with both groups receiving standard-of-care treatment. The primary endpoint was the need for invasive ventilation after 28 days (ClinicalTrials.gov NCT04379271; EudraCT 2020-001264-28). RESULTS: Between June 12, 2020 and December 10, 2020, a total of 223 were randomized to receive either placebo (n = 112) or vidofludimus calcium (n = 111); three patients withdrew consent and were not treated. Eight (9%) patients in the placebo group and 12 (11%) patients in the vidofludimus calcium group needed invasive ventilation during the 28-day study period, which was lower than the assumed rate of 40%. Time to clinical improvement was shorter by approximately 1 day in the vidofludimus calcium group (15.0 days [90% CI 14.8-15.9]) compared to the placebo group (15.9 days [90% CI 14.9-19.9]). This effect was greatest in patients who initiated therapy within 9 days of symptom onset (3.8 days shorter in the vidofludimus calcium group). Higher trough concentrations of vidofludimus calcium were associated with quicker time to clinical recovery. The rate and timing of appearance of anti-SARS-CoV-2 antibodies were not different between groups. Serious adverse events occurred in 4 (4%) patients in the placebo group and 2 (2%) patients in the vidofludimus calcium group; treatment-emergent adverse events of increased severity related to COVID-19 occurred in 13 (12%) patients in the placebo group and 8 (7%) patients in the vidofludimus calcium group. Overall mortality was low (2%). CONCLUSIONS: These findings support vidofludimus calcium being safe and well tolerated in patients with COVID-19.

Inhibitors of dihydroorotate dehydrogenase cooperate with molnupiravir and N4-hydroxycytidine to suppress SARS-CoV-2 replication.

The nucleoside analog N4-hydroxycytidine (NHC) is the active metabolite of the prodrug molnupiravir, which has been approved for the treatment of COVID-19. SARS-CoV-2 incorporates NHC into its RNA, resulting in defective virus genomes. Likewise, inhibitors of dihydroorotate dehydrogenase (DHODH) reduce virus yield upon infection, by suppressing the cellular synthesis of pyrimidines. Here, we show that NHC and DHODH inhibitors strongly synergize in the inhibition of SARS-CoV-2 replication in vitro. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC into nascent viral RNA. This concept is supported by the rescue of virus replication upon addition of pyrimidine nucleosides to the media. DHODH inhibitors increased the antiviral efficiency of molnupiravir not only in organoids of human lung, but also in Syrian Gold hamsters and in K18-hACE2 mice. Combining molnupiravir with DHODH inhibitors may thus improve available therapy options for COVID-19.

Clinical relevance of intestinal barrier dysfunction in common gastrointestinal diseases.

The intestinal barrier is a complex and well-controlled physiological construct designed to separate luminal contents from the bowel wall. In this review, we focus on the intestinal barrier's relationship with the host's immune system interaction and the external environment, specifically the microbiome. The bowel allows the host to obtain nutrients vital to survival while protecting itself from harmful pathogens, luminal antigens, or other pro-inflammatory factors. Control over barrier function and the luminal milieu is maintained at the biochemical, cellular, and immunological level. However, disruption to this highly regulated environment can cause disease. Recent advances to the field have progressed the mechanistic understanding of compromised intestinal barrier function in the context of gastrointestinal pathology. There are numerous examples where bowel barrier dysfunction and the resulting interaction between the microbiome and the immune system has disease-triggering consequences. The purpose of this review is to summarize the clinical relevance of intestinal barrier dysfunction in common gastrointestinal and related diseases. This may help highlight the importance of restoring barrier function as a therapeutic mechanism of action in gastrointestinal pathology.

IMU-838, a Developmental DHODH Inhibitor in Phase II for Autoimmune Disease, Shows Anti-SARS-CoV-2 and Broad-Spectrum Antiviral Efficacy In Vitro.

The ongoing pandemic spread of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) demands skillful strategies for novel drug development, drug repurposing and cotreatments, in particular focusing on existing candidates of host-directed antivirals (HDAs). The developmental drug IMU-838, currently being investigated in a phase 2b trial in patients suffering from autoimmune diseases, represents an inhibitor of human dihydroorotate dehydrogenase (DHODH) with a recently proven antiviral activity in vitro and in vivo. Here, we established an analysis system for assessing the antiviral potency of IMU-838 and DHODH-directed back-up drugs in cultured cell-based infection models. By the use of SARS-CoV-2-specific immunofluorescence, Western blot, in-cell ELISA, viral yield reduction and RT-qPCR methods, we demonstrated the following: (i) IMU-838 and back-ups show anti-SARS-CoV-2 activity at several levels of viral replication, i.e., protein production, double-strand RNA synthesis, and release of infectious virus; (ii) antiviral efficacy in Vero cells was demonstrated in a micromolar range (IMU-838 half-maximal effective concentration, EC50, of 7.6 ± 5.8 µM); (iii) anti-SARS-CoV-2 activity was distinct from cytotoxic effects (half-cytotoxic concentration, CC50, >100 µM); (iv) the drug in vitro potency was confirmed using several Vero lineages and human cells; (v) combination with remdesivir showed enhanced anti-SARS-CoV-2 activity; (vi) vidofludimus, the active determinant of IMU-838, exerted a broad-spectrum activity against a selection of major human pathogenic viruses. These findings strongly suggest that developmental DHODH inhibitors represent promising candidates for use as anti-SARS-CoV-2 therapeutics.

Novel Dihydroorotate Dehydrogenase Inhibitors with Potent Interferon-Independent Antiviral Activity against Mammarenaviruses In Vitro.

Mammarenaviruses cause chronic infections in rodents, which are their predominant natural hosts. Human infection with some of these viruses causes high-consequence disease, posing significant issues in public health. Currently, no FDA-licensed mammarenavirus vaccines are available, and anti-mammarenavirus drugs are limited to an off-label use of ribavirin, which is only partially efficacious and associated with severe side effects. Dihydroorotate dehydrogenase (DHODH) inhibitors, which block de novo pyrimidine biosynthesis, have antiviral activity against viruses from different families, including Arenaviridae, the taxonomic home of mammarenaviruses. Here, we evaluate five novel DHODH inhibitors for their antiviral activity against mammarenaviruses. All tested DHODH inhibitors were potently active against lymphocytic choriomeningitis virus (LCMV) (half-maximal effective concentrations [EC50] in the low nanomolar range, selectivity index [SI] > 1000). The tested DHODH inhibitors did not affect virion cell entry or budding, but rather interfered with viral RNA synthesis. This interference resulted in a potent interferon-independent inhibition of mammarenavirus multiplication in vitro, including the highly virulent Lassa and Junín viruses.

Vidofludimus calcium, a next generation DHODH inhibitor for the Treatment of relapsing-remitting multiple sclerosis.

BACKGROUND: Inhibition of dihydroorotate dehydrogenase (DHODH) is an established mechanism for the treatment of relapsing-remitting multiple sclerosis (RRMS). Currently approved treatments have several shortcomings. Consequently, new and effective treatments with improved safety and convenience profiles are sought after by patients. OBJECTIVE: To explore the overall profile of vidofludimus for the treatment of RRMS. METHODS: Preclinical investigations were done exploring the species-dependency of DHODH inhibition of vidofludimus. In addition, the preclinical efficacy in a rat experimental autoimmune encephalomyelitis (EAE) model and the inhibition of cytokine release from activated PBMC were investigated. Pharmacokinetic data were also obtained in a Phase 1 multiple ascending dose trial of the formulation IMU-838 (vidofludimus calcium). RESULTS: It was shown that vidofludimus is 2.6 times more potent in inhibiting DHO oxidation by human DHODH compared to teriflunomide. Although both compounds increased cell apoptosis, vidofludimus was more efficacious in the inhibition of T-lymphocyte proliferation compared to teriflunomide. The same was also observed for the secretion of IL-17 and IFN-γ. Interestingly, the potency or vidofludimus to inhibit rat or mouse DHODH is 7.5 and 64.4 time lower than the for the human DHODH, respectively. The rat EAE study clearly exhibited a dose-dependent inhibition of cumulative disease scores by vidofludimus. In the multiple ascending dose Phase 1 clinical trial, the serum half-life of about 30 h provides a favorable profile for once daily dosing of IMU-838, with quick dosing to steady state through levels within 5 days and the ability to wash out drug quickly, if required. CONCLUSIONS: The investigations highlighted that the desired selective immunomodulatory properties can be separated from general antiproliferative effects seen and related adverse events in first-generation DHODH inhibitors. Based on data obtained from a series of pre-clinical as well as phase 1 and phase 2 studies, IMU-838 is a promising next-generation candidate for the oral treatment of RRMS. However, this will need to be confirmed in the currently ongoing Phase 2 study in RRMS patients.

Safety, Tolerability and Pharmacokinetics of Vidofludimus calcium (IMU-838) After Single and Multiple Ascending Oral Doses in Healthy Male Subjects.

BACKGROUND AND OBJECTIVE: Vidofludimus is a potent and selective inhibitor of human mitochondrial enzyme dihydroorotate dehydrogenase (DHODH). The clinical efficacy and safety profile of vidofludimus has been analyzed in patients suffering from rheumatoid arthritis and Crohn's disease and ulcerative colitis. In previous sudies, hematuria at higher doses occurred in close temporal relationship to vidofludimus administration and appeared to be dose related. The present report describes the results from two phase 1 studies conducted in healthy male subjects to investigate the safety, tolerability and pharmacokinetics after single and multiple ascending (SAD and MAD) oral doses of IMU-838 (vidofludimus calcium, tablets containing a specific polymorph). The effect of food on the pharmacokinetics of IMU-838 was also assessed in the SAD study. METHODS: In the SAD study, 12 subjects received single doses of IMU-838 under fasting (10-40 mg) or fed (10 mg) condition in an open-label, partial parallel group design. In the MAD study, 52 subjects received multiple doses of IMU-838 (30-50 mg) in a double-blind, placebo-controlled, parallel group design. RESULTS: IMU-838 showed dose-proportional pharmacokinetics after single and multiple oral dosing in both SAD and MAD studies. IMU-838 was well absorbed after single daily doses. Food did not impact the pharmacokinetics of IMU-838. The accumulation factor for multiple daily dosing was approximately 2. Steady-state concentrations were reached within about 6-8 days for 30-50 mg groups. The geometric mean plasma half-life of IMU-838 at steady state was approximately 30 h, which supports its use for once-daily dosing regimen. Single and multiple oral doses of IMU-838 were safe and well tolerated. CONCLUSION: Overall, oral IMU-838 was generally well tolerated in SAD and MAD studies in healthy subjects over a wide dose range of 10-50 mg. IMU-838 was well absorbed after single daily doses. IMU-838 showed dose proportional pharmacokinetics after single and multiple oral dosing.

The Selective Oral Immunomodulator Vidofludimus in Patients with Active Rheumatoid Arthritis: Safety Results from the COMPONENT Study.

INTRODUCTION: The dihydroorotate dehydrogenase (DHODH) inhibitors leflunomide and teriflunomide are immunomodulatory agents approved to treat rheumatoid arthritis (RA) and multiple sclerosis, respectively, and are actively being investigated as therapeutic agents for other immune-related diseases; however, both structurally related compounds have a number of potentially serious adverse effects. Vidofludimus, a new selective second-generation DHODH inhibitor, is chemically distinct from leflunomide/teriflunomide and appears to exhibit a distinct safety profile. OBJECTIVE: The aim of the COMPONENT study was to assess the efficacy, safety, and pharmacokinetics of vidofludimus in the treatment of patients with active RA on a background therapy of methotrexate. This report focuses solely on the safety results of the COMPONENT trial. METHODS: Patients received once-daily oral vidofludimus (N = 122) or placebo (N = 119) along with their standard of care methotrexate treatment for 13 weeks. Efficacy endpoints were assessed. Safety parameters were monitored throughout treatment and at follow-up. Plasma concentrations of vidofludimus were measured. RESULTS: The primary efficacy endpoint, American College of Rheumatology 20 (ACR20) responder rate at 13 weeks, demonstrated numerical superiority in the treatment group compared with placebo; however, it did not reach statistical significance. Nonetheless, the COMPONENT study yielded important safety and pharmacokinetic data that could provide important information regarding the use of vidofludimus in other clinical trials, not only for RA but also for other autoimmune diseases. A safety profile for vidofludimus similar to placebo was obtained in this RA patient population. This includes similar rates of the adverse events of diarrhea, alopecia, neutropenia, and elevated liver enzymes, all of which are known drug-related adverse events reported for leflunomide and teriflunomide. A potential pharmacokinetic interaction between vidofludimus and methotrexate was observed. CONCLUSIONS: Vidofludimus demonstrated a positive safety profile, making it a promising candidate for the treatment of a variety of immune-related diseases. TRIAL REGISTRATIONS: ClinicalTrials.gov identifier: NCT01010581.

Targeting class I histone deacetylases by the novel small molecule inhibitor 4SC-202 blocks oncogenic hedgehog-GLI signaling and overcomes smoothened inhibitor resistance.

Aberrant activation of Hedgehog (HH)/GLI signaling is causally involved in numerous human malignancies, including basal cell carcinoma (BCC) and medulloblastoma. HH pathway antagonists targeting smoothened (SMO), an essential effector of canonical HH/GLI signaling, show significant clinical success in BCC patients and have recently been approved for the treatment of advanced and metastatic BCC. However, rapid and frequent development of drug resistance to SMO inhibitors (SMOi) together with severe side effects caused by prolonged SMOi treatment call for alternative treatment strategies targeting HH/GLI signaling downstream of SMO. In this study, we report that 4SC-202, a novel clinically validated inhibitor of class I histone deacetylases (HDACs), efficiently blocks HH/GLI signaling. Notably, 4SC-202 treatment abrogates GLI activation and HH target gene expression in both SMOi-sensitive and -resistant cells. Mechanistically, we propose that the inhibition of HDACs 1/2/3 is crucial for targeting oncogenic HH/GLI signaling, and that class I HDAC inhibitors either in combination with SMOi or as second-line therapy may improve the treatment options for HH-associated malignancies with SMOi resistance.

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.

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.

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.

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.

Assessment of drug candidates for broad-spectrum antiviral therapy targeting cellular pyrimidine biosynthesis.

Currently available antiviral drugs frequently induce side-effects or selection of drug-resistant viruses. We describe a novel antiviral principle based on targeting the cellular enzyme dihydroorotate dehydrogenase (DHODH). In silico drug design and biochemical evaluation identified Compound 1 (Cmp1) as a selective inhibitor of human DHODH in vitro (IC50 1.5±0.2nM). Crystallization data specified the mode of drug-target interaction. Importantly, Cmp1 displayed a very potent antiviral activity that could be reversed by co-application of uridine or other pyrimidine precursors, underlining the postulated DHODH-directed mode of activity. Human and animal cytomegaloviruses as well as adenoviruses showed strong sensitivity towards Cmp1 in cell culture-based infection systems with IC50 values in the low micromolar to nanomolar range. Particularly, broad inhibitory activity was demonstrated for various types of laboratory and clinically relevant adenoviruses. For replication of human cytomegalovirus in primary fibroblasts, antiviral mode of activity was attributed to the early stage of gene expression. A mouse in vivo model proved reduced replication of murine cytomegalovirus in various organs upon Cmp1 treatment. These findings suggested Cmp1 as drug candidate and validated DHODH as a promising cellular target for antiviral therapy.

The NF-kappaB inhibitor SC75741 protects mice against highly pathogenic avian influenza A virus.

The appearance of pandemic H1N1 and highly pathogenic avian H5N1 viruses in humans as well as the emergence of seasonal H1N1 variants resistant against neuraminidase inhibitors highlight the urgent need for new and amply available antiviral drugs. We and others have demonstrated that influenza virus misuses the cellular IKK/NF-kappaB signaling pathway for efficient replication suggesting that this module may be a suitable target for antiviral intervention. Here, we show that the novel NF-kappaB inhibitor SC75741 significantly protects mice against infection with highly pathogenic avian influenza A viruses of the H5N1 and H7N7 subtypes. Treatment was efficient when SC75741 was given intravenously in a concentration of 5mg/kg/day. In addition, application of SC75741 via the intraperitoneal route resulted in a high bioavailability and was also efficient against influenza when given 15 mg/kg/day or 7.5 mg/kg/twice a day. Protection was achieved when SC75741 was given for seven consecutive days either prior to infection or as late as four days after infection. SC75741 treatment showed no adverse effects in the concentrations required to protect mice against influenza virus infection. Although more pre-clinical studies are needed SC75741 might be a promising candidate for a novel antiviral drug against influenza viruses that targets the host cell rather than the virus itself.

The NF-κB inhibitor SC75741 efficiently blocks influenza virus propagation and confers a high barrier for development of viral resistance.

Ongoing human infections with highly pathogenic avian H5N1 viruses and the emergence of the pandemic swine-origin influenza viruses (IV) highlight the permanent threat elicited by these pathogens. Occurrence of resistant seasonal and pandemic strains against the currently licensed antiviral medications points to the urgent need for new and amply available anti-influenza drugs. The recently identified virus-supportive function of the cellular IKK/NF-κB signalling pathway suggests this signalling module as a potential target for antiviral intervention. We characterized the NF-κB inhibitor SC75741 as a broad and efficient blocker of IV replication in non-toxic concentrations. The underlying molecular mechanism of SC75741 action involves impaired DNA binding of the NF-κB subunit p65, resulting in reduced expression of cytokines, chemokines, and pro-apoptotic factors, subsequent inhibition of caspase activation and block of caspase-mediated nuclear export of viralribonucleoproteins. SC75741 reduces viral replication and H5N1-induced IL-6 and IP-10 expression in the lung of infected mice. Besides its virustatic effect the drug suppresses virus-induced overproduction of cytokines and chemokines, suggesting that it might prevent hypercytokinemia that is discussed to be an important pathogenicity determinant of highly pathogenic IV. Importantly the drug exhibits a high barrier for development of resistant virus variants. Thus, SC75741-derived drugs may serve as broadly non-toxic anti-influenza agents.