Cyclin-Dependent Kinases (CDKs) and the Human Cytomegalovirus-Encoded CDK Ortholog pUL97 Represent Highly Attractive Targets for Synergistic Drug Combinations.

Human cytomegalovirus (HCMV) is a pathogenic human herpesvirus associated with serious, potentially life-threatening symptoms in the immunocompromised or immunonaïve host. The limitations encountered by antiviral therapy options currently available include a narrow panel of accessible targets, the induction of viral drug resistance as well as severe drug dosage-mediated side-effects. Improved drug-targeting strategies to resolve these issues are the focus of our investigations. In particular, pharmaceutical kinase inhibitors (PKIs), either directed to host kinases or directed to the viral protein kinase pUL97, have been considered to overcome these restrictions. Recently, we reported the identification of a synergistic combination of two PKIs directed to host cyclin-dependent kinase 7 (CDK7) and viral CDK ortholog pUL97. Here, we substantiate these findings with the following results: (i) true drug synergy was exhibited by various chemical classes of PKI pairs directed to pUL97 and CDK7; (ii) no putative amplification of cytotoxicity by these drug combinations was observed; (iii) a reduction in drug dosage levels for synergistic combinations was defined on a quantitative basis and compared to monotreatments; (iv) the quantities of target proteins CDK7 and pUL97 expressed in HCMV-infected cells were assessed by confocal imaging, indicating a strong down-modulation of CDK7 levels as a result of synergistic drug treatment; (v) the functional importance of these target kinases, both binding to cyclin H, was illustrated by assessing HCMV replication under the viral genomic deletion of ORF-UL97 or cellular cyclin knock-out; (vi) new combinations of HCMV-specific drug synergy were demonstrated for solely host-directed treatments using PKIs against CDK2, CDK7, CDK8 and/or CDK9 and (vii) a triple PKI combination provided further support for the synergy approach. With these combined findings, this study highlights the potential of therapeutic drug combinations of approved, developmental and preclinical PKIs for expanding future options for anti-HCMV therapy.

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.

The pan-histone deacetylase inhibitor CR2408 disrupts cell cycle progression, diminishes proliferation and causes apoptosis in multiple myeloma cells.

In view of the fact that histone deacetylases (HDACs) are promising targets for myeloma therapy, we investigated the effects of the HDAC inhibitor CR2408 on multiple myeloma (MM) cells in vitro. CR2408 is a direct pan-HDAC inhibitor and inhibits all known 11 HDACs with a 50% inhibitory concentration (IC(50) ) of 12 nmol/l (HDAC 6) to 520 nmol/l (HDAC 8). Correspondingly, CR2408 induces hyperacetylation of histone H4, inhibits cell growth and strongly induces apoptosis (IC(50) =0.1-0.5 µmol/l) in MM cell lines and primary MM cells. CR2408 leads to fragmentation of cells and induces an accumulation in the subG1 phase accompanied with moderately decreased levels of cyclin D1 and cdk4 and strongly decreased levels of cdc25a, pRb and p53. Interruption of the cell cycle is reflected by inhibition of cell proliferation and is accompanied by decreased phosphorylation of 4E-BP1 and p70S6k. Treatment with CR2408 results in increased protein levels of Bim and pJNK and downregulation of Bad and Bcl-xL and activation of Caspases 3, 8 and 9. Furthermore, as HDAC inhibitors have shown synergism with other drugs, these effects were investigated and synergism was observed for combinations of CR2408 with doxorubicin and bortezomib. In conclusion, we have identified potent anti-myeloma activity for this novel HDAC inhibitor that gives further insights into the biological sequelae of HDAC inhibition in MM.

2-Benzamido-N-(1H-benzo[d]imidazol-2-yl)thiazole-4-carboxamide derivatives as potent inhibitors of CK1δ/ε.

In this study we identified two heterocyclic compounds (5 and 6) as potent and specific inhibitors of CK1δ (IC(50) = 0.040 and 0.042 µM, respectively). Whereas compound 5 exhibited fivefold higher affinity towards CK1δ than to CK1ε (IC(50) CK1ε = 0.199 µM), compound 6 also inhibited CK1ε (IC(50) = 0.0326 µM) in the same range as CK1δ. Selected compound 5 was screened over 442 kinases identifying 5 as a highly potent and selective inhibitor of CK1δ. X-ray analysis of 5 bound to CK1δ demonstrated its binding mode. In addition, characterization of 5 and 6 in a cell biological approach revealed the ability of both compounds to inhibit proliferation of tumor cell lines in a dose and cell line specific manner. In summary, our optimizations lead to the development of new highly selective CK1δ and ε specific inhibitors with biological activity.

Impact of drought on the temporal dynamics of wood formation in Pinus sylvestris.

We determined the temporal dynamics of cambial activity and xylem cell differentiation of Scots pine (Pinus sylvestris L.) within a dry inner Alpine valley (750 m a.s.l., Tyrol, Austria), where radial growth is strongly limited by drought in spring. Repeated micro-sampling of the developing tree ring of mature trees was carried out during two contrasting years at two study plots that differ in soil water availability (xeric and dry-mesic sites). In 2007, when air temperature at the beginning of the growing season in April exceeded the long-term mean by 6.4 degrees C, cambial cell division started in early April at both study plots. A delayed onset of cambial activity of c. 2 weeks was found in 2008, when average climate conditions prevailed in spring, indicating that resumption of cambial cell division after winter dormancy is temperature controlled. Cambial cell division consistently ended about the end of June/early July in both study years. Radial enlargement of tracheids started almost 3 weeks earlier in 2007 compared with 2008 at both study plots. At the xeric site, the maximum rate of tracheid production in 2007 and 2008 was reached in early and mid-May, respectively, and c. 2 weeks later at the dry-mesic site. Since in both study years more favorable growing conditions (i.e., an increase in soil water content) were recorded during summer, we suggest a strong sink competition for carbohydrates to mycorrhizal root and shoot growth. Wood formation stopped c. 4 weeks earlier at the xeric compared with the dry-mesic site in both years, indicating a strong influence of drought stress on cell differentiation. This is supported by radial widths of earlywood cells, which were found to be significantly narrower at the xeric than at the dry-mesic site (P < 0.05). Repeated cellular analyses during the two growing seasons revealed that, although spatial variability in the dynamics and duration of cell differentiation processes in P. sylvestris exposed to drought is strongly influenced by water availability, the onset of cambial activity and cell differentiation is controlled by temperature.

Intravenous delivery of the toll-like receptor 7 agonist SC1 confers tumor control by inducing a CD8+ T cell response.

TLR7 agonists are considered promising drugs for cancer therapy. The currently available compounds are not well tolerated when administered intravenously and therefore are restricted to disease settings amenable for topical application. Here we present the preclinical characterization of SC1, a novel synthetic agonist with exquisite specificity for TLR7. We found that intravenously administered SC1 mediates systemic release of type I interferon, but not of proinflammatory cytokines such as TNFα and IL6, and results in activation of circulating immune cells. Tumors of SC1-treated mice have brisk immune cell infiltrates and are polarized towards a Th1 type signature. Intratumoral CD8+ T cells and CD11b+ conventional dendritic cells (cDCs) are significantly increased, plasmacytoid dendritic cells (pDCs) are strongly activated and macrophages are M1 phenotype polarized, whereas myeloid-derived suppressor cells (MDSCs) are decreased. We further show that treatment of mice with SC1 profoundly inhibits the growth of established syngeneic tumors and results in significantly prolonged survival. Mice, which are tumor-free after SC1 treatment are protected from subsequent tumor rechallenge. The antitumor effect of SC1 depends on antigen-specific CD8+ T cells, which we found to be strongly enriched in the tumors of SC1-treated mice. In conclusion, this study shows that systemically administered SC1 mobilizes innate and adaptive immunity and is highly potent as single agent in mice and thereby provides a rationale for clinical testing of this compound.

A new promising candidate to overcome drug resistant herpes simplex virus infections.

Infections with Herpes simplex viruses (HSV) belong to the most common human diseases worldwide, resulting in symptoms ranging from painful, but commonly self-limiting lesions of the orofacial or genital tract to severe infections of the eye or life-threatening generalized infections. Frequent HSV-reactivations at the eye may lead to the development of herpetic stromal keratitis, which is one of the major causes of infectious blindness in developed countries. The vast majority of life-threatening generalized infections occur in immunocompromised individuals, such as transplant recipients or patients suffering from advanced human immunodeficiency virus (HIV) infection with concurrent HSV-reactivation. Over the past decades, Acyclovir (ACV) became the golden standard for the treatment of HSV infections. However, long-term antiviral treatment, as it is required mainly in immunocompromised patients, led to the emergence of resistances towards ACV and other antivirals. Therefore, there is a clear need for the development of new potent antivirals which combine good oral bioavailability and tolerability with low side effects. In the current study we present SC93305 as a novel potent antiviral substance that proved to be highly effective not only against different HSV-1 and HSV-2 strains but also towards ACV- and multi-resistant HSV-1 and HSV-2 isolates. SC93305 shows comparable antiviral activity as reported for ACV and very importantly it does not interfere with the activation of specific immune cells. Here we report that SC93305 does not affect the biological function of dendritic cells (DC), the most potent antigen presenting cells of the immune system to induce antiviral immune responses, nor T cell stimulation or the release of inflammatory cytokines. Thus, SC93305 is a new and promising candidate for the treatment of HSV-1 and HSV-2 infections and in particular also for the inhibition of drug-resistant HSV-1/2 strains.

Novel cytomegalovirus-inhibitory compounds of the class pyrrolopyridines show a complex pattern of target binding that suggests an unusual mechanism of antiviral activity.

Human cytomegalovirus (HCMV) is a major human pathogen with seropositivity rates in the adult population ranging between 40% and 95%. HCMV infection is associated with severe pathology, such as life-threatening courses of infection in immunocompromised individuals and neonates. Current standard therapy with valganciclovir has the disadvantage of adverse side effects and viral drug resistance. A novel anti-HCMV drug, letermovir, has been approved recently, so that improved therapy options are available. Nevertheless, even more so far unexploited classes of compounds and molecular modes of action will be required for a next generation of antiherpesviral treatment strategies. In this study, we focused on the analysis of the antiviral potency of a novel class of compounds, i.e. pyrrolopyridine analogs, and identified both hit compounds and their target protein candidates. In essence, we provide novel evidence as follows: (i) screening hit SC88941 is highly active in inhibiting HCMV replication in primary human fibroblasts with an EC50 value of 0.20 ±â€¯0.01 µM in the absence of cytotoxicity, (ii) inhibition occurs at the early-late stage of viral protein production and shows reinforcing effects upon LMV cotreatment, (iii) among the viruses analyzed, antiviral activity was most pronounced against ß-herpesviruses (HCMV, HHV-6A) and intermediate against adenovirus (HAdV-2), (iv) induction of SC88941 resistance was not detectable, thus differed from the induction of ganciclovir resistance, (v) a linker-coupled model compound was used for mass spectrometry-based target identification, thus yielding several drug-binding target proteins and (vi) a first confocal imaging approach used for addressing intracellular effects of SC88941 indicated qualitative and quantitative alteration of viral protein expression and localization. Thus, our findings suggest a multifaceted pattern of compound-target binding in connection with an unusual mode of action, opening up further opportunities of antiviral drug development.

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.

SC83288 is a clinical development candidate for the treatment of severe malaria.

Severe malaria is a life-threatening complication of an infection with the protozoan parasite Plasmodium falciparum, which requires immediate treatment. Safety and efficacy concerns with currently used drugs accentuate the need for new chemotherapeutic options against severe malaria. Here we describe a medicinal chemistry program starting from amicarbalide that led to two compounds with optimized pharmacological and antiparasitic properties. SC81458 and the clinical development candidate, SC83288, are fast-acting compounds that can cure a P. falciparum infection in a humanized NOD/SCID mouse model system. Detailed preclinical pharmacokinetic and toxicological studies reveal no observable drawbacks. Ultra-deep sequencing of resistant parasites identifies the sarco/endoplasmic reticulum Ca2+ transporting PfATP6 as a putative determinant of resistance to SC81458 and SC83288. Features, such as fast parasite killing, good safety margin, a potentially novel mode of action and a distinct chemotype support the clinical development of SC83288, as an intravenous application for the treatment of severe malaria.

A novel TLR7 agonist reverses NK cell anergy and cures RMA-S lymphoma-bearing mice.

Toll-like receptor 7 (TLR7) agonists are potent immune stimulants able to overcome cancer-associated immune suppression. Due to dose-limiting systemic toxicities, only the topically applied TLR7 agonist (imiquimod) has been approved for therapy of skin tumors. There is a need for TLR7-activating compounds with equivalent efficacy but less toxicity. SC1, a novel small molecule agonist for TLR7, is a potent type-1 interferon inducer, comparable to the reference TLR7 agonist resiquimod, yet with lower induction of proinflammatory cytokines. In vivo, SC1 activates NK cells in a TLR7-dependent manner. Mice bearing the NK cell-sensitive lymphoma RMA-S are cured by repeated s. c. administrations of SC1 as efficiently as by the administration of resiquimod. No relevant toxicities were observed. Mechanistically, SC1 reverses NK cell anergy and restores NK cell-mediated tumor cell killing in an IFN-α-dependent manner. TLR7 targeting by SC1-based compounds may form an attractive strategy to activate NK cell responses for cancer therapy.

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.

RPPApipe: a pipeline for the analysis of reverse-phase protein array data.

BACKGROUND AND SCOPE: Today, web-based data analysis pipelines exist for a wide variety of microarray platforms, such as ordinary gene-centered arrays, exon arrays and SNP arrays. However, most of the available software tools provide only limited support for reverse-phase protein arrays (RPPA), as relevant inherent properties of the corresponding datasets are not taken into account. Thus, we developed the web-based data analysis pipeline RPPApipe, which was specifically tailored to suit the characteristics of the RPPA platform and encompasses various tools for data preprocessing, statistical analysis, clustering and pathway analysis. IMPLEMENTATION AND PERFORMANCE: All tools which are part of the RPPApipe software were implemented using R/Bioconductor. The software was embedded into our web-based ZBIT Bioinformatics Toolbox which is a customized instance of the Galaxy platform. AVAILABILITY: RPPApipe is freely available under GNU Public License from http://webservices.cs.uni-tuebingen.de. A full documentation of the tool can be found on the corresponding website http://www.cogsys.cs.uni-tuebingen.de/software/RPPApipe.

Profiling of the kinome of cytomegalovirus-infected cells reveals the functional importance of host kinases Aurora A, ABL and AMPK.

Human cytomegalovirus infection can lead to life-threatening clinical manifestations particularly in the immunocompromised host. Current therapy options face severe limitations leading to a continued search for alternative drug candidates. Viral replication is dependent on a balanced interaction between viral and cellular proteins. Especially protein kinases are important regulators of virus-host interaction indicated by remarkable kinome alterations induced upon HCMV infection. Here we report a novel approach of kinome profiling with an outcome that suggests an important role of specific cellular protein kinases, such as AMPK, ABL2 and Aurora A. Inhibition of AMPK and ABL kinases showed a significant reduction, whereas inhibition of Aurora A kinase led to a slight activation of HCMV replication, as measured in a GFP reporter-based replication assay. Furthermore, analysis of the mode of antiviral action suggested a substantial benefit for the efficiency of viral replication at the immediate early (AMPK) or early-late (ABL) phases of HCMV gene expression. In contrast, inhibition of Aurora A kinase promoted an enhancement of viral early-late gene expression, suggesting a putative role of Aurora A signaling in host defense. Thus, the combined data provide new information on host cell kinases involved in viral replication and uncovered potential targets for future antiviral strategies.

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.

Flexibility analysis and structure comparison of two crystal forms of calcium-free human m-calpain.

The calpains form a growing family of structurally related intracellular multidomain cysteine proteinases containing a papain-related catalytic domain, whose activity depends on calcium. The calpains are believed to play important roles in cytoskelatel remodeling processes, cell differentiation, apoptosis and signal transduction, but are also implicated in a number of diseases. Recent crystal structures of truncated rat and full-length human apo-m-calpain revealed the domain arrangement and explained the inactivity of m-calpain in the absence of calcium by a disrupted catalytic domain. Proteolysis studies have indicated several susceptible sites, in particular in the catalytic subdomain IIb and in the following domain III, which are more accessible to attacking proteinases in the presence than in the absence of calcium. The current view is that m-calpain exhibits a number of calcium binding sites, which upon calcium binding cooperatively interact, triggering the reformation of a papain-like catalytic domain, accompanied by enhanced mobilisation of the whole structure. To further analyse the flexibility of m-calpain, we have determined and refined the human full-length apo-m-calpain structure of a second crystal form to 3.15 A resolution. Here we present this new structure, compare it with our first structure now re-refined with tighter constrain parameters, discuss the flexibility in context with the proteolysis and calcium binding data available, and suggest implications for the calcium-induced activation process.