Identification of 2-aminopyrimidine derivatives as inhibitors of the canonical Wnt signaling pathway.

The canonical Wnt signaling pathway plays a fundamental role in embryonic as well as in adult development. Consequently, dysregulation of the pathway has been linked to a wide spectrum of pathological conditions. In a program aimed at the identification of small molecule inhibitors of the canonical Wnt pathway we identified a series of 2-aminopyrimidine derivatives which specifically inhibited the pathway with minimal or no sign of cellular toxicity. The hit molecules 1 and 2 showed promising inhibitory activity with IC50 values of approximately 10 µM, but low solubility and metabolic stability. During the early stage of the hit series exploration, the pyrimidine core was variously decorated to obtain active compounds with a better physico-chemical profile. In particular, compound 13 showed Wnt inhibition activity comparable to hit molecules 1 and 2, with improved physico-chemical properties. Therefore, this series of compounds may be considered a promising starting point for the design of novel small molecule inhibitors of the canonical Wnt pathway.

Probing Fluorinated Motifs onto Dual AChE-MAO B Inhibitors: Rational Design, Synthesis, Biological Evaluation, and Early-ADME Studies.

Bioisosteric H/F or CH2OH/CF2H replacement was introduced in coumarin derivatives previously characterized as dual AChE-MAO B inhibitors to probe the effects on both inhibitory potency and drug-likeness. Along with in vitro screening, we investigated early-ADME parameters related to solubility and lipophilicity (Sol7.4, CHI7.4, log D7.4), oral bioavailability and central nervous system (CNS) penetration (PAMPA-HDM and PAMPA-blood-brain barrier (BBB) assays, Caco-2 bidirectional transport study), and metabolic liability (half-lives and clearance in microsomes, inhibition of CYP3A4). Both specific and nonspecific tissue toxicities were determined in SH-SY5Y and HepG2 lines, respectively. Compound 15 bearing a -CF2H motif emerged as a water-soluble, orally bioavailable CNS-permeant potent inhibitor of both human AChE (IC50 = 550 nM) and MAO B (IC50 = 8.2 nM, B/A selectivity > 1200). Moreover, 15 behaved as a safe and metabolically stable neuroprotective agent, devoid of cytochrome liability.

Synthesis, physicochemical properties, and biological activity of bile acids 3-glucuronides: Novel insights into bile acid signalling and detoxification.

Glucuronidation is considered an important detoxification pathway of bile acids especially in cholestatic conditions. Glucuronides are less toxic than the parent free forms and are more easily excreted in urine. However, the pathophysiological significance of bile acid glucuronidation is still controversial and debated among the scientific community. Progress in this field has been strongly limited by the lack of appropriate methods for the preparation of pure glucuronides in the amount needed for biological and pharmacological studies. In this work, we have developed a new synthesis of bile acid C3-glucuronides enabling the convenient preparation of gram-scale quantities. The synthesized compounds have been characterized in terms of physicochemical properties and abilities to modulate key nuclear receptors including the farnesoid X receptor (FXR). In particular, we found that C3-glucuronides of chenodeoxycholic acid and lithocholic acid, respectively the most abundant and potentially cytotoxic species formed in patients affected by cholestasis, behave as FXR agonists and positively regulate the gene expression of transporter proteins, the function of which is critical in human conditions related to imbalances of bile acid homeostasis.

T-cell immune reconstitution after hematopoietic stem cell transplantation for HIV-associated lymphoma.

BACKGROUND: One of the major concern for high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT) for HIV-associated lymphoma is that posttransplant immunosuppression might worsen immune defects of HIV individuals. Since the introduction of highly active antiretroviral therapy has made HSCT possible also in these patients, we analyzed whether the immune system already compromised by HIV infection might support an efficient T-cell recovery after HSCT. METHODS: The kinetics and the extent of T-cell reconstitution were investigated before and after HSCT in four patients with HIV-related lymphoma (one with Hodgkin's Disease and three with non-Hodgkin's lymphoma) by measuring the thymic output, the level of IL-7 and the heterogeneity of T-cell repertoire. T-cell competence was gauged at two functional levels: by determining the number of T-cell divisions and by measuring IFN-gamma production. RESULTS: The thymus of transplanted patients can be capable of generating new T cells, but there is no relationship between increasing number of newly produced lymphocytes and modification of IL-7 level. Various T-cell subsets, expressing different T-cell receptor variable beta genes, were preferentially expanded in CD8 population and most of them showed a restricted diversity. Furthermore, CD3 lymphocytes showed heterogeneous behaviors in terms of proliferative capability and IFN-gamma production. CONCLUSIONS: High-dose therapy and HSCT in HIV patients under highly active antiretroviral therapy does not worsen the immune defects. On the contrary, in the presence of some conditions (including the type of hematologic malignancy, the therapy compliance, and the immune status before transplantation), high-dose therapy and HSCT might support the improvement of immune conditions.

Development and characterization of a monoclonal antibody directed against human telomerase reverse transcriptase (hTERT).

Telomerase activity plays an important role in the two complementary processes of cellular immortalization and senescence. This enzyme is active in almost all tumors, but also in inflammatory and many normal proliferating cells. Therefore, the main limits of molecular determinations, such as telomeric repeat amplification protocol assay is that they are not able to discriminate between the enzymatic activity of tumor and normal cells. The most appropriate technique for this would be immunohistochemical determination using monoclonal antibodies. Very few monoclonal antibodies (Mabs) directed against the human telomerase reverse transcriptase (hTERT) are commercially available and in the present study, we developed a new Mab directed against this protein (TERT-3 36-10) to investigate the possibility of detecting immunoreactivity to this Mab by immunohistochemical and flow cytometric approaches. Immunohistochemical determination showed a lack of reactivity to the Mab in highly differentiated striated muscle tissue, a variable reactivity in dysplastic cervical epithelial tissue and similar and widespread immunoreactivity in cell lines and clinical tumors. Furthermore, we demonstrated the ability of this Mab to inhibit enzyme activity in cell extract from MCR bladder tumor cell line.

Structure-activity relationship and properties optimization of a series of quinazoline-2,4-diones as inhibitors of the canonical Wnt pathway.

Wnt signaling pathway plays a critical role in numerous cellular processes, including tumor initiation, proliferation, invasion/infiltration, metastasis formation and resistance to chemotherapy. In a drug discovery project aimed at the identification of inhibitors of the canonical Wnt pathway, we selected a series of quinazoline 2,4-diones as starting point for the therapeutic treatment of glioblastoma multiforme. Despite of poor physico-chemical properties of hit compound 1, our medicinal chemistry effort allowed the discovery and characterization of lead compound 33 (SEN461), with improved ADME profile, good bioavailability and active in vitro and in vivo in glioblastoma, gastric and sarcoma tumors.

A phenotypic screening assay for modulators of huntingtin-induced transcriptional dysregulation.

Huntington's Disease is a rare neurodegenerative disease caused by an abnormal expansion of CAG repeats encoding polyglutamine in the first exon of the huntingtin gene. N-terminal fragments containing polyglutamine (polyQ) sequences aggregate and can bind to cellular proteins, resulting in several pathophysiological consequences for affected neurons such as changes in gene transcription. One transcriptional pathway that has been implicated in HD pathogenesis is the CREB binding protein (CBP)/cAMP responsive element binding (CREB) pathway. We developed a phenotypic assay to screen for compounds that can reverse the transcriptional dysregulation of the pathway caused by induced mutated huntingtin protein (µHtt). 293/T-REx cells were stably co-transfected with an inducible full-length mutated huntingtin gene containing 138 glutamine repeats and with a reporter gene under control of the cAMP responsive element (CRE). One clone, which showed reversible inhibition of µHtt-induced reporter activity upon treatment with the neuroprotective Rho kinase inhibitor Y27632, was used for the development of a high-throughput phenotypic assay suitable for a primary screening campaign, which was performed on a library of 24,000 compounds. Several hit compounds were identified and validated further in a cell viability adenosine triphosphate assay. The assay has the potential for finding new drug candidates for the treatment of HD.

Fused 3-Hydroxy-3-trifluoromethylpyrazoles Inhibit Mutant Huntingtin Toxicity.

Here, we describe the selection and optimization of a chemical series active in both a full-length and a fragment-based Huntington's disease (HD) assay. Twenty-four thousand small molecules were screened in a phenotypic HD assay, identifying a series of compounds bearing a 3-hydroxy-3-trifluoromethylpyrazole moiety as able to revert the toxicity induced by full-length mutant Htt by up to 50%. A chemical exploration around the series led to the identification of compound 4f, which demonstrated to be active in a Htt171-82Q rat primary striatal neuron assay and a PC12-Exon-1 based assay. This compound was selected for testing in R6/2 mice, in which it was well-tolerated and showed a positive effect on body weight and a positive trend in preventing ventricular volume enlargment. These studies provide strong rationale for further testing the potential benefits of 3-hydroxy-3-trifluoromethylpyrazoles in treating HD.

Identification and characterization of a small-molecule inhibitor of Wnt signaling in glioblastoma cells.

Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than 1 year. Although canonical Wnt pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, ß-catenin, or Axin proteins), an increasing number of studies suggest that elevated Wnt signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of Wnt signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to Wnt inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical Wnt signaling and associated proliferative responses in GBM cells. Here, we show that the small molecule SEN461 inhibits the canonical Wnt signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced Axin stabilization, increased ß-catenin phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized Wnt signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small-molecule-mediated inhibition of Wnt signaling may be a potential approach for GBM therapeutics.

A homogeneous HTRF assay for the identification of inhibitors of the TWEAK-Fn14 protein interaction.

The TWEAK-Fn14 pathway is upregulated in models of inflammation, autoimmune diseases, and cancer. Both TWEAK and Fn14 show increased expression also in the CNS in response to different stimuli, particularly astrocytes, microglia, and neurons, leading to activation of NF-κB and release of proinflammatory cytokines. Although neutralizing antibodies against these proteins have been shown to have therapeutic efficacy in animal models of inflammation, no small-molecule therapeutics are yet available. Here, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF)-based screening assay together with several counterassays for the identification of small-molecule inhibitors of this protein-protein interaction. Recombinant HIS-TWEAK and Fn14-Fc proteins as well as FLAG-TWEAK and Fn14-FLAG proteins and an anti-Fn14 antibody were used to establish and validate these assays and to screen a library of 60 000 compounds. Two HTRF counterassays with unrelated proteins in the same assay format, an antiaggregation assay and a redox assay, were applied to filter out potential false-positive compounds. The novel assay and associated screening cascade should be useful for the discovery of small-molecule inhibitors of the TWEAK-Fn14 protein interaction.

Reduced thymic output, increased spontaneous apoptosis and oligoclonal B cells in polyethylene glycol-adenosine deaminase-treated patients.

Impairment of purine metabolism due to adenosine deaminase (ADA) deficiency is associated with a severe combined immunodeficiency (SCID). Polyethylene glycol-modified ADA (PEG-ADA) has provided noncurative, life-saving treatment for these patients, but full immune recovery is not achieved with this therapy. Since ADA-SCID is perhaps the most difficult form of SCID to handle clinically, understanding the benefits and limitations of PEG-ADA therapy may be relevant for treatment selection. To this purpose, we analyzed the rate of thymic output, T and B cell repertoires, number of T cell divisions, IFN-gamma and IL-4 production, and the extent of cell death in five ADA-SCID patients following a prolonged period of treatment with PEG-ADA. We found that thymic output was low in these patients. However, their T cell repertoire was heterogeneous, and their T lymphocytes produced cytokines upon activation and responded to mitogen stimulation, although with different kinetics. Furthermore, a high number of peripheral T lymphocytes were committed to apoptosis. Anomalies were also observed in the B cell compartment, with oligoclonal expansions of B cell clonotypes in two patients. Our data indicate that decreased thymic function, B cell oligoclonality, and increased spontaneous apoptosis may be the mechanisms by which the immunodeficiency of ADA-SCID patients persists in spite of treatment with PEG-ADA.