Identification and characterization of inhibitors of hedgehog-induced osteoblast differentiation

Hedgehog (Hh) signaling is one of the major pathways that is essentially required during embryonic development in vertebrates. Dysregulation during embryogenesis has been linked to severe developmental malformations including cyclopia and holoprosencephaly. Moreover, aberrant Hh signaling has been associated with several types of cancer, such as medulloblastoma and basal cell carcinoma. Over the past 40 years, studies have provided many insights into the current understanding of the Hh signaling pathway. Due promising potential for therapeutic modulation of this pathway, identification of small molecule modulators of Hh signaling is in high demand. Throughout this thesis, six compounds were identified as potent inhibitors of Hh-dependent osteogenesis during a phenotypic screening. Five of these compounds inhibited GLI2/3-dependent reporter activity and expression of Hh target genes and were thus classified as novel Hh pathway inhibitors. While four compounds, a Furo[3,2-b]pyridine, quinoline, pyrroloquinoline and 20-membered macrocycle derivative bind to the key signaling molecule smoothened (SMO), one compound, a 4-arylisoquinolone does not bind to the heptahelical bundle of this protein. An 8-oxotetrahydroprotoberberine derivate, termed Picoberin, inhibited Hh-dependent osteogenesis with a single-digit picomolar IC50 but did not modulate canonical Hh signaling. Despite its remarkable bioactivity, this small molecule was not cytotoxic at high concentrations in several cell lines. Global transcriptome and proteome profiling revealed that Picoberin activates Aryl hydrocarbon Receptor (AhR) signaling and led to the identification of AhR as the potential protein target. Subsequent functional evaluation of the influence of Picoberin on AhR signaling in several cell lines confirmed this target hypothesis. Moreover, chemical validations and genetic AhR depletion linked Picoberin mediated activation of AhR signaling to inhibition of Hh-dependent osteogenesis. Additionally, results obtained in this thesis provide evidence for crosstalk of AhR and Hh signaling and a functional role for AhR during osteoblast differentiation. AhR regulates xenobiotic metabolism as well as numerous physiological processes such as immune cell function, stem cell maintenance, and differentiation. Dysregulation of this ligand-activated transcription factor is observed in several diseases, including cancer. Historically, AhR is linked to mainly toxic effects. However, recent approval of the AhR agonist Tapinarof for treatment of plaque psoriasis and several studies exploring AhR in cancer provide evidence for potential therapeutic applications of AhR modulators. Picoberin could not only serve as a tool compound for AhR research but may potentially also provide a starting point for the development of further therapeutic AhR agonists

Mutações no gene HFE (C282Y, H63D, S65C) em uma população brasileira

Hereditary hemochromatosis (HH) is the most common genetic disorder occurring in individuals of northern European descent. The clinical characteristic of this disease is the gradual accumulation of iron in internal organs, which ultimately leads to organ failure and death. The defective gene in the majority of cases, HFE, was identified in 1996. Three allelic variants of the HFE gene have been correlated with HH: C282Y is significantly associated with HH; H63D and S65C have unclear relationships. In this report, these mutations were analyzed in 8 patients with HH and in 148 healthy individuals (blood donors). To detect the mutations, exons 2 and 4 of the HFE gene were amplified by PCR followed by restriction endonucleases cleavage. In patients with HH, three individuals were homozygous for the C282Y mutation, one showed compound heterozygous (C282Y/H63D), one was heterozygous for the C282Y and 3 presented with no mutations. In healthy individuals, the allele frequency observed was 0.014 for C282Y, 0.108 for H63D and 0.010 for S65C. The frequency of mutations was significantly higher in Caucasians compared with non-Caucasians. These data are concordant with the previous literature and with the ethnical origin of the population studied.A Hemocromatose hereditária (HH) é a alteração genética mais comumente encontrada em descendentes de Europeus, em especial da região Norte. A alteração clínica característica é a acúmulo gradual do ferro em órgãos internos, que evolui para lesão orgânica e morte. Na maioria dos casos, o gene alterado é o HFE, que foi identificado em 1996. Três variantes alélicas do gene HFE foram correlacionados com a HH: a C282Y, significativamente associada com a HH; e a H63D e a S65C, que apresentam uma relação obscura com esta doença. Neste relato, foi analisada a presença destas mutações em 8 pacientes com HH e em 148 indivíduos saudáveis (doadores de sangue). Para detecção das mutações, foi realizado PCR dos exons 2 e 4 do gene HFE, seguido pela clivagem com endonucleases específicas. No grupo de pacientes com HH, observou-se 3 indivíduos homozigotos para a mutação de C282Y, um heterozigoto composto (C282Y/H63D), um heterozigoto para C282Y e ausência de mutações nos outros 3 pacientes. Nos indivíduos saudáveis, a freqüência observada foi de 0,014 para o alelo C282Y, 0,108 para o H63D e 0,010 para o S65C. A presença de mutações foi significantemente maior nos indivíduos brancos, comparando com os não brancos. Estes dados são concordantes com a literatura prévia e com a origem étnica da população estudada.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo (UNIFESP) EPMUNIFESP, EPMSciEL

Focus on therapy of hypnic headache

Hypnic headache (HH) is a primary headache disorder, which occurs exclusively during sleep and usually begins after 50 years of age. There are no controlled trials for the treatment of HH. We reviewed all the available papers, including 119 cases published in literature up to date, reporting the efficacy of the medications used to treat HH. Acute treatment is not recommended, since no drug proved to be clearly effective and also because the intensity and the duration of the attacks do not require the intake of a medication in most cases. As for prevention, a wide variety of medications were reported to be of benefit in HH. The drugs that were found to be effective in at least five cases are: lithium, indomethacin, caffeine and flunarizine. Lithium was the most extensively studied compound and demonstrated to be an efficacious treatment in 32 cases. Unfortunately, despite its efficacy, significant adverse effects and poor tolerability are not rare, mainly in elderly patients. Many patients reported a good response to indomethacin, but some could not tolerate it. Caffeine and melatonin treatments did not yield robust evidence to recommend their use as single preventive agents. Nevertheless, their association with lithium or indomethacin seems to produce an additional therapeutic efficacy. A course of lithium should be tried first, followed 3–4 months later by tapering. If headache recurs during tapering, a longer duration of therapy may be needed. If lithium treatment does not provide a significant response, indomethacin can be commenced as second-line approach. If these treatments prove to be ineffective or poorly tolerated, other agents, such as caffeine and melatonin, can be administered

Inhibition of Hedgehog-dependent tumors and cancer stem cells by a newly identified naturally occurring chemotype

Hedgehog (Hh) inhibitors have emerged as valid tools in the treatment of a wide range of cancers. Indeed, aberrant activation of the Hh pathway occurring either by ligand-dependent or -independent mechanisms is a key driver in tumorigenesis. The smoothened (Smo) receptor is one of the main upstream transducers of the Hh signaling and is a validated target for the development of anticancer compounds, as underlined by the FDA-approved Smo antagonist Vismodegib (GDC-0449/Erivedge) for the treatment of basal cell carcinoma. However, Smo mutations that confer constitutive activity and drug resistance have emerged during treatment with Vismodegib. For this reason, the development of new effective Hh inhibitors represents a major challenge for cancer therapy. Natural products have always represented a unique source of lead structures in drug discovery, and in recent years have been used to modulate the Hh pathway at multiple levels. Here, starting from an in house library of natural compounds and their derivatives, we discovered novel chemotypes of Hh inhibitors by mean of virtual screening against the crystallographic structure of Smo. Hh functional based assay identified the chalcone derivative 12 as the most effective Hh inhibitor within the test set. The chalcone 12 binds the Smo receptor and promotes the displacement of Bodipy-Cyclopamine in both Smo WT and drug-resistant Smo mutant. Our molecule stands as a promising Smo antagonist able to specifically impair the growth of Hh-dependent tumor cells in vitro and in vivo and medulloblastoma stem-like cells and potentially overcome the associated drug resistance

A Smo/Gli multitarget hedgehog pathway inhibitor impairs tumor growth

Pharmacological Hedgehog (Hh) pathway inhibition has emerged as a valuable anticancer strategy. A number of small molecules able to block the pathway at the upstream receptor Smoothened (Smo) or the downstream effector glioma-associated oncogene 1 (Gli1) has been designed and developed. In a recent study, we exploited the high versatility of the natural isoflavone scaffold for targeting the Hh signaling pathway at multiple levels showing that the simultaneous targeting of Smo and Gli1 provided synergistic Hh pathway inhibition stronger than single administration. This approach seems to effectively overcome the drug resistance, particularly at the level of Smo. Here, we combined the pharmacophores targeting Smo and Gli1 into a single and individual isoflavone, compound 22, which inhibits the Hh pathway at both upstream and downstream level. We demonstrate that this multitarget agent suppresses medulloblastoma growth in vitro and in vivo through antagonism of Smo and Gli1, which is a novel mechanism of action in Hh inhibition

A comparison of the physical and chemical composition of UK waste streams based on hypothetical compound structure

The suitability and effectiveness of a waste treatment process or strategy depends upon a waste stream’s physical and chemical composition. Chemical properties of UK waste streams, particularly MSW, are limited while physical properties are better documented. Consequently this presents a degree of uncertainty with the waste’s properties, manifesting itself as financial risk in the investment of new treatment or disposal plant. To mitigate this uncertainty, a number of UK waste surveys have been reviewed to determine if there is significant difference in the calorific value between waste streams. Ultimate and proximate analysis data from a number of sources have been collected and used to approximate the chemical composition of each waste fraction. To facilitate the comparison of each waste stream, a hypothetical compound of the form C6HaOb has been determined for each. Based on this analysis, all UK waste streams share the hypothetical formula C6H10O3, indicating that on a dry basis, the composition of waste in the UK is fairly consistent. Monte Carlo analysis of the hypothetical compound structure revealed that for both household and civic amenity waste streams, the hydrogen and oxygen content only deviate slightly from the mean values. Since MSW is predominantly comprised of household and civic amenity waste, the hypothetical compound C6H10O3 can be used to approximate UK MSW