Innovative 3D proteome-wide scale identification of ALKBH5 target for MV1035 small molecule able to reduce migration and invasiveness in U87 glioblastoma cell lines by SPILLO-PBSS

The innovative in silico technologies developed at SPILLOproject,1 e.g., the SPILLO potential binding sites searcher (SPILLO-PBSS) software,2,3 allow to identify targets and off-targets of any small molecule on a multiple-organism proteomewide scale, and to perform an accurate multilevel cross-organism transferability analysis (MCOTA) aimed at rationalising animal testing. SPILLO-PBSS has been successfully used in several research projects, such as a study in which a compound (MV1035) was found to reduce migration and invasiveness in U87 glioblastoma (GBM) cell lines: the human structural proteome was analyzed and the RNA demethylase ALKBH5 has been identified as a target responsible for the observed effects (target experimentally validated). Another top-ranked target identified by SPILLO-PBSS, the DNA repair protein AlkB homolog 2 (ALKBH2), abundantly expressed in GBM cell lines, resulted particularly interesting for its pivotal role in the onset of resistance to Temozolomide (TMZ), the standard firstline treatment for GBM.

Phonon-Mediated KIDs as Light Detectors for Rare-Event Search: The CALDER Project

Background suppression plays a crucial role in experiments searching for rare events, like neutrino-less double beta decay (0 $$\nu$$ DBD) and dark matter. Large mass bolometers that are among the most competitive devices in this field would largely benefit from the development of ultrasensitive light detectors, as the combined readout of the bolometric and light signals enables the particle identification. The CALDER collaboration is developing cryogenic light detectors that will match the requirements of next generation experiments: noise lower than 20 eV RMS, large active area (several cm $$^{2}$$ ), wide temperature range of operation, and ease in fabricating and operating a thousand of detectors. For this purpose, we are exploiting the excellent energy resolution and the natural multiplexed read-out provided by kinetic inductance detectors (KIDs). These devices can be operated in a phonon-mediated approach, in which KIDs are coupled to a large insulating substrate in order to increase the active surface from a few mm $$^{2}$$ to 25 cm $$^{2}$$ . Our current best prototype, based on aluminum LEKIDs, reached a baseline sensitivity of 80 eV with an overall efficiency of about 20 %

Fraisinib: a calixpyrrole derivative reducing A549 cell-derived NSCLC tumor in vivo acts as a ligand of the glycine-tRNA synthase, a new molecular target in oncology

Background and purpose: Lung cancer is the leading cause of death in both men and women, constituting a major public health problem worldwide. Non-small-cell lung cancer accounts for 85%–90% of all lung cancers. We propose a compound that successfully fights tumor growth in vivo by targeting the enzyme GARS1.Experimental approach: We present an in-depth investigation of the mechanism through which Fraisinib [meso-(p-acetamidophenyl)-calix(4)pyrrole] affects the human lung adenocarcinoma A549 cell line. In a xenografted model of non-small-cell lung cancer, Fraisinib was found to reduce tumor mass volume without affecting the vital parameters or body weight of mice. Through a computational approach, we uncovered that glycyl-tRNA synthetase is its molecular target. Differential proteomics analysis further confirmed that pathways regulated by Fraisinib are consistent with glycyl-tRNA synthetase inhibition.Key results: Fraisinib displays a strong anti-tumoral potential coupled with limited toxicity in mice. Glycyl-tRNA synthetase has been identified and validated as a protein target of this compound. By inhibiting GARS1, Fraisinib modulates different key biological processes involved in tumoral growth, aggressiveness, and invasiveness.Conclusion and implications: The overall results indicate that Fraisinib is a powerful inhibitor of non-small-cell lung cancer growth by exerting its action on the enzyme GARS1 while displaying marginal toxicity in animal models. Together with the proven ability of this compound to cross the blood–brain barrier, we can assess that Fraisinib can kill two birds with one stone: targeting the primary tumor and its metastases “in one shot.” Taken together, we suggest that inhibiting GARS1 expression and/or GARS1 enzymatic activity may be innovative molecular targets for cancer treatment

AMPK and Diseases: State of the Art Regulation by AMPK-Targeting Molecules

5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an enzyme that regulates cellular energy homeostasis, glucose, fatty acid uptake, and oxidation at low cellular ATP levels. AMPK plays an important role in several molecular mechanisms and physiological conditions. It has been shown that AMPK can be dysregulated in different chronic diseases, such as inflammation, diabetes, obesity, and cancer. Due to its fundamental role in physiological and pathological cellular processes, AMPK is considered one of the most important targets for treating different diseases. Over decades, different AMPK targeting compounds have been discovered, starting from those that activate AMPK indirectly by altering intracellular AMP:ATP ratio to compounds that activate AMPK directly by binding to its activation sites. However, indirect altering of intracellular AMP:ATP ratio influences different cellular processes and induces side effects. Direct AMPK activators showed more promising results in eliminating side effects as well as the possibility to engineer drugs for specific AMPK isoforms activation. In this review, we discuss AMPK targeting drugs, especially concentrating on those compounds that activate AMPK by mimicking AMP. These compounds are poorly described in the literature and still, a lot of questions remain unanswered about the exact mechanism of AMP regulation. Future investigation of the mechanism of AMP binding will make it possible to develop new compounds that, in combination with others, can activate AMPK in a synergistic manner

Potential in-vitro antiviral activity of MV1035 on SARS-CoV-2 wild type viruses

Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, single stranded RNA virus, and is responsible for the pandemic outbreak called COVID-19. The pandemic, still ongoing, had presented unprecedented challenges in terms of finding appropriate pharmacological treatments. Methods Starting from the recent literature that demonstrates how ALKBH5 inhibitors could be used as a new strategy to reduce SARS-CoV-2 replication, we decided to repurpose our newly discovered ALKBH5 inhibitor MV1035, previously tested and proved effective against glioblastoma, for its putative antiviral activity against SARS-CoV-2. We demonstrated a reduction in SARS-CoV-2-induced CPE after 72 h incubation using MV1035 (50 µM), for SARS-CoV-2 wild type (Wuhan strain) and South African variant. Results and conclusion The results show how MV1035 seems to be able to reduce SARS-CoV-2 replication through an indirect mechanism of action, which might involve an interaction with the host cell rather than with a virus protein

3D proteome-wide scale screening and activity evaluation of a new ALKBH5 inhibitor in U87 glioblastoma cell line

The imidazobenzoxazin-5-thione MV1035, synthesized as a new sodium channel blocker, has been tested on tumoral cells that differ for origin and for expressed NaV pool (U87-MG, H460 and A549). In this paper we focus on the effect of MV1035 in reducing U87 glioblastoma cell line migration and invasiveness. Since the effect of this compound on U87-MG cells seemed not dependent on its sodium channel blocking capability, alternative off-target interaction for MV1035 have been identified using SPILLO-PBSS software. This software performs a structure-based in silico screening on a proteome-wide scale, that allows to identify off-target interactions. Among the top-ranked off-targets of MV1035, we focused on the RNA demethylase ALKBH5 enzyme, known for playing a key role in cancer. In order to prove the effect of MV1035 on ALKBH5 in vitro coincubation of MV1035 and ALKBH5 has been performed demonstrating a consequent increase of N6-methyladenosine (m6A) RNA. To further validate the pathway involving ALKBH5 inhibition by MV1035 in U87-MG reduced migration and invasiveness, we evaluated CD73 as possible downstream protein. CD73 is an extrinsic protein involved in the generation of adenosine and is overexpressed in several tumors including glioblastoma. We have demonstrated that treating U87-MG with MV1035, CD73 protein expression was reduced without altering CD73 transcription. Our results show that MV1035 is able to significantly reduce U87 cell line migration and invasiveness inhibiting ALKBH5, an RNA demethylase that can be considered an interesting target in fighting glioblastoma aggressiveness. Our data encourage to further investigate the MV1035 inhibitory effect on glioblastoma

Cognitive-Behavioral Treatment of Non-alcoholic Fatty Liver Disease: A Propensity Score-Adjusted Observational Study

Abstract The effectiveness of cognitive-behavior treatment (CBT) in nonalcoholic fatty liver disease (NAFLD), largely related to overweight/obesity and considered the hepatic expression of the metabolic syndrome (MS), has so far been tested in very limited samples. In a tertiary referral center, consecutively-observed NAFLD subjects were offered a CBT program aimed at weight loss and increased physical activity, based on 13 group sessions; 68 cases entered the treatment protocol, those who refused (n=82) were given recommendations for diet and physical activity. Treatment goals (weight loss ≥ 7% initial b.w., normalization of liver enzymes and improved parameters of MS) were tested by logistic regression at 6 months (all cases) and at 2 years, both on intention-to-treat (ITT) and in completers (Diet, 78; CBT, 65). The results were adjusted for the propensity score of attending the CBT program, based on civil, anthropometric and clinical variables. At baseline the CBT group had a larger prevalence of obesity and more severe insulin resistance (HOMA assessment). At follow-up, CBT was associated with a higher probability of weight loss and normal liver enzymes (6-month: odds ratio (OR), 2.56; 95% confidence interval (CI), 1.15-5.69; 2-year ITT: OR, 3.57, 95% CI, 1.59-8.00), after adjustment for propensity and changes in body weight. A similar trend was observed in the outcome goals of insulin resistance and the score of MS, which were both reduced. In conclusion, subjects with NAFLD participating in a CBT program significantly improve their general and liver parameters. The beneficial effects are largely maintained at 2-year follow-up, in keeping with the lifestyle-related pathogenesis of disease

MV1035 Overcomes Temozolomide Resistance in Patient-Derived Glioblastoma Stem Cell Lines

Glioblastoma (GBM, grade IV glioma) represents the most aggressive brain tumor and patients with GBM have a poor prognosis. Until now surgical resection followed by radiotherapy and temozolomide (TMZ) treatment represents the standard strategy for GBM. We showed that the imidazobenzoxazin-5-thione MV1035 is able to significantly reduce GBM U87-MG cells migration and invasiveness through inhibition of the RNA demethylase ALKBH5. In this work, we focus on the DNA repair protein ALKBH2, a further MV1035 target resulting from SPILLO-PBSS proteome-wide scale in silico analysis. Our data demonstrate that MV1035 inhibits the activity of ALKBH2, known to be involved in GBM TMZ resistance. MV1035 was used on both U87-MG and two patient-derived (PD) glioma stem cells (GSCs): in combination with TMZ, it has a significant synergistic effect in reducing cell viability and sphere formation. Moreover, MV1035 induces a reduction in MGMT expression in PD-GSCs cell lines most likely through a mechanism that acts on MGMT promoter methylation. Taken together our data show that MV1035 could act as an inhibitor potentially helpful to overcome TMZ resistance and able to reduce GBM migration and invasiveness

Cognitive-behavioral treatment of nonalcoholic fatty liver disease: A propensity score-adjusted observational study

The effectiveness of cognitive-behavior treatment (CBT) in nonalcoholic fatty liver disease (NAFLD), largely related to overweight/obesity and considered the hepatic expression of the metabolic syndrome (MS), has so far been tested in very limited samples. In a tertiary referral center, consecutively observed NAFLD subjects were offered a CBT program aimed at weight loss and increased physical activity, based on 13 group sessions; 68 cases entered the treatment protocol, those who refused (n = 82) were given recommendations for diet and physical activity. Treatment goals (weight loss >= 7% initial body weight, normalization of liver enzymes, and improved parameters of MS) were tested by logistic regression at 6 months (all cases) and at 2 years, both on intention-to-treat and in completers (Diet, 78; CBT, 65). The results were adjusted for the propensity score of attending the CBT program, based on civil, anthropometric and clinical variables. At baseline the CBT group had a larger prevalence of obesity and more severe insulin resistance (homeostasis model assessment (HOMA)). At follow-up, CBT was associated with a higher probability of weight loss and normal liver enzymes (6-month: odds ratio (OR), 2.56; 95% confidence interval (CI), 1.15-5.69; 2-year intention-to-treat: OR, 3.57, 95% CI, 1.59-8.00), after adjustment for propensity and changes in body weight. A similar trend was observed in the outcome goals of insulin resistance and the score of MS, which were both reduced. In conclusion, subjects with NAFLD participating in a CBT program significantly improve their general and liver parameters. The beneficial effects are largely maintained at 2-year follow-up, in keeping with the lifestyle-related pathogenesis of disease