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A Morita-Baylis-Hillman Adduct (MBHA) derivative bearing a triphenylamine moiety was found to react with human serum albumin (HSA) shifting its emission from the blue to the green-yellow thus leading to green fluorescent albumin (GFA) derivatives and enlarging the platform of probes for aggregation-induced fluorescent-based detection techniques. A possible interaction of MBHA derivative 7 with a lipophilic pocket within the HSA structure was suggested by docking studies. DLS experiments showed that the reaction with HSA induce a conformational change of the protein contributing to the aggregation process of GFA derivatives. The results of investigations on the biological properties suggested that GFA retained the ability of binding drug molecules such as warfarin and diazepam. Finally, cytotoxicity evaluation studies suggested that, although the MBHA derivative 7 at 0.1â µg/mL affected the percentage of cell viability in comparison to the negative control, it cannot be considered cytotoxic, whereas at all the other concentrations≥0.5â µg/mL resulted cytotoxic at different extent.
Assuntos
Albumina Sérica Humana , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas/metabolismo , Albumina Sérica Humana/química , Espectrometria de FluorescênciaRESUMO
Riluzole, the first clinically approved treatment for amyotrophic lateral sclerosis (ALS), represents a successful example of a drug endowed with a multimodal mechanism of action. In recent years, different series of riluzole-based compounds have been reported, including several agents acting as Multi-Target-Directed Ligands (MTLDs) endowed with neuroprotective effects. Aiming at identical twin structures inspired by riluzole (2a-c), a synthetic procedure was planned, but the reactivity of the system took a different path, leading to the serendipitous isolation of benzo[b][1,4]thiazepines 3a-c and expanded intermediates N-cyano-benzo[b][1,4]thiazepines 4a-c, which were fully characterized. The newly obtained structures 3a-c, bearing riluzole key elements, were initially tested in an in vitro ischemia/reperfusion injury protocol, simulating the cerebral stroke. Results identified compound 3b as the most effective in reverting the injury caused by an ischemia-like condition, and its activity was comparable, or even higher than that of riluzole, exhibiting a concentration-dependent neuroprotective effect. Moreover, derivative 3b completely reverted the release of Lactate Dehydrogenase (LDH), lowering the values to those of the control slices. Based on its very promising pharmacological properties, compound 3b was then selected to assess its effects on voltage-dependent Na+ and K+ currents. The results indicated that derivative 3b induced a multifaceted inhibitory effect on voltage-gated currents in SH-SY5Y differentiated neurons, suggesting its possible applications in epilepsy and stroke management, other than ALS. Accordingly, brain penetration was also measured for 3b, as it represents an elegant example of a MTDL and opens the way to further ex-vivo and/or in-vivo characterization.
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Fármacos Neuroprotetores , Riluzol , Animais , Humanos , Relação Dose-Resposta a Droga , Ligantes , Estrutura Molecular , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Riluzol/farmacologia , Riluzol/síntese química , Riluzol/química , Relação Estrutura-Atividade , Tiazepinas/síntese química , Tiazepinas/química , Tiazepinas/farmacologiaRESUMO
Glioblastoma multiforme (GBM) is one of the most aggressive malignancies with a high recurrence rate and poor prognosis. Theranostic, combining therapeutic and diagnostic approaches, arises as a successful strategy to improve patient outcomes through personalized medicine. Src is a non-receptor tyrosine kinase (nRTK) whose involvement in GBM has been extensively demonstrated. Our previous research highlighted the effectiveness of the pyrazolo[3,4-d]pyrimidine SI306 and its more soluble prodrug CMP1 as Src inhibitors both in in vitro and in vivo GBM models. In this scenario, we decided to develop a theranostic prodrug of SI306, ProSI-DOTA(68 Ga) 1, which was designed to target GBM cells after hydrolysis and follow-up on the disease's progression and improve the therapy's outcome. First, the corresponding nonradioactive prodrug 2 was tested to evaluate its ADME profile and biological activity. It showed good metabolic stability, no inhibition of CYP3A4, suboptimal aqueous solubility, and slight gastrointestinal and blood-brain barrier passive permeability. Compound 2 exhibited a drastic reduction of cell vitality after 72 h on two different GBM cell lines (GL261 and U87MG). Then, 2 was subjected to complexation with the radionuclide Gallium-68 to give ProSI-DOTA(68 Ga) 1. The cellular uptake of 1 was evaluated on GBM cells, highlighting a slight but significant time-dependent uptake. The data obtained from our preliminary studies reflect the physiochemical properties of 1. The use of an alternative route of administration, such as the intranasal route, could overcome the physiochemical limitations and enhance the pharmacokinetic properties of 1, paving the way for its future development.
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Glioblastoma , Pró-Fármacos , Humanos , Medicina de Precisão , Glioblastoma/diagnóstico por imagem , Glioblastoma/tratamento farmacológico , Barreira Hematoencefálica , Linhagem Celular , Pró-Fármacos/farmacologiaRESUMO
Globalization and climate change are both contributing to an increase in the number of potentially invasive algae in coastal areas. In terms of biodiversity and financial losses, the invasiveness of algae has become a significant issue in Orbetello Lagoon. Indeed, studies from the Tuscany Regional Agency for Environmental Protection show that the reduction in dissolved oxygen caused by algal diffusion is detrimental to fisheries and biodiversity. Considering that wakame and numerous other potentially invasive seaweeds are consumed as food in Asia, we assess the nutritional and nutraceutical qualities of two potentially invasive seaweeds: Valonia aegagrophila and Chaetomorpha linum. We found that both algae are a valuable source of proteins and essential amino acids. Even if the fat content accounts for less than 2% of the dried weight, its quality is high, due to the presence of unsaturated fatty acids. Both algae are rich in antioxidants pigments and polyphenols, which can be exploited as nutraceuticals. Most importantly, human gastrointestinal digestion increased the quantity of polyphenols and originated secondary metabolites with ACE inhibitory activity. Taken together, our data strongly promote the use of Valonia aegagrophila and Chaetomorpha linum as functional foods, with possible application in the treatment of hypertension and cardiovascular diseases.
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Inibidores da Enzima Conversora de Angiotensina , Antioxidantes , Alimento Funcional , Alga Marinha , Antioxidantes/farmacologia , Antioxidantes/química , Alga Marinha/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Humanos , Nutrientes/análise , Suplementos Nutricionais , Polifenóis/análise , Polifenóis/farmacologia , Polifenóis/química , Valor NutritivoRESUMO
The use of privileged scaffolds as a starting point for the construction of libraries of bioactive compounds is a widely used strategy in drug discovery and development. Scaffold decoration, morphing and hopping are additional techniques that enable the modification of the chosen privileged framework and better explore the chemical space around it. In this study, two series of highly functionalized pyrimidine and pyridine derivatives were synthesized using a scaffold morphing approach consisting of triazine compounds obtained previously as antiviral agents. Newly synthesized azines were evaluated against lymphoma, hepatocarcinoma, and colon epithelial carcinoma cells, showing in five cases acceptable to good anticancer activity associated with low cytotoxicity on healthy fibroblasts. Finally, ADME in vitro studies were conducted on the best derivatives of the two series showing good passive permeability and resistance to metabolic degradation.
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Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Antineoplásicos/farmacologia , Antivirais/farmacologia , Compostos AzoRESUMO
Aberrant activation of Hedgehog (HH) signaling in cancer is the result of genetic alterations of upstream pathway components (canonical) or other oncogenic mechanisms (noncanonical), that ultimately concur to activate the zinc-finger transcription factors GLI1 and GLI2. Therefore, inhibition of GLI activity is a good therapeutic option to suppress both canonical and noncanonical activation of the HH pathway. However, only a few GLI inhibitors are available, and none of them have the profile required for clinical development due to poor metabolic stability and aqueous solubility, and high hydrophobicity. Two promising quinoline inhibitors of GLI were selected by virtual screening and subjected to hit-to-lead optimization, thus leading to the identification of the 4-methoxy-8-hydroxyquinoline derivative JC19. This molecule impaired GLI1 and GLI2 activities in several cellular models interfering with the binding of GLI1 and GLI2 to DNA. JC19 suppressed cancer cell proliferation by enhancing apoptosis, inducing a strong anti-tumor response in several cancer cell lines in vitro. Specificity towards GLI1 and GLI2 was demonstrated by lower activity of JC19 in GLI1- or GLI2-depleted cancer cells. JC19 showed excellent metabolic stability and high passive permeability. Notably, JC19 inhibited GLI1-dependent melanoma xenograft growth in vivo, with no evidence of toxic effects in mice. These results highlight the potential of JC19 as a novel anti-cancer agent targeting GLI1 and GLI2.
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Neoplasias , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de Zinco , Animais , Humanos , Camundongos , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Proteína Gli2 com Dedos de Zinco/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/patologiaRESUMO
In recent decades, vaccines have been extraordinary resources to prevent pathogen diffusion and cancer. Even if they can be formed by a single antigen, the addition of one or more adjuvants represents the key to enhance the response of the immune signal to the antigen, thus accelerating and increasing the duration and the potency of the protective effect. Their use is of particular importance for vulnerable populations, such as the elderly or immunocompromised people. Despite their importance, only in the last forty years has the search for novel adjuvants increased, with the discovery of novel classes of immune potentiators and immunomodulators. Due to the complexity of the cascades involved in immune signal activation, their mechanism of action remains poorly understood, even if significant discovery has been recently made thanks to recombinant technology and metabolomics. This review focuses on the classes of adjuvants under research, recent mechanism of action studies, as well as nanodelivery systems and novel classes of adjuvants that can be chemically manipulated to create novel small molecule adjuvants.
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Adjuvantes Imunológicos , Vacinas , Humanos , Idoso , Adjuvantes Imunológicos/farmacologia , Fatores Imunológicos , Adjuvantes Farmacêuticos , Antivirais/farmacologiaRESUMO
The progression of drugs into clinical phases requires proper toxicity assessment in animals and the correct identification of possible metabolites. Accordingly, different animal models are used to preliminarily evaluate toxicity and biotransformations. Rodents are the most common models used to preliminarily evaluate the safety of drugs; however, their use is subject to ethical consideration and elevated costs, and strictly regulated by national legislations. Herein, we developed a novel, cheap and convenient toxicity model using Tenebrio molitor coleoptera (TMC). A panel of 15 drugs-including antivirals and antibacterials-with different therapeutic applications was administered to TMC and the LD50 was determined. The values are comparable with those already determined in mice and rats. In addition, a TMC model was used to determine the presence of the main metabolites and in vivo pharmacokinetics (PK), and results were compared with those available from in vitro assays and the literature. Taken together, our results demonstrate that TMC can be used as a novel and convenient preliminary toxicity model to preliminarily evaluate the safety of experimental compounds and the formation of main metabolites, and to reduce the costs and number of rodents, according to 3R principles.
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Besouros , Tenebrio , Animais , Camundongos , Ratos , Tenebrio/metabolismoRESUMO
The Bcr-Abl tyrosine kinase (TK) is the molecular hallmark of chronic myeloid leukemia (CML). Src is another TK kinase whose involvement in CML was widely demonstrated. Small molecules active as dual Src/Bcr-Abl inhibitors emerged as effective targeted therapies for CML and a few compounds are currently in clinical use. In this study, we applied a target-oriented approach to identify a family of pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl inhibitors as anti-leukemia agents. Considering the high homology between Src and Bcr-Abl, in-house Src inhibitors 8a-l and new analogue compounds 9a-n were screened as dual Src/Bcr-Abl inhibitors. The antiproliferative activity on K562 CML cells and the ADME profile were determined for the most promising compounds. Molecular modeling studies elucidated the binding mode of the inhibitors into the Bcr-Abl (wt) catalytic pocket. Compounds 8j and 8k showed nanomolar activities in enzymatic and cellular assays, together with favorable ADME properties, emerging as promising candidates for CML therapy. Finally, derivatives 9j and 9k, emerging as valuable inhibitors of the most aggressive Bcr-Abl mutation, T315I, constitute a good starting point in the search for compounds able to treat drug-resistant forms of CML. Overall, this study allowed us to identify more potent compounds than those previously reported by the group, marking a step forward in searching for new antileukemic agents.
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Antineoplásicos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/químicaRESUMO
Current therapy against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are based on the use of Remdesivir 1, Molnupiravir 2, and the recently identified Nirmatrelvir 3. Unfortunately, these three drugs showed some limitations regarding potency and possible drug-drug interactions. A series of derivatives coming from a decoration approach of the privileged scaffold s-triazines were synthesized and evaluated against SAR-CoV-2. One derivative emerged as the hit of the series for its micromolar antiviral activity and low cytotoxicity. Mode of action and pharmacokinetic in vitro preliminary studies further confirm the role as candidates for a future optimization campaign of the most active derivative identified with this work.
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COVID-19 , SARS-CoV-2 , Humanos , Antivirais/farmacologiaRESUMO
The proto-oncogene c-Src is a non-receptor tyrosine kinase which is involved in the regulation of many cellular processes, such as differentiation, adhesion and survival. c-Src hyperactivation has been detected in many tumors, including neuroblastoma (NB), one of the major causes of death from neoplasia in infancy. We already reported a large family of pyrazolo[3,4-d]pyrimidines active as c-Src inhibitors. Interestingly, some of these derivatives resulted also active on SH-SY5Y NB cell line. Herein, starting from our previous Free Energy Perturbation/Monte Carlo calculations, we report an optimization study which led to the identification of a new series of derivatives endowed with nanomolar Ki values against c-Src, interesting antiproliferative activity on SH-SY5Y cells and a suitable ADME profile.
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Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proto-Oncogene Mas , Pirazóis/síntese química , Pirazóis/química , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
A stimuli-sensitive linker is one of the indispensable components of prodrugs for cancer therapy as it covalently binds the drug and releases it upon external stimulation at the tumour site. Quinone methide elimination has been widely used as the key transformation to release drugs based on their nucleofugacity. The usual approach is to bind the drug to the linker as a carbamate and release it as a free amine after a self-immolative 1,6-elimination. Although this approach is very efficient, it is limited to amines (as carbamates), alcohols or phenols (as carbonates) or other acidic functional groups. We report here a self-immolative spacer capable of directly linking and releasing amines, phenols, thiols, sulfonamides and carboxyamides after a reductive stimulus. The spacer is based on the structure of (5-nitro-2-pyrrolyl)methanol (NPYM-OH), which was used for the direct alkylation of the functional groups mentioned above. The spacer is metabolically stable and has three indispensable sites for bioconjugation: the bioresponsive trigger, the conjugated 1,6 self-immolative system and a third arm suitable for conjugation with a carrier or other modifiers. Release was achieved by selective reduction of the nitro group over Fe/Pd nanoparticles (NPs) in a micellar aqueous environment (H2O/TPGS-750-M), or by NADH mediated nitroreductase activation. A DFT study demonstrates that, during the 1,6 elimination, the transition state formed from 5-aminopyrrole has a lower activation energy compared to other 5-membered heterocycles or p-aminobenzyl derivatives. The NPYM scaffold was validated by late-stage functionalisation of approved drugs such as celecoxib, colchicine, vorinostat or ciprofloxacin. A hypoxia-activated NPYM-based prodrug (HAP) derived from HDAC inhibitor ST7612AA1 was also produced, which was active in cancer cells under hypoxic conditions.
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Bioconjugation is one of the most promising strategies to improve drug delivery, especially in cancer therapy. Biomolecules such as bile acids (BAs) have been intensively explored as carriers, due to their peculiar physicochemical properties and biocompatibility. BAs trafficking is regulated by intracellular lipid-binding proteins and their transport in the liver can be studied using chicken liver Bile Acid-Binding Proteins (cL-BABPs) as a reference model. Therefore, we conceived the idea of developing a BA-conjugate with Mirin, an exonuclease inhibitor of Mre11 endowed with different anticancer activities, to direct its transport to the liver. Following computational analysis of various BAs in complex with cL-BABP, we identified cholic acid (CA) as the most promising candidate as carrier, leading to the synthesis of a novel bioconjugate named CA-M11. As predicted by computational data and confirmed by X-ray crystallographic studies, CA-M11 was able to accommodate into the binding pocket of BABP. Hence, it can enter BAs trafficking in the hepatic compartment and here release Mirin. The effect of CA-M11, evaluated in combination with varying concentrations of Doxorubicin on HepG2 cell line, demonstrated a significant increase in cell mortality compared to the use of the cytotoxic drug or Mirin alone, thus highlighting chemo-sensitizing properties. The promising results regarding plasma stability for CA-M11 validate its potential as a valuable agent or adjuvant for hepatic cancer therapy.
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Proteínas de Transporte , Ácido Cólico , Neoplasias Hepáticas , Humanos , Ácido Cólico/química , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Células Hep G2 , Animais , Proteínas de Transporte/metabolismo , Doxorrubicina/farmacologia , Galinhas , Antineoplásicos/farmacologia , Antineoplásicos/química , Fígado/metabolismo , Fígado/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Glicoproteínas de MembranaRESUMO
Influenza viruses (IV) are single-stranded RNA viruses with a negative-sense genome and have the potential to cause pandemics. While vaccines exist for influenza, their protection is only partial. Additionally, there is only a limited number of approved anti-IV drugs, which are associated to emergence of drug resistance. To address these issues, for years we have focused on the development of small-molecules that can interfere with the heterodimerization of PA and PB1 subunits of the IV RNA-dependent RNA polymerase (RdRP). In this study, starting from a cycloheptathiophene-3-carboxamide compound that we recently identified, we performed iterative cycles of medicinal chemistry optimization that led to the identification of compounds 43 and 45 with activity in the nanomolar range against circulating A and B strains of IV. Mechanistic studies demonstrated the ability of 43 and 45 to interfere with viral RdRP activity by disrupting PA-PB1 subunits heterodimerization and to bind to the PA C-terminal domain through biophysical assays. Most important, ADME studies of 45 also showed an improvement in the pharmacokinetic profile with respect to the starting hit.
Assuntos
Antivirais , RNA Polimerase Dependente de RNA , Antivirais/farmacologia , Antivirais/química , Antivirais/síntese química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , Humanos , Animais , Relação Estrutura-Atividade , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Proteínas Virais/química , Estrutura Molecular , Multimerização Proteica/efeitos dos fármacos , Relação Dose-Resposta a Droga , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/enzimologia , Testes de Sensibilidade Microbiana , CãesRESUMO
Hepatocellular carcinoma (HCC) is the most common type of liver solid tumor and the second leading cause of cancer-related deaths worldwide. Although new treatment options have been recently approved, the development of tumor resistance and the poor prognosis for advanced HCC make the current standard of care unsatisfying. In this scenario, the non-receptor tyrosine kinase (TK) c-Src emerged as a promising target for developing new anti-HCC agents. Our group reported a large library of pyrazolo[3,4-d]pyrimidines active as potent c-Src inhibitors. Starting from these data, we applied a molecular hybridization approach to combine the in-house pyrazolo[3,4-d]pyrimidine SI192 with the approved TK inhibitor (TKI) dasatinib, with the aim of identifying a new generation of Src inhibitors. Enzymatic results prompted us to design second-generation compounds with a better binding profile based on a hit optimization protocol comprised of molecular modeling and on-paper rational design. This investigation led to the identification of a few nanomolar Src inhibitors active toward two HCC cell lines (HepG2 and HUH-7) selected according to their high and low c-Src expression, respectively. In particular, 7e showed an IC50 value of 0.7 nM toward Src and a relevant antiproliferative efficacy on HepG2 cells after 72h (IC50 = 2.47 µM). Furthermore, 7e exhibited a cytotoxic profile better than dasatinib. The ADME profile suggested that 7e deserves further investigation as a promising TKI in cancer therapies. Finally, 7e's ability to inhibit HepG2 cell proliferation, elicit an irreversible cytotoxic effect, arrest cellular migration, and induce apoptotic-mediated cell death was assessed.
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Although patients would rather oral therapies to injections, the gastrointestinal tract's low permeability makes this method limiting for most compounds, including anticancer drugs. Due to their low bioavailability, oral antitumor therapies suffer from significant variability in pharmacokinetics and efficacy. The improvement of their pharmacokinetic profiles can be achieved by a new approach: the use of natural extracts enriched with polyphenolic compounds that act as intestinal permeability enhancers. Here, we propose a safe sweet cherry extract capable of enhancing oral absorption. The extract was characterized by the HPLC-UV/MS method, evaluated for in vitro antioxidant activity, safety on the Caco-2 cell line, and as a potential permeation enhancer. The sweet cherry extract showed a high antioxidant capacity (ABTS and DPPH assays were 211.74 and 48.65 µmol of Trolox equivalent/g dried extract, respectively), high content of polyphenols (8.44 mg of gallic acid per gram of dry extract), and anthocyanins (1.80 mg of cyanidin-3-glucoside equivalent per g of dry extract), reassuring safety profile (cell viability never lower than 98%), and a significant and fully reversible ability to alter the integrity of the Caco-2 monolayer (+81.5% of Lucifer yellow permeability after 2 h). Furthermore, the ability of the sweet cherry extract to improve the permeability (Papp) and modify the efflux ratio (ER) of reference compounds (atenolol, propranolol, and dasatinib) and selected pyrazolo[3,4-d]pyrimidine derivatives was investigated. The obtained results show a significant increase in apparent permeability across the Caco-2 monolayer (tripled and quadrupled in most cases), and an interesting decrease in efflux ratio when compounds were co-incubated with sweet cherry extract.
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Short-chain fatty acids (SCFAs), the end products of gut microbial fermentation of dietary fibers and non-digestible polysaccharides, act as a link between the microbiome, immune system, and inflammatory processes. The importance of accurately quantifying SCFAs in plasma has recently emerged to understand their biological role. In this work, a sensitive and reproducible LC-MS/MS method is reported for SCFAs quantification in three different matrices such as human, rat and mouse plasma via derivatization, using as derivatizing agent O-benzylhydroxylamine (O-BHA), coupled with liquid-liquid extraction. First, the instrumental parameters of the mass spectrometer and then the chromatographic conditions were optimized using previously SCFAs derivatives synthetized and used as standards. After that, the best conditions for derivatization and extraction from plasma were studied and a series of determinations were performed on human, rat, and mouse plasma aliquots to validate the overall method (derivatization, extraction, and LC-MS/MS determination). The method showed good performance in terms of recovery (> 80%), precision (RSD <14%), accuracy (RE < ± 10%) and sensitivity (LOQ of 0.01 µM for acetic, butyric, propionic and isobutyric acid) in all plasma samples. The method thus developed and validated was applied to the quantification of major SCFAs in adult and aged mice, germ-free mice and in germ-free recipient mice subjected to fecal transplant from adult and aged donors. Results highlighted how plasma concentrations of SCFAs are correlated with age further highlighting the importance of developing a method that is reliable for the quantification of SCFAs to study their biological role.
Assuntos
Microbioma Gastrointestinal , Espectrometria de Massas em Tandem , Camundongos , Ratos , Humanos , Animais , Idoso , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Fezes/química , Ácidos Graxos Voláteis/análiseRESUMO
The therapeutic use of tyrosine kinase inhibitors (TKIs) represents one of the successful strategies for the treatment of glioblastoma (GBM). Pyrazolo[3,4-d]pyrimidines have already been reported as promising small molecules active as c-Src/Abl dual inhibitors. Herein, we present a series of pyrazolo[3,4-d]pyrimidine derivatives, selected from our in-house library, to identify a promising candidate active against GBM. The inhibitory activity against c-Src and Abl was investigated, and the antiproliferative profile against four GBM cell lines was studied. For the most active compounds endowed with antiproliferative efficacy in the low-micromolar range, the effects toward nontumoral, healthy cell lines (fibroblasts FIBRO 2-93 and keratinocytes HaCaT) was investigated. Lastly, the in silico and in vitro ADME properties of all compounds were also assessed. Among the tested compounds, the promising inhibitory activity against c-Src and Abl (Ki 3.14 µM and 0.44 µM, respectively), the irreversible, apoptotic-mediated death toward U-87, LN18, LN229, and DBTRG GBM cell lines (IC50 6.8 µM, 10.8 µM, 6.9 µM, and 8.5 µM, respectively), the significant reduction in GBM cell migration, the safe profile toward FIBRO 2-93 and HaCaT healthy cell lines (CC50 91.7 µM and 126.5 µM, respectively), the high metabolic stability, and the excellent passive permeability across gastrointestinal and blood-brain barriers led us to select compound 5 for further in vivo assays.
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Our research group has been involved for a long time in the development of macrocyclic amidinoureas (MCAs) as antifungal agents. The mechanistic investigation drove us to perform an in silico target fishing study, which allowed the identification of chitinases as one of their putative targets, with 1a showing a submicromolar inhibition of Trichoderma viride chitinase. In this work, we investigated the possibility to further inhibit the corresponding human enzymes, acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1), involved in several chronic inflammatory lung diseases. Thus, we first validated the inhibitory activity of 1a against AMCase and CHIT1 and then designed and synthesized new derivatives aimed at improving the potency and selectivity against AMCase. Among them, compound 3f emerged for its activity profile along with its promising in vitro ADME properties. We also gained a good understanding of the key interactions with the target enzyme through in silico studies.
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In recent years, globalization, global warming, and population aging have contributed to the spread of emerging viruses, such as coronaviruses (COVs), West Nile (WNV), Dengue (DENV), and Zika (ZIKV). The number of reported infections is increasing, and considering the high viral mutation rate, it is conceivable that it will increase significantly in the coming years. The risk caused by viruses is now more evident due to the COVID-19 pandemic, which highlighted the need to find new broad-spectrum antiviral agents able to tackle the present pandemic and future epidemics. DDX3X helicase is a host factor required for viral replication. Selective inhibitors have been identified and developed into broad-spectrum antivirals active against emerging pathogens, including SARS-CoV-2 and most importantly against drug-resistant strains. This perspective describes the inhibitors identified in the last years, highlighting their therapeutic potential as innovative broad-spectrum antivirals.