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1.
Front Pharmacol ; 12: 664608, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34421588

RESUMO

Phenazopyridine is a widely used drug against urinary tract pain. The compound has also been shown to enhance neural differentiation of pluripotent stem cells. However, its mechanism of action is not understood. Based on its chemical structure, we hypothesized that phenazopyridine could be a kinase inhibitor. Phenazopyridine was investigated in the following experimental systems: 1) activity of kinases in pluripotent stem cells; 2) binding to recombinant kinases, and 3) functional impact on pluripotent stem cells. Upon addition to pluripotent stem cells, phenazopyridine induced changes in kinase activities, particularly involving Mitogen-Activated Protein Kinases, Cyclin-Dependent Kinases, and AKT pathway kinases. To identify the primary targets of phenazopyridine, we screened its interactions with 401 human kinases. Dose-inhibition curves showed that three of these kinases interacted with phenazopyridine with sub-micromolar binding affinities: cyclin-G-associated kinase, and the two phosphatidylinositol kinases PI4KB and PIP4K2C, the latter being known for participating in pain induction. Docking revealed that phenazopyridine forms strong H-bonds with the hinge region of the ATP-binding pocket of these kinases. As previous studies suggested increased autophagy upon inhibition of the phosphatidyl-inositol/AKT pathway, we also investigated the impact of phenazopyridine on this pathway and found an upregulation. In conclusion, our study demonstrates for the first time that phenazopyridine is a kinase inhibitor, impacting notably phosphatidylinositol kinases involved in nociception.

2.
Front Chem ; 9: 672267, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33959589

RESUMO

Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional degraders that specifically eliminate targeted proteins by hijacking the ubiquitin-proteasome system (UPS). This modality has emerged as an orthogonal approach to the use of small-molecule inhibitors for knocking down classic targets and disease-related proteins classified, until now, as "undruggable." In early 2019, the first targeted protein degraders reached the clinic, drawing attention to PROTACs as one of the most appealing technology in the drug discovery landscape. Despite these promising results, PROTACs are often affected by poor cellular permeability due to their high molecular weight (MW) and large exposed polar surface area (PSA). Herein, we report a comprehensive record of PROTAC design, pharmacology and thermodynamic challenges and solutions, as well as some of the available strategies to enhance cellular uptake, including suggestions of promising biological tools for the in vitro evaluation of PROTACs permeability toward successful protein degradation.

3.
Cells ; 10(3)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803230

RESUMO

Macrophages are involved in tissue homeostasis. They participate in inflammatory episodes and are involved in tissue repair. Macrophages are characterized by a phenotypic heterogeneity and a profound cell plasticity. In the kidney, and more particularly within glomeruli, macrophages are thought to play a maintenance role that is potentially critical for preserving a normal glomerular structure. Literature on the glomerular macrophage role in human crescentic glomerulonephritis and renal transplantation rejection with glomerulitis, is sparse. Evidence from preclinical models indicates that macrophages profoundly modulate disease progression, both in terms of number-where depletion has resulted in a reduced glomerular lesion-and sub-phenotype-M1 being more profoundly detrimental than M2. This evidence is corroborated by better outcomes in patients with a lower number of glomerular macrophages. However, due to the very limited biopsy sample size, the type and role of macrophage subpopulations involved in human proliferative lesions is more difficult to precisely define and synthesize. Therefore, specific biomarkers of macrophage activation may enhance our ability to assess their role, potentially enabling improved monitoring of drug activity and ultimately allowing the development of novel therapeutic strategies to target these elusive cellular players.


Assuntos
Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Glomérulos Renais/patologia , Macrófagos/patologia , Animais , Modelos Animais de Doenças , Rejeição de Enxerto/patologia , Humanos , Fenótipo
4.
PLoS One ; 16(2): e0245372, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33534816

RESUMO

Strict storage recommendations for insulin are difficult to follow in hot tropical regions and even more challenging in conflict and humanitarian emergency settings, adding an extra burden to the management of people with diabetes. According to pharmacopeia unopened insulin vials must be stored in a refrigerator (2-8°C), while storage at ambient temperature (25-30°C) is usually permitted for the 4-week usage period during treatment. In the present work we address a critical question towards improving diabetes care in resource poor settings, namely whether insulin is stable and retains biological activity in tropical temperatures during a 4-week treatment period. To answer this question, temperature fluctuations were measured in Dagahaley refugee camp (Northern Kenya) using log tag recorders. Oscillating temperatures between 25 and 37°C were observed. Insulin heat stability was assessed under these specific temperatures which were precisely reproduced in the laboratory. Different commercialized formulations of insulin were quantified weekly by high performance liquid chromatography and the results showed perfect conformity to pharmacopeia guidelines, thus confirming stability over the assessment period (four weeks). Monitoring the 3D-structure of the tested insulin by circular dichroism confirmed that insulin monomer conformation did not undergo significant modifications. The measure of insulin efficiency on insulin receptor (IR) and Akt phosphorylation in hepatic cells indicated that insulin bioactivity of the samples stored at oscillating temperature during the usage period is identical to that of the samples maintained at 2-8°C. Taken together, these results indicate that insulin can be stored at such oscillating ambient temperatures for the usual four-week period of use. This enables the barrier of cold storage during use to be removed, thereby opening up the perspective for easier management of diabetes in humanitarian contexts and resource poor settings.


Assuntos
Temperatura Alta/efeitos adversos , Insulina/química , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Quênia , Refrigeração , Clima Tropical/efeitos adversos
5.
J Pharm Biomed Anal ; 189: 113469, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32688211

RESUMO

One of the crucial roles played in the context of human physiology by the human gut microbiota is to ferment resistant polysaccharides and dietary fibres in the colon. Even though it has long been presumed that these processes play fundamental roles in regulating human health, we remain unable to treat or even diagnose deficiencies in microbial fermentation. In part, this relatively slow progress can be attributed to the fact that studying the gut microbiota and its metabolic properties has until now heavily relied on next generation sequencing and case-control cohorts to identify differentially abundant genes, pathways or organisms in the context of a particular clinical indication. Unfortunately, these methods and studies do not allow us to rigorously probe the functional and metabolic phenotype of a microbiota, or for elucidating its mechanisms of action on the host. To improve our clinical control over these fermentation processes, it is critical that we improve our quantitative, mechanistic understanding of their impact on host physiology. In this review, we provide an overview of our current understanding of the roles microbial fermentation processes play in human health in the context of disease prevention. We then describe the evidence linking these processes with depression and anxiety-related conditions, and use these complex disorders as a framework for illustrating the fact that achieving a clinical vision that exploits microbial fermentation towards human health will depend on thoughtful multi-disciplinary collaboration between clinical research, systems biology, and the pharmaceutical and analytical sciences.


Assuntos
Microbioma Gastrointestinal , Microbiota , Estudos de Casos e Controles , Colo , Fermentação , Humanos
6.
Molecules ; 25(12)2020 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-32545835

RESUMO

In this review, we retraced the '40-year evolution' of molecular docking algorithms. Over the course of the years, their development allowed to progress from the so-called 'rigid-docking' searching methods to the more sophisticated 'semi-flexible' and 'flexible docking' algorithms. Together with the advancement of computing architecture and power, molecular docking's applications also exponentially increased, from a single-ligand binding calculation to large screening and polypharmacology profiles. Recently targeting nucleic acids with small molecules has emerged as a valuable therapeutic strategy especially for cancer treatment, along with bacterial and viral infections. For example, therapeutic intervention at the mRNA level allows to overcome the problematic of undruggable proteins without modifying the genome. Despite the promising therapeutic potential of nucleic acids, molecular docking programs have been optimized mostly for proteins. Here, we have analyzed literature data on nucleic acid to benchmark some of the widely used docking programs. Finally, the comparison between proteins and nucleic acid targets docking highlighted similarity and differences, which are intrinsically related to their chemical and structural nature.


Assuntos
Algoritmos , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/química
7.
J Biol Chem ; 295(28): 9299-9312, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32371390

RESUMO

Pleckstrin homology domain-containing A7 (PLEKHA7) is a cytoplasmic protein at adherens junctions that has been implicated in hypertension, glaucoma, and responses to Staphylococcus aureus α-toxin. Complex formation between PLEKHA7, PDZ domain-containing 11 (PDZD11), tetraspanin 33, and the α-toxin receptor ADAM metallopeptidase domain 10 (ADAM10) promotes junctional clustering of ADAM10 and α-toxin-mediated pore formation. However, how the N-terminal region of PDZD11 interacts with the N-terminal tandem WW domains of PLEKHA7 and how this interaction promotes tetraspanin 33 binding to the WW1 domain is unclear. Here, we used site-directed mutagenesis, glutathione S-transferase pulldown experiments, immunofluorescence, molecular modeling, and docking experiments to characterize the mechanisms driving these interactions. We found that Asp-30 of WW1 and His-75 of WW2 interact through a hydrogen bond and, together with Thr-35 of WW1, form a binding pocket that accommodates a polyproline stretch within the N-terminal PDZD11 region. By strengthening the interactions of the ternary complex, the WW2 domain stabilized the WW1 domain and cooperatively promoted the interaction with PDZD11. Modeling results indicated that, in turn, PDZD11 binding induces a conformational rearrangement, which strengthens the ternary complex, and contributes to enlarging a "hydrophobic hot spot" region on the WW1 domain. The last two lipophilic residues of tetraspanin 33, Trp-283 and Tyr-282, were required for its interaction with PLEKHA7. Docking of the tetraspanin 33 C terminus revealed that it fits into the hydrophobic hot spot region of the accessible surface of WW1. We conclude that communication between the two tandem WW domains of PLEKHA7 and the PLEKHA7-PDZD11 interaction modulate the ligand-binding properties of PLEKHA7.


Assuntos
Proteínas de Transporte/química , Tetraspaninas/química , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Humanos , Ligação de Hidrogênio , Camundongos , Camundongos Knockout , Simulação de Acoplamento Molecular , Domínios Proteicos , Estrutura Quaternária de Proteína , Tetraspaninas/genética , Tetraspaninas/metabolismo
8.
Cells ; 9(5)2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32466100

RESUMO

The roles and interactions of platelets and liver sinusoidal endothelial cells in liver regeneration are unclear, and the trigger that initiates hepatocyte proliferation is unknown. We aimed to identify the key factors released by activated platelets that induce liver sinusoidal endothelial cells to produce interleukin-6 (IL-6), a cytokine implicated in the early phase of liver regeneration. We characterized the releasate of activated platelets inducing the in vitro production of IL-6 by mouse liver sinusoidal endothelial cells and observed that the stimulating factor was a thermolabile protein. Following gel filtration, a single fraction of activated platelet releasate induced a maximal IL-6 secretion by liver sinusoidal endothelial cells (90.2 ± 13.9 versus control with buffer, 9.0 ± 0.8 pg/mL, p < 0.05). Mass spectroscopy analysis of this fraction, followed by in silico processing, resulted in a reduced list of 18 candidates. Several proteins from the list were tested, and only recombinant transforming growth factor ß1 (TGF-ß1) resulted in an increased IL-6 production up to 242.7 ± 30.5 pg/mL, which was comparable to non-fractionated platelet releasate effect. Using neutralizing anti-TGF-ß1 antibody or a TGF-ß1 receptor inhibitor, IL-6 production by liver sinusoidal endothelial cells was dramatically reduced. These results support a role of platelet TGF-ß1 ß1 in the priming phase of liver regeneration.


Assuntos
Plaquetas/metabolismo , Células Endoteliais/metabolismo , Interleucina-6/metabolismo , Fígado/citologia , Fator de Crescimento Transformador beta1/farmacologia , Animais , Bovinos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Macrófagos do Fígado/efeitos dos fármacos , Macrófagos do Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Ativação Plaquetária/efeitos dos fármacos , Solubilidade , Fator de von Willebrand/metabolismo
9.
Chimia (Aarau) ; 74(4): 274-277, 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32331546

RESUMO

In the context of dysregulated ubiquitylation, the accumulation of oncogenic substrates can lead to tumorigenesis. In particular, mutations in Von Hippel-Lindau (VHL) E3 ubiquitin ligase are related to overexpression of hypoxia-inducible factors (HIF-1α and HIF-2α) which is evolving into renal cell carcinoma (RCC). The classical approach of drug discovery focuses on the development of highly selective small molecules able to bind and to inhibit enzymatic active sites. This strategy faces limitations in the context of ' undruggable ' proteins, which are challenging to target. The discovery of Proteolysis Targeting Chimeras (PROTACs) as an alternative strategy to induce selective protein degradation is presented as a working hypothesis to understand further the UbiquitinProteasome System (UPS) and eventually counteract RCC cancer lacking VHL ubiquitin ligase.


Assuntos
Ubiquitina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos , Complexo de Endopeptidases do Proteassoma , Ubiquitina-Proteína Ligases , Proteína Supressora de Tumor Von Hippel-Lindau
10.
Front Microbiol ; 11: 266, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32153546

RESUMO

Tubercular Mycobacteria and Legionella pneumophila are the causative agents of potentially fatal respiratory diseases due to their intrinsic pathogenesis but also due to the emergence of antibiotic resistance that limits treatment options. The aim of our study was to explore the antimicrobial activity of a small ligand-based chemical library of 1255 structurally diverse compounds. These compounds were screened in a combination of three assays, two monitoring the intracellular growth of the pathogenic bacteria, Mycobacterium marinum and L. pneumophila, and one assessing virulence of M. marinum. We set up these assays using two amoeba strains, the genetically tractable social amoeba Dictyostelium discoideum and the free-living amoeba Acanthamoeba castellanii. In summary, 64 (5.1%) compounds showed anti-infective/anti-virulence activity in at least one of the three assays. The intracellular assays hit rate varied between 1.7% (n = 22) for M. marinum and 2.8% (n = 35) for L. pneumophila with seven compounds in common for both pathogens. In parallel, 1.2% (n = 15) of the tested compounds were able to restore D. discoideum growth in the presence of M. marinum spiked in a lawn of food bacteria. We also validated the generality of the hits identified in the A. castellanii-M. marinum anti-infective screen using the D. discoideum-M. marinum host-pathogen model. The characterization of anti-infective and antibacterial hits in the latter infection model revealed compounds able to reduce intracellular growth more than 50% at 30 µM. Moreover, the chemical space and physico-chemical properties of the anti-M. marinum hits were compared to standard and candidate Mycobacterium tuberculosis (Mtb) drugs using ChemGPS-NP. A principle component analysis identified separate clusters for anti-M. marinum and anti-L. pneumophila hits unveiling the potentially new physico-chemical properties of these hits compared to standard and candidate M. tuberculosis drugs. Our studies underscore the relevance of using a combination of low-cost and low-complexity assays with full 3R compliance in concert with a rationalized focused library of compounds to identify new chemical scaffolds and to dissect some of their properties prior to taking further steps toward compound development.

11.
Chem Biol Drug Des ; 94(2): 1545-1555, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31063658

RESUMO

The need for new antibacterial agents is increasingly becoming of great importance as bacterial resistance to current drugs is quickly spreading. Enoyl-acyl carrier protein reductases (FabI) are important enzymes for fatty acid biosynthesis in bacteria and other micro-organisms. In this project, we conducted structure-based virtual screening against the FabI enzyme, and accordingly, 37 compounds were selected for experimental testing. Interestingly, five compounds were able to demonstrate antimicrobial effect with variable inhibition activity against various strains of bacteria and fungi. Minimum inhibitory concentrations of the active compounds were determined and showed to be in low to medium micromolar range. Subsequently, enzyme inhibition assay was carried out for our five antimicrobial hits to confirm their biological target and determine their IC50 values. Three of these tested compounds exhibited inhibition activity for the FabI enzyme where our best hit MN02 had an IC50 value of 7.8 µM. Furthermore, MN02 is a small bisphenolic compound that is predicted to have all required features to firmly bind with the target enzyme. To sum up, hits discovered in this work can act as a good starting point for the future development of new and potent antimicrobial agents.


Assuntos
Antibacterianos , Bactérias/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Inibidores Enzimáticos , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia
12.
Biochim Biophys Acta Mol Cell Res ; 1866(11): 118474, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30954571

RESUMO

Discoidin domain receptor1 (DDR1) is a collagen activated receptor tyrosine kinase and an attractive anti-fibrotic target. Its expression is mainly limited to epithelial cells located in several organs including skin, kidney, liver and lung. DDR1's biology is elusive, with unknown downstream activation pathways; however, it may act as a mediator of the stromal-epithelial interaction, potentially controlling the activation state of the resident quiescent fibroblasts. Increased expression of DDR1 has been documented in several types of cancer and fibrotic conditions including skin hypertrophic scars, idiopathic pulmonary fibrosis, cirrhotic liver and renal fibrosis. The present review article focuses on: a) detailing the evidence for a role of DDR1 as an anti-fibrotic target in different organs, b) clarifying DDR1 tissue distribution in healthy and diseased tissues as well as c) exploring DDR1 protective mode of action based on literature evidence and co-authors experience; d) detailing pharmacological efforts attempted to drug this subtle anti-fibrotic target to date.


Assuntos
Receptor com Domínio Discoidina 1/efeitos dos fármacos , Receptor com Domínio Discoidina 1/metabolismo , Fibrose/metabolismo , Animais , Aterosclerose/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/tratamento farmacológico , Humanos , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Neoplasias/metabolismo , Nefrite Intersticial/patologia , Plasmócitos , Receptores Proteína Tirosina Quinases , Pele/metabolismo , Pele/patologia , Doenças Vasculares/metabolismo , Cicatrização
13.
Biochim Biophys Acta Rev Cancer ; 1871(2): 434-454, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31034926

RESUMO

The strategy of using existing drugs originally developed for one disease to treat other indications has found success across medical fields. Such drug repurposing promises faster access of drugs to patients while reducing costs in the long and difficult process of drug development. However, the number of existing drugs and diseases, together with the heterogeneity of patients and diseases, notably including cancers, can make repurposing time consuming and inefficient. The key question we address is how to efficiently repurpose an existing drug to treat a given indication. As drug efficacy remains the main bottleneck for overall success, we discuss the need for machine-learning computational methods in combination with specific phenotypic studies along with mechanistic studies, chemical genetics and omics assays to successfully predict disease-drug pairs. Such a pipeline could be particularly important to cancer patients who face heterogeneous, recurrent and metastatic disease and need fast and personalized treatments. Here we focus on drug repurposing for colorectal cancer and describe selected therapeutics already repositioned for its prevention and/or treatment as well as potential candidates. We consider this review as a selective compilation of approaches and methodologies, and argue how, taken together, they could bring drug repurposing to the next level.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Reposicionamento de Medicamentos/métodos , Aprendizado de Máquina , Animais , Humanos , Oncologia/métodos , Fenótipo
14.
Hum Mol Genet ; 28(12): 1931-1946, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-30590522

RESUMO

Mutations in OCRL encoding the inositol polyphosphate 5-phosphatase OCRL (Lowe oculocerebrorenal syndrome protein) disrupt phosphoinositide homeostasis along the endolysosomal pathway causing dysfunction of the cells lining the kidney proximal tubule (PT). The dysfunction can be isolated (Dent disease 2) or associated with congenital cataracts, central hypotonia and intellectual disability (Lowe syndrome). The mechanistic understanding of Dent disease 2/Lowe syndrome remains scarce due to limitations of animal models of OCRL deficiency. Here, we investigate the role of OCRL in Dent disease 2/Lowe syndrome by using OcrlY/- mice, where the lethal deletion of the paralogue Inpp5b was rescued by human INPP5B insertion, and primary culture of proximal tubule cells (mPTCs) derived from OcrlY/- kidneys. The OcrlY/- mice show muscular defects with dysfunctional locomotricity and present massive urinary losses of low-molecular-weight proteins and albumin, caused by selective impairment of receptor-mediated endocytosis in PT cells. The latter was due to accumulation of phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 in endolysosomes, driving local hyper-polymerization of F-actin and impairing trafficking of the endocytic LRP2 receptor, as evidenced in OcrlY/- mPTCs. The OCRL deficiency was also associated with a disruption of the lysosomal dynamic and proteolytic activity. Partial convergence of disease-pathways and renal phenotypes observed in OcrlY/- and Clcn5Y/- mice suggest shared mechanisms in Dent diseases 1 and 2. These studies substantiate the first mouse model of Lowe syndrome and give insights into the role of OCRL in cellular trafficking of multiligand receptors. These insights open new avenues for therapeutic interventions in Lowe syndrome and Dent disease.


Assuntos
Doença de Dent/genética , Endossomos/metabolismo , Túbulos Renais Proximais/metabolismo , Lisossomos/metabolismo , Síndrome Oculocerebrorrenal/genética , Monoéster Fosfórico Hidrolases/genética , Actinas/metabolismo , Animais , Células Cultivadas , Canais de Cloreto/genética , Doença de Dent/metabolismo , Doença de Dent/fisiopatologia , Modelos Animais de Doenças , Endocitose/genética , Humanos , Rim/fisiopatologia , Túbulos Renais Proximais/fisiopatologia , Locomoção/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Síndrome Oculocerebrorrenal/metabolismo , Síndrome Oculocerebrorrenal/fisiopatologia , Fosfatidilinositol 4,5-Difosfato/metabolismo
15.
Nat Commun ; 9(1): 4848, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451843

RESUMO

X-linked myotubular myopathy (XLMTM, also known as XLCNM) is a severe congenital muscular disorder due to mutations in the myotubularin gene, MTM1. It is characterized by generalized hypotonia, leading to neonatal death of most patients. No specific treatment exists. Here, we show that tamoxifen, a well-known drug used against breast cancer, rescues the phenotype of Mtm1-deficient mice. Tamoxifen increases lifespan several-fold while improving overall motor function and preventing disease progression including lower limb paralysis. Tamoxifen corrects functional, histological and molecular hallmarks of XLMTM, with improved force output, myonuclei positioning, myofibrillar structure, triad number, and excitation-contraction coupling. Tamoxifen normalizes the expression level of the XLMTM disease modifiers DNM2 and PI3KC2B, likely contributing to the phenotypic rescue. Our findings demonstrate that tamoxifen is a promising candidate for clinical evaluation in XLMTM patients.


Assuntos
Atividade Motora/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Miopatias Congênitas Estruturais/tratamento farmacológico , Substâncias Protetoras/farmacologia , Proteínas Tirosina Fosfatases não Receptoras/genética , Tamoxifeno/farmacologia , Animais , Classe II de Fosfatidilinositol 3-Quinases/genética , Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Dinamina II/genética , Dinamina II/metabolismo , Estimulação Elétrica , Acoplamento Excitação-Contração/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Genes Letais , Humanos , Longevidade/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miofibrilas/efeitos dos fármacos , Miofibrilas/metabolismo , Miofibrilas/ultraestrutura , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Miopatias Congênitas Estruturais/patologia , Proteínas Tirosina Fosfatases não Receptoras/deficiência
16.
Mol Cell ; 71(2): 343-351.e4, 2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-30029007

RESUMO

Class II phosphoinositide 3-kinases (PI3K-C2) are large multidomain enzymes that control cellular functions ranging from membrane dynamics to cell signaling via synthesis of 3'-phosphorylated phosphoinositides. Activity of the alpha isoform (PI3K-C2α) is associated with endocytosis, angiogenesis, and glucose metabolism. How PI3K-C2α activity is controlled at sites of endocytosis remains largely enigmatic. Here we show that the lipid-binding PX-C2 module unique to class II PI3Ks autoinhibits kinase activity in solution but is essential for full enzymatic activity at PtdIns(4,5)P2-rich membranes. Using HDX-MS, we show that the PX-C2 module folds back onto the kinase domain, inhibiting its basal activity. Destabilization of this intramolecular contact increases PI3K-C2α activity in vitro and in cells, leading to accumulation of its lipid product, increased recruitment of the endocytic effector SNX9, and facilitated endocytosis. Our studies uncover a regulatory mechanism in which coincident binding of phosphoinositide substrate and cofactor selectively activate PI3K-C2α at sites of endocytosis.


Assuntos
Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Classe II de Fosfatidilinositol 3-Quinases/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Animais , Domínios C2/fisiologia , Células COS , Chlorocebus aethiops , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/fisiologia , Clatrina/fisiologia , Endocitose/fisiologia , Células HEK293 , Homeostase , Humanos , Lipídeos/fisiologia , Espectrometria de Massas , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , Transdução de Sinais
17.
Nat Commun ; 9(1): 2032, 2018 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-29795225

RESUMO

Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5' splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.


Assuntos
Imidazóis/farmacologia , Indóis/farmacologia , Atrofia Muscular Espinal/tratamento farmacológico , RNA Mensageiro/metabolismo , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Drosophila , Avaliação Pré-Clínica de Medicamentos , Éxons/genética , Células HeLa , Humanos , Imidazóis/química , Imidazóis/uso terapêutico , Indóis/química , Indóis/uso terapêutico , Terapia de Alvo Molecular/métodos , Atrofia Muscular Espinal/genética , Fenótipo , Sítios de Splice de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Elementos Reguladores de Transcrição/efeitos dos fármacos , Proteína 2 de Sobrevivência do Neurônio Motor/genética
18.
Chimia (Aarau) ; 72(4): 238-240, 2018 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-29720316

RESUMO

Drug discovery is a long, expensive and risky process. Evaluating drugs that have already been proved safe for use in humans and testing them for a new indication greatly reduces the time and monetary costs involved in finding treatments for life-threatening conditions. Here tamoxifen, a drug that is used for the treatment of breast cancer, is investigated in a mouse model of Duchenne muscular dystrophy. Tamoxifen was efficacious in countering the symptoms of the disease without affecting the underlying genetic cause. Based on these results, tamoxifen has been tested in other forms of muscle disease with success. Drug repurposing may not only be a cost-effective manner for treating a variety of diseases, it may also help us uncover common mechanisms between conditions that were previously thought to be unrelated.


Assuntos
Distrofia Muscular de Duchenne/tratamento farmacológico , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Tamoxifeno/uso terapêutico , Animais , Feminino , Humanos , Masculino , Camundongos
19.
EMBO J ; 37(7)2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29519896

RESUMO

Toxoplasma gondii aspartyl protease 3 (TgASP3) phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X (PfPMIX, PfPMX). These proteases are essential for parasite survival, acting as key maturases for secreted proteins implicated in invasion and egress. A potent antimalarial peptidomimetic inhibitor (49c) originally developed against Plasmepsin II selectively targets TgASP3, PfPMIX, and PfPMX To unravel the molecular basis for the selectivity of 49c, we constructed homology models of PfPMIX, PfPMX, and TgASP3 that were first validated by identifying the determinants of microneme and rhoptry substrate recognition. The flap and flap-like structures of several reported Plasmepsins are highly flexible and critically modulate the access to the binding cavity. Molecular docking of 49c to TgASP3, PfPMIX, and PfPMX models predicted that the conserved phenylalanine residues in the flap, F344, F291, and F305, respectively, account for the sensitivity toward 49c. Concordantly, phenylalanine mutations in the flap of the three proteases increase twofold to 15-fold the IC50 values of 49c. Compellingly the selection of mutagenized T. gondii resistant strains to 49c reproducibly converted F344 to a cysteine residue.


Assuntos
Antimaláricos/farmacologia , Ácido Aspártico Proteases/antagonistas & inibidores , Ácido Aspártico Proteases/metabolismo , Resistência a Medicamentos/fisiologia , Inibidores de Proteases/farmacologia , Proteínas de Protozoários/química , Antimaláricos/química , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Cisteína , Relação Dose-Resposta a Droga , Resistência a Medicamentos/efeitos dos fármacos , Concentração Inibidora 50 , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutação , Testes de Sensibilidade Parasitária , Fenilalanina/efeitos dos fármacos , Fenilalanina/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Alinhamento de Sequência , Toxoplasma/efeitos dos fármacos , Toxoplasma/genética
20.
Sci Rep ; 8(1): 3939, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29500372

RESUMO

Tuberculosis remains a serious threat to human health world-wide, and improved efficiency of medical treatment requires a better understanding of the pathogenesis and the discovery of new drugs. In the present study, we performed a whole-cell based screen in order to complete the characterization of 168 compounds from the GlaxoSmithKline TB-set. We have established and utilized novel previously unexplored host-model systems to characterize the GSK compounds, i.e. the amoeboid organisms D. discoideum and A. castellanii, as well as a microglial phagocytic cell line, BV2. We infected these host cells with Mycobacterium marinum to monitor and characterize the anti-infective activity of the compounds with quantitative fluorescence measurements and high-content microscopy. In summary, 88.1% of the compounds were confirmed as antibiotics against M. marinum, 11.3% and 4.8% displayed strong anti-infective activity in, respectively, the mammalian and protozoan infection models. Additionally, in the two systems, 13-14% of the compounds displayed pro-infective activity. Our studies underline the relevance of using evolutionarily distant pathogen and host models in order to reveal conserved mechanisms of virulence and defence, respectively, which are potential "universal" targets for intervention. Subsequent mechanism of action studies based on generation of over-expresser M. bovis BCG strains, generation of spontaneous resistant mutants and whole genome sequencing revealed four new molecular targets, including FbpA, MurC, MmpL3 and GlpK.


Assuntos
Acanthamoeba castellanii/microbiologia , Antituberculosos/farmacologia , Dictyostelium/microbiologia , Descoberta de Drogas/métodos , Mycobacterium marinum/efeitos dos fármacos , Animais , Linhagem Celular , Farmacorresistência Bacteriana/genética , Camundongos , Testes de Sensibilidade Microbiana , Microglia/citologia , Microglia/efeitos dos fármacos , Mutação , Mycobacterium marinum/genética , Mycobacterium marinum/crescimento & desenvolvimento
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