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1.
Acta Physiol (Oxf) ; 240(2): e14078, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38205922

RESUMO

AIM: To determine whether the crustacean Rh1 protein functions as a dual CO2 /ammonia transporter and investigate its role in branchial ammonia excretion and acid-base regulation. METHODS: Sequence analysis of decapod Rh1 proteins was used to determine the conservation of amino acid residues putatively involved in ammonia transport and CO2 binding in human and bacterial Rh proteins. Using the Carcinus maenas Rh1 protein (CmRh1) as a representative of decapod Rh1 proteins, we test the ammonia and CO2 transport capabilities of CmRh1 through heterologous expression in yeast and Xenopus oocytes coupled with site-directed mutagenesis. Quantitative PCR was used to assess the distribution of CmRh1 mRNA in various tissues. Western blotting was used to assess CmRh1 protein expression changes in response to high environmental ammonia and CO2 . Further, immunohistochemistry was used to assess sub-cellular localization of CmRh1 and a membrane-bound carbonic anhydrase (CmCAg). RESULTS: Sequence analysis of decapod Rh proteins revealed high conservation of several amino acid residues putatively involved in conducting ammonia transport and CO2 binding. Expression of CmRh1 in Xenopus oocytes enhanced both ammonia and CO2 transport which was nullified in CmRh1 D180N mutant oocytes. Transport of the ammonia analog methylamine by CmRh1 is dependent on both ionized and un-ionized ammonia/methylamine species. CmRh1 was co-localized with CmCAg to the apical membrane of the crustacean gill and only experienced decreased protein expression in the anterior gills when exposed to high environmental ammonia. CONCLUSION: CmRh1 is the first identified apical transporter-mediated route for ammonia and CO2 excretion in the crustacean gill. Our findings shed further light on the potential universality of dual ammonia and CO2 transport capacity of Rhesus glycoproteins in both vertebrates and invertebrates.


Assuntos
Amônia , Dióxido de Carbono , Animais , Humanos , Dióxido de Carbono/metabolismo , Amônia/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Aminoácidos , Metilaminas
2.
mBio ; 13(2): e0291321, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35196127

RESUMO

Ammonium translocation through biological membranes, by the ubiquitous Amt-Mep-Rh family of transporters, plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of the pore of these transporters, forming the family's characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology on Escherichia coli AmtB, in silico molecular dynamics simulations, and in vivo yeast functional complementation assays, we demonstrate that variations in the Twin-His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs specificity in Amt-Mep-Rh transporters, but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We show that coexistence of both mechanisms in single Twin-His variants of yeast Mep2 transceptors disrupts the signaling function and so impairs fungal filamentation. These data support a signaling process driven by the transport mechanism of the fungal Mep2 transceptors. IMPORTANCE Fungal infections represent a significant threat to human health and cause huge damage to crop yields worldwide. The dimorphic switch between yeast and filamentous growth is associated with the virulence of pathogenic fungi. Of note, fungal Mep2 proteins of the conserved Amt-Mep-Rh family play a transceptor role in the induction of filamentation; however, the signaling mechanism remains largely unknown. Amt-Mep-Rh proteins ensure the specific scavenging of NH4+ through a mechanism relying on substrate deprotonation, thereby preventing competition and translocation of similar-sized K+. Our multidisciplinary approaches using E. coli AmtB, Saccharomyces cerevisiae, and Candida albicans Mep2 show that double variation of the family-defining Twin-His motif triggers a mechanistic switch from a specific transporter to an unspecific ion channel with both mechanisms still coexisting in single variants. Moreover, we show that this mechanistic alteration is associated with loss of signaling ability of Mep2, supporting a transport mechanism-driven process in filamentation induction.


Assuntos
Compostos de Amônio , Proteínas de Transporte de Cátions , Proteínas de Escherichia coli , Proteínas de Saccharomyces cerevisiae , Candida albicans/genética , Proteínas de Transporte de Cátions/genética , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas Fúngicas/genética , Humanos , Proteínas de Membrana Transportadoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Translocação Genética
4.
J Enzyme Inhib Med Chem ; 36(1): 1874-1883, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34340614

RESUMO

A library of variously decorated N-phenyl secondary sulphonamides featuring the bicyclic tetrahydroquinazole scaffold was synthesised and biologically evaluated for their inhibitory activity against human carbonic anhydrase (hCA) I, II, IV, and IX. Of note, several compounds were identified showing submicromolar potency and excellent selectivity for the tumour-related hCA IX isoform. Structure-activity relationship data attained for various substitutions were rationalised by molecular modelling studies in terms of both inhibitory activity and selectivity.


Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Biologia Computacional/métodos , Isoenzimas/antagonistas & inibidores , Quinazolinas/química , Sulfonamidas/farmacologia , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Avaliação Pré-Clínica de Medicamentos , Simulação de Acoplamento Molecular , Espectroscopia de Prótons por Ressonância Magnética , Relação Estrutura-Atividade , Sulfonamidas/química
5.
J Enzyme Inhib Med Chem ; 36(1): 1783-1797, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34340630

RESUMO

Carbonic Anhydrase Activators (CAAs) could represent a novel approach for the treatment of Alzheimer's disease, ageing, and other conditions that require remedial achievement of spatial learning and memory therapy. Within a research project aimed at developing novel CAAs selective for certain isoforms, three series of indole-based derivatives were investigated. Enzyme activation assay on human CA I, II, VA, and VII isoforms revealed several effective micromolar activators, with promising selectivity profiles towards the brain-associated cytosolic isoform hCA VII. Molecular modelling studies suggested a theoretical model of the complex between hCA VII and the new activators and provide a possible explanation for their modulating as well as selectivity properties. Preliminary biological evaluations demonstrated that one of the most potent CAA 7 is not cytotoxic and is able to increase the release of the brain-derived neurotrophic factor (BDNF) from human microglial cells, highlighting its possible application in the treatment of CNS-related disorders.


Assuntos
Anidrases Carbônicas/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Indóis/farmacologia , Isoenzimas/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Anidrases Carbônicas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Ativadores de Enzimas/química , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Indóis/química , Isoenzimas/metabolismo , Microglia/citologia , Microglia/efeitos dos fármacos , Modelos Moleculares , Espectroscopia de Prótons por Ressonância Magnética , Especificidade por Substrato
6.
Eur J Med Chem ; 220: 113490, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33975138

RESUMO

Carbonic Anhydrases (CAs) are pharmaceutically relevant targets for the treatment of several disease conditions. The ubiquitous localization of these enzymes and the high homology shared by the different isoforms represent substantial impediments for the discovery of potential drugs devoid of off-target side effects. As a consequence, substantial efforts are still needed to allow for the full realization of the pharmacological potential of CA modulators. In this contribution, starting from our previous studies, we describe the synthesis of a set of new bicyclic tetrahydroindazoles featuring a secondary sulfonamide. Biological evaluation of the inhibitory activity against the hCA I, II, IV, and IX isoforms allowed drawing a structure-activity relationship profile that was rationalized through theoretical studies. This allowed dissecting the new molecules into the single portions influencing the zinc chelation properties and the selectivity profile thereby offering a new platform for the discovery of new isotype selective CA inhibitors.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Indazóis/farmacologia , Sulfonamidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Humanos , Indazóis/química , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química
7.
Eur J Pharm Sci ; 156: 105594, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33059042

RESUMO

DNA Topoisomerases (Topos) are ubiquitous nuclear enzymes involved in regulating the topological state of DNA and, in eukaryotic organisms, Topos can be classified into two structurally and functionally different main classes: TopoI and TopoII. Both these enzymes proved to be excellent targets of clinically significant classes of anticancer drugs. Actually, TopoI or II inhibitors show considerable wide spectrum antitumor activities, an important feature to be included in many chemotherapeutic protocols. Despite their clinical efficacy, the use of inhibitors targeting only one of the two enzymes can increase the levels of the other one, favouring the onset of unwanted phenomena such as drug resistance. Therefore, targeting both TopoI and TopoII can reduce the probability of developing resistance, as well as side effects thanks to the use of lower doses, given the synergistic effect of the dual activity. Moreover, since drug resistance is also due to DNA repair systems such as tyrosyl-DNA phosphodiesterases I and II, inhibiting Topoisomerases concomitantly to Tyrosyl-DNA phosphodiesterase enzymes could allow more efficient and safe drugs. This review represents an update of previous works reporting about dual TopoI and TopoII inhibitors, but also an overview of the new strategy regarding the development of derivatives able to simultaneously inhibit Topo and TDP enzymes, with particular attention to structure-affinity relationship studies. The newly collected derivatives are described focusing attention on their chemical structures and their biological profiles. The final aim is to highlight the structural requirements necessary for the development of potent multiple modulators of these targets, thus providing new potential antitumor agents for the clinical usage.


Assuntos
Antineoplásicos , DNA Topoisomerases Tipo I , Antineoplásicos/farmacologia , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA , Diester Fosfórico Hidrolases , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase II/farmacologia
8.
Elife ; 92020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32662768

RESUMO

The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+ transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea. The pathway underpins a mechanism by which charged H+ and neutral NH3 are carried separately across the membrane after NH4+ deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process.


Assuntos
Amônia/metabolismo , Compostos de Amônio/metabolismo , Escherichia coli/metabolismo , Transporte de Íons , Nitrosomonas europaea/metabolismo
9.
Mol Med Rep ; 22(3): 1910-1920, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32583004

RESUMO

To the best of our knowledge, the vertebrate apolipoprotein L (APOL) family has not previously been ascribed to any definite pathophysiological function, although the conserved BH3 protein domain suggests a role in programmed cell death or an interference with mitochondrial processes. In the present study, the human APOL1 was expressed in the yeast Saccharomyces cerevisiae in order to determine the molecular action of APOL1. APOL1 inhibited cell proliferation in a non­fermentable carbon source, such as glycerol, while it had no effect on proliferation in fermentable carbon sources, such as galactose. APOL1, expressed in yeast, is localized in the mitochondrial fraction, as determined via western blotting. APOL1 induced a loss of mitochondrial function, demonstrated by a loss of respiratory index, and mitochondrial membrane potential. Green fluorescent protein tagging of mitochondrial protein revealed that APOL1 was associated with abnormal mitochondrial and lysosomal morphologies, observed by a loss of the normal mitochondrial tubular network. Thus, the results of the present study suggest that APOL1 could be a physiological regulator of mitochondrial function.


Assuntos
Apolipoproteína L1/genética , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Apolipoproteína L1/metabolismo , Fermentação , Glicerol/metabolismo , Humanos , Potencial da Membrana Mitocondrial , Viabilidade Microbiana , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
10.
Eur J Pharm Sci ; 149: 105337, 2020 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-32311457

RESUMO

The oncogene KRAS is involved in the pathogenesis of many tumors such as pancreatic, lung and colorectal cancers, thereby representing a relevant target for the treatment of these diseases. The KRAS P1 promoter contains a nuclease hypersensitive, guanine-rich sequence able to fold into a G-quadruplex motif (G4). The stabilization of this G4 structure by small molecules is emerging as a feasible approach to downregulate KRAS expression. Here, a set of novel stabilizing molecules was identified through a virtual screening campaign on the NMR structure of the 22-mer KRAS G4. The most promising hits were then submitted to structure-activity relationships studies which allowed improving their binding affinity and selectivity over double helix DNA and different G4 topologies. The best derivative (19) underwent fluorescence titration experiments and further computational studies to disclose its binding mechanism to KRAS G4. Finally, biological assays showed that this compound is capable to reduce the viability of colorectal cancer cells in which mutated KRAS acts as a driver oncogene. Thus, 19 might represent the prototype of a new class of drugs for the treatment of tumors that, expressing mutated forms of KRAS, are refractory to current therapeutic regimens.

11.
PLoS Genet ; 16(2): e1008634, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32069286

RESUMO

The dimorphic transition from the yeast to the filamentous form of growth allows cells to explore their environment for more suitable niches and is often crucial for the virulence of pathogenic fungi. In contrast to their Mep1/3 paralogues, fungal Mep2-type ammonium transport proteins of the conserved Mep-Amt-Rh family have been assigned an additional receptor role required to trigger the filamentation signal in response to ammonium scarcity. Here, genetic, kinetic and structure-function analyses were used to shed light on the poorly characterized signaling role of Saccharomyces cerevisiae Mep2. We show that Mep2 variants lacking the C-terminal tail conserve the ability to induce filamentation, revealing that signaling can proceed in the absence of exclusive binding of a putative partner to the largest cytosolic domain of the protein. Our data support that filamentation signaling requires the conformational changes accompanying substrate translocation through the pore crossing the hydrophobic core of Mep2. pHluorin reporter assays show that the transport activity of Mep2 and of non-signaling Mep1 differently affect yeast cytosolic pH in vivo, and that the unique pore variant Mep2H194E, with apparent uncoupling of transport and signaling functions, acquires increased ability of acidification. Functional characterization in Xenopus oocytes reveals that Mep2 mediates electroneutral substrate translocation while Mep1 performs electrogenic transport. Our findings highlight that the Mep2-dependent filamentation induction is connected to its specific transport mechanism, suggesting a role of pH in signal mediation. Finally, we show that the signaling process is conserved for the Mep2 protein from the human pathogen Candida albicans.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Hifas/metabolismo , Domínios Proteicos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Compostos de Amônio/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Genes Reporter/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Mutação , Oócitos , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/fisiologia , Xenopus
12.
iScience ; 20: 415-433, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31622882

RESUMO

To adjust cell growth and metabolism according to environmental conditions, the conserved TORC1 signaling network controls autophagy, protein synthesis, and turnover. Here, we dissected the signals controlling phosphorylation and activity of the TORC1-effector kinase Npr1, involved in tuning the plasma membrane permeability to nitrogen sources. By evaluating a role of pH as a signal, we show that, although a transient cytosolic acidification accompanies nitrogen source entry and is correlated to a rapid TORC1-dependent phosphorylation of Npr1, a pH drop is not a prerequisite for TORC1 activation. We show that the Gtr1/Gtr2 and Pib2 regulators of TORC1 both independently and differently contribute to regulate Npr1 phosphorylation and activity. Finally, our data reveal that Npr1 mediates nitrogen-dependent phosphorylation of Pib2, as well as a Pib2-dependent inhibition of TORC1. This work highlights a feedback control loop likely enabling efficient downregulation and faster re-activation of TORC1 in response to a novel stimulating signal.

13.
ACS Med Chem Lett ; 10(4): 457-462, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30996779

RESUMO

Protein kinases dysregulation is extremely common in cancer cells, and the development of new agents able to simultaneously target multiple kinase pathways involved in angiogenesis and tumor growth may offer several advantages in the treatment of cancer. Herein we report the discovery of new pyridothiopyranopyrimidine derivatives (2-4) showing high potencies in VEGFR-2 KDR inhibition as well as antiproliferative effect on a panel of human tumor cell lines. Investigation on the selectivity profile of the representative 2-anilino-substituted compounds 3b, 3i, and 3j revealed a multiplicity of kinase targets that should account for the potent antiproliferative effect produced by these pyridothiopyranopyrimidine derivatives.

14.
J Biol Chem ; 294(18): 7231-7244, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30872403

RESUMO

Ethanol causes dysregulated muscle protein homeostasis while simultaneously causing hepatocyte injury. Because hepatocytes are the primary site for physiological disposal of ammonia, a cytotoxic cellular metabolite generated during a number of metabolic processes, we determined whether hyperammonemia aggravates ethanol-induced muscle loss. Differentiated murine C2C12 myotubes, skeletal muscle from pair-fed or ethanol-treated mice, and human patients with alcoholic cirrhosis and healthy controls were used to quantify protein synthesis, mammalian target of rapamycin complex 1 (mTORC1) signaling, and autophagy markers. Alcohol-metabolizing enzyme expression and activity in mouse muscle and myotubes and ureagenesis in hepatocytes were quantified. Expression and regulation of the ammonia transporters, RhBG and RhCG, were quantified by real-time PCR, immunoblots, reporter assays, biotin-tagged promoter pulldown with proteomics, and loss-of-function studies. Alcohol and aldehyde dehydrogenases were expressed and active in myotubes. Ethanol exposure impaired hepatocyte ureagenesis, induced muscle RhBG expression, and elevated muscle ammonia concentrations. Simultaneous ethanol and ammonia treatment impaired protein synthesis and mTORC1 signaling and increased autophagy with a consequent decreased myotube diameter to a greater extent than either treatment alone. Ethanol treatment and withdrawal followed by ammonia exposure resulted in greater impairment in muscle signaling and protein synthesis than ammonia treatment in ethanol-naive myotubes. Of the three transcription factors that were bound to the RhBG promoter in response to ethanol and ammonia, DR1/NC2 indirectly regulated transcription of RhBG during ethanol and ammonia treatment. Direct effects of ethanol were synergistic with increased ammonia uptake in causing dysregulated skeletal muscle proteostasis and signaling perturbations with a more severe sarcopenic phenotype.


Assuntos
Amônia/metabolismo , Etanol/farmacologia , Músculo Esquelético/efeitos dos fármacos , Animais , Linhagem Celular , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hiperamonemia/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Proteostase/efeitos dos fármacos , Transdução de Sinais , Ureia/metabolismo
15.
Eur J Med Chem ; 165: 46-58, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30660826

RESUMO

New benzothiopyranoindoles (5a-l) and pyridothiopyranoindoles (5m-t), featuring different combinations of substituents (H, Cl, OCH3) at R2-R4 positions and protonatable R1-dialkylaminoalkyl chains, were synthesized and biologically assayed on three human tumor cell lines, showing significant antiproliferative activity (GI50 values spanning from 0.31 to 6.93 µM) and pro-apoptotic effect. Linear flow dichroism experiments indicate the ability of both chromophores to form a molecular complex with DNA, following an intercalative mode of binding. All compounds displayed a moderate ability to inhibit the relaxation activity of both topoisomerases I and II, reasonably correlated to their intercalative capacities. Cleavable assay performed with topoisomerase I revealed a significant poisoning effect for compounds 5g, 5h, 5s, and 5t. A theoretical model provided by hydrated docking calculations clarified the role of the R1-R4 substituents on the topoisomerase I poison activity, revealing a crucial role of the R2-OCH3 group.


Assuntos
Antineoplásicos/síntese química , Indóis/farmacologia , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase II/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Humanos , Indóis/síntese química , Indóis/química , Indóis/metabolismo , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase II/síntese química
16.
J Med Chem ; 61(13): 5765-5770, 2018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-29912561

RESUMO

As a part of our efforts to expand chemical diversity in the carbonic anhydrases inhibitors (CAIs), three small series of polyheterocyclic compounds (4-6) featuring the primary benzenesulfonamide moiety linked to bi/tricyclic scaffolds were investigated. Highly effective inhibitors against the target tumor-associated hCA IX (low nanomolar/subnanomolar potency levels) showing significant functional selectivity profile toward hCA I, II, and IV isozymes were identified. Molecular docking studies clarified the reasons behind the activity and selectivity of the new compounds.


Assuntos
Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica IX/antagonistas & inibidores , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/farmacologia , Desenho de Fármacos , Sulfonamidas/química , Sulfonamidas/farmacologia , Anidrase Carbônica II/química , Anidrase Carbônica IX/química , Domínio Catalítico , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Modelos Moleculares , Benzenossulfonamidas
17.
Eur J Med Chem ; 150: 446-456, 2018 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-29547832

RESUMO

Inhibition of angiogenesis via blocking vascular endothelial growth factor receptor (VEGFR) signaling pathway emerged as an established approach in anticancer therapy. So far, many monoclonal antibodies and ATP-competitive small molecule inhibitors have been clinically validated and approved. In this study, structure-activity relationships (SAR) within the 2-phenylamino-substituted benzothiopyrano[4,3-d]pyrimidine class of kinase inhibitors were further refined by the synthesis and biological evaluation of new compounds 1-21 featuring different substitution patterns on the pendant phenyl moiety, combined with H, OCH3, or Cl at 8-position. Most compounds showed a promising human kinase insert domain receptor (KDR) inhibition profile, with IC50 values in the submicromolar/low micromolar range, and promising antiproliferative activity on human umbilical vein endothelial cells (HUVECs) as well as on a panel of three human tumor cell lines. The angio-kinase selectivity profile was assessed for the most promising compound 16 against a set of six human kinases. Finally, computational studies allowed clarifying at molecular level the interaction pattern established by the compounds with KDR, highlighting key stable cation-π interactions, and thus providing the basis for further designing novel inhibitors.


Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piranos/farmacologia , Pirimidinas/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Compostos de Anilina/química , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Piranos/síntese química , Piranos/química , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
18.
Sci Rep ; 7: 44602, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-28303961

RESUMO

Two structurally and functionally distinct mammalian TOR complexes control cell growth and metabolism in physiological and pathological contexts including cancer. Upregulated glutaminolysis is part of the metabolic reprogramming occurring in cancer, providing fuels for growth but also liberating ammonium, a potent neurotoxic waste product. Here, we identify ammonium as a novel dose-dependent signal mediating rapid mTORC2 activation and further regulating mTORC1. We show that ammonium induces rapid RICTOR-dependent phosphorylation of AKT-S473, a process requiring the PI3K pathway and further involving the Src-family kinase YES1, the FAK kinase and the ITGß1 integrin. Release of calcium from the endoplasmic reticulum store triggers rapid mTORC2 activation, similar to ammonium-induced activation, the latter being conversely prevented by calcium chelation.Moreover, in analogy to growth factors, ammonium triggers the AKT-dependent phosphoinhibition of the TSC complex and of PRAS40, two negative regulators of mTORC1. Consistent with mTORC1 stimulation, ammonium induces the inhibitory phosphorylation of 4EBP1, a negative regulator of protein biogenesis. Ammonium however dually impacts on the phosphorylation of p70S6K1 triggering a transient AKT-independent decrease in the phosphorylation of this second mTORC1 readout. Finally, we reveal ammonium as a dose-dependent stimulator of proliferation. This study underscores an mTORC2 and mTORC1 response to the so-called ammonium waste.


Assuntos
Compostos de Amônio/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Transdução de Sinais , Cálcio/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina beta1/metabolismo , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-yes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
19.
J Med Chem ; 59(13): 6547-52, 2016 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-27305384

RESUMO

Three novel series of 1,2-benzisothiazole derivatives have been developed as inhibitors of carbonic anhydrase isoform IX. Compounds 5c and 5j, tested in vitro on the human colon cell line HT-29, blocked the growth of cells cultured under chemically induced hypoxic conditions, displaying a specific activity against cancer cells characterized by CAIX up-regulation. Moreover, a synergistic activity of 5c with SN-38 (the active metabolite of irinotecan) and 5-fluorouracil on cell proliferation under hypoxic conditions was demonstrated.


Assuntos
Antineoplásicos/farmacologia , Anidrase Carbônica IX/antagonistas & inibidores , Inibidores da Anidrase Carbônica/farmacologia , Tiazóis/farmacologia , Antígenos de Neoplasias/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HT29 , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/química
20.
Bioorg Med Chem ; 24(4): 921-7, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26796953

RESUMO

Three series of polycyclic compounds possessing either primary sulfonamide or carboxylic acid moieties as zinc-binding groups were investigated as inhibitors of four physiologically relevant CA isoforms, the cytosolic hCA I and II, as well as the transmembrane hCA IX and XII. Most of the new sulfonamides reported here showed excellent inhibitory effects against isoforms hCA II, IX and XII, but no highly isoform-selective inhibition profiles. On the other hand, the carboxylates selectively inhibited hCA IX (KIs ranging between 40.8 and 92.7nM) without inhibiting significantly the other isoforms. Sulfonamides/carboxylates incorporating polycyclic ring systems such as benzothiopyranopyrimidine, pyridothiopyranopyrimidine or dihydrobenzothiopyrano[4,3-c]pyrazole may be considered as interesting candidates for exploring the design of isoform-selective CAIs with various pharmacologic applications.


Assuntos
Anidrase Carbônica I/química , Inibidores da Anidrase Carbônica/síntese química , Ácidos Carboxílicos/química , Pirazóis/química , Pirimidinas/química , Sulfonamidas/síntese química , Inibidores da Anidrase Carbônica/química , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Cinética , Ligação Proteica , Relação Estrutura-Atividade , Sulfonamidas/química
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