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1.
Methods Mol Biol ; 2312: 237-251, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228294

RESUMO

Chemical control of protein localization is a powerful approach for manipulating mammalian cellular processes. Self-localizing ligand-induced protein translocation (SLIPT) is an emerging platform that enables control of protein localization in living mammalian cells using synthetic self-localizing ligands (SLs). We recently established a chemogenetic SLIPT system, in which any protein of interest fused to an engineered variant of Escherichia coli dihydrofolate reductase, DHFRiK6, can be rapidly and specifically translocated from the cytoplasm to the inner leaflet of the plasma membrane (PM) using a trimethoprim (TMP)-based PM-targeting SL, mDcTMP. The mDcTMP-mediated PM recruitment of DHFRiK6-fusion proteins can be efficiently returned to the cytoplasm by subsequent addition of free TMP, enabling temporal and reversible control over the protein localization. Here we describe the use of this mDcTMP/DHFRiK6-based SLIPT system for inducing (1) reversible protein translocation and (2) synthetic activation of the Raf/ERK pathway. This system provides a simple and versatile tool in mammalian synthetic biology for temporally manipulating various signaling molecules and pathways at the PM.


Assuntos
Engenharia Celular , Proteínas de Escherichia coli/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas Genéticas , Biologia Sintética , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/farmacologia , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tetra-Hidrofolato Desidrogenase/metabolismo , Quinases raf/metabolismo
2.
Mol Biochem Parasitol ; 244: 111393, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34197864

RESUMO

Mitochondrial protein import depends on heterooligomeric translocases in the outer and inner membranes. Using import substrates consisting of various lengths of the N-terminal part of mitochondrial dihydrolipoamide dehydrogenase (LDH) fused to dihydrofolate reductase we present an in vivo analysis showing that in Trypanosoma brucei at least 96 aa of mature LDH are required to efficiently produce an import intermediate that spans both translocases. This is different to yeast, where around 50 aa are sufficient to achieve the same task and likely reflects the different arrangement and architecture of the trypanosomal mitochondrial translocases. Furthermore, we show that formation of the stuck import intermediate leads to a strong growth inhibition suggesting that, depending on the length of the LDH, the import channels in the translocases are quantitatively blocked.


Assuntos
Di-Hidrolipoamida Desidrogenase/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Sistemas de Translocação de Proteínas/genética , Proteínas de Protozoários/genética , Tetra-Hidrofolato Desidrogenase/genética , Trypanosoma brucei brucei/genética , Sequência de Aminoácidos , Di-Hidrolipoamida Desidrogenase/metabolismo , Regulação da Expressão Gênica , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Sistemas de Translocação de Proteínas/metabolismo , Transporte Proteico , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade da Espécie , Tetra-Hidrofolato Desidrogenase/metabolismo , Trypanosoma brucei brucei/enzimologia
3.
J Chem Phys ; 154(19): 195103, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34240890

RESUMO

Interactions among ions and their specific interactions with macromolecular solutes are known to play a central role in biomolecular stability. However, similar effects in the conformational stability of protein loops that play functional roles, such as binding ligands, proteins, and DNA/RNA molecules, remain relatively unexplored. A well-characterized enzyme that has such a functional loop is Escherichia coli dihydrofolate reductase (ecDHFR), whose so-called M20 loop has been observed in three ordered conformations in crystal structures. To explore how solution ionic strengths may affect the M20 loop conformation, we proposed a reaction coordinate that could quantitatively describe the loop conformation and used it to classify the loop conformations in representative ecDHFR x-ray structures crystallized in varying ionic strengths. The Protein Data Bank survey indicates that at ionic strengths (I) below the intracellular ion concentration-derived ionic strength in E. coli (I ≤ 0.237M), the ecDHFR M20 loop tends to adopt open/closed conformations, and rarely an occluded loop state, but when I is >0.237M, the loop tends to adopt closed/occluded conformations. Distance-dependent electrostatic potentials around the most mobile M20 loop region from molecular dynamics simulations of ecDHFR in equilibrated CaCl2 solutions of varying ionic strengths show that high ionic strengths (I = 0.75/1.5M) can preferentially stabilize the loop in closed/occluded conformations. These results nicely correlate with conformations derived from ecDHFR structures crystallized in varying ionic strengths. Altogether, our results suggest caution in linking M20 loop conformations derived from crystal structures solved at ionic strengths beyond that tolerated by E. coli to the ecDHFR function.


Assuntos
Cloreto de Cálcio/química , Escherichia coli/enzimologia , Tetra-Hidrofolato Desidrogenase/química , Simulação de Dinâmica Molecular , Concentração Osmolar , Conformação Proteica , Soluções , Tetra-Hidrofolato Desidrogenase/metabolismo
4.
Nat Commun ; 12(1): 3486, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108489

RESUMO

The metabolome represents a complex network of biological events that reflects the physiologic state of the organism in health and disease. Additionally, specific metabolites and metabolic signaling pathways have been shown to modulate animal ageing, but whether there are convergent mechanisms uniting these processes remains elusive. Here, we used high resolution mass spectrometry to obtain the metabolomic profiles of canonical longevity pathways in C. elegans to identify metabolites regulating life span. By leveraging the metabolomic profiles across pathways, we found that one carbon metabolism and the folate cycle are pervasively regulated in common. We observed similar changes in long-lived mouse models of reduced insulin/IGF signaling. Genetic manipulation of pathway enzymes and supplementation with one carbon metabolites in C. elegans reveal that regulation of the folate cycle represents a shared causal mechanism of longevity and proteoprotection. Such interventions impact the methionine cycle, and reveal methionine restriction as an underlying mechanism. This comparative approach reveals key metabolic nodes to enhance healthy ageing.


Assuntos
Carbono/metabolismo , Ácido Fólico/metabolismo , Longevidade/fisiologia , Redes e Vias Metabólicas , Animais , Caenorhabditis elegans , Insulina/metabolismo , Longevidade/genética , Redes e Vias Metabólicas/genética , Metaboloma , Metionina/metabolismo , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Peptídeos/metabolismo , Transdução de Sinais , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Tetra-Hidrofolatos/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismo
5.
J Chem Inf Model ; 61(6): 2537-2541, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34138546

RESUMO

Drug resistance impacts the effectiveness of many new therapeutics. Mutations in the therapeutic target confer resistance; however, deciphering which mutations, often remote from the enzyme active site, drive resistance is challenging. In a series of Pneumocystis jirovecii dihydrofolate reductase variants, we elucidate which interactions are key bellwethers to confer resistance to trimethoprim using homology modeling, molecular dynamics, and machine learning. Six molecular features involving mainly residues that did not vary were the best indicators of resistance.


Assuntos
Farmacorresistência Fúngica , Pneumocystis carinii , Aprendizado de Máquina , Simulação de Dinâmica Molecular , Pneumocystis carinii/efeitos dos fármacos , Pneumocystis carinii/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
6.
PLoS Negl Trop Dis ; 15(4): e0009377, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33905412

RESUMO

Our understanding of folate metabolism in Leishmania has greatly benefited from studies of resistance to the inhibitor methotrexate (MTX). Folates are reduced in Leishmania by the bifunctional dihydrofolate reductase thymidylate synthase (DHFR-TS) and by pteridine reductase (PTR1). To further our understanding of folate metabolism in Leishmania, a Cos-seq genome-wide gain of function screen was performed against MTX and against the two thymidylate synthase (TS) inhibitors 5-fluorouracil and pemetrexed. The screen revealed DHFR-TS and PTR1 but also the nucleoside transporter NT1 and one hypothetical gene derived from chromosome 31. For MTX, the concentration of folate in the culture medium affected the enrichment pattern for genes retrieved by Cos-seq. We generated a L. infantum DHFR-TS null mutant that was thymidine auxotroph, a phenotype that could be rescued by the addition of thymidine or by transfection of the flavin dependent bacterial TS gene ThyX. In these DHFR-TS null mutants it was impossible to obtain a chromosomal null mutant of PTR1 except if DHFR-TS or PTR1 were provided episomally. The transfection of ThyX however did not allow the elimination of PTR1 in a DHFR-TS null mutant. Leishmania can survive without copies of either DHFR-TS or PTR1 but not without both. Provided that our results observed with the insect stage parasites are also replicated with intracellular parasites, it would suggest that antifolate therapy in Leishmania would only work if both DHFR-TS and PTR1 would be targeted simultaneously.


Assuntos
Deleção de Genes , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/genética , Metotrexato/farmacologia , Complexos Multienzimáticos/genética , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética , Animais , DNA de Protozoário/genética , DNA Recombinante/genética , Resistência a Medicamentos , Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Leishmania infantum/enzimologia , Metotrexato/metabolismo , Complexos Multienzimáticos/metabolismo , Fenótipo , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/metabolismo , Transfecção
7.
Biochem Mol Biol Educ ; 49(4): 560-569, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33830617

RESUMO

Student feedback from an undergraduate biochemistry lab course suggested the use of visibly traceable proteins may assist learning. Based on this feedback, we used guided inquiry lab exercises where students developed and characterized a suite of fluorescent protein-dihydrofolate reductase (DHFR) fusions as tools for a biochemistry teaching lab. In contrast to the unfused versions, members of this suite are well-expressed, soluble, visible, highly stable, and easily characterized. The color of mCherry and EGFP fluorescent fusions with microbial DHFR allows students to visibly track their target protein from expression through purification under ambient light, while fusions with BFP are visible under UV-light. Fusions were made to both wild-type and kinetically enhanced DHFR variants. Importantly, we found that fluorescent protein fusions with DHFR did not kinetically interfere as the KM and kcat values were not remarkably altered from the unfused variant. With these fusions, students can easily measure kinetic parameters under steady-state conditions with readily available substrate and common laboratory spectrophotometers. Additionally, students also determined IC50 values of trimethoprim for DHFR. These exercises can be completed in a series of up to six lab periods and we have included the protocols for instructors who wish undertake a similar series of experiments in their biochemistry teaching labs. Using these visible fusion enzymes with subsequent students, we observed potential learning gains on a course assessment and received positive student feedback. We suggest that the often over-looked element of visual cues in a biochemistry lab may be an exploitable component of learning.


Assuntos
Bioquímica/educação , Pesquisa Biomédica/educação , Fluorescência , Aprendizagem , Proteínas Recombinantes de Fusão/metabolismo , Estudantes/psicologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Humanos , Laboratórios , Proteínas Recombinantes de Fusão/análise , Projetos de Pesquisa , Ensino , Tetra-Hidrofolato Desidrogenase/análise , Universidades
8.
J Vis Exp ; (170)2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33900288

RESUMO

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a chemically-induced protein dimerization system to create condensates on telomeres. The chemical dimerizer consists of two linked ligands that can each bind to a protein: Halo ligand to Halo-enzyme and trimethoprim (TMP) to E. coli dihydrofolate reductase (eDHFR), respectively. Fusion of Halo enzyme to a telomere protein anchors dimerizers to telomeres through covalent Halo ligand-enzyme binding. Binding of TMP to eDHFR recruits eDHFR-fused phase separating proteins to telomeres and induces condensate formation. Because TMP-eDHFR interaction is non-covalent, condensation can be reversed by using excess free TMP to compete with the dimerizer for eDHFR binding. An example of inducing promyelocytic leukemia (PML) nuclear body formation on telomeres and determining condensate growth, dissolution, localization and composition is shown. This method can be easily adapted to induce condensates at other genomic locations by fusing Halo to a protein that directly binds to the local chromatin or to dCas9 that is targeted to the genomic locus with a guide RNA. By offering the temporal resolution required for single cell live imaging while maintaining phase separation in a population of cells for biochemical assays, this method is suitable for probing both the formation and function of chromatin-associated condensates.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Multimerização Proteica , Telômero/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Trimetoprima/metabolismo , Proteínas de Escherichia coli/química , Humanos , Ligantes , Ligação Proteica , Tetra-Hidrofolato Desidrogenase/química , Trimetoprima/química
9.
Acta Crystallogr D Struct Biol ; 77(Pt 3): 293-299, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33645533

RESUMO

Methotrexate (MTX) is an anticancer and anti-rheumatoid arthritis drug that is considered to block nucleotide synthesis and the cell cycle mainly by inhibiting the activity of dihydrofolate reductase (DHFR). Using affinity-matrix technology and X-ray analysis, the present study shows that MTX also interacts with macrophage migration inhibitory factor (MIF). Fragment molecular-orbital calculations quantified the interaction between MTX and MIF based on the structure of the complex and revealed the amino acids that are effective in the interaction of MTX and MIF. It should be possible to design new small-molecule compounds that have strong inhibitory activity towards both MIF and DHFR by structure-based drug discovery.


Assuntos
Antimetabólitos Antineoplásicos/química , Antirreumáticos/química , Oxirredutases Intramoleculares/química , Fatores Inibidores da Migração de Macrófagos/química , Metotrexato/química , Antimetabólitos Antineoplásicos/metabolismo , Antirreumáticos/metabolismo , Cristalografia por Raios X , Humanos , Metotrexato/metabolismo , Modelos Moleculares , Tetra-Hidrofolato Desidrogenase/metabolismo
10.
Cancer Lett ; 503: 129-137, 2021 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-33545223

RESUMO

Brain tumors are a heterogeneous group of benign and malignant tumors arising from the brain parenchyma and its surrounding structures, with in general a poor clinical outcome due to high recurrence. One of the underlying causes for this somber prognostic is the presence of brain tumor initiating cells (BTIC) endowed with self-renewal potential, multi-lineage differentiation and resistance to treatment. One promising therapeutic avenue for brain tumors is targeting BTIC self-renewal potential and forcing their differentiation. A compelling candidate is one-carbon metabolism shown to play a key role in maintaining stem cell self-renewal in several lineages. Here, we focus on dihydrofolate reductase (DHFR), a key enzyme in one-carbon metabolism, and demonstrate this enzyme's overexpression in several human brain tumors and its expression in human BTIC. We show that DHFR inhibition, either by Methotrexate (MTX) or EphB activation with synthetic ligands, reduces the tumorigenic potential of 4 human BTIC lines, by reducing their self-renewal capacities both in vitro and in a cerebral organoid glioma (GLICO) model. Our data indicate that driving BTIC differentiation by inhibiting DHFR may provide a new therapeutic approach to treating highly refractory aggressive tumors.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Metotrexato/farmacologia , Células-Tronco Neoplásicas/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Regulação para Cima/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/tratamento farmacológico , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/patologia , Prognóstico
11.
J Enzyme Inhib Med Chem ; 36(1): 198-206, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33530764

RESUMO

In various malaria-endemic regions, the appearance of resistance has precluded the use of pyrimidine-based antifolate drugs. Here, a three-step fragment screening was used to identify new non-pyrimidine Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors. Starting from a 1163-fragment commercial library, a two-step differential scanning fluorimetry screen identified 75 primary fragment hits. Subsequent enzyme inhibition assay identified 11 fragments displaying IC50 in the 28-695 µM range and selectivity for PfDHFR. In addition to the known pyrimidine, three new anti-PfDHFR chemotypes were identified. Fragments from each chemotype were successfully co-crystallized with PfDHFR, revealing a binding in the active site, in the vicinity of catalytic residues, which was confirmed by molecular docking on all fragment hits. Finally, comparison with similar non-hit fragments provides preliminary input on available growth vectors for future drug development.


Assuntos
Antimaláricos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Antimaláricos/síntese química , Antimaláricos/química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Plasmodium falciparum/enzimologia , Proguanil/síntese química , Proguanil/química , Proguanil/farmacologia , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Pirimetamina/síntese química , Pirimetamina/química , Pirimetamina/farmacologia , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/isolamento & purificação , Tetra-Hidrofolato Desidrogenase/metabolismo , Triazinas/síntese química , Triazinas/química , Triazinas/farmacologia
12.
J Phys Chem B ; 125(5): 1369-1377, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33522797

RESUMO

Calculation of temperature-dependent kinetic isotope effects (KIE) in enzymes presents a significant theoretical challenge. Additionally, it is not trivial to identify enzymes with available experimental accurate intrinsic KIEs in a range of temperatures. In the current work, we present a theoretical study of KIEs in the primitive R67 dihydrofolate reductase (DHFR) enzyme and compare with experimental work. The advantage of R67 DHFR is its significantly lower kinetic complexity compared to more evolved DHFR isoforms. We employ mass-perturbation-based path-integral simulations in conjunction with umbrella sampling and a hybrid quantum mechanics-molecular mechanics Hamiltonian. We obtain temperature-dependent KIEs in good agreement with experiments and ascribe the temperature-dependent KIEs primarily to zero-point energy effects. The active site in the primitive enzyme is found to be poorly preorganized, which allows excessive water access to the active site and results in loosely bound reacting ligands.


Assuntos
Isótopos , Tetra-Hidrofolato Desidrogenase , Cinética , Simulação de Dinâmica Molecular , Temperatura , Tetra-Hidrofolato Desidrogenase/metabolismo
13.
J Enzyme Inhib Med Chem ; 36(1): 295-306, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33404277

RESUMO

Five series of novel carbazole derivatives containing an aminoguanidine, dihydrotriazine, thiosemicarbazide, semicarbazide or isonicotinic moiety were designed, synthesised and evaluated for their antimicrobial activities. Most of the compounds exhibited potent inhibitory activities towards different bacterial strains (including one multidrug-resistant clinical isolate) and one fungal strain with minimum inhibitory concentrations (MICs) between 0.5 and 16 µg/ml. Compounds 8f and 9d showed the most potent inhibitory activities (MICs of 0.5-2 µg/ml). Furthermore, compounds 8b, 8d, 8f, 8k, 9b and 9e with antimicrobial activities were not cytotoxic to human gastric cancer cell lines (SGC-7901 and AGS) or a normal human liver cell line (L-02). Structure-activity relationship analyses and docking studies implicated the dihydrotriazine group in increasing the antimicrobial potency and reducing the toxicity of the carbazole compounds. In vitro enzyme activity assays suggested that compound 8f binding to dihydrofolate reductase might account for the antimicrobial effect.


Assuntos
Anti-Infecciosos/síntese química , Proteínas de Bactérias/química , Carbazóis/síntese química , Inibidores Enzimáticos/síntese química , Escherichia coli/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/química , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida albicans/crescimento & desenvolvimento , Carbazóis/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Guanidinas/química , Hepatócitos/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Ácidos Isonicotínicos/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/enzimologia , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Semicarbazidas/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Staphylococcus aureus/crescimento & desenvolvimento , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/enzimologia , Streptococcus mutans/crescimento & desenvolvimento , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/metabolismo , Triazinas/química
14.
Diagn Microbiol Infect Dis ; 99(4): 115296, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33387894

RESUMO

The objective of this pilot study was to examine the activity of iclaprim, a diaminopyrimidine dihydrofolate reducatase inhibitor, in an in vitro infection model of infection with Toxoplasma gondii. Toxoplasma growth was assessed by enzyme linked immunoassay (ELISA) performed directly on the fixed cultures using a peroxidase labeled monoclonal antibody directed against the SAG-l surface protein of T. gondii. For each well, the results were expressed as optical density (OD) values. Iclaprim inhibited T. gondii growth at concentrations between 0.1 and 10 mg/L; the IC50 was estimated at 0.26 mg/L (95% confidence interval 0.22-0.33). Iclaprim was about 10 times more active than trimethoprim, which had an IC50 of 2.3 mg/L. Iclaprim demonstrated synergistic effects at concentrations of 0.02, 0.05 and 0.1 mg/L when combined with subinhibitory concentrations of sulfamethoxazole (0.1 or 0.02 mg/L). These results show that iclaprim is a potent inhibitor of T. gondii growth in vitro. In addition, iclaprim exhibited synergy in vitro when tested in the presence of sulfamethoxazole. Iclaprim should be further investigated as an agent for the treatment or prophylaxis of toxoplasmosis.


Assuntos
Fibroblastos/parasitologia , Antagonistas do Ácido Fólico/farmacologia , Pirimidinas/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Toxoplasma/efeitos dos fármacos , Toxoplasma/enzimologia , Animais , Linhagem Celular , Projetos Piloto
15.
Curr Med Chem ; 28(5): 910-939, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-31622199

RESUMO

BACKGROUND: Dihydrofolate reductase (DHFR) has been known for decades as a molecular target for antibacterial, antifungal and anti-malarial treatments. This enzyme is becoming increasingly important in the design of new anticancer drugs, which is confirmed by numerous studies including modelling, synthesis and in vitro biological research. This review aims to present and discuss some remarkable recent advances in the research of new DHFR inhibitors with potential anticancer activity. METHODS: The scientific literature of the last decade on the different types of DHFR inhibitors has been searched. The studies on design, synthesis and investigation structure-activity relationships were summarized and divided into several subsections depending on the leading molecule and its structural modification. Various methods of synthesis, potential anticancer activity and possible practical applications as DHFR inhibitors of new chemical compounds were described and discussed. RESULTS: This review presents the current state of knowledge on the modification of known DHFR inhibitors and the structures and searches for about eighty new molecules, designed as potential anticancer drugs. In addition, DHFR inhibitors acting on thymidylate synthase (TS), carbon anhydrase (CA) and even DNA-binding are presented in this paper. CONCLUSION: Thorough physicochemical characterization and biological investigations highlight the structure-activity relationship of DHFR inhibitors. This will enable even better design and synthesis of active compounds, which would have the expected mechanism of action and the desired activity.


Assuntos
Antineoplásicos , Antagonistas do Ácido Fólico , Antineoplásicos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Humanos , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/metabolismo
16.
Eur J Med Chem ; 210: 112986, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33187806

RESUMO

To tackle leishmaniasis, search for efficient therapeutic drug targets should be pursued. Dihydrofolate reductase (DHFR) is considered as a key target for the treatment of leishmaniasis. In current study, we are interested in the design and synthesis of selective antifolates targeting DHFR from L. major. We focused on the development of new antifolates based on 3,4-dihydropyrimidine-2-one and 5-(3,5-dimethoxybenzyl)pyrimidine-2,4-diamine motif. Structure activity relationship (SAR) studies were performed on 4-phenyl ring of dihydropyrimidine (26-30) template. While for 5-(3,5-dimethoxybenzyl)pyrimidine-2,4-diamine, the impact of different amino acids (valine, tryptophan, phenylalanine, and glutamic acid) and two carbon linkers were explored (52-59). The synthesized compounds were assayed against LmDHFR. Compound 59 with the IC50 value of 0.10 µM appeared as potent inhibitors of L. major. Selectivity for parasite DHFR over human DHFR was also determined. Derivatives 55-59 demonstrated excellent selectivity for LmDHFR. Highest selectivity for LmDHFR was shown by compounds 56 (SI = 84.5) and 58 (SI = 87.5). Compounds Antileishmanial activity against L. major and L. donovani promastigotes was also performed. To explore the interaction pattern of the synthesized compounds with biological macromolecules, the docking studies were carried out against homology modelled LmDHFR and hDHFR targets.


Assuntos
Antiprotozoários/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Leishmania/efeitos dos fármacos , Pirimidinas/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Antiprotozoários/síntese química , Antiprotozoários/química , Relação Dose-Resposta a Droga , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Leishmania/enzimologia , Estrutura Molecular , Testes de Sensibilidade Parasitária , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade
17.
Appl Microbiol Biotechnol ; 105(1): 67-76, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33191460

RESUMO

Our previous work showed that there is a limitation in the use of dihydrofolate reductase (dhfr)/methotrexate (MTX)-mediated gene amplification systems in dhfr-non-deficient HEK293 cells, as endogenous dhfr may interfere with the amplification process. In the present study, we successfully generated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-amplified HEK293 cells in a dhfr-non-deficient HEK293 cell background using a single-plasmid vector-based gene amplification system with shRNA targeting the 3'-UTR of endogenous dhfr. The introduction of this shRNA efficiently downregulated the expression of endogenous dhfr in the HEK293 cells without affecting exogenous dhfr expression. The downregulation of endogenous dhfr improved the efficiency of EBNA-1 amplification, as evidenced by a comparison with the amplification extent in cells lacking shRNA expression at the same MTX concentration. The EBNA-1 expression levels from the EBNA-1-amplified clones selected in this study were higher than those obtained from EBNA-1-amplified clones that were generated using the conventional amplification in our previous study. Consistent with previous studies, EBNA-1 amplification improved the production of the Fc-fusion protein through a specific protein productivity (qp)-enhancing effect, rather than by improving cell growth or transfection efficiency. In addition, the N-glycan profiles in the Fc-fusion protein produced using this transient gene expression (TGE) system were not affected by EBNA-1 amplification. These results indicate the potential utility of EBNA-1-amplified mammalian cells, developed using a single-plasmid vector-based gene amplification system, for efficient protein production. KEY POINTS: • EBNA-1-amplified HEK293 cells were established using gene amplification system. • EBNA-1 amplification in TGE system can increase the Fc-fusion protein productivity. • EBNA-1 amplification does not affect the N-glycan profile in the Fc-fusion protein.


Assuntos
Infecções por Vírus Epstein-Barr , Amplificação de Genes , Animais , Células CHO , Cricetinae , Antígenos Nucleares do Vírus Epstein-Barr/genética , Expressão Gênica , Células HEK293 , Herpesvirus Humano 4/genética , Humanos , Metotrexato , Plasmídeos/genética , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
18.
Methods Mol Biol ; 2253: 185-219, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33315225

RESUMO

Protein motions play a fundamental role in enzyme catalysis and ligand binding. The relationship between protein motion and function has been extensively investigated in the model enzyme dihydrofolate reductase (DHFR). DHFR is an essential enzyme that catalyzes the reduction of dihydrofolate to tetrahydrofolate. Numerous experimental and computational methods have been used to probe the motions of DHFR through the catalytic cycle and to investigate the effect of distal mutations on DHFR motions and ligand binding. These experimental investigations have pushed forward the study of protein motions and their role in protein-ligand interactions. The introduction of mutations distal to the active site has been shown to have profound effects on ligand binding, hydride transfer rates and catalytic efficacy and these changes are captured by enzyme kinetics measurements. Distal mutations have been shown to exert their effects through a network of correlated amino acids and these effects have been investigated by NMR, protein dynamics, and analysis of coupled amino acids. The experimental methods and the findings that are reviewed here have broad implications for our understanding of enzyme mechanisms, ligand binding and for the future design and discovery of enzyme inhibitors.


Assuntos
Mutação , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Tetra-Hidrofolato Desidrogenase/genética
19.
Malar J ; 19(1): 434, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33238987

RESUMO

BACKGROUND: Anti-malarial drug resistance is a severe challenge for eventual control and global elimination of malaria. Resistance to sulfadoxine-pyrimethamine (SP) increases as mutations accumulate in the Pfdhfr and Pfdhps genes. This study aimed to assess the polymorphisms and prevalence of mutation in these genes in the Plasmodium falciparum infecting migrant workers returning to Wuhan, China. METHODS: Blood samples were collected for 9 years (2011-2019). Parasite genomic DNA was extracted from blood spots on filter paper. The mutations were evaluated by nested PCR and sequencing. The single-nucleotide polymorphisms (SNPs) and haplotypes of the Pfdhfr and Pfdhps genes were analysed. RESULTS: Pfdhfr codon 108 showed a 94.7% mutation rate, while for Pfdhps, the rate for codon 437 was 79.0%. In total, five unique haplotypes at the Pfdhfr locus and 11 haplotypes at the Pfdhps locus were found while the Pfdhfr-Pfdhps combined loci revealed 28 unique haplotypes. A triple mutant (IRNI) of Pfdhfr was the most prevalent haplotype (84.4%). For Pfdhps, a single mutant (SGKAA) and a double mutant (SGEAA) were detected at frequencies of 37.8 and 22.3%, respectively. Among the combined haplotypes, a quadruple mutant (IRNI-SGKAA) was the most common, with a 30.0% frequency, followed by a quintuplet mutant (IRNI-SGEAA) with a frequency of 20.4%. CONCLUSION: The high prevalence and saturation of Pfdhfr haplotypes and the medium prevalence of Pfdhps haplotypes demonstrated in the present data will provide support for predicting the status and progression of antifolate resistance in malaria-endemic regions and imported malaria in nonendemic areas. Additional interventions to evaluate and prevent SP resistance should be continuously considered.


Assuntos
Antimaláricos/farmacologia , Doenças Transmissíveis Importadas/parasitologia , Resistência a Medicamentos/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Polimorfismo Genético , Proteínas de Protozoários/genética , Tetra-Hidrofolato Desidrogenase/genética , África , Ásia Sudeste , China , Combinação de Medicamentos , Humanos , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Pirimetamina/farmacologia , Sulfadoxina/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo
20.
Sci Rep ; 10(1): 19844, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199757

RESUMO

This study aimed to investigate the influence of chronic ischemia on nitric oxide biosynthesis in the bladder and the effect of administering tetrahydrobiopterin (BH4), a cofactor for endothelial nitric oxide synthase (eNOS), on chronic ischemia-related lower urinary tract dysfunction (LUTD). This study divided male Sprague-Dawley rats into Control, chronic bladder ischemia (CBI) and CBI with oral BH4 supplementation (CBI/BH4) groups. In the CBI group, bladder capacity and bladder muscle strip contractility were significantly lower, and arterial wall was significantly thicker than in Controls. Significant improvements were seen in bladder capacity, muscle strip contractility and arterial wall thickening in the CBI/BH4 group as compared with the CBI group. Western blot analysis of bladder showed expressions of eNOS (p = 0.043), HIF-1α (p < 0.01) and dihydrofolate reductase (DHFR) (p < 0.01), which could regenerate BH4, were significantly higher in the CBI group than in Controls. In the CBI/BH4 group, HIF-1α (p = 0.012) and DHFR expressions (p = 0.018) were significantly decreased compared with the CBI group. Our results suggest that chronic ischemia increases eNOS and DHFR in the bladder to prevent atherosclerosis progression. However, DHFR could not synthesize sufficient BH4 relative to the increased eNOS, resulting in LUTD. BH4 supplementation protects lower urinary tract function by promoting eNOS activity.


Assuntos
Biopterina/análogos & derivados , Isquemia/prevenção & controle , Óxido Nítrico/biossíntese , Bexiga Urinária/irrigação sanguínea , Animais , Disponibilidade Biológica , Biopterina/administração & dosagem , Biopterina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/etiologia , Isquemia/metabolismo , Masculino , Contração Muscular/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Oxirredução , Ratos , Ratos Sprague-Dawley , Tetra-Hidrofolato Desidrogenase/metabolismo , Bexiga Urinária/efeitos dos fármacos
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