RESUMEN

A new superior bacteria complementation model was achieved for testing antifolate compounds and investigating antifolate resistance in the dihydrofolate reductase (DHFR) enzyme of the malaria parasite. Earlier models depended on the addition of trimethoprim (TMP) to chemically suppress the host Escherichia coli (Ec) DHFR function. However, incomplete suppression of EcDHFR and potential interference of antibiotics needed to maintain plasmids for complementary gene expression can complicate the interpretations. To overcome such limitations, the folA (F) and thyA (T) genes were genetically knocked out (Δ) in E. coli BL21(DE3). The resulting EcΔFΔT cells were thymidine auxotroph where thymidine supplementation or functional complementation with heterologous DHFR-thymidylate synthase (TS) is needed to restore the loss of gene functions. When tested against pyrimethamine (PYR) and its analogs designed to target Plasmodium falciparum (Pf) DHFR-TS, the 50 % inhibitory concentration values obtained from EcΔFΔT surrogates expressing wildtype (PfTM4) or double mutant (PfK1) DHFR-TS showed strong correlations to the results obtained from the standard in vitro P. falciparum growth inhibition assay. Interestingly, while TMP had little effect on the susceptibility to PYR and analogs in EcΔFΔT expressing PfDHFR-TS, it hypersensitized the chemically knockdown E. coli BL21(DE3) expressing PfTM4 DHFR-TS but desensitized the one carrying PfK1 DHFR-TS. The low intrinsic expression level of PfTM4 in E. coli BL21(DE3) by western blot analysis may explain the hypersensitive to antifolates of chemical knockdown bacteria surrogate. These results demonstrated the usefulness of EcΔFΔT surrogate as a new tool for antifolate antimalarial screening with potential application for investigation of antifolate resistance mechanism.

Asunto(s)

Escherichia coli , Antagonistas del Ácido Fólico , Técnicas de Inactivación de Genes , Plasmodium falciparum , Pirimetamina , Tetrahidrofolato Deshidrogenasa , Timidilato Sintasa , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Antagonistas del Ácido Fólico/farmacología , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Pirimetamina/farmacología , Antimaláricos/farmacología , Concentración 50 Inhibidora , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Resistencia a Medicamentos/genética , Prueba de Complementación Genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complejos Multienzimáticos

RESUMEN

Parkinson's disease is a progressive neurodegenerative disorder marked by the death of dopaminergic neurons in the substantia nigra region of the brain. Aggregation of alpha-synuclein (α-synuclein) is a contributing factor to Parkinson's disease pathogenesis. The objective of this study is to investigate the neuroprotective effects of gut microbes on α-synuclein aggregation using both in silico and in vivo approaches. We focussed on the interaction between α-synuclein and metabolites released by gut bacteria that protect from PD. We employed three probiotic microbe strains against α-synuclein protein: Lactobacillus casei, Escherichia coli, and Bacillus subtilis, with their chosen PDB IDs being Dihydrofolate reductase (3DFR), methionine synthetase (6BM5), and tryptophanyl-tRNA synthetase (3PRH), respectively. Using HEX Dock 6.0 software, we examined the interactions between these proteins. Among the various metabolites, methionine synthetase produced by E. coli showed potential interactions with α-synuclein. To further evaluate the neuroprotective benefits of E. coli, an in vivo investigation was performed using a rotenone-induced Parkinsonian mouse model. The motor function of the animals was assessed through behavioural tests, and oxidative stress and neurotransmitter levels were also examined. The results demonstrated that, compared to the rotenone-induced PD mouse model, the rate of neurodegeneration was considerably reduced in mice treated with E. coli. Additionally, histopathological studies provided evidence of the neuroprotective effects of E. coli. In conclusion, this study lays the groundwork for future research, suggesting that gut bacteria may serve as potential therapeutic agents in the development of medications to treat Parkinson's disease. fig. 1.

Asunto(s)

Bacillus subtilis , Escherichia coli , Microbioma Gastrointestinal , Simulación del Acoplamiento Molecular , Estrés Oxidativo , Probióticos , Rotenona , alfa-Sinucleína , Animales , Ratones , Microbioma Gastrointestinal/fisiología , Probióticos/uso terapéutico , Probióticos/farmacología , alfa-Sinucleína/metabolismo , Estrés Oxidativo/efectos de los fármacos , Rotenona/toxicidad , Lacticaseibacillus casei/fisiología , Metionina-ARNt Ligasa , Triptófano-ARNt Ligasa/fisiología , Masculino , Tetrahidrofolato Deshidrogenasa/metabolismo , Simulación por Computador , Trastornos Parkinsonianos/microbiología , Humanos , Fármacos Neuroprotectores/uso terapéutico , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Enfermedad de Parkinson Secundaria/inducido químicamente , Neuronas Dopaminérgicas/efectos de los fármacos , Enfermedad de Parkinson/microbiología

RESUMEN

Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (1) and 2-guanidino benzimidazoles (2), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of TbDHFR and TbPTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent (Ki = 9 nM) and highly selective TbDHFR inhibitor, 6000-fold over TbPTR1 and 394-fold over hDHFR. The 5,6-dimethyl tricyclic analogue 1g, despite showing a lower potency and selectivity profile than 2g, shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.

Asunto(s)

Antagonistas del Ácido Fólico , Tetrahidrofolato Deshidrogenasa , Triazinas , Trypanosoma brucei brucei , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimología , Humanos , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/química , Antagonistas del Ácido Fólico/farmacología , Antagonistas del Ácido Fólico/química , Triazinas/farmacología , Triazinas/química , Tripanocidas/farmacología , Tripanocidas/química , Proguanil/farmacología , Proguanil/química , Timidilato Sintasa/antagonistas & inhibidores , Timidilato Sintasa/química , Timidilato Sintasa/metabolismo , Leishmania infantum/efectos de los fármacos , Leishmania infantum/enzimología , Bencimidazoles/farmacología , Bencimidazoles/química , Relación Estructura-Actividad , Antiprotozoarios/farmacología , Antiprotozoarios/química , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Oxidorreductasas

RESUMEN

Viral communities exist in a variety of ecosystems and play significant roles in mediating biogeochemical processes, whereas viruses inhabiting strongly alkaline geochemical systems remain underexplored. In this study, the viral diversity, potential functionalities, and virus-host interactions in a strongly alkaline environment (pH = 10.4-12.4) exposed to the leachates derived from the serpentinization-like reactions of smelting slags were investigated. The viral populations (e.g., Herelleviridae, Queuovirinae, and Inoviridae) were closely associated with the dominating prokaryotic hosts (e.g., Meiothermus, Trueperaceae, and Serpentinomonas) in this ultrabasic environment. Auxiliary metabolic genes (AMGs) suggested that viruses may enhance hosts' fitness by facilitating cofactor biosynthesis, hydrogen metabolism, and carbon cycling. To evaluate the activity of synthesis of essential cofactor vitamin B9 by the viruses, a viral folA (vfolA) gene encoding dihydrofolate reductase (DHFR) was introduced into a thymidine-auxotrophic strain Escherichia coli MG1655 ΔfolA mutant, which restored the growth of the latter in the absence of thymidine. Notably, the homologs of the validated vDHFR were globally distributed in the viromes across various ecosystems. The present study sheds new light on the unique viral communities in hyperalkaline ecosystems and their potential beneficial impacts on the coexisting microbial consortia by supplying essential cofactors. IMPORTANCE: This study presents a comprehensive investigation into the diversity, potential functionalities, and virus-microbe interactions in an artificially induced strongly alkaline environment. Functional validation of the detected viral folA genes encoding dihydrofolate reductase substantiated the synthesis of essential cofactors by viruses, which may be ubiquitous, considering the broad distribution of the viral genes associated with folate cycling.

Asunto(s)

Microbiota , Concentración de Iones de Hidrógeno , Viroma/genética , Virus/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Bacterias/genética , Bacterias/metabolismo , Bacterias/clasificación

RESUMEN

Cystathionine beta-synthase-deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. HCU can be treated by using betaine to lower tissue and plasma levels of homocysteine (Hcy). Here, we show that mice with severely elevated Hcy and potentially deficient in the folate species tetrahydrofolate (THF) exhibit a very limited response to betaine indicating that THF plays a critical role in treatment efficacy. Analysis of a mouse model of HCU revealed a 10-fold increase in hepatic levels of 5-methyl -THF and a 30-fold accumulation of formiminoglutamic acid, consistent with a paucity of THF. Neither of these metabolite accumulations were reversed or ameliorated by betaine treatment. Hepatic expression of the THF-generating enzyme dihydrofolate reductase (DHFR) was significantly repressed in HCU mice and expression was not increased by betaine treatment but appears to be sensitive to cellular redox status. Expression of the DHFR reaction partner thymidylate synthase was also repressed and metabolomic analysis detected widespread alteration of hepatic histidine and glutamine metabolism. Many individuals with HCU exhibit endothelial dysfunction. DHFR plays a key role in nitric oxide (NO) generation due to its role in regenerating oxidized tetrahydrobiopterin, and we observed a significant decrease in plasma NOx (NO2 + NO3) levels in HCU mice. Additional impairment of NO generation may also come from the HCU-mediated induction of the 20-hydroxyeicosatetraenoic acid generating cytochrome CYP4A. Collectively, our data shows that HCU induces dysfunctional one-carbon metabolism with the potential to both impair betaine treatment and contribute to multiple aspects of pathogenesis in this disease.

Asunto(s)

Homocistinuria , Hígado , Oxidación-Reducción , Tetrahidrofolato Deshidrogenasa , Tetrahidrofolatos , Animales , Homocistinuria/metabolismo , Homocistinuria/tratamiento farmacológico , Homocistinuria/genética , Ratones , Tetrahidrofolatos/metabolismo , Hígado/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Betaína/metabolismo , Betaína/farmacología , Homocisteína/metabolismo , Ratones Endogámicos C57BL , Cistationina betasintasa/metabolismo , Cistationina betasintasa/genética , Carbono/metabolismo , Masculino , Ácido Fólico/metabolismo , Femenino

RESUMEN

The Escherichia coli GroEL/ES chaperonin system facilitates protein folding in an ATP-driven manner. There are <100 obligate clients of this system in E. coli although GroEL can interact and assist the folding of a multitude of proteins in vitro. It has remained unclear, however, which features distinguish obligate clients from all the other proteins in an E. coli cell. To address this question, we established a system for selecting mutations in mouse dihydrofolate reductase (mDHFR), a GroEL interactor, that diminish its dependence on GroEL for folding. Strikingly, both synonymous and non-synonymous codon substitutions were found to reduce mDHFR's dependence on GroEL. The non-synonymous substitutions increase the rate of spontaneous folding whereas computational analysis indicates that the synonymous substitutions appear to affect translation rates at specific sites.

Asunto(s)

Chaperonina 60 , Codón , Escherichia coli , Pliegue de Proteína , Tetrahidrofolato Deshidrogenasa , Chaperonina 60/genética , Chaperonina 60/química , Chaperonina 60/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Codón/genética , Codón/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ratones , Mutación Silenciosa

RESUMEN

Malaria is an intracellular protozoan parasitic disease caused by Plasmodium species with significant morbidity and mortality in endemic regions. The complex lifecycle of the parasite and the emergence of drug-resistant Plasmodium falciparum have hampered the efficacy of current anti-malarial agents. To circumvent this situation, the present study attempts to demonstrate the blood-stage anti-plasmodial action of 26 hybrid compounds containing the three privileged bioactive scaffolds (sulfonamide, chalcone, and nitro group) with synergistic and multitarget action. These three parent scaffolds exhibit divergent activities, such as antibacterial, anti-malarial, anti-fungal, anti-inflammatory, and anticancer. All the synthesised compounds were characterised using various spectroscopic techniques. The in vitro blood-stage inhibitory activity of 26 hybrid compounds was evaluated against mixed-stage culture (asynchronize) of human malarial parasite P. falciparum, Pf 3D7 at different concentrations ranging from 25.0 µg/mL to 0.78 µg/mL using SYBR 1 green assay, with IC50 values determined after 48 h of treatment based on the drug-response curves. Two potent compounds (11 and 10), with 2-Br and 2,6-diCl substitutions, showed pronounced activity with IC50 values of 5.4 µg/mL and 5.6 µg/mL, whereas others displayed varied activity with IC50 values ranging from 7.0 µg/mL to 22.0 µg/mL. Both 11 and 10 showed greater susceptibility towards mature-stage trophozoites than ring-stage parasites. The hemolytic and in vitro cytotoxicity assays revealed that compounds 11 and 10 did not cause any toxic effects on host red blood cells (uninfected), human-derived Mo7e cells, and murine-derived BA/F3 cells. The in vitro observations are consistent with the in silico studies using P. falciparum-dihydrofolate reductase, where 11 and 10 showed a binding affinity of -10.4 Kcal/mol. This is the first report of the hybrid scaffold, 4-nitrobenzenesulfonamide chalcones, demonstrating its potential as an anti-plasmodial agent.

Asunto(s)

Antimaláricos , Chalconas , Diseño de Fármacos , Plasmodium falciparum , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/farmacología , Antimaláricos/síntesis química , Antimaláricos/química , Chalconas/farmacología , Chalconas/síntesis química , Chalconas/química , Humanos , Simulación del Acoplamiento Molecular , Sulfonamidas/farmacología , Sulfonamidas/química , Sulfonamidas/síntesis química , Simulación por Computador , Relación Estructura-Actividad , Tetrahidrofolato Deshidrogenasa/metabolismo

RESUMEN

New imidazo[2,1-b]thiazole analogs were designed, synthesized, and biologically evaluated as anticancer agents. In vitro biological evaluation of the anticancer properties of the compounds was performed against different cancer cell lines. Compounds 23 and 39 showed remarkable broad -spectrum cytotoxic potency on most of the tested cell lines. Compounds 23 and 39 exhibited potent activity against the MCF-7 breast cancer cell line, with IC50 values of 1.81 and 4.95 µM, respectively, compared to DOX and SOR (IC50 values of 4.17 and 7.26 µM, respectively). An enzyme inhibition assay was carried out to clarify the possible mode of action of the tested compounds. Compounds 23 and 39 were identified as possible EGFR, HER-2, and DHFR inhibitors. Cell cycle arrest results indicated that compound 23 caused cell cycle arrest at the G0/G1 phase in the MCF-7 cells and at the G2/M phase in the Hep G2 cells. Compound 39 induced cell cycle arrest at the G2/M phase in Hela cells. In vivo testing of the anticancer activity of the two most promising molecules in this study was conducted, and the results indicated that they possess considerable in vivo anticancer activity in mice. Data obtained from the molecular modeling simulation study were consistent with the biological evaluation results.

Asunto(s)

Antineoplásicos , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales , Receptores ErbB , Antagonistas del Ácido Fólico , Receptor ErbB-2 , Tetrahidrofolato Deshidrogenasa , Tiazoles , Humanos , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Tiazoles/química , Tiazoles/farmacología , Tiazoles/síntesis química , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Relación Estructura-Actividad , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Proliferación Celular/efectos de los fármacos , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Estructura Molecular , Antagonistas del Ácido Fólico/farmacología , Antagonistas del Ácido Fólico/síntesis química , Antagonistas del Ácido Fólico/química , Relación Dosis-Respuesta a Droga , Ratones , Imidazoles/química , Imidazoles/farmacología , Imidazoles/síntesis química , Modelos Moleculares , Línea Celular Tumoral

RESUMEN

BACKGROUND/AIM: Methotrexate (MTX) resistance in osteosarcoma leads to a very poor prognosis. In the present study, in order to further understand the basis and ramifications of MTX resistance in osteosarcoma, we selected an osteosarcoma cell line that has a 5,500-fold-increased MTX IC50 Materials and Methods: The super MTX-resistant 143B osteosarcoma cells (143B-MTXSR) were selected from MTX-sensitive parental human 143B osteosarcoma cells (143B-P) by continuous culture with step-wise increased amounts of MTX. To compare the malignancy of 143B-MTXSR and 143B-P, colony-formation capacity was compared with clonogenic assays on plastic and in soft agar. In addition, tumor growth was compared with orthotopic xenograft mouse models of osteosarcoma. Expression of dihydrofolate reductase (DHFR), phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), and myelocytomatosis oncogene (MYC) was examined with western immunoblotting and compared in 143B-MTXSR and 143B-P cells. RESULTS: 143B-MTXSR had a 5,500-fold increase in the MTX IC50 compared to the parental 143B-P cells. Expression of DHFR was increased 10-fold in 143B-MTXSR compared to 143B-P (p<0.01). 143B-MTXSR cells had reduced colony-formation capacity on plastic (p=0.032) and in soft agar (p<0.01) compared to 143B-P and reduced tumor growth in orthotopic xenograft mouse models (p<0.001). These results demonstrate that 143B-MTXSR had reduced malignancy. 143B-MTXSR also showed an increased expression of PI3K (p<0.01), phosphorylated (activated) AKT (p=0.031), phosphorylated mTOR (p=0.043), and c-MYC (p=0.024) compared to 143B-P. CONCLUSION: The present study demonstrates that the increased expression of DHFR, PI3K/AKT/mTOR and c-MYC appears to be linked to super MTX resistance and, paradoxically, to reduced malignancy. The present results suggest that DHFR may be a powerful tumor suppressor when highly amplified.

Asunto(s)

Resistencia a Antineoplásicos , Metotrexato , Osteosarcoma , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-myc , Serina-Treonina Quinasas TOR , Tetrahidrofolato Deshidrogenasa , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/patología , Osteosarcoma/metabolismo , Osteosarcoma/genética , Metotrexato/farmacología , Humanos , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Animales , Resistencia a Antineoplásicos/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/patología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/genética , Amplificación de Genes , Transducción de Señal/efectos de los fármacos , Ratones Desnudos , Antimetabolitos Antineoplásicos/farmacología

RESUMEN

Launaea sarmentosa, also known as Sa Sam Nam, is a widely used remedy in Vietnamese traditional medicine and cuisine. However, the chemical composition and bioactivity of its essential oil have not been elucidated yet. In this study, we identified 40 compounds (98.6% of total peak area) in the essential oil via GC-MS analysis at the first time. Among them, five main compounds including Thymohydroquinone dimethyl ether (52.4%), (E)-α-Atlantone (9.0%), Neryl isovalerate (6.6%), Davanol D2 (isomer 2) (3.9%), and trans-Sesquisabinene hydrate (3.9%) have accounted for 75.8% of total peak area. The anti-bacterial activity of the essential oil against 4 microorganisms including Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa has also investigated via agar well diffusion assay. The results showed that the essential oil exhibited a strong antibacterial activity against Bacillus subtilis with the inhibition zones ranging from 8.2 to 18.7 mm. To elucidate the anti-bacterial effect mechanism of the essential oil, docking study of five main compounds of the essential oil (Thymohydroquinone dimethyl ether, (E)-α-Atlantone, Neryl isovalerate, Davanol D2 (isomer 2), and trans-Sesquisabinene hydrate) against some key proteins for bacterial growth such as DNA gyrase B, penicillin binding protein 2A, tyrosyl-tRNA synthetase, and dihydrofolate reductase were performed. The results showed that the main constituents of essential oil were highly bound with penicillin binding protein 2A with the free energies ranging -27.7 to -44.8 kcal/mol, which suggests the relationship between the antibacterial effect of essential oil and the affinity of main compounds with penicillin binding protein. In addition, the free energies of main compounds of the essential oil with human cyclooxygenase 1, cyclooxygenase 2, and phospholipase A2, the crucial proteins related with inflammatory response were less than diclofenac, a non-steroidal antiinflammatory drug. These findings propose the essential oil as a novel and promising anti-bacterial and anti-inflammatory medicine or cosmetic products.

Asunto(s)

Antibacterianos , Bacillus subtilis , Hemiterpenos , Simulación del Acoplamiento Molecular , Aceites Volátiles , Ácidos Pentanoicos , Antibacterianos/farmacología , Antibacterianos/aislamiento & purificación , Antibacterianos/química , Aceites Volátiles/farmacología , Aceites Volátiles/química , Aceites Volátiles/aislamiento & purificación , Bacillus subtilis/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Tetrahidrofolato Deshidrogenasa/metabolismo , Girasa de ADN/metabolismo , Sesquiterpenos/aislamiento & purificación , Sesquiterpenos/farmacología , Pruebas de Sensibilidad Microbiana , Cromatografía de Gases y Espectrometría de Masas

RESUMEN

In this study, new series of N'-(2-(substitutedphenoxy)acetyl)-4-(1H-pyrrol-1-yl)benzohydrazides (3a-j) 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N'-(2-(substitutedphenoxy)acetyl)benzohydrazides (5a-j) were synthesized, characterized and assessed as inhibitors of enoyl ACP reductase and DHFR. Most of the compounds exhibited dual inhibition against the enzymes enoyl ACP reductase and DHFR. Several synthesized substances also demonstrated significant antibacterial and antitubercular properties. A molecular docking analysis was conducted in order to determine the potential mechanism of action of the synthesized compounds. The results indicated that there were binding interactions seen with the active sites of dihydrofolate reductase and enoyl ACP reductase. Additionally, important structural details were identified that play a critical role in sustaining the dual inhibitory activity. These findings were useful for the development of future dual inhibitors. Therefore, this study provided strong evidence that several synthesized molecules could exert their antitubercular properties at the cellular level through multi-target inhibition. By shedding light on the mechanisms through which these compounds exert their inhibitory effects, this research opens up promising avenues for the future development of dual inhibitors with enhanced antibacterial and antitubercular properties. The study's findings underscore the importance of multi-target approaches in drug design, providing a strong foundation for the design and optimization of novel compounds that can effectively target bacterial infections at the cellular level.

Asunto(s)

Antituberculosos , Pirroles , Tetrahidrofolato Deshidrogenasa , Humanos , Antituberculosos/farmacología , Antituberculosos/química , Antituberculosos/síntesis química , Dominio Catalítico , Enoil-ACP Reductasa (NADH)/antagonistas & inhibidores , Enoil-ACP Reductasa (NADH)/metabolismo , Enoil-ACP Reductasa (NADH)/química , Antagonistas del Ácido Fólico/farmacología , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/síntesis química , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Pirroles/síntesis química , Pirroles/química , Pirroles/farmacología , Relación Estructura-Actividad , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/química

RESUMEN

New thienopyrimidine derivatives 2-16 have been synthesized and their in vitro cytotoxicity was evaluated against five different human cancer cell lines HCT-116, Hela, MDA-MB-231, MCF7 and PC3. Compounds 6e, 7a, 7b, 7d, 10c and 10e displayed the highest antitumor activity against all tested cell lines compared to Doxorubicin. Enzyme inhibition assay revealed that compounds 6e and 10e showed high inhibitory activity against EGFR-TK, with IC50 values of 0.133 and 0.151 µM, compared to Olmutinib (IC50 = 0.028 µM); while the highest DHFR inhibitory activity was shown by compounds 7d and 10e with IC50 values of 0.462 and 0.541 µM, compared to Methotrexate (IC50 = 0.117 µM). Cell cycle analysis following a flow cytometric study using colorectal HCT-116 cancer cell line proved that compound 6e induced cell cycle arrest in G0-G1 phase, while compound 10e arrested the cell cycle at both G0-G1 and S phases. Additionally, both compounds (6e and 10e) were potently able to induce apoptosis in HCT-116 cell line. Docking results of compounds 6e and 10e into the pocket of EGFR active site showed their similar main binding features with Olmutinib, while compounds 7d and 10e showed only moderate fitting into DHFR compared to methotrexate. In silico studies revealed that most of the tested compounds obeyed Lipinski's RO5 and showed positive drug likeness scores.

Asunto(s)

Antineoplásicos , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales , Receptores ErbB , Antagonistas del Ácido Fólico , Simulación del Acoplamiento Molecular , Pirimidinas , Tetrahidrofolato Deshidrogenasa , Humanos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Pirimidinas/farmacología , Pirimidinas/química , Pirimidinas/síntesis química , Tetrahidrofolato Deshidrogenasa/metabolismo , Antagonistas del Ácido Fólico/farmacología , Antagonistas del Ácido Fólico/síntesis química , Antagonistas del Ácido Fólico/química , Relación Estructura-Actividad , Proliferación Celular/efectos de los fármacos , Estructura Molecular , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química

RESUMEN

BACKGROUND: Mutations in the Plasmodium falciparum dhfr gene confer resistance to pyrimethamine, which is widely used for malaria chemoprevention in Africa. We aimed to evaluate the frequency and evolution of dhfr mutations in Plasmodium ovale spp in Africa and their functional consequences, which are incompletely characterised. METHODS: We analysed dhfr mutations and their frequencies in P ovale spp isolates collected between Feb 1, 2004, and Aug 31, 2023, from the French National Malaria Reference Centre collection and from field studies in Benin, Gabon, and Kenya. Genetic patterns of positive selection were investigated. Full-length recombinant wild-type and mutant DHFR enzymes from both P ovale curtisi and P ovale wallikeri were expressed in bacteria to test whether the most common mutations reduced pyrimethamine susceptibility. FINDINGS: We included 518 P ovale spp samples (314 P ovale curtisi and 204 P ovale wallikeri). In P ovale curtisi, Ala15Ser-Ser58Arg was the most common dhfr mutation (39%; 124 of 314 samples). In P ovale wallikeri, dhfr mutations were less frequent, with Phe57Leu-Ser58Arg reaching 17% (34 of 204 samples). These two mutants were the most prevalent in central and east Africa and were fixed in Kenyan isolates. We detected six and four other non-synonymous mutations, representing 8% (24 isolates) and 2% (five isolates) of the P ovale curtisi and P ovale wallikeri isolates, respectively. Whole-genome sequencing and microsatellite analyses revealed reduced genetic diversity around the mutant pocdhfr and powdhfr genes. The mutant DHFR proteins showed structural changes at the pyrimethamine binding site in-silico, confirmed by a 4-times increase in pyrimethamine half-maximal inhibitory concentration in an Escherichia coli growth assay for the Phe57Leu-Ser58Arg mutant and 50-times increase for the Ala15Ser-Ser58Arg mutant, compared with the wild-type counterparts. INTERPRETATION: The widespread use of sulfadoxine-pyrimethamine for malaria chemoprevention might have exerted fortuitous selection pressure for dhfr mutations in P ovale spp. This calls for closer monitoring of dhfr and dhps mutations in P ovale spp. FUNDING: French Ministry of Health, Agence Nationale de la Recherche, and Global Emerging Infections Surveillance branch of the Armed Forces Health Surveillance Division.

Asunto(s)

Antimaláricos , Resistencia a Medicamentos , Malaria , Mutación , Plasmodium ovale , Pirimetamina , Tetrahidrofolato Deshidrogenasa , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Pirimetamina/farmacología , Pirimetamina/uso terapéutico , Resistencia a Medicamentos/genética , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Plasmodium ovale/genética , Plasmodium ovale/efectos de los fármacos , Humanos , Malaria/epidemiología , Estudios Retrospectivos , África del Sur del Sahara/epidemiología , Proteínas Protozoarias/genética , Kenia/epidemiología

RESUMEN

The agricultural fungicide cymoxanil (CMX) is commonly used in the treatment of plant pathogens, such as Phytophthora infestans. Although the use of CMX is widespread throughout the agricultural industry and internationally, the exact mechanism of action behind this fungicide remains unclear. Therefore, we sought to elucidate the biocidal mechanism underlying CMX. This was accomplished by first performing a large-scale chemical-genomic screen comprising the 4000 haploid non-essential gene deletion array of the yeast Saccharomyces cerevisiae. We found that gene families related to de novo purine biosynthesis and ribonucleoside synthesis were enriched in the presence of CMX. These results were confirmed through additional spot-test and colony counting assays. We next examined whether CMX affects RNA biosynthesis. Using qRT-PCR and expression assays, we found that CMX appears to target RNA biosynthesis possibly through the yeast dihydrofolate reductase (DHFR) enzyme Dfr1. To determine whether DHFR is a target of CMX, we performed an in-silico molecular docking assay between CMX and yeast, human, and P. infestans DHFR. The results suggest that CMX directly interacts with the active site of all tested forms of DHFR using conserved residues. Using an in vitro DHFR activity assay we observed that CMX inhibits DHFR activity in a dose-dependent relationship.

Asunto(s)

Fungicidas Industriales , Simulación del Acoplamiento Molecular , Proteínas de Saccharomyces cerevisiae , Tetrahidrofolato Deshidrogenasa , Humanos , Antagonistas del Ácido Fólico/farmacología , Fungicidas Industriales/farmacología , ARN/biosíntesis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/genética

RESUMEN

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

Asunto(s)

Farmacorresistencia Fúngica , Antagonistas del Ácido Fólico , Metotrexato , Mutación , Pneumocystis carinii , Tetrahidrofolato Deshidrogenasa , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/química , Pneumocystis carinii/genética , Pneumocystis carinii/enzimología , Pneumocystis carinii/efectos de los fármacos , Antagonistas del Ácido Fólico/farmacología , Farmacorresistencia Fúngica/genética , Metotrexato/farmacología , Regulación Alostérica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Humanos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Dominio Catalítico/genética

RESUMEN

BACKGROUND: Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. METHODS: We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. RESULTS: The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. CONCLUSION: The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

Asunto(s)

Resistencia a Antineoplásicos , Amplificación de Genes , Metotrexato , Tetrahidrofolato Deshidrogenasa , Humanos , Metotrexato/farmacología , Resistencia a Antineoplásicos/genética , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Antimetabolitos Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genómica/métodos

RESUMEN

The observation of multiple conformations of a functional loop (termed M20) in the Escherichia coli dihydrofolate reductase (ecDHFR) enzyme triggered the proposition that large-scale motions of protein structural elements contribute to enzyme catalysis. The transition of the M20 loop from a closed conformation to an occluded conformation was thought to aid the rate-limiting release of the products. However, the influence of charged species in the solution environment on the observed M20 loop conformations, independent of charged ligands bound to the enzyme, had not been considered. Molecular dynamics simulations of ecDHFR in model CaCl2 solutions of varying molar ionic strengths IM reveal a substantial free energy barrier between occluded and closed M20 loop states at IM exceeding the E. coli threshold (∼0.24 M). This barrier may facilitate crystallization of ecDHFR in the occluded state, consistent with ecDHFR structures obtained at IM exceeding 0.3 M. At lower IM (≤0.15 M), the M20 loop can explore the occluded state, but prefers an open/partially closed conformation, again consistent with ecDHFR structures. Our findings caution against using ecDHFR structures obtained at nonphysiological ionic strengths in interpreting catalytic events or in structure-based drug design.

Asunto(s)

Escherichia coli , Simulación de Dinámica Molecular , Conformación Proteica , Tetrahidrofolato Deshidrogenasa , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/química , Escherichia coli/enzimología , Concentración Osmolar , Soluciones , Cloruro de Calcio/química , Cloruro de Calcio/metabolismo

RESUMEN

In order to develop novel antimicrobial agents, we prepared quinoline bearing pyrimidine analogues 2-7, 8 a-d and 9 a-d and their structures were elucidated by spectroscopic techniques. Furthermore, our second aim was to predict the interactions between the active compounds and enzymes (DNA gyrase and DHFR). In this work, fourteen pyrimido[4,5-b]quinoline derivatives were prepared and assessed for their antimicrobial potential by estimating zone of inhibition. All the screened candidates displayed antibacterial potential with zone of inhibition range of 9-24 mm compared with ampicillin (20-25 mm) as a reference drug. Moreover, the target derivatives 2 (ZI=16), 9 c (ZI=17 mm) and 9 d (ZI=16 mm) recorded higher antifungal activity against C. albicans to that exhibited by the antifungal drug amphotericin B (ZI=15 mm). Finally, the most potent pyrimidoquinoline compounds (2, 3, 8 c, 8 d, 9 c and 9 d) were docked inside DHFR and DNA gyrase active sites and they recorded excellent fitting within the active regions of DNA gyrase and DHFR. These outcomes revealed us that compounds (2, 3, 8 c, 8 d, 9 c and 9 d) could be lead compounds to discover novel antibacterial candidates.

Asunto(s)

Antibacterianos , Candida albicans , Girasa de ADN , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Quinolinas , Tetrahidrofolato Deshidrogenasa , Quinolinas/química , Quinolinas/farmacología , Girasa de ADN/metabolismo , Girasa de ADN/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/química , Candida albicans/efectos de los fármacos , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Relación Estructura-Actividad , Antifúngicos/farmacología , Antifúngicos/química , Antifúngicos/síntesis química , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinas/síntesis química , Estructura Molecular , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/síntesis química , Relación Dosis-Respuesta a Droga

RESUMEN

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections for which the development of antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks enzymes required to scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa is to disrupt thymidine synthesis while providing exogenous thymine. However, known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa.Here we characterize fluorofolin, a dihydrofolate reductase (DHFR) inhibitor derived from Irresistin-16, that exhibits significant activity against P. aeruginosa in culture and in a mouse thigh infection model. Fluorofolin is active against a wide range of clinical P. aeruginosa isolates resistant to known antibiotics. Metabolomics and in vitro assays using purified folA confirm that fluorofolin inhibits P. aeruginosa DHFR. Importantly, in the presence of thymine supplementation, fluorofolin activity is selective for P. aeruginosa. Resistance to fluorofolin can emerge through overexpression of the efflux pumps MexCD-OprJ and MexEF-OprN, but these mutants also decrease pathogenesis. Our findings demonstrate how understanding species-specific genetic differences can enable selective targeting of important pathogens while revealing trade-offs between resistance and pathogenesis.

Asunto(s)

Antibacterianos , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Tetrahidrofolato Deshidrogenasa , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Animales , Ratones , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/tratamiento farmacológico , Antibacterianos/farmacología , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Antagonistas del Ácido Fólico/farmacología , Ácido Fólico/metabolismo , Farmacorresistencia Bacteriana , Modelos Animales de Enfermedad , Timina/metabolismo , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Femenino

RESUMEN

Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.

Asunto(s)

Sistemas CRISPR-Cas , Cricetulus , Edición Génica , Células CHO , Animales , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Anticuerpos Monoclonales/genética , Proteínas Recombinantes/genética , Técnicas de Inactivación de Genes/métodos , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Cricetinae , Ingeniería Genética/métodos