RESUMO
Infections caused by Staphylococcus aureus pose a significant global public problem. Therefore, new antibiotics and therapeutic strategies are needed to combat this pathogen. This investigation delves into the effects of iclaprim, a newly discovered inhibitor of folic acid synthesis, on S. aureus virulence. The phenotypic and genotypic effects of iclaprim were thoroughly examined in relation to virulence factors, biofilm formation, and dispersal, as well as partial virulence-encoding genes associated with exoproteins, adherence, and regulation in S. aureus MW2, N315, and ATCC 25923. Then, the in vivo effectiveness of iclaprim on S. aureus pathogenicity was explored by a Galleria mellonella larvae infection model. The use of iclaprim at sub-inhibitory concentrations (sub-MICs) resulted in a reduction of α-hemolysin (Hla) production and a differential effect on the activity of coagulase in S. aureus strains. The results of biofilm formation and eradication assay showed that iclaprim was highly effective in depolymerizing the mature biofilm of S. aureus strains at concentrations of 1 MIC or greater, however, inhibited the biofilm-forming ability of only strains N315 and ATCC 25923 at sub-MICs. Interestingly, treatment of strains with sub-MICs of iclaprim resulted in significant stimulation or suppression of most virulence-encoding genes expression. Iclaprim did not affect the production of δ-hemolysin or staphylococcal protein A (SpA), nor did it impact the total activity of proteases, nucleases, and lipases. In vivo testing showed that sub-MICs of iclaprim significantly improves infected larvae survival. The present study offered valuable insights towards a better understating of the influence of iclaprim on different strains of S. aureus. The findings suggest that iclaprim may have potential as an anti-virulence and antibiofilm agent, thus potentially mitigating the pathogenicity of S. aureus and improving clinical outcomes associated with infections caused by this pathogen. KEY POINTS: ⢠Iclaprim effectively inhibits α-hemolysin production and biofilm formation in a strain-dependent manner and was an excellent depolymerizing agent of mature biofilm ⢠Iclaprim affected the mRNA expression of virulence-encoding genes associated with exoproteins, adherence, and regulation ⢠In vivo study in G. mellonella larvae challenged with S. aureus exhibited that iclaprim improves larvae survival.
Assuntos
Antibacterianos , Biofilmes , Larva , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas , Staphylococcus aureus , Fatores de Virulência , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/genética , Biofilmes/efeitos dos fármacos , Animais , Fatores de Virulência/genética , Antibacterianos/farmacologia , Virulência/efeitos dos fármacos , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Larva/microbiologia , Mariposas/microbiologia , Proteínas Hemolisinas/genética , Ácido Fólico/farmacologia , Ácido Fólico/biossíntese , Antagonistas do Ácido Fólico/farmacologia , Coagulase/metabolismo , Modelos Animais de Doenças , PirimidinasRESUMO
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.
Assuntos
Antagonistas do Ácido Fólico , Tetra-Hidrofolato Desidrogenase , Triazinas , Trypanosoma brucei brucei , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Humanos , Tetra-Hidrofolato Desidrogenase/metabolismo , Tetra-Hidrofolato Desidrogenase/química , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/química , Triazinas/farmacologia , Triazinas/química , Tripanossomicidas/farmacologia , Tripanossomicidas/química , Proguanil/farmacologia , Proguanil/química , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/química , Timidilato Sintase/metabolismo , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/enzimologia , Benzimidazóis/farmacologia , Benzimidazóis/química , Relação Estrutura-Atividade , Antiprotozoários/farmacologia , Antiprotozoários/química , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , OxirredutasesRESUMO
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.
Assuntos
Antineoplásicos , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB , Antagonistas do Ácido Fólico , Receptor ErbB-2 , Tetra-Hidrofolato Desidrogenase , Tiazóis , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Tiazóis/química , Tiazóis/farmacologia , Tiazóis/síntese química , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Relação Estrutura-Atividade , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Proliferação de Células/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Estrutura Molecular , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Relação Dose-Resposta a Droga , Camundongos , Imidazóis/química , Imidazóis/farmacologia , Imidazóis/síntese química , Modelos Moleculares , Linhagem Celular TumoralRESUMO
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.
Assuntos
Fungicidas Industriais , Simulação de Acoplamento Molecular , Proteínas de Saccharomyces cerevisiae , Tetra-Hidrofolato Desidrogenase , Humanos , Antagonistas do Ácido Fólico/farmacologia , Fungicidas Industriais/farmacologia , RNA/biossíntese , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Tetra-Hidrofolato Desidrogenase/genéticaRESUMO
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.
Assuntos
Antituberculosos , Pirróis , Tetra-Hidrofolato Desidrogenase , Humanos , Antituberculosos/farmacologia , Antituberculosos/química , Antituberculosos/síntese química , Domínio Catalítico , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/síntese química , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Pirróis/síntese química , Pirróis/química , Pirróis/farmacologia , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/metabolismo , Tetra-Hidrofolato Desidrogenase/químicaRESUMO
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.
Assuntos
Antineoplásicos , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB , Antagonistas do Ácido Fólico , Simulação de Acoplamento Molecular , Pirimidinas , Tetra-Hidrofolato Desidrogenase , Humanos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Pirimidinas/farmacologia , Pirimidinas/química , Pirimidinas/síntese química , Tetra-Hidrofolato Desidrogenase/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Relação Estrutura-Atividade , Proliferação de Células/efeitos dos fármacos , Estrutura Molecular , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/químicaRESUMO
Malaria is caused by parasites of the Plasmodium genus and remains one of the most pressing human health problems. The spread of parasites resistant to or partially resistant to single or multiple drugs, including frontline antimalarial artemisinin and its derivatives, poses a serious threat to current and future malaria control efforts. In vitro drug assays are important for identifying new antimalarial compounds and monitoring drug resistance. Due to its robustness and ease of use, the [3H]-hypoxanthine incorporation assay is still considered a gold standard and is widely applied, despite limited sensitivity and the dependence on radioactive material. Here, we present a first-of-its-kind chemiluminescence-based antimalarial drug screening assay. The effect of compounds on P. falciparum is monitored by using a dioxetane-based substrate (AquaSpark ß-D-galactoside) that emits high-intensity luminescence upon removal of a protective group (ß-D-galactoside) by a transgenic ß-galactosidase reporter enzyme. This biosensor enables highly sensitive, robust, and cost-effective detection of asexual, intraerythrocytic P. falciparum parasites without the need for parasite enrichment, washing, or purification steps. We are convinced that the ultralow detection limit of less than 100 parasites of the presented biosensor system will become instrumental in malaria research, including but not limited to drug screening.
Assuntos
Antimaláricos , Antagonistas do Ácido Fólico , Malária Falciparum , Malária , Humanos , Antimaláricos/farmacologia , Plasmodium falciparum , Malária/tratamento farmacológico , Malária Falciparum/parasitologia , Antagonistas do Ácido Fólico/farmacologia , Galactosídeos/farmacologia , Galactosídeos/uso terapêuticoRESUMO
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.
Assuntos
Farmacorresistência Fúngica , Antagonistas do Ácido Fólico , Metotrexato , Mutação , Pneumocystis carinii , Tetra-Hidrofolato Desidrogenase , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Tetra-Hidrofolato Desidrogenase/química , Pneumocystis carinii/genética , Pneumocystis carinii/enzimologia , Pneumocystis carinii/efeitos dos fármacos , Antagonistas do Ácido Fólico/farmacologia , Farmacorresistência Fúngica/genética , Metotrexato/farmacologia , Regulação Alostérica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Humanos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Domínio Catalítico/genéticaRESUMO
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.
Assuntos
Antibacterianos , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas , Pseudomonas aeruginosa , Tetra-Hidrofolato Desidrogenase , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Animais , Camundongos , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/tratamento farmacológico , Antibacterianos/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Antagonistas do Ácido Fólico/farmacologia , Ácido Fólico/metabolismo , Farmacorresistência Bacteriana , Modelos Animais de Doenças , Timina/metabolismo , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , FemininoRESUMO
p16 is a tumor suppressor encoded by the CDKN2A gene whose expression is lost in approximately 50% of all human cancers. In its canonical role, p16 inhibits the G1-S-phase cell cycle progression through suppression of cyclin-dependent kinases. Interestingly, p16 also has roles in metabolic reprogramming, and we previously published that loss of p16 promotes nucleotide synthesis via the pentose phosphate pathway. However, the broader impact of p16/CDKN2A loss on other nucleotide metabolic pathways and potential therapeutic targets remains unexplored. Using CRISPR knockout libraries in isogenic human and mouse melanoma cell lines, we determined several nucleotide metabolism genes essential for the survival of cells with loss of p16/CDKN2A. Consistently, many of these genes are upregulated in melanoma cells with p16 knockdown or endogenously low CDKN2A expression. We determined that cells with low p16/CDKN2A expression are sensitive to multiple inhibitors of de novo purine synthesis, including antifolates. Finally, tumors with p16 knockdown were more sensitive to the antifolate methotrexate in vivo than control tumors. Together, our data provide evidence to reevaluate the utility of these drugs in patients with p16/CDKN2Alow tumors as loss of p16/CDKN2A may provide a therapeutic window for these agents. SIGNIFICANCE: Antimetabolites were the first chemotherapies, yet many have failed in the clinic due to toxicity and poor patient selection. Our data suggest that p16 loss provides a therapeutic window to kill cancer cells with widely-used antifolates with relatively little toxicity.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina , Purinas , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Metotrexato/farmacologia , Purinas/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/uso terapêuticoRESUMO
The high lethality of Staphylococcus aureus infections and the emergence of antibiotic resistance make the development of new antibiotics urgent. Our previous work identified a hit compound h1 (AF-353) as a novel Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR) inhibitor. Herein, we analyzed the antimicrobial profile of h1 and performed a comprehensive structure-activity relationship (SAR) assay based on h1. The representative compound j9 exhibited potent antibacterial activity against S. aureus without cross-resistance to other antimicrobial classes. Multiple genetic and biochemical approaches showed that j9 directly binds to SaDHFR, resulting in strong inhibition of its enzymatic activity (IC50 = 0.97 nM). Additionally, j9 had an acceptable in vivo safety profile and oral bioavailability (F = 40.7%) and also showed favorable efficacy in a mouse model of methicillin-resistant S. aureus (MRSA) skin infection. Collectively, these findings identified j9 as a novel SaDHFR inhibitor with the potential to combat drug-resistant S. aureus infections.
Assuntos
Antagonistas do Ácido Fólico , Staphylococcus aureus Resistente à Meticilina , Éteres Fenílicos , Pirimidinas , Infecções Estafilocócicas , Animais , Camundongos , Staphylococcus aureus , Antagonistas do Ácido Fólico/farmacologia , Antibacterianos/química , Infecções Estafilocócicas/tratamento farmacológico , Testes de Sensibilidade MicrobianaRESUMO
Malaria parasites have adaptive mechanisms to modulate their intracellular redox status to tolerate the enhanced oxidizing effects created by malaria fever, hemoglobinopathies and other stress conditions, including antimalaria drugs. Emerging artemisinin (ART) resistance in Plasmodium falciparum is a complex phenotype linked to the parasite's tolerance of the activated drug's oxidative damage along with changes in vesicular transport, lipid metabolism, DNA repair, and exported proteins. In an earlier study, we discovered that many of these metabolic processes are induced in P. falciparum to respond to the oxidative damage caused by artemisinin, which exhibited a highly significant overlap with the parasite's adaptive response mechanisms to survive febrile temperatures. In addition, there was a significant overlap with the parasite's survival responses to oxidative stress. In this study, we investigated these relationships further using an in vitro model to evaluate if oxidative stress and heat-shock conditions could alter the parasite's response to artemisinin. The results revealed that compared to ideal culture conditions, the antimalarial efficacy of artemisinin was significantly reduced in parasites growing in intraerythrocytic oxidative stress but not in heat-shock condition. In contrast, heat shock significantly reduced the efficacy of lumefantrine that is an important ART combination therapy partner drug. We propose that prolonged exposure to intraerythrocytic microenvironmental oxidative stress, as would occur in endemic regions with high prevalence for sickle trait and other hemoglobinopathies, can predispose malaria parasites to develop tolerance to the oxidative damage caused by antimalarial drugs like artemisinin. IMPORTANCE: Emerging resistance to the frontline antimalarial drug artemisinin represents a significant threat to worldwide malaria control and elimination. The patterns of parasite changes associated with emerging resistance represent a complex array of metabolic processes evident in various genetic mutations and altered transcription profiles. Genetic factors identified in regulating P. falciparum sensitivity to artemisinin overlap with the parasite's responses to malarial fever, sickle trait, and other types of oxidative stresses, suggesting conserved inducible survival responses. In this study we show that intraerythrocytic stress conditions, oxidative stress and heat shock, can significantly decrease the sensitivity of the parasite to artemisinin and lumefantrine, respectively. These results indicate that an intraerythrocytic oxidative stress microenvironment and heat-shock condition can alter antimalarial drug efficacy. Evaluating efficacy of antimalarial drugs under ideal in vitro culture conditions may not accurately predict drug efficacy in all malaria patients.
Assuntos
Anemia Falciforme , Antimaláricos , Artemisininas , Antagonistas do Ácido Fólico , Hemoglobinopatias , Malária Falciparum , Malária , Humanos , Antimaláricos/farmacologia , Plasmodium falciparum/genética , Artemisininas/farmacologia , Malária Falciparum/tratamento farmacológico , Malária/tratamento farmacológico , Lumefantrina/farmacologia , Lumefantrina/uso terapêutico , Combinação de Medicamentos , Proteínas de Protozoários/genética , Antagonistas do Ácido Fólico/farmacologia , Estresse Oxidativo , Hemoglobinopatias/tratamento farmacológico , Anemia Falciforme/tratamento farmacológico , Resistência a Medicamentos/genéticaRESUMO
Malaria remains a leading cause of morbidity and mortality in Burkina Faso, which utilizes artemether-lumefantrine as the principal therapy to treat uncomplicated malaria and seasonal malaria chemoprevention with monthly sulfadoxine-pyrimethamine plus amodiaquine in children during the transmission season. Monitoring the activities of available antimalarial drugs is a high priority. We assessed the ex vivo susceptibility of Plasmodium falciparum to 11 drugs in isolates from patients presenting with uncomplicated malaria in Bobo-Dioulasso in 2021 and 2022. IC50 values were derived using a standard 72 h growth inhibition assay. Parasite DNA was sequenced to characterize known drug resistance-mediating polymorphisms. Isolates were generally susceptible, with IC50 values in the low-nM range, to chloroquine (median IC5010 nM, IQR 7.9-24), monodesethylamodiaquine (22, 14-46) piperaquine (6.1, 3.6-9.2), pyronaridine (3.0, 1.3-5.5), quinine (50, 30-75), mefloquine (7.1, 3.7-10), lumefantrine (7.1, 4.5-12), dihydroartemisinin (3.7, 2.2-5.5), and atovaquone (0.2, 0.1-0.3) and mostly resistant to cycloguanil (850, 543-1,290) and pyrimethamine (33,200, 18,400-54,200), although a small number of outliers were seen. Considering genetic markers of resistance to aminoquinolines, most samples had wild-type PfCRT K76T (87%) and PfMDR1 N86Y (95%) sequences. For markers of resistance to antifolates, established PfDHFR and PfDHPS mutations were highly prevalent, the PfDHPS A613S mutation was seen in 19% of samples, and key markers of high-level resistance (PfDHFR I164L; PfDHPS K540E) were absent or rare (A581G). Mutations in the PfK13 propeller domain known to mediate artemisinin partial resistance were not detected. Overall, our results suggest excellent susceptibilities to drugs now used to treat malaria and moderate, but stable, resistance to antifolates used to prevent malaria.
Assuntos
Antimaláricos , Antagonistas do Ácido Fólico , Malária Falciparum , Malária , Criança , Humanos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Plasmodium falciparum , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Combinação Arteméter e Lumefantrina/uso terapêutico , Antagonistas do Ácido Fólico/farmacologia , Burkina Faso , Artemeter/uso terapêutico , Pirimetamina/farmacologia , Pirimetamina/uso terapêutico , Malária/tratamento farmacológico , Lumefantrina/farmacologia , Lumefantrina/uso terapêutico , Combinação de Medicamentos , Polimorfismo Genético/genética , Resistência a Medicamentos/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/uso terapêuticoRESUMO
One-carbon (C1) metabolism is compartmentalized between the cytosol and mitochondria with the mitochondrial C1 pathway as the major source of glycine and C1 units for cellular biosynthesis. Expression of mitochondrial C1 genes including SLC25A32, serine hydroxymethyl transferase (SHMT) 2, 5,10-methylene tetrahydrofolate dehydrogenase 2, and 5,10-methylene tetrahydrofolate dehydrogenase 1-like was significantly elevated in primary epithelial ovarian cancer (EOC) specimens compared with normal ovaries. 5-Substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF347, AGF359, AGF362) inhibited proliferation of cisplatin-sensitive (A2780, CaOV3, IGROV1) and cisplatin-resistant (A2780-E80, SKOV3) EOC cells. In SKOV3 and A2780-E80 cells, colony formation was inhibited. AGF347 induced apoptosis in SKOV3 cells. In IGROV1 cells, AGF347 was transported by folate receptor (FR) α. AGF347 was also transported into IGROV1 and SKOV3 cells by the proton-coupled folate transporter (SLC46A1) and the reduced folate carrier (SLC19A1). AGF347 accumulated to high levels in the cytosol and mitochondria of SKOV3 cells. By targeted metabolomics with [2,3,3-2H]L-serine, AGF347, AGF359, and AGF362 inhibited SHMT2 in the mitochondria. In the cytosol, SHMT1 and de novo purine biosynthesis (i.e., glycinamide ribonucleotide formyltransferase, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase) were targeted; AGF359 also inhibited thymidylate synthase. Antifolate treatments of SKOV3 cells depleted cellular glycine, mitochondrial NADH and glutathione, and showed synergistic in vitro inhibition toward SKOV3 and A2780-E80 cells when combined with cisplatin. In vivo studies with subcutaneous SKOV3 EOC xenografts in SCID mice confirmed significant antitumor efficacy of AGF347. Collectively, our studies demonstrate a unique metabolic vulnerability in EOC involving mitochondrial and cytosolic C1 metabolism, which offers a promising new platform for therapy.
Assuntos
Cisplatino , Citosol , Resistencia a Medicamentos Antineoplásicos , Mitocôndrias , Neoplasias Ovarianas , Humanos , Feminino , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Citosol/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/genética , Animais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Cisplatino/farmacologia , Camundongos , Linhagem Celular Tumoral , Carbono/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Glicina Hidroximetiltransferase/metabolismo , Glicina Hidroximetiltransferase/genética , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/genética , Antagonistas do Ácido Fólico/farmacologiaRESUMO
Dihydrofolate reductase (DHFR) is a ubiquitous enzyme that regulates the biosynthesis of tetrahydrofolate among various species of Plasmodium parasite. It is a validated target of the antifolate drug pyrimethamine (Pyr) in Plasmodium falciparum (Pf), but its clinical efficacy has been hampered due to the emergence of drug resistance. This has made the attempt to screen Food & Drug Administration-approved drugs against wild- and mutant PfDHFR by employing an in-silico pipeline to identify potent candidates. The current study has followed a virtual screening approach for identifying potential DHFR inhibitors from DrugBank database, based on a structure similarity search of candidates, followed by absorption, distribution, metabolism, and excretion estimation. The screened drugs were subjected to various parameters like docking, molecular mechanics with generalized born and surface area solvation calculations, and molecular simulations. We have thus identified two potential drug candidates, duloxetine and guanethidine, which can be repurposed to be tested for their efficacy against wild type and drug resistant falciparum malaria.
Assuntos
Antimaláricos , Antagonistas do Ácido Fólico , Malária , Humanos , Antimaláricos/farmacologia , Antimaláricos/química , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Preparações Farmacêuticas , Reposicionamento de Medicamentos , Malária/tratamento farmacológico , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/química , Resistência a Medicamentos , Ácido FólicoRESUMO
Toxic bacterial modules such as toxin-antitoxin systems hold antimicrobial potential, though successful applications are rare. Here we show that in Vibrio cholerae the cyclic-oligonucleotide-based anti-phage signalling system (CBASS), another example of a toxic module, increases sensitivity to antifolate antibiotics up to 10×, interferes with their synergy and ultimately enables bacterial lysis by these otherwise classic bacteriostatic antibiotics. Cyclic-oligonucleotide production by the CBASS nucleotidyltransferase DncV upon antifolate treatment confirms full CBASS activation under these conditions, and suggests that antifolates release DncV allosteric inhibition by folates. Consequently, the CBASS-antifolate interaction is specific to CBASS systems with closely related nucleotidyltransferases and similar folate-binding pockets. Last, antifolate resistance genes abolish the CBASS-antifolate interaction by bypassing the effects of on-target antifolate activity, thereby creating potential for their coevolution with CBASS. Altogether, our findings illustrate how toxic modules can impact antibiotic activity and ultimately confer bactericidal activity to classical bacteriostatic antibiotics.
Assuntos
Bacteriófagos , Antagonistas do Ácido Fólico , Vibrio cholerae , Antagonistas do Ácido Fólico/farmacologia , Bacteriófagos/genética , Antibacterianos/farmacologia , Vibrio cholerae/genética , Bactérias , OligonucleotídeosRESUMO
Approximately 619,000 malaria deaths were reported in 2021, and resistance to recommended drugs, including artemisinin-combination therapies (ACTs), threatens malaria control. Treatment failure with ACTs has been found to be as high as 93% in northeastern Thailand, and parasite mutations responsible for artemisinin resistance have already been reported in some African countries. Therefore, there is an urgent need to identify alternative treatments with novel targets. In this Perspective, we discuss some promising antimalarial drug targets, including enzymes involved in proteolysis, DNA and RNA metabolism, protein synthesis, and isoprenoid metabolism. Other targets discussed are transporters, Plasmodium falciparum acetyl-coenzyme A synthetase, N-myristoyltransferase, and the cyclic guanosine monophosphate-dependent protein kinase G. We have outlined mechanistic details, where these are understood, underpinning the biological roles and hence druggability of such targets. We believe that having a clear understanding of the underlying chemical interactions is valuable to medicinal chemists in their quest to design appropriate inhibitors.
Assuntos
Antimaláricos , Artemisininas , Antagonistas do Ácido Fólico , Malária Falciparum , Malária , Humanos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Antimaláricos/metabolismo , Malária/tratamento farmacológico , Plasmodium falciparum , Descoberta de Drogas , Antagonistas do Ácido Fólico/farmacologia , Artemisininas/metabolismo , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Resistência a MedicamentosRESUMO
Methotrexate (MTX) is a tight-binding dihydrofolate reductase (DHFR) inhibitor, used as both an antineoplastic and immunosuppressant therapeutic. MTX, like folate undergoes folylpolyglutamate synthetase-mediated γ-glutamylation, which affects cellular retention and target specificity. Mechanisms of MTX resistance in cancers include a decrease in MTX poly-γ-glutamylation and an upregulation of DHFR. Here, we report a series of potent MTX-based proteolysis targeting chimeras (PROTACs) to investigate DHFR degradation pharmacology and one-carbon biochemistry. These on-target, cell-active PROTACs show proteasome- and E3 ligase-dependent activity, and selective degradation of DHFR in multiple cancer cell lines. By comparison, treatment with MTX increases cellular DHFR protein expression. Importantly, these PROTACs produced distinct, less-lethal phenotypes compared to MTX. The chemical probe set described here should complement conventional DHFR inhibitors and serve as useful tools for studying one-carbon biochemistry and dissecting complex polypharmacology of MTX and related drugs. Such compounds may also serve as leads for potential autoimmune and antineoplastic therapeutics.
Assuntos
Antineoplásicos , Antagonistas do Ácido Fólico , Neoplasias , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carbono , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/uso terapêutico , Metotrexato/farmacologia , Metotrexato/metabolismo , Metotrexato/uso terapêutico , Neoplasias/tratamento farmacológico , Quimera de Direcionamento de Proteólise , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
The Mycobacterium abscessus drug development pipeline is poorly populated, with particularly few validated target-lead couples to initiate de novo drug discovery. Trimethoprim, an inhibitor of dihydrofolate reductase (DHFR) used for the treatment of a range of bacterial infections, is not active against M. abscessus. Thus, evidence that M. abscessus DHFR is vulnerable to pharmacological intervention with a small molecule inhibitor is lacking. Here, we show that the pyrrolo-quinazoline PQD-1, previously identified as a DHFR inhibitor active against Mycobacterium tuberculosis, exerts whole cell activity against M. abscessus. Enzyme inhibition studies showed that PQD-1, in contrast to trimethoprim, is a potent inhibitor of M. abscessus DHFR and over-expression of DHFR causes resistance to PQD-1, providing biochemical and genetic evidence that DHFR is a vulnerable target and mediates PQD-1's growth inhibitory activity in M. abscessus. As observed in M. tuberculosis, PQD-1 resistant mutations mapped to the folate pathway enzyme thymidylate synthase (TYMS) ThyA. Like trimethoprim in other bacteria, PQD-1 synergizes with the dihydropteroate synthase (DHPS) inhibitor sulfamethoxazole (SMX), offering an opportunity to exploit the successful dual inhibition of the folate pathway and develop similarly potent combinations against M. abscessus. PQD-1 is active against subspecies of M. abscessus and a panel of clinical isolates, providing epidemiological validation of the target-lead couple. Leveraging a series of PQD-1 analogs, we have demonstrated a dynamic structure-activity relationship (SAR). Collectively, the results identify M. abscessus DHFR as an attractive target and PQD-1 as a chemical starting point for the discovery of novel drugs and drug combinations that target the folate pathway in M. abscessus.
Assuntos
Antagonistas do Ácido Fólico , Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Mycobacterium tuberculosis , Humanos , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Trimetoprima/farmacologia , Mycobacterium tuberculosis/metabolismo , Inibidores Enzimáticos/farmacologia , Ácido Fólico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológicoRESUMO
Pharmacological inhibition of dihydrofolate reductase (DHFR) is an established approach for treating a variety of human diseases, including foreign infections and cancer. However, treatment with classic DHFR inhibitors, such as methotrexate (MTX), are associated with negative side-effects and resistance mechanisms that have prompted the search for alternatives. The DHFR inhibitor pyrimethamine (Pyr) has compelling anti-cancer activity in in vivo models, but lacks potency compared to MTX, thereby requiring higher concentrations to induce therapeutic responses. The purpose of this work was to investigate structural analogues of Pyr to improve its in vitro and cellular activity. A series of 36 Pyr analogues were synthesized and tested in a sequence of in vitro and cell-based assays to monitor their DHFR inhibitory activity, cellular target engagement, and impact on breast cancer cell viability. Ten top compounds were identified, two of which stood out as potential lead candidates, 32 and 34. These functionalized Pyr analogues potently engaged DHFR in cells, at concentrations as low as 1 nM and represent promising DHFR inhibitors that could be further explored as potential anti-cancer agents.