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1.
Nat Commun ; 12(1): 2949, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011959

RESUMO

The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4'-desmethyltrimethoprim (4'-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4'-DTMP than in the presence of TMP. We find that 4'-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.


Assuntos
Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Resistência a Trimetoprima/genética , Trimetoprima/análogos & derivados , Substituição de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Cristalografia por Raios X , Evolução Molecular Direcionada , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Genes Bacterianos , Genótipo , Humanos , Modelos Moleculares , Mutação , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/química , Trimetoprima/farmacologia
2.
Nat Commun ; 12(1): 1676, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33723254

RESUMO

The recently identified Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. How this novel beta-coronavirus virus, and coronaviruses more generally, alter cellular metabolism to support massive production of ~30 kB viral genomes and subgenomic viral RNAs remains largely unknown. To gain insights, transcriptional and metabolomic analyses are performed 8 hours after SARS-CoV-2 infection, an early timepoint where the viral lifecycle is completed but prior to overt effects on host cell growth or survival. Here, we show that SARS-CoV-2 remodels host folate and one-carbon metabolism at the post-transcriptional level to support de novo purine synthesis, bypassing viral shutoff of host translation. Intracellular glucose and folate are depleted in SARS-CoV-2-infected cells, and viral replication is exquisitely sensitive to inhibitors of folate and one-carbon metabolism, notably methotrexate. Host metabolism targeted therapy could add to the armamentarium against future coronavirus outbreaks.


Assuntos
COVID-19/metabolismo , Carbono/metabolismo , Ácido Fólico/metabolismo , SARS-CoV-2/fisiologia , Replicação Viral , Células A549 , Animais , COVID-19/virologia , Chlorocebus aethiops , Efeito Citopatogênico Viral , Antagonistas do Ácido Fólico/farmacologia , Glucose/metabolismo , Humanos , Metotrexato/farmacologia , RNA Viral/biossíntese , SARS-CoV-2/efeitos dos fármacos , Serina/metabolismo , Transcrição Genética , Células Vero , Proteínas Virais/genética , Replicação Viral/efeitos dos fármacos
3.
PLoS Negl Trop Dis ; 14(8): e0008506, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745103

RESUMO

Plasmodium vivax has become the predominant malaria parasite and a major challenge for malaria elimination in the Greater Mekong Subregion (GMS). Yet, our knowledge about the evolution of P. vivax populations in the GMS is fragmental. We performed whole genome sequencing on 23 P. vivax samples from the China-Myanmar border (CMB) and used 21 high-coverage samples to compare to over 200 samples from the rest of the GMS. Using genome-wide single nucleotide polymorphisms (SNPs), we analyzed population differentiation, genetic structure, migration and potential selection using an array of methods. The CMB parasites displayed a higher proportion of monoclonal infections, and 52% shared over 90% of their genomes in identity-by-descent segments with at least one other sample from the CMB, suggesting preferential expansion of certain parasite strains in this region, likely resulting from the P. vivax outbreaks occurring during this study period. Principal component, admixture, fixation index and phylogenetic analyses all identified that parasites from the CMB were genetically distinct from parasites from eastern parts of the GMS (Cambodia, Laos, Vietnam, and Thailand), whereas the eastern GMS parasite populations were largely undifferentiated. Such a genetic differentiation pattern of the P. vivax populations from the GMS parasite was largely explainable through geographic distance. Using the genome-wide SNPs, we narrowed down to a set of 36 SNPs for differentiating parasites from different areas of the GMS. Genome-wide scans to determine selection in the genome with two statistical methods identified genes potentially under drug selection, including genes associated with antifolate resistance and genes linked to chloroquine resistance in Plasmodium falciparum.


Assuntos
Malária Vivax/epidemiologia , Malária Vivax/parasitologia , Plasmodium vivax/genética , Polimorfismo de Nucleotídeo Único , Antimaláricos/farmacologia , China , Surtos de Doenças , Resistência a Medicamentos/genética , Antagonistas do Ácido Fólico/farmacologia , Genômica , Humanos , Mianmar , Filogenia , Plasmodium vivax/efeitos dos fármacos
4.
Malar J ; 19(1): 107, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32127009

RESUMO

BACKGROUND: Resistance to anti-malarials is a major threat to the control and elimination of malaria. Sulfadoxine-pyrimethamine (SP) anti-malarial treatment was used as a national policy for treatment of uncomplicated falciparum malaria in Thailand from 1973 to 1990. In order to determine whether withdrawal of this antifolate drug has led to restoration of SP sensitivity, the prevalence of genetic markers of SP resistance was assessed in historical Thai samples. METHODS: Plasmodium falciparum DNA was collected from the Thailand-Myanmar, Thailand-Malaysia and Thailand-Cambodia borders during 2008-2016 (N = 233). Semi-nested PCR and nucleotide sequencing were used to assess mutations in Plasmodium falciparum dihydrofolate reductase (pfdhfr), P. falciparum dihydropteroate synthase (pfdhps). Gene amplification of Plasmodium falcipaurm GTP cyclohydrolase-1 (pfgch1) was assessed by quantitative real-time PCR. The association between pfdhfr/pfdhps mutations and pfgch1 copy numbers were evaluated. RESULTS: Mutations in pfdhfr/pfdhsp and pfgch1 copy number fluctuated overtime through the study period. Altogether, 14 unique pfdhfr-pdfhps haplotypes collectively containing quadruple to octuple mutations were identified. High variation in pfdhfr-pfdhps haplotypes and a high proportion of pfgch1 multiple copy number (51% (73/146)) were observed on the Thailand-Myanmar border compared to other parts of Thailand. Overall, the prevalence of septuple mutations was observed for pfdhfr-pfdhps haplotypes. In particular, the prevalence of pfdhfr-pfdhps, septuple mutation was observed in the Thailand-Myanmar (50%, 73/146) and Thailand-Cambodia (65%, 26/40) border. In Thailand-Malaysia border, majority of the pfdhfr-pfdhps haplotypes transaction from quadruple (90%, 9/10) to quintuple (65%, 24/37) during 2008-2016. Within the pfdhfr-pfdhps haplotypes, during 2008-2013 the pfdhps A/S436F mutation was observed only in Thailand-Myanmar border (9%, 10/107), while it was not identified later. In general, significant correlation was observed between the prevalence of pfdhfr I164L (ϕ = 0.213, p-value = 0.001) or pfdhps K540E/N (ϕ = 0.399, p-value ≤ 0.001) mutations and pfgch1 gene amplification. CONCLUSIONS: Despite withdrawal of SP as anti-malarial treatment for 17 years, the border regions of Thailand continue to display high prevalence of antifolate and anti-sulfonamide resistance markers in falciparum malaria. Significant association between pfgch1 amplification and pfdhfr (I164L) or pfdhps (K540E) resistance markers were observed, suggesting a compensatory mutation.


Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos/genética , Antagonistas do Ácido Fólico/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Pirimetamina/farmacologia , Sulfadoxina/farmacologia , Camboja/epidemiologia , DNA de Protozoário/genética , Teste em Amostras de Sangue Seco , Combinação de Medicamentos , Genótipo , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Mutação , Prevalência , Tailândia/epidemiologia
5.
Parasitol Res ; 119(1): 165-175, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31807868

RESUMO

A novel library of synthetic piperidine derivatives was used to screen against human lymphatic filarial parasite Brugia malayi. Piperidine has earlier been reported to have effect against parasites including rodent filarial nematodes. Compounds with hydroxyl substitutions (4Q and 4H) showed marked antifilarial effect. Molecular docking of 4H derivative showed more favorable thermodynamic parameters against thymidylate synthase of B. malayi than human counterpart. A wide difference between IC50 and LD50 ensured the therapeutic safety of the candidates against the filarial parasites. Addition of thymidine to the treatment regimen led to a significant reversal of antifilarial effect of 4H that confirmed inhibition of thymidylate synthase as pharmacological rationale. Apoptosis induced in the parasite as a consequence of probable inhibition of thymidylate synthase was studied by acridine orange/ethidium bromide fluorescent staining and poly (ADP-ribose) polymerase activity inhibition. Involvement of mitochondria was confirmed by decreased 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) conversion and increased cytosolic cytochrome c level in 4H treated microfilariae, compared with the untreated microfilariae. Moreover, Michael adduct of chalcone targeting dihydrofolate reductase and piperidine targeting thymidylate synthase demonstrated synergistic effect on the parasite, indicating the importance of inhibition of DNA synthesis by combined effect. In conclusion, piperidine derivatives with hydroxyl substitution have a great therapeutic potential with an apoptotic rationale involving mitochondrial pathway, due to possible inhibition of parasitic thymidylate synthase.


Assuntos
Brugia Malayi/efeitos dos fármacos , Filariose Linfática/tratamento farmacológico , Filaricidas/farmacologia , Piperidinas/farmacologia , Timidilato Sintase/antagonistas & inibidores , Animais , Chalcona/farmacologia , Replicação do DNA/efeitos dos fármacos , Filariose Linfática/parasitologia , Antagonistas do Ácido Fólico/farmacologia , Humanos , Microfilárias/efeitos dos fármacos , Simulação de Acoplamento Molecular , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , Sais de Tetrazólio , Timidina/farmacologia
6.
Arch Pharm (Weinheim) ; 353(2): e1900287, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31867798

RESUMO

Inhibition of dihydrofolate reductase from Mycobacterium tuberculosis-dihydrofolate reductase (Mtb-DHFR) has emerged as a promising approach for the treatment of tuberculosis. To identify novel Mtb-DHFR inhibitors, structure-based virtual screening (SBVS) of the Molecular Diversity Preservation International (MolMall) database was performed using Glide against the Mtb-DHFR and h-DHFR enzymes. On the basis of SBVS, receptor fit, drug-like filters, and ADMET (absorption, distribution, metabolism, excretion, and toxicity) analysis, 16 hits were selected and tested for their antitubercular activity against the H37 RV strain of M. tuberculosis. Five compounds showed promising activity with compounds 11436 and 15275 as the most potent hits with IC50 values of 0.65 and 12.51 µM, respectively, against the H37 RV strain of M. tuberculosis. The two compounds were further tested in the Mtb-DHFR and h-DHFR enzymatic assay for selectivity and were found to be three- to eight-fold selective towards Mtb-DHFR over h-DHFR with minimum inhibitory concentration values of 5.50, 73.89 µM and 42.00, 263.00 µM, respectively. In silico simulation studies also supported the stability of the protein-ligand complex formation. The present study demonstrates the successful utilization of in silico SBVS tools for the identification of novel and potential Mtb-DHFR inhibitors and compound 11436 ((2,4-dihydroxyphenyl)(3,4,5-trihydroxyphenyl)methanone) as a potential lead for the development of novel Mtb-DHFR inhibitors.


Assuntos
Antituberculosos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/metabolismo , Antituberculosos/síntese química , Antituberculosos/química , Relação Dose-Resposta a Droga , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Relação Estrutura-Atividade
7.
Cancer Lett ; 470: 134-140, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31733288

RESUMO

Cancer is a disease of uncontrolled cell growth and a major cause of death worldwide. Many molecular events characterize tumor initiation and progression. Global gene expression analyses using next-generation sequencing, proteomics and metabolomics show genomic, epigenetic, and metabolite concentration changes in various tumors. Molecular alterations identified include multiple cancer-driving mutations, gene fusions, amplifications, deletions, and post-translational modifications. Data integration from many high-throughput platforms unraveled dysregulation in many metabolic pathways in cancer. Since cancer cells are fast-growing, their metabolic needs are enhanced, hence the requirement for de novo synthesis of essential metabolites. One critical requirement of fast-growing cells and a historically important pathway in cancer is the nucleotide biosynthetic pathway and its enzymes are valuable targets for small molecule inhibition. Purines and pyrimidines are building blocks of DNA synthesis and due to their excessive growth, cancer cells extensively utilize de novo pathways for nucleotide biosynthesis. Methotrexate, one of the early chemotherapeutic agents, targets dihydrofolate reductase of the folate metabolic pathway that is involved in nucleotide biosynthesis. In this review, we discuss the nucleotide biosynthetic pathways in cancer and targeting opportunities.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Neoplasias/patologia , Nucleotídeos de Purina/biossíntese , Nucleotídeos de Pirimidina/biossíntese , Antimetabólitos Antineoplásicos/uso terapêutico , Vias Biossintéticas/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/uso terapêutico , Humanos , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Neoplasias/tratamento farmacológico , Processamento de Proteína Pós-Traducional , S-Adenosilmetionina/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Tetra-Hidrofolatos/metabolismo
8.
J Antibiot (Tokyo) ; 73(1): 5-27, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31578455

RESUMO

The development of new mechanisms of resistance among pathogens, the occurrence and transmission of genes responsible for antibiotic insensitivity, as well as cancer diseases have been a serious clinical problem around the world for over 50 years. Therefore, intense searching of new leading structures and active substances, which may be used as new drugs, especially against strain resistant to all available therapeutics, is very important. Dihydrofolate reductase (DHFR) has attracted a lot of attention as a molecular target for bacterial resistance over several decades, resulting in a number of useful agents. Trimethoprim (TMP), (2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine) is the well-known dihydrofolate reductase inhibitor and one of the standard antibiotics used in urinary tract infections (UTIs). This review highlights advances in design, synthesis, and biological evaluations in structural modifications of TMP as DHFR inhibitors. In addition, this report presents the differences in the active site of human and pathogen DHFR. Moreover, an excellent review of DHFR inhibition and their relevance to antimicrobial and parasitic chemotherapy was presented.


Assuntos
Desenvolvimento de Medicamentos/métodos , Antagonistas do Ácido Fólico/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Trimetoprima/farmacologia , Antibacterianos , Desenho de Fármacos , Descoberta de Drogas , Antagonistas do Ácido Fólico/uso terapêutico , Humanos , Trimetoprima/uso terapêutico
9.
Bioorg Med Chem ; 27(24): 115158, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31685330

RESUMO

The series of des-Cl (unsubstituted) and m-Cl phenyl analogues of PYR with various flexible 6-substituents were synthesized and studied for the binding affinities with highly resistant quadruple mutant (QM) DHFR. The derivatives carrying 4 atoms linker with a terminal carboxyl substituted on the aromatic ring exhibited good inhibition to the QM enzyme and also showed effective antimalarial activities against resistant P. falciparum bearing the mutant enzymes with relatively low cytotoxicity to mammalian cells. The X-ray crystallographic analysis of the enzyme-inhibitor complexes suggested that the hydrophobic substituent at 6-position was accommodated well in the hydrophobic pocket and the optimal length of the flexible linker could effectively promote the binding of the terminal carboxyl group to the key amino acid residues, Arg59 and Arg122.


Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Pirimetamina/análogos & derivados , Animais , Antimaláricos/química , Chlorocebus aethiops , Desenho de Fármacos , Resistência a Medicamentos , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Pirimetamina/química , Pirimetamina/farmacologia , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Células Vero
10.
Bioorg Med Chem ; 27(23): 115125, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31679978

RESUMO

We previously showed that classical 6-substituted pyrrolo[2,3-d]pyrimidine antifolates bind to folate receptor (FR) α and the target purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFTase) with different cis and trans conformations. In this study, we designed novel analogs of this series with an amide moiety in the bridge region that can adopt both the cis and trans lowest energy conformations. This provides entropic benefit, by restricting the number of side-chain conformations of the unbound ligand to those most likely to promote binding to FRα and the target enzyme required for antitumor activity. NMR of the most active compound 7 showed both cis and trans amide bridge conformations in ~1:1 ratio. The bridge amide group in the best docked poses of 7 in the crystal structures of FRα and GARFTase adopted both cis and trans conformations, with the lowest energy conformations predicted by Maestro and evidenced by NMR within 1 kcal/mol. Compound 7 showed ~3-fold increased inhibition of FRα-expressing cells over its non-restricted parent analog 1 and was selectively internalized by FRα over the reduced folate carrier (RFC), resulting in significant in vitro antitumor activity toward FRα-expressing KB human tumor cells. Antitumor activity of 7 was abolished by treating cells with adenosine but was incompletely protected by 5-aminoimidazole-4-carboxamide (AICA) at higher drug concentrations, suggesting GARFTase and AICA ribonucleotide formyltransferase (AICARFTase) in de novo purine biosynthesis as the likely intracellular targets. GARFTase inhibition by compound 7 was confirmed by an in situ cell-based activity assay. Our results identify a "first-in-class" classical antifolate with a novel amide linkage between the scaffold and the side chain aryl L-glutamate that affords exclusive selectivity for transport via FRα over RFC and antitumor activity resulting from inhibition of GARFTase and likely AICARFTase. Compound 7 offers significant advantages over clinically used inhibitors of this class that are transported by the ubiquitous RFC, resulting in dose-limiting toxicities.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Piridinas/química , Piridinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Amidas/química , Amidas/farmacocinética , Amidas/farmacologia , Animais , Antineoplásicos/farmacocinética , Vias Biossintéticas/efeitos dos fármacos , Células CHO , Linhagem Celular Tumoral , Cricetulus , Receptor 1 de Folato/metabolismo , Antagonistas do Ácido Fólico/farmacocinética , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Nucleotídeos de Purina/metabolismo , Piridinas/farmacocinética , Pirróis/farmacocinética
11.
Sci Rep ; 9(1): 15625, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31666629

RESUMO

Antibiotic resistance in Acinetobacter baumannii is a major global health threat. New drugs with novel chemical structures are needed to overcome a myriad of resistance mechanisms in A. baumannii. In this study, we screened an open-source Pathogen Box library for anti-A. baumannii compounds. Compound MMV675968 (a diaminoquinazoline analog) was the only non-reference compound found to inhibit the growth of all four A. baumannii test strains with IC50 of 0.6-2.7 µM, IC90 of 0.7-3.9 µM, and MIC of 1.6-10 µM. We showed that MMV675968 targeted A. baumannii dihydrofolate reductase (AbDHFR) as determined by an E. coli surrogate whose growth was dependent on AbDHFR function and by an in vitro DHFR activity assay. Additionally, chemical scaffolds of DHFR inhibitors that are effective as antibiotics against A. baumannii were identified using an in vitro DHFR activity assay and A. baumannii growth inhibition. MMV675968 was the most potent among DHFR inhibitors tested in inhibiting A. baumannii growth. This study shows for the first time that MMV675968 inhibits A. baumannii growth via selective inhibition of AbDHFR and is therefore a promising scaffold for further antibiotic development against A. baumannii.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/crescimento & desenvolvimento , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Antagonistas do Ácido Fólico/farmacologia , Quinazolinas/farmacologia , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/enzimologia , Antibacterianos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Quinazolinas/química , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
12.
Int J Mol Sci ; 20(20)2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31601031

RESUMO

: Bacterial, protozoan and other microbial infections share an accelerated metabolic rate. In order to ensure a proper functioning of cell replication and proteins and nucleic acids synthesis processes, folate metabolism rate is also increased in these cases. For this reason, folic acid antagonists have been used since their discovery to treat different kinds of microbial infections, taking advantage of this metabolic difference when compared with human cells. However, resistances to these compounds have emerged since then and only combined therapies are currently used in clinic. In addition, some of these compounds have been found to have an immunomodulatory behavior that allows clinicians using them as anti-inflammatory or immunosuppressive drugs. Therefore, the aim of this review is to provide an updated state-of-the-art on the use of antifolates as antibacterial and immunomodulating agents in the clinical setting, as well as to present their action mechanisms and currently investigated biomedical applications.


Assuntos
Anti-Infecciosos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Fatores Imunológicos/farmacologia , Animais , Anti-Infecciosos/química , Resistência a Medicamentos , Antagonistas do Ácido Fólico/química , Humanos , Fatores Imunológicos/química , Imunomodulação/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade
13.
ACS Chem Biol ; 14(12): 2841-2850, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31609568

RESUMO

Various riboswitch classes are being discovered that precisely monitor the status of important biological processes, including metabolic pathway function, signaling for physiological adaptations, and responses to toxic agents. Biochemical components for some of these processes might make excellent targets for the development of novel antibacterial molecules, which can be broadly sought by using phenotypic drug discovery (PDD) methods. However, PDD data do not normally provide clues regarding the target for each hit compound. We have developed and validated a robust fluorescent reporter system based on a ZTP riboswitch that identifies numerous folate biosynthesis inhibitors with high sensitivity and precision. The utility of the riboswitch-based PDD strategy was evaluated using Escherichia coli bacteria by conducting a 128 310-compound high-throughput screen, which identified 78 sulfanilamide derivatives among the many initial hits. Similarly, representatives of other riboswitch classes could be employed to rapidly match antibacterial hits with the biological processes they target.


Assuntos
Escherichia coli/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Ácido Fólico/biossíntese , Ensaios de Triagem em Larga Escala/métodos , Riboswitch , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Descoberta de Drogas , Antagonistas do Ácido Fólico/química , Reprodutibilidade dos Testes , Relação Estrutura-Atividade
14.
ACS Infect Dis ; 5(11): 1896-1906, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31565920

RESUMO

The spread of plasmid borne resistance enzymes in clinical Staphylococcus aureus isolates is rendering trimethoprim and iclaprim, both inhibitors of dihydrofolate reductase (DHFR), ineffective. Continued exploitation of these targets will require compounds that can broadly inhibit these resistance-conferring isoforms. Using a structure-based approach, we have developed a novel class of ionized nonclassical antifolates (INCAs) that capture the molecular interactions that have been exclusive to classical antifolates. These modifications allow for a greatly expanded spectrum of activity across these pathogenic DHFR isoforms, while maintaining the ability to penetrate the bacterial cell wall. Using biochemical, structural, and computational methods, we are able to optimize these inhibitors to the conserved active sites of the endogenous and trimethoprim resistant DHFR enzymes. Here, we report a series of INCA compounds that exhibit low nanomolar enzymatic activity and potent cellular activity with human selectivity against a panel of clinically relevant TMP resistant (TMPR) and methicillin resistant Staphylococcus aureus (MRSA) isolates.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Antagonistas do Ácido Fólico/química , Staphylococcus aureus Resistente à Meticilina/enzimologia , Infecções Estafilocócicas/microbiologia , Tetra-Hidrofolato Desidrogenase/química , Trimetoprima/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Antagonistas do Ácido Fólico/farmacologia , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
15.
EBioMedicine ; 48: 289-300, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31648989

RESUMO

BACKGROUND: Thymidylate synthase (TYMS) is a successful chemotherapeutic target for anticancer therapy. Numerous TYMS inhibitors have been developed and used for treating gastrointestinal cancer now, but they have limited clinical benefits due to the prevalent unresponsiveness and toxicity. It is urgent to identify a predictive biomarker to guide the precise clinical use of TYMS inhibitors. METHODS: Genome-scale CRISPR-Cas9 knockout screening was performed to identify potential therapeutic targets for treating gastrointestinal tumours as well as key regulators of raltitrexed (RTX) sensitivity. Cell-based functional assays were used to investigate how MYC regulates TYMS transcription. Cancer patient data were used to verify the correlation between drug response and MYC and/or TYMS mRNA levels. Finally, the role of NIPBL inactivation in gastrointestinal cancer was evaluated in vitro and in vivo. FINDINGS: TYMS is essential for maintaining the viability of gastrointestinal cancer cells, and is selectively inhibited by RTX. Mechanistically, MYC presets gastrointestinal cancer sensitivity to RTX through upregulating TYMS transcription, supported by TCGA data showing that complete response cases to TYMS inhibitors had significantly higher MYC and TYMS mRNA levels than those of progressive diseases. NIPBL inactivation decreases the therapeutic responses of gastrointestinal cancer to RTX through blocking MYC. INTERPRETATION: Our study unveils a mechanism of how TYMS is transcriptionally regulated by MYC, and provides rationales for the precise use of TYMS inhibitors in the clinic. FUNDING: This work was financially supported by grants of NKRDP (2016YFC1302400), STCSM (16JC1406200), NSFC (81872890, 81322034, 81372346) and CAS (QYZDB-SSW-SMC034, XDA12020210).


Assuntos
Resistência a Medicamentos/genética , Antagonistas do Ácido Fólico/farmacologia , Neoplasias Gastrointestinais/genética , Regulação Neoplásica da Expressão Gênica , Genes myc , Timidilato Sintase/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Neoplasias Gastrointestinais/metabolismo , Humanos , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/metabolismo , Transcrição Genética
16.
Eur J Pharmacol ; 863: 172665, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31542479

RESUMO

Glioma is one of the most lethal malignancies and molecular regulators driving gliomagenesis are incompletely understood. Although temozolomide (TMZ) has been applied for malignant gliomas as a canonical chemotherapy, the treatment of glioma still remains limited due to frequently developed resistance to TMZ. Therefore, promising strategies that sensitize glioma cells to temozolomide are overwhelming to develop. Here we found that the expression of dihydrofolate reductase (DHFR) and thymidylate synthetase (TYMS), which played an essential role in folate metabolism and several types of tumors, were up-regulated in both human glioma tissues and cell lines, and overexpression of DHFR/TYMS promoted the proliferation of glioma cells. Notably, inhibition of DHFR/TYMS by pemetrexed exhibited synergistic anti-glioma activity with TMZ in both cell lines and U251 xenografts, which suggested potential combined chemotherapy for glioma. Mechanistically, the synergistic effect of inhibition of DHFR/TYMS with TMZ was due to activated AMPK and subsequently suppressed mTOR signaling pathway. Taken together, these findings identify an uncharacterized role of DHFR/TYMS in glioma growth and TMZ sensitivity mediated by AMPK-mTOR signal pathway, and provide a prospective approach for improving the anti-tumor activity of TMZ in glioma.


Assuntos
Glioma/patologia , Temozolomida/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Antagonistas do Ácido Fólico/farmacologia , Glioma/tratamento farmacológico , Glioma/enzimologia , Humanos , Camundongos , Terapia de Alvo Molecular , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Timidilato Sintase/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Arch Pharm (Weinheim) ; 352(9): e1900099, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31381192

RESUMO

Optimization of a modified Grimmel's method for N-heterocyclization of a leucine-linked sulfonamide side-arm at position 2 leading to 2,3-disustituted-4-quinazolin-(3H)-ones was accomplished. Further, 22 hybrid quinazolinone motifs (4a-v) were synthesized by N-heterocyclization reaction under microwave irradiation using the ionic liquid [Bmim][BF4 ]-H2 O as green solvent as well as the catalyst. The in vitro screening of the hybrid entities against the malarial species Plasmodium falciparum yielded five potent molecules 4l, 4n, 4o, 4t, and 4u owning antimalarial activity comparable to those of the reference drugs. In continuation, an in silico study was carried out to obtain a pharmacophoric model and quantitative structure-activity relationship. We also built a 3D-QSAR model to procure more information that could be applied to design new molecules with more potent Pf-DHFR inhibitory activity. The designed pharmacophore was recognized to be more potent for the selected molecules, exhibiting five pharmacophoric features. The active scaffolds were further evaluated for enzyme inhibition efficacy against alleged receptor Pf-DHFR computationally and in vitro, proving their candidature as lead dihydrofolate reductase inhibitors, and the selectivity of the test candidates was ascertained by toxicity study against Vero cells. Good oral bioavailability was also proved by studying pharmacokinetic properties.


Assuntos
Antimaláricos/síntese química , Desenho de Fármacos , Antagonistas do Ácido Fólico/síntese química , Ácido Fólico/metabolismo , Leucina/química , Quinazolinas/química , Sulfonamidas/síntese química , Animais , Antimaláricos/farmacocinética , Antimaláricos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Antagonistas do Ácido Fólico/farmacocinética , Antagonistas do Ácido Fólico/farmacologia , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Estrutura Molecular , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Relação Quantitativa Estrutura-Atividade , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Células Vero
18.
Postgrad Med ; 131(8): 589-596, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31399001

RESUMO

No new drugs for treatment of toxoplasmosis have been approved in over 60 years, despite the burden of toxoplasmosis on human society. The small selection of effective drugs is limited by important side effects, often limiting patient use. This perspective highlights promising late-stage drug candidates in the treatment of toxoplasmosis. Presently, drugs target the tachyzoite form of the parasite Toxoplasma gondii responsible for the acute infection but do not eradicate the tissue cyst form underlying chronic infection. Pyrimethamine - the first-line and only approved drug for treatment of toxoplasmosis in the United States - inhibits parasite DNA synthesis by inhibiting dihydrofolate reductase (DHFR). Two novel DHFR inhibitors with improved potency and selectivity for parasite DHFR over human DHFR are in clinical-stage development. One of the most advanced and promising therapeutic targets, demonstrating potential to treat both acute and chronic toxoplasmosis, is the calcium-dependent protein kinase 1 (CDPK1) which plays an essential role in the intracellular replicative cycle of the parasite, and has no direct mammalian homolog. Two CDPK1 inhibitor programs have identified potent and selective lead series, demonstrating acceptable systemic and CNS exposure, and in vivo efficacy in animal models of acute and chronic infection. Physicians need a better arsenal of parasiticidal drugs for the treatment of toxoplasmosis, particularly those active against tissue cysts.


Assuntos
Antiprotozoários/uso terapêutico , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Antagonistas do Ácido Fólico/uso terapêutico , Proteínas de Protozoários/antagonistas & inibidores , Toxoplasmose/tratamento farmacológico , Doença Aguda , Animais , Antiprotozoários/efeitos adversos , Antiprotozoários/farmacologia , Doença Crônica , Antagonistas do Ácido Fólico/efeitos adversos , Antagonistas do Ácido Fólico/farmacologia , Humanos , Proteínas Quinases , Pirimetamina/uso terapêutico , Tetra-Hidrofolato Desidrogenase , Toxoplasma , Estados Unidos
19.
Nanoscale ; 11(32): 15224-15233, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31385577

RESUMO

One of the frontiers of nanomedicine is the rational design of theranostic nanovectors. These are nanosized materials combining diagnostic and therapeutic capabilities, i.e. capable of tracking cancer cells and tissues in complex environments, and of selectively acting against them. We herein report on the preparation and application of antifolate plasmonic nanovectors, made of functionalized gold nanoparticles conjugated with the folic acid competitors aminopterin and methotrexate. Due to the overexpression of folate binding proteins on many types of cancer cells, these nanosystems can be exploited for selective cancer cell targeting. The strong surface enhanced Raman scattering (SERS) signature of these nanovectors acts as a diagnostic tool, not only for tracing their presence in biological samples, but also, through a careful spectral analysis, to precisely quantify the amount of drug loaded on a single nanoparticle, and therefore delivered to the cells. Meanwhile, the therapeutic action is implemented based on the strong toxicity of antifolate drugs. Remarkably, supplying the drug in the nanostructured form, rather than as a free molecule, enhances its specific toxicity. The selectivity of the antifolate nanovectors can be optimized by the design of a hybrid folate/antifolate coloaded nanovector for the specific targeting of folate receptor α, which is overexpressed on numerous cancer cell types.


Assuntos
Antagonistas do Ácido Fólico/química , Nanoestruturas/química , Análise Espectral Raman , Nanomedicina Teranóstica , Aminopterina/química , Aminopterina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácido Fólico/química , Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Ouro/química , Humanos , Nanopartículas Metálicas/química , Metotrexato/química , Metotrexato/farmacologia
20.
Molecules ; 24(17)2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31466322

RESUMO

A series of Schiff bases 14-25 were designed and synthesized for evaluation of their antibacterial properties against multi-drug resistant bacteria (MDRB). The antibacterial activities of Schiff bases 14-25 showed that most of the synthesized compounds displayed a significant antibacterial activity. Assessment of in silico ADMET properties (absorption, distribution, metabolism, excretion and toxicity) of Schiff bases illustrates that all derivatives showed agreement to the Lipinski's rule of five. Further enzymatic assay aided by molecular docking study demonstrated that compound 18 is a potent inhibitor of staphylococcus aureus DNA gyrase and dihydrofolate reductase kinases. This study could be valuable in the discovery of new potent antimicrobial agents.


Assuntos
Antibacterianos/síntese química , Pirazóis/química , Bases de Schiff/síntese química , Staphylococcus aureus/enzimologia , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Simulação por Computador , DNA Girase/metabolismo , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Bases de Schiff/química , Bases de Schiff/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/metabolismo , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia
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