RESUMO
Human thymidylate synthase (hTS) is pivotal for cell survival and proliferation, indeed it provides the only synthetic source of dTMP, required for DNA biosynthesis. hTS represents a validated target for anticancer chemotherapy. However, active site-targeting drugs towards hTS have limitations connected to the onset of resistance. Thus, new strategies have to be applied to effectively target hTS without inducing resistance in cancer cells. Here, we report the generation and the functional and structural characterization of a new hTS interface variant in which Arg175 is replaced by a cysteine. Arg175 is located at the interface of the hTS obligate homodimer and protrudes inside the active site of the partner subunit, in which it provides a fundamental contribution for substrate binding. Indeed, the R175C variant results catalytically inactive. The introduction of a cysteine at the dimer interface is functional for development of new hTS inhibitors through innovative strategies, such as the tethering approach. Structural analysis, performed through X-ray crystallography, has revealed that a cofactor derivative is entrapped inside the catalytic cavity of the hTS R175C variant. The peculiar binding mode of the cofactor analogue suggests new clues exploitable for the design of new hTS inhibitors.
Assuntos
Timidilato Sintase/química , Timidilato Sintase/metabolismo , Substituição de Aminoácidos , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Domínio Catalítico , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Variação Genética , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/genéticaRESUMO
Thymidylate synthase (TS) is an enzyme of paramount importance as it provides the only de novo source of deoxy-thymidine monophosphate (dTMP). dTMP, essential for DNA synthesis, is produced by the TS-catalyzed reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) using N5,N10-methylenetetrahydrofolate (mTHF) as a cofactor. TS is ubiquitous and a validated drug target. TS enzymes from different organisms differ in sequence and structure, but are all obligate homodimers. The structural and mechanistic differences between the human and bacterial enzymes are exploitable to obtain selective inhibitors of bacterial TSs that can enrich the currently available therapeutic tools against bacterial infections. Enterococcus faecalis is a pathogen fully dependent on TS for dTMP synthesis. In this study, we present four new crystal structures of Enterococcus faecalis and human TSs in complex with either the substrate dUMP or the inhibitor FdUMP. The results provide new clues about the half-site reactivity of Enterococcus faecalis TS and the mechanisms underlying the conformational changes occurring in the two enzymes. We also identify relevant differences in cofactor and inhibitor binding between Enterococcus faecalis and human TS that can guide the design of selective inhibitors against bacterial TSs.
Assuntos
Enterococcus faecalis/enzimologia , Fluordesoxiuridilato/química , Conformação Proteica , Timidina Monofosfato/química , Timidilato Sintase/química , Sítios de Ligação , Domínio Catalítico , Fluordesoxiuridilato/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Timidina Monofosfato/metabolismo , Timidilato Sintase/metabolismoRESUMO
Thymidylate synthase (TS) is a prominent drug target for different cancer types. However, the prolonged use of its classical inhibitors, substrate analogs that bind at the active site, leads to TS overexpression and drug resistance in the clinic. In the effort to identify anti-TS drugs with new modes of action and able to overcome platinum drug resistance in ovarian cancer, octapeptides with a new allosteric inhibition mechanism were identified as cancer cell growth inhibitors that do not cause TS overexpression. To improve the biological properties, 10 cyclic peptides (cPs) were designed from the lead peptides and synthesized. The cPs were screened for the ability to inhibit recombinant human thymidylate synthase (hTS), and peptide 7 was found to act as an allosteric inhibitor more potent than its parent open-chain peptide [Pro3]LR. In cytotoxicity studies on three human ovarian cancer cell lines, IGROV-1, A2780, and A2780/CP, peptide 5 and two other cPs, including 7, showed IC50 values comparable with those of the reference drug 5-fluorouracil, of the open-chain peptide [d-Gln4]LR, and of another seven prolyl derivatives of the lead peptide LR. These promising results indicate cP 7 as a possible lead compound to be chemically modified with the aim of improving both allosteric TS inhibitory activity and anticancer effectiveness.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Sítio Alostérico , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ativação Enzimática , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clinically important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The "LR" peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concentrations in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.
Assuntos
Inibidores Enzimáticos/farmacologia , Terapia de Alvo Molecular , Neoplasias Ovarianas/enzimologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Feminino , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Peptídeos/química , Peptídeos/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Multimerização Proteica/efeitos dos fármacos , Termodinâmica , Timidilato Sintase/química , Timidilato Sintase/metabolismoRESUMO
Pteridine reductase 1 (PTR1) is a catalytic protein belonging to the folate metabolic pathway in Trypanosmatidic parasites. PTR1 is a known target for the medicinal chemistry development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. In previous studies, new nitro derivatives were elaborated as PTR1 inhibitors. The compounds showing a diamino-pyrimidine core structure were previously developed but they showed limited efficacy. Therefore, a new class of phenyl-, heteroaryl- and benzyloxy-nitro derivatives based on the 2-nitroethyl-2,4,6-triaminopyrimidine scaffold were designed and tested. The compounds were assayed for their ability to inhibit T. brucei and L. major PTR1 enzymes and for their antiparasitic activity towards T. brucei and L. infantum parasites. To understand the structure-activity relationships of the compounds against TbPTR1, the X-ray crystallographic structure of the 2,4,6-triaminopyrimidine (TAP) was obtained and molecular modelling studies were performed. As a next step, only the most effective compounds against T. brucei were then tested against the amastigote cellular stage of T. cruzi, searching for a broad-spectrum antiprotozoal agent. An early ADME-Tox profile evaluation was performed. The early toxicity profile of this class of compounds was investigated by measuring their inhibition of hERG and five cytochrome P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), cytotoxicity towards A549 cells and mitochondrial toxicity. Pharmacokinetic studies (SNAP-PK) were performed on selected compounds using hydroxypropyl-ß-cyclodextrins (50 % w/v) to preliminarily study their plasma concentration when administered per os at a dose of 20 mg/kg. Compound 1p, showed the best pharmacodynamic and pharmacokinetic properties, can be considered a good candidate for further bioavailability and efficacy studies.
Assuntos
Antiprotozoários , Doença de Chagas , Trypanosoma brucei brucei , Trypanosoma cruzi , Humanos , Relação Estrutura-Atividade , Antiprotozoários/química , Modelos Moleculares , Antiparasitários/farmacologia , Doença de Chagas/tratamento farmacológicoRESUMO
Broad-spectrum anti-infective chemotherapy agents with activity against Trypanosomes, Leishmania, and Mycobacterium tuberculosis species were identified from a high-throughput phenotypic screening program of the 456 compounds belonging to the Ty-Box, an in-house industry database. Compound characterization using machine learning approaches enabled the identification and synthesis of 44 compounds with broad-spectrum antiparasitic activity and minimal toxicity against Trypanosoma brucei, Leishmania Infantum, and Trypanosoma cruzi. In vitro studies confirmed the predictive models identified in compound 40 which emerged as a new lead, featured by an innovative N-(5-pyrimidinyl)benzenesulfonamide scaffold and promising low micromolar activity against two parasites and low toxicity. Given the volume and complexity of data generated by the diverse high-throughput screening assays performed on the compounds of the Ty-Box library, the chemoinformatic and machine learning tools enabled the selection of compounds eligible for further evaluation of their biological and toxicological activities and aided in the decision-making process toward the design and optimization of the identified lead.
Assuntos
Leishmania infantum , Trypanosoma brucei brucei , Trypanosoma cruzi , Ensaios de Triagem em Larga Escala , AntiparasitáriosRESUMO
Drug resistance to therapeutic antibiotics poses a challenge to the identification of novel targets and drugs for the treatment of infectious diseases. Infections caused by Enterococcus faecalis are a major health problem. Thymidylate synthase (TS) from E. faecalis is a potential target for antibacterial therapy. The X-ray crystallographic structure of E. faecalis thymidylate synthase (EfTS), which was obtained as a native binary complex composed of EfTS and 5-formyltetrahydrofolate (5-FTHF), has been determined. The structure provides evidence that EfTS is a half-of-the-sites reactive enzyme, as 5-FTHF is bound to two of the four independent subunits present in the crystal asymmetric unit. 5-FTHF is a metabolite of the one-carbon transfer reaction catalysed by 5-formyltetrahydrofolate cyclo-ligase. Kinetic studies show that 5-FTHF is a weak inhibitor of EfTS, suggesting that the EfTS-5-FTHF complex may function as a source of folates and/or may regulate one-carbon metabolism. The structure represents the first example of endogenous 5-FTHF bound to a protein involved in folate metabolism.
Assuntos
Enterococcus faecalis/enzimologia , Ácido Fólico/química , Timidilato Sintase/química , Sequência de Aminoácidos , Animais , Ácido Fólico/metabolismo , Humanos , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência , Timidilato Sintase/metabolismoRESUMO
The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective antiparasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure-activity relationship for multitarget inhibition. The approach yielded compounds showing apparent picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei while retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds, and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.
Assuntos
Leishmania major , Oxirredutases , Tetra-Hidrofolato Desidrogenase , Trypanosoma brucei brucei , Leishmania major/efeitos dos fármacos , Leishmania major/enzimologia , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Pteridinas/química , Pteridinas/farmacologia , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologiaRESUMO
Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anticancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, E7, we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.
Assuntos
Neoplasias Ovarianas , Timidilato Sintase , Feminino , Animais , Camundongos , Humanos , Sítios de Ligação , Timidilato Sintase/química , Timidilato Sintase/metabolismo , Fluoruracila/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Inibidores Enzimáticos/farmacologiaRESUMO
Three open-source anti-kinetoplastid chemical boxes derived from a whole-cell phenotypic screening by GlaxoSmithKline (Tres Cantos Anti-Kinetoplastid Screening, TCAKS) were exploited for the discovery of a novel core structure inspiring new treatments of parasitic diseases targeting the trypansosmatidic pteridine reductase 1 (PTR1) and dihydrofolate reductase (DHFR) enzymes. In total, 592 compounds were tested through medium-throughput screening assays. A subset of 14 compounds successfully inhibited the enzyme activity in the low micromolar range of at least one of the enzymes from both Trypanosoma brucei and Lesihmania major parasites (pan-inhibitors), or from both PTR1 and DHFR-TS of the same parasite (dual inhibitors). Molecular docking studies of the protein-ligand interaction focused on new scaffolds not reproducing the well-known antifolate core clearly explaining the experimental data. TCMDC-143249, classified as a benzenesulfonamide derivative by the QikProp descriptor tool, showed selective inhibition of PTR1 and growth inhibition of the kinetoplastid parasites in the 5 µM range. In our work, we enlarged the biological profile of the GSK Kinetobox and identified new core structures inhibiting selectively PTR1, effective against the kinetoplastid infectious protozoans. In perspective, we foresee the development of selective PTR1 and DHFR inhibitors for studies of drug combinations.
RESUMO
In the process of drug discovery the lead-identification phase may be critical due to the likely poor safety profile of the candidates, causing the delay or even the abandonment of a certain project. Nowadays, combining molecular modeling and in vivo cellular evaluation can help to identify compounds with an enhanced safety profile. Previously, two quinoxalines have been identified as inhibitors of the folate-dependent proteins belonging to the thymidylate synthase cycle. Unfortunately, cytotoxic activity against a panel of cisplatin(cDDP)-sensitive ovarian carcinoma cell lines and their resistant counterparts was coupled with toxicity to non-tumorigenic Vero cells. Here we describe the application of a ligand-based virtual screening, and several [1,2,4]triazolo[4,3-a]quinoxalines were optimized to improve their ADME-tox profile. The resulting 4-(trifluoromethyl)-1-p-tolyl-[1,2,4]triazolo[4,3-a]quinoxaline (24), which interferes intracellularly with DHFR and TS reducing the protein levels like 5-FU, but without inducing TS ternary complex formation, was 2-times less toxic in vitro than cisplatin and 5-FU.
Assuntos
Antagonistas do Ácido Fólico/química , Ácido Fólico/metabolismo , Ligantes , Quinoxalinas/química , Triazóis/química , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/toxicidade , Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacocinética , Antagonistas do Ácido Fólico/toxicidade , Humanos , Quinoxalinas/farmacocinética , Quinoxalinas/toxicidade , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/metabolismo , Triazóis/farmacocinética , Triazóis/toxicidadeRESUMO
Fourteen lots of cooked ham in modified atmosphere packaging (CH) were analyzed within a few days from packaging (S) and at the end of the shelf-life (E), after storage at 7⯰C to simulate thermal abuse. Five more lots, rejected from the market because spoiled (R), were included in the study. Quality of the products was generally compromised during the shelf life, with only 4 lots remaining unaltered. Analysis of 16S rRNA gene amplicons resulted in 801 OTUs. S samples presented a higher diversity than E and R ones. At the beginning of the shelf life, Proteobacteria and Firmicutes dominated the microbiota, with Acinetobacter, Brochothrix, Carnobacterium, Lactobacillus, Prevotella, Pseudomonas, Psychrobacter, Weissella, Vibrio rumoiensis occurring frequently and/or abundantly. E and R samples were dominated by Firmicutes mostly ascribed to Lactobacillales. It is noteworthy the appearance of abundant Leuconostoc, negligible in S samples, in some E and R samples, while in other LAB were outnumbered by V. rumoiensis or Brochothrix thermosphacta. The microbiota of spoiled and R samples could not be clustered on the basis of specific defects (discoloration, presence of slime, sourness, and swollen packages) or supplemented additives. LAB population of S samples, averaging 2.9 log10(cfu/g), increased to 7.7 log10(cfu/g) in the E and R samples. Dominant cultivable LAB belonged to the species Lactobacillus sakei and Leuconostoc carnosum. The same biotypes ascribed to different species where often found in the corresponding S and R samples, and sometime in different batches provided from the same producer, suggesting a recurrent contamination from the plant of production. Consistently with growth of LAB, initial pH (6.26) dropped to 5.74 in E samples. Volatiles organic compound (VOCs) analysis revealed that ethanol was the major metabolite produced during the shelf life. The profile of volatile compounds got enriched with other molecules (e.g. 2-butanone, ethyl acetate, acetic acid, acetoin, butanoic acid, ethyl ester, butanoic acid, and 2,3-butanediol) mainly ascribed to microbial metabolism.
Assuntos
Bactérias/classificação , Biodiversidade , Culinária , Microbiologia de Alimentos , Embalagem de Alimentos , Carne Vermelha/microbiologia , Ácido Acético/análise , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Contagem de Colônia Microbiana , RNA Ribossômico 16S/genética , Suínos , Fatores de TempoRESUMO
2-Amino-benzo[ d]thiazole was identified as a new scaffold for the development of improved pteridine reductase-1 (PTR1) inhibitors and anti-trypanosomatidic agents. Molecular docking and crystallography guided the design and synthesis of 42 new benzothiazoles. The compounds were assessed for Trypanosoma brucei and Leishmania major PTR1 inhibition and in vitro activity against T. brucei and amastigote Leishmania infantum. We identified several 2-amino-benzo[ d]thiazoles with improved enzymatic activity ( TbPTR1 IC50 = 0.35 µM; LmPTR1 IC50 = 1.9 µM) and low µM antiparasitic activity against T. brucei. The ten most active compounds against TbPTR1 were able to potentiate the antiparasitic activity of methotrexate when evaluated in combination against T. brucei, with a potentiating index between 1.2 and 2.7. The compound library was profiled for early ADME toxicity, and 2-amino- N-benzylbenzo[ d]thiazole-6-carboxamide (4c) was finally identified as a novel potent, safe, and selective anti-trypanocydal agent (EC50 = 7.0 µM). Formulation of 4c with hydroxypropyl-ß-cyclodextrin yielded good oral bioavailability, encouraging progression to in vivo studies.
Assuntos
Antiprotozoários/química , Benzotiazóis/química , Inibidores Enzimáticos/química , Leishmania major/enzimologia , Oxirredutases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Trypanosoma brucei brucei/enzimologia , Animais , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Benzotiazóis/metabolismo , Benzotiazóis/farmacologia , Benzotiazóis/uso terapêutico , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Meia-Vida , Leishmania major/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Oxirredutases/metabolismo , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase/tratamento farmacológico , Tripanossomíase/patologiaRESUMO
Chemical modulation of the flavonol 2-(benzo[d][1,3]dioxol-5-yl)-chromen-4-one (1), a promising anti-Trypanosomatid agent previously identified, was evaluated through a phenotypic screening approach. Herein, we have performed structure-activity relationship studies around hit compound 1. The pivaloyl derivative (13) showed significant anti-T. brucei activity (EC50 = 1.1 µM) together with a selectivity index higher than 92. The early in vitro ADME-tox properties (cytotoxicity, mitochondrial toxicity, cytochrome P450 and hERG inhibition) were determined for compound 1 and its derivatives, and these led to the identification of some liabilities. The 1,3-benzodioxole moiety in the presented compounds confers better in vivo pharmacokinetic properties than those of classical flavonols. Further studies using different delivery systems could lead to an increase of compound blood levels.
RESUMO
Leishmaniasis, a major health problem worldwide, has a limited arsenal of drugs for its control. The appearance of resistance to first- and second-line anti-leishmanial drugs confirms the need to develop new and less toxic drugs that overcome spontaneous resistance. In the present study, we report the design and synthesis of a novel library of 38 flavonol-like compounds and their evaluation in a panel of assays encompassing parasite killing, pharmacokinetics, genomics and ADME-Toxicity resulting in the progression of a compound in the drug discovery value chain. Compound 19, 2-(benzo[b]thiophen-3-yl)-3-hydroxy-6-methoxy-4H-chromen-4-one, exhibited a broad-spectrum activity against Leishmania spp. (EC50 1.9⯵M for Leishmania infantum, 3.4⯵M for L. donovani, 6.7⯵M for L. major), Trypanosoma cruzi (EC50 7.5⯵M) and T. brucei (EC50 0.8⯵M). Focusing on anti-Leishmania activity, compound 19 challenge in vitro did not select for resistance markers in L. donovani, while a Cos-Seq screening for dominant resistance genes identified a gene locus on chromosome 36 that became ineffective at concentrations beyond EC50. Thus, compound 19 is a promising scaffold to tackle drug resistance in Leishmania infection. In vivo pharmacokinetic studies indicated that compound 19 has a long half-life (intravenous (IV): 63.2â¯h; per os (PO): 46.9â¯h) with an acceptable ADME-Toxicity profile. When tested in Leishmania infected hamsters, no toxicity and limited efficacy were observed. Low solubility and degradation were investigated spectroscopically as possible causes for the sub-optimal pharmacokinetic properties. Compound 19 resulted a specific compound based on the screening against a protein set, following the intrinsic fluorescence changes.
Assuntos
Antiprotozoários , Flavonóis , Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Fosforilcolina/análogos & derivados , Tiofenos , Animais , Antiprotozoários/síntese química , Antiprotozoários/química , Antiprotozoários/farmacologia , Cricetinae , Avaliação Pré-Clínica de Medicamentos , Resistência a Medicamentos/efeitos dos fármacos , Flavonóis/síntese química , Flavonóis/química , Flavonóis/farmacologia , Genômica , Humanos , Fosforilcolina/química , Fosforilcolina/farmacologia , Tiofenos/síntese química , Tiofenos/química , Tiofenos/farmacologiaRESUMO
LR and [d-Gln4]LR peptides bind the monomer-monomer interface of human thymidylate synthase and inhibit cancer cell growth. Here, proline-mutated LR peptides were synthesized. Molecular dynamics calculations and circular dichroism spectra have provided a consistent picture of the conformational propensities of the [Pro n]-peptides. [Pro3]LR and [Pro4]LR show improved cell growth inhibition and similar intracellular protein modulation compared with LR. These represent a step forward to the identification of more rigid and metabolically stable peptides.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Peptídeos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Antineoplásicos/química , Linhagem Celular Tumoral , Dicroísmo Circular , Inibidores Enzimáticos/química , Feminino , Humanos , Simulação de Dinâmica Molecular , Mutação , Neoplasias Ovarianas/patologia , Peptídeos/química , Peptídeos/genética , Prolina/genética , Conformação Proteica , Timidilato Sintase/genética , Timidilato Sintase/metabolismoRESUMO
Basing on a library of thiadiazole derivatives showing anti-trypanosomatidic activity, we have considered the thiadiazoles opened forms and reaction intermediates, thiosemicarbazones, as compounds of interest for phenotypic screening against Trypanosoma brucei (Tb), intracellular amastigote form of Leishmania infantum (Li) and Trypanosoma cruzi (Tc). Similar compounds have already shown interesting activity against the same organisms. The compounds were particularly effective against T. brucei and T. cruzi. Among the 28 synthesized compounds, the best one was (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene) hydrazinecarbothioamide (A14) yielding a comparable anti-parasitic activity against the three parasitic species (TbEC50â¯=â¯2.31⯵M, LiEC50â¯=â¯6.14⯵M, TcEC50â¯=â¯1.31⯵M) and a Selectivity Index higher than 10 with respect to human macrophages, therefore showing a pan-anti-trypanosomatidic activity. (E)-2-((3'.4'-dimethoxy-[1.1'-biphenyl]-3-yl)methyle ne) hydrazinecarbothioamide (A12) and (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene)hydrazine carbothioamide (A14) were able to potentiate the anti-parasitic activity of methotrexate (MTX) when evaluated in combination against T. brucei, yielding a 6-fold and 4-fold respectively Dose Reduction Index for MTX. The toxicity profile against four human cell lines and a panel of in vitro early-toxicity assays (comprising hERG, Aurora B, five cytochrome P450 isoforms and mitochondrial toxicity) demonstrated the low toxicity for the thosemicarbazones class in comparison with known drugs. The results confirmed thiosemicarbazones as a suitable chemical scaffold with potential for the development of properly decorated new anti-parasitic drugs.
Assuntos
Antiprotozoários/farmacologia , Doença de Chagas/tratamento farmacológico , Tiossemicarbazonas/farmacologia , Trypanosoma/efeitos dos fármacos , Antiprotozoários/síntese química , Antiprotozoários/química , Relação Dose-Resposta a Droga , Humanos , Macrófagos/efeitos dos fármacos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Tiossemicarbazonas/síntese química , Tiossemicarbazonas/químicaRESUMO
Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 µM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.
RESUMO
Repurposing and repositioning drugs has become a frequently pursued and successful strategy in the current era, as new chemical entities are increasingly difficult to find and get approved. Herein we report an integrated BioGPS/FLAPdock pipeline for rapid and effective off-target identification and drug repurposing. Our method is based on the structural and chemical properties of protein binding sites, that is, the ligand image, encoded in the GRID molecular interaction fields (MIFs). Protein similarity is disclosed through the BioGPS algorithm by measuring the pockets' overlap according to which pockets are clustered. Co-crystallized and known ligands can be cross-docked among similar targets, selected for subsequent inâ vitro binding experiments, and possibly improved for inhibitory potency. We used human thymidylate synthase (TS) as a test case and searched the entire RCSB Protein Data Bank (PDB) for similar target pockets. We chose casein kinaseâ IIα as a control and tested a series of its inhibitors against the TS template. Ellagic acid and apigenin were identified as TS inhibitors, and various flavonoids were selected and synthesized in a second-round selection. The compounds were demonstrated to be active in the low-micromolar range.
Assuntos
Reposicionamento de Medicamentos/métodos , Simulação de Acoplamento Molecular/métodos , Timidilato Sintase/antagonistas & inibidores , Algoritmos , Apigenina/química , Sítios de Ligação , Caseína Quinase II/química , Nucleotídeos de Desoxiuracil/química , Ácido Elágico/química , Humanos , Ligação de Hidrogênio , Ligantes , Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Timidilato Sintase/química , Água/químicaRESUMO
Infections caused by Enterococcus faecalis (Ef) represent nowadays a relevant health problem. We selected Thymidylate synthase (TS) from this organism as a potential specific target for antibacterial therapy. We have previously demonstrated that species-specific inhibition of the protein can be achieved despite the relatively high structural similarity among bacterial TSs and human TS. We had previously obtained the EfTS crystal structure of the protein in complex with the metabolite 5-formyl-tetrahydrofolate (5-FTHF) suggesting the protein role as metabolite reservoir; however, protein-inhibitors complexes were still missing. In the present work we identified some inhibitors bearing the phthalimidic core from our in-house library and we performed crystallographic screening towards EfTS. We obtained two X-ray crystallographic structures: the first with a weak phthalimidic inhibitor bound in one subunit and 5-hydroxymethylene-6-hydrofolic acid (5-HMHF) in the other subunit; a second X-ray structure complex with methotrexate. The structural information achieved confirm the role of EfTS as an enzyme involved in the folate pool system and provide a structural basis for structure-based drug design.