RESUMO
Human African trypanosomiasis is among the World Health Organization's designated neglected tropical diseases. Repurposing strategies are often employed in academic drug discovery programs due to financial limitations, and in this instance, we used human kinase inhibitor chemotypes to identify substituted 4-aminoazaindoles, exemplified by 1. Structure-activity and structure-property relationship analysis, informed by cheminformatics, identified 4s as a potent inhibitor of Trypanosoma brucei growth. While 4s appeared to be fast acting and cidal in the in vitro assays, it failed to cure a murine model of infection. Preliminary efforts to identify the potential mechanism of action of the series pointed to arginine kinase, though, as we demonstrate, this does not appear to be the sole target of our compounds. This comprehensive approach to drug discovery, encompassing cheminformatics, structure-potency and structure-property analysis, and pharmacophore identification, highlights our multipronged efforts to identify novel lead compounds for this deadly disease.
Assuntos
Indóis , Tripanossomicidas , Trypanosoma brucei brucei , Trypanosoma brucei brucei/efeitos dos fármacos , Relação Estrutura-Atividade , Animais , Tripanossomicidas/farmacologia , Tripanossomicidas/química , Tripanossomicidas/síntese química , Indóis/química , Indóis/farmacologia , Indóis/síntese química , Humanos , Camundongos , Tripanossomíase Africana/tratamento farmacológico , Compostos Aza/química , Compostos Aza/farmacologia , Compostos Aza/síntese química , Estrutura Molecular , FarmacóforoRESUMO
The strasseriolide macrolides show promising in vitro and in vivo activities against P. falciparum and T. cruzi, the parasites causing malaria and Chagas disease, respectively. However, the as yet poor understanding of structure/activity relationships and the fact that one family member proved systemically toxic for unknown reasons render a more detailed assessment of these potential lead compounds difficult. To help overcome these issues, a collective total synthesis was devised. The key steps consisted of a ring closing alkyne metathesis (RCAM) reaction to forge a common macrocyclic intermediate followed by a hydroxy-directed ruthenium catalyzed trans-hydrostannation of the propargyl alcohol site thus formed. The resulting alkenyltin derivative served as the central node of the synthesis blueprint, which could be elaborated into the natural products themselves as well as into a set of non-natural analogues according to the concept of diverted total synthesis. The recorded biological data confirmed the potency of the compounds and showed the lack of any noticeable cytotoxicity. The "northern" allylic alcohol subunit was recognized as an integral part of the pharmacophore, yet it provides opportunities for chemical modification.
Assuntos
Macrolídeos , Plasmodium falciparum , Trypanosoma cruzi , Macrolídeos/farmacologia , Macrolídeos/síntese química , Macrolídeos/química , Plasmodium falciparum/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Relação Estrutura-Atividade , Antimaláricos/farmacologia , Antimaláricos/síntese química , Antimaláricos/química , Tripanossomicidas/farmacologia , Tripanossomicidas/síntese química , Tripanossomicidas/química , Humanos , Testes de Sensibilidade ParasitáriaRESUMO
IMPORTANCE: The COVID-19 pandemic has revealed the lack of effective treatments against betacoronaviruses and the urgent need for new broad-spectrum antivirals. Natural products are a valuable source of bioactive compounds with pharmaceutical potential that may lead to the discovery of new antiviral agents. Specifically, compared to conventional synthetic molecules, microbial natural extracts possess a unique and vast chemical diversity and are amenable to large-scale production. The implementation of a high-throughput screening platform using the betacoronavirus OC43 in a human cell line infection model has provided proof of concept of the approach and has allowed for the rapid and efficient evaluation of 1,280 microbial extracts. The identification of several active compounds validates the potential of the platform for the search for new compounds with antiviral capacity.
Assuntos
Produtos Biológicos , Coronavirus Humano OC43 , Humanos , Produtos Biológicos/farmacologia , Produtos Biológicos/metabolismo , Pandemias , Linhagem Celular , Antivirais/farmacologiaRESUMO
[This corrects the article DOI: 10.3389/fmicb.2023.1149145.].
RESUMO
Neglected diseases caused by kinetoplastid parasites are a health burden in tropical and subtropical countries. The need to create safe and effective medicines to improve treatment remains a priority. Microbial natural products are a source of chemical diversity that provides a valuable approach for identifying new drug candidates. We recently reported the discovery and bioassay-guided isolation of a novel family of macrolides with antiplasmodial activity. The novel family of four potent antimalarial macrolides, strasseriolides A-D, was isolated from cultures of Strasseria geniculata CF-247251, a fungal strain obtained from plant tissues. In the present study, we analyze these strasseriolides for activity against kinetoplastid protozoan parasites, namely, Trypanosoma brucei brucei, Leishmania donovani and Trypanosoma cruzi. Compounds exhibited mostly low activities against T. b. brucei, yet notable growth inhibition and selectivity were observed for strasseriolides C and D in the clinically relevant intracellular T. cruzi and L. donovani amastigotes with EC50 values in the low micromolar range. Compound C is fast-acting and active against both intracellular and trypomastigote forms of T. cruzi. While cell cycle defects were not identified, prominent morphological changes were visualized by differential interference contrast microscopy and smaller and rounded parasites were visualized upon exposure to strasseriolide C. Moreover, compound C lowers parasitaemia in vivo in acute models of infection of Chagas disease. Hence, strasseriolide C is a novel natural product active against different forms of T. cruzi in vitro and in vivo. The study provides an avenue for blocking infection of new cells, a strategy that could additionally contribute to avoid treatment failure.
Assuntos
Doença de Chagas , Parasitos , Trypanosoma brucei brucei , Trypanosoma cruzi , Animais , Doença de Chagas/tratamento farmacológico , Macrolídeos/farmacologiaRESUMO
Acanthamoeba species, Naegleria fowleri, and Balamuthia mandrillaris are opportunistic pathogens that cause a range of brain, skin, eye, and disseminated diseases in humans and animals. These pathogenic free-living amoebae (pFLA) are commonly misdiagnosed and have sub-optimal treatment regimens which contribute to the extremely high mortality rates (>90%) when they infect the central nervous system. To address the unmet medical need for effective therapeutics, we screened kinase inhibitor chemotypes against three pFLA using phenotypic drug assays involving CellTiter-Glo 2.0. Herein, we report the activity of the compounds against the trophozoite stage of each of the three amoebae, ranging from nanomolar to low micromolar potency. The most potent compounds that were identified from this screening effort were: 2d (A. castellanii EC50: 0.92 ± 0.3 µM; and N. fowleri EC50: 0.43 ± 0.13 µM), 1c and 2b (N. fowleri EC50s: <0.63 µM, and 0.3 ± 0.21 µM), and 4b and 7b (B. mandrillaris EC50s: 1.0 ± 0.12 µM, and 1.4 ± 0.17 µM, respectively). With several of these pharmacophores already possessing blood-brain barrier (BBB) permeability properties, or are predicted to penetrate the BBB, these hits present novel starting points for optimization as future treatments for pFLA-caused diseases.
RESUMO
Memnoniella is a fungal genus from which a wide range of diverse biologically active compounds have been isolated. A Memnoniella dichroa CF-080171 extract was identified to exhibit potent activity against Plasmodium falciparum 3D7 and Trypanosoma cruzi Tulahuen whole parasites in a high-throughput screening (HTS) campaign of microbial extracts from the Fundación MEDINA's collection. Bioassay-guided isolation of the active metabolites from this extract afforded eight new meroterpenoids of varying potencies, namely, memnobotrins C-E (1-3), a glycosylated isobenzofuranone (4), a tricyclic isobenzofuranone (5), a tetracyclic benzopyrane (6), a tetracyclic isobenzofuranone (7), and a pentacyclic isobenzofuranone (8). The structures of the isolated compounds were established by (+)-ESI-TOF high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Compounds 1, 2, and 4 exhibited potent antiparasitic activity against P. falciparum 3D7 (EC50 0.04-0.243 µM) and T. cruzi Tulahuen (EC50 0.266-1.37 µM) parasites, as well as cytotoxic activity against HepG2 tumoral liver cells (EC50 1.20-4.84 µM). The remaining compounds (3, 5-8) showed moderate or no activity against the above-mentioned parasites and cells.
RESUMO
In this study, we explored a fungal strain UIAU-3F identified as Aspergillus fumigatus isolated from soil samples collected from the River Oyun in Kwara State, Nigeria. In order to explore its chemical diversity, the fungal strain UIAU-3F was cultured in three different fermentation media, which resulted in different chemical profiles, evidenced by LC-ESI-MS-based metabolomics and multivariate analysis. The methanolic extract afforded two known compounds, fumitremorgin C (1) and pseurotin D (2). The in vitro antiparasitic assays of 1 against Trypanosoma cruzi and Plasmodium falciparum showed moderate activity with IC50 values of 9.6 µM and 2.3 µM, respectively, while 2 displayed IC50 values > 50 µM. Molecular docking analysis was performed on major protein targets to better understand the potential mechanism of the antitrypanosomal and antiplasmodial activities of the two known compounds.
RESUMO
BACKGROUND: Malaria is a global health problem for which novel therapeutic compounds are needed. To this end, a recently published novel family of antiplasmodial macrolides, strasseriolides A-D, was herein subjected to in vivo efficacy studies and preclinical evaluation in order to identify the most promising candidate(s) for further development. METHODS: Preclinical evaluation of strasseriolides A-D was performed by MTT-based cytotoxicity assay in THLE-2 (CRL-2706) liver cells, cardiotoxicity screening using the FluxOR™ potassium assay in hERG expressed HEK cells, LC-MS-based analysis of drug-drug interaction involving CYP3A4, CYP2D6 and CYP2C9 isoforms inhibition and metabolic stability assays in human liver microsomes. Mice in vivo toxicity studies were also accomplished by i.v. administration of the compounds (vehicle: 0.5% HPMC, 0.5% Tween 80, 0.5% Benzyl alcohol) in mice at 25 mg/kg dosage. Plasma were prepared from mice blood samples obtained at different time points (over a 24-h period), and analysed by LC-MS to quantify compounds. The most promising compounds, strasseriolides C and D, were subjected to a preliminary in vivo efficacy study in which transgenic GFP-luciferase expressing Plasmodium berghei strain ANKA-infected Swiss Webster female mice (n = 4-5) were treated 48 h post-infection with an i.p. dosage of strasseriolide C at 50 mg/kg and strasseriolide D at 22 mg/kg for four days after which luciferase activity was quantified on day 5 in an IVIS® Lumina II imager. RESULTS: Strasseriolides A-D showed no cytotoxicity, no carditoxicity and no drug-drug interaction problems in vitro with varying intrinsic clearance (CLint). Only strasseriolide B was highly toxic to mice in vivo (even at 1 mg/kg i.v. dosage) and, therefore, discontinued in further in vivo studies. Strasseriolide D showed statistically significant activity in vivo giving rise to lower parasitaemia levels (70% lower) compared to the controls treated with vehicle. CONCLUSIONS: Animal efficacy and preclinical evaluation of the recently discovered potent antiplasmodial macrolides, strasseriolides A-D, led to the identification of strasseriolide D as the most promising compound for further development. Future studies dealing on structure optimization, formulation and establishment of optimal in vivo dosage explorations of this novel compound class could enhance their clinical potency and allow for progress to later stages of the developmental pipeline.
Assuntos
Antimaláricos , Ascomicetos/química , Macrolídeos , Malária/tratamento farmacológico , Plasmodium berghei/efeitos dos fármacos , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Antimaláricos/toxicidade , Avaliação Pré-Clínica de Medicamentos , Feminino , Macrolídeos/química , Macrolídeos/farmacologia , Macrolídeos/toxicidade , CamundongosRESUMO
In the fight against Malaria, new strategies need to be developed to avoid resistance of the parasite to pharmaceutics and other prevention barriers. Recently, a Host Directed Therapy approach based on the suppression of the starting materials uptake from the host by the parasite has provided excellent results. In this article, we propose the synthesis of bioisosteric compounds that are capable of inhibiting Plasmodium falciparum Choline Kinase and therefore to reduce choline uptake, which is essential for the development of the parasite. Of the 41 bioisosteric compounds reported herein, none showed any influence of the linker on the antimalarial and enzyme inhibitory activity, whereas an effect of the type of cationic heads used could be observed. SARs determined that the thienopyrimidine substituted in 4 by a pyrrolidine is the best scaffold, independently of the chosen linker. The decrease in lipophilicity seems to improve the antimalarial activity but to cause an opposite effect on the inhibition of the enzyme. While potent compounds with similar good inhibitory values have been related to the proposed mechanism of action, some of them still show discrepancies and further studies are needed to determine their specific molecular target.
RESUMO
Neglected tropical diseases such as human African trypanosomiasis (HAT) are prevalent primarily in tropical climates and among populations living in poverty. Historically, the lack of economic incentive to develop new treatments for these diseases has meant that existing therapeutics have serious shortcomings in terms of safety, efficacy, and administration, and better therapeutics are needed. We now report a series of 3,5-disubstituted-7-azaindoles identified as growth inhibitors of Trypanosoma brucei, the parasite that causes HAT, through a high-throughput screen. We describe the hit-to-lead optimization of this series and the development and preclinical investigation of 29d, a potent antitrypanosomal compound with promising pharmacokinetic (PK) parameters. This compound was ultimately not progressed beyond in vivo PK studies due to its inability to penetrate the blood-brain barrier (BBB), critical for stage 2 HAT treatments.
Assuntos
Indóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Relação Dose-Resposta a Droga , Humanos , Indóis/síntese química , Indóis/química , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/químicaRESUMO
A novel family of four potent antimalarial macrolides, strasseriolides A-D (1-4), has been isolated from cultures of Strasseria geniculata CF-247251, a fungal strain obtained from plant tissues. The structures of these compounds, including their absolute configurations, were elucidated by HRMS, NMR spectroscopy, and X-ray single-crystal diffraction. The four compounds gave respective IC50 values of 9.810, 0.013, 0.123, and 0.128 µM against Plasmodium falciparum 3D7 parasites with no significant cytotoxicity against the HepG2 cell line.
Assuntos
Antibacterianos/farmacologia , Antimaláricos/farmacologia , Macrolídeos/farmacologia , Inibidores da Síntese de Proteínas/análise , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antimaláricos/química , Antimaláricos/isolamento & purificação , Ascomicetos , Fungos , Macrolídeos/química , Macrolídeos/isolamento & purificação , Estrutura Molecular , Inibidores da Síntese de Proteínas/químicaRESUMO
Ten bromotyrosine alkaloids were isolated and characterised from the marine sponge Aplysinella rhax (de Laubenfels 1954) collected from the Fiji Islands, which included one new bromotyrosine analogue, psammaplin P and two other analogues, psammaplin O and 3-bromo-2-hydroxy-5-(methoxycarbonyl)benzoic acid, which have not been previously reported from natural sources. HR-ESI-MS, 1D and 2D NMR spectroscopic methods were used in the elucidation of the compounds. Bisaprasin, a biphenylic dimer of psammaplin A, showed moderate activity with IC50 at 19±5 and 29±6â µM against Trypanzoma cruzi Tulahuen C4, and the lethal human malaria species Plasmodium falciparum clone 3D7, respectively, while psammaplins A and D exhibited low activity against both parasites. This is the first report of the antimalarial and antitrypanosomal activity of the psammaplin-type compounds. Additionally, the biosynthesis hypotheses of three natural products were proposed.
Assuntos
Alcaloides/farmacologia , Antiprotozoários/farmacologia , Produtos Biológicos/farmacologia , Poríferos/química , Trypanosoma cruzi/efeitos dos fármacos , Tirosina/análogos & derivados , Alcaloides/química , Alcaloides/isolamento & purificação , Animais , Antiprotozoários/química , Antiprotozoários/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Estrutura Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/efeitos dos fármacos , Tirosina/química , Tirosina/isolamento & purificação , Tirosina/farmacologiaRESUMO
Humanity faces great challenges, such as the rise of bacterial antibiotic resistance and cancer incidence. Thus, the discovery of novel therapeutics from underexplored environments, such as marine habitats, is fundamental. In this study, twelve strains from the phylum Firmicutes and thirty-four strains from the phylum Proteobacteria, isolated from marine sponges of the Erylus genus, collected in Portuguese waters, were tested for bioactivities and the secondary metabolites were characterised. Bioactivity screenings comprised antimicrobial, anti-fungal, anti-parasitic and anti-cancer assays. Selected bioactive extracts were further analysed for already described molecules through high performance liquid chromatography and mass spectrometry. Several bioactivities were observed against the fungus Aspergillusfumigatus, the bacteria (methicillin-resistant Staphylococcus aureus and Escherichia coli), the human liver cancer cell line HepG2 and the parasite Trypanosoma cruzi. Medium scale-up volume extracts confirmed anti-fungal activity by strains Proteus mirabilis #118_13 and Proteus sp. (JX006497) strain #118_20. Anti-parasitic activity was also confirmed in Enterococcus faecalis strain #118_3. Moreover, P. mirabilis #118_13 showed bioactivity in human melanoma cell line A2058 and the human hepatocellular carcinoma cell line HepG2. The dereplication of bioactive extracts showed the existence of a variety of secondary metabolites, with some unidentifiable molecules. This work shows that bacterial communities of sponges are indeed good candidates for drug discovery and, as far as we know, we describe anti-parasitic activity of a strain of E. faecalis and the presence of diketopiperazines in Proteus genus for the first time.
Assuntos
Bactérias/metabolismo , Dicetopiperazinas/isolamento & purificação , Dicetopiperazinas/metabolismo , Dicetopiperazinas/farmacologia , Poríferos/microbiologia , Animais , Antibacterianos/isolamento & purificação , Antifúngicos , Antineoplásicos/farmacologia , Antiparasitários/farmacologia , Bactérias/classificação , Linhagem Celular Tumoral , Dicetopiperazinas/química , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Firmicutes/classificação , Firmicutes/metabolismo , Fungos/efeitos dos fármacos , Células Hep G2/efeitos dos fármacos , Humanos , Neoplasias Hepáticas , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Simbiose , Trypanosoma cruzi/efeitos dos fármacosRESUMO
BACKGROUND: The proliferation and resistance of microorganisms area serious threat against humankind and the search for new therapeutics is needed. The present report describes the antiplasmodial and anticancer activities of samples isolated from the methanol extract of Albizia zygia (Mimosaseae). MATERIAL: The plant extract was prepared by maceration in methanol. Standard chromatographic, HPLC and spectroscopic methods were used to isolate and identify six compounds (1-6). The acetylated derivatives (7-10) were prepared by modifying 2-O-ß-D-glucopyranosyl-4-hydroxyphenylacetic acid and quercetin 3-O-α-L-rhamnopyranoside, previously isolated from A. zygia (Mimosaceae). A two-fold serial micro-dilution method was used to determine the IC50s against five tumor cell lines and Plasmodium falciparum. RESULTS: In general, compounds showed moderate activity against the human pancreatic carcinoma cell line MiaPaca-2 (10 < IC50 < 20 µM) and weak activity against other tumor cell lines such as lung (A-549), hepatocarcinoma (HepG2) and human breast adenocarcinoma (MCF-7and A2058) (IC50 > 20 µM). Additionally, the two semi-synthetic derivatives of quercetin 3-O-α-L-rhamnopyranoside exhibited significant activity against P. falciparum with IC50 of 7.47 ± 0.25 µM for compound 9 and 6.77 ± 0.25 µM for compound 10, higher than that of their natural precursor (IC50 25.1 ± 0.25 µM). CONCLUSION: The results of this study clearly suggest that, the appropriate introduction of acetyl groups into some flavonoids could lead to more useful derivatives for the development of an antiplasmodial agent.
Assuntos
Albizzia/química , Antimaláricos/farmacologia , Citotoxinas/farmacologia , Extratos Vegetais/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Camarões , Linhagem Celular Tumoral , Cromatografia Líquida , Humanos , Espectrometria de Massas , Estrutura Molecular , Folhas de Planta/química , Plasmodium falciparum/efeitos dos fármacosRESUMO
Streptomyces remains one of the prolific sources of structural diversity, and a reservoir to mine for novel natural products. Continued screening for new Streptomyces strains in our laboratory led to the isolation of Streptomyces sp. RK44 from the underexplored areas of Kintampo waterfalls, Ghana, Africa. Preliminary screening of the metabolites from this strain resulted in the characterization of a new 2-alkyl-4-hydroxymethylfuran carboxamide (AHFA) 1 together with five known compounds, cyclo-(L-Pro-Gly) 2, cyclo-(L-Pro-L-Phe) 3, cyclo-(L-Pro-L-Val) 4, cyclo-(L-Leu-Hyp) 5, and deferoxamine E 6. AHFA 1, a methylenomycin (MMF) homolog, exhibited anti-proliferative activity (EC50 = 89.6 µM) against melanoma A2058 cell lines. This activity, albeit weak is the first report amongst MMFs. Furthermore, the putative biosynthetic gene cluster (ahfa) was identified for the biosynthesis of AHFA 1. DFO-E 6 displayed potent anti-plasmodial activity (IC50 = 1.08µM) against P. falciparum 3D7. High-resolution electrospray ionization mass spectrometry (HR ESIMS) and molecular network assisted the targeted-isolation process, and tentatively identified six AHFA analogues, 7-12 and six siderophores 13-18.
Assuntos
Streptomyces/metabolismo , Antimaláricos/efeitos adversos , Antineoplásicos/efeitos adversos , Linhagem Celular Tumoral , Humanos , Família Multigênica/genética , Peptídeos/efeitos adversos , Peptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Human African trypanosomiasis is a neglected tropical disease (NTD) that is fatal if left untreated. Although approximately 13 million people live in moderate- to high-risk areas for infection, current treatments are plagued by problems with safety, efficacy, and emerging resistance. In an effort to fill the drug development pipeline for HAT, we have expanded previous work exploring the chemotype represented by the compound NEU-1090, with a particular focus on improvement of absorption, distribution, metabolism and elimination (ADME) properties. These efforts resulted in several compounds with substantially improved aqueous solubility, although these modifications typically resulted in a loss of trypanosomal activity. We herein report the results of our investigation into the antiparasitic activity, toxicity, and ADME properties of this class of compounds in the interest of informing the NTD drug discovery community and avoiding duplication of effort.
RESUMO
From a high-throughput screen of 42â¯444 known human kinases inhibitors, a pyrazolo[1,5-b]pyridazine scaffold was identified to begin optimization for the treatment of human African trypanosomiasis. Previously reported data for analogous compounds against human kinases GSK-3ß, CDK-2, and CDK-4 were leveraged to try to improve the selectivity of the series, resulting in 23a which showed selectivity for T. b. brucei over these three human enzymes. In parallel, properties known to influence the absorption, distribution, metabolism, and excretion (ADME) profile of the series were optimized resulting in 20g being progressed into an efficacy study in mice. Though 20g showed toxicity in mice, it also demonstrated CNS penetration in a PK study and significant reduction of parasitemia in four out of the six mice.
Assuntos
Piridazinas/síntese química , Piridazinas/farmacologia , Tripanossomicidas/síntese química , Tripanossomicidas/farmacologia , Tripanossomíase Africana/tratamento farmacológico , Animais , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Reposicionamento de Medicamentos , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Leishmania donovani/efeitos dos fármacos , Camundongos , Modelos Moleculares , Piridazinas/farmacocinética , Ratos , Relação Estrutura-Atividade , Especificidade por Substrato , Distribuição Tecidual , Tripanossomicidas/farmacocinética , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/parasitologiaRESUMO
To maintain dNTP pool homeostasis and preserve genetic integrity of nuclear and mitochondrial genomes, the synthesis and degradation of DNA precursors must be precisely regulated. Human all-alpha dCTP pyrophosphatase 1 (DCTPP1) is a dNTP pyrophosphatase with high affinity for dCTP and 5'-modified dCTP derivatives, but its contribution to overall nucleotide metabolism is controversial. Here, we identify a central role for DCTPP1 in the homeostasis of dCTP, dTTP and dUTP. Nucleotide pools and the dUTP/dTTP ratio are severely altered in DCTPP1-deficient cells, which exhibit an accumulation of uracil in genomic DNA, the activation of the DNA damage response and both a mitochondrial and nuclear hypermutator phenotype. Notably, DNA damage can be reverted by incubation with thymidine, dUTPase overexpression or uracil-DNA glycosylase suppression. Moreover, DCTPP1-deficient cells are highly sensitive to down-regulation of nucleoside salvage. Our data indicate that DCTPP1 is crucially involved in the provision of dCMP for thymidylate biosynthesis, introducing a new player in the regulation of pyrimidine dNTP levels and the maintenance of genomic integrity.
Assuntos
Nucleotídeos de Desoxicitosina/metabolismo , Nucleotídeos de Desoxiuracil/metabolismo , Pirofosfatases/metabolismo , Nucleotídeos de Timina/metabolismo , Linhagem Celular , Proliferação de Células , Dano ao DNA , Nucleotídeos de Desoxicitosina/genética , Nucleotídeos de Desoxiuracil/genética , Técnicas de Inativação de Genes , Instabilidade Genômica , Humanos , Células MCF-7 , Mutação , Pirofosfatases/genética , Nucleotídeos de Timina/genéticaRESUMO
Human African trypanosomiasis (HAT) is a neglected tropical disease caused by infection with either of two subspecies of the parasite Trypanosoma brucei. Due to a lack of economic incentive to develop new drugs, current treatments have severe limitations in terms of safety, efficacy, and ease of administration. In an effort to develop new HAT therapeutics, we report the structure-activity relationships around T. brucei for a series of benzoxazepinoindazoles previously identified through a high-throughput screen of human kinase inhibitors, and the subsequent in vivo experiments for HAT. We identified compound 18, which showed an improved kinase selectivity profile and acceptable pharmacokinetic parameters, as a promising lead. Although treatment with 18 cured 60% of mice in a systemic model of HAT, the compound was unable to clear parasitemia in a CNS model of the disease. We also report the results of cross-screening these compounds against T. cruzi, L. donovani, and S. mansoni.