RESUMEN
Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.
Asunto(s)
Proteínas de Transporte de Nucleósidos/metabolismo , Nucleósidos/metabolismo , Purinonas/metabolismo , Toxoplasma/fisiología , Toxoplasmosis/parasitología , Fibroblastos , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de Transporte de Nucleósidos/genética , Nucleósidos/química , Filogenia , Unión Proteica , Purinonas/química , Toxoplasma/clasificaciónRESUMEN
We report the design, synthesis, and validation of the novel compound photocaged N6-cyclopentyladenosine (cCPA) to achieve precisely localized and timed release of the parent adenosine A1 receptor agonist CPA using 405 nm light. Gi protein-coupled A1 receptors (A1Rs) modulate neurotransmission via pre- and post-synaptic routes. The dynamics of the CPA-mediated effect on neurotransmission, characterized by fast activation and slow recovery, make it possible to implement a closed-loop control paradigm. The strength of neurotransmission is monitored as the amplitude of stimulus-evoked local field potentials. It is used for feedback control of light to release CPA. This system makes it possible to regulate neurotransmission to a pre-defined level in acute hippocampal brain slices incubated with 3 µM cCPA. This novel approach of closed-loop photopharmacology holds therapeutic potential for fine-tuned control of neurotransmission in diseases associated with neuronal hyperexcitability.
Asunto(s)
Agonistas del Receptor de Adenosina A1 , Receptor de Adenosina A1 , Agonistas del Receptor de Adenosina A1/farmacología , Retroalimentación , Hipocampo/metabolismo , Receptor de Adenosina A1/metabolismo , Transmisión Sináptica , Xantinas/farmacologíaRESUMEN
Chagas disease, caused by Trypanosoma cruzi (T. cruzi), is a serious public health problem. Current treatment is restricted to two drugs, benznidazole and nifurtimox, displaying serious efficacy and safety drawbacks. Nucleoside analogues represent a promising alternative as protozoans do not biosynthesize purines and rely on purine salvage from the hosts. Protozoan transporters often present different substrate specificities from mammalian transporters, justifying the exploration of nucleoside analogues as therapeutic agents. Previous reports identified nucleosides with potent trypanocidal activity; therefore, two 7-derivatized tubercidins (FH11706, FH10714) and a 3'-deoxytubercidin (FH8513) were assayed against T. cruzi. They were highly potent and selective, and the uptake of the tubercidin analogues appeared to be mediated by the nucleoside transporter TcrNT2. At 10 µM, the analogues reduced parasitemia >90% in 2D and 3D cardiac cultures. The washout assays showed that FH10714 sterilized the infected cultures. Given orally, the compounds did not induce noticeable mouse toxicity (50 mg/kg), suppressed the parasitemia of T. cruzi-infected Swiss mice (25 mg/kg, 5 days) and presented DNA amplification below the limit of detection. These findings justify further studies with longer treatment regimens, as well as evaluations in combination with nitro drugs, aiming to identify more effective and safer therapies for Chagas disease.
Asunto(s)
Enfermedad de Chagas , Tripanocidas , Trypanosoma cruzi , Ratones , Animales , Nucleósidos/farmacología , Nucleósidos/uso terapéutico , Tripanocidas/farmacología , Tripanocidas/uso terapéutico , Tripanocidas/química , Parasitemia/tratamiento farmacológico , Enfermedad de Chagas/tratamiento farmacológico , MamíferosRESUMEN
A series of Mycobacterium tuberculosis TMPK (MtbTMPK) inhibitors based on a reported compound 3 were synthesized and evaluated for their capacity to inhibit MtbTMPK catalytic activity and the growth of a virulent M. tuberculosis strain (H37Rv). Modifications of the scaffold of 3 failed to afford substantial improvements in MtbTMPK inhibitory activity and antimycobacterial activity. Optimization of the substitution pattern of the D ring of 3 resulted in compound 21j with improved MtbTMPK inhibitory potency (three-fold) and H37Rv growth inhibitory activity (two-fold). Moving the 3-chloro substituent of 21j to the para-position afforded isomer 21h, which, despite a 10-fold increase in IC50-value, displayed promising whole cell activity (minimum inhibitory concentration (MIC) = 12.5 µM).
Asunto(s)
Antituberculosos , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos , Mycobacterium tuberculosis/enzimología , Nucleósido-Fosfato Quinasa/antagonistas & inhibidores , Timina , Antituberculosos/síntesis química , Antituberculosos/química , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Moleculares , Estructura Molecular , Nucleósido-Fosfato Quinasa/metabolismo , Relación Estructura-Actividad , Timina/análogos & derivados , Timina/síntesis química , Timina/química , Timina/farmacologíaRESUMEN
A series of new nucleoside analogues based on a C-3 branched ethynyl sugar derivative as present in 3'-C-ethynylcytidine (ECyd) and -adenosine (EAdo), combined with modified purine bases was synthetized and evaluated against a broad array of viruses and tumour cell lines. The pronounced cytostatic activity of EAdo was confirmed. EAdo and its 2,6-diaminopurine analogue showed inhibitory activity against vaccinia virus (EC50: 0.31 and 51 µM, respectively). Derivative 10 on the other hand was found active against varicella zoster virus (EC50: 4.68 µM).
Asunto(s)
Adenosina/análogos & derivados , Antineoplásicos/química , Antineoplásicos/farmacología , Antivirales/química , Antivirales/farmacología , Herpesvirus Humano 3/efectos de los fármacos , Purinas/química , Adenosina/síntesis química , Adenosina/química , Adenosina/farmacología , Antineoplásicos/síntesis química , Antivirales/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Purinas/farmacología , Relación Estructura-ActividadRESUMEN
Trypanosoma cruzi is a protozoan parasite and the etiological agent of Chagas disease, a debilitating and sometimes fatal disease that continues to spread to new areas. Yet, Chagas disease is still only treated with two related nitro compounds that are insufficiently effective and cause severe side effects. Nucleotide metabolism is one of the known vulnerabilities of T. cruzi, as they are auxotrophic for purines, and nucleoside analogues have been shown to have genuine promise against this parasite in vitro and in vivo. Since purine antimetabolites require efficient uptake through transporters, we here report a detailed characterisation of the T. cruzi NB1 nucleobase transporter with the aim of elucidating the interactions between TcrNB1 and its substrates and finding the positions that can be altered in the design of novel antimetabolites without losing transportability. Systematically determining the inhibition constants (Ki) of purine analogues for TcrNB1 yielded their Gibbs free energy of interaction, ΔG0. Pairwise comparisons of substrate (hypoxanthine, guanine, adenine) and analogues allowed us to determine that optimal binding affinity by TcrNB1 requires interactions with all four nitrogen residues of the purine ring, with N1 and N9, in protonation state, functioning as presumed hydrogen bond donors and unprotonated N3 and N7 as hydrogen bond acceptors. This is the same interaction pattern as we previously described for the main nucleobase transporters of Trypanosoma brucei spp. and Leishmania major and makes it the first of the ENT-family genes that is functionally as well as genetically conserved between the three main kinetoplast pathogens.
Asunto(s)
Guanina , Hipoxantina , Trypanosoma cruzi , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/química , Guanina/metabolismo , Hipoxantina/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/química , Proteínas de Transporte de Nucleobases/metabolismo , Proteínas de Transporte de Nucleobases/genética , Proteínas de Transporte de Nucleobases/química , Transporte Biológico , Especificidad por Sustrato , Unión Proteica , Nucleósidos/metabolismoRESUMEN
Profiling of the illicit fentanyl supply is invaluable from surveillance and intelligence perspectives. An important strategy includes the study of chemical attribution signatures (e.g., trace amounts of synthesis precursors, impurities/byproducts in seized material and metabolites in biological samples). This information provides valuable insight into the employed synthesis routes at the heart of illicit fentanyl manufacture (previously mainly the so-called Janssen or Siegfried methods), allowing to track and ultimately regulate crucial precursors. This report focuses on phenethyl-4-anilino-N-phenethylpiperidine (phenethyl-4-ANPP), a formerly unknown compound that was identified for the first time in a fentanyl powder sample seized in April 2019, followed by its identification in a biological sample in December 2019. Between 2019-Q4 and 2020-Q3, phenethyl-4-ANPP was detected in 25/1,054 fentanyl cases in the USA. There are currently no reports on how this compound may have ended up in illicit drug preparations and whether its presence may have potential in vivo relevance. We propose three possible fentanyl synthesis routes that, when badly executed in a single reaction vessel, may involve the formation of phenethyl-4-ANPP. We hypothesize that the presence of the latter is the result of a shift in fentanyl synthesis routes in an attempt to circumvent restrictions on previously used precursors. Using a cell-based µ-opioid receptor recruitment assay, we show that the extent of MOR activation caused by 100 µM phenethyl-4-ANPP is comparable to that exerted by a roughly 100,000-fold lower concentration of fentanyl (0.001 µM or 0.336 ng/mL). Negligible in vitro opioid activity, combined with its low abundance in fentanyl preparations, most likely renders phenethyl-4-ANPP biologically irrelevant in vivo. However, as clandestine operations are constantly changing shape, monitoring of fentanyl attributions remains pivotal in our understanding and control of illicit fentanyl manufacture and supply.
Asunto(s)
Analgésicos Opioides , FentaniloRESUMEN
Leishmaniasis causes high mortality and morbidity in tropical and subtropical regions of Africa, Asia, the Americas and southern Europe, and is characterized by diverse clinical manifestations. As a neglected tropical disease, limited resources are allocated for antileishmanial drug discovery. The Leishmania parasite is deficient in de novo purine synthesis, and therefore acquires purines from the host and processes these using a purine salvage pathway. By making use of purine transport systems and interfering with this salvage pathway, purine (nucleoside) analogues might exert a selective detrimental impact on its growth and survival. In vitro screening of an in-house purine nucleoside library and analogue synthesis afforded the 6-methyl-7-(2-pyridyl)-7-deazapurine ribonucleoside analogue 18 as a promising hit. Optimization of the 7-substituent afforded 31 and 32 which displayed potent activity against wild-type and resistant L. infantum, intracellular amastigote and extracellular promastigote forms, and favorable selectivity versus primary mouse macrophages (Mφ) and MRC-5 cells. Encouraged by the favorable in vitro metabolic stability of 32, an in vivo study was performed using an early curative L. infantum hamster model. When orally administrated at 50 mg/kg once daily (s.i.d) for 10 days, 32 was devoid of side effects, however, it only poorly reduced amastigote burdens in the major target organs.
Asunto(s)
Antiprotozoarios , Leishmania , Leishmaniasis , Purinas , Ribonucleósidos , Animales , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Cricetinae , Leishmania/efectos de los fármacos , Leishmania/metabolismo , Leishmaniasis/tratamiento farmacológico , Ratones , Nucleósidos/farmacología , Nucleósidos/uso terapéutico , Nucleósidos de Purina/farmacología , Nucleósidos de Purina/uso terapéutico , Purinas/farmacología , Purinas/uso terapéutico , Ribonucleósidos/farmacología , Ribonucleósidos/uso terapéuticoRESUMEN
Animal trypanosomiasis (AT) is a parasitic disease with high socio-economic impact. Given the limited therapeutic options and problems of toxicity and drug resistance, this study assessed redirecting our previously identified antitrypanosomal nucleosides for the treatment of AT. Promising hits were identified with excellent in vitro activity across all important animal trypanosome species. Compound 7, an inosine analogue, and our previously described lead compound, 3'-deoxytubercidin (8), showed broad spectrum anti-AT activity, metabolic stability in the target host species and absence of toxicity, but with variable efficacy ranging from limited activity to full cure in mouse models of Trypanosoma congolense and T. vivax infection. Several compounds show promise against T. evansi (surra) and T. equiperdum (dourine). Given the preferred target product profile for a broad-spectrum compound against AT, this study emphasizes the need to include T. vivax in the screening cascade given its divergent susceptibility profile and provides a basis for lead optimization towards such broad spectrum anti-AT compound.
Asunto(s)
Trypanosoma congolense , Trypanosoma , Tripanosomiasis , Animales , Modelos Animales de Enfermedad , Resistencia a Medicamentos , Ratones , Nucleósidos/uso terapéutico , Tripanosomiasis/tratamiento farmacológico , Tripanosomiasis/parasitologíaRESUMEN
Chagas disease and leishmaniasis are two poverty-related neglected tropical diseases that cause high mortality and morbidity. Current treatments suffer from severe limitations and novel, safer and more effective drugs are urgently needed. Both Trypanosoma cruzi and Leishmania are auxotrophic for purines and absolutely depend on uptake and assimilation of host purines. This led us to successfully explore purine nucleoside analogues as chemotherapeutic agents against these and other kinetoplastid infections. This study extensively explored the modification of the 6-amino group of tubercidin, a natural product with trypanocidal activity but unacceptable toxicity for clinical use. We found that mono-substitution of the amine with short alkyls elicits potent and selective antitrypanosomal and antileishmanial activity. The methyl analogue 15 displayed the best in vitro activity against both T. cruzi and L. infantum and high selectivity versus host cells. Oral administration for five consecutive days in an acute Chagas disease mouse model resulted in significantly reduced peak parasitemia levels (75, 89 and 96% with 12.5, 25 and 50 mg/kg/day, respectively). as well as increased animal survival rates with the lower doses (83 and 67% for 12.5 and 25 mg/kg/day, respectively).
Asunto(s)
Enfermedad de Chagas , Leishmania , Tripanocidas , Trypanosoma cruzi , Animales , Enfermedad de Chagas/tratamiento farmacológico , Ratones , Nucleósidos/farmacología , Purinas/farmacología , Purinas/uso terapéutico , Relación Estructura-Actividad , Tripanocidas/farmacología , Tripanocidas/uso terapéuticoRESUMEN
Kinetoplastid parasites are the causative agents of neglected tropical diseases with an unmet medical need. These parasites are unable to synthesize the purine ring de novo, and therefore rely on purine salvage to meet their purine demand. Evaluating purine nucleoside analogs is therefore an attractive strategy to identify antikinetoplastid agents. Several anti-Trypanosoma cruzi and anti-Trypanosoma brucei 7-deazapurine nucleosides were previously discovered, with the removal of the 3'-hydroxyl group resulting in a significant boost in activity. In this work we therefore decided to assess the effect of the introduction of a 3'-fluoro substituent in 7-deazapurine nucleosides on the anti-kinetoplastid activities. Hence, we synthesized two series of 3'-deoxy-3'-fluororibofuranosyl and 3'-deoxy-3'-fluoroxylofuranosyl nucleosides comprising 7-deazaadenine and -hypoxanthine bases and assayed these for antiparasitic activity. Several analogs with potent activity against T. cruzi and T. brucei were discovered, indicating that a fluorine atom in the 3'-position is a promising modification for the discovery of antiparasitic nucleosides.
Asunto(s)
Nucleósidos de Purina/química , Purinas/química , Tripanocidas/síntesis química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Nucleósidos de Purina/síntesis química , Nucleósidos de Purina/farmacología , Purinas/síntesis química , Purinas/farmacología , Relación Estructura-Actividad , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacosRESUMEN
New psychoactive substances continue to appear on the drug market. Until recently, new synthetic opioids, which are among the most dangerous new psychoactive substances, primarily encompassed analogs of the potent analgesic fentanyl. Lately, also other new synthetic opioids have increasingly started to surface. This is the first report on the identification and full chemical characterization of brorphine, a novel potent synthetic opioid with a piperidine benzimidazolone structure. A powder, identified as brorphine, was obtained from a patient seeking medical help for detoxification. Brorphine was also found in a serum sample of the patient. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) identified an exact mass of m/z 400.1020 and 402.1005 for the compound, corresponding to both bromine isotopes. Further chemical characterization was performed by gas chromatography-mass spectrometry, liquid chromatography-diode array detection and Fourier-transform infrared spectroscopy analyses. Finally, the structure was confirmed by performing 1H-NMR and 13C-NMR spectroscopy. In vitro biological activity of brorphine was determined by a cell-based µ-opioid receptor activation assay, resulting in an EC50 of 30.9 nM (13.5 ng/mL) and an Emax of 209% relative to hydromorphone, confirming the high potency and efficacy of this compound. In a serum sample of the patient, brorphine and a hydroxy-metabolite were found using the LC-HRMS screening method. The presence of opioid activity in the serum was also confirmed via the activity-based opioid screening assay. The occurrence of brorphine is yet another example of how the illicit drug market is continuously evolving in an attempt to escape international legislation. Its high potency poses a serious and imminent health threat for any user.
Asunto(s)
Analgésicos Opioides/sangre , Drogas Ilícitas/sangre , Imidazoles/sangre , Piperidinas/sangre , Psicotrópicos/sangre , Analgésicos Opioides/química , Cromatografía Liquida , Drogas de Diseño/análisis , Fentanilo/análogos & derivados , Cromatografía de Gases y Espectrometría de Masas , Humanos , Drogas Ilícitas/química , Imidazoles/química , Piperidinas/química , Psicotrópicos/química , Detección de Abuso de Sustancias , Espectrometría de Masas en TándemRESUMEN
Kinetoplastid parasites are the causative agents of Chagas disease (CD), leishmaniasis and human African trypanosomiasis (HAT). Despite a sustained decrease in the number of HAT cases, more efforts are needed to discover safe and effective therapies against CD and leishmaniasis. Kinetoplastid parasites lack the capability to biosynthesize purines de novo and thus critically depend on uptake and processing of purines from host cells. As such, modified purine nucleoside analogues may act as broad-spectrum antikinetoplastid agents. This study assessed the in vitro activity profile of 7-modified 6-methyl tubercidin derivatives against Trypanosoma cruzi, Leishmania infantum, Trypanosoma brucei brucei and T. b. rhodesiense, and led to the identification of analogues that display activity against all these species, such as 7-ethyl (13) and 7-chloro (7) analogues. These selected analogues also proved sufficiently stable in liver microsomes to warrant in vivo follow-up evaluation.
Asunto(s)
Antiprotozoarios , Trypanosoma brucei brucei , Trypanosoma cruzi , Antiprotozoarios/farmacología , Humanos , Nucleósidos/farmacología , Purinas , Relación Estructura-ActividadRESUMEN
In this paper, the identification and full characterization of a novel non-fentanyl opioid sourced online, which is a member of the 1-substituted-4-(1,2-diphenylethyl)piperazine derivatives related to MT-45, is reported. The sample was sold under the name "diphenpipenol," (3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenylethyl]phenol), although extensive NMR analysis showed that the product obtained was actually a diphenpipenol structural isomer, (2-[4-(2-methoxyphenyl)piperazin-1-yl]-1,2-diphenylethanol). Liquid chromatography time-of-flight mass spectrometry identified an exact mass for the protonated molecule of m/z 389.2264, with two prominent fragment ions (m/z 91.0567 and 150.0937), which were not reported in earlier literature describing MT-45 derivatives. The chemical characterization was finalized by gas chromatography-mass spectrometry, high-performance liquid chromatography diode array detector and Fourier-transform infrared spectroscopy analyses. This product is a clear example of the trend that new non-fentanyl opioids are reappearing on the recreational drug market to escape the recent changes in (inter)national legislation concerning fentanyl analogues. Although in this particular case, the product's potency and efficacy were relatively low, other new non-fentanyl opioids might possess stronger potencies and therefore pose greater health risks for ignorant users. The fact that the product was sold under the wrong name further demonstrates the well-known problematic issue of a mismatch between the adverted and true identity, confirming the irregularities of the online new psychoactive substances market.
Asunto(s)
Drogas de Diseño/análisis , Analgésicos Opioides , Cromatografía Líquida de Alta Presión , Cromatografía de Gases y Espectrometría de Masas , Drogas Ilícitas , PiperazinasRESUMEN
Human African trypanosomiasis is a neglected parasitic disease for which the current treatment options are quite limited. Trypanosomes are not able to synthesize purines de novo and thus solely depend on purine salvage from the host environment. This characteristic makes players of the purine salvage pathway putative drug targets. The activity of known nucleoside analogues such as tubercidin and cordycepin led to the development of a series of C7-substituted nucleoside analogues. Here, we use RNA interference (RNAi) libraries to gain insight into the mode-of-action of these novel nucleoside analogues. Whole-genome RNAi screening revealed the involvement of adenosine kinase and 4E interacting protein into the mode-of-action of certain antitrypanosomal nucleoside analogues. Using RNAi lines and gene-deficient parasites, 4E interacting protein was found to be essential for parasite growth and infectivity in the vertebrate host. The essential nature of this gene product and involvement in the activity of certain nucleoside analogues indicates that it represents a potential novel drug target.
RESUMEN
Trichomoniasis is the most common nonviral sexually transmitted disease in humans, but treatment options are limited. Here, we report a resorufin-based drug sensitivity assay for high-throughput microplate-based screening under hypoxic conditions. A 5203-compound enamine kinase library and several specialized compound series were tested for the inhibition of Trichomonas growth at 10 µM with Z' values of >0.5. Hits were rescreened in serial dilution to establish an IC50 concentration. A series of 7-substituted 7-deazaadenosine analogues emerged as highly potent anti-T. vaginalis agents, with EC50 values in the low double digit nanomolar range. These analogues exhibited excellent selectivity indices. Follow-up medicinal chemistry efforts identified an optimal ribofuranose and C7 substituent. Several nucleosides rapidly cleared cultures of T. vaginalis at a concentrations of just 2 × EC50. Preliminary in vivo evaluation in a murine trichomoniasis model (Tritrichomonas foetus) revealed promising activity upon topical administration, validating purine nucleoside analogues as a new class of antitrichomonal agents.
Asunto(s)
Enfermedades de Transmisión Sexual , Trichomonas vaginalis , Animales , Resistencia a Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Nucleósidos/farmacologíaRESUMEN
Chagas disease is a tropical infectious disease resulting in progressive organ-damage and currently lacks efficient treatment and vaccine options. The causative pathogen, Trypanosoma cruzi, requires uptake and processing of preformed purines from the host because it cannot synthesize these de novo, instigating the evaluation of modified purine nucleosides as potential trypanocides. By modifying the pyrimidine part of a previously identified 7-aryl-7-deazapurine nucleoside, we found that substitution of a 6-methyl for a 6-amino group allows retaining T. cruzi amastigote growth inhibitory activity but confers improved selectivity towards mammalian cells. By keeping the 6-methyl group unaltered, and introducing different 7-aryl groups, we identified several analogues with sub-micromolar antitrypanosomal activity. The 7-(4-chlorophenyl) analogue 14, which was stable in microsomes, was evaluated in an acute mouse model. Oral administration of 25â mg/kg b.i.d. suppressed peak parasitemia and protected mice from infection-related mortality, gave similar reductions as the reference drug of blood parasite loads determined by qPCR, but as benznidazole failed to induce sterile cure in the short time period of drug exposure (5â days).
Asunto(s)
Nucleósidos/farmacología , Purinas/farmacología , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Estructura Molecular , Nucleósidos/síntesis química , Nucleósidos/química , Pruebas de Sensibilidad Parasitaria , Purinas/síntesis química , Purinas/química , Relación Estructura-Actividad , Tripanocidas/síntesis química , Tripanocidas/químicaRESUMEN
Swapping the substituents in positions 2 and 4 of the previously synthesized but yet undisclosed 5-cyano-4-(methylthio)-2-arylpyrimidin-6-ones 4, ring closure, and further optimization led to the identification of the potent antitubercular 2-thio-substituted quinazolinone 26. Structure-activity relationship (SAR) studies indicated a crucial role for both meta-nitro substituents for antitubercular activity, while the introduction of polar substituents on the quinazolinone core allowed reduction of bovine serum albumin (BSA) binding (63c, 63d). While most of the tested quinazolinones exhibited no cytotoxicity against MRC-5, the most potent compound 26 was found to be mutagenic via the Ames test. This analogue exhibited moderate inhibitory potency against Mycobacterium tuberculosis thymidylate kinase, the target of the 3-cyanopyridones that lies at the basis of the current analogues, indicating that the whole-cell antimycobacterial activity of the present S-substituted thioquinazolinones is likely due to modulation of alternative or additional targets. Diminished antimycobacterial activity was observed against mutants affected in cofactor F420 biosynthesis (fbiC), cofactor reduction (fgd), or deazaflavin-dependent nitroreductase activity (rv3547), indicating that reductive activation of the 3,5-dinitrobenzyl analogues is key to antimycobacterial activity.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Nitrorreductasas/metabolismo , Quinazolinonas/farmacología , Riboflavina/análogos & derivados , Antituberculosos/química , Ensayos Analíticos de Alto Rendimiento , Pruebas de Sensibilidad Microbiana , Pruebas de Mutagenicidad , Mycobacterium tuberculosis/crecimiento & desarrollo , Quinazolinonas/química , Riboflavina/metabolismo , Relación Estructura-ActividadRESUMEN
Mycobacterium tuberculosis thymidylate kinase (MtTMPK) has emerged as an attractive target for rational drug design. We recently investigated new families of non-nucleoside MtTMPK inhibitors in an effort to diversify MtTMPK inhibitor chemical space. We here report a new series of MtTMPK inhibitors by combining the Topliss scheme with rational drug design approaches, fueled by two co-crystal structures of MtTMPK in complex with developed inhibitors. These efforts furnished the most potent MtTMPK inhibitors in our assay, with two analogues displaying low micromolar MIC values against H37Rv Mtb. Prepared inhibitors address new sub-sites in the MtTMPK nucleotide binding pocket, thereby offering new insights into its druggability. We studied the role of efflux pumps as well as the impact of cell wall permeabilizers for selected compounds to potentially provide an explanation for the lack of correlation between potent enzyme inhibition and whole-cell activity.
Asunto(s)
Antituberculosos/farmacología , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Nucleósido-Fosfato Quinasa/antagonistas & inhibidores , Piperidinas/farmacología , Timina/farmacología , Antituberculosos/síntesis química , Antituberculosos/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Mycobacterium tuberculosis/enzimología , Nucleósido-Fosfato Quinasa/metabolismo , Piperidinas/síntesis química , Piperidinas/química , Relación Estructura-Actividad , Timina/síntesis química , Timina/químicaRESUMEN
BACKGROUND: The protozoan Trypanosoma cruzi is auxotrophic for purines and causes Chagas' disease (CD), a neglected illness affecting >6 million people. Combining the 3-deoxyribofuranose part of cordycepin with the modified purine ring of a nucleoside 'hit' led to the discovery of 4-amino-5-(4-chlorophenyl)-N7-(3'-deoxy-ß-d-ribofuranosyl)-pyrrolo[2,3-d]pyrimidine (Cpd1), revealing promising anti-T. cruzi activity. OBJECTIVES: To further evaluate Cpd1 in vitro and in vivo to fully assess its therapeutic potential against CD, covering cell culture sterilization through washout assays, drug combination with benznidazole and long-term administration in T. cruzi-infected mice. RESULTS: Although less susceptible to Cpd1 than amastigotes, trypomastigotes present an impaired capacity to successfully establish intracellular infection of cardiac cultures. Combination of benznidazole with Cpd1 indicated no interaction (additive effect) (FIC index = 0.72) while administration to mice at one-tenth of the optimal dose (2.5 mg/kg and 10 mg/kg for Cpd1 and benznidazole, respectively) suppressed parasitaemia but failed to avoid mortality. Long-term treatment (60 days) gave a rapid drop of the parasitaemia (>98% decline) and 100% mice survival but only 16% cure. In vitro washout experiments demonstrated that although parasite release into the supernatant of infected cardiac cultures was reduced by >94%, parasite recrudescence did occur after treatment. CONCLUSIONS: Parasite recrudescence did occur after treatment corroborating the hypothesis of therapeutic failure due to subpopulations of dormant forms and/or genetic factors in persister parasites involved in natural drug resistance.