RESUMEN
The protozoan parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for the severely debilitating neglected Tropical diseases of African sleeping sickness, Chagas disease and leishmaniasis, respectively. As part of our ongoing programme exploring the potential of simplified analogues of the acetogenin chamuvarinin we identified the T. brucei FoF1-ATP synthase as a target of our earlier triazole analogue series. Using computational docking studies, we hypothesised that the central triazole heterocyclic spacer could be substituted for a central 2,5-substituted furan moiety, thus diversifying the chemical framework for the generation of compounds with greater potency and/or selectivity. Here we report the design, docking, synthesis and biological evaluation of new series of trypanocidal compounds and demonstrate their on-target inhibitory effects. Furthermore, the synthesis of furans by the modular coupling of alkyne- and aldehyde-THPs to bis-THP 1,4-alkyne diols followed by ruthenium/xantphos-catalysed heterocyclisation described here represents the most complex use of this method of heterocyclisation to date.
RESUMEN
Adenosine 5'-triphosphate phosphoribosyltransferase (ATPPRT) catalyzes the first step in histidine biosynthesis, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to generate N1-(5-phospho-ß-d-ribosyl)-ATP and inorganic pyrophosphate. The enzyme is allosterically inhibited by histidine. Two forms of ATPPRT, encoded by the hisG gene, exist in nature, depending on the species. The long form, HisGL, is a single polypeptide chain with catalytic and regulatory domains. The short form, HisGS, lacks a regulatory domain and cannot bind histidine. HisGS instead is found in complex with a regulatory protein, HisZ, constituting the ATPPRT holoenzyme. HisZ triggers HisGS catalytic activity while rendering it sensitive to allosteric inhibition by histidine. Until recently, HisGS was thought to be catalytically inactive without HisZ. Here, recombinant HisGS and HisZ from the psychrophilic bacterium Psychrobacter arcticus were independently overexpressed and purified. The crystal structure of P. arcticus ATPPRT was determined at 2.34 Å resolution, revealing an equimolar HisGS-HisZ hetero-octamer. Steady-state kinetics indicate that both the ATPPRT holoenzyme and HisGS are catalytically active. Surprisingly, HisZ confers only a modest 2-4-fold increase in kcat. Reaction profiles for both enzymes cannot be distinguished by 31P nuclear magnetic resonance, indicating that the same reaction is catalyzed. The temperature dependence of kcat shows deviation from Arrhenius behavior at 308 K with the holoenzyme. Interestingly, such deviation is detected only at 313 K with HisGS. Thermal denaturation by CD spectroscopy resulted in Tm's of 312 and 316 K for HisZ and HisGS, respectively, suggesting that HisZ renders the ATPPRT complex more thermolabile. This is the first characterization of a psychrophilic ATPPRT.
Asunto(s)
ATP Fosforribosil Transferasa/química , Aminoacil-ARNt Sintetasas/química , Proteínas Bacterianas/química , Histidina/química , Proteínas de Transporte de Monosacáridos/química , Psychrobacter/enzimología , ATP Fosforribosil Transferasa/genética , ATP Fosforribosil Transferasa/metabolismo , Aclimatación , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Frío , Cristalografía por Rayos X , Difosfatos/química , Difosfatos/metabolismo , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Histidina/biosíntesis , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Fosforribosil Pirofosfato/química , Fosforribosil Pirofosfato/metabolismo , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína , Psychrobacter/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinámicaRESUMEN
The need for new treatments for the neglected tropical diseases African sleeping sickness, Chagas disease and Leishmaniasis remains urgent with the diseases widespread in tropical regions, affecting the world's very poorest. We have previously reported bis-tetrahydropyran 1,4-triazole analogues designed as mimics of the annonaceous acetogenin natural product chamuvarinin, which maintained trypanocidal activity. Building upon these studies, we here report related triazole compounds with pendant heterocycles, mimicking the original butenolide of the natural product. Analogues were active against T. brucei, with a nitrofuran compound displaying nanomolar trypanocidal activity. Several analogues also showed strong activity against T. cruzi and L. major. Importantly, select compounds gave excellent selectivity over mammalian cells with a furan-based analogue highly selective while remaining active against all three cell lines, thus representing a potential lead for a new broad spectrum kinetoplastid inhibitor.
Asunto(s)
Acetogeninas/química , Diseño de Fármacos , Tripanocidas/química , Acetogeninas/síntesis química , Acetogeninas/farmacología , Supervivencia Celular/efectos de los fármacos , Furanos/química , Células HeLa , Humanos , Leishmania major/efectos de los fármacos , Relación Estructura-Actividad , Triazoles/química , Tripanocidas/síntesis química , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacosRESUMEN
Neglected tropical diseases caused by parasitic infections are an ongoing and increasing concern that have a devastating effect on the developing world due to their burden on human and animal health. In this work, we detail the preparation of a focused library of substituted-tetrahydropyran derivatives and their evaluation as selective chemical tools for trypanosomatid inhibition and the follow-on development of photoaffinity probes capable of labeling target protein(s) in vitro. Several of these functionalized compounds maintain low micromolar activity against Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, and Leishmania donovani. In addition, we demonstrate the utility of the photoaffinity probes for target identification through preliminary cellular localization studies.
Asunto(s)
Tripanocidas/aislamiento & purificación , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos , Reactivos de Enlaces Cruzados/síntesis química , Reactivos de Enlaces Cruzados/aislamiento & purificación , Reactivos de Enlaces Cruzados/farmacología , Leishmania donovani/efectos de los fármacos , Leishmania major/efectos de los fármacos , Microscopía Fluorescente , Estructura Molecular , Coloración y Etiquetado/métodos , Tripanocidas/síntesis químicaRESUMEN
Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and ß-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model.
Asunto(s)
Productos Biológicos/farmacología , Descubrimiento de Drogas/métodos , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Sondas Moleculares , Etiquetas de Fotoafinidad , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Productos Biológicos/análisis , Productos Biológicos/química , Productos Biológicos/metabolismo , Diseño de Fármacos , Humanos , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Fosforilación Oxidativa , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Coloración y Etiquetado/métodos , Tripanocidas/análisis , Tripanocidas/química , Tripanocidas/metabolismo , Rayos UltravioletaRESUMEN
Neglected tropical diseases caused by parasitic infections are an ongoing and increasing concern. They are a burden to human and animal health, having the most devastating effect on the world's poorest countries. Building upon our previously reported triazole analogues, in this study we describe the synthesis and biological testing of other novel heterocyclic acetogenin-inspired derivatives, namely 3,5-isoxazoles, furoxans, and furazans. Several of these compounds maintain low-micromolar levels of inhibition against Trypanosoma brucei, whilst having no observable inhibitory effect on mammalian cells, leading to the possibility of novel lead compounds for selective treatment.
Asunto(s)
Acetogeninas/farmacología , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Acetogeninas/síntesis química , Reacción de Cicloadición , Células HeLa , Humanos , Isoxazoles/síntesis química , Isoxazoles/farmacología , Oxadiazoles/síntesis química , Oxadiazoles/farmacología , Oximas/síntesis química , Oximas/química , Tripanocidas/síntesis químicaRESUMEN
Neglected tropical diseases remain a serious global health concern. Here, a series of novel bis-tetrahydropyran 1,4-triazole analogues based on the framework of chamuvarinin, a polyketide natural product isolated from the annonaceae plant species are detailed. The analogues synthesized display low micromolar trypanocidal activities towards both bloodstream and insect forms of Trypanosoma brucei, the causative agent of African sleeping sickness, also known as Human African Trypanosomiasis (HAT). A divergent synthetic strategy was adopted for the synthesis of the key tetrahydropyran intermediates to enable rapid access to diastereochemical variation either side of the 1,4-triazole core. The resulting diastereomeric analogues displayed varying degrees of trypanocidal activity and selectivity in structure-activity relationship studies. Together, the biological potency and calculated lipophilicity values indicate that while there is room for improvement, these derivatives may represent a promising novel class of anti-HAT agents.