RESUMEN
IMPORTANCE: Our study leverages gene editing techniques in Plasmodium falciparum asexual blood stage parasites to profile novel mutations in mutant PfCRT, an important mediator of piperaquine resistance, which developed in Southeast Asian field isolates or in parasites cultured for long periods of time. We provide evidence that increased parasite fitness of these lines is the primary driver for the emergence of these PfCRT variants. These mutations differentially impact parasite susceptibility to piperaquine and chloroquine, highlighting the multifaceted effects of single point mutations in this transporter. Molecular features of drug resistance and parasite physiology were examined in depth using proteoliposome-based drug uptake studies and peptidomics, respectively. Energy minimization calculations, showing how these novel mutations might impact the PfCRT structure, suggested a small but significant effect on drug interactions. This study reveals the subtle interplay between antimalarial resistance, parasite fitness, PfCRT structure, and intracellular peptide availability in PfCRT-mediated parasite responses to changing drug selective pressures.
Asunto(s)
Antimaláricos , Malaria Falciparum , Parásitos , Piperazinas , Quinolinas , Animales , Plasmodium falciparum , Quinolinas/farmacología , Quinolinas/química , Cloroquina/farmacología , Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Mutación , Proteínas Protozoarias/genética , Proteínas Protozoarias/química , Malaria Falciparum/parasitologíaRESUMEN
In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase δ. Mutation accumulation assays reveal a ~5-8 fold elevation in the mutation rate, with an increase of 13-28 fold in drug-pressured lines. Upon challenge with the spiroindolone PfATP4-inhibitor KAE609, high-level resistance is obtained more rapidly and at lower inocula than wild-type parasites. Selections also yield mutants with resistance to an "irresistible" compound, MMV665794 that failed to yield resistance with other strains. We validate mutations in a previously uncharacterised gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), as causal for resistance to MMV665794 and a panel of quinoxaline analogues. The increased genetic repertoire available to this "mutator" parasite can be leveraged to drive P. falciparum resistome discovery.
Asunto(s)
Antimaláricos , Malaria Falciparum , Parásitos , Animales , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Parásitos/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Antimaláricos/uso terapéutico , Mutación , Resistencia a Medicamentos/genética , Proteínas Protozoarias/metabolismoRESUMEN
Antimalarial drug discovery has until recently been driven by high-throughput phenotypic cellular screening, allowing millions of compounds to be assayed and delivering clinical drug candidates. In this review, we will focus on target-based approaches, describing recent advances in our understanding of druggable targets in the malaria parasite. Targeting multiple stages of the Plasmodium lifecycle, rather than just the clinically symptomatic asexual blood stage, has become a requirement for new antimalarial medicines, and we link pharmacological data clearly to the parasite stages to which it applies. Finally, we highlight the IUPHAR/MMV Guide to MALARIA PHARMACOLOGY, a web resource developed for the malaria research community that provides open and optimized access to published data on malaria pharmacology.
Asunto(s)
Antimaláricos , Malaria , Humanos , Malaria/tratamiento farmacológico , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Descubrimiento de Drogas , Ensayos Analíticos de Alto RendimientoRESUMEN
Development of antimalarial compounds into clinical candidates remains costly and arduous without detailed knowledge of the target. As resistance increases and treatment options at various stages of disease are limited, it is critical to identify multistage drug targets that are readily interrogated in biochemical assays. Whole-genome sequencing of 18 parasite clones evolved using thienopyrimidine compounds with submicromolar, rapid-killing, pan-life cycle antiparasitic activity showed that all had acquired mutations in the P. falciparum cytoplasmic isoleucyl tRNA synthetase (cIRS). Engineering two of the mutations into drug-naïve parasites recapitulated the resistance phenotype, and parasites with conditional knockdowns of cIRS became hypersensitive to two thienopyrimidines. Purified recombinant P. vivax cIRS inhibition, cross-resistance, and biochemical assays indicated a noncompetitive, allosteric binding site that is distinct from that of known cIRS inhibitors mupirocin and reveromycin A. Our data show that Plasmodium cIRS is an important chemically and genetically validated target for next-generation medicines for malaria.
Asunto(s)
Antimaláricos , Malaria Falciparum , Malaria , Humanos , Antimaláricos/química , Isoleucina-ARNt Ligasa/metabolismo , Plasmodium falciparum/metabolismo , Malaria Falciparum/parasitología , Malaria/tratamiento farmacológico , Resistencia a MedicamentosRESUMEN
Structural modification of existing chemical scaffolds to afford new molecules able to circumvent drug resistance constitutes one of the rational approaches to antimalarial drug discovery. Previously synthesized compounds based on the 4-aminoquinoline core hybridized with a chemosensitizing dibenzylmethylamine side group showed in vivo efficacy in Plasmodium berghei-infected mice despite low microsomal metabolic stability, suggesting a contribution from their pharmacologically active metabolites. Here, we report on a series of these dibemequine (DBQ) metabolites with low resistance indices against chloroquine-resistant parasites and improved metabolic stability in liver microsomes. The metabolites also exhibit improved pharmacological properties including lower lipophilicity, cytotoxicity, and hERG channel inhibition. Using cellular heme fractionation experiments, we also demonstrate that these derivatives inhibit hemozoin formation by causing a buildup of toxic "free" heme in a similar manner to chloroquine. Finally, assessment of drug interactions also revealed synergy between these derivatives and several clinically relevant antimalarials, thus highlighting their potential interest for further development.
Asunto(s)
Antimaláricos , Animales , Ratones , Antimaláricos/farmacología , Antimaláricos/química , Plasmodium falciparum , Cloroquina/farmacología , Hemo/metabolismoRESUMEN
The emergence of Plasmodium falciparum parasite resistance to dihydroartemisinin + piperaquine (PPQ) in Southeast Asia threatens plans to increase the global use of this first-line antimalarial combination. High-level PPQ resistance appears to be mediated primarily by novel mutations in the P. falciparum chloroquine resistance transporter (PfCRT), which enhance parasite survival at high PPQ concentrations in vitro and increase the risk of dihydroartemisinin + PPQ treatment failure in patients. Using isogenic Dd2 parasites expressing contemporary pfcrt alleles with differential in vitro PPQ susceptibilities, we herein characterize the molecular and physiological adaptations that define PPQ resistance in vitro. Using drug uptake and cellular heme fractionation assays we report that the F145I, M343L, and G353V PfCRT mutations differentially impact PPQ and chloroquine efflux. These mutations also modulate proteolytic degradation of host hemoglobin and the chemical inactivation of reactive heme species. Peptidomic analyses reveal significantly higher accumulation of putative hemoglobin-derived peptides in the PPQ-resistant mutant PfCRT isoforms compared to parental PPQ-sensitive Dd2. Joint transcriptomic and metabolomic profiling of late trophozoites from PPQ-resistant or -sensitive isogenic lines reveals differential expression of genes involved in protein translation and cellular metabolism. PPQ-resistant parasites also show increased susceptibility to an inhibitor of the P. falciparum M17 aminopeptidase that operates on short globin-derived peptides. These results reveal unique physiological changes caused by the gain of PPQ resistance and highlight the potential therapeutic value of targeting peptide metabolism in P. falciparum.
Asunto(s)
Antimaláricos , Artemisininas , Malaria Falciparum , Parásitos , Animales , Humanos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Cloroquina/farmacología , Cloroquina/metabolismo , Parásitos/metabolismo , Proteínas Protozoarias/metabolismo , Resistencia a Medicamentos/genética , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/genética , Malaria Falciparum/parasitología , Antimaláricos/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Artemisininas/farmacología , Mutación , Hemoglobinas/metabolismo , Hemo/metabolismoRESUMEN
Widespread Plasmodium falciparum resistance to first-line antimalarials underscores the vital need to develop compounds with novel modes of action and identify new druggable targets. Here, we profile five compounds that potently inhibit P. falciparum asexual blood stages. Resistance selection studies with three carboxamide-containing compounds, confirmed by gene editing and conditional knockdowns, identify point mutations in the parasite transporter ABCI3 as the primary mediator of resistance. Selection studies with imidazopyridine or quinoline-carboxamide compounds also yield changes in ABCI3, this time through gene amplification. Imidazopyridine mode of action is attributed to inhibition of heme detoxification, as evidenced by cellular accumulation and heme fractionation assays. For the copy-number variation-selecting imidazopyridine and quinoline-carboxamide compounds, we find that resistance, manifesting as a biphasic concentration-response curve, can independently be mediated by mutations in the chloroquine resistance transporter PfCRT. These studies reveal the interconnectedness of P. falciparum transporters in overcoming drug pressure in different parasite strains.
Asunto(s)
Antimaláricos , Antagonistas del Ácido Fólico , Malaria Falciparum , Parásitos , Quinolinas , Transportadoras de Casetes de Unión a ATP/genética , Animales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Hemo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Proteínas de Transporte de Membrana/genética , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Quinolinas/farmacologíaRESUMEN
There is a shift in antimalarial drug discovery from phenotypic screening toward target-based approaches, as more potential drug targets are being validated in Plasmodium species. Given the high attrition rate and high cost of drug discovery, it is important to select the targets most likely to deliver progressible drug candidates. In this paper, we describe the criteria that we consider important for selecting targets for antimalarial drug discovery. We describe the analysis of a number of drug targets in the Malaria Drug Accelerator (MalDA) pipeline, which has allowed us to prioritize targets that are ready to enter the drug discovery process. This selection process has also highlighted where additional data are required to inform target progression or deprioritization of other targets. Finally, we comment on how additional drug targets may be identified.
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Antimaláricos , Malaria , Plasmodium , Descubrimiento de Drogas , Humanos , Malaria/tratamiento farmacológicoRESUMEN
Recent progress in genomics and molecular genetics has empowered novel approaches to study gene functions in disease-causing pathogens. In the human malaria parasite Plasmodium falciparum, the application of genome-based analyses, site-directed genome editing, and genetic systems that allow for temporal and quantitative regulation of gene and protein expression have been invaluable in defining the genetic basis of antimalarial resistance and elucidating candidate targets to accelerate drug discovery efforts. Using examples from recent studies, we review applications of some of these approaches in advancing our understanding of Plasmodium biology and illustrate their contributions and limitations in characterizing parasite genomic loci associated with antimalarial drug responses.
Asunto(s)
Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Genoma de Protozoos/genética , Genómica , Biología Molecular , Plasmodium falciparum/fisiología , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/genéticaRESUMEN
3-Hydroxypropanamidines are a new promising class of highly active antiplasmodial agents. The most active compound 22 exhibited excellent antiplasmodial in vitro activity with nanomolar inhibition of chloroquine-sensitive and multidrug-resistant parasite strains ofPlasmodium falciparum (with IC50 values of 5 and 12 nM against 3D7 and Dd2 strains, respectively) as well as low cytotoxicity in human cells. In addition, 22 showed strong in vivo activity in thePlasmodium berghei mouse model with a cure rate of 66% at 50 mg/kg and a cure rate of 33% at 30 mg/kg in the Peters test after once daily oral administration for 4 consecutive days. A quick onset of action was indicated by the fast drug absorption shown in mice. The new lead compound was also characterized by a high barrier to resistance and inhibited the heme detoxification machinery in P. falciparum.
Asunto(s)
Amidinas/química , Amidinas/farmacología , Antimaláricos/química , Antimaláricos/farmacología , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Amidinas/farmacocinética , Amidinas/uso terapéutico , Animales , Antimaláricos/farmacocinética , Antimaláricos/uso terapéutico , Línea Celular , Diseño de Fármacos , Humanos , Malaria/tratamiento farmacológico , Ratones , Pruebas de Sensibilidad Parasitaria , Plasmodium berghei/efectos de los fármacos , Propano/química , Propano/farmacocinética , Propano/farmacología , Propano/uso terapéuticoRESUMEN
Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized Plasmodium falciparum parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 (pfmdr1). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.
Asunto(s)
Amidas/farmacología , Antimaláricos/farmacología , Hemoglobinas/metabolismo , Plasmodium falciparum/efectos de los fármacos , Tetrazoles/farmacología , Amidas/síntesis química , Amidas/farmacocinética , Antimaláricos/síntesis química , Antimaláricos/farmacocinética , Farmacorresistencia Microbiana/efectos de los fármacos , Hemoproteínas/antagonistas & inhibidores , Células Hep G2 , Humanos , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacocinética , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Tetrazoles/síntesis química , Tetrazoles/farmacocinéticaRESUMEN
A series of 2,4-disubstituted imidazopyridines, originating from a SoftFocus Kinase library, was identified from a high throughput phenotypic screen against the human malaria parasite Plasmodium falciparum. Hit compounds showed moderate asexual blood stage activity. During lead optimization, several issues were flagged such as cross-resistance against the multidrug-resistant K1 strain, in vitro cytotoxicity, and cardiotoxicity and were addressed through structure-activity and structure-property relationship studies. Pharmacokinetic properties were assessed in mice for compounds showing desirable in vitro activity, a selectivity window over cytotoxicity, and microsomal metabolic stability. Frontrunner compound 37 showed good exposure in mice combined with good in vitro activity against the malaria parasite, which translated into in vivo efficacy in the P. falciparum NOD-scid IL-2Rγnull (NSG) mouse model. Preliminary mechanistic studies suggest inhibition of hemozoin formation as a contributing mode of action.
Asunto(s)
Antimaláricos/química , Hemoproteínas/antagonistas & inhibidores , Imidazoles/química , Plasmodium falciparum/fisiología , Proteínas Protozoarias/antagonistas & inhibidores , Piridinas/química , Animales , Antimaláricos/metabolismo , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Modelos Animales de Enfermedad , Semivida , Hemoproteínas/metabolismo , Imidazoles/metabolismo , Imidazoles/farmacología , Imidazoles/uso terapéutico , Estadios del Ciclo de Vida/efectos de los fármacos , Malaria/tratamiento farmacológico , Malaria/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Microsomas Hepáticos/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Piridinas/metabolismo , Piridinas/farmacología , Piridinas/uso terapéutico , Relación Estructura-ActividadRESUMEN
Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; however, resistance threatens to undermine global control efforts. To broadly explore artemisinin susceptibility in apicomplexan parasites, we employ genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncover the putative transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provide evidence that mitochondrial metabolism can modulate resistance. We show that disrupting a top candidate from the screens, the mitochondrial protease DegP2, lowers porphyrin levels and decreases DHA susceptibility, without significantly altering parasite fitness in culture. Deleting the homologous gene in P. falciparum, PfDegP, similarly lowers heme levels and DHA susceptibility. These results expose the vulnerability of heme metabolism to genetic perturbations that can lead to increased survival in the presence of DHA.
Asunto(s)
Antimaláricos/farmacología , Artemisininas/farmacología , Resistencia a Medicamentos/genética , Pruebas Genéticas/métodos , Hemo/genética , Hemo/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Técnicas de Inactivación de Genes , Humanos , Malaria Falciparum/tratamiento farmacológico , Proteínas de Transporte de Membrana/metabolismo , Mutación , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Toxoplasma/efectos de los fármacos , Toxoplasma/genéticaRESUMEN
Fusidic acid (FA) is a potent congener of the fusidane triterpenoid class of antibiotics. Structure-activity relationship (SAR) studies suggest the chemical structure of FA is optimal for its antibacterial activity. SAR studies from our group within the context of a drug repositioning approach in tuberculosis (TB) suggest that, as with its antibacterial activity, the C-21 carboxylic acid group is indispensable for its anti-mycobacterial activity. Further studies have led to the identification of 16-deacetoxy-16ß-ethoxyfusidic acid (58), an analog which exhibited comparable activity to FA with an in vitro MIC99 value of 0.8 µM. Preliminary SAR studies around the FA scaffold suggested that the hydrophobic side chain at C-20, like the C-11 OH group, was required for activity. The C-3 OH group, however, can be functionalized to obtain more potent compounds.
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Antibacterianos/química , Ácido Fusídico/química , Mycobacterium/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Animales , Antibacterianos/farmacología , Cricetulus , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Ácido Fusídico/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Relación Estructura-ActividadRESUMEN
Fusidic acid (FA) has previously been shown to be rapidly metabolized in rodents to its C-3 epimer, which has significantly lower antimycobacterial activity relative to FA. This was in part hypothesized to account for FA's lack of in vivo efficacy in a mouse model of tuberculosis despite potent in vitro antimycobacterial activity. In the current work, we hypothesized that C-3 alkyl ester prodrugs of FA would deliver higher levels of the drug and prevent the rapid metabolism observed upon administration of FA in its original form. Pharmacokinetic analysis of FA and its 3-ketofusidic acid metabolite as well as novel C-3 alkyl ester prodrugs of FA revealed that FA has low exposure in mice due to rapid metabolism to a species-specific metabolite, 3-epifusidic acid. The C-3 alkyl ester prodrugs showed improved absorption and tissue distribution in pharmacokinetic and organ distribution experiments. These results support the original objective of the FA C-3 ester prodrugs to improve drug concentrations and tissue distribution.
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Antibacterianos/farmacocinética , Ésteres/farmacocinética , Ácido Fusídico/farmacocinética , Profármacos/farmacocinética , Alquilación , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Plasmepsins represent novel antimalarial drug targets. However, plasmepsin-based antimalarial drug discovery efforts in the past 2 decades have generally suffered some drawbacks including lack of translatability of target inhibition to potent parasite inhibition in vitro and in vivo as well as poor selectivity over the related human aspartic proteases. Most studies reported in this period have over-relied on the use of hemoglobinase plasmepsins I-IV (particularly I and II) as targets for the new inhibitors even though these are known to be nonessential at the asexual stage of parasite development. Therefore, future antimalarial drug discovery efforts seeking to identify plasmepsin inhibitors should focus on incorporating non-hemoglobinase plasmepsins such as V, IX, and X in their screening in order to maximize chances of success. Additionally, there is need to go beyond just target enzymatic activity profiling to establishing cellular activity, physicochemical as well as drug metabolism and pharmacokinetics properties and finally in vivo proof-of-concept while ensuring selectivity over related human host proteases.
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Antimaláricos/farmacología , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Peptidomiméticos/farmacología , Inhibidores de Proteasas/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Antimaláricos/química , Química Farmacéutica , Descubrimiento de Drogas , Humanos , Ratones , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Peptidomiméticos/química , Plasmodium falciparum/enzimología , Inhibidores de Proteasas/química , Relación Estructura-ActividadRESUMEN
The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite3-9. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.
Asunto(s)
Microscopía por Crioelectrón , Resistencia a Medicamentos/efectos de los fármacos , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/ultraestructura , Plasmodium falciparum/química , Proteínas Protozoarias/química , Proteínas Protozoarias/ultraestructura , Cloroquina/metabolismo , Cloroquina/farmacología , Resistencia a Medicamentos/genética , Concentración de Iones de Hidrógeno , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Mutación , Plasmodium falciparum/genética , Plasmodium falciparum/ultraestructura , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Quinolinas/metabolismo , Quinolinas/farmacologíaRESUMEN
Fusidic acid (FA), a natural product fusidane triterpene-based antibiotic with unique structural features, is active in vitro against Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). While possessing good pharmacokinetics in man, FA is rapidly metabolized in rodents, thus complicating proof-of-concept studies in this model. Toward the repositioning of FA as an anti-TB agent, we herein describe the synthesis, activity, and metabolism of FA and semisynthesized ester derivatives in rat liver microsomes, rat plasma, and mycobacterial cell culture. FA and derivative molecules with a free C-3 OH underwent species-specific metabolism to the corresponding 3-OH epimer, 3-epifusidic acid (3-epiFA). FA was also metabolized in rat plasma to form FA lactone. These additional routes of metabolism may contribute to the more rapid clearance of FA observed in rodents. C-3 alkyl and aryl esters functioned as classic prodrugs of FA, being hydrolyzed to FA in microsomes, plasma, and Mycobacterium tuberculosis culture. In contrast, C-3 silicate esters and C-21 esters were inert to hydrolysis and so did not act as prodrugs. The antimycobacterial activity of the C-3 silicate esters was comparable to that of FA, and these compounds were stable in microsomes and plasma, identifying them as potential candidates for evaluation in a rodent model of tuberculosis.