RESUMO
Among the various components of the protozoan Plasmodium mitochondrial respiratory chain, only Complex III is a validated cellular target for antimalarial drugs. The compound CK-2-68 was developed to specifically target the alternate NADH dehydrogenase of the malaria parasite respiratory chain, but the true target for its antimalarial activity has been controversial. Here, we report the cryo-EM structure of mammalian mitochondrial Complex III bound with CK-2-68 and examine the structure-function relationships of the inhibitor's selective action on Plasmodium. We show that CK-2-68 binds specifically to the quinol oxidation site of Complex III, arresting the motion of the iron-sulfur protein subunit, which suggests an inhibition mechanism similar to that of Pf-type Complex III inhibitors such as atovaquone, stigmatellin, and UHDBT. Our results shed light on the mechanisms of observed resistance conferred by mutations, elucidate the molecular basis of the wide therapeutic window of CK-2-68 for selective action of Plasmodium vs. host cytochrome bc1, and provide guidance for future development of antimalarials targeting Complex III.
Assuntos
Antimaláricos , Plasmodium , Animais , Antimaláricos/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium/metabolismo , Citocromos/metabolismo , Mamíferos/metabolismoRESUMO
Protein ubiquitination is an important posttranslational regulation mechanism that mediates Plasmodium development and modifies parasite responses to antimalarial drugs. Although mutations in several parasite ubiquitination enzymes have been linked to increased drug tolerance, the molecular mechanisms by which ubiquitination pathways mediate these parasite responses remain largely unknown. Here, we investigate the roles of a Plasmodium falciparum ring finger ubiquitin ligase (PfRFUL) in parasite development and in responses to antimalarial drugs. We engineered a transgenic parasite having the Pfrful gene tagged with an HA-2A-NeoR-glmS sequence to knockdown (KD) Pfrful expression using glucosamine (GlcN). A Western blot analysis of the proteins from GlcN-treated pSLI-HA-NeoR-glmS-tagged (PfRFULg) parasites, relative to their wild-type (Dd2) controls, showed changes in the ubiquitination of numerous proteins. PfRFUL KD rendered the parasites more sensitive to multiple antimalarial drugs, including mefloquine, piperaquine, amodiaquine, and dihydroartemisinin. PfRFUL KD also decreased the protein level of the P. falciparum multiple drug resistance 1 protein (PfMDR1) and altered the ratio of two bands of the P. falciparum chloroquine resistance transporter (PfCRT), suggesting contributions to the changed drug responses by the altered ubiquitination of these two molecules. The inhibition of proteasomal protein degradation by epoxomicin increased the PfRFUL level, suggesting the degradation of PfRFUL by the proteasome pathways, whereas the inhibition of E3 ubiquitin ligase activities by JNJ26854165 reduced the PfRFUL level. This study reveals the potential mechanisms of PfRFUL in modifying the expression of drug transporters and their roles in parasite drug responses. PfRFUL could be a potential target for antimalarial drug development.
Assuntos
Antimaláricos , Plasmodium falciparum , Proteínas de Protozoários , Ubiquitina-Proteína Ligases , Humanos , Antimaláricos/farmacologia , Cloroquina/farmacologia , Resistência a Medicamentos/genética , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Malaria control is threatened by a limited pipeline of effective pharmaceuticals against drug-resistant strains of Plasmodium falciparum Components of the mitochondrial electron transport chain (ETC) are attractive targets for drug development, owing to exploitable differences between the parasite and human ETC. Disruption of ETC function interferes with metabolic processes including de novo pyrimidine synthesis, essential for nucleic acid replication. We investigated the effects of ETC inhibitor selection on two distinct P. falciparum clones, Dd2 and 106/1. Compounds CK-2-68 and RYL-552, substituted quinolones reported to block P. falciparum NADH dehydrogenase 2 (PfNDH2; a type II NADH:quinone oxidoreductase), unexpectedly selected mutations at the quinol oxidation (Qo) pocket of P. falciparum cytochrome B (PfCytB). Selection experiments with atovaquone (ATQ) on 106/1 parasites yielded highly resistant PfCytB Y268S mutants seen in clinical infections that fail ATQ-proguanil treatment. In contrast, ATQ pressure on Dd2 yielded moderately resistant parasites carrying a PfCytB M133I or K272R mutation. Strikingly, all ATQ-selected mutants demonstrated little change or slight increase of sensitivity to CK-2-68 or RYL-552. Molecular docking studies demonstrated binding of all three ETC inhibitors to the Qo pocket of PfCytB, where Y268 forms strong van der Waals interactions with the hydroxynaphthoquinone ring of ATQ but not the quinolone ring of CK-2-68 or RYL-552. Our results suggest that combinations of suitable ETC inhibitors may be able to subvert or delay the development of P. falciparum drug resistance.
Assuntos
Citocromos b/genética , NADH Desidrogenase/antagonistas & inibidores , Plasmodium falciparum/genética , Antimaláricos/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/genética , Malária Falciparum/parasitologia , Simulação de Acoplamento Molecular/métodos , Mutação/genética , Plasmodium falciparum/efeitos dos fármacos , Quinolonas/farmacologiaRESUMO
Protozoan Plasmodium parasites are the causative agents of malaria, a deadly disease that continues to afflict hundreds of millions of people every year. Infections with malaria parasites can be asymptomatic, with mild or severe symptoms, or fatal, depending on many factors such as parasite virulence and host immune status. Malaria can be treated with various drugs, with artemisinin-based combination therapies (ACTs) being the first-line choice. Recent advances in genetics and genomics of malaria parasites have contributed greatly to our understanding of parasite population dynamics, transmission, drug responses, and pathogenesis. However, knowledge gaps in parasite biology and host-parasite interactions still remain. Parasites resistant to multiple antimalarial drugs have emerged, while advanced clinical trials have shown partial efficacy for one available vaccine. Here we discuss genetic and genomic studies of Plasmodium biology, host-parasite interactions, population structures, mosquito infectivity, antigenic variation, and targets for treatment and immunization. Knowledge from these studies will advance our understanding of malaria pathogenesis, epidemiology, and evolution and will support work to discover and develop new medicines and vaccines.
Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos/genética , Evolução Molecular , Genoma de Protozoário/genética , Malária/epidemiologia , Malária/parasitologia , Plasmodium/efeitos dos fármacos , Plasmodium/genética , Humanos , Plasmodium/classificação , Plasmodium/patogenicidadeRESUMO
WR99210, a former antimalarial drug candidate now widely used for the selection of Plasmodium transfectants, selectively targets the parasite's dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing the human dhfr gene have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds 1 and 2). Compound 1 proved effective at low nanomolar concentrations against Plasmodium falciparum parasites, whereas compound 2 was ineffective, even at micromolar concentrations. Intact and fragmented mass spectra indicated identical molecular formulae of the unprotonated (free base) structures of compounds 1 and 2; however, the compounds displayed differences by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and UV-visible spectroscopy, indicating important isomeric differences. Structural evaluations by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy confirmed compound 1 as WR99210 and compound 2 as a dihydrotriazine regioisomer. Induced fit computational docking models showed that compound 1 binds tightly and specifically in the P. falciparum DHFR active site, whereas compound 2 fits poorly to the active site in loose and varied orientations. Stocks and concentrates of WR99210 should be monitored for the presence of regioisomer 2, particularly when they are not supplied as the hydrochloride salt or are exposed to basic conditions that may promote rearrangement. Absorption spectroscopy can serve for assays of the unrearranged and rearranged triazines.
Assuntos
Antimaláricos , Antagonistas do Ácido Fólico , Malária Falciparum , Antimaláricos/farmacologia , Resistência a Medicamentos , Antagonistas do Ácido Fólico/farmacologia , Humanos , Plasmodium falciparum/genética , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase , TriazinasRESUMO
Preventing and delaying the emergence of drug resistance is an essential goal of antimalarial drug development. Monotherapy and highly mutable drug targets have each facilitated resistance, and both are undesirable in effective long-term strategies against multi-drug-resistant malaria. Haem remains an immutable and vulnerable target, because it is not parasite-encoded and its detoxification during haemoglobin degradation, critical to parasite survival, can be subverted by drug-haem interaction as in the case of quinolines and many other drugs. Here we describe a new antimalarial chemotype that combines the haem-targeting character of acridones, together with a chemosensitizing component that counteracts resistance to quinoline antimalarial drugs. Beyond the essential intrinsic characteristics common to deserving candidate antimalarials (high potency in vitro against pan-sensitive and multi-drug-resistant Plasmodium falciparum, efficacy and safety in vivo after oral administration, inexpensive synthesis and favourable physicochemical properties), our initial lead, T3.5 (3-chloro-6-(2-diethylamino-ethoxy)-10-(2-diethylamino-ethyl)-acridone), demonstrates unique synergistic properties. In addition to 'verapamil-like' chemosensitization to chloroquine and amodiaquine against quinoline-resistant parasites, T3.5 also results in an apparently mechanistically distinct synergism with quinine and with piperaquine. This synergy, evident in both quinoline-sensitive and quinoline-resistant parasites, has been demonstrated both in vitro and in vivo. In summary, this innovative acridone design merges intrinsic potency and resistance-counteracting functions in one molecule, and represents a new strategy to expand, enhance and sustain effective antimalarial drug combinations.
Assuntos
Acridonas/farmacologia , Antimaláricos/farmacologia , Descoberta de Drogas , Plasmodium falciparum/efeitos dos fármacos , Acridonas/análise , Acridonas/metabolismo , Animais , Antimaláricos/análise , Antimaláricos/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Sinergismo Farmacológico , Heme/antagonistas & inibidores , Heme/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação/genética , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Plasmodium yoelii/efeitos dos fármacos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Quinina/farmacologia , Quinolinas/farmacologia , Trofozoítos/metabolismo , Verapamil/farmacologiaRESUMO
Staphylococcal pathogenicity islands (SaPIs) carry superantigen and resistance genes and are extremely widespread in Staphylococcus aureus and in other Gram-positive bacteria. SaPIs represent a major source of intrageneric horizontal gene transfer and a stealth conduit for intergeneric gene transfer; they are phage satellites that exploit the life cycle of their temperate helper phages with elegant precision to enable their rapid replication and promiscuous spread. SaPIs also interfere with helper phage reproduction, blocking plaque formation, sharply reducing burst size and enhancing the survival of host cells following phage infection. Here, we show that SaPIs use several different strategies for phage interference, presumably the result of convergent evolution. One strategy, not described previously in the bacteriophage microcosm, involves a SaPI-encoded protein that directly and specifically interferes with phage DNA packaging by blocking the phage terminase small subunit. Another strategy involves interference with phage reproduction by diversion of the vast majority of virion proteins to the formation of SaPI-specific small infectious particles. Several SaPIs use both of these strategies, and at least one uses neither but possesses a third. Our studies illuminate a key feature of the evolutionary strategy of these mobile genetic elements, in addition to their carriage of important genes-interference with helper phage reproduction, which could ensure their transferability and long-term persistence.
Assuntos
Antibiose/genética , Transferência Genética Horizontal/genética , Ilhas Genômicas/genética , Fagos de Staphylococcus/fisiologia , Staphylococcus aureus/genética , Replicação Viral/fisiologia , Clonagem Molecular , Escherichia coli , Microscopia Eletrônica , Reação em Cadeia da Polimerase em Tempo Real , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/virologia , Técnicas do Sistema de Duplo-Híbrido , Ensaio de Placa ViralRESUMO
The SaPIs and their relatives are a family of genomic islands that exploit helper phages for high frequency horizontal transfer. One of the mechanisms used by SaPIs to accomplish this molecular piracy is the redirection of the helper phage DNA packaging machinery. SaPIs encode a small terminase subunit that can be substituted for that of the phage. In this study we have determined the initial packaging cleavage sites for helper phage 80α, which uses the phage-encoded small terminase subunit, and for SaPI1, which uses the SaPI-encoded small terminase subunit. We have identified a 19nt SaPI1 sequence that is necessary and sufficient to allow high frequency 80α transduction of a plasmid by a terminase carrying the SaPI1-encoded small subunit. We also show that the hybrid enzyme with the SaPI1 small terminase subunit is capable of generalized transduction.
Assuntos
Empacotamento do DNA , Ilhas Genômicas , Fagos de Staphylococcus/genética , Staphylococcus aureus/genética , Mapeamento Cromossômico , DNA Bacteriano/genética , DNA Viral/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Plasmídeos , Análise de Sequência de DNA , Transdução GenéticaRESUMO
BACKGROUND: Lumefantrine and mefloquine are used worldwide in artemisinin-based combination therapy (ACT) of malaria. Better understanding of drug susceptibility and resistance is needed and can be obtained from studies of genetic crosses. METHODS: Drug response phenotypes of a cross between Plasmodium falciparum lines 803 (Cambodia) and GB4 (Ghana) were obtained as half-maximal effective concentrations (EC50s) and days to recovery (DTR) after 24 h exposure to 500 nM lumefantrine. EC50s of mefloquine, halofantrine, chloroquine, and dihydroartemisinin were also determined. Quantitative trait loci (QTL) analysis and statistical tests with candidate genes were used to identify polymorphisms associated with response phenotypes. RESULTS: Lumefantrine EC50s averaged 5.8-fold higher for the 803 than GB4 parent, and DTR results were 3-5 and 16-18 days, respectively. In 803 × GB4 progeny, outcomes of these two lumefantrine assays showed strong inverse correlation; these phenotypes also correlated strongly with mefloquine and halofantrine EC50s. By QTL analysis, lumefantrine and mefloquine phenotypes mapped to a chromosome 5 region containing codon polymorphisms N86Y and Y184F in the P. falciparum multidrug resistance 1 protein (PfMDR1). Statistical tests of candidate genes identified correlations between inheritance of PfK13 Kelch protein polymorphism C580Y (and possibly K189T) and lumefantrine and mefloquine susceptibilities. Correlations were detected between lumefantrine and chloroquine EC50s and polymorphisms N326S and I356T in the CVIET-type P. falciparum chloroquine resistance transporter (PfCRT) common to 803 and GB4. CONCLUSIONS: Correlations in this study suggest common mechanisms of action in lumefantrine, mefloquine, and halofantrine responses. PfK13 as well as PfMDR1 and PfCRT polymorphisms may affect access and/or action of these arylaminoalcohol drugs at locations of hemoglobin digestion and heme metabolism. In endemic regions, pressure from use of lumefantrine or mefloquine in ACTs may drive selection of PfK13 polymorphisms along with versions of PfMDR1 and PfCRT associated with lower susceptibility to these drugs.
Assuntos
Antimaláricos , Malária Falciparum , Plasmodium falciparum/genética , Antimaláricos/farmacologia , Camboja , Resistência a Medicamentos , Etanolaminas/uso terapêutico , Fluorenos/uso terapêutico , Gana , Humanos , Lumefantrina , Malária Falciparum/tratamento farmacológico , Mefloquina/farmacologia , Mefloquina/uso terapêutico , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Plasmodium falciparum/efeitos dos fármacos , Proteínas de ProtozoáriosRESUMO
A Plasmodium falciparum gene closely linked to the chloroquine resistance locus encodes PfCG2, a predicted 320-330kDa protein. In the parasitized erythrocyte, PfCG2 expression rises sharply in the trophozoite stage and is detected in electron-dense patches along the parasitophorous vacuolar membrane (PVM), in the cytoplasm and in the digestive vacuole (DV). Results of extraction and partitioning experiments show that PfCG2 is a peripheral membrane protein. Exposure of trophozoite-infected erythrocytes to trypsin-containing buffer after streptolysin O permeabilization indicates that PfCG2 is exposed to the erythrocyte cytosol at the outer face of the PVM. PfCG2 is highly susceptible to hydrolysis by aspartic and cysteine proteases and shows dose-dependent accumulation in the presence of protease inhibitors. These results suggest that PfCG2 is delivered from the outside face of the PVM to the DV, where it is broken down by parasite proteases. PfCG2 interacts with erythrocyte cytoplasm and may be associated with processes of hemoglobin uptake and digestion by erythrocytic-stage parasites.
Assuntos
Hemoglobinas/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Ácido Aspártico Endopeptidases/farmacologia , Membrana Celular/metabolismo , Cisteína Endopeptidases/farmacologia , Citosol/metabolismo , Resistência a Medicamentos , Eritrócitos/química , Eritrócitos/citologia , Eritrócitos/parasitologia , Humanos , Estágios do Ciclo de Vida , Microscopia Eletrônica , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
Herpesviruses commandeer distinct cellular pathways to enter target cells. The mechanism by which herpes simplex virus (HSV) selects a pH-dependent, endocytic route or a pH-independent route remains to be elucidated. We investigated the role of the non-glycosylated viral envelope protein UL45 in HSV entry via endocytosis. UL45 plays a role in mediating cell-cell fusion and has been proposed to functionally interact with gB to regulate membrane fusion. Thus, we also probed the impact of UL45 on the structure and function of gB present in virions. A UL45 deletion virus successfully entered cells via low pH, endocytic pathway with wild type kinetics. In the absence or presence of UL45, the antigenic conformation of virion gB appeared unaltered. Antibodies to gB neutralized infection of the UL45-deletion virus and wild type virus to a similar extent, regardless of whether the target cells supported low pH endocytic or non-endocytic entry routes. Lastly, HSV virions were inactivated by low pH regardless of the presence of UL45. The results, together with previous studies, suggest that UL45 plays distinct roles in cell-cell fusion and virus-cell fusion during acid-dependent entry.
Assuntos
Endocitose , Simplexvirus/fisiologia , Proteínas do Envelope Viral/fisiologia , Proteínas Virais/fisiologia , Internalização do Vírus , Animais , Células CHO , Cricetinae , Cricetulus , Deleção de Genes , Concentração de Íons de Hidrogênio , Conformação Proteica , Simplexvirus/genética , Proteínas do Envelope Viral/química , Proteínas Virais/genéticaRESUMO
Mutations in the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT) can result in verapamil-reversible CQ resistance and altered susceptibility to other antimalarials. PfCRT contains 10 membrane-spanning domains and is found in the digestive vacuole (DV) membrane of intraerythrocytic parasites. The mechanism by which PfCRT mediates CQ resistance is unclear although it is associated with decreased accumulation of drug within the DV. On the permissive background of the P. falciparum 106/1(K76) parasite line, we used single-step drug selection to generate isogenic clones containing unique pfcrt point mutations that resulted in amino acid changes in PfCRT transmembrane domains 1 (C72R, K76N, K76I and K76T) and 9 (Q352K, Q352R). The resulting changes of charge and hydropathy affected quantitative CQ susceptibility and accumulation as well as the stereospecific responses to quinine and quinidine. These results, together with a previously described S163R mutation in transmembrane domain 4, indicate that transmembrane segments 1, 4 and 9 of PfCRT provide important structural components of a substrate recognition and translocation domain. Charge-affecting mutations within these segments may affect the ability of PfCRT to bind different quinoline drugs and determine their net accumulation in the DV.
Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/genética , Animais , Cloroquina/farmacologia , Resistência a Medicamentos/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/química , Plasmodium falciparum/genética , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/química , Quinidina/farmacologia , Quinina/farmacologiaRESUMO
A series of novel 10-N-substituted acridones, bearing alkyl side chains with tertiary amine groups at the terminal position, were designed, synthesized, and evaluated for the ability to enhance the potency of quinoline drugs against multidrug-resistant (MDR) Plasmodium falciparum malaria parasites. A number of acridone derivatives, with side chains bridged three or more carbon atoms apart between the ring nitrogen and terminal nitrogen, demonstrated chloroquine (CQ)-chemosensitizing activity against the MDR strain of P. falciparum (Dd2). Isobologram analysis revealed that selected candidates demonstrated significant synergy with CQ in the CQ-resistant (CQR) parasite Dd2 but only additive (or indifferent) interaction in the CQ-sensitive (CQS) D6. These acridone derivatives also enhanced the sensitivity of other quinoline antimalarials, such as desethylchloroquine (DCQ) and quinine (QN), in Dd2. The patterns of chemosensitizing effects of selected acridones on CQ and QN were similar to those of verapamil against various parasite lines with mutations encoding amino acid 76 of the P. falciparum CQ resistance transporter (PfCRT). Unlike other known chemosensitizers with recognized psychotropic effects (e.g., desipramine, imipramine, and chlorpheniramine), these novel acridone derivatives exhibited no demonstrable effect on the uptake or binding of important biogenic amine neurotransmitters. The combined results indicate that 10-N-substituted acridones present novel pharmacophores for the development of chemosensitizers against P. falciparum.