RESUMO
Infections caused by malaria parasites place an enormous burden on the world's poorest communities. Breakthrough drugs with novel mechanisms of action are urgently needed. As an organism that undergoes rapid growth and division, the malaria parasite Plasmodium falciparum is highly reliant on protein synthesis, which in turn requires aminoacyl-tRNA synthetases (aaRSs) to charge tRNAs with their corresponding amino acid. Protein translation is required at all stages of the parasite life cycle; thus, aaRS inhibitors have the potential for whole-of-life-cycle antimalarial activity. This review focuses on efforts to identify potent plasmodium-specific aaRS inhibitors using phenotypic screening, target validation, and structure-guided drug design. Recent work reveals that aaRSs are susceptible targets for a class of AMP-mimicking nucleoside sulfamates that target the enzymes via a novel reaction hijacking mechanism. This finding opens up the possibility of generating bespoke inhibitors of different aaRSs, providing new drug leads.
Assuntos
Aminoacil-tRNA Sintetases , Antimaláricos , Malária , Humanos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Plasmodium falciparum/genética , Malária/tratamento farmacológico , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência/uso terapêuticoRESUMO
The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.
Assuntos
Compostos de Boro/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Administração Oral , Animais , Compostos de Boro/administração & dosagem , Compostos de Boro/química , Domínio Catalítico , Humanos , Malária Falciparum/enzimologia , Malária Falciparum/parasitologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Plasmodium falciparum/enzimologia , Inibidores de Proteassoma/administração & dosagem , Inibidores de Proteassoma/químicaRESUMO
The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the ß2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum ß2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this ß2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next-generation anti-malarial agents.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Desenho de Fármacos , Plasmodium/efeitos dos fármacos , Plasmodium/enzimologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Animais , Antimaláricos/efeitos adversos , Antimaláricos/toxicidade , Artemisininas/farmacologia , Domínio Catalítico , Microscopia Crioeletrônica , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Sinergismo Farmacológico , Ativação Enzimática , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Plasmodium/crescimento & desenvolvimento , Plasmodium chabaudi/efeitos dos fármacos , Plasmodium chabaudi/enzimologia , Plasmodium chabaudi/fisiologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Inibidores de Proteassoma/efeitos adversos , Inibidores de Proteassoma/toxicidade , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade da Espécie , Especificidade por Substrato/efeitos dos fármacosRESUMO
Current first-line artemisinin antimalarials are threatened by the emergence of resistant Plasmodium falciparum. Decreased sensitivity is evident in the initial (early ring) stage of intraerythrocytic development, meaning that it is crucial to understand the action of artemisinins at this stage. Here, we examined the roles of iron (Fe) ions and haem in artemisinin activation in early rings using Fe ion chelators and a specific haemoglobinase inhibitor (E64d). Quantitative modelling of the antagonism accounted for its complex dependence on the chemical features of the artemisinins and on the drug exposure time, and showed that almost all artemisinin activity in early rings (>80%) is due to haem-mediated activation. The surprising implication that haemoglobin uptake and digestion is active in early rings is supported by identification of active haemoglobinases (falcipains) at this stage. Genetic down-modulation of the expression of the two main cysteine protease haemoglobinases, falcipains 2 and 3, renders early ring stage parasites resistant to artemisinins. This confirms the important role of haemoglobin-degrading falcipains in artemisinin activation, and shows that changes in the rate of artemisinin activation could mediate high-level artemisinin resistance.
Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Avaliação Pré-Clínica de Medicamentos , Resistência a Medicamentos , Sinergismo Farmacológico , Hemoglobinas , Humanos , Dose Letal Mediana , Leucina/análogos & derivados , Leucina/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/enzimologia , Proteólise , Proteínas de Protozoários/metabolismoRESUMO
Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.
Assuntos
Artemisininas/farmacologia , Plasmodium falciparum/fisiologia , Estresse Fisiológico , Animais , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Genoma de Protozoário , Mutação , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genéticaRESUMO
Artemisinin resistance constitutes a major threat to the continued success of control programs for malaria, particularly in light of developing resistance to partner drugs. Improving our understanding of how artemisinin-based drugs act and how resistance manifests is essential for the optimization of dosing regimens and the development of strategies to prolong the life span of current first-line treatment options. Recent short-drug-pulse in vitro experiments have shown that the parasite killing rate depends not only on drug concentration but also the exposure time, challenging the standard pharmacokinetic-pharmacodynamic (PK-PD) paradigm in which the killing rate depends only on drug concentration. Here, we introduce a dynamic stress model of parasite killing and show through application to 3D7 laboratory strain viability data that the inclusion of a time-dependent parasite stress response dramatically improves the model's explanatory power compared to that of a traditional PK-PD model. Our model demonstrates that the previously reported hypersensitivity of early-ring-stage parasites of the 3D7 strain to dihydroartemisinin compared to other parasite stages is due primarily to a faster development of stress rather than a higher maximum achievable killing rate. We also perform in vivo simulations using the dynamic stress model and demonstrate that the complex temporal features of artemisinin action observed in vitro have a significant impact on predictions for in vivo parasite clearance. Given the important role that PK-PD models play in the design of clinical trials for the evaluation of alternative drug dosing regimens, our novel model will contribute to the further development and improvement of antimalarial therapies.
Assuntos
Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Resistência a Medicamentos/fisiologia , Humanos , Modelos BiológicosRESUMO
Fully synthetic endoperoxide antimalarials, namely, OZ277 (RBx11160; also known as arterolane) and OZ439 (artefenomel), have been approved for marketing or are currently in clinical development. We undertook an analysis of the kinetics of the in vitro responses of Plasmodium falciparum to the new ozonide antimalarials. For these studies we used a K13 mutant (artemisinin resistant) isolate from a region in Cambodia and a genetically matched (artemisinin sensitive) K13 revertant. We used a pulsed-exposure assay format to interrogate the time dependence of the response. Because the ozonides have physicochemical properties different from those of the artemisinins, assay optimization was required to ensure that the drugs were completely removed following the pulsed exposure. Like that of artemisinins, ozonide activity requires active hemoglobin degradation. Short pulses of the ozonides were less effective than short pulses of dihydroartemisinin; however, when early-ring-stage parasites were exposed to drugs for periods relevant to their in vivo exposure, the ozonide antimalarials were markedly more effective.
Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Compostos Heterocíclicos/farmacologia , Testes de Sensibilidade Parasitária , Plasmodium falciparum/efeitos dos fármacosRESUMO
Reports of emerging resistance to first-line artemisinin antimalarials make it critical to define resistance mechanisms and identify in vitro correlates of resistance. Here we combine unique in vitro experimental and analytical approaches to mimic in vivo drug exposure in an effort to provide insight into mechanisms of drug resistance. Tightly synchronized parasites exposed to short drug pulses exhibit large stage-dependent differences in their drug response that correlate with hemoglobin digestion throughout most of the asexual cycle. As a result, ring-stage parasites can exhibit >100-fold lower sensitivity to short drug pulses than trophozoites, although we identify a subpopulation of rings (2-4 h postinvasion) that exhibits hypersensitivity. We find that laboratory strains that show little differences in drug sensitivity in standard in vitro assays exhibit substantial (>95-fold) difference in sensitivity when exposed to short drug pulses. These stage- and strain-dependent differences in drug sensitivity reflect differential response lag times with rings exhibiting lag times of up to 4 h. A simple model that assumes that the parasite experiences a saturable effective drug dose describes the complex dependence of parasite viability on both drug concentration and exposure time and is used to demonstrate that small changes in the parasite's drug response profile can dramatically alter the sensitivity to artemisinins. This work demonstrates that effective resistance can arise from the interplay between the short in vivo half-life of the drug and the stage-specific lag time and provides the framework for understanding the mechanisms of drug action and parasite resistance.
Assuntos
Artemisininas/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Lactonas/farmacologia , Modelos Biológicos , Plasmodium falciparum/metabolismo , Resistência a Medicamentos/fisiologia , Plasmodium falciparum/citologia , Especificidade da EspécieRESUMO
Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism.
Assuntos
Antimaláricos , Aspartato-tRNA Ligase , Animais , Humanos , Plasmodium falciparum/genética , Asparagina/metabolismo , Aspartato-tRNA Ligase/genética , Aminoacil-RNA de Transferência/metabolismo , Antimaláricos/farmacologia , Mamíferos/genéticaRESUMO
Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure activity relationship and the selectivity mechanism.
RESUMO
Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.
Assuntos
Antimaláricos , Malária Falciparum , Terapia de Alvo Molecular , Plasmodium falciparum , Biossíntese de Proteínas , Proteínas de Protozoários , Tirosina-tRNA Ligase , Adenosina/análogos & derivados , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Cristalografia por Raios X , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Camundongos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Ácidos Sulfônicos/química , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/metabolismoRESUMO
The Ubiquitin Proteasome System is the main proteolytic pathway in eukaryotic cells, playing a role in key cellular processes. The essentiality of the Plasmodium falciparum proteasome is well validated, underlying its potential as an antimalarial target, but selective compounds are required to avoid cytotoxic effects in humans. Almost 550000 compounds were tested for the inhibition of the chymotrypsin-like activity of the P. falciparum proteasome using a Proteasome-GLO luminescence assay. Hits were confirmed in an orthogonal enzyme assay using Rho110-labeled peptides, and selectivity was assessed against the human proteasome. Four nonpeptidomimetic chemical families with some selectivity for the P. falciparum proteasome were identified and characterized in assays of proteasome trypsin and caspase activities and in parasite growth inhibition assays. Target engagement studies were performed, validating our approach. Hits identified are good starting points for the development of new antimalarial drugs and as tools to better understand proteasome function in P. falciparum.
Assuntos
Antimaláricos , Malária Falciparum , Antimaláricos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum , Inibidores de Proteassoma/farmacologiaRESUMO
Artemisinins - the frontline antimalarial drug class - are compromised by emerging resistance, putting at risk the lives of hundreds of thousands of people each year. Resistance is associated with mutations in a malaria parasite protein, called Kelch 13 (K13). Recent work suggests that K13 is located at the cytostome (cell mouth) that the parasite uses to take up hemoglobin. Here we explore the proposal that K13 mutations confer artemisinin resistance by dampening hemoglobin endocytosis. This model suggests that the resultant decrease in hemoglobin-derived heme reduces artemisinin activation, which is sufficient to enable parasite survival in the early ring stage of infection. A fuller understanding of the resistance mechanism will underpin efforts to develop alternative antimalarial strategies.
Assuntos
Artemisininas/farmacologia , Resistência a Medicamentos/genética , Plasmodium/efeitos dos fármacos , Plasmodium/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Antimaláricos/farmacologia , Heme/metabolismo , Humanos , Mutação , Plasmodium/metabolismoRESUMO
Introduction: The proteasome is a multi-subunit enzyme complex responsible for the turnover of short-lived, abnormal or damaged proteins in eukaryotic cells. As organisms that undergo rapid growth and cell division, protozoan parasites exist on the knife-edge of proteotoxic catastrophe and thus rely heavily on their protein quality control machinery for survival. Because of this, the proteasome has recently emerged as a desirable drug target.Area covered: This review focuses on efforts to identify protozoan parasite-specific proteasome inhibitors using substrate profiling, library screening, and in vitro evolution of resistance approaches to inform medicinal chemistry. Targeting the parasite's 20S proteasome chymotrypsin-like (ß5) activity and selectively inhibiting protein turnover in parasites compared to human cells are critical properties of potent, selective inhibitors.Expert opinion: Proteasome inhibitors have the potential for rapid action against all stages, all species and all strains of plasmodium and kinetoplastid parasites. Given the high level of conservation of proteasome active sites in eukaryotes, an important challenge is achieving inhibitors that show sufficient selectivity while maintaining properties consistent with drug development.
Assuntos
Antiprotozoários/farmacologia , Inibidores de Proteassoma/farmacologia , Infecções por Protozoários/tratamento farmacológico , Animais , Desenvolvimento de Medicamentos , Humanos , Parasitos/isolamento & purificação , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Infecções por Protozoários/parasitologiaRESUMO
The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.
Assuntos
Plasmodium falciparum/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Artemisininas/farmacologia , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Simulação de Dinâmica Molecular , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Complexo de Endopeptidases do Proteassoma/genética , Conformação Proteica , Multimerização Proteica , Proteostase , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Increased tolerance of Plasmodium falciparum to front-line artemisinin antimalarials (ARTs) is associated with mutations in Kelch13 (K13), although the precise role of K13 remains unclear. Here, we show that K13 mutations result in decreased expression of this protein, while mislocalization of K13 mimics resistance-conferring mutations, pinpointing partial loss of function of K13 as the relevant molecular event. K13-GFP is associated with â¼170 nm diameter doughnut-shaped structures at the parasite periphery, consistent with the location and dimensions of cytostomes. Moreover, the hemoglobin-peptide profile of ring-stage parasites is reduced when K13 is mislocalized. We developed a pulse-SILAC approach to quantify protein turnover and observe less disruption to protein turnover following ART exposure when K13 is mislocalized. Our findings suggest that K13 regulates digestive vacuole biogenesis and the uptake/degradation of hemoglobin and that ART resistance is mediated by a decrease in heme-dependent drug activation, less proteotoxicity, and increased survival of parasite ring stages.
Assuntos
Artemisininas/metabolismo , Hemoglobinas/genética , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/genética , Humanos , MutaçãoRESUMO
Artemisinin and its derivatives (collectively referred to as ARTs) rapidly reduce the parasite burden in Plasmodium falciparum infections, and antimalarial control is highly dependent on ART combination therapies (ACTs). Decreased sensitivity to ARTs is emerging, making it critically important to understand the mechanism of action of ARTs. Here we demonstrate that dihydroartemisinin (DHA), the clinically relevant ART, kills parasites via a two-pronged mechanism, causing protein damage, and compromising parasite proteasome function. The consequent accumulation of proteasome substrates, i.e., unfolded/damaged and polyubiquitinated proteins, activates the ER stress response and underpins DHA-mediated killing. Specific inhibitors of the proteasome cause a similar build-up of polyubiquitinated proteins, leading to parasite killing. Blocking protein synthesis with a translation inhibitor or inhibiting the ubiquitin-activating enzyme, E1, reduces the level of damaged, polyubiquitinated proteins, alleviates the stress response, and dramatically antagonizes DHA activity.
RESUMO
The Plasmodium proteasome represents a potential antimalarial drug target for compounds with activity against multiple life cycle stages. We screened a library of human proteasome inhibitors (peptidyl boronic acids) and compared activities against purified P. falciparum and human 20S proteasomes. We chose four hits that potently inhibit parasite growth and show a range of selectivities for inhibition of the growth of P. falciparum compared with human cell lines. P. falciparum was selected for resistance in vitro to the clinically used proteasome inhibitor, bortezomib, and whole genome sequencing was applied to identify mutations in the proteasome ß5 subunit. Active site profiling revealed inhibitor features that enable retention of potent activity against the bortezomib-resistant line. Substrate profiling reveals P. falciparum 20S proteasome active site preferences that will inform attempts to design more selective inhibitors. This work provides a starting point for the identification of antimalarial drug leads that selectively target the P. falciparum proteasome.
Assuntos
Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Desenho de Fármacos , Plasmodium falciparum/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Resistência a Medicamentos/efeitos dos fármacos , Humanos , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/químicaRESUMO
Recent reports demonstrate that failure of artemisinin-based antimalarial therapies is associated with an altered response of early blood stage Plasmodium falciparum. This has led to increased interest in the use of pulse assays that mimic clinical drug exposure for analysing artemisinin sensitivity of highly synchronised ring stage parasites. We report a methodology for the reliable execution of drug pulse assays and detail a synchronisation strategy that produces well-defined tightly synchronised ring stage cultures in a convenient time-frame.