RESUMEN
To contribute to the global effort to develop new antimalarial therapies, we previously disclosed initial findings on the optimization of the dihydroquinazolinone-3-carboxamide class that targets PfATP4. Here we report on refining the aqueous solubility and metabolic stability to improve the pharmacokinetic profile and consequently in vivo efficacy. We show that the incorporation of heterocycle systems in the 8-position of the scaffold was found to provide the greatest attainable balance between parasite activity, aqueous solubility, and metabolic stability. Optimized analogs, including the frontrunner compound S-WJM992, were shown to inhibit PfATP4-associated Na+-ATPase activity, gave rise to a metabolic signature consistent with PfATP4 inhibition, and displayed altered activities against parasites with mutations in PfATP4. Finally, S-WJM992 showed appreciable efficacy in a malaria mouse model and blocked gamete development preventing transmission to mosquitoes. Importantly, further optimization of the dihydroquinazolinone class is required to deliver a candidate with improved pharmacokinetic and risk of resistance profiles.
Asunto(s)
Antimaláricos , Plasmodium falciparum , Quinazolinonas , Antimaláricos/farmacología , Antimaláricos/química , Antimaláricos/farmacocinética , Animales , Plasmodium falciparum/efectos de los fármacos , Quinazolinonas/farmacología , Quinazolinonas/química , Quinazolinonas/farmacocinética , Ratones , Administración Oral , Relación Estructura-Actividad , Humanos , Malaria/tratamiento farmacológico , Femenino , SolubilidadRESUMEN
Emerging resistance to current antimalarials is reducing their effectiveness and therefore there is a need to develop new antimalarial therapies. Toward this goal, high throughput screens against the P. falciparum asexual parasite identified the pyrazolopyridine 4-carboxamide scaffold. Structure-activity relationship analysis of this chemotype defined that the N1-tert-butyl group and aliphatic foliage in the 3- and 6-positions were necessary for activity, while the inclusion of a 7'-aza-benzomorpholine on the 4-carboxamide motif resulted in potent anti-parasitic activity and increased aqueous solubility. A previous report that resistance to the pyrazolopyridine class is associated with the ABCI3 transporter was confirmed, with pyrazolopyridine 4-carboxamides showing an increase in potency against parasites when the ABCI3 transporter was knocked down. The low metabolic stability intrinsic to the pyrazolopyridine scaffold and the slow rate by which the compounds kill asexual parasites resulted in poor performance in a P. berghei asexual blood stage mouse model. Lowering the risk of resistance and mitigating the metabolic stability and cytochrome P450 inhibition will be challenges in the future development of the pyrazolopyrimidine antimalarial class.
Asunto(s)
Antimaláricos , Plasmodium falciparum , Pirazoles , Piridinas , Antimaláricos/farmacología , Antimaláricos/química , Antimaláricos/síntesis química , Plasmodium falciparum/efectos de los fármacos , Relación Estructura-Actividad , Pirazoles/química , Pirazoles/farmacología , Pirazoles/síntesis química , Animales , Piridinas/farmacología , Piridinas/química , Piridinas/síntesis química , Ratones , Pruebas de Sensibilidad Parasitaria , Estructura Molecular , Resistencia a Medicamentos/efectos de los fármacos , Relación Dosis-Respuesta a Droga , HumanosRESUMEN
Vortioxetine (VTX) is a recently approved antidepressant that targets a variety of serotonin receptors. Here, we investigate the drug's molecular mechanism of operation at the serotonin 5-HT3 receptor (5-HT3R), which features two properties: VTX acts differently on rodent and human 5-HT3R, and VTX appears to suppress any subsequent response to agonists. Using a combination of cryo-EM, electrophysiology, voltage-clamp fluorometry and molecular dynamics, we show that VTX stabilizes a resting inhibited state of the mouse 5-HT3R and an agonist-bound-like state of human 5-HT3R, in line with the functional profile of the drug. We report four human 5-HT3R structures and show that the human receptor transmembrane domain is intrinsically fragile. We also explain the lack of recovery after VTX administration via a membrane partition mechanism.
Asunto(s)
Antidepresivos , Microscopía por Crioelectrón , Receptores de Serotonina 5-HT3 , Vortioxetina , Vortioxetina/farmacología , Vortioxetina/química , Humanos , Receptores de Serotonina 5-HT3/metabolismo , Receptores de Serotonina 5-HT3/química , Animales , Ratones , Antidepresivos/farmacología , Antidepresivos/química , Piperazinas/farmacología , Piperazinas/química , Sulfuros/química , Sulfuros/farmacología , Simulación de Dinámica Molecular , Células HEK293RESUMEN
Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.
Asunto(s)
Antimaláricos , Proteínas Portadoras , Malaria Falciparum , Malaria , Ratas , Ratones , Humanos , Animales , Antimaláricos/química , Plasmodium falciparum , Malaria Falciparum/tratamiento farmacológico , Malaria/tratamiento farmacológico , Acetamidas/farmacología , LípidosRESUMEN
There is an urgent need to populate the antimalarial clinical portfolio with new candidates because of resistance against frontline antimalarials. To discover new antimalarial chemotypes, we performed a high-throughput screen of the Janssen Jumpstarter library against the Plasmodium falciparum asexual blood-stage parasite and identified the 2,3-dihydroquinazolinone-3-carboxamide scaffold. We defined the SAR and found that 8-substitution on the tricyclic ring system and 3-substitution of the exocyclic arene produced analogues with potent activity against asexual parasites equivalent to clinically used antimalarials. Resistance selection and profiling against drug-resistant parasite strains revealed that this antimalarial chemotype targets PfATP4. Dihydroquinazolinone analogues were shown to disrupt parasite Na+ homeostasis and affect parasite pH, exhibited a fast-to-moderate rate of asexual kill, and blocked gametogenesis, consistent with the phenotype of clinically used PfATP4 inhibitors. Finally, we observed that optimized frontrunner analogue WJM-921 demonstrates oral efficacy in a mouse model of malaria.
Asunto(s)
Antimaláricos , Malaria Falciparum , Malaria , Animales , Ratones , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Plasmodium falciparum , Homeostasis , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitologíaRESUMEN
The development of new antimalarials is required because of the threat of resistance to current antimalarial therapies. To discover new antimalarial chemotypes, we screened the Janssen Jumpstarter library against the P. falciparum asexual parasite and identified the 7-N-substituted-3-oxadiazole quinolone hit class. We established the structure-activity relationship and optimized the antimalarial potency. The optimized analog WJM228 (17) showed robust metabolic stability in vitro, although the aqueous solubility was limited. Forward genetic resistance studies uncovered that WJM228 targets the Qo site of cytochrome b (cyt b), an important component of the mitochondrial electron transport chain (ETC) that is essential for pyrimidine biosynthesis and an established antimalarial target. Profiling against drug-resistant parasites confirmed that WJM228 confers resistance to the Qo site but not Qi site mutations, and in a biosensor assay, it was shown to impact the ETC via inhibition of cyt b. Consistent with other cyt b targeted antimalarials, WJM228 prevented pre-erythrocytic parasite and male gamete development and reduced asexual parasitemia in a P. berghei mouse model of malaria. Correcting the limited aqueous solubility and the high susceptibility to cyt b Qo site resistant parasites found in the clinic will be major obstacles in the future development of the 3-oxadiazole quinolone antimalarial class.
Asunto(s)
Antimaláricos , Antagonistas del Ácido Fólico , Malaria Falciparum , Quinolonas , Animales , Ratones , Antimaláricos/farmacología , Citocromos b , Antagonistas del Ácido Fólico/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Plasmodium falciparum , Quinolonas/farmacologíaRESUMEN
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.
Asunto(s)
Compuestos de Boro/farmacología , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/química , Inhibidores de Proteasoma/farmacología , Administración Oral , Animales , Compuestos de Boro/administración & dosificación , Compuestos de Boro/química , Dominio Catalítico , Humanos , Malaria Falciparum/enzimología , Malaria Falciparum/parasitología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Moleculares , Plasmodium falciparum/enzimología , Inhibidores de Proteasoma/administración & dosificación , Inhibidores de Proteasoma/químicaRESUMEN
The emergence and spread of Plasmodium falciparum resistance to first-line antimalarials creates an imperative to identify and develop potent preclinical candidates with distinct modes of action. Here, we report the identification of MMV688533, an acylguanidine that was developed following a whole-cell screen with compounds known to hit high-value targets in human cells. MMV688533 displays fast parasite clearance in vitro and is not cross-resistant with known antimalarials. In a P. falciparum NSG mouse model, MMV688533 displays a long-lasting pharmacokinetic profile and excellent safety. Selection studies reveal a low propensity for resistance, with modest loss of potency mediated by point mutations in PfACG1 and PfEHD. These proteins are implicated in intracellular trafficking, lipid utilization, and endocytosis, suggesting interference with these pathways as a potential mode of action. This preclinical candidate may offer the potential for a single low-dose cure for malaria.
Asunto(s)
Antimaláricos , Malaria Falciparum , Malaria , Parásitos , Animales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Endocitosis , Malaria/tratamiento farmacológico , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparumRESUMEN
The aspartic proteases plasmepsin IX/X are important antimalarial drug targets due to their specificity to the malaria parasite and their vital role as mediators of disease progression. Focusing on parasite-specific targets where no human homologue exists reduces the possibility of on-target drug toxicity. However, there is a risk of toxicity driven by inadequate selectivity for plasmepsins IX/X in Plasmodium over related mammalian aspartic proteases. Of these, CatD/E may be of most toxicological relevance as CatD is a ubiquitous lysosomal enzyme present in most cell types and CatE is found in the gut and in erythrocytes, the clinically significant site of malarial infection. Based on mammalian aspartic protease physiology and adverse drug reactions (ADRs) to FDA-approved human immunodeficiency virus (HIV) aspartic protease inhibitors, we predicted several potential toxicities including ß-cell and congenital abnormalities, hypotension, hypopigmentation, hyperlipidaemia, increased infection risk and respiratory, renal, gastrointestinal, dermatological, and other epithelial tissue toxicities. These ADRs to the HIV treatments are likely to be a result of host aspartic protease inhibition due a lack of specificity for the HIV protease; plasmepsins are much more closely related to human CatD than to HIV proteinase. Plasmepsin IX/X inhibition presents an opportunity to specifically target Plasmodium as an effective antimalarial treatment, providing adequate selectivity can be obtained. Potential plasmepsin IX/X inhibitors should be assayed for inhibitory activity against the main human aspartic proteases and particularly CatD/E. An investigative rodent study conducted early in drug discovery would serve as an initial risk assessment of the potential hazards identified.
RESUMEN
Mitochondrial Carriers (MCs) are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism. The ADP/ATP Carriers (AACs) are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP. In human, four different AACs isoforms are described which are expressed in tissue-specific manner. They are involved in different genetic diseases and play a role in cancerogenesis. Up to now only the structures of the bovine (isoform 1) and yeast (isoforms 2 and 3) AAC have been determined in one particular conformation, obtained in complex with the CATR inhibitor. Herein, we report that full-length human ADP/ATP Carriers isoform 1 and 3 were successfully expressed in cell-free system and purified in milligram amounts in detergent-solubilized state. The proteins exhibited the expected secondary structure content. Thermostability profiles showing stabilization by the CATR inhibitor suggest that the carriers are well folded.
Asunto(s)
Translocasas Mitocondriales de ADP y ATP/aislamiento & purificación , Sistema Libre de Células , Expresión Génica , Humanos , Translocasas Mitocondriales de ADP y ATP/genética , Translocasas Mitocondriales de ADP y ATP/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Human plasma paraoxonase (HuPON1) is a high density lipoprotein (HDL)-bound enzyme exhibiting antiatherogenic properties. The molecular basis for the binding specificity of HuPON1 to HDL has not been established. Isolation of HuPON1 from HDL requires the use of detergents. We have determined the activity, dispersity, and oligomeric states of HuPON1 in solutions containing mild detergents using nondenaturing electrophoresis, size exclusion chromatography, and cross-linking. HuPON1 was active whatever its oligomeric state. In nonmicellar solutions, HuPON1 was polydisperse. In contrast, HuPON1 exhibited apparent homogeneity in micellar solutions, except with CHAPS. The enzyme apparent hydrodynamic radius varied with the type of detergent and protein concentration. In C(12)E(8) micellar solutions, from sedimentation velocity, equilibrium analytical ultracentrifugation, and radioactive detergent binding, HuPON1 was described as monomers and dimers in equilibrium. A decrease of the detergent concentration shifted this equilibrium toward the formation of dimers. About 100 detergent molecules were associated per monomer and dimer. The assembly of amphiphilic molecules, phospholipids in vivo, in sufficiently large aggregates could be a prerequisite for anchoring of HuPON1 and then allowing stabilization of the enzyme activity. Changes of HDL size and shape could strongly affect the binding affinity and stability of HuPON1 and result in reduced antioxidative capacity of the lipoprotein.