RESUMEN
Malaria, a mosquito-borne disease caused by several parasites of the Plasmodium genus, remains a huge threat to global public health. There are an estimated 0.5 million malaria deaths each year, mostly among African children. Unlike humans, Plasmodium parasites and a number of important pathogenic bacteria employ the methyl erythritol phosphate (MEP) pathway for isoprenoid synthesis. Thus, the MEP pathway represents a promising set of drug targets for antimalarial and antibacterial compounds. Here, we present new unsaturated MEPicide inhibitors of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway. A number of these compounds have demonstrated robust inhibition of Plasmodium falciparum DXR, potent antiparasitic activity, and low cytotoxicity against HepG2 cells. Parasites treated with active compounds are rescued by isopentenyl pyrophosphate, the product of the MEP pathway. With higher levels of DXR substrate, parasites acquire resistance to active compounds. These results further confirm the on-target inhibition of DXR in parasites by the inhibitors. Stability in mouse liver microsomes is high for the phosphonate salts, but remains a challenge for the prodrugs. Taken together, the potent activity and on-target mechanism of action of this series further validate DXR as an antimalarial drug target and the α,ß-unsaturation moiety as an important structural component.
Asunto(s)
Antimaláricos , Fosfomicina , Niño , Humanos , Animales , Ratones , Plasmodium falciparum , Fosfomicina/farmacología , Fosfomicina/química , Pentosafosfatos/metabolismo , Antimaláricos/farmacología , Antimaláricos/químicaRESUMEN
The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last â¼10 years, with numerous lead compounds identified, including several tRNA synthetase inhibitors. Here, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (Cryptosporidium parvum MetRS [CpMetRS]) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day 5. Parasites shed by each recrudescent calf had different amino acid-altering mutations in the gene encoding CpMetRS (CpMetRS), yielding either an aspartate 243-to-glutamate (D243E) or a threonine 246-to-isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant-expressing parasites, respectively, had 2093 half-maximal effective concentrations (EC50s) that were 613- and 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.
Asunto(s)
Enfermedades de los Bovinos , Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animales , Bovinos , Enfermedades de los Bovinos/tratamiento farmacológico , Niño , Preescolar , Criptosporidiosis/tratamiento farmacológico , Cryptosporidium/genética , Cryptosporidium parvum/genética , Resistencia a Medicamentos/genética , Heces , HumanosRESUMEN
With the rising prevalence of multidrug resistance, there is an urgent need to develop novel antibiotics. Many putative antibiotics demonstrate promising in vitro potency but fail in vivo due to poor drug-like qualities (e.g., serum half-life, oral absorption, solubility, and toxicity). These drug-like properties can be modified through the addition of chemical protecting groups, creating "prodrugs" that are activated prior to target inhibition. Lipophilic prodrugging techniques, including the attachment of a pivaloyloxymethyl group, have garnered attention for their ability to increase cellular permeability by masking charged residues and the relative ease of the chemical prodrugging process. Unfortunately, pivaloyloxymethyl prodrugs are rapidly activated by human sera, rendering any membrane permeability qualities absent during clinical treatment. Identification of the bacterial prodrug activation pathway(s) will allow for the development of host-stable and microbe-targeted prodrug therapies. Here, we use two zoonotic staphylococcal species, Staphylococcus schleiferi and S. pseudintermedius, to establish the mechanism of carboxy ester prodrug activation. Using a forward genetic screen, we identify a conserved locus in both species encoding the enzyme hydroxyacylglutathione hydrolase (GloB), whose loss-of-function confers resistance to carboxy ester prodrugs. We enzymatically characterize GloB and demonstrate that it is a functional glyoxalase II enzyme, which has the capacity to activate carboxy ester prodrugs. As GloB homologues are both widespread and diverse in sequence, our findings suggest that GloB may be a useful mechanism for developing species- or genus-level prodrug targeting strategies.
Asunto(s)
Profármacos , Antibacterianos/farmacología , Ésteres , Humanos , Profármacos/farmacología , StaphylococcusRESUMEN
Coagulase-positive staphylococci, which frequently colonize the mucosal surfaces of animals, also cause a spectrum of opportunistic infections including skin and soft tissue infections, urinary tract infections, pneumonia, and bacteremia. However, recent advances in bacterial identification have revealed that these common veterinary pathogens are in fact zoonoses that cause serious infections in human patients. The global spread of multidrug-resistant zoonotic staphylococci, in particular the emergence of methicillin-resistant organisms, is now a serious threat to both animal and human welfare. Accordingly, new therapeutic targets that can be exploited to combat staphylococcal infections are urgently needed. Enzymes of the methylerythritol phosphate pathway (MEP) of isoprenoid biosynthesis represent potential targets for treating zoonotic staphylococci. Here we demonstrate that fosmidomycin (FSM) inhibits the first step of the isoprenoid biosynthetic pathway catalyzed by deoxyxylulose phosphate reductoisomerase (DXR) in staphylococci. In addition, we have both enzymatically and structurally determined the mechanism by which FSM elicits its effect. Using a forward genetic screen, the glycerol-3-phosphate transporter GlpT that facilitates FSM uptake was identified in two zoonotic staphylococci, Staphylococcus schleiferi and Staphylococcus pseudintermedius. A series of lipophilic ester prodrugs (termed MEPicides) structurally related to FSM were synthesized, and data indicate that the presence of the prodrug moiety not only substantially increased potency of the inhibitors against staphylococci but also bypassed the need for GlpT-mediated cellular transport. Collectively, our data indicate that the prodrug MEPicides selectively and robustly inhibit DXR in zoonotic staphylococci, and further, that DXR represents a promising, druggable target for future development.
Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana Múltiple , Profármacos , Infecciones Estafilocócicas , Staphylococcus , Zoonosis , Animales , Antibacterianos/química , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Humanos , Profármacos/química , Profármacos/farmacología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/metabolismo , Staphylococcus/genética , Staphylococcus/crecimiento & desarrollo , Zoonosis/tratamiento farmacológico , Zoonosis/genética , Zoonosis/metabolismo , Zoonosis/microbiologíaRESUMEN
BACKGROUND: Cryptosporidiosis, an enteric protozoon, causes substantial morbidity and mortality associated with diarrhea in children <2 years old in low- to middle-income countries. There is no vaccine and treatments are inadequate. A piperazine-based compound, MMV665917, has in vitro and in vivo efficacy against Cryptosporidium parvum. In this study, we evaluated the efficacy of MMV665917 in gnotobiotic piglets experimentally infected with Cryptosporidium hominis, the species responsible for >75% of diarrhea reported in these children. METHODS: Gnotobiotic piglets were orally challenged with C hominis oocysts, and oral treatment with MMV665917 was commenced 3 days after challenge. Oocyst excretion and diarrhea severity were observed daily, and mucosal colonization and lesions were recorded after necropsy. RESULTS: MMV665917 significantly reduced fecal oocyst excretion, parasite colonization and damage to the intestinal mucosa, and peak diarrheal symptoms, compared with infected untreated controls. A dose of 20 mg/kg twice daily for 7 days was more effective than 10 mg/kg. There were no signs of organ toxicity at either dose, but 20 mg/kg was associated with slightly elevated blood cholesterol and monocytes at euthanasia. CONCLUSIONS: These results demonstrate the effectiveness of this drug against C hominis. Piperazine-derivative MMV665917 may potentially be used to treat human cryptosporidiosis; however, further investigations are required.
Asunto(s)
Criptosporidiosis/tratamiento farmacológico , Cryptosporidium parvum/efectos de los fármacos , Diarrea/tratamiento farmacológico , Piperazinas/farmacología , Animales , Criptosporidiosis/parasitología , Diarrea/parasitología , Modelos Animales de Enfermedad , Mucosa Intestinal/parasitología , Monocitos/parasitología , Oocistos/efectos de los fármacos , PorcinosRESUMEN
Severe malaria due to Plasmodium falciparum remains a significant global health threat. DXR, the second enzyme in the MEP pathway, plays an important role to synthesize building blocks for isoprenoids. This enzyme is a promising drug target for malaria due to its essentiality as well as its absence in humans. In this study, we designed and synthesized a series of α,ß-unsaturated analogues of fosmidomycin, a natural product that inhibits DXR in P. falciparum. All compounds were evaluated as inhibitors of P. falciparum. The most promising compound, 18a, displays on-target, potent inhibition against the growth of P. falciparum (IC50 = 13 nM) without significant inhibition of HepG2 cells (IC50 > 50 µM). 18a was also tested in a luciferase-based Plasmodium berghei mouse model of malaria and showed exceptional in vivo efficacy. Together, the data support MEPicide 18a as a novel, potent, and promising drug candidate for the treatment of malaria.
Asunto(s)
Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Antimaláricos/farmacología , Fosfomicina/análogos & derivados , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/crecimiento & desarrollo , Profármacos/farmacología , Animales , Antimaláricos/química , Femenino , Fosfomicina/química , Fosfomicina/farmacología , Malaria Falciparum/enzimología , Malaria Falciparum/parasitología , Ratones , Plasmodium falciparum/efectos de los fármacos , Profármacos/química , Relación Estructura-ActividadRESUMEN
The ability to measure chemical gradients surrounding single cells provides novel insights into several areas of cell dynamics--particularly metabolism. Detection of metabolic oxygen consumption can be achieved from a single mammalian cell using a modulated amperometric sensor in a self-referencing mode. To date, however, apart from visual cues, we do not have a reliable and cell-compatible method for determining and stabilizing the position of such probes. In this paper, we report on having successfully measured the increase in the uncompensated resistance of an electrochemical cell upon approach to single, living, biological cells, while simultaneously measuring the metabolic oxygen consumption. This was accomplished by applying an ac and a dc excitation signal to the electrode. The applied ac waveform was a 100-kHz sine wave with an amplitude of 10 mV rms, while the dc voltage applied was -600 mV. The two signals were shown not to interfere with one another. Furthermore, it is shown that the sample-probe distance can be measured for approach to single cells on the order of 10-15-microm diameter and 5-microm height, with 100-nm resolution.
Asunto(s)
Técnicas Citológicas/métodos , Consumo de Oxígeno/fisiología , Animales , Línea Celular , Técnicas Citológicas/instrumentación , Impedancia Eléctrica , Insulinoma , RatasRESUMEN
A rapid and sensitive method to determine 8-oxoguanine (8oxoG) and 8-hydroxydeoxyguanosine (8OHdG), biomarkers for oxidative DNA damage, in cerebral cortex microdialysate samples using capillary electrophoresis (CE) with electrochemical detection (CEEC) was developed. Samples were concentrated on-column using pH-mediated stacking for anions. On-column anodic detection was performed with a carbon fiber working electrode and laser-etched decoupler. The method is linear over the expected extracellular concentration range for 8oxoG and 8-OHdG during induced ischemia-reperfusion, with R.S.D. values Asunto(s)
Biomarcadores/análisis
, Corteza Cerebral/química
, Desoxiadenosinas/análisis
, Guanina/análogos & derivados
, Animales
, Cromatografía Liquida/métodos
, Electroquímica
, Electroforesis Capilar/métodos
, Femenino
, Guanina/análisis
, Espectrometría de Masas/métodos
, Microdiálisis/métodos
, Ratas
, Ratas Sprague-Dawley
RESUMEN
The development of a cellulose acetate decoupler for on-column electrochemical detection in microchip capillary electrophoresis is presented. The capillary based laser-etched decoupler is translated to the planar format to isolate the detector circuit from the separation circuit. The decoupler is constructed by aligning a series of 20 30-microm holes through the coverplate of the microchip with the separation channel and casting a thin film of cellulose acetate within the holes. The decoupler shows excellent isolation of the detection circuit for separation currents up to 60 microA, with noise levels at or below 1 pA at a carbon fiber electrode. Detection limits of 25 nM were achieved for dopamine. This decoupler design combines excellent mechanical stability, effective shunting of high separation currents, and ease of manufacture.