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1.
Cell ; 185(19): 3520-3532.e26, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-36041435

RESUMO

We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6-12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6-12 residue size range cross membranes with an apparent permeability greater than 1 × 10-6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.


Assuntos
Amidas , Peptídeos , Amidas/química , Hidrogênio , Ligação de Hidrogênio , Lipídeos , Peptídeos/química
2.
Cell ; 184(21): 5432-5447.e16, 2021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34619077

RESUMO

Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle (RBD-NP) vaccine protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NPs in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested, although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic.

3.
J Infect Dis ; 229(2): 558-566, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-37889572

RESUMO

Congenital toxoplasmosis in humans and in other mammalian species, such as small ruminants, is a well-known cause of abortion and fetal malformations. The calcium-dependent protein kinase 1 (CDPK1) inhibitor BKI-1748 has shown a promising safety profile for its use in humans and a good efficacy against Toxoplasma gondii infection in vitro and in mouse models. Ten doses of BKI-1748 given every other day orally in sheep at 15 mg/kg did not show systemic or pregnancy-related toxicity. In sheep experimentally infected at 90 days of pregnancy with 1000 TgShSp1 oocysts, the BKI-1748 treatment administered from 48 hours after infection led to complete protection against abortion and congenital infection. In addition, compared to infected/untreated sheep, treated sheep showed a drastically lower rectal temperature increase and none showed IgG seroconversion throughout the study. In conclusion, BKI-1748 treatment in pregnant sheep starting at 48 hours after infection was fully effective against congenital toxoplasmosis.


Assuntos
Aborto Espontâneo , Doenças Transmissíveis , Toxoplasma , Toxoplasmose Congênita , Toxoplasmose , Gravidez , Humanos , Feminino , Camundongos , Ovinos , Animais , Toxoplasmose Congênita/tratamento farmacológico , Toxoplasmose Congênita/prevenção & controle , Mamíferos
4.
Int J Mol Sci ; 25(5)2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38473953

RESUMO

Cryptosporidium parvum is an apicomplexan parasite causing persistent diarrhea in humans and animals. Issuing from target-based drug development, calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs), with excellent efficacies in vitro and in vivo have been generated. Some BKIs including BKI-1748 share a core structure with similarities to the first-generation antiprotozoal drug quinine, which is known to exert notorious side effects. Unlike quinine, BKI-1748 rapidly interfered with C. parvum proliferation in the human colon tumor (HCT) cell line HCT-8 cells and caused dramatic effects on the parasite ultrastructure. To identify putative BKI targets in C. parvum and in host cells, we performed differential affinity chromatography with cell-free extracts from non-infected and infected HCT-8 cells using BKI-1748 and quinine epoxy-activated sepharose columns followed by mass spectrometry. C. parvum proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from both BKI-1748 and quinine columns. However, no C. parvum proteins could be identified binding exclusively to BKI-1748. In contrast, 25 BKI-1748-specific binding proteins originating from HCT-8 cells were detected. Moreover, 29 C. parvum and 224 host cell proteins were identified in both BKI-1748 as well as in quinine eluates. In both C. parvum and host cells, the largest subset of binding proteins was involved in RNA binding and modification, with a focus on ribosomal proteins and proteins involved in RNA splicing. These findings extend previous results, showing that BKI-1748 interacts with putative targets involved in common, essential pathways such as translation and RNA processing.


Assuntos
Antineoplásicos , Antiprotozoários , Criptosporidiose , Cryptosporidium parvum , Cryptosporidium , Animais , Humanos , Quinina/farmacologia , Antiprotozoários/farmacologia , Antineoplásicos/farmacologia
6.
Antimicrob Agents Chemother ; 67(4): e0142522, 2023 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-36920244

RESUMO

Recent advances on the development of bumped kinase inhibitors for treatment of cryptosporidiosis have focused on the 5-aminopyrazole-4-carboxamide scaffold, due to analogs that have less hERG inhibition, superior efficacy, and strong in vitro safety profiles. Three compounds, BKI-1770, -1841, and -1708, showed strong efficacy in C. parvum infected mice. Both BKI-1770 and BKI-1841 had efficacy in the C. parvum newborn calf model, reducing diarrhea and oocyst excretion. However, both compounds caused hyperflexion of the limbs seen as dropped pasterns. Toxicity experiments in rats and calves dosed with BKI-1770 showed enlargement of the epiphyseal growth plate at doses only slightly higher than the efficacious dose. Mice were used as a screen to check for bone toxicity, by changes to the tibia epiphyseal growth plate, or neurological causes, by use of a locomotor activity box. These results showed neurological effects from both BKI-1770 and BKI-1841 and bone toxicity in mice from BKI-1770, indicating one or both effects may be contributing to toxicity. However, BKI-1708 remains a viable treatment candidate for further evaluation as it showed no signs of bone toxicity or neurological effects in mice.


Assuntos
Antineoplásicos , Antiprotozoários , Criptosporidiose , Cryptosporidium parvum , Animais , Bovinos , Camundongos , Ratos , Criptosporidiose/tratamento farmacológico , Antiprotozoários/farmacologia , Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Oocistos
7.
J Nat Prod ; 86(6): 1596-1605, 2023 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-37276438

RESUMO

Xanthoquinodins make up a distinctive class of xanthone-anthraquinone heterodimers reported as secondary metabolites from several fungal species. Through a collaborative multi-institutional screening program, a fungal extract prepared from a Trichocladium sp. was identified that exhibited strong inhibitory effects against several human pathogens (Mycoplasma genitalium, Plasmodium falciparum, Cryptosporidium parvum, and Trichomonas vaginalis). This report focuses on one of the unique samples that exhibited a desirable combination of biological effects: namely, it inhibited all four test pathogens and demonstrated low levels of toxicity toward HepG2 (human liver) cells. Fractionation and purification of the bioactive components and their congeners led to the identification of six new compounds [xanthoquinodins NPDG A1-A5 (1-5) and B1 (6)] as well as several previously reported natural products (7-14). The chemical structures of 1-14 were determined based on interpretation of their 1D and 2D NMR, HRESIMS, and electronic circular dichroism (ECD) data. Biological testing of the purified metabolites revealed that they possessed widely varying levels of inhibitory activity against a panel of human pathogens. Xanthoquinodins A1 (7) and A2 (8) exhibited the most promising broad-spectrum inhibitory effects against M. genitalium (EC50 values: 0.13 and 0.12 µM, respectively), C. parvum (EC50 values: 5.2 and 3.5 µM, respectively), T. vaginalis (EC50 values: 3.9 and 6.8 µM, respectively), and P. falciparum (EC50 values: 0.29 and 0.50 µM, respectively) with no cytotoxicity detected at the highest concentration tested (HepG2 EC50 > 25 µM).


Assuntos
Anti-Infecciosos , Criptosporidiose , Cryptosporidium , Fungos Mitospóricos , Humanos , Anti-Infecciosos/farmacologia , Antibacterianos/farmacologia , Estrutura Molecular
8.
Exp Parasitol ; 255: 108655, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37981259

RESUMO

In previous studies, the artemisinin derivatives artemisone, its pro-drug artemiside and the bumped-kinase inhibitor BKI-1748 were effective against T. gondii via different modes of action. This suggests that they may act synergistically resulting in improved efficacies in vitro and in vivo. To test this hypothesis, the compounds were applied alone and in combination to T. gondii infected human fibroblast host cells in order to determine their inhibition constants and effects on cellular ultrastructure. In addition, the efficacy of either single- or combined treatments were assessed in an acute TgShSp1-oocyst infection model based on CD1 outbred mice. Whereas the IC50 of the compounds in combination (42 nM) was close to the IC50 of BKI-1748 alone (46 nM) and half of the IC50 of artemisone alone (92 nM), the IC90 of the combination was half of the values found with the single compounds (138 nM vs. ca. 270 nM). Another indication for synergistic effects in vitro were distinct alterations of the cellular ultrastructure of tachyzoites observed in combination, but not with the single compounds. These promising results could not be reproduced in vivo. There was no decrease in number of T. gondii positive brains by either treatment. However, the levels of infection in these brains, i. e. the number of tachyzoites, was significantly decreased upon BKI-1748 treatment alone, and the combination with artemiside did not produce any further decrease. The treatment with artemiside alone had no significant effects. A vertical transmission model could not be established since artemiside strongly interfered with pregnancy and caused abortion. These results show that is difficult to extrapolate from promising in vitro results to the situation in vivo.


Assuntos
Antineoplásicos , Artemisininas , Toxoplasma , Toxoplasmose , Gravidez , Feminino , Camundongos , Humanos , Animais , Toxoplasmose/tratamento farmacológico , Artemisininas/farmacologia , Artemisininas/uso terapêutico , Transmissão Vertical de Doenças Infecciosas , Antineoplásicos/farmacologia
9.
Antimicrob Agents Chemother ; 66(1): e0156021, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-34748385

RESUMO

Infection with Cryptosporidium spp. can cause severe diarrhea, leading to long-term adverse impacts and even death in malnourished children and immunocompromised patients. The only FDA-approved drug for treating cryptosporidiosis, nitazoxanide, has limited efficacy in the populations impacted the most by the diarrheal disease, and safe, effective treatment options are urgently needed. Initially identified by a large-scale phenotypic screening campaign, the antimycobacterial therapeutic clofazimine demonstrated great promise in both in vitro and in vivo preclinical models of Cryptosporidium infection. Unfortunately, a phase 2a clinical trial in HIV-infected adults with cryptosporidiosis did not identify any clofazimine treatment effect on Cryptosporidium infection burden or clinical outcomes. To explore whether clofazimine's lack of efficacy in the phase 2a trial may have been due to subtherapeutic clofazimine concentrations, a pharmacokinetic/pharmacodynamic modeling approach was undertaken to determine the relationship between clofazimine in vivo concentrations and treatment effects in multiple preclinical infection models. Exposure-response relationships were characterized using Emax and logistic models, which allowed predictions of efficacious clofazimine concentrations for the control and reduction of disease burden. After establishing exposure-response relationships for clofazimine treatment of Cryptosporidium infection in our preclinical model studies, it was unmistakable that the clofazimine levels observed in the phase 2a study participants were well below concentrations associated with anti-Cryptosporidium efficacy. Thus, despite a dosing regimen above the highest doses recommended for mycobacterial therapy, it is very likely the lack of treatment effect in the phase 2a trial was at least partially due to clofazimine concentrations below those required for efficacy against cryptosporidiosis. It is unlikely that clofazimine will provide a remedy for the large number of cryptosporidiosis patients currently without a viable treatment option unless alternative, safe clofazimine formulations with improved oral absorption are developed. (This study has been registered in ClinicalTrials.gov under identifier NCT03341767.).


Assuntos
Antiprotozoários , Criptosporidiose , Cryptosporidium , Adulto , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Criança , Clofazimina/farmacologia , Clofazimina/uso terapêutico , Criptosporidiose/tratamento farmacológico , Diarreia/tratamento farmacológico , Humanos
10.
Int J Mol Sci ; 23(4)2022 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-35216497

RESUMO

Neospora caninum is an apicomplexan parasite closely related to Toxoplasma gondii, and causes abortions, stillbirths and/or fetal malformations in livestock. Target-based drug development has led to the synthesis of calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs). Previous studies have shown that several BKIs have excellent efficacy against neosporosis in vitro and in vivo. However, several members of this class of compounds impair fertility in pregnant mouse models and cause embryonic malformation in a zebrafish (Danio rerio) model. Similar to the first-generation antiprotozoal drug quinine, some BKIs have a quinoline core structure. To identify common targets in both organisms, we performed differential affinity chromatography with cell-free extracts from N. caninum tachyzoites and D. rerio embryos using the 5-aminopyrazole-4-carboxamide (AC) compound BKI-1748 and quinine columns coupled to epoxy-activated sepharose followed by mass spectrometry. BKI-binding proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from BKI-1748 as well as quinine columns. In N. caninum, 12 proteins were bound specifically to BKI-1748 alone, and 105 proteins, including NcCDPK1, were bound to both BKI-1748 and quinine. For D. rerio, the corresponding numbers were 13 and 98 binding proteins, respectively. In both organisms, a majority of BKI-1748 binding proteins was involved in RNA binding and modification, in particular, splicing. Moreover, both datasets contained proteins involved in DNA binding or modification and key steps of intermediate metabolism. These results suggest that BKI-1748 interacts with not only specific targets in apicomplexans, such as CDPK1, but also with targets in other eukaryotes, which are involved in common, essential pathways.


Assuntos
Neospora/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Quinolonas/metabolismo , Peixe-Zebra/metabolismo , Animais , Antiprotozoários/metabolismo , Células Cultivadas , Quinolinas/metabolismo
11.
Clin Infect Dis ; 71(6): 1481-1490, 2020 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31621832

RESUMO

BACKGROUND: Chemoprophylaxis vaccination with sporozoites (CVac) with chloroquine induces protection against a homologous Plasmodium falciparum sporozoite (PfSPZ) challenge, but whether blood-stage parasite exposure is required for protection remains unclear. Chloroquine suppresses and clears blood-stage parasitemia, while other antimalarial drugs, such as primaquine, act against liver-stage parasites. Here, we evaluated CVac regimens using primaquine and/or chloroquine as the partner drug to discern whether blood-stage parasite exposure impacts protection against homologous controlled human malaria infection. METHODS: In a Phase I, randomized, partial double-blind, placebo-controlled study of 36 malaria-naive adults, all CVac subjects received chloroquine prophylaxis and bites from 12-15 P. falciparum-infected mosquitoes (CVac-chloroquine arm) at 3 monthly iterations, and some received postexposure primaquine (CVac-primaquine/chloroquine arm). Drug control subjects received primaquine, chloroquine, and uninfected mosquito bites. After a chloroquine washout, subjects, including treatment-naive infectivity controls, underwent homologous, PfSPZ controlled human malaria infection and were monitored for parasitemia for 21 days. RESULTS: No serious adverse events occurred. During CVac, all but 1 subject in the study remained blood-smear negative, while only 1 subject (primaquine/chloroquine arm) remained polymerase chain reaction-negative. Upon challenge, compared to infectivity controls, 3/3 chloroquine arm subjects displayed delayed patent parasitemia (P = .01) but not sterile protection, while 3/11 primaquine/chloroquine subjects remained blood-smear negative. CONCLUSIONS: CVac-primaquine/chloroquine is safe and induces sterile immunity to P. falciparum in some recipients, but a single 45 mg dose of primaquine postexposure does not completely prevent blood-stage parasitemia. Unlike previous studies, CVac-chloroquine did not produce sterile immunity. CLINICAL TRIALS REGISTRATION: NCT01500980.


Assuntos
Antimaláricos , Malária Falciparum , Adulto , Animais , Antimaláricos/uso terapêutico , Quimioprevenção , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/prevenção & controle , Plasmodium falciparum , Esporozoítos , Vacinação
12.
Molecules ; 25(12)2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32560085

RESUMO

Transmission of human malaria parasites (Plasmodium spp.) by Anopheles mosquitoes is a continuous process that presents a formidable challenge for effective control of the disease. Infectious gametocytes continue to circulate in humans for up to four weeks after antimalarial drug treatment, permitting prolonged transmission to mosquitoes even after clinical cure. Almost all reported malaria cases are transmitted to humans by mosquitoes, and therefore decreasing the rate of Plasmodium transmission from humans to mosquitoes with novel transmission-blocking remedies would be an important complement to other interventions in reducing malaria incidence.


Assuntos
Anopheles/parasitologia , Antimaláricos/uso terapêutico , Malária , Animais , Humanos , Malária/epidemiologia , Malária/prevenção & controle , Malária/transmissão
13.
J Infect Dis ; 220(7): 1188-1198, 2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31180118

RESUMO

Recent studies have illustrated the burden Cryptosporidium infection places on the lives of malnourished children and immunocompromised individuals. Treatment options remain limited, and efforts to develop a new therapeutic are currently underway. However, there are unresolved questions about the ideal pharmacokinetic characteristics of new anti-Cryptosporidium therapeutics. Specifically, should drug developers optimize therapeutics and formulations to increase drug exposure in the gastrointestinal lumen, enterocytes, or systemic circulation? Furthermore, how should researchers interpret data suggesting their therapeutic is a drug efflux transporter substrate? In vivo drug transporter-mediated alterations in efficacy are well recognized in multiple disease areas, but the impact of intestinal transporters on therapeutic efficacy against enteric diseases has not been established. Using multiple in vitro models and a mouse model of Cryptosporidium infection, we characterized the effect of P-glycoprotein efflux on bumped kinase inhibitor pharmacokinetics and efficacy. Our results demonstrated P-glycoprotein decreases bumped kinase inhibitor enterocyte exposure, resulting in reduced in vivo efficacy against Cryptosporidium. Furthermore, a hollow fiber model of Cryptosporidium infection replicated the in vivo impact of P-glycoprotein on anti-Cryptosporidium efficacy. In conclusion, when optimizing drug candidates targeting the gastrointestinal epithelium or gastrointestinal epithelial infections, drug developers should consider the adverse impact of active efflux transporters on efficacy.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Criptosporidiose/tratamento farmacológico , Cryptosporidium/efeitos dos fármacos , Enteropatias Parasitárias/tratamento farmacológico , Naftalenos/metabolismo , Naftalenos/uso terapêutico , Piperidinas/metabolismo , Piperidinas/uso terapêutico , Pirazóis/metabolismo , Pirazóis/uso terapêutico , Pirimidinas/metabolismo , Pirimidinas/uso terapêutico , Quinolinas/metabolismo , Quinolinas/uso terapêutico , Animais , Transporte Biológico Ativo , Células CACO-2 , Permeabilidade da Membrana Celular/efeitos dos fármacos , Criptosporidiose/parasitologia , Modelos Animais de Doenças , Descoberta de Drogas/métodos , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Enterócitos/parasitologia , Feminino , Absorção Gastrointestinal/efeitos dos fármacos , Humanos , Interferon gama/genética , Camundongos , Camundongos Knockout , Naftalenos/química , Piperidinas/química , Pirazóis/química , Pirimidinas/química , Quinolinas/química , Resultado do Tratamento
14.
J Infect Dis ; 219(9): 1464-1473, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30423128

RESUMO

Bumped kinase inhibitors (BKIs) have been shown to be potent inhibitors of Toxoplasma gondii calcium-dependent protein kinase 1. Pyrazolopyrimidine and 5-aminopyrazole-4-carboxamide scaffold-based BKIs are effective in acute and chronic experimental models of toxoplasmosis. Through further exploration of these 2 scaffolds and a new pyrrolopyrimidine scaffold, additional compounds have been identified that are extremely effective against acute experimental toxoplasmosis. The in vivo efficacy of these BKIs demonstrates that the cyclopropyloxynaphthyl, cyclopropyloxyquinoline, and 2-ethoxyquinolin-6-yl substituents are associated with efficacy across scaffolds. In addition, a broad range of plasma concentrations after oral dosing resulted from small structural changes to the BKIs. These select BKIs include anti-Toxoplasma compounds that are effective against acute experimental toxoplasmosis and are not toxic in human cell assays, nor to mice when administered for therapy. The BKIs described here are promising late leads for improving anti-Toxoplasma therapy.


Assuntos
Inibidores de Proteínas Quinases/uso terapêutico , Proteínas de Protozoários/antagonistas & inibidores , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Toxoplasmose Animal/tratamento farmacológico , Toxoplasmose Cerebral/tratamento farmacológico , Administração Oral , Animais , Área Sob a Curva , Feminino , Técnicas In Vitro , Camundongos , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/sangue , Pirazóis/farmacologia , Pirimidinas/sangue , Pirimidinas/farmacologia
15.
Artigo em Inglês | MEDLINE | ID: mdl-30783001

RESUMO

Infection with the free-living amoeba Naegleria fowleri leads to life-threatening primary amoebic meningoencephalitis. Efficacious treatment options for these infections are limited, and the mortality rate is very high (∼98%). Parasite metabolism may provide suitable targets for therapeutic design. Like most other organisms, glucose metabolism is critical for parasite viability, being required for growth in culture. The first enzyme required for glucose metabolism is typically a hexokinase (HK), which transfers a phosphate from ATP to glucose. The products of this enzyme are required for both glycolysis and the pentose phosphate pathway. However, the N. fowleri genome lacks an obvious HK homolog and instead harbors a glucokinase (Glck). The N. fowleri Glck (NfGlck) shares limited (25%) amino acid identity with the mammalian host enzyme (Homo sapiens Glck), suggesting that parasite-specific inhibitors with anti-amoeba activity can be generated. Following heterologous expression, NfGlck was found to have a limited hexose substrate range, with the greatest activity observed with glucose. The enzyme had apparent Km values of 42.5 ± 7.3 µM and 141.6 ± 9.9 µM for glucose and ATP, respectively. The NfGlck structure was determined and refined to 2.2-Å resolution, revealing that the enzyme shares greatest structural similarity with the Trypanosoma cruzi Glck. These similarities include binding modes and binding environments for substrates. To identify inhibitors of NfGlck, we screened a small collection of inhibitors of glucose-phosphorylating enzymes and identified several small molecules with 50% inhibitory concentration values of <1 µM that may prove useful as hit chemotypes for further leads and therapeutic development against N. fowleri.


Assuntos
Glucoquinase/química , Glucoquinase/metabolismo , Naegleria fowleri/enzimologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Glucose/metabolismo , Humanos , Trypanosoma cruzi/enzimologia
16.
Artigo em Inglês | MEDLINE | ID: mdl-31061151

RESUMO

Previous studies on drug efficacy showed low protection against abortion and vertical transmission of Toxoplasma gondii in pregnant sheep. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. Here, we present the safety and efficacy of BKI-1294 treatment (dosed orally at 100 mg/kg of body weight 5 times every 48 h) initiated 48 h after oral infection of sheep at midpregnancy with 1,000 TgShSp1 oocysts. BKI-1294 demonstrated systemic exposure in pregnant ewes, with maximum plasma concentrations of 2 to 3 µM and trough concentrations of 0.4 µM at 48 h after each dose. Oral administration of BKI-1294 in uninfected sheep at midpregnancy was deemed safe, since there were no changes in behavior, fecal consistency, rectal temperatures, hematological and biochemical parameters, or fetal mortality/morbidity. In ewes infected with a T. gondii oocyst dose lethal for fetuses, BKI-1294 treatment led to a minor rectal temperature increase after infection and a decrease in fetal/lamb mortality of 71%. None of the lambs born alive in the treated group exhibited congenital encephalitis lesions, and vertical transmission was prevented in 53% of them. BKI-1294 treatment during infection led to strong interferon gamma production after cell stimulation in vitro and a low humoral immune response to soluble tachyzoite antigens but high levels of anti-SAG1 antibodies. The results demonstrate a proof of concept for the therapeutic use of BKI-1294 to protect ovine fetuses from T. gondii infection during pregnancy.


Assuntos
Aborto Espontâneo/etiologia , Aborto Espontâneo/prevenção & controle , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Naftalenos/farmacologia , Piperidinas/farmacologia , Substâncias Protetoras/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Toxoplasmose Animal/complicações , Animais , Feminino , Oocistos , Gravidez , Proteínas Quinases/metabolismo , Ovinos , Toxoplasma/patogenicidade
17.
Artigo em Inglês | MEDLINE | ID: mdl-30745384

RESUMO

Cryptosporidiosis is one of the leading causes of moderate to severe diarrhea in children in low-resource settings. The therapeutic options for cryptosporidiosis are limited to one drug, nitazoxanide, which unfortunately has poor activity in the most needy populations of malnourished children and HIV-infected persons. We describe here the discovery and early optimization of a class of imidazopyridine-containing compounds with potential for treating Cryptosporidium infections. The compounds target the Cryptosporidium methionyl-tRNA synthetase (MetRS), an enzyme that is essential for protein synthesis. The most potent compounds inhibited the enzyme with Ki values in the low picomolar range. Cryptosporidium cells in culture were potently inhibited with 50% effective concentrations as low as 7 nM and >1,000-fold selectivity over mammalian cells. A parasite persistence assay indicates that the compounds act by a parasiticidal mechanism. Several compounds were demonstrated to control infection in two murine models of cryptosporidiosis without evidence of toxicity. Pharmacological and physicochemical characteristics of compounds were investigated to determine properties that were associated with higher efficacy. The results indicate that MetRS inhibitors are excellent candidates for development for anticryptosporidiosis therapy.


Assuntos
Antiprotozoários/farmacologia , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Imidazóis/farmacologia , Metionina tRNA Ligase/antagonistas & inibidores , Piridinas/farmacologia , Animais , Cryptosporidium parvum/genética , Ciclo-Oxigenase 1/efeitos dos fármacos , Modelos Animais de Doenças , Descoberta de Drogas/métodos , Feminino , Células Hep G2 , Humanos , Imidazóis/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Piridinas/química
18.
Clin Microbiol Rev ; 30(3): 647-669, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28446445

RESUMO

In the last 2 decades, renewed attention to neglected tropical diseases (NTDs) has spurred the development of antiparasitic agents, especially in light of emerging drug resistance. The need for new drugs has required in vitro screening methods using parasite culture. Furthermore, clinical laboratories sought to correlate in vitro susceptibility methods with treatment outcomes, most notably with malaria. Parasites with their various life cycles present greater complexity than bacteria, for which standardized susceptibility methods exist. This review catalogs the state-of-the-art methodologies used to evaluate the effects of drugs on key human parasites from the point of view of drug discovery as well as the need for laboratory methods that correlate with clinical outcomes.


Assuntos
Antiparasitários/farmacologia , Parasitos/efeitos dos fármacos , Animais , Descoberta de Drogas , Humanos , Doenças Negligenciadas/parasitologia
19.
Artigo em Inglês | MEDLINE | ID: mdl-29555627

RESUMO

In Toxoplasma gondii, calcium-dependent protein kinase 1 (CDPK1) is an essential protein kinase required for invasion of host cells. We have developed several hundred CDPK1 inhibitors, many of which block invasion. Inhibitors with similar 50% inhibitory concentrations (IC50s) were tested in thermal shift assays for their ability to stabilize CDPK1 in cell lysates, in intact cells, or in purified form. Compounds that inhibited parasite growth stabilized CDPK1 in all assays. In contrast, two compounds that showed poor growth inhibition stabilized CDPK1 in lysates but not in cells. Thus, cellular exclusion could explain exceptions in the correlation between the action on the target and cellular activity. We used thermal shift assays to examine CDPK1 in two clones that were independently selected by growth in the CDPK1 inhibitor RM-1-132 and that had increased 50% effective concentrations (EC50s) for the compound. The A and C clones had distinct point mutations in the CDPK1 kinase domain, H201Q and L96P, respectively, residues that lie near one another in the inactive isoform. Purified mutant proteins showed RM-1-132 IC50s and thermal shifts similar to those shown by wild-type CDPK1. Reduced inhibitor stabilization (and a presumed reduced interaction) was observed only in cellular thermal shift assays. This highlights the utility of cellular thermal shift assays in demonstrating that resistance involves reduced on-target engagement (even if biochemical assays suggest otherwise). Indeed, similar EC50s were observed upon overexpression of the mutant proteins, as in the corresponding drug-selected parasites, although high levels of CDPK1(H201Q) only modestly increased resistance compared to that achieved with high levels of wild-type enzyme.


Assuntos
Quinase 2 de Adesão Focal/antagonistas & inibidores , Naftalenos/farmacologia , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasmose/tratamento farmacológico , Animais , Resistência a Medicamentos/genética , Quinase 2 de Adesão Focal/genética , Humanos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Toxoplasma/genética
20.
Artigo em Inglês | MEDLINE | ID: mdl-29661877

RESUMO

Recent reports highlighting the global significance of cryptosporidiosis among children have renewed efforts to develop control measures. We evaluated the efficacy of bumped kinase inhibitor (BKI) 1369 in the gnotobiotic piglet model of acute diarrhea caused by Cryptosporidium hominis, the species responsible for most human cases. Five-day treatment with BKI 1369 reduced signs of disease early during treatment compared to those of untreated animals. Piglets treated with BKI 1369 exhibited significant reductions of oocyst excretion, mucosal colonization by C. hominis, and mucosal lesions, which resulted in considerable symptomatic improvement. BKI 1369 reduced the parasite burden and disease severity in the gnotobiotic pig model. Together these data suggest that a BKI-mediated therapeutic may be an effective treatment against cryptosporidiosis.


Assuntos
Antiprotozoários/uso terapêutico , Criptosporidiose/tratamento farmacológico , Cryptosporidium/efeitos dos fármacos , Diarreia/tratamento farmacológico , Piperidinas/uso terapêutico , Pirimidinas/uso terapêutico , Quinolinas/uso terapêutico , Doença Aguda , Animais , Animais Recém-Nascidos , Criptosporidiose/parasitologia , Diarreia/parasitologia , Modelos Animais de Doenças , Vida Livre de Germes , Oocistos/metabolismo , Carga Parasitária , Suínos
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