RESUMO
BACKGROUND: Recent whole cell in vitro screening campaigns identified thousands of compounds that are active against asexual blood stages of Plasmodium falciparum at submicromolar concentrations. These hits have been made available to the public, providing many novel chemical starting points for anti-malarial drug discovery programmes. Knowing which of these hits are fast-acting compounds is of great interest. Firstly, a fast action will ensure rapid relief of symptoms for the patient. Secondly, by rapidly reducing the parasitaemia, this could minimize the occurrence of mutations leading to new drug resistance mechanisms.An in vitro assay that provides information about the speed of action of test compounds has been developed by researchers at GlaxoSmithKline (GSK) in Spain. This assay also provides an in vitro measure for the ratio between parasitaemia at the onset of drug treatment and after one intra-erythrocytic cycle (parasite reduction ratio, PRR). Both parameters are needed to determine in vitro killing rates of anti-malarial compounds. A drawback of the killing rate assay is that it takes a month to obtain first results. METHODS: The approach described in the present study is focused only on the speed of action of anti-malarials. This has the advantage that initial results can be achieved within 4-7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly. It is expected that this new assay can be used as a filter in the early drug discovery phase, which will reduce the number of compounds progressing to secondary, more time-consuming assays like the killing rate assay. RESULTS: The speed of action of a selection of seven anti-malarial compounds was measured with two independent experimental procedures using modifications of the standard [3H]hypoxanthine incorporation assay. Depending on the outcome of both assays, the tested compounds were classified as either fast or non-fast-acting. CONCLUSION: The results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds. Another advantage of the approach is its ability to discriminate between static or cidal compound effects.
Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Concentração Inibidora 50 , Testes de Sensibilidade Parasitária/métodos , Fatores de TempoRESUMO
Four double-drug HIV NRTI/NNRTI inhibitors 15a-d of the type [d4U]-spacer-[HI-236] in which the spacer is varied as 1-butynyl (15a), propargyl-1-PEG (15b), propargyl-2-PEG (15c) and propargyl-4-PEG (15d) have been synthesized and biologically evaluated as RT inhibitors against HIV-1. The key step in their synthesis involved a Sonogashira coupling of 5-iodo d4U's benzoate with an alkynylated tethered HI-236 precursor followed by introduction of the HI-236 thiourea functionality. Biological evaluation in both cell-culture (MT-2 cells) as well as using an in vitro RT assay revealed 15a-c to be all more active than d4T. However, overall the results indicate the derivatives are acting as chain-extended NNRTIs in which for 15b-d the nucleoside component is likely situated outside of the pocket but with no evidence for any synergistic double binding between the NRTI and NNRTI sites. This is attributed, in part, to the lack of phosphorylation of the nucleoside component of the double-drug as a result of kinase recognition failure, which is not improved upon with the phosphoramidate of 15d incorporating a 4-PEG spacer.
Assuntos
Fármacos Anti-HIV/química , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/enzimologia , Piridinas/química , Inibidores da Transcriptase Reversa/química , Tioureia/análogos & derivados , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/toxicidade , Sítios de Ligação , Linhagem Celular , Simulação por Computador , Desenho de Fármacos , Transcriptase Reversa do HIV/metabolismo , Humanos , Polietilenoglicóis/química , Piridinas/síntese química , Piridinas/farmacologia , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Tioureia/síntese química , Tioureia/química , Tioureia/farmacologiaRESUMO
Several novel thiourea derivatives of the NNRTI HI-236 substituted at the C-2 oxygen of the phenyl ring have been synthesized and evaluated for their inhibitory activity against HIV-1 (IIIB) replication in MT-2 cell cultures. The compounds were synthesized in order to fine-tune the activity of HI-236 as well as to gain insight into spatial characteristics in the pocket pertaining to the positional choice of tether in the design of [NRTI]-tether-[HI-236] bifunctional inhibitors. Two of the thiourea derivatives bearing a butynyl (6c) or hydroxyethyl tether (6n) were endowed with improved anti-HIV activity compared to HI-236. NNRTI activity was confirmed by a cell-free RT assay on six of the derivatives in which 6c returned an IC(50) of 3.8 nM compared to 28 nM for HI-236, establishing it as an improved lead for HI-236. The structure-activity profile is discussed in terms of potential interactions in the NNRTI pocket as suggested by a docking model using AutoDock, which have a bearing on the bifunctional drug design.
Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Piridinas/química , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Tioureia/análogos & derivados , Fármacos Anti-HIV/química , Células Cultivadas , Simulação por Computador , Desenho de Fármacos , HIV-1/enzimologia , Concentração Inibidora 50 , Estrutura Molecular , Inibidores da Transcriptase Reversa/química , Relação Estrutura-Atividade , Tioureia/químicaRESUMO
As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.
Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Aminopiridinas/uso terapêutico , Antimaláricos/uso terapêutico , Sulfonas/uso terapêutico , Aminopiridinas/farmacologia , Animais , Antimaláricos/farmacologia , Feminino , Malária/tratamento farmacológico , Malária/enzimologia , Masculino , Camundongos , Camundongos SCID , Testes de Sensibilidade Parasitária , Plasmodium/efeitos dos fármacos , Plasmodium/patogenicidade , Sulfonas/farmacologiaRESUMO
Introduction of water-solubilizing groups on the 5-phenyl ring of a 2-aminopyrazine series led to the identification of highly potent compounds against the blood life-cycle stage of the human malaria parasite Plasmodium falciparum. Several compounds displayed high in vivo efficacy in two different mouse models for malaria, P. berghei-infected mice and P. falciparum-infected NOD-scid IL-2Rγnull mice. One of the frontrunners, compound 3, was identified to also have good pharmacokinetics and additionally very potent activity against the liver and gametocyte parasite life-cycle stages.
Assuntos
Antimaláricos/farmacologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária/tratamento farmacológico , Doenças Parasitárias em Animais/tratamento farmacológico , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Pirazinas/farmacologia , Animais , Antimaláricos/química , Antimaláricos/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Células Hep G2 , Humanos , Camundongos , Camundongos SCID , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Doenças Parasitárias em Animais/parasitologia , Testes de Sensibilidade Parasitária , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Pirazinas/química , Pirazinas/metabolismo , Solubilidade , Relação Estrutura-Atividade , Água/químicaRESUMO
Following a structure-based virtual screening, a series of 2,4 thiazolidinediones was synthesized in order to explore structure activity relationships for inhibition of the Plasmodium falciparum cysteine protease falcipain-2 (FP-2) and of whole cell antiparasitic activity. Most compounds exhibited low micromolar antiplasmodial activities against the P. falciparum drug resistant W2 strain. The most active compounds of the series were tested for in vitro microsomal metabolic stability and found to be susceptible to hepatic metabolism. Subsequent metabolite identification studies highlighted the metabolic hot spots. Molecular docking studies of a frontrunner inhibitor were carried out to determine the probable binding mode of this class of inhibitors in the active site of FP-2.
Assuntos
Antimaláricos/farmacologia , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Antimaláricos/síntese química , Antimaláricos/química , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/enzimologia , Relação Estrutura-Atividade , Tiazolidinedionas/síntese química , Tiazolidinedionas/químicaRESUMO
On the basis of our recent results on a novel series of imidazopyridazine-based antimalarials, we focused on identifying compounds with improved aqueous solubility and hERG profile while maintaining metabolic stability and in vitro potency. Toward this objective, 41 compounds were synthesized and evaluated for antiplasmodial activity against NF54 (sensitive) and K1 (multidrug resistant) strains of the malaria parasite Plasmodium falciparum and evaluated for both aqueous solubility and metabolic stability. Selected compounds were tested for in vitro hERG activity and in vivo efficacy in the P. berghei mouse model. Several compounds were identified with significantly improved aqueous solubility, good metabolic stability, and a clean hERG profile relative to a previous frontrunner lead compound. A sulfoxide-based imidazopyridazine analog 45, arising from a prodrug-like strategy, was completely curative in the Plasmodium berghei mouse model at 4 × 50 mg/kg po.
Assuntos
Antimaláricos/síntese química , Piridazinas/síntese química , Sulfonas/síntese química , Animais , Antimaláricos/metabolismo , Antimaláricos/farmacocinética , Antimaláricos/farmacologia , Resistência a Múltiplos Medicamentos , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Humanos , Malária Falciparum/parasitologia , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Testes de Sensibilidade Parasitária , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Piridazinas/metabolismo , Piridazinas/farmacologia , Ratos Sprague-Dawley , Solubilidade , Relação Estrutura-Atividade , Sulfonas/metabolismo , Sulfonas/farmacologiaRESUMO
A novel class of imidazopyridazines identified from whole cell screening of a SoftFocus kinase library was synthesized and evaluated for antiplasmodial activity against K1 (multidrug resistant strain) and NF54 (sensitive strain). Structure-activity relationship studies led to the identification of highly potent compounds against both strains. Compound 35 was highly active (IC50: K1 = 6.3 nM, NF54 = 7.3 nM) and comparable in potency to artesunate, and 35 exhibited 98% activity in the in vivo P. berghei mouse model (4-day test by Peters) at 4 × 50 mg/kg po. Compound 35 was also assessed against P. falciparum in the in vivo SCID mouse model where the efficacy was found to be more consistent with the in vitro activity. Furthermore, 35 displayed high (78%) rat oral bioavailability with good oral exposure and plasma half-life. Mice exposure at the same dose was 10-fold lower than in rat, suggesting lower oral absorption and/or higher metabolic clearance in mice.
Assuntos
Antimaláricos/síntese química , Antimaláricos/farmacologia , Imidazóis/síntese química , Imidazóis/farmacologia , Plasmodium/efeitos dos fármacos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Piridazinas/síntese química , Piridazinas/farmacologia , Animais , Antimaláricos/farmacocinética , Disponibilidade Biológica , Desenho de Fármacos , Resistência a Medicamentos , Estabilidade de Medicamentos , Biblioteca Gênica , Meia-Vida , Ensaios de Triagem em Larga Escala , Malária/tratamento farmacológico , Malária/parasitologia , Malária Falciparum/tratamento farmacológico , Malária Falciparum/psicologia , Camundongos , Camundongos SCID , Testes de Sensibilidade Parasitária , Plasmodium berghei , Plasmodium falciparum/efeitos dos fármacos , Ratos , Relação Estrutura-AtividadeRESUMO
A novel series of 2,4-diaminothienopyrimidines with potential as antimalarials was identified from whole-cell high-throughput screening of a SoftFocus ion channel library. Synthesis and structure-activity relationship studies identified compounds with potent antiplasmodial activity and low in vitro cytotoxicity. Several of these analogues exhibited in vivo activity in the Plasmodium berghei mouse model when administered orally. However, inhibition of the hERG potassium channel was identified as a liability for this series.
Assuntos
Antimaláricos/síntese química , Pirimidinas/síntese química , Tiofenos/síntese química , Administração Oral , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Linhagem Celular , Bases de Dados de Compostos Químicos , Resistência a Múltiplos Medicamentos , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Humanos , Malária/tratamento farmacológico , Malária/parasitologia , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Plasmodium berghei , Plasmodium falciparum/efeitos dos fármacos , Pirimidinas/química , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacologiaRESUMO
In the second part of this Miniperspectives series, we highlight our medicinal chemistry efforts involving progression of hits from whole cell high-throughput screening (HTS) of a SoftFocus kinase library against the malaria parasite Plasmodium falciparum . Successful SAR exploration in Hit-to-Lead and Lead Optimization efforts leading to the selection of a preclinical development candidate are demonstrated. Related efforts by researchers from Broad/Genzyme, Anacor, and GSK are briefly covered.
Assuntos
Antimaláricos/uso terapêutico , Química Farmacêutica/métodos , Descoberta de Drogas/métodos , Administração Oral , Antimaláricos/administração & dosagem , Antimaláricos/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Malária Falciparum/parasitologia , Malária Falciparum/prevenção & controle , Estrutura Molecular , Plasmodium falciparum/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Replacement of the pyridine core of antimalarial 3,5-diaryl-2-aminopyridines led to the identification of a novel series of pyrazine analogues with potent oral antimalarial activity. However, other changes to the pyridine core and replacement or substitution of the 2-amino group led to loss of antimalarial activity. The 3,5-diaryl-2-aminopyrazine series showed impressive in vitro antiplasmodial activity against the K1 (multidrug resistant) and NF54 (sensitive) strains of Plasmodium falciparum in the nanomolar IC50 range of 6-94 nM while also demonstrating good in vitro metabolic stability in human liver microsomes. In the Plasmodium berghei mouse model, this series generally exhibited good efficacy at low oral doses. One of the frontrunner compounds, 4, displayed potent in vitro antiplasmodial activity with IC50 values of 8.4 and 10 nM against the K1 and NF54 strains, respectively. When evaluated in P. berghei -infected mice, compound 4 was completely curative at an oral dose of 4 × 10 mg/kg.
Assuntos
Aminopiridinas/farmacologia , Antimaláricos/farmacologia , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Administração Oral , Aminopiridinas/administração & dosagem , Aminopiridinas/química , Animais , Antimaláricos/administração & dosagem , Antimaláricos/química , Células CHO , Cricetulus , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Camundongos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Testes de Sensibilidade Parasitária , Ratos , Relação Estrutura-AtividadeRESUMO
In an effort to address potential cardiotoxicity liabilities identified with earlier frontrunner compounds, a number of new 3,5-diaryl-2-aminopyridine derivatives were synthesized. Several compounds exhibited potent antiplasmodial activity against both the multidrug resistant (K1) and sensitive (NF54) strains in the low nanomolar range. Some compounds displayed a significant reduction in potency in the hERG channel inhibition assay compared to previously reported frontrunner analogues. Several of these new analogues demonstrated promising in vivo efficacy in the Plasmodium berghei mouse model and will be further evaluated as potential clinical candidates. The SAR for in vitro antiplasmodial and hERG activity was delineated.
Assuntos
Aminopiridinas/síntese química , Antimaláricos/síntese química , Administração Oral , Aminopiridinas/química , Aminopiridinas/farmacologia , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Resistência a Múltiplos Medicamentos , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Humanos , Malária/tratamento farmacológico , Camundongos , Microssomos Hepáticos/metabolismo , Plasmodium berghei , Plasmodium falciparum/efeitos dos fármacos , Solubilidade , Relação Estrutura-AtividadeRESUMO
A novel class of orally active antimalarial 3,5-diaryl-2-aminopyridines has been identified from phenotypic whole cell high-throughput screening of a commercially available SoftFocus kinase library. The compounds were evaluated in vitro for their antiplasmodial activity against K1 (chloroquine and drug-resistant strain) and NF54 (chloroquine-susceptible strain) as well as for their cytotoxicity. Synthesis and structure-activity studies identified a number of promising compounds with selective antiplasmodial activity. One of these frontrunner compounds, 15, was equipotent across the two strains (K1 = 25.0 nM, NF54 = 28.0 nM) and superior to chloroquine in the K1 strain (chloroquine IC(50) K1 = 194.0 nM). Compound 15 completely cured Plasmodium berghei-infected mice with a single oral dose of 30 mg/kg. Dose-response studies generated ED(50) and ED(90) values of 0.83 and 1.74 mg/kg for 15 in the standard four-dose Peters test. Pharmacokinetic studies in the rat indicated that this compound has good oral bioavailability (51% at 20 mg/kg) and a reasonable half-life (t(1/2) â¼ 7-8 h).
Assuntos
Aminopiridinas/síntese química , Antimaláricos/síntese química , Administração Oral , Aminopiridinas/farmacocinética , Aminopiridinas/farmacologia , Animais , Antimaláricos/farmacocinética , Antimaláricos/farmacologia , Disponibilidade Biológica , Linhagem Celular , Cloroquina/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Resistência a Medicamentos , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Feminino , Humanos , Isoenzimas/antagonistas & inibidores , Malária/tratamento farmacológico , Camundongos , Microssomos Hepáticos/metabolismo , Plasmodium berghei , Plasmodium falciparum/efeitos dos fármacos , Coelhos , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
An aminomethylthiazole pyrazole carboxamide lead 3 with good in vitro antiplasmodial activity [IC(50): 0.08 µM (K1, chloroquine and multidrug resistant strain) and 0.07 µM (NF54, chloroquine sensitive strain)] and microsomal metabolic stability was identified from whole cell screening of a SoftFocus kinase library. Compound 3 also exhibited in vivo activity in the P. berghei mouse model at 4 × 50 mg/kg administration via the oral route, showing 99.5% activity and 9 days survival and showed low in vitro cytotoxicity. Pharmacokinetic studies in rats revealed good oral bioavailability (51% at 22 mg/kg) with a moderate rate of absorption, reasonable half-life (t(1/2) 3 h), and high volume of distribution with moderately high plasma and blood clearance after IV administration. Toward toxicity profiling, 3 exhibited moderate potential to inhibit CYP1A2 (IC(50) = 1.5 µM) and 2D6 (IC(50) = 0.4 µM) as well as having a potential hERG liability (IC(50) = 3.7 µM).