RESUMO
BACKGROUND: Deficiency of lysosomal acid lipase (LAL) causes Wolman disease and cholesterol ester storage disease. With the recent introduction of enzyme replacement therapy to manage LAL deficiency comes the need for a reliable assay of LAL enzymatic activity that can be applied to dried blood spots (DBS). METHODS: We prepared and tested a library of analogs of palmitoyl 4-methylumbelifferyl esters to find a highly active and specific substrate for LAL in DBS. The LAL assay was optimized leading to both LC-MS/MS and fluorometric assay of LAL. We tested the new assay on DBS from healthy and LAL-deficient patients. RESULTS: The ester formed between palmitic acid and 4-propyl-8-methyl-7-hydroxycoumarin (P-PMHC) was found to be >98% selective for LAL in DBS based on the sensitivity of its activity to the LAL-specific inactivator Lalistat-2 and the fact that the activity was close to zero using DBS from patients previously shown to be LAL-deficient. Use of P-PMHC and heavy isotope-labeled internal standard with optimized assay conditions led to an approximately 2-fold increase in the specific activity of LAL compared with the previously reported LAL assay. Patients deficient in LAL were readily distinguished from normal persons with the new LAL assay using UPLC-MS/MS or fluorometric assay platforms. CONCLUSIONS: The new assay can measure LAL in DBS with a single measurement compared with the previous method involving 2 assays done in parallel.
Assuntos
Esterol Esterase/sangue , Adulto , Estudos de Casos e Controles , Pré-Escolar , Doença do Armazenamento de Colesterol Éster/enzimologia , Cromatografia Líquida/métodos , Teste em Amostras de Sangue Seco , Fluorometria , Humanos , Reprodutibilidade dos Testes , Especificidade por Substrato , Espectrometria de Massas em Tandem/métodos , Doença de Wolman/enzimologiaRESUMO
We previously reported that the cancer drug clinical candidate tipifarnib kills the causative agent of Chagas disease, Trypanosoma cruzi, by blocking ergosterol biosynthesis at the level of inhibition of lanosterol 14alpha-demethylase. Tipifarnib is an inhibitor of human protein farnesyltransferase. We synthesized tipifarnib analogues that no longer bind to protein farnesyltransferase and display increased potency for killing parasites. This was achieved in a structure-guided fashion by changing the substituents attached to the phenyl group at the 4-position of the quinoline ring of tipifarnib and by replacing the amino group by OMe. Several compounds that kill Trypanosoma cruzi at subnanomolar concentrations and are devoid of protein farnesyltransferase inhibition were discovered. The compounds are shown to be advantageous over other lanosterol 14alpha-demethylase inhibitors in that they show only modest potency for inhibition of human cytochrome P450 (3A4). Since tipifarnib displays high oral bioavailability and acceptable pharmacokinetic properties, the newly discovered tipifarnib analogues are ideal leads for the development of drugs to treat Chagas disease.
Assuntos
Doença de Chagas/tratamento farmacológico , Quinolonas/síntese química , Tripanossomicidas/síntese química , Células 3T3 , Alquil e Aril Transferases/metabolismo , Animais , Antineoplásicos/química , Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/química , Humanos , Isoenzimas/antagonistas & inibidores , Camundongos , Modelos Moleculares , Ligação Proteica , Quinolonas/química , Quinolonas/farmacologia , Ratos , Esterol 14-Desmetilase , Relação Estrutura-Atividade , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
We developed a synthetic route to prepare isoquinoline analogs of the cancer drug clinical candidate tipifarnib. We show that these compounds kill Trypanosoma cruzi amastigotes grown in mammalian host cells at concentrations in the low nanomolar range. These isoquinolines represent new leads for the development of drugs to treat Chagas disease.