RESUMO
Acanthamoeba castellanii is an amoeba that inhabits soil and water in every part of the world. Acanthamoeba infection of the eye causes keratitis and can lead to a loss of vision. Current treatment options are only moderately effective, have multiple harmful side effects, and are tedious. In our study, we developed a novel drug screening method to define the inhibitory properties of potential new drugs against A. castellanii in vitro. We found that the clinically used carbonic anhydrase inhibitors, acetazolamide, ethoxzolamide, and dorzolamide, have promising antiamoebic properties.
Assuntos
Acanthamoeba castellanii , Amoeba , Inibidores da Anidrase Carbônica/farmacologia , Avaliação Pré-Clínica de MedicamentosRESUMO
Acanthamoeba keratitis is a devastating infectious disease of the cornea caused by an opportunistic amoeba, Acanthamoeba castellanii. It is poorly recognized, and diagnostic delays can lead to irreversible damage to the vision. The gold standard for diagnosis has been a sample culture that lasts approximately 2 weeks. Nevertheless, the essence of time has led to the need for an accurate and fast technique to detect A. castellanii from a sample. We developed both traditional and quantitative real-time-PCR-based methods to detect A. castellanii in less than 3 hours and with the sensitivity of one amoeba. Diagnostic laboratories can select the best-suited method for their purposes from 2 comparable methods. The correct treatment can be initiated from the emergency room when the diagnosis has been made quickly within a few hours, hence saving the patient from long-term complications.
Assuntos
Ceratite por Acanthamoeba , Acanthamoeba castellanii , Humanos , Acanthamoeba castellanii/genética , Testes de Diagnóstico Rápido , Ceratite por Acanthamoeba/diagnóstico , Córnea , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Schistosoma mansoni is an intestinal parasite with one ß-class carbonic anhydrase, SmaBCA. We report the sequence enhancing, production, catalytic activity, and inhibition results of the recombinant SmaBCA. It showed significant catalytic activity on CO2 hydration in vitro with kcat 1.38 × 105 s-1 and kcat/Km 2.33 × 107 M-1 s-1. Several sulphonamide inhibitors, from which many are clinically used, showed submicromolar or nanomolar inhibitory effects on SmaBCA. The most efficient inhibitor with a KI of 43.8 nM was 4-(2-amino-pyrimidine-4-yl)-benzenesulfonamide. Other effective inhibitors with KIs in the range of 79.4-95.9 nM were benzolamide, brinzolamide, topiramate, dorzolamide, saccharin, epacadostat, celecoxib, and famotidine. The other tested compounds showed at least micromolar range inhibition against SmaBCA. Our results introduce SmaBCA as a novel target for drug development against schistosomiasis, a highly prevalent parasitic disease.
Assuntos
Anidrases Carbônicas , Parasitos , Animais , Schistosoma mansoni , Benzolamida , Clonagem MolecularRESUMO
The ß-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic protozoan Entamoeba histolytica, EhiCA, was investigated for its activation with a panel of natural and non-natural amino acids and amines. EhiCA was potently activated by D-His, D-Phe, D-DOPA, L- and D-Trp, L- and D-Tyr, 4-amino-L-Tyr, histamine and serotonin, with KAs ranging between 1.07 and 10.1 M. The best activator was D-Tyr (KA of 1.07 µM). L-Phe, L-DOPA, L-adrenaline, L-Asn, L-Asp, L-Glu and L-Gln showed medium potency activation, with KAs of 16.5â»25.6 µM. Some heterocyclic- alkyl amines, such as 2-pyridyl-methyl/ethyl-amine and 4-(2-aminoethyl)-morpholine, were devoid of EhiCA activating properties with KAs > 100 µM. As CA activators have poorly been investigated for their interaction with protozoan CAs, our study may be relevant for an improved understanding of the role of this enzyme in the life cycle of E. histolytica.
RESUMO
A newly described ß-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic protozoan Entamoeba histolytica, EhiCA, was recently shown to possess a significant catalytic activity for the physiologic CO2 hydration reaction (kcat of 6.7 × 105 s-1 and a kcat/Km of 8.9 × 107 M-1 s-1). A panel of sulphonamides and one sulfamate, some of which are clinically used drugs, were investigated for their inhibitory properties against EhiCA. The best inhibitors detected in the study were 4-hydroxymethyl/ethyl-benzenesulfonamide (KIs of 36â»89 nM), whereas some sulfanilyl-sulfonamides showed activities in the range of 285â»331 nM. Acetazolamide, methazolamide, ethoxzolamide, and dichlorophenamide were less effective inhibitors (KIs of 509â»845 nM) compared to other sulfonamides investigated here. As ß-CAs are not present in vertebrates, the present study may be useful for detecting lead compounds for the design of more effective inhibitors with potential to develop anti-infectives with alternative mechanisms of action.
Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Entamoeba histolytica/enzimologia , Sulfonamidas/farmacologia , Sequência de Aminoácidos , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/química , Humanos , Cinética , Relação Estrutura-AtividadeRESUMO
We report the cloning and catalytic activity of a ß-carbonic anhydrase (CA, EC 4.2.1.1), isolated from the pathogenic protozoan Entamoeba histolytica, EhiCA. This enzyme has a high catalytic activity for the physiologic CO2 hydration reaction, with a kcat of 6.7 × 105 s-1 and a kcat/Km of 8.9 × 107 M-1 × s-1. An anion inhibition study of EhiCA with inorganic/organic anions and small molecules revealed that fluoride, chloride, cyanide, azide, pyrodiphosphate, perchlorate, tetrafluoroborate and sulfamic acid did not inhibit the enzyme activity, whereas pseudohalides (cyanate and thiocyanate), bicarbonate, nitrate, nitrite, diethyldithiocarbamate, and many complex inorganic anions showed inhibition in the millimolar range (KIs of 0.51â»8.4 mM). The best EhiCA inhibitors were fluorosulfonate, sulfamide, phenylboronic acid and phenylarsonic acid (KIs in the range of 28â»86 µM). Since ß-CAs are not present in vertebrates, the present study may be useful for detecting lead compounds for the design of effective enzyme inhibitors, with potential to develop anti-infectives with alternative mechanisms of action.
Assuntos
Anidrases Carbônicas , Clonagem Molecular , Entamoeba histolytica , Proteínas de Protozoários , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Anidrases Carbônicas/isolamento & purificação , Entamoeba histolytica/enzimologia , Entamoeba histolytica/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificaçãoRESUMO
Carbonic anhydrases (CAs) are metalloenzymes that are omnipresent in nature. CAs catalyze the basic reaction of the reversible hydration of CO2 to HCO3- and H⺠in all living organisms. Photosynthetic organisms contain six evolutionarily different classes of CAs, which are namely: α-CAs, ß-CAs, γ-CAs, δ-CAs, ζ-CAs, and θ-CAs. Many of the photosynthetic organisms contain multiple isoforms of each CA family. The model alga Chlamydomonas reinhardtii contains 15 CAs belonging to three different CA gene families. Of these 15 CAs, three belong to the α-CA gene family; nine belong to the ß-CA gene family; and three belong to the γ-CA gene family. The multiple copies of the CAs in each gene family may be due to gene duplications within the particular CA gene family. The CAs of Chlamydomonas reinhardtii are localized in different subcellular compartments of this unicellular alga. The presence of a large number of CAs and their diverse subcellular localization within a single cell suggests the importance of these enzymes in the metabolic and biochemical roles they perform in this unicellular alga. In the present review, we update the information on the molecular biology of all 15 CAs and their metabolic and biochemical roles in Chlamydomonas reinhardtii. We also present a hypothetical model showing the known functions of CAs and predicting the functions of CAs for which precise metabolic roles are yet to be discovered.