RESUMO
Ribose 5-phosphate isomerase type B (RPI-B) is a key enzyme of the pentose phosphate pathway that catalyzes the isomerization of ribose 5-phosphate (R5P) and ribulose 5-phosphate (Ru5P). Trypanosoma cruzi RPI-B (TcRPI-B) appears to be a suitable drug-target mainly due to: (i) its essentiality (as previously shown in other trypanosomatids), (ii) it does not present a homologue in mammalian genomes sequenced thus far, and (iii) it participates in the production of NADPH and nucleotide/nucleic acid synthesis that are critical for parasite cell survival. In this survey, we report on the competitive inhibition of TcRPI-B by a substrate - analogue inhibitor, Compound B (Ki = 5.5 ± 0.1 µM), by the Dixon method. This compound has an iodoacetamide moiety that is susceptible to nucleophilic attack, particularly by the cysteine thiol group. Compound B was conceived to specifically target Cys-69, an important active site residue. By incubating TcRPI-B with Compound B, a trypsin digestion LC-MS/MS analysis revealed the identification of Compound B covalently bound to Cys-69. This inhibitor also exhibited notable in vitro trypanocidal activity against T. cruzi infective life-stages co-cultured in NIH-3T3 murine host cells (IC50 = 17.40 ± 1.055 µM). The study of Compound B served as a proof-of-concept so that next generation inhibitors can potentially be developed with a focus on using a prodrug group in replacement of the iodoacetamide moiety, thus representing an attractive starting point for the future treatment of Chagas' disease.
Assuntos
Aldose-Cetose Isomerases/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/química , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/síntese química , Trypanosoma cruzi/enzimologia , Células 3T3 , Aldose-Cetose Isomerases/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Cinética , Camundongos , Simulação de Dinâmica Molecular , Proteínas de Protozoários/metabolismo , Especificidade por Substrato , Tripanossomicidas/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
A high-throughput screening (HTS) campaign was carried out for Trypanosoma cruzi glucokinase (TcGlcK), a potential drug-target of the pathogenic protozoan parasite. Glycolysis and the pentose phosphate pathway (PPP) are important metabolic pathways for T. cruzi and the inhibition of the glucose kinases (i.e. glucokinase and hexokinase) may be a strategic approach for drug discovery. Glucose kinases phosphorylate d-glucose with co-substrate ATP to yield G6P, and moreover, the produced G6P enters both pathways for catabolism. The TcGlcK - HTS campaign revealed 25 novel enzyme inhibitors that were distributed in nine chemical classes and were discovered from a primary screen of 13,040 compounds. Thirteen of these compounds were found to have low micromolar IC50 enzyme - inhibition values; strikingly, four of those compounds exhibited low toxicity towards NIH-3T3 murine host cells and notable in vitro trypanocidal activity. These compounds were of three chemical classes: (a) the 3-nitro-2-phenyl-2H-chromene scaffold, (b) the N-phenyl-benzenesulfonamide scaffold, and (c) the gossypol scaffold. Two compounds from the 3-nitro-2-phenyl-2H-chromene scaffold were determined to be hit-to-lead candidates that can proceed further down the early-stage drug discovery process.
Assuntos
Doença de Chagas/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Glucoquinase/uso terapêutico , Ensaios de Triagem em Larga Escala/métodos , Inibidores Enzimáticos/farmacologia , Glucoquinase/farmacologia , Trypanosoma cruziRESUMO
Glucokinase from pathogenic protozoa of the genus Leishmania is a potential drug target for the chemotherapeutic treatment against leishmaniasis because this enzyme is located at a nodal point between two critically important metabolic pathways, glycolysis and the pentose phosphate pathway (PPP). L. braziliensis glucokinase (LbGlcK) was evaluated for its structural characterization and enzymatic performance. The enzyme catalyzes the phosphorylation of d-glucose with co-substrate ATP to yield the products G6P and ADP. LbGlcK had KM values determined as 6.61 ± 2.63 mM and 0.338 ± 0.080 mM for d-glucose and ATP, respectively. The 1.85 Å resolution X-ray crystal structure of the apo form of LbGlcK was determined and a homodimer was revealed where each subunit (both in open conformations) included the typical small and large domains. Structural comparisons were assessed in relationship to Homo sapiens hexokinase IV and Trypanosoma cruzi glucokinase. Comparisons revealed that all residues important for making hydrogen bonding interactions with d-glucose in the active site and catalysis were strictly conserved. LbGlcK was screened against four glucosamine analogue inhibitors and the stronger inhibitor of the series, HPOP-GlcN, had a Ki value of 56.9 ± 16.6 µM that exhibited competitive inhibition. For the purpose of future structure-based drug design experimentation, L. braziliensis glucokinase was observed to be very similar to T. cruzi glucokinase even though there was a 44% protein sequence identity between the two enzymes.
Assuntos
Glucoquinase/química , Glucoquinase/metabolismo , Leishmania braziliensis/enzimologia , Leishmaniose Cutânea/parasitologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Glucoquinase/genética , Glucose/metabolismo , Humanos , Cinética , Leishmania braziliensis/química , Leishmania braziliensis/genética , Modelos Moleculares , Fosforilação , Proteínas de Protozoários/genética , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Arginase from parasitic protozoa belonging to the genus Leishmania is a potential drug target for the treatment of leishmaniasis because this binuclear manganese metalloenzyme catalyzes the first committed step in the biosynthesis of polyamines that enable cell growth and survival. The high resolution X-ray crystal structures of the unliganded form of Leishmania mexicana arginase (LmARG) and four inhibitor complexes are now reported. These complexes include the reactive substrate analogue 2(S)-amino-6-boronohexanoic acid (ABH) and the hydroxylated substrate analogue nor-N(ω)-hydroxy-l-arginine (nor-NOHA), which are the most potent arginase inhibitors known to date. Comparisons of the LmARG structure with that of the archetypal arginase, human arginase I, reveal that all residues important for substrate binding and catalysis are strictly conserved. However, three regions of tertiary structure differ between the parasitic enzyme and the human enzyme corresponding to the G62 - S71, L161 - C172, and I219 - V230 segments of LmARG. Additionally, variations are observed in salt link interactions that stabilize trimer assembly in LmARG. We also report biological studies in which we demonstrate that localization of LmARG to the glycosome, a unique subcellular organelle peculiar to Leishmania and related parasites, is essential for robust pathogenesis.