RESUMO
Estimation of the precursors of nucleic acids in the presence of anti-leukaemic developing agents is a valid approach towards seeking a cure for leukaemia. Therefore, in this study the focus was on primary inhibition of the early de novo purine pathway. A radiolabel protocol for optimum hot intermediates of the de novo purine biosynthesis produced in human leukaemia cells CCRF-CEM was developed in this study. According to recent earlier finding in this lab, methotrexate [MTX] and lome-trexol [LTX] showed blockage potency for the de novo synthesis ofpurines due to an accumulation of intracellular 5-phospho-ribosyl-1-pyrophosphate [PRPP]. Therefore, in the current study, the mechanism of action of these two antifolates on de novo purine biosynthesis in human CCRF-CEM leukaemia cells [labelled various metabolites with 14C-glycine] was elucidated by using high-pressure liquid chromatographic [HPLC] technique for separation of acid soluble metabolites and quantified using an ultraviolet detector along with a radioactivity monitor. Total level of nucleotides indicated that both MTX and LTX induced depletion ofATP, ADP, AMP, GTP, GMP, sAMP, IMP and NAD, while GDP was decreased by 63% of the control level only in the MTX [0.1 microM] treated sample. A level of nucleotides synthesized in the de novo purine biosynthesis indicated that both antifolates [0.1 microM] also induced depletion ofATP, ADP, AMP, GTP, GDP, IMP and NAD in treated human leukaemia cells. All mono-, di- and triphosphates of N-formylglycinamide ribotide [FGAR] were also decreased by MTX and LTX in the presence ofazaserine [25 microM]. These results support the finding that two tested antifolates in the present study induced primary inhibition of the early de novo purine pathway in human leukaemia cells CCRF-CEM, i.e. the inhibition of amido phosphoribosyl-transfemse [APRT] catalyses the first committed step of the de novo purine pathway
RESUMO
Tolserine is a novel and highly selective synthetic inhibitor of the enzyme acetylcholinesterase [AchE] that is being developed towards clinical trials for the treatment of Alzheimer's disease. We recently characterized its enzyme kinetics for inhibiting purified human erythrocyte AChE by utilizing classical methodology. In the current study, we describe a new approach to analyze its mechanism of inhibition of AChE. This was undertaken with purified human erythrocyte AChE using low to higher substrate concentrations in both the absence and presence of dual tolserine concentrations. The optical density of the generated reaction-product then was monitored during the initial reaction time after addition of each concentration of substrate. Thereafter, the new kinetic parameters [Kneidc, Kesic, Kslxx, Krss, Kslm and Kslx] were calculated from the resulting experimental data. These kinetic constants will hopefully aid our understanding of the mechanism of inhibittion and kinetic analysis of a variety of critical physiological enzymes by a wide assortment of inhibitors in vitro, during health, aging and disease. A low Kneidc value of 2.31 nM for tolserine indicates that it is a highly potent inhibitor of human erythrocyte AChE, which supports previous reports of its unusually high potency for inhibiting AChE both in vitro and in vivo, as compared to its structural analogues, physostigmine and phenserine