Identification of methylglyoxal-lowering agents for the treatment of advanced glycation end products-related diseases

Advanced glycation end products (AGEs) play a major role in age-related diseases such as Alzheimer’s disease (AD), as well as in complications of diabetes such as retinopathy, nephropathy and neuropathy. With ageing the amounts of AGEs naturally increase, however, they are even more highly elevated in AD and diabetes. ALT-711 (Alagebrium) is an AGE lowering agent that has shown beneficial effects in decreasing diabetes-accelerated atherosclerosis and reversal of age-related myocardial stiffening. However, ALT-711 exhibits a structural homology to thiamine (TF) and therefore, it was assessed in this work whether ALT-711 is an inhibitor of the enzyme thiamine diphosphokinase (TDPK), which converts TF to thiamine diphosphate (TDP). TDP is an essential co-factor for different enzymes such as transketolase, pyruvate dehydrogenase (E1 subunit) and α-ketoglutarate dehydrogenase. A decrease in transketolase activity due to TDP deficiency results in less degradation of methylglyoxal (MGO) precursors during the pentose phosphate pathway. High levels of MGO are involved in elevated formation of AGEs. Thus, inhibition of the enzyme TDPK would counteract with ALT-711‟s main AGE lowering effect. To determine the inhibitory effect of ALT-711 and its metabolite ALT-1235 on TDPK an enzyme extract from bovine kidney was incubated with TF following pre-incubation with ALT-711 or ALT-1235, respectively. The concentration of TDP was measured by using a high performance liquid chromatography (HPLC)-method with fluorescence detection. Furthermore, molecular modelling was used to identify the inhibitory binding sites of ALT-711 and ALT-1235 with TDPK. With increasing ALT-711 concentrations, a dose-dependent decrease in TDPK activity was observed (Ki = 760 μM), whilst ALT-1235 showed a much lower effect (Ki = 2.0 mM). Although the difference in affinity between ALT-711 and TF (KM = 0.18 μM) is more than 3 orders of magnitude, concentrations of the drug in the highest clinically used dosage of 420 mg/day greatly exceed the recommended TF uptake of approximately 1 mg/day. This suggests that TDP blood levels should be investigated in vivo after treatment with ALT-711. Additionally, the competitive binding sites of ALT-711 and ALT-1235 were identified by performing molecular modelling studies. These studies revealed that the interactions between the pyrimidine ring of TF and TDPK are lost, as this structural element is replaced in ALT-711 and ALT-1235 by a phenyl ring. However, the interactions between the thiazolium ring and the enzyme - 4 - are still possible, as well as the parallel stacking. It is proposed that these interactions allow binding of ALT-711 and ALT-1235 to the TF binding site of TDPK, thus explaining the competitive inhibition of TDPK. These studies will assist in the development of AGE lowering agents which do not interfere with TF metabolism. Furthermore, dicarbonyl compounds are involved in the formation and cross-linking of AGEs. Therefore, the second aim of this work was to determine the potential of different agents – aminoguanidine, (-)-epigallocatechin-3-gallate (EGCG), ALT-711, phloretin and phloridzin – to lower the concentration of the dicarbonyl compounds glyoxal (GO) and MGO. Changes in GO and MGO concentrations were detected by using a HPLC-method with fluorescence detection. The experiments were performed in vitro in a reaction tube, as well as in mouse neuroblastoma (Neuro-2a) cells. At high GO and MGO concentrations of 18.90 μM a dicarbonyl scavenging effect could be observed from the reaction tube experiments for aminoguanidine and EGCG. However, when performing the same experiment with lower GO and MGO concentrations of 2.48 μM this effect could not been observed. Furthermore, when conducting the cell culture experiments with all 5 agents also no change in GO and MGO concentration was detected. According to the literature GO and MGO concentrations below 3 μM are expected per 106 cells. These results suggest that the limit of detection of this HPLC-method, which has been previously determined, had been reached. Therefore, further method establishment is required to assure accurate detection of the small amounts of GO and MGO from cells. In summary, the development of effective AGE lowering agents, as well as the identification of safe and potent dicarbonyl scavengers is a promising goal for slowing down the progression of AD and other AGE-related diseases