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

Advanced glycation end products as biomarkers and gerontotoxins : a basis to explore methylglyoxal-lowering agents for Alzheimer's disease?

Alzheimer's disease (AD) is the most common dementing disorder of late life. Although there might be various different triggering events in the early stages of the disease, they seem to converge on a few characteristic final pathways in the late stages, characterized by inflammation and neurodegeneration. In this review, we put forward the hypothesis that advanced glycation end products (AGEs) and their precursors, including methylglyoxal, are both biomarkers and causative agents (" gerontotoxins" ) characteristic for this disorder. Accumulation of AGEs is a normal feature of aging, but is accelerated in AD, where AGEs can be detected in amyloid plaques and neurofibrillary tangles. AGE modification may explain many of the neuropathological and biochemical features of AD such as extensive protein cross-linking, inflammation, oxidative stress and neuronal cell death. We suggest that methylglyoxal is one of the major carbonyl species responsible for the formation of AGEs. We propose that one promising pharmacological approach to prevent the formation of AGEs would be to lower the methylglyoxal concentration. This can be achieved, for example, by decreasing the concentration of methylglyoxal precursors such as d-glyceraldehyde-3-phosphate by allowing a higher flux through the pentose phosphate pathway or by increasing methylglyoxal detoxification through the glyoxalase system. Alternatively, methylglyoxal could be scavenged by various types of carbonyl scavengers

The thiamine analogue and advanced glycation endproducts crosslink breaker ALT-711 does not interfere with transketolase activity

The enzyme transketolase (sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) is involved in the pentose phosphate pathway (PPP) and catalyses the transfer of a 2-carbon fragment from a 5-carbon keto sugar (xylulose-5-P) to a 5-carbon aldo sugar (ribose-5-P) to form a 7-carbon keto sugar (sedoheptulose- 7-P) and a 3-carbon aldo sugar (glyceraldehyde-3-P). Transketolase requires thiamine pyrophosphate as a co-factor. Advanced glycation endproducts (AGEs) are implicated in the complications of diabetes and aging, primarily via adventitious and crosslinking of tissue proteins. ALT-711 is an AGE crosslink breaker and has been tested as an intervention therapy in established complications of diabetes. It has been noticed that it has a similar structure to that of thiamine and it was hypothesized that it might inhibit transketolase by replacing the active co-factor rendering the enzyme inactive. In this study, we have established a novel microtiter plate format transketolase assay which determines the concentration of NADH by measuring its fluorescence. Using this assay, it was found that ALT-711 does not inhibit the activity of transketolase up to concentration of 5 mM. We conclude that ALT-711 does not interfere with transketolase activity at clinically relevant concentrations

The advanced glycation end product-lowering agent ALT-711 is a low-affinity inhibitor of Thiamine Diphosphokinase

Advanced glycation end products (AGEs) are involved in age-related diseases, including the complications of diabetes and chronic renal impairment with arterial stiffening. Alagebrium chloride (ALT-711) is an AGE-lowering agent with beneficial effects in renal structural and functional parameters in diabetes, decreased diabetes-accelerated atherosclerosis, and age-related myocardial stiffening. ALT-711 exhibits a structural homology to thiamine, and it was suggested to interfere with thiamine metabolism. Thiamine is converted to thiamine diphosphate (TDP) by thiamine diphosphokinase (TDPK). TDP is a cofactor for pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase. A decreased activity of these enzymes due to TDP deficiency results in disorders such as beriberi and Wernicke–Korsakoff syndrome. Therefore, we investigated whether ALT-711 is an inhibitor of TDPK. Molecular modeling studies showed that ALT-711 fits into the thiamine-binding pocket of TDPK, and there are three interactions between the thiazolium ring and the enzyme, as well as parallel stacking between the phenyl ring and the indole ring of Trp222B. Enzyme kinetic experiments also showed that ALT-711 dose-dependently decreased TDPK activity with Kis, calculated by different experiments and fitting models ranging from 0.88 to 1.09 mM. Fitting of the kinetic data favored mixed-mode inhibition with a major role for competitive inhibition. In summary, our results suggest that ALT-711 is a low-affinity inhibitor of TDPK, but is unlikely to interfere with thiamine metabolism at therapeutic concentrations. However, when new AGE-crosslink breakers based on thiamine are designed, care should be taken that they do not act as more potent competitive inhibitors than ALT-711

Impact of Selected Factors on the Occurrence of Contact Dermatitis in Turkeys on Commercial Farms in Germany

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A versatile high throughput screening system for the simultaneous identification of anti-inflammatory and neuroprotective compounds

In many chronic neurodegenerative diseases including Frontotemporal Dementia and Alzheimer's disease (AD), microglial activation is suggested to be involved in pathogenesis or disease progression. Activated microglia secrete a variety of cytokines, including interleukin-1β, interleukin-6, and tumor necrosis factor as well as reactive oxygen and nitrogen species (ROS/RNS). ROS and RNS contribute to alterations in neuronal glucose uptake, inhibition of mitochondrial enzymes, a decrease in mitochondrial membrane potential, impaired axonal transport, and synaptic signaling. In addition, ROS act as signaling molecules in pro-inflammatory redox-active signal transduction pathways. To establish a high throughput screening system for anti-inflammatory and neuroprotective compounds, we have constructed an "Enhanced Green Fluorescent protein" (EGFP) expressing neuronal cell line and set up a murine microglia/neuron co-culture system with these EGFP expressing neuronal cells. We show that microglia activation leads to neuronal cell death, which can be conveniently measured by loss of neuronal EGFP fluorescence. Moreover, we used this system to test selected polyphenolic compounds for their ability to downregulate inflammatory markers and to protect neurons against microglial insult. We suggest that this system might allow accelerated drug discovery for the treatment of inflammation-mediated neurodegenerative diseases