br Experimental br Acknowledgment br Introduction The NADPH

Experimental
Acknowledgment
Introduction The NADPH-dependent reduction of d-glucose catalyzed by aldose reductase (E.C.1.1.1.21) (AR) is considered as one of the phenomena leading to the onset of long term diabetic complications [as review see: [1], [2]. In fact, the reduction of the sugar, which occurs mainly in hyperglycemic conditions, is associated with a number of deleterious events. Stressful and damaging cell conditions are caused by a number of factors: the osmotic imbalance due to sorbitol accumulation [3], the loss of reducing, and thus, antioxidant power associated with NADPH oxidation [4] and the induction of more favorable conditions for protein glycation [5]. This led a strong impulse in the search for molecules able to efficiently inhibit the enzyme (aldose reductase inhibitors, ARIs) with the aim to develop active drugs to antagonize the onset of diabetic complications. Several powerful ARIs have been proposed on the basis of their in vitro action, however the promotion of those molecules for effective drugs was not very successful; to date Japan and India are the only countries where an Epalrestat-based drug is distributed. This is likely due to the ability of AR to act on toxic STK393606 aldehydes, derived from lipid peroxidation processes, such as trans-4-hydroxy-2,3-nonenal (HNE) [6], [7], whose reduction attenuates their cytotoxicity. Thus, AR inhibition would no longer appear to be a useful approach in favoring cell health [8], [9], [10]. Nevertheless, several experimental evidences suggest an anti-inflammatory action of ARIs [11]; this on the basis of the ability of AR to reduce the adduct between HNE and glutathione, generating a pro-inflammatory signal [6], [7], [12], [13], [14]. More recently a new approach to control AR activity by differential inhibition was proposed [15]. In this case, the inhibitors (AR differential inhibitors, ARDIs) should be able to inhibit AR while acting on glucose, but not on molecules such as HNE, whose breakdown is part of the detoxifying action exerted by AR. The reaction rate of AR as a function of substrate concentration has been reported to display, in some instances, a negative cooperative type of behavior [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26]. As AR is unequivocally a monomeric enzyme, the observed negative cooperativity was associated with the presence, in the enzyme preparations, of two enzyme forms, which were often referred to as the “unactivated” and the “activated” enzyme and displayed different kinetic properties, including a different susceptibility to inhibition by ARIs and by substrates [16], [18], [21], [25], [26]. Indeed, a fascinating and intriguing mechanistic explanation has been proposed to justify, despite the presence of the two enzyme forms, the hyperbolic behavior occasionally observed with glyceraldehyde or glycol aldehyde [27]. None of the above considerations are easy to rationalize, since in the literature it is difficult to find a common established procedure for enzyme preparation and manipulation in order to define which form of AR is present. Although it has been reported that AR from the human psoas muscle could be modified by treatment with pyridoxal phosphate followed by NaBH4 treatment [28], the only reversible covalent modification assessed for the enzyme to date has been the thiolation of Cys298. This is induced by different thiol compounds, including 2-mercaptoethanol (2-ME), in oxidative conditions, with consequent possible intramolecular trans-thiolation arrangements [29], [30], [31], [32], [33], [34]. In addition, a S-glutathionyl-modified AR, a relatively stable enzyme form, not susceptible to Sorbinil inhibition and still sensitive to dithiothreitol (DTT) reduction, has been generated in situ in intact bovine lenses undergoing oxidative stress [35], [36]. On this basis, the occurrence of an apparent negative cooperative behavior that has been reported for the reduction of different substrates could be associated with the presence in pure enzyme preparations of enzyme forms with an altered cysteine redox status, deriving from inappropriate thiol-reducing conditions (i.e., the use of 2-ME) adopted during enzyme purification and storage [29].