Identification of molecules acting as inhibitors of members of the aldo-keto reductase superfamily

AKR1B1 and AKR1B10 are NADPH-dependent reductases belonging to Aldo-Keto Reductase superfamily. AKR1B1 and AKR1B10 act as detoxicant enzymes. However, plenty of evidence suggest the involvement of both enzymes in pathological conditions. AKR1B10 emerged as a cancer biomarker since its overexpression in several non-gastrointestinal cancers. AKR1B10 activity supports cancer progression through several mechanisms including cytotoxic aldehydes detoxification, chemoresistance, regulation of retinoids homeostasis, and polycyclic aromatic hydrocarbon (PAH) derivatives metabolism. Therefore, AKR1B10 is considered a target for antineoplastic treatments. However, AKR1B10 shares kinetic and structural features with AKR1B1. Inhibitors that discriminate between AKR1B10 and AKR1B1, and minimize the interference with the detoxifying ability of the ubiquitous AKR1B1, may be relevant to develop novel treatment for cancer. Several natural products were proven to be inhibitors of AKRs. In this regard, zolfino bean extract is known for being rich in AKR1B1 inhibitors. Here results on isolation and characterization of selective inhibitors of AKR1B10 from zolfino bean extract are reported. The workflow includes fractionation of a crude extract of zolfino bean on a C18 column, evaluation of inhibitory ability of the fractions, and LC-MS characterization of relevant sets of fractions. Correlation between results from LC-MS and kinetic analyses allowed the identification of promising candidates for selective enzyme inhibition. In addition, this study was focused on the detrimental effects of AKR1B1 in diabetes. The role of AKR1B1 as the first enzyme of the polyol-pathway is generally accepted as the responsible of several cell damaging processes including oxidative stress and accumulation of both glycating agents and sorbitol, which contribute to the onset of secondary diabetes complications. However, several inhibitors of AKR1B1 failed clinical trials because of side effects. AKR1B1 is a multispecific but non-permissive enzyme, i.e. it reduces a variety of aldehydes with different chemical properties still discriminating between compounds belonging to the same chemical class. This peculiar feature of AKR1B1 paved the way for differential inhibition (DI), an alternative inhibition strategy based on compounds able to inhibit AKR1B1 depending on the substrate it is working on. These compounds, namely differential inhibitors, have to intervene on AKR1B1 glucose-reductase activity leaving unaffected or affecting at lesser extent its detoxifying activity. In this study, novel synthetic compounds were screened for their ability to act as differential inhibitors of AKR1B1. In addition, these compounds were tested for their ability to discriminate between AKR1B1 and AKR1B10. 8 Finally, this study aimed to optimize a fluorescent assay for the detection of AKR1B10. The characterization of a substrate with fluorescent properties and preliminary tests for its application to detect AKR1B10 reductase activity in biological samples are also reported.