Discovery of new inhibitors of aldose reductase from molecular docking and database screening

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Abstract

Aldose reductase (ALR2) is a target enzyme for the treatment of diabetic complications. Owing to the limited number of currently available drugs for the treatment of diabetic complications, the discovery of new inhibitors of ALR2 that can potentially be optimized as drugs appears highly desirable. In this study, a molecular docking analysis of the structures of more than 127,000 organic compounds contained in the National Cancer Institute database was performed to find and score molecules that are complementary to ALR2. Besides retrieving several carboxylic acid derivatives, which are known to generally inhibit aldose reductase, docking proposed other families of putative inhibitors such as sulfonic acids, nitro-derivatives, sulfonamides and carbonyl derivatives. Twenty-five compounds, chosen as the highest-scoring representatives of each of these families, were tested as aldose reductase inhibitors. Five of them were found to inhibit aldose reductase in the micromolar range. For these active compounds, selectivity with respect to the closely-related aldehyde reductase was determined by measuring the corresponding inhibitory activities. The structures of the complexes between the new lead inhibitors and aldose reductase, here refined with molecular mechanics and molecular dynamics calculations, suggest that new pharmacophoric groups can bind aldose reductase very efficiently. In the case of the family of the nitro-derivative inhibitors, a class of particularly interesting compounds, a round of optimizations was performed with the synthesis and biological evaluation of a series of derivatives aimed at testing the proposed binding mode and at improving interaction with active site residues. Starting from a hit compound having an IC50 of 42 μM, the most potent compound synthesized showed a 10-fold increase in inhibitory activity and 10-fold selectivity with respect to ALR1, and structure–activity relationships of the designed compounds were in agreement with the proposed mode of binding at the active site.

Docking screening of the NCI database of compounds identified novel inhibitors of aldose reductase. Synthesis and optimization of inhibitory activity was undertaken for the class of the nitro derivatives.

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Introduction

Aldose reductase (alditol/NADP+ oxidoreductase, E.C.1.1.1.21, ALR2) is the first enzyme of the polyol pathway which reduces excess d-glucose into d-sorbitol with concomitant conversion of NADPH to NADP+.1, 2, 3, 4 The polyol pathway plays an important role in the development of degenerative complications of diabetes. The association between hyperglycaemia and the development of long-term diabetic complications such as neuropathy, retinopathy and cataract is well documented.5 At elevated blood glucose levels, such as those occurring during diabetes, a significant flux of glucose through the polyol pathway is induced in tissues like nerves, retina, lens and kidney, and the activation of this pathway is believed to induce the appearance of diabetic complications affecting various pathogenic factors. Aldose reductase inhibitors therefore offer the possibility of safely preventing or arresting the progression of long-term diabetic complications, despite imperfect control of blood glucose and with no risk of hyperglycaemia.

To date, two main classes of orally-active aldose reductase inhibitors (ARIs) have been reported: spirohydantoins and carboxylic acids, with Sorbinil and Tolrestat being the most representative members of each family, respectively. In general, while the in vitro activity of spirohydantoins and carboxylic acid ARIs is similar, their in vivo activity is very different. In vivo, carboxylic acids possess lower activity than spirohydantoins,6, 7 a finding that is likely attributable to their lower pKa and to a resultant impairment in penetrating physiological membranes. Spirohydantoins, on the other hand, which show higher activity in vivo, have clearly proved to cause hypersensitivity reactions.4, 8 On these grounds, there is an urgent need to discover and develop new lead ARIs not related to spirohydantoins or carboxylic acids, and preferably with pKa values higher than those of carboxylic acids.

Molecular docking and ‘de-novo’ design are widely applied to the discovery of enzyme inhibitors with specific and desired chemical properties.9, 10, 11, 12 One efficient approach to the design of new lead inhibitors is to select, within structural databases of several thousands of known and available organic molecules, those compounds that display the highest steric and electrostatic complementarity with the site of action.13, 14, 15 In this context, the docking of 3-D-databases of organic molecules into the crystal structures of enzymes provides an efficient way of rationally selecting small subsets of interesting and promising candidates for biological testing.

Here, we report the results of a molecular docking study of the National Cancer Institute (NCI) database of organic molecules into the crystal structure of aldose reductase. After the screening of the entire database, which contained more than 127,000 compounds, only 25 were selected and evaluated as aldose reductase inhibitors. As will be shown, this study led to the discovery of families of ARIs not belonging to the classes of carboxylic acids or spirohydantoins. For one family of particular interest, a series of derivatives was designed and synthesized in order to test the proposed binding mode and to improve the interaction with active site residues. Finally, the selectivity for aldose reductase with respect to aldehyde reductase (ALR1),4 an enzyme closely related to aldose reductase but not involved in diabetic complications, was determined by measuring the ALR1-inhibitory activity of the more active compounds here discovered.

Section snippets

Strategy

Docking requires that the 3-D-structures of both the target enzyme and the ligands are known and available. To date, the Protein Data Bank contains several crystal structures of ALR2, both alone and complexed with known inhibitors. Crystallographic analysis of human ALR2 complexed with two carboxylic acid inhibitors, Zopolrestat16 and citrate,17 has provided important insights into the identification of the inhibitor binding site. Both complexes show that the inhibitors bind at the substrate

Results and discussion

As a matter of validation of the docking procedure used throughout, Zopolrestat was first docked into the structure of ALR2. The highest-score orientation found by docking (with a score of −49 kcal/mol) was found to be in full agreement with the crystal structure of the ALR2-Zopolrestat complex,16 the docked orientation and the orientation in the crystal structure being closely superimposable. Docking of the entire NCI database of compounds into the ALR2 structure was then performed.

Before

Conclusions

We have presented a structure-based design of new inhibitors of aldose reductase. Using the DOCK program, the NCI database of compounds was searched for molecules complementary to the ALR2 active site. This search led to the discovery of five novel compounds that inhibited ALR2 in the micromolar range and showed selectivity with respect to ALR1. Remarkably, these compounds do not belong to the classes of the carboxylic acid or spirohydantoins, which have long been studied and optimized as ARIs.

Docking

The aldose reductase structure used for docking is the one previously obtained by docking and energy minimization of a complex between ALR2 and a carboxylic acid inhibitor carrying a benzothiazole substituent.22

Docking of inhibitors was performed using the program DOCK 3.5,40 which consists of several modules. The module SPHGEN41 was used to generate clusters of overlapping spheres that describe the accessible surface of the active site. Seventy four spheres were used to describe the active

Acknowledgements

Financial support from Consiglio Nazionale delle Ricerche (grant CNR no. 98.01911.CT03) is gratefully acknowledged. Thanks are due to Prof. Stefano Manfredini (University of Ferrara) for allowing us to use Sybyl to convert the NCI-3-D database in a format suitable for DOCK3.5.

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