Identification of Novel Aldose Reductase Inhibitors Based on

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Identification of Novel Aldose Reductase Inhibitors Based on Carboxymethylated Mercapto-Triazino-Indole Scaffold Milan Stefek, Marta Soltesova Prnova, Magdalena Majekova, Chris Rechlin, Andreas Heine, and Gerhard Klebe J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5015814 • Publication Date (Web): 19 Feb 2015 Downloaded from http://pubs.acs.org on February 22, 2015

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Journal of Medicinal Chemistry

Identification of Novel Aldose Reductase Inhibitors Based on Carboxymethylated Mercapto-Triazino-Indole Scaffold

Milan Stefek,*1 Marta Soltesova Prnova,1 Magdalena Majekova,1 Chris Rechlin,2 Andreas Heine,2 Gerhard Klebe2

1

Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences,

Dubravska cesta 9, 841 04 Bratislava, Slovakia, 2

Department of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6,

35032 Marburg, Germany

ABSTRACT: Fifteen compounds, sharing an indole-1-acetic acid moiety as a common fragment, were selected from commercial databases for testing aldose reductase inhibition. 3Mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid (13) was the most promising inhibitor, with an IC50 in submicromolar range and high selectivity, relative to aldehyde reductase. Crystal structure of aldose reductase complexed with 13 revealed an interaction pattern explaining its high affinity. Physicochemical parameters underline the excellent „leadlikeness“ of 13, as a promising candidate for further structure optimizations.

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INTRODUCTION

In diabetic patients, under conditions of hyperglycemia, glucose becomes a substrate of aldose reductase (ALR2), the first enzyme of the polyol pathway. Increased flux of glucose through the polyol pathway results in overproduction of the organic osmolyte sorbitol.

Depletion of

NADPH cell stores by aldose reductase may eventually increase the susceptibility of cells to damage by reactive oxygen species. In addition, the polyol pathway contributes to the glycation process supplying fructose, a reactive glycation agent. Through these mechanisms glucose exerts its toxicity with diabetic complications as a consequence.1,2 Aldose reductase, due to its physiological role, metabolizes lipid peroxidation products, contributing in some settings to the inflammatory response. Inhibition of aldose reductase was found to prevent significantly the inflammatory signals induced by cytokines, growth factors, endotoxins, high glucose, allergens and auto-immune reactions in cellular as well as animal models.3,4 The binding pocket of Aldose Reductase consists of a rigid anion binding pocket and a flexible specificity pocket (Figure 1a). The anion binding pocket is formed mostly by His110, Tyr48, Trp 111 and the positively charged NADP+. Different classes of inhibitors are described in literature which exhibit varying head groups such as carboxylic acids, succinimides, hydantoins and phenolic OH groups which bind with their negatively charge to this pocket5. This specificity pocket may only be opened by inhibitors that offer the right interactions. Phe 122 and Leu 300 serve as gatekeepers and can seal the pocket. The main flexibility is shown by the Ala 299- Leu 300 backbone and is best visible in different conformations of the Leu 300 side chain which determines whether the pocket is open or not6. In Figure 1b the protein is shown complexed with citrate (pdb 1x96)7 with the specificity pocket in closed conformation. In Figure 1c, structure of ALR2 complexed with the highly


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potent

aldose

reductase

inhibitor

2-[(4-Bromo-2-fluorobenzyl)carbamothioyl]-5-

fluorophenoxy acetic acid (IDD5948, Fig. 1d), is shown with the specificity pocket in open conformation (pdb: 1US0). Aldose reductase inhibitors (ARIs), such as acetic acid derivatives (tolrestat, zenarestat), spiro hydantoins (sorbinil), or the succinimide class of compounds (ranirestat) have been primarily investigated for their role against diabetic complications.9-11 None of them has demonstrated sufficient efficacy in human clinical trials without undesirable side effects. The involvement of ALR2 in inflammatory pathologies apparently renewed the interest in the field of ARIs.12 In the search for better ARIs, the focus has been shifted towards novel chemotypes. Emphasis was given not only to the inhibitory activities but also to the physicochemical properties determining biological availability in target tissues.13,14 Recently presented examples of a new generation of aldose reductase "differential" inhibitors were found to preferentially inhibit the reduction of either hydrophilic or hydrophobic substrates.15,16 Substituted indole-1-acetic acids represent a group of ARIs of high activity and selectivity17-21, with lidorestat as a promising candidate for clinical studies. Yet lidorestat was withdrawn from these studies owing to its side effects. Nonetheless, we still consider the indole-1-acetic acid moiety as a promising starting fragment for the design of efficient, selective and pharmacologically applicable ARIs. In line with the above findings, we used a ligand-based drug design strategy to search for novel ARIs in databases of commercially available compounds. Fifteen compounds with an indole-1-acetic acid moiety as a common fragment in their structure were selected for experimental testing of their inhibitory potency against aldose reductase. Among the compounds studied, 3-mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid (compound 13) exhibited the highest biological activities both at the level of isolated enzyme and at organ


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level, in a model of isolated rat eye lenses. The stoichiometry of the binding reaction was determined by isothermal titration calorimetry. To identify crucial interactions within the aldose reductase binding site, the crystal structure of ALR2 in complex with 13 was determined. Selectivity in relation to the closely related rat kidney aldehyde reductase (ALR1) was evaluated. Structure–activity relationships along with a computer-based physicochemical profiling of the compounds are discussed.

RESULTS AND DISCUSSION

Compounds. ChemSpider database was screened for compounds exhibiting the structural requirements to embed an indole-1-acetic and indoline-1-acetic acid moiety. An initial database of 5813 compounds was thus obtained. Coincidence of four additional requirements, namely experimental/ACD calculated logP = 0-3.5, free N-carboxymethyl group, up to 6 hydrogen bond donors and 2-6 hydrogen bond acceptors reduced the set to 1147 compounds. Then applying LASSO similarity value of 0.99 for ALR2 inhibition in combination with commercial availability requirement gave 335 compounds. In our further search we focused on indole-1-acetic acids bearing a neutral substituent (compound 3) or a basic substituent (compounds 4-8) in position 3 (structural analogues of lidorestat). In addition, we were interested in the effect of embedding the substituent into a fused ring (compounds 9-17). Vendor accessibility was taken into consideration in selection of the compounds for experimental evaluation (Table 1, Zinc codes and MolPort IDs are also listed). Their structures are shown in Figure 2. For comparison, two indole-1-propionic acid congeners (2, 5) were included into the experimental sample set. In addition, two structural variants lacking indole-1-acetic acid moiety were selected (compounds 18, 19).


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ALR2 inhibition. The compounds 1 - 19 were evaluated for their potential to inhibit the in vitro reduction of D,L-glyceraldehyde by partially purified ALR2 extracted from rat eye lenses, using zopolrestat and epalrestat as reference inhibitors (Table 2). It has been shown that human ALR2 exhibits 85% sequence homology to rat lens ALR222, while the catalytic active sites of both enzymes are considered identical23. Unsubstituted indole-1-acetic acid (1) inhibited ALR2 in low micromolar range as suggested by the measured IC50 values. Insertion of a substituent at position 3 (compounds 3,4, 6-8) resulted in reduced inhibitory power towards ALR2 compared to the unsubstituted indole-1-acetic acid (1). Variation of the basic substituents in compounds 4, 6-8 had only mild effect on the inhibitory potential. For the propionic acid congeners 2 and 5, a profound decrease of inhibition was observed, with IC50 ~50 µM for 2 and IC50 >100 µM for 5. The polycyclic derivatives 9 and 10, with one or two additional fused aromatic rings, respectively, revealed moderately increased inhibitory power compared to 1. Yet, the addition of a third ring in 11 resulted in a substantial drop of inhibitory potential. Increased polarity of the fused ring in 12 resulted in a similar reduction of the inhibitory power. On the other hand, profoundly increased inhibition was recorded for the mercapto triazine derivatives 13 and 17, with IC50 values in submicromolar range. Values of IC50 in low micromolar range were recorded for the remaining mercapto triazine derivatives (14-16). The strongest inhibition of ALR2 was found for 13 with IC50 ~ 0.1 µM. In addition, we obtained a closely related value (IC50 = 0.057 ± 0.002 µM) for the inhibition of human recombinant AKR1B1 by 13, measured under identical reaction conditions as applied for rat ALR2 assay, with the protein concentration of 0.28 µM. The structure-activity relationship in the series of 12, 13, 18 and 19 points to the necessity of a concurrent presence of both, the carboxymethyl group in position 5 and the terminal sulfur to achieve best affinity. Shifting of


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the carboxymethyl group from indole nitrogen to sulfur in 18 or its absence in 19 resulted in a loss of activity. Selectivity. An important feature of pharmacologically applicable ARIs is their high selectivity over other members of the aldo-keto reductase family. The co-inhibition of structurally related physiologically relevant oxido-reductases may create undesired side effects. To test for selectivity, the closely related aldehyde reductase (ALR1), is routinely studied.24 The ALR1 inhibitory potencies, expressed as IC50 values for the inhibition of the reduction of the substrate D-glucuronate by partially purified ALR1 from rat kidney, are shown in Table 2. For comparison, the reference valproic acid is included. All tested compounds were found to be less active on ALR1 compared to ALR2. In the series of the most efficient ARIs, the selectivity indices were found to be the highest for the mercapto triazines. It should be noted that the most selective compound 13, with a selectivity factor above 400, is also the most potent ARI in this series. For compound 13, AKR1B10 inhibition was assayed yielding an IC50 value of 21.4 ± 2.0 µM and the corresponding selectivity factor with respect to ALR2 was above 200. Enzyme kinetics. In the next step, the enzyme kinetics for the most efficient ARI 13 was analyzed. Uncompetitive inhibition was observed in relation to D,L-glyceraldehyde as a substrate (Fig. 3) with a corresponding inhibition constant Ki(13) = 0.089 ± 0.018 µmol/l. Conformation analysis. For the most efficient ALR2 inhibitor 13, tautomeric forms were first estimated by the Marvin calculator25 in the range of pH = 6.2 – 8.0. As a result, three tautomers were selected (Fig. 4) and investigated by means of quantum-chemical calculations. The most preferred tautomer in solution was found to be 13-1 followed by the forms 13-2 and 13-3 with relative energy stabilities of 16.4 kJ/mol and 72.9 kJ/mol, respectively (see Table S1 in Supporting material).


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Crystal structure and isothermal titration calorimetry (ITC) measurements. The crystal structure of ALR2 in complex with 13 was determined at pH 8.0 with a resolution of 1.26 Å. Two molecules of the inhibitor are visible in the determined difference electron density, one as expected in the binding pocket and a second in the vicinity of the entrance of the pocket (Fig. 5a). The latter is in contact with the binding site inhibitor molecule (first molecule) and refines to an occupancy of 70 % whereas the active site molecule shows 100 % occupancy. The oxygen atom of the carboxylate group of the second inhibitor molecule and N2 of the first one form an H-bond (2.8 Å, Fig. 5c). The proton is most likely contributed by the nitrogen of the triazine ring of the first molecule of 13, as we assume that the carboxylate is deprotonated under the applied conditions. In our structure (Fig. 5b), the first inhibitor molecule which occupies the anion binding pocket forms similar interactions as described for related inhibitors exhibiting a carboxylate head group, for example the inhibitor 20 (IDD594), with which the so far best resolved crystal structure of an ALR2-inhibitor complex could be determined.8 One carboxylate oxygen atom of the inhibitor forms an H-bond to NE2 of His 110 (2.7 Å) and to the Tyr 48 (OH) group (2.7 Å), while the second oxygen is in contact with NE1 of Trp 111 (3.0 Å). The most likely negatively charged carboxylate group of 13 makes an electrostatic interaction with the oxidized and then positively charged NADP+ (3.1 Å). A water molecule is connected to ND1 of His 110 (3.0 Å). One further water molecule is interacting with N1 of the triazine ring of the inhibitor (3.0 Å). The remaining contacts to the protein are of hydrophobic nature, e.g. to Trp 219 (3.5 Å), Trp 20 (3.5 Å), Trp 111 (4.0 Å), Trp 79 (5.0 Å), Val 47 (3.8 Å), Phe 122 (4.2 Å). Presumably the inhibitor binds favorably to this aromatic environment due to its own aromatic character. We analyzed the crystal structure to rationalize the good selectivity of 13 for ALR2 compared to ALR1. The conformation adopted in the binding pocket is surprising. Previously


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it has been proposed that addressing the protein with the specificity pocket in open state is a prerequisite for a high selectivity index,26 however we found in the present complex this pocket to be closed (Fig. 5a). The distance of the sulfur atom of 13 to the backbone nitrogen of Leu 300 (3.2 Å) indicates a putative H-bond to this residue (Fig. 5b) even though the geometry of this interaction is not ideal for an H-bond. This requires the sulfur atom to be in the tautomeric state to act as an H-bond acceptor. To obtain accurate values for the bond lengths and angles with corresponding standard deviations in order to support our assumption about the tautomeric state actually adopted for binding to ALR2, we performed a refinement with SHELXL without imposing any geometry restraints for 13. However, no conclusive results could be obtained. This is most likely due to the insufficient resolution of the dataset to accurately determine the bond length with reasonable estimated standard deviations. Thus based on the bond lengths, we cannot unambigously assign which tautomer of 13 binds to the protein. Nonetheless we assume that tautomer 13-2 is present as it would allow to establish the observed H-bond to the second molecule of 13. In tautomer 13-1, N2 cannot serve as an H-bond donor to interact with the most likely deprotonated carboxylate group of the second molecule of 13 (pH 8.0). The quantum-chemical calculations however suggested the tautomer 13-1 to be the most likely one in water. The energies of 13-1 and 13-2 in vacuum were very similar, slightly shifted in favor of 13-2 (Table S1). We even assume the equilibrium between different tautomers might be changed upon ligand binding to the protein as the inhibitor molecule in the anion binding pocket is largely desolvated. The second inhibitor molecule binds (Fig. 5c) via the described H-bond to the first one and the other carboxylate oxygen atom is in H-bond distance to the backbone nitrogen of Ser 302 (2.8 Å). Furthermore, the aromatic core skeleton makes a face to face π-stacking to Trp 219 (3.4 Å).


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We perfomed ITC measurements to determine the stoichiometry of the binding reaction, which reveals a value in agreement with a 1:1 binding, similarly observed for other inhibitors. Accordingly, the second inhibitor molecule is only weakly bound and possibly results from the concentrated crystal soaking conditions. It is well accommodated in the protein structure and likely suggests an additional binding position that could intermediately be adopted during the penetration of the inhibitor into the catalytic center. Also the occupancy of 70 % indicates that the second molecule is only weakly bound. In agreement with the first molecule of 13, we decided to build it as tautomer 13-2. Our ITC experiments were performed from three buffer conditions (Fig. S1) suggesting a proton entrappment upon protein binding (Fig. S2). This method has been described previously27-29. Therefore the enthalpy of binding had to be corrected for buffer dependencies. The thermodynamic parameters are ∆G0= -35.6 kJ/mol, ∆H0= -62.8 kJ/mol and -T∆S0= +27.2 kJ/mol (Fig. S3). The binding process is strongly dominated by the enthalpy and comprises an unfavorable entropic contribution. Solely considering the rigid molecule an enthalpic signature appears surprising. However, the entire binding process including changes of the protein and the residual solvation structure have to be regarded. On the other hand, the strong exothermic signal suggests the formed H-bonds to be quite attractive. The contribution resulting from the displacement of water molecules is difficult to estimate in the present case since there is no apo-structure of ALR2 available. The uptake of 0.65 mol/L protons falls into the same range as already described by Steuber et al.27 for carboxylate-type ALR2 inhibitors. In their study Tyr 48 was identified as the proton acceptor. Comparison with the binding mode of lidorestat.The complex crystal structure of lidorestat18,19 was aligned with the one of 13 (Fig. 5d). The carboxylate groups of 13 and lidorestat align perfectly well. Lidorestat opens the specificity pocket with its 4,5,7-trifluoro1,3-benzothiazole side chain and thus induces the conformational changes in the loop next to


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Leu 300. The aromatic core structure of 13 coincides with the plane as present in lidorestat. Lidorestat makes several interactions in the specificity pocket18,19 which explains the higher IC50 value (5 nM) compared to 13. Physicochemical properties. Values of logP and log D7.4 calculated by the ACD/Labs software30 for 13 were found to be 1.68 and -2.03, respectively. The distribution ratio of 13 was also determined experimentally by a standard shake-flask technique which gave a closely similar value of log D7.4 = -1.90 ± 0.03. Considering the high inhibition efficacy/selectivity along with the structural parameters fitting „the rule of three“, (MW

Identification of Novel Aldose Reductase Inhibitors Based on

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