Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia (high blood sugar). This disease, which is highly prevalent worldwide, is a major health problem that significantly negatively impacts public health. Hyperglycemia plays a significant role in the development of diabetes-related complications, such as nerve damage (neuropathy), eye damage (retinopathy), kidney disease (nephropathy), and cataracts. This is because, when blood sugar is high (hyperglycemia), more glucose than normal is diverted to the polyol pathway [1] (Figure 1).

Although considered a minor pathway in normal glucose metabolism, the polyol pathway becomes overactive under conditions of hyperglycemia, leading to osmotic stress and redox imbalance due to the formation of reactive oxygen species (ROS) [2]. Aldose reductase (ALR2; EC 1.1.1.21), a metabolic enzyme in the polyol pathway, is a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzyme that facilitates the reduction of diverse ketones and aldehydes to alcohols. [1]. In hyperglycemia, glucose is converted to sorbitol using the auxiliary cofactor nicotinamide adenine dinucleotide (NAD⁺). When NAD⁺ and NADPH levels in the cell decrease or are insufficient, excess sorbitol cannot be broken down and begins to accumulate within the cell. This buildup raises oxidative stress and throws off cells' osmotic equilibrium, which damages organs and cells and eventually results in consequences from diabetes. In addition, when sorbitol and its metabolites cannot be broken down sufficiently, they accumulate in the kidneys, retina and nerve tissue, contributing to the formation of diabetic complications [3].

Figure 1. Polyol pathway mechanism in the formation of diabetic complications due to hyperglycemia

Aldose reductase (ALR2; EC 1.1.1.21) is a NADPH-dependent oxidoreductase enzyme that is part of the aldo-keto reductase (AKR) superfamily. The enzyme catalyzes the reduction of carbonyl compounds (e.g., aldehydes and ketones) to the corresponding alcohols. In humans, the ALR2 gene is called AKR1B1 and is located in chromosome 9q22.3. ALR2 is a protein of approximately 36 kDa, consisting of 315 amino acids. In its crystal structure, the enzyme exhibits a β/α-barrel (TIM-barrel) fold, and its active site contains the critical catalytic residues Tyr48, His110, and Asp43 [4].

It uses NADPH as a cofactor, which provides the electrons necessary for the reduction of the carbonyl group. Under normal physiological conditions, aldose reductase is involved in the detoxification of various endogenous aldehydes. It is known to be particularly active on sugars such as glucose, galactose, and ribose, as well as lipid peroxidation products (e.g., 4-hydroxy-nonenal). The enzyme is highly expressed in the kidney, liver, retina, nervous tissue, and lymphocytes. Furthermore, as an enzyme involved in the stress response, it reduces the accumulation of reactive carbonyl compounds in the cell [5].

ALR2 catalyzes the first and rate-limiting step of the polyol pathway in the pathogenesis of diabetic complications. In the polyol pathway, aldose reductase changes glucose into sorbitol, and then sorbitol dehydrogenase changes sorbitol into fructose. Under hyperglycemia, excess glucose is directed toward the polyol pathway; this process consumes NADPH and creates intracellular osmotic stress [2]. Decreased NADPH levels reduce glutathione reductase (GR) enzyme activity, leading to increased oxidative stress. Consequently, complications such as retinopathy, nephropathy, and neuropathy develop in the retina, kidney, and nerve tissues [3].

ALR2 is overexpressed not only in diabetic complications but also in many malignancies. Increased ALR2 expression has been reported, particularly in hepatocellular, prostate, and lung carcinomas. This increase is due to ALR2's influence on redox homeostasis and apoptotic signaling. Furthermore, ALR2 acts as a bridge between oxidative stress and inflammation by mediating the increase in inflammatory cytokines (e.g., TNF-α, IL-6) [6].

In recent years, various ALR2 inhibitors (ALR2Is) have been studied, including epalrestat [7], fidarestat [8], ranirestat [9], and sorbinil [10]. These molecules (Figure 2) exhibit pharmacokinetic performance with different levels of inhibitory activity. Also, their medicinal development has often been slowed down by problems including limited oral bioavailability, poor membrane permeability, metabolic instability, or lack of target selectivity [11].

Figure 2. Chemical structures of some known ALR2 inhibitors

Heterocyclic compounds are of great importance in medicinal chemistry, agricultural chemicals, polymer materials, and various industrial products [12]. Nitrogen-containing heterocyclic compounds are abundant in nature and are also found in many natural products such as various alkaloids, vitamin derivatives, antibiotics, and hormones [13].  It is also known that approximately 60% of currently approved medical drugs contain heterocyclic nitrogen in their structures. Pyrimidine and its various analogs have long been a focus of interest due to their diverse biological activities. In addition to their medicinal properties, pyrimidine constitutes an important class of heterocyclic compounds because it is the basic molecule in the presence of the pyrimidine base, which consists of uracil, cytosine, and thymine, which form the structures of DNA and RNA [14].

Many chemicals are known to act on various enzymes. In fact, drugs exert their therapeutic effects by significantly altering the activities of enzymes. In this context, examining the effects of pyrimidine and some derivatives (Figure 3), known to have an impact on many metabolic diseases, on ALR2 activity is of critical importance. In summary, this study was designed to characterize novel ALR2 inhibitors and investigated the in vitro inhibitory effects of commercially available pyrimidines and some derivatives on human ALR2 enzyme activity.

Figure 3. The pyrimidine and some derivatives used in the inhibition study

MATERIALS AND METHODS

All compounds used in the investigation were purchased from Sigma-Aldrich. The inhibitory profile of pyrimidine and its derivative compounds against ALR2 was examined using a cuvette-based spectrophotometric approach derived from the validated kinetic assay procedure by Cerelli [15] and all measurements were done in triplicate for consistency.  NADPH oxidation at 340 nm (ε = 3.4 × 102 mM−1·cm−1) in a final assay volume of 1.0 mL was used to measure enzyme activity. Sodium phosphate buffer (10 mM, pH 7.4), NADPH (0.11 mM), and DL-glyceraldehyde as substrate at different concentrations (0.071–0.376 mM) made up the reaction mixture [16]. To achieve a final DMSO concentration of at least 1%, inhibitor stock solutions were made in DMSO and added. Before adding the substrate, the enzyme and inhibitors were preincubated on ice for one minute. All analyses were carried out for three minutes. The reference inhibitor was EPR. The kinetic mechanism of inhibition was identified using Lineweaver-Burk plots, and inhibition constants were computed as a result [17]. These plots were used to categorize inhibition types.

RESULTS

In this study, the effects of pyrimidine and some of its derivatives on ALR2 were investigated. The physiological inhibitory properties of pyrimidine and some of its derivatives against the ALR2 enzyme were analyzed in vitro and compared with the reference compound EPR. Inhibition data for pyrimidine and some of its derivatives (1a-g) are presented in Table 1.

Table 1. IC50 and KI values for EPR used as standard inhibitor with pyrimidine and its derivatives (1a–g) on ALR2

Regarding the inhibition of ALR2, it was observed that the analyzed pyrimidines (1a–g) exhibited inhibition with K_I values ​​ranging from 4.49 ± 1.45 μM to 244.73 ± 3.36 μM. In addition, compounds (1f) and (1c), which showed better inhibition than EPR, were found to be quite strong inhibitors compared to the clinically used reference agent EPR (15.05 ± 1.51 μM K_I). While (1f) (K_I value 4.49 ± 1.45 μM) was found to be the strongest agent, 4-amino-2,6-dimethylpyrimidine (1g) (K_I value 244.73 ± 3.36 μM) was determined to have less effect against ALR2. The inhibitory activities of pyrimidine and some of its derivatives on ALR2 are as follows; 4-amino-6-hydroxypyrimidine (1f) > 4-amino-2,6-dichloropyrimidine (1c) > EPR > 4-amino-6-chloropyrimidine (1e) > 4-amino-5,6-dichloropyrimidine (1d) > 4-amino-2-chloropyrimidine (1b) > Pyrimidine (1a) > 4-amino-2,6-dimethylpyrimidine (1g).

IC₅₀ and K_I plots for EPR, used as standard inhibitors, with pyrimidine and its derivatives (1a–g) on ​​the ALR2 enzyme are shown in figure 4 and figure 5.

Figure 4. The IC50 graphs for EPR used as standard inhibitors with pyrimidine and its derivatives on the ALR2 enzyme

Figure 5. KI plots for EPR using pyrimidine and its derivatives (1a–1g) as standard inhibitors on the ALR2 enzyme