Structural re-positioning, in silico molecular modelling, oxidative degradation, and biological screening of linagliptin as adenosine 3 receptor (ADORA3) modulators targeting hepatocellular carcinoma

Abstract

Chemical entities with structural diversity were introduced as candidates targeting adenosine receptor with different clinical activities, containing 3,7-dihydro-1H-purine-2,6-dione, especially adenosine 3 receptors (ADORA3). Our initial approach started with pharmacophore screening of ADORA3 modulators; to choose linagliptin (LIN), approved anti-diabetic drug as Dipeptidyl peptidase-4 inhibitors, to be studied for its modulating effect towards ADORA3. This was followed by generation, purification, analytical method development, and structural elucidation of oxidative degraded product (DEG). Both of LIN and DEG showed inhibitory profile against hepatocellular carcinoma cell lines with induction of apoptosis at G2/M phase with increase in caspase-3 levels, accompanied by a downregulation in gene and protein expression levels of ADORA3 with a subsequent increase in cAMP. Quantitative in vitro assessment of LIN binding affinity against ADORA3 was also performed to exhibit inhibitory profile at Ki of 37.7 nM. In silico molecular modelling showing binding affinity of LIN and DEG towards ADORA3 was conducted.

Keywords: Drug repositioning; adenosine 3 receptor; hepatocellular carcinoma; linagliptin.

Figure 1.

Chemical structures of adenosine receptor modulators with 3,7-dihydro-1H-purine-2,6-dione.

Figure 2.

Two dimensional structure for (a) Linagliptin (b) Proposed chemical structure of linagliptin degradation product (DEG).

Figure 3.

HPLC chromatograms of (a) Linagliptin (10 µg mL⁻¹) and the proposed degradation product (2 µg mL⁻¹). (b) Multiple reaction monitoring (MRM) chromatogram (a) of linagliptin (m/z = 473.11–420.07) and the degradation product (m/z = 285.05–156.93) and HPLC chromatogram (b) of a lab prepared mixture of linagliptin (80 µg mL⁻¹) and the degradation product (50 µg mL⁻¹).

Figure 4.

Full scan mass spectrum and daughter ion mass spectrum in positive ESI ion detection mode with the proposed fragment. (a) Linagliptin and (b) linagliptin major degradation product (DEG).

Figure 5.

Effect of linagliptin and its major oxidative degradation product on viability in HepG2 and Huh7 cell lines and cell cycle progression in HepG2 cell line. (a) Dose-response plots of linagliptin (LIN) and its degradation product (DEG) on HepG2 and Huh7 cell lines after 72 h exposure, as detected by MTT assay. (b,c) DNA content-based cell cycle analysis in HepG2 cells treated with either LIN or DEG.

Figure 6.

Effect of linagliptin and its major oxidative degradation product on caspase-3, ADORA3, and intracellular cAMP levels. (a) Active caspase-3 protein levels in control, linagliptin (LIN)-treated, and the degradation product (DEG)-treated HepG2 cells. (b) ADORA3 relative mRNA expression levels in LIN- and DEG-treated HepG2 cells. (c) ADORA3 protein expression levels in LIN-, DEG-, adenosine-, & caffeine-treated HepG2 cells. (d) Intracellular cAMP protein levels in control, LIN-, and DEG-treated HepG2 cells. Gene expression levels were estimated using real-time qPCR. Relative mRNA expression level (fold change) was determined using the 2^−ΔΔCt analysis method. Protein levels were estimated using ELISA. Values are presented as means ± SD Statistical analysis was performed using one-way analysis of variance (One-way ANOVA) followed by Bonferroni post hoc test while Student’s t-test was used for relative mRNA data analysis. *Significantly different (at p < .05) versus control untreated cells (a, c, and d) or LIN-treated group (b).

Figure 7.

Visual representation of ADORA3 crystal structure. (a) Lingaliptin is showing hydrophobic–hydrophobic interactions along with the receptor surface and one hydrogen bond along with ASN:250:A. (c) Degradation Product (DEG) is showing potential hydrophobic–hydrophobic interactions along with the receptor surface and one hydrogen bond along with SER:247:A.