The potential inhibitory activity of the compounds 11, 11l, 12 and 12a on the evaluated Cytochrome P450 enzyme isoforms was measured using the corresponding specific fluorimetric screening kits CYP1A2 Inhibitor Screening Kit (Fluorometric) (Abcam) with 3-cyano-7-hydroxycoumarin (3-CHC) applied as substrate and α-naphthoflavone used as an inhibitor. The CYP2D6 Inhibitor Screening Kit (Fluorometric) (Abcam) with 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin applied as a substrate and quinidine as an inhibitor, and CYP3A4 Inhibitor Screening Kit (Fluorometric) (Abcam) with resorufin applied as a substrate and ketoconazole as an inhibitor. The fluorescence for the different isoforms was measured at Ex/Em = 406/468 nm for CYP1A2; Ex/Em = 535/587 nm for CYP3A4 and Ex/Em = 390/468 nm for CYP2D6, respectively.

The different CYP450 isoform activity were normalized as percentage of the untreated control set as 100% and the results were expressed as mean values and standard deviation (±SD) (Graph Pad Prizm). Statistical analysis was performed by one-way analysis of variance (ANOVA) with post hoc multiple comparisons procedure (Dunnet’s test) to assess the statistical differences in case of normal distribution. Values of p < 0.05 and p < 0.001 were considered statistically significant.

Preliminary evaluations of hepatotoxicity and antiproliferative activity of two series of synthesized by us pyrrole-based hydrazide-hydrazones were recently published (Georgieva et al 2022) and outlined four representatives with low toxic effects and promising activity, identified as 11, 11l, 12 and 12a. The structures of the evaluated compounds is presented on Fig. 1.

The evaluated compounds 11, 11l, 12 and 12a were tested for possible inhibitory activity on some human isoforms of Cytochrome P450 at concentration 1µM. Three enzymes were chosen: the localized to the endoplasmic reticulum CYP1A2, the primarily expressed in the liver and highly expressed in areas of central nervous system (CNS) CYP2D6 and the available in the liver and in the intestine CYP3A4.

The target compounds were tested as CYP1A2 and CYP2D6 potential inhibitors. The results from the performed evaluations are presented on Figs 2, 3, respectively.

The results indicated that the compounds didn’t show any statistically significant inhibitory effects on CYP1A2 and CYP2D6 (Figs 2, 3).

In addition all evaluated structures were analyzed for potential inhibitory effects on CYP3A4, and the results are presented on Fig. 4.

The observations identified that only compound 12 shows low statistically significant inhibitory effect by decreasing the enzyme activity by 20%, compared to the control (pure CYP3A4). The other 3 tested derivatives didn’t reveal any effects on the CYP3A4 enzymatic activity (Fig. 4).

The effects on each isoform of Cytochrome P450 were compared with the relevant enzyme inhibitor. For CYP1A2 is α-naphthoflavone, for CYP2D6 is quinidine and CYP3A4 is ketoconazole. These inhibitors decreased statistically significant the enzyme activity by 55%, compared to the control (pure enzyme) (Figs 2–4).

The performed analysis identified the evaluated compounds as poor inhibitors to the discussed CYP’s, with 12 performing highest inhibitory activity among others, but still lower than the applied comparative agents. This results are a prerequisite to consider all of the evaluated compounds as substances with low effect of hepatic metabolism and decreased possibility for drug-drug interactions performance.

The docking module of Maestro – Glide was implemented for the current study considering the recent success rate when CYP enzymes were applied in the docking with the latter software (Ridhwan et al. 2022). Initially, self-docking simulations were carried out to assess the reliability of the docking software to correctly generate the conformations of the co-crystallized ligands back into the active sites of CYP1A2 (PDB:2HI4), CYP2D6 (PDB:4WNU) and CYP3A4 (PDB:2V0M). The Extra-Precision docking led to root mean square deviation (RMSD) values under 1 Å in all crystallographic structures. Thus, the Glide’s docking protocols demonstrate reliable results when CYP1A2, CYP2D6 and CYP3A4 are employed.

After the validation of the crystallographic structures, all four ligands were docked into the three CYP isoforms to observe the potential correlation between the in vitro and the in silico results, and also to discuss the active conformation of the active CYP3A4 inhibitor – 12. The data from the docking simulations is provided in Table 1. The co-crystallized ligands were docked in their native enzymes for reference.

In the active site of CYP1A2 (PDB: 2HI4) only 11 and 12 demonstrated moderate binding affinities of -7.80 and -8.21, respectively. After employing the more exhausting search in the active site with the IFD, the binding energies dropped to -5.57 and -6.36. The binding free energies calculated with the MM/GBSA were drastically lower compared to the value of the co-crystallized ligand. Subsequent docking in the CYP2D6 isoform agreed with the experimental data. In the latter case 11 and 12 showed the best results, however their binding scores were negligible compared to the scores of the native CYP2D6 inhibitor quinidine.

Interestingly, the results from the simulations in the active site of CYP3A4 demonstrated similar results for all four ligands. When the Extra-precision option in Glide was applied, the binding affinities were in the range of -6.57 to -8.28. In comparison, the co-crystallized ligand – ketoconazole, displayed XP score of -10.14. After the additional recalculations with IFD and MM/GBSA the most prominent results were demonstrated by compounds 12 and 12a. However, the key difference between the two conformations was the distance between the het group and the ligands. In the case of 12a the distance was calculated to be 5.64 Å while 12 was in close vicinity to the het structure (2.88 Å). The length between the heme Fe ion and the observed ligand could be used as a criterion to determine the robustness of the binding orientation. Typically, a pose is considered as reliable if the distance is shorter than 6 Å (Bonomo et al. 2017).

To further observe the intermolecular interactions of the complex 12-2V0M, both the 2D and 3D visualizations are presented (Fig. 5).

Based on the visualized stabilization forces, the hem group and Phe304 were the major drug-binding residues in CYP3A4. The latter formed strong p-p bonds with the pyrrole ring and the benzene moiety. Several weak hydrophobic interactions were formed by the active amino residues Ile120, Leu210, Leu211, Phe241, Ala305, Ile369, Leu482 and Leu483. After the XP docking, the active pose of 12 demonstrated that the carbonyl group from the ester group was in close proximity to the hem structure. In contrast, the more hardware demanding, and more precise IFD displayed different orientation in the active site of CYP3A4. During the latter simulations the hydrazide-hydrazone moiety was facing the hem moiety. Therefore, the implementation of IFD for virtual simulations in various CYPs isoforms is essential considering the enhanced reliability of the acquired results (Ridhwan et al. 2022).