Research Article |
Corresponding author: Emilio Mateev ( e.mateev@pharmfac.mu-sofia.bg ) Academic editor: Plamen Peikov
© 2024 Emilio Mateev, Ali Irfan, Alexandrina Mateeva, Magdalena Kondeva-Burdina, Maya Georgieva, Alexander Zlatkov.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Mateev E, Irfan A, Mateeva A, Kondeva-Burdina M, Georgieva M, Zlatkov A (2024) In silico and in vitro screening of pyrrole-based Hydrazide-Hydrazones as novel acetylcholinesterase inhibitors. Pharmacia 71: 1-7. https://doi.org/10.3897/pharmacia.71.e114120
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Virtual screening is emerging as a highly applied technique and gained prominence as widely used method for the search and identification of potential hits, significantly reducing the time needed to discover novel and effective compounds compared to high-throughput screening. Recently, the superiority of simulations with multiple programs compared to a single software docking has been discussed. The aim of this work was to apply consensus docking, molecular mechanics/generalized Born surface area (MM/GBSA) free binding energy recalculations, and in vitro evaluations on an in-house dataset of recently synthesized pyrrole-based hydrazide-hydrazones in the search for novel acetylcholinesterase (AChE) inhibitors. Two licensed softwares – GOLD 5.3 and Glide, were employed for the virtual screenings, and several chemotherapeutic potential hits were identified. Furthermore, MM/GBSA free binding energy recalculations were provided to enhance the robustness of the in silico results. The MM/GBSA scores of the top ten pyrrole-based hydrazide-hydrazones were ranging from -60.44 to -70.93 Kcal/mol. Subsequent, in vitro evaluations of the top ranked compounds revealed that 12d exhibited the highest AChE inhibitory activity, with a 55% inhibition rate at a concentration of 10 μM. Moreover, this prominent pyrrole-based AChE inhibitor formed stable complex with the active site of the enzyme. Interactions with the active amino residues Tyr72 and Tyr286 indicated that 12d was located near the peripheral anionic site of the enzyme. Additionally, in silico ADME investigations using QikProp demonstrated that 12d possesses optimal pharmacokinetic properties. In conclusion, this study identified a novel pyrrole-based AChE inhibitor 12d through a combination of computational and experimental findings.
Acetylcholinesterase inhibitor, pyrrole-hydrazide-hydrazones, consensus docking, MM/GBSA, biological evaluation, ADME
The positive role of acetylcholine (ACh) in the memory processes has been examined in details (
Pyrrole is a five-atom N-containing heterocycle introduced in many biological compounds, such as chlorophyll, heme, vitamin B12, bile pigments, and alkaloids. Among the N-containing heterocycles, the derivatives of pyrroles possess various biological activities, such as antituberculosis, antifungal, antioxidant, antidiabetic, anti-inflammatory, analgesic, and anticancer effects (
Structure-based drug design (SBDD) is successfully applied when a 3D structure of the corresponding protein is available. The major purpose of the SBDD is to distinguish false-positives from true inhibitors by employing scoring and searching algorithms (
Considering the vast pharmacological profile of the pyrrole-based compounds (
The employed dataset comprised recently synthesized pyrrole-based hydrazide-hydrazones with various biological activities (Fig.
The exact structures of the utilized database are provided in Supplementary Table
Compound | XP docking score (Kcal/mol) | ChemPLP (fitness score) | MM/GBSA (Kcal/mol) |
---|---|---|---|
11b | -8.159 | 132.51 | -70.93 |
12d | -8.738 | 132.68 | -69.29 |
11c | -7.152 | 131.50 | -66.04 |
12b | -7.684 | 141.20 | -64.06 |
12a | -8.259 | 132.51 | -62.68 |
TZ4 | -20.189 | 122.92 | -192.06 |
Donepezil | -18.41 | 114.06 | -187.64 |
The docking simulations were performed with both Glide (Schrödinger Release 2021-3: Glide, Schrödinger, LLC, New York, NY, 2021) and GOLD 5.3 on an AMD Ryzen 9 5950× 16-core 4.0 GHz CPU, NVIDIA GeForce RTX 3060 12GB GPU, 64 GB RAM installed memory and 64-bit Operating system Windows 11 Pro. The default GOLD 5.3 docking protocol was constructed out of a 8 Å binding gorge around the co-crystallized ligand, ChemPLP as a scoring function, addition of active waters in the active site and no rotatable side chain residues. For Glide, we selected the Extra-Precision (XP) option and created the enzyme’s active site using Receptor Grid Generation based on the conformation of the co-crystallized ligand. The crystal structure with PDB code 1Q84 was selected, which includes the highly active AChE co-crystallized ligand TZ4 (
In this study, Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) recalculations with Prime were employed to assess the free binding energies of the obtained through the docking studies complexes. The calculations were performed by the incorporation of the OPLS4 force field and VSGB dissolvable model (
Donepezil, Dimethyl sulfoxide (DMSO), 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), acetylthiocholine iodide (ATChI), and acetylcholinesterase were purchased from Sigma-Aldrich in analytically pure or chemically pure grades. No further purifications were applied.
The inhibitory AChE potential of the top ranked pyrrole-based agents was measured according to a modified Ellman’s method (
The significant physicochemical and pharmacokinetic properties of the most prominent AChE inhibitor in the current database were calculated with the QikProp module in Schrödinger (Schrödinger Release 2021-2: QikProp, Schrödinger, LLC, New York, NY, 2021.). The simulation provides ranges based on the properties of 95% of the known drugs and also evaluates outliners based on the Lipinski’s rule of five.
The docking protocols of GOLD 5.3 and Glide were initially validated through re-docking simulations. The re-docking procedures are essential for the preliminary assessments of the softwares’ reliability and robustness (
The re-docking of the co-crystallized ligand TZ4 back into the active site of 1Q84 was carried out with the ChemPLP docking score of GOLD 5.3 and the XP score of Glide. Notably, GOLD 5.3 demonstrated RMSD value of 1.21 Å, whereas Glide re-docked the native ligand with RMSD of 0.54 Å, which indicated the slightly better performance of Glide. The re-docking conformations obtained with both docking softwares are provided in Fig.
To increase the reliability of the structure-based drug design simulations, a consensus docking technique was implemented. Several studies have reported the superiority of simulations with multiple programs when compared to a single software docking (
Initial molecular docking simulations were carried out with the XP option in Glide, and the ChemPLP scoring function in GOLD 5.3 for the whole dataset (Supplementary Table
The final recalculations of the binding free energies of the complexes were conducted with the MM/GBSA method considering the enhanced hit rate of the latter (Table
However, the major concern is the immense gap of the binding free energy related to the native co-crystallized ligand – TZ4. In the former case the complex protein-ligand demonstrated drastically elevated stability (MM/GBSA score of -192.06). Thus, the expected in vitro experimental values of the title compounds may not achieve the IC50 value of TZ4. Nevertheless, the docking simulations suggest that pyrroles condensed with a short, non-bulky aminoacids could potentially be used as AChE inhibitors.
Pharmacologically active drugs acting as acetylcholinesterase (AChE) inhibitors are frequently employed for patients suffering from AD. Drugs such as Donepezil, Rivastigmine and Galanthamine are registered as AChE inhibitors (
The in vitro inhibitory capacity of the top ranked through docking compounds was measured against eeAChE (electric eel acetylcholinesterase) according to the method of Ellman et al. (
The most active pyrrole-based compound was the hydrazide-hydrazone condensed with 2-nitrofuran 12d which inhibited the enzyme with 55% at 10 μM. In comparison, the standard drug Donepezil revealed blocking capacity of 93% at the same concentration. The enhanced AChE inhibitory effects could be related to the nitro moiety (
Subsequently, the major intermolecular interactions between 12d and the active site of 1Q84 were examined (Fig.
The molecular docking simulations showed that 12d is located near the opening of the hydrophobic pocket. Hydrophobic interactions with the active amino residues Tyr286 and π−π bond with Tyr72 (5.25 Å) indicate that the most active pyrrole-base inhibitor was located near the PAS site (above the active site triad and near the gorge entrance) of the protein (
Compound | XP Glide | ChemPLP | MM/GBSA | Amino residues participating in stabilization | In vitro AChE activity |
---|---|---|---|---|---|
12d | -8.73 | 132.68 | -69.29 | Tyr72, Thr75 (H-bond), Trp86, Trp286, Leu289, Ser293, Ile294, Phe297, Tyr337, Phe338, and Tyr341 | 55% inhibition (10 μM concentration) |
The residues Trp86 and Tyr341 were also found to stabilize the active conformation of recently synthesized pyrrole-based AChE inhibitor (
As a final stage of our study, we carried out an in silico ADME analysis to examine the pharmaceutically relevant properties of the most prominent compound in our dataset – 12d. The QikProp module in Maestro 11.8 was employed for the virtual determination of the absorption, distribution, metabolism and excretion (ADME) (Table
Compound | a)MW | b)Donor HB | c)Accept HB | d)QPLog Po/w | e)QPLog BB | f)Percent human oral absorption | g)PSA | h)Rule of five | i)Metab |
---|---|---|---|---|---|---|---|---|---|
12d | 517.35 | 1 | 6 | 4.942 | -2.445 | 77 % | 141.39 | 1 | 3 |
Notably, 12d exerted excellent physicochemical properties related to 95 % of the existing drugs. None of the calculated descriptors felt out of range during the conducted simulations. However, the observed molecular mass of 517.35 violated one of the Lipinski’s Rule of 5. Importantly, the calculated brain/blood partition coefficient (QPlogBB) of the examined compound was in the optimal range of -3.0–1.2, which is essential for potential AChE inhibitors (
In the current study, the utilization of two widely employed docking software programs, namely GOLD 5.3 and Glide, coupled with MM/GBSA recalculations, a set of pyrrole-based scaffolds with potential binding affinity was identified. In vitro evaluations demonstrated a moderate correlation between the results of theoretical predictions and experimental tests. Notably, among the pyrrole-hydrazide-hydrazone tested compounds, the scaffold 12d emerged as the most prominent AChE inhibitor, forming a stable complex with the active site of AChE. In silico ADME investigations using the QikProp module in Maestro revealed that 12d exhibits favorable pharmacokinetic properties. To validate the findings of this study, further in vivo tests could be conducted.
This study was financed by the European Union-Next Generation EU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project No. BG-RRP-2.004-0004-C01.
Docking scores and MM/GBSA recalculation of the applied dataset
Data type: pdf