Molecular modeling piloted analysis for semicarbazone derivative of curcumin as a potent Abl-kinase inhibitor targeting colon cancer

The human Abl kinases comprise a family of proteins that are known to be key stimulus drivers in the signaling pathways modulating cell growth, cell survival, cell adhesion, and apoptosis. Recent collative studies have indicated the role of activation of Abl and Abl-related genes in solid tumors; further terming the Abl kinases as molecular switches which promote proliferation, tumorigenesis, and metastasis. The up-regulated Abl-kinase expression in colorectal cancer (CRC) and the role of Abl tyrosine kinase activity in the Matrigel invasion of CRC cells have cemented its significance in CRC advancement. Therefore, the requisite of identifying small molecules which serve as Abl selective inhibitors and designing anti-Abl therapies, particularly for CRC tumors, has driven this study. Curcumin has been touted as an effective inhibitor of cancer cells; however, it is limited by its physicochemical inadequacies. Hence, we have studied the behavior of heterocyclic derivatives of curcumin via computational tools such as pharmacophore-based virtual screening, molecular docking, free-energy binding, and ADME profiling. The most actively docked molecule, 3,5-bis(4-hydroxy-3-methylstyryl)-1H-pyrazole-1-carboxamide, was comparatively evaluated against Curcumin via molecular dynamics simulation using Desmond, Schrödinger. The study exhibited the improved stability of the derivative as compared to Curcumin in the tested protein pocket and displayed the interaction bonds with the contacted key amino acids. To further establish the claim, the derivatives were synthesized via the mechanism of cyclization of Curcumin and screened in vitro using SRB assay against human CRC cell line, HCT 116. The active derivative indicated an IC50 value of 5.85 µM, which was sevenfold lower as compared to Curcumin’s IC50 of 35.40 µM. Hence, the results base the potential role of the curcumin derivative in modulating Abl-kinase activity and in turn may have potential therapeutic value as a lead for CRC therapy. Supplementary Information The online version contains supplementary material available at 10.1007/s13205-021-03051-9.


Protein-Ligand RMSD
The Root Mean Square Deviation (RMSD) is used to measure the average change in displacement of a selection of atoms for a particular frame with respect to a reference frame. It is calculated for all frames in the trajectory. The RMSD for frame x is: where N is the number of atoms in the atom selection; t ref is the reference time, (typically the first frame is used as the reference and it is regarded as time t=0); and r' is the position of the selected atoms in frame x after superimposing on the reference frame, where frame x is recorded at time t x . The procedure is repeated for every frame in the simulation trajectory.
Protein RMSD: The above plot shows the RMSD evolution of a protein (left Y-axis). All protein frames are first aligned on the reference frame backbone, and then the RMSD is calculated based on the atom selection. Monitoring the RMSD of the protein can give insights into its structural conformation throughout the simulation. RMSD analysis can indicate if the simulation has equilibrated -its fluctuations towards the end of the simulation are around some thermal average structure. Changes of the order of 1-3 Å are perfectly acceptable for small, globular proteins. Changes much larger than that, however, indicate that the protein is undergoing a large conformational change during the simulation. It is also important that your simulation converges -the RMSD values stabilize around a fixed value. If the RMSD of the protein is still increasing or decreasing on average at the end of the simulation, then your system has not equilibrated, and your simulation may not be long enough for rigorous analysis.
Ligand RMSD: Ligand RMSD (right Y-axis) indicates how stable the ligand is with respect to the protein and its binding pocket. In the above plot, 'Lig fit Prot' shows the RMSD of a ligand when the protein-ligand complex is first aligned on the protein backbone of the reference and then the RMSD of the ligand heavy atoms is measured. If the values observed are significantly larger than the RMSD of the protein, then it is likely that the ligand has diffused away from its initial binding site.

Protein RMSF
The Root Mean Square Fluctuation (RMSF) is useful for characterizing local changes along the protein chain. The RMSF for residue i is: where T is the trajectory time over which the RMSF is calculated, t ref is the reference time, r i is the position of residue i; r' is the position of atoms in residue i after superposition on the reference, and the angle brackets indicate that the average of the square distance is taken over the selection of atoms in the residue.
On this plot, peaks indicate areas of the protein that fluctuate the most during the simulation. Typically you will observe that the tails (N-and C-terminal) fluctuate more than any other part of the protein. Secondary structure elements like alpha helices and beta strands are usually more rigid than the unstructured part of the protein, and thus fluctuate less than the loop regions. Protein secondary structure elements (SSE) like alpha-helices and beta-strands are monitored throughout the simulation. The plot above reports SSE distribution by residue index throughout the protein structure. The plot below summarizes the SSE composition for each trajectory frame over the course of the simulation, and the plot at the bottom monitors each residue and its SSE assignment over time.

Ligand RMSF
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Protein-Ligand Contacts
Protein interactions with the ligand can be monitored throughout the simulation. These interactions can be categorized by type and summarized, as shown in the plot above. Protein-ligand interactions (or 'contacts') are categorized into four types: Hydrogen Bonds, Hydrophobic, Ionic and Water Bridges. Each interaction type contains more specific subtypes, which can be explored through the 'Simulation Interactions Diagram' panel. The stacked bar charts are normalized over the course of the trajectory: for example, a value of 0.7 suggests that 70% of the simulation time the specific interaction is maintained. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with the ligand.
Hydrogen Bonds: (H-bonds) play a significant role in ligand binding. Consideration of hydrogen-bonding properties in drug design is important because of their strong influence on drug specificity, metabolization and adsorption. Hydrogen bonds between a protein and a ligand can be further broken down into four subtypes: backbone acceptor; backbone donor; side-chain acceptor; side-chain donor. The current geometric criteria for protein-ligand H-bond is: distance of 2.5 Å between the donor and acceptor atoms (D-H···A); a donor angle of ≥120° between the donor-hydrogen-acceptor atoms (D-H···A); and an acceptor angle of ≥90° between the hydrogen-acceptor-bonded_atom atoms (H···A-X).
Hydrophobic contacts: fall into three subtypes: π-Cation; π-π; and Other, non-specific interactions. Generally these type of interactions involve a hydrophobic amino acid and an aromatic or aliphatic group on the ligand, but we have extended this category to also include π-Cation interactions. The current geometric criteria for hydrophobic interactions is as follows: π-Cation -Aromatic and charged groups within 4.5 Å; π-π -Two aromatic groups stacked face-to-face or face-to-edge; Other -A non-specific hydrophobic sidechain within 3.6 Å of a ligand's aromatic or aliphatic carbons.
Ionic interactions: or polar interactions, are between two oppositely charged atoms that are within 3.7 Å of each other and do not involve a hydrogen bond. We also monitor Protein-Metal-Ligand interactions, which are defined by a metal ion coordinated within 3.4 Å of protein's and ligand's heavy atoms (except carbon). All ionic interactions are broken down into two subtypes: those mediated by a protein backbone or side chains.
Water Bridges: are hydrogen-bonded protein-ligand interactions mediated by a water molecule. The hydrogen-bond geometry is slightly relaxed from the standard H-bond definition. The current geometric criteria for a protein-water or water-ligand H-bond are: a distance of 2.8 Å between the donor and acceptor atoms (D-H···A); a donor angle of ≥110° between the donor-hydrogen-acceptor atoms (D-H···A); and an acceptor angle of ≥90° between the hydrogen-acceptor-bonded_atom atoms (H···A-X).

Protein-Ligand Contacts (cont.)
A timeline representation of the interactions and contacts (H-bonds, Hydrophobic, Ionic, Water bridges) summarized in the previous page. The top panel shows the total number of specific contacts the protein makes with the ligand over the course of the trajectory. The bottom panel shows which residues interact with the ligand in each trajectory frame. Some residues make more than one specific contact with the ligand, which is represented by a darker shade of orange, according to the scale to the right of the plot.

Ligand Torsion Profile
The ligand torsions plot summarizes the conformational evolution of every rotatable bond (RB) in the ligand throughout the simulation trajectory ( 0.00 through 50.05 nsec). The top panel shows the 2d schematic of a ligand with color-coded rotatable bonds. Each rotatable bond torsion is accompanied by a dial plot and bar plots of the same color.
Dial (or radial) plots describe the conformation of the torsion throughout the course of the simulation. The beginning of the simulation is in the center of the radial plot and the time evolution is plotted radially outwards.
The bar plots summarize the data on the dial plots, by showing the probability density of the torsion. If torsional potential information is available, the plot also shows the potential of the rotatable bond (by summing the potential of the related torsions). The values of the potential are on the left Y-axis of the chart, and are expressed in kcal/mol. Looking at the histogram and torsion potential relationships may give insights into the conformational strain the ligand undergoes to maintain a protein-bound conformation.