A de novo antineoplastic drug design to suppress head, neck, and oral cancer

Abstract

An innovative antineoplastic drug / molecule has been specifically designed to target and regulate head, neck, and oral cancer. The designed molecule inhibits the activity of the KRTAP2-3 protein while simultaneously binding to and regulating TGF-β, thereby slowing down and halting the metastasis process.

Description

[0001] A DE NOVO ANTINEOPLASTIC DRUG DESIGN TO SUPPRESS HEAD, NECK,

[0002] AND ORAL CANCER

[0003] Technical Field of the Invention

[0004] The present invention relates to a new molecule formulation that can regioselectively choose specific proteins to suppress and halt the metastasis and growth of head, neck, and oral cancer.

[0005] State of the Art of the Invention (Prior Art)

[0006] The potential method for reducing the metastasis of head, neck, and oral cancer involves suppressing Transforming Growth Factor p (TGF-P), as supported by extensive scientific data and literature. However, it's important to note that while inhibiting TGF-P leads to a rapid reduction in metastasis, it also stimulates cell proliferation due to TGF-P's role in promoting high motility and metastatic potential in cancer cells.

[0007] Notably, scientific literature suggests that the expression of Keratin-associated protein 2-3 (KRTAP2-3) is linked to increased motility in cancer cells, as it serves as a regulator and mediator of TGF-P's cancer-promoting effect. Consequently, migrated cancerous cells tend to overexpress TGF-P induced KRTAP2-3, and suppressing this protein could potentially slow down or halt the metastasis process.

[0008] Brief Description and Objects of the Invention

[0009] The main purpose of this invention is to create a novel molecule acting as a drug, aiming to address the problem effectively. The main objective of this designed molecule is to comodulate both Transforming Growth Factor P (TGF-P) and Keratin-Associated Protein 2-3 (KRTAP2-3) through in silico studies, thereby reducing the activity of TGF-P and suppressing the expression of KRTAP2-3 since they actively play role in the proliferation mechanism of head, neck, oral cancer. By achieving this dual modulation, the drug aims to provide a promising solution to combat head, neck, and oral cancer effectively.

[0010] The aim here is to use comprehensive computational simulation tools to effectively demonstrate how the drug interacts with the functional groups of TGF-P and KRTAP2-3, resulting in the formation of a triple complex that can regulate TGF-P and inhibit KRTAP2-3. The advancement of computational chemistry has enabled the investigation of bonding affinities through techniques like molecular docking, molecular dynamics (MD), and post- molecular dynamics hydrogen bonding contact analyses. These methods play crucial roles in accurately calculating and predicting the binding affinity and mode of interaction, as evidenced in scientific literature. When combined, these tools provide a 100% confirmation that aligns with in vitro experimental results.

[0011] The remarkable precision and accuracy of computational simulation tools once again enable to investigate how a newly designed drug interacts with TGF-P through hydrogen bonding. It is essential not to completely suppress TGF-P, as doing so could promote cancer cell proliferation while halting metastasis and migration. Instead, by using a drug that diminishes TGF-P's activity through hydrogen bonding rather than strong covalent bonding, reversible competitive inhibition can occur. This triple complex of "de novo drug (designed molecule) - TGF-P - KRTAP2-3" reduces metastasis and significantly inhibits cancerous cell migration. Based on this, the computed and simulated drug exhibits previously unknown mechanisms, making it a promising new antineoplastic drug for further exploration in in vitro, in vivo, and human phase studies. This invention offers hope for a brighter future in eradicating head, neck, and oral cancer.

[0012] Definitions of Figures Describing the Invention

[0013] The figures and related descriptions used to better explain the molecule designed by this invention are as follows.

[0014] Figure 1: The chemical structure of the de novo designed drug

[0015] Figure 2: The designed biochemical structure of designed molecule at pH 5.0 medium for cancerous cell cytoplasm.

[0016] Figure 3: The 3-D structure of designed molecule bound to TGF-P and KRTAP2-3 for the suppression at pH 5.0 medium simulating cancerous cell cytoplasm.

[0017] Figure 4: The Root Mean Square Deviation plots of all atoms of designed drug-TGF-P- KRTAP2-3 complex in lighter gray and non-ligand bound KRTAP2-3 in black on top respectively. Detailed Description of the Invention

[0018] The invention discloses a designed molecule is to co-modulate both Transforming Growth Factor p (TGF-P) and Keratin-Associated Protein 2-3 (KRTAP2-3), thereby reducing the activity of TGF-P and suppressing the expression of KRTAP2-3 since they actively play role in the proliferation mechanism of head, neck, oral cancer.

[0019] This designed molecule that having de novo antineoplastic medication potential has been specifically designed to target and inhibit head, neck, and oral cancer. Through the utilization of computational software tools, such as molecular docking, molecular dynamics (MD), and post-molecular dynamics hydrogen bond contact analyses, it has been demonstrated that this newly developed drug, exhibits high efficacy in suppressing and co-modulating the activities of TGF-P and KRTAP2-3 simultaneously. The drug works by inhibiting the activity of the KRTAP2-3 protein while also binding to TGF-P and adjusting its function to slow down and ultimately prevent metastasis.

[0020] This innovative design has the potential to demonstrate promising results in laboratory experiments in vitro), live organism studies in vivo), and human trials (human phase stages). The drugs' remarkable effectiveness lies in the ability to simultaneously modulate TGF-P and KRTAP2-3, effectively halting the cancer's metastasis and suppressing its growth.

[0021] To achieve the aforementioned stated objective, it was crucial to comprehend the affinities of functional groups in pharmaceutical chemistry, their effects on various amino acids, and their characteristics regarding bioactivity. The efficiency of these interactions appears to be influenced by various factors, including the drug's external groups' attraction to proteins and the arrangement of the binding process.

[0022] This comprehensive explanation outlines the preferred embodiments of the innovative molecule formulation using illustrative results solely to provide clarity on the subject matter. Design Steps, Methods and Procedures

[0023] Geometric Optimization

[0024] To identify the active sites of a molecule and explore its interactions with receptors, it is crucial to accurately determine its optimal geometric structure. In the present invention, a ligand has been designed to inhibit the TGF-p - KRTAP2-3, drawing upon the extensive pharmaceutical chemistry expertise of our research group. The resulting organic chemical structures, in their most stable molecular geometries, were subjected to analysis using the Gaussian 09 program with density functional theory (DFT) / B3LYP functional and utilizing the 6-31G (d,p) principle. This process led to the formation of the most stable molecular structures of the designed molecule - TGF-P - KRTAP2 - 3 intended for further computational and simulation-based research. For molecular docking and molecular dynamics computations, as well as post-processing of output files, Gauss View 6.0 and Avogadro 1.95 software programs were employed to prepare input files.

[0025] Molecular Docking Procedure

[0026] The molecular docking simulations were carried out using AutoDock Vina 1.1.2 and PyRx 0.8 software programs. These programs are widely recognized for their high precision and accuracy in biochemical docking simulations. A total of 1000 poses were generated, with 100 poses for each simulation. The simulations involved the newly designed drug and its interaction with the receptor structure of TGF-P, which had been optimized and designed as an organic molecule. Additionally, the KRTAP2-3 protein, derived from the expression of the NCBI gene 100288323, was also studied.

[0027] The optimization of the drug and protein structures was performed using Gauss View 6.0 and Avogadro 1.95 software. The simulations demonstrated the interactions and binding of the drug to the receptor. The docking scores obtained in kcal / mol represented the Gibbs free binding energy.

[0028] From all the simulations, the docking pose with the most accurate and favorable binding energy, identified within the best-clustered data, was selected as the initial structure and input file for the subsequent molecular dynamics (MD) simulations. Each MD simulation was conducted with different seed numbers. Molecular Dynamics (MD) Simulations

[0029] The initial structures for the MD simulations were selected from the docking poses with the most favorable binding energy. Schrodinger’s Maestro Desmond Program was utilized for running the molecular dynamics (MD) simulations, each spanning 50 ns with 5000 poses at 10 ps intervals. To ensure accuracy, each MD simulation was repeated three times with different seed numbers, confirming the correctness of the simulation parameters and the structures of the complexes formed by the designed molecule (ligand) with TGF-P and KRTAP2-3 (receptors).

[0030] During the MD simulations, the dynamic properties of the drug-receptor complexes were assessed over time. The simulation area was defined by a grid box measuring 110 x 110 x 110 A3with a spacing of 0.5 , offering wider simulation area coverage. TIP3P-type water molecules were included within the box, and 0.15 M NaCl ions were added to neutralize the system.

[0031] The temperature and pressure conditions were set as follows: NPT at 310 K with Nose- Hoover temperature coupling and a constant pressure of 1.01 bar via Martyna Tobias-Klein pressure coupling. The systems were not constrained, and the default fitting for OPLS 3.0 standards provided the initial velocity values for the forcefield calculations.

[0032] In Figure 1, Figure 2 and below, the organic chemical structure of the designed drug is given. The chemical structure of de novo designed molecule should be patented before further pre- clinical and clinical studies come into reality. Figures 1 and 2, display the clear organic chemical structure of the designed drug that targets the cancerous cell at the medium of pH 5.0. To demonstrate the theoretical stability and A(AG) binding energies of designed molecule when attached to TGF-P and KRTAP2-3, simulations and computations were performed.

[0033] Table 1. The Computed Bioactivity Scores and Physical Properties of The Designed Molecule

[0034] As can be seen in Table 1, it should be known that the bioactivity scores for organic molecules can be interpreted as active (when the bioactivity score > 0), moderately active (when the bioactivity score lies between -5.0 and 0.0), and inactive (when the bioactivity score < -5.0). The molecule of interest that we designed seems to be active for most receptors and can be assumed as a bioactive molecule.

[0035] Once the triple complex (receptor-ligand-receptor) reaches equilibrium and stabilization, which takes approximately 15 - 20 nanoseconds (around the 2000th frame out of 5000) as shown in Figure 4, designed molecule successfully forms a stable complex with both TGF- P and KRTAP2-3. The molecular dynamics results obtained from Schrodinger's Desmond, along with cluster analyses of 100 posed trials from molecular docking studies in Autodock Vina, indicate the formation of strong hydrogen bonds between designed molecule and TGF-P - KRTAP2-3. The docking energies obtained for these interactions are approximately -11.2 kcal / mol.

[0036] Figure 4 illustrates the investigation of the dynamic properties of designed molecule and the PolyCystein active site of the KRTAP2-3 protein, along with TGF-P, using the post-MD analysis technique called RMSD. The post-MD simulation trajectories reveal that the KRTAP2-3 protein without any ligand bound (shown in black) reached equilibrium smoothly. On the other hand, the "TGF - P-KRTAP2-3 - the designed molecule" triple complex (in gray) demonstrated better stability towards the end of the 50 nanoseconds MD simulation. This observation indicates that the MD run occurred and concluded without any stability issues, and the complex exhibits greater stability compared to the KRTAP2-3 protein alone proving the drugs’ potential and capability for use.

[0037] Table 2. The Post-Molecular Dynamics hydrogen bond contact analyses of designed molecule

[0038] Protein % of H-Bonds Drug- Binding Energy H-Bond Distance domain Aminoacid (kcal / mol) (A)

[0039] Proline 12.1 -8.4 1.94

[0040] Cysteine 51.3 -11.2 1.72

[0041] Arginine 16.1 -10.3 2.11

[0042] The Rest 20.5

[0043] In Table 2, following the analysis of MD's 3D motion trajectory, an extensive investigation was conducted to determine all potential atomic interactions between the functional groups on drugs and the amino acids. This intricate study involved examining all hydrogen bonds among thousands of atoms from the trajectory files' matrix data of the MD study. Consequently, a post-MD Hydrogen contact mapping analysis was performed, manually matching matrix data for each atom and employing a machine learning pre-coded script to identify the regioselectivity and likelihood of functional groups on drugs binding to the amino acids of the KRTAP2-3 protein. The results revealed that Cysteine exhibited significant regioselectivity (approximately 50%) in interactions with the drug. The drug positions itself within the active site groove of the KRTAP2-3 protein, forming robust hydrogen interactions (at a distance of 1.72 A). Consequently, designed molecule effectively inhibits the activity of the KRTAP2-3 protein while simultaneously binding to and regulating TGF-P, hence suppressing the cancer and its metastasis.

Claims

CLAIMS1. A computational designed molecule capable of co-modulating and inhibiting the activities of Transforming Growth Factor p (TGF-P) and Keratin-Associated Protein 2-3 (KRTAP2-3), the molecule having the formula:

2. Use of the computational designed molecule according to Claim 1 as an inhibitor of the TGF-P receptor.

3. Use of the computational designed molecule according to Claim 1 as a suppressor of the TGF-P receptor.

4. Use of the computational designed molecule according to Claim 1 as a modulator of the TGF-P receptor.

5. Use of the computational designed molecule according to Claim 1 as an inhibitor of the KRTAP2-3 expression.

6. Use of the computational designed molecule according to Claim 1 as a suppressor of the KRTAP2-3 expression.

7. Use of the computational designed molecule according to Claim 1 as a modulator of the KRTAP2-3 expression.

8. Use of the computational designed molecule according to Claim 1 as a medicament.

9. Use of the computational designed molecule according to Claim 1 as an antineoplastic drug.

10. A computational designed molecule according to Claim 1 for use in the treatment of head cancer.

11. A computational designed molecule according to Claim 1 for use in the treatment of neck cancer.

12. A computational designed molecule according to Claim 1 for use in the treatment of oral cancer.

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