META-TYROSINE CONJUGATES LABELED 64 CU AND 68 GA AS A CANCER RADIODIAGNOSIS AGENT USING MOLECULAR DOCKING SIMULATION ON LAT-1

Objective: This in silico study aims to determine the most potential compound of meta-tyrosine (JX-075, JX-078, and JX-119) 64 Cu and 68 Ga conjugated with various bifunctional chelating agents, NOTA, DOTA, and NODAGA, against the antiporter site of the LAT1 as conduct to develop a cancer diagnostic compound. Methods: Molecular docking simulation was performed to investigate the interactions between meta-tyrosine compounds and LAT-1. Ligand compounds were drawn in 2D structures using ChemDraw Professional 16.0 and then labeled with 64 Cu and 68 Ga to build a radiopharmaceutical scaffold. The docking process was validated, characterized, and evaluated the interaction using several docking protocols in MOE 2020, a license owned by Gadjah Mada University. A visualization of the protein with the ligand was carried out on the BIOVIA Discovery Studio 2020. Results: Docking simulation results show that JX119 has greater potential due to lower bond energy, JX119_NODAGA_ 68 Ga of-9.22 kcal/mol and JX119_NODAGA_ 64 Cu of-9.09 kcal/mol. This compound showed interactions with transporter amino acid sites Tyr259 and Phe252, both JX-119_NODAGA 68 Ga and JX119_NODAGA_ 64 Cu. Conclusion: The compounds [ 64 Cu]Cu-NODAGA-JX119 and [ 68 Ga]Ga-NODAGA-JX119 are the most potential compounds with the lowest (most negative) Gibbs energy as conduct to develop a diagnostic compound.


INTRODUCTION
Cancer is the abnormal growth of cells that are not controlled, then attacks the body's physiological functions and can spread to other normal organs in the body.Cancer is the second leading cause of death in the world [1].According to data reported by the Global Burden of Cancer (GLOBOCAN), in 2020, cancer cases in the world reached 19,292,789 new cases [2].GLOBOCAN predictions for the next 20 to 30 y, the trend of cancer cases will continue to increase.This presents a challenge to develop an innovation that can improve the quality of life and the quality of treatment for cancer.
The best strategy to decrease cancer morbidity is early identification.Unfortunately, the methods used for cancer detection are currently ineffective, including overdiagnosis, inaccurate, less sensitive, and damage to normal tissues [3].Thus, a renewable detection that can accurately assess the growth of cancerous or precancerous tissue is needed.The currently developing method in cancer detection is "molecular targeted therapy," which detects specific molecules expressed in cancer growth pathways [4].Large Neutral Amino Acid Transporters type-1) LAT-1 can potentially be used as a diagnostic and therapeutic target due to its specific and overexpressing ability on cancer cells [5].LAT-1 is a transporter protein that specifically plays a role in supplying growing tumor cells with essential amino acids and using amino acids as nutrients to build biomass and signaling molecules through activating progrowth pathways [6].
Several LAT-1 inhibitors like BCH (2-amino-2-norbornene carboxylic acid) and JPH 203 had been developed.BCH has disadvantages because it is not specific for LAT-1, has poor affinity, and requires large doses for antitumor effects (>10 mmol), leading to increased toxicity [7,8].A study by Yan et al. (2021) reported types of meta-tyrosine compounds JX-075, JX-078, and JX-119, and examination results on colon cancer cell lines (HT-29) showed that JPH203, as gold standard LAT-1 inhibitor, had an IC50 of 0.06µM while the meta-tyrosine compounds had an IC50 that was not too much different (IC50 0.10-0.25 µM).These showed that the three compounds have an effective IC50 to be used as LAT-1 inhibitors [9].
Based on these findings, meta-tyrosine seemed appealing as a carrier agent for radiopharmaceuticals to target LAT-1.Due to their lower concentrations and greater accuracy for diagnostic reasons, radiopharmaceuticals have additional benefits.Radiopharmaceuticals consist of a radionuclide (radioactive metal) and a carrier substance.Radiopharmaceuticals for diagnostic purposes must be able to emit positron or gamma rays since they have less destructive energy (0.3 keV/m), which is necessary for diagnostic purposes [10].Gamma rays are more secure and, have powerful penetration strength (33-164 mm) and are interpreted by gamma cameras like SPECT (Single-Photon Emission Computed Tomography) and PET (Positron Emission Tomography) [11].The imaging result could reveal details about the location and severity of the malignancy.
Radiopharmaceuticals conducted using the radioactive metals 64 Cu and 68 Ga, which generate gamma rays having energies of 511 KeV (43%) with half-lives of 12.7 h and 890 KeV (90%) 68 min, respectively.However, it is not possible to directly label these metatyrosine molecules with these metals. 64Cu and 68 Ga need a bifunctional chelating agent (BFCA) to bind the metal into the chelator to form a stable complex and enhance its stability.Several chelators have been known to build suitable complexes with 64 Cu and 68 Ga and the complexes have been coupled with biological compounds to form targeting agents [12].Based on this background, research was conducted to develop a diagnostic compound with a carrier agent for meta-tyrosine compounds and to find the best chelator that can compile 64 Cu and 68 Ga metal and interact well with LAT-1 using the in silico method.
The LAT-1 receptor (PDB ID: 7DSQ) included with the native ligand was downloaded from the Protein Data Bank website (www.rcsb.org).The native ligand (3,5-diiodotyrosine) and all small molecules around were removed from the protein, then electronic and stericity parameters were corrected using the 'quick-prep' tool (located on the protein toolbar) in the MOE application 2020.The resulting LAT-1 protein was then stored in.mdb format and continued for method validation and molecular docking simulations [13].

Validation of docking poses
The docking pose was validated using several docking protocols in MOE 2020.The docking protocol with the best Root-Mean-Square Deviation (RMSD) result was chosen to be applied in the molecular docking simulation of ligands.The results of the validation are marked with the RMSD value less than 2 Å [14].

Molecular docking simulation
MOE software version 2020 was used in this study to help visualize, characterize, and evaluate the interaction of the protein with the test ligand.A database is created between ligands and JPH as comparison ligands using MOE 2020 for facilitating the molecular docking process.The database was saved in.mdb file format and continued running a procedure similar to the validation that was carried out.Then ΔG (Gibbs free energy) between the ligand and the receptor will be observed.Molecular docking simulation is accomplished by using the best docking protocol, specifically ligand atom (site), triangle matcher (placement), and induced fit (refinement).The top five docking poses out of 30 attempts were selected for data analysis.Each one of the ligands had docked, and their interactions had been observed.Aiming to observe hydrogen bonds between the ligand and the amino acid on the receptor, a visualization was carried out on the BIOVIA Discovery Studio application 2020 (https://discover.3ds.com/discovery-studio-visualizer-download).The visualization results are stored for later data analysis to determine whether ligands have the potential to be radio diagnostic for cancer [15].

Radiopharmaceutical ligands preparation
The meta-tyrosine compound consisting of JX-075, JX-078, and JX-119 was conjugated with the NOTA and NODAGA chelators using the ChemDraw Professional 16.0 program which resulted in a 2dimensional structure (table 1).An amide bond is formed between the primary amine group of the carrier compound and the carboxylic acid in the chelator.

Validation of docking poses
The LAT-1 protein was prepared by protonating and correcting the amino acid sequence according to the sequence listed on PDB.The LAT-1 protein and its native ligand (3,5diiodotyrosine) were redocked using several molecular docking protocols to obtain the protocol with the best RMSD results (table 2).

Docking result
Based on the molecular docking simulation process, Gibbs energy data was obtained, which illustrates the energy required for the interaction between the test ligand and LAT-1 protein as the target protein.The more negative the Gibbs energy value obtained, the interaction that occurs between the test ligand and the LAT-1 protein is more spontaneous and easier to occur [16] In addition, interactions of amino acids in the LAT-1 protein with functional groups in the ligands were also obtained.The results of the molecular docking simulation can be seen in table 3.
Based on the conjugation with the active metal of the JX119 compound, the molecular docking simulation results show that JX119 has greater potential due to lower bond energy, [ 68 Ga]Ga-NODAGA-JX119 of-9.22 kcal/mol and [ 64 Cu]Cu-NODAGA-JX119 of-9.09kcal/mol.When compared to the three chelators, both 64 Cu and 68 Ga metals, the compound with the NODAGA chelator showed the best potential with the lowest bond energy, while the compound with the NOTA chelator had the worst bond energy.Compounds [ 68 Ga]Ga-NODAGA-JX119 and [ 64 Cu]Cu-NODAGA-JX119 can bind to Tyr259 and Phe252 residues through Van Der Walls bonds (fig.2).

DISCUSSION
The target molecule and the novel therapeutic compound may interact, and the molecular docking simulation technique will be able to discover and predict these interactions.Molecular docking simulation can aid in the more effective and efficient screening of many compounds during the research and development of new medicine.
JX-075, JX-078, and JX-119 are new compounds derived from bicyclic meta-tyrosine developed as LAT-1 inhibitors [9].In the meta-tyrosine compound, the acidic part of JX will bind to the binding site in the unwound region of LAT-1 (TM1 and TM6), while the hydrophobic tail will bind to the TM10 region with detailed LAT-1 regions explained in Yan et al., 2021 [8] and Singh et al., 2018 [9].This causes the prevention of the cycle of transport of amino acids into the cell.The JX inhibitor binds to LAT-1 in the same way as BCH through the mechanism of the oxygen atom on the carboxyl and ammonium groups which form hydrogen bonds with the main chain atoms in the TM1 and TM6 regions.The interaction of JX compounds with LAT-1 can occur through the bonds of key amino acids such as Tyr289, Phe252, Val396 or Tyr117, Trp257, Asn258, Tyr259, and Arg348 [17].
The crystal structure of the protein target (fig.3), LAT-1, was downloaded from the Protein Data Bank (PDB ID: 7DSQ) and prepared with MOE 2020 [13].LAT-1 was prepared by removing all small molecules and water molecules around the protein molecule, except for the native ligand molecule, 3,5-diiodotyrosine [18].Target proteins are also prepared by balancing polar hydrogens and protein charges to increase the susceptibility of the protein to the electronegative atoms of the ligands.Minimization of energy in the target protein is carried out to stabilize the protein so that molecular docking is achieved in a stable protein phase [19].[20].Molecular docking simulation using MOE aims to find a favorable bond configuration between the ligand and the target protein.The process is carried out in a 3D docking box whose size depends on the selected atoms.The docking process begins with a random conformation of a predetermined conformation.In this study, the docking process was carried out using the conformation that had been determined at the pose validation step.The protocol that gives the lowest RMSD value in the pose validation stage is used for the molecular docking step [21].
In MOE, the receptor-ligand interaction affinities of all possible binding geometries are prioritized based on a numerical value called the S-score or it can be represented as a receptor-ligand binding energy value commonly interpreted as Gibbs energy [20].Gibbs energy is obtained by calculating using the built-in potential function or grid-based potential field.The Gibbs energy calculated using the grid-based potential field method was calculated using electrostatic and van der Waals potential fields sampled on the grid lining the docking box.The grid-based method calculates the grid potential energy only at the start of the docking calculation [21,22].This method is faster than the built-in potential energy function.
Table 3 shows the results of the molecular docking simulation of the 18 test compounds.The JX119 compound conjugated with the NODAGA chelator has the most negative Gibbs free energy.Based on in vitro research by Yan et al. (2021), the JX078 compound is the compound with the smallest IC50 (121 nM) when compared to JX075 and JX119 with IC50 165nM and 234nM respectively [9].Because IC50 correlates with bond energy, the smaller the free energy of the Gibbs, the greater the potential of IC50 [23].
Chelators play a role in bond stability and influence pharmacokinetics.The NODAGA chelator is a derived macrocyclic chelator used to chelate 64 Cu and 68 Ga.NODAGA can provide thermodynamic stability and has a suitable cavity size. 64Cu has a tendency for high pharmacokinetic stability when conjugated to NODAGA.The NODAGA bond also forms a neutral bond with 68 Ga through conjugation with 3 carboxylates.Neutral conjugation causes lower hepatic uptake and better contrast against cancer cells [24].

CONCLUSION
Based on the results of molecular docking using MOE software, it was found that the compounds [ 64 Cu]Cu-NODAGA-JX119 (S =-9.09 kcal/mol) and [ 68 Ga]Ga-NODAGA-JX119 (S =-9.22 kcal/mol) are the compounds with the lowest (most negative) Gibbs energy.The more negative the Gibbs energy produced, the better and more spontaneous the interaction that can occur between the ligand compound and the target protein.Therefore, [ 68 Ga]Ga-NODAGA-JX119 and [ 64 Cu]Cu-NODAGA-JX119 are assumed to be able to interact better with LAT-1 protein when compared to other compounds.In addition, [ 68 Ga]Ga-NODAGA-JX119 interacts with three of the six key amino acids in the LAT-1 protein, namely the amino acids Phe252; Tyr259.Meanwhile, [ 64 Cu]Cu-NODAGA-JX119 interacts with two of the six key amino acids in the LAT-1 protein, namely the amino acids Phe252; Tyr259.