Bioisosteric replacements of tyrosine kinases inhibitors to make potent and safe chemotherapy against malignant cells

Abstract The liver function test is an imperative element in chemotherapy management due to the idiosyncratic reaction of chemotherapy drugs. This study primly aimed to replace the toxic fragments of known protein tyrosine kinases inhibitors (PTKi) to develop safe and effective chemotherapy. All the current PTKi’s were docked with the tyrosine kinases and metabolic enzymes to study the affinities on the target. It resulted from most of the PTKi’s found higher affinity and efficacy with metabolic enzymes lead the hepatic cells damage. To overcome this limitation of PTKi’s, a bioisosteric replacement strategy was achieved and conceptual analogs were designed. Specifically, the Generated pose of the Axitinib molecule showed that axitinib fragments C = C-, -C = O and NH2 produced clashes with active site residues of tyrosine kinases protein and good affinity with metabolic enzyme primes to the liver toxicity. The above said fragments were replaced with various bioisosteric groups and efficacy was measured. The resulting molecule shows improved affinity with tyrosine kinases enzyme and less interactions with metabolic enzyme were imminent molecule for the treatment of malignant cells with outside effects. Communicated by Ramaswamy H. Sarma


Introduction
Malignance is the 2nd prominent cause of life loss universally and is accountable for the value of 9.6 million Jules loss in 2018 (The International Agency for Research on Cancer (IARC) report W. Latest global cancer data: Cancer burden rises to 18.1 million new cases and 9.6 million cancer deaths in 2018, 2018).Among the 6 deaths, about 1 is due to various types of tumor growth specifically, in low-and middleincome countries contribute 70%.World Health Organization (WHO) recently released a report which showed that the universal cancer encumbrance is climbed to 18.1 million firsthand cases and India's share in it will be a worrying 8.17% (Cooper & Hausman, 2000).Chemotherapy is radical in treating and management of malignant cells and is currently a powerful medication used to kill cancer cells.In the process of cell generation, protein tyrosine kinases (PTK) are shown a significant function to displace the phosphate assemblies on ATP to the tyrosine moiety of various key proteins, make proteins phosphorylation, transfer cell signals to regulate cell growth, differentiation, a series of physiological and biochemical processes and death (Dhillon et al., 2018;Wang & Cole, 2014).Atypical expression of PTK will prime to cell proliferation, regulation disorders and tumorigenesis (Drake et al., 2014).Besides, PTK unusual manifestation is related to tumor neovascularization, tumor incursion and metastasis, and tumor chemotherapy resistance.Hence the aim of PTK is a promising target for a searing spot of anti-tumor drug research.
An intracellular juxtamembrane (JM) region, a tyrosine kinases domain, a large extracellular area, a single-spanning transmembrane (TM) domain, and a C-terminal regulatory region make up the PTK family protein.Members of the EGFR family have two homologous ligand-binding domains (domains I and III) and two cystine-rich domains in their extracellular regions (domains II and IV).Only the insulin receptor (IR) family of RTKs shares the identical domain I/II/III organization with extracellular domains; however, the membrane-proximal cysteine-rich domain IV of ErbB receptors is substituted by fibronectin type III domains in the IR family.Most other RTKs have extracellular portions made up of immunoglobulin or fibronectin type III domains, in contrast to the EGFR and IR families.The IR and ErbB families differ from other RTKs in their modes of ligand activation just as they do in the composition of their domains (Kn€ osel et al., 2014).
The monooxygenases enzyme known as cytochrome P450 proteins catalyzes numerous drug metabolic processes as well as the formation of biomolecules such as lipids, steroids, and other cholesterol.The PTKi's bind inside the cavity of this enzyme and degrade the regular functionality.Which leads the hepatic cell necrosis and many other side effects.More than 50% of medications are metabolized by CYP3A4.At birth, CYP3A4 activity is nonexistent; however, by the age of one year, it reaches adult levels.The highest CYP3A4 activity is seen in the liver and small intestine (Thigpen et al., 2019).
The present treatment with PTKi's plays a significant role in cancer management/treatment and it's unbearable to avoid causing some harm to other cells and tissues in the body.Public Health England and Cancer Research UK studies found that 8.4% (lung cancer) and 2.4% (breast cancer) died within a month after being treated with PTK (Knapton, 2016).The drug is used for cancer treatment or management, alone or in combinations possible to lead to direct hepatic toxicity or hypersensitivity retorts and altered liver function (Breast Cancer: Types of Treatment, 2019).Hence, this work aimed to identify the toxicity-producing fragment in the known tyrosine kinases inhibitors and the biotransformation of those toxic fragments was identified using the computational methodology.It leads to a better, safer and more effective drug without side effects.BIOVIA Discovery Studio (2017) was used to apply the force field and minimize the macro and micro molecules.BIOSOLVE IT software such as SeeSAR (9.2) and Star Drop modules were used for multiparameter optimization to maximize the likelihood of success, rather than affinity alone and pharmacokinetic-based parameter drug design (BioSolveIT GmbH, 2019; SeeSAR version 9.2).

PTKi's to breed new molecules
First, 23 PTKi's were downloaded from the PubChem database and the Pharmacodynamic/Kinetic properties details of inhibitors were calculated using the SWISSADME online module (Daina et al., 2017;Kim et al., 2019).All the molecules were drawn in ChemDraw and saved as a structural data format (Table 1).Those drugs were subjected to the potential energy calculation using the CHARMM Force field protocol and minimized the energy of the molecule by a smart minimizer algorithm of 2000 steps and an RMS cutoff of 0.01 (Balasubramaniyan et al., 2018;Puratchikody et al., 2019).

Proteins, binding site definition, and selection
Crystal structure of 3 D PTK (PDB ID: 5YU9) and human metabolic enzyme CYP3A4 (PDB ID: 6BCZ) are collected from the RCSB database with a resolution of 1.95 Å and 2.23 Å, respectively (Samuels & Sevrioukova, 2018;Wang et al., 2016).PTK was loaded in SeeSAR protein mode and defined the PDB ligand in protein.The binding site mode identifies the basis of unoccupied pockets of proteins including the binding site around a molecule.The residues in the PDB ligand bind site appeared in pink color and confirm the docking pocket.The coordinates of binding the bioisosteric molecule were fixed at À 3.367030, À 26.397727, and 52.148727.

Docking mode
PTK (PDB ID: 5YU9) was transmitted to the docking mode of the SeeSAR module and the ligands were loaded in the pocket.The number of poses generation was fixed at 200 conformations and the docking protocol was executed.The final resulting complex of protein and drug molecule affinity were calculated for the HYDE algorithm.Also, the inter/intramolecular clashes, torsion energy and multiple optimum properties of the molecules were measured (Nagendran et al., 2022;Navabshan et al., 2021;Puratchikody et al., 2019).Similarly, all the docking protocol was performed with PTKi's and cytochrome P450 enzyme.The energy of docked complex was calculated using the following formula

Bioisosteric replacement of toxic fragments in PTKi's
After the interpretation of clashes and interactions between the PTK protein and metabolic protein clearly, describe the fragments responsible for the hepatotoxicity of the known inhibitors.These fragment molecules are transferred to the inspiratory mode and the replacing core and linkers will be  analyzed from the various database.Multiple breed molecules will be generated and all properties were calculated (Blay et al., 2020).The final molecule which shows better affinity and better pharmacodynamics/kinetics properties will be submitted for the synthesis and biological evaluation studies.

Preparation of PTKi's and proteins (PTK &CYP450)
The energy of all 23 PTKi's was shown in Table 2.The energy was brought up to the local minimum range of À 72.4598 to 57.2442 kcal/mol.Two proteins were collected and cleaned to remove the alternative conformation and incomplete amino acids.The report of cleaned protein was enclosed in supplementary 1 for PTK and CYP450 proteins.The energy of the protein was minimized to the local minima of À 18797.07475and À 32048.61316kcal/mol for 5YU9, and 6BCZ, respectively (Table 2).The binding pockets of both the proteins were identified and analyzed for the binding interaction studies of PTKi's (Figure 1).The binding site of PTK contains 41 active site amino acids (Figure 1(a)) and the 6BCZ binding site possesses 30 amino acids which are significant for the binding of drug molecule (Figure 1(b)).
The pharmacodynamic properties of all PTKi's were studied and analyzed for their metabolic enzyme degradation properties as well as the violations of rules (Table 3).
After the definition of the binding site, all the PTKi are docked using the FlexX software to identify the binding affinity of the molecules with tyrosine kinases and CYP450.The results of docking showed that the PTKi's made less affinity with the tyrosine kinases protein (Figure 2) and more affinity with the metabolic enzyme (Figure 3) (Ali et al., 2011).This proved the reason behind the hepatotoxicity of the known drugs.Based on these results, the toxicityproducing fragments of known drug molecules were replaced with suitable bioisosteres from the library using the StarDrop module.
Further, Figure 4 shows the binding affinity of axitinib with tyrosine kinase proteins.N 6 atom of Axitinib formed one hydrogen bond interaction with CYS 797 and other carbon atoms forming the electrostatic interaction with the active site amino acids of PTK.It favors the binding of axitinib inside the cavity of protein but the atoms N 3 and N 4 developed a negative environment inside the cavity.Also, the O 2 generates strong repulsive force with THR 854 and ASP 855 which spawns the improper fit inside the active space.Further irregular torsion angles of C 16 -C 21 , N 5 -C 21 > and S 1 -C 11 not allow the conformational changes of the molecule to fit inside the binding pocket of tyrosine kinases protein (Morando et al., 2016).Overall, this effect leads to less antagonistic activity against the PTK, which causes less activity against preventing cancer cells.
Figure 5 revealed the affinity and clashes of axitinib with the active site amino acids of a metabolic enzyme (6BCZ) and proved the higher affinity with the metabolic enzyme.Which is the reason behind the liver toxic properties of most  Only the two Nitrogen's such as N 3 and N 6 developed unfavor binding, which is not forceful as the above affinities of other atoms.These describe the cause of hepatotoxicity of axitinib drug administration to cancer patients.Similarly, other PTKi's are studied and interaction and clashes generating fragments are identified and subjected to replacement studies.

Bioisosteric replacements and affinity studies of designed axitinib analog
Toxicity-producing fragments or the fragment reducing the binding with the PTK were replaced using suitable bioisosteres from the libraries using chemistry transformation knowledge.The SMARTS pattern compound filters are used to check the transformation with the libraries such as Glaxo Wellcome Reactive Groups, NTD Screening Library, BMS HTS Deck Filters, and MLSMR Functionality (Hevener, 2018).Initially, the Axitinib molecule was submitted to the NOVA platform to fabricate the new molecule.This protocol run resulted in a maximum of 11-381 biotransformed molecules from the parent compounds.All the designed molecules docked with the PTK and good interacting bioisosteric replaced molecules only shown in Table 4 and other   molecules are displayed in the supplementary file.The following transformation in the axitinib molecule modulates the affinity and ligand efficiency, as well as the torsion angle and the devoid of interresidual clashes.The following Figures 6  and 7 clearly explain the SAR of the designed axitinib molecules.Bioisosteric replacement of the amide bond as a keto group developed two hydrogen bonds and increase the steric circle of the alkyl (-CH 3 ) group.Additionally, it facilitates the Stilbene skeleton perpendicular (99 � ) to the benzene ring system to fit well orientation inside the cavity (Figure 8).
In the meanwhile, keto group replacement increased the repulsive force inside the binding site of the metabolic enzyme CYP450 (6BCZ).Electronegativity of the oxygen atom increases the positive energy of carbon atoms of the benzene ring.These electron clouds produce steric repulsions with the pyridine ring of the hem coenzyme and ILE 301 residues (Figure 9) which leads the less affinity with metabolic enzyme and reduces the possibility of hepatotoxicity of the designed molecule.
The following Table 4 showed the number of newly generated molecules, transferred bioisosteric fragments, affinities, and efficacy of the bioisosteric replaced analogs of known PTKi's.
The table clearly illustrates the biotransformation groups in basic skeletons of know PTKi's and the modification of affinities on the metabolic enzyme and the tyrosine kinases enzyme.Specifically, modification of the amide linker in axitinib into keto will increase the affinities with tyrosine kinases     enzyme and the possibilities of higher potency against malignant cells.The affinities with the CYP450 enzyme decrease due to the clashes of active site amino acid residues reducing the toxicity of the axitinib.

Conclusion
Newly generated PTKi's are one of the preeminent ways to treat and manage various types of cancer but the significant problem behind the chemotherapy with these drugs was the development of moderate to severe hepatotoxicity frequently reported.This study describes the conceptual explanation of hepatic cell necrosis due to the PTKi's.The results of this study conclude the biotransformation of specific fragments of known PTKi leads the effective and safe molecules.Conversion of the amide group as a keto group increased the binding affinity with tyrosine kinases protein and denature of the protein leads to higher inhibition of cancer cells.Also, the keto group unfavor the binding with the metabolic enzyme reduces the toxicity properties of axitinib.Similarly, all the drug's PTKi's structures were modified using better effective fragments and reported.These molecules will be the lead molecules devoid of toxicity in the chemotherapy.
's.The affinity of C 26 atom of axitinib made good hydrophobic interaction with the pyridine moiety of Hemo coenzyme and O 2 atom produce hydrogen bond interaction with ARG 105 residue.In addition, all the aromatic and aliphatic carbons of axitinib produce favorable electrostatic interactions, which assemble the orientation match of the molecule with the active site of the CYP450 enzyme.There are no inter/intra atomic clashes and unfitting torsion angles chief the axitinib to bind well with the metabolic enzyme.

Figure 2 .
Figure 2. Binding affinity and the active site interaction characteristics of drugs with the PTK (5YU6).

Figure 3 .
Figure 3. Binding affinity and the active site interaction characteristics of drugs with the cytochrome p450 (6BCZ) enzyme.

Figure 5 .
Figure 5.The affinity and clashes of axitinib with the active site amino acids of a metabolic enzyme (6BCZ).

Figure 7 .
Figure 7. Transforming groups of axitinib and the interaction with tyrosine kinases protein.

Figure 8 .
Figure 8. (a).Interaction of designed Axitnib derivative with tyrosine kinases protein after substitution of Keto (-C ¼ O) group.(b).Binding cavity configuration of the Axitinib derivative.

Table 2 .
Known PTKi's and the minimized energies of the molecules indicated in kcal/mol.

Table 4 .
Bio-transformed PTKi's and their binding properties with tyrosine kinases protein.