Development of New Src Inhibitors

 

Samar Alnukari¹*, Faten Sliman²

¹Master Student in Pharmaceutics and Pharmaceutical Technology Department, Faculty of Pharmacy,

Tishreen University, Syria.

²Doctor in Pharmaceutical Chemistry Department, Faculty of Pharmacy, Tartous University, Syria.

 

ABSTRACT:

Protein kinase, especially tyrosine kinase Src, is considered as an important target for cancer therapy such as lung, prostate and central nervous system cancers, as well as its involvement in tumor metastasis. The aim of this study was to find effective and selective inhibitors of Src protein kinase using molecular modeling study, a library of chemical compounds derived from purine and Theophylline cores was then docked within the active site of the Src enzyme. The link affinity and the attitudes of binding of the suggested compounds with the active site of the previously prepared Src enzyme were determined of these compounds. The study led to three effective and highly selective compounds on the Src enzyme; two compounds derived from the Purine core Pur46 and Pur49 and one compound derived from Theophylline core Theo21. The study was completed by chemical synthesis of Theo23 molecule.

 

 

 

INTRODUCTION:

Protein kinases (Pk) play an important role in cell growth, proliferation and differentiation [9]. These enzymes transfer a phosphate group from the energy molecule ATP to specific residues to stimulate signaling pathways in cells and organisms [1]. Protein kinases include many groups, one of the most important groups is protein tyrosine kinases PTK_S that catalyze phosphorylation of tyrosine in several proteins from ATP [2]. PTKS are included in many cellular signaling pathways and affect some biological processes such as mutagenesis, migration and survival [2]. Tks are divided into two essential parts, receptor tyrosine kinases [3] and non-receptor tyrosine kinases [4]. Several Non-receptor Tks have been identified, among them the Src family which has been related to many diseases, including cancer [4,5]. In order to develop a wider view of Src family kinases SFK_s, these enzymes are classified into nine members that have some specifications in structure and function: Blk, Brk, Fyn, Frk, Hck, Lyn, Lck, Srm, Yes and Src itself [5].

 

Looking at the structure for Src enzyme, it consists of Src homology domain 1 (SH₁), Src homology domain 2 Src homology domain 3 (SH₃), Src homology domain 4 (SH₄) [6]. The SH₁ domain includes kinase domain with tyrosine residues which are responsible for the activity of Src enzyme by phosphorylation and dephosphorylation while the SH₂ contains 100 amino acids and its conformation is crucial for the regulation of Src activity and binding to other protein kinases. SH₃ is a linker which is intermediated protein – protein interaction with Src itself, while the SH₄ domain function is the membrane attachment [6]. Src enzyme is 60 KDa cytoplasmic protein that plays various roles in signaling pathways via interaction with specific proteins which are responsible for signal transduction [7]. Src activity is regulated by two essential tyrosine residues, the first is Y527 which is dephosphorylated to convert Src enzyme from the close conformation to the open one then Y416 is autophosphorylated to catalyze Src enzyme by converting it to the active form [8]. Src is implicated in several cancers such as prostate cancers and breast cancers in addition to its role in metastasis when it has been over expressed [9]. Though both the design and synthesis of a vast number of selective inhibitors for Src enzyme, still there is an inability of finding critical chemical features of Src inhibitors [10], this leads to the importance of discovering new inhibitors by studying new molecules by molecular modeling and synthesizing them. Src has several sites to be inhibited, for example, some researchers studied the inhibition of Src by synthesis of SH₂, SH₃ domains inhibitors while others were interested in studying ATP competitive inhibitors which bind to ATP binding site [10,11]. Simply stated, ATP binds to the ATP binding site through several bonds, two hydrogen bonds between the purine core of ATP and the amino acids Glu339 and Met 341, in need of Mg⁺² which helps to transfer the phosphate from ATP to the peptide substrate [10]. Moreover, the ATP site of Src includes a hydrophobic specificity pocket [10].  Depending on the structure and mechanism of binding of ATP in the active site, this research has been conducted to study new inhibitors of Src enzyme by molecular modeling.

 

METHODS AND MATERIALS:

According to the importance of the inhibition of Src enzyme and the crucial role of molecular modeling at this time in designing new inhibitors for various kinds of enzyme , this research followed molecular modeling in designing inhibitors of Src enzyme using Accelry Discovery Studio 2.5 (DS) program and protein Data Bank (PDB) which is a huge bank involves a big number of crystalline proteins and macromolecules with or without compounds attached to them.

 

1. Data set:

When preparing a set of data, all the crystalline forms available to the Src are collected from the PDB, they are classified according to active form (open) or inactive form (closed) and by its association with an inhibitor to SH2 / SH3 domains or an ATP inhibitor. An active form of the enzyme in complex with an inhibitor was chosen for the study (pdb code: 2BDJ). Different amino acids residues are important for binding like Lys295 conserved from N-terminal domain, Glu339, Met341 from ATP site and Asp404 from DFG motif (DFG in). Src also has Thr 338 as a small gatekeeper residue. This crystalline form of the Src protein, was effectively selected and associated with the AP23464 inhibitor at the site of ATP derived from the purine core. The shape is highly accurate R = 2.5°A and the standard deviation is less than 2 [12] as seen later.

2. Preparation of crystalline form of the SRC protein:

Protein structure used in study (2BDJ) was prepared. For docking, a CHARMm force field was applied and the hydrogen atoms were added. Global system was then minimized to reduce its energy without affecting the remains of a structure of the protein (as heavy atoms)> After minimization a 10-angstrom sphere around the binding site was determined in order to study the association of the new inhibitors inside (Fig1).

 

 

Fig1: The Crystalline form of Src enzyme including 10°A sphere

 

3. Set a library of new chemical compounds:

Depending on the nature of the core commonly used in the design of Src inhibitors, which showed good IC₅₀ values, two cores were proposed to develop Src inhibitors. The first core purine was the same core of the ATP molecule. Although several inhibitors derived from this core were studied but they were not designed to docked into the additional pocket at the site of the ATP, which was mentioned the importance of selectivity. The second chosen core was Theophylline which has a proximate shape of purine core and it is available with low price, generally. After drawing and designing the compounds derived from these two cores, the study of the docking process of these compounds was carried out within the active site of the Src enzyme.

 

Theophylline core,                          Purine core

Fig 2: The used cores in the study

 

 

 

 

 

 

 

Table1:  The designed compounds derived from purine core

 

 

 

Table2:  The designed compounds derived from Theophylline core

 

4. Docking studies:

The docking process of both the 2BDJ structure of the protein and the compounds drawn from the cores required docking in DS program using the CDocker process. The validation of the docking method was first investigated by comparing the standard deviation of the basic inhibitor of AP23464 crystalline and deviation placement according to the CDocker method provided where the standard deviation RMSD must be less than 2°A. This was followed by a docking procedure between the 2BDJ protein and the compounds drawn according to the protocol followed in the program.

 

After the completion of the docking process, the binding mode was studied and the affinity of binding which is expressed as score function was measured as the value of CDocker energy. In other words, the binding of the CDocker method is indicated by the negative value of the CDocker energy.

 

RESULTS AND DISCUSSION:

As shown in Table1 and Table2, several potential variants of the R1, R2, R3 substituents were included for the purine core and the R substituents for Theophylline core. Many of the chemical substituents, including hydrogen and alkyl groups, were analyzed and their affinity was determined based on two basic principles. The first was the expression of the correlation between position and type of substituents and the affinity of binding, the second was the best binding with essential amino acids such as Thr338, Met341, Asp404 and Glu310. Selection of the best compound was depending on two parameters, the first was score function and the second was the ability of binding with important amino acids in the active site of Src. Docked compound finally filtered by Lipinski rules to find the best compound. In addition to that, the Src enzyme has the additional extra hydrophobic pocket (Thr338 as gatekeeper), which can be exploited thus can add an extra feature of the selective inhibitor of Src enzyme.

 

For the purine core, three substituents were included for the R1, R2, and R3 in order to carry out a detailed study of the potential inhibitors within the active pocket based on the principles mentioned above. The results were discussed in detail. Table3 shows -CDoker energy values ​​for the designed compounds.

 

 

Table3: CDocker energy values of the compounds derived from purine core

 

It has been noted that the compounds from Pur1 to Pur6 contribute one hydrogen bond with Met341 and were characterized by a significant decrease in the value of the CDocker energy, indicating that the compounds containing one chemical substituent on N7, whatever this substituent and the hydrogen on N9 and C6 were low-correlation compounds (low-energy CDocker values) and formed few bonds with the Src active site.

 

Accordingly, different substituents were added to C6 while maintaining the presence of hydrogen on N7 to study the effect of these substituents on the affinity and extent of association with the active pocket of the Src enzyme. Addition of the phenyl amine resulted a hydrogen bond between NH of phenyl amine and Met341 amino acid in most compounds, knowing that these simple compounds cannot be adopted because of their low affinity, as well as the non-exploitation of these compounds for the additional hydrophobic pocket of the Src.

 

The changes in the associated substituents in N9 showed great importance in affinity where the para hydroxyphenyl substituent had an obvious importance in improving the correlation. The hydroxyl group contributed to Lys295 and this significantly improved the association with important amino acids within the active Src envelope as in the compound Pur22, but the correlation was still low (-Cdocker energy = 9.9), which showed a low affinity. Therefore, the substituent of the N9 must be lengthened. When adding a para hydroxyphenyl ethyl substituent, the extra pocket was exploited, as well as an additional hydrogen bonding between hydroxyl and Glu310 was formed as seen in Pur38.

 

All previously studied compounds had a hydrogen in R2 substituent, to study the importance of the substituents here, it was necessary to test different alkyl substituents of C2 such as cyclopentane, hydroxylamine and cyclohexane. However, there was a significant increase in the CDocker energy of these compounds (from Pur44 to Pur50) to show the importance of the presence of alkyl substituent R2 for affinity (-CDocker energy = 46 for Pur44). This high value was often found in the Pur45 compound due to the internal hydrogen bond between the OH on the R2 substituent and the N3 nitrogen, which solidifies and stabilizes the compound to become more stable and less flexibility to take multiple forms. Moreover, it was detected that the presence of peripheral substituent, such as cyclopentane substituent and hydroxyethyl amine substituent in R2, with the presence of meta methyl hydroxyethyl amine in R3 showed a significant improvement in the affinity of the binding by changing the placement of the resulting compounds, this was observed in the Pur46 and Pur49 compounds, in addition to the hydrogen bond between OH and Glu310, two hydrogen bonds between N7 and NH from the phenyl root on the C6 were formed with the amino acid Met341 and one hydrogen bond with Lys295, as well as the Pi bond between the aromatic ring and the hydrophobic amino acids of the active site, compound Pur46 possess high score function (51) and exploits Src-specific additional pocket, noting that meta position for hydroxyl group was the best for affinity see Fig3. On the other hand, the presence of heterogeneous groups in R3 showed a significant reduction in affinity and a significant decrease in the number of bonds with the active pocket, showing the negative impact of the presence of these substituents in R3. Thus, the optimal inhibitor of the Src enzyme had three main components: phenylamine in C6, cyclopentane or hydroxylamine in C2, and meta hydroxyphenyl ethyl in N9, where these inhibitors meet the three conditions in terms of high values of the CDocker energy and thus the high affinity of these compounds on the active pocket and the formation of important bonds with multiple amino acids of the active site, in addition to the exploitation of the additional pocket of Src active site thus ensured high selective of these inhibitors on the enzyme.

 

Fig3: Binding mode of compounds Pur46 (a) and Pur49 (b) within the active site

 

The core of Theophylline differs from purine by the number of replaceable substituents. Theophylline core has a single adjustable substituent on N7, unlike the three-substituents purine core as mentioned earlier. As the compounds derived of the purine core, the results of –Cdocker energy the compounds derived from the core of Theophylline were presented in Table4 due to the importance of these values in detecting the affinity of these compounds to the active site of Src enzyme.

 

 

Table4: -CDocker energy values of the compounds derived from Theophylline core

 

Derivatives with small hydrophobic substituents were not suitable for the binding. For example, the nephthyl substituent in the Theo7 did not contribute to any correlation and the value of the CDocker energy was very low and therefore very weak. The presence of a meta-hydroxyphenyl substituent played an important role in the correlation. Theo5 compound formed two hydrogen bonds with Glu310 and Thr338 but did not show any importance in the exploitation of the additional hydrophobic pocket of Src enzyme with a low affinity for this compound (-CDocker energy = 10.5). On the other hand, the presence of the aryl group with para hydroxyl group as Theo4 or two hydroxyl groups at the para and meta sites as Theo6 could not have a good affinity within the Src pocket.

 

Through studying the compounds Theo14,21,22,23, a significant increase in the values ​​of CDocker energy was noticed, which showed that the presence of a long carrier link with an aromatic ring affected positively the affinity of the compound and the stability within the active site, but the composition of these compounds in the active site were different according to the groups carried on the aromatic ring, Theo22 and Theo23 demonstrated great importance for the presence of a hydroxyl group on the aromatic ring, providing important hydrogen bonds with the active site. However, para hydroxyphenyl ethyl formed a single hydrogen bond between Theo22 and the amino acid Met341 within the active site of the Src enzyme while meta hydroxyphenyl ethyl was preferable and it formed two hydrogen bonds with Glu310 and Asp404. In the case where there was an absence of a hydroxyl group on the aromatic ring carried on a long arm, the compounds lose any type of binding on the active site of the Src enzyme, despite the fact that there was a high CDocker energy value for these compounds, such as Theo21, Theo14.

 

On the other hand, compound with phenylethyl substituent was very important in exploiting the extra hydrophobic pocket of src active site, these compounds such as Theo23 showed a distinct position within the active pocket of the Src enzyme and a clear exploitation of the extra pocket, led to a clear selective selection of the enzyme, this relatively large size of phenylethyl substituent and the hydroxyl group in meta site, as compound Theo23, had a high affinity for the active site of Src enzyme (-CDocker energy = 44.6) and formed two important bonds with the amino acids Glu310 and Asp404, as well as, the ethyl holder for the aromatic ring allowed to occupy the extra pocket, making it a selective inhibitor for the Src enzyme. (Fig4).

 

Fig4: The binding mode between Theo23 and the active site of Src enzyme (-CDocker energy = 44.6)

These types of purine and theophylline derivatives compounds could be synthesized by N-alkylation of the core unit with an alkyl group such as ethylphenyl or hydroxyl groups in presence of strong base [13] (Fig5).

 

Fig5: The alkylation of Theophylline to synthesize caffeine

 

CONCLUSION:

Molecular modeling was affected on Src tyrosine kinase. Docking study using purine and theophylline cores led to identify different hits which had high affinity to the Src active pocket, in addition to that, these compounds formed bonds with essential amino acids such as Glu310 and Asp404.

 

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Received on 16.01.2019          Modified on 14.02.2019

Accepted on 01.03.2019        © RJPT All right reserved