Net Charge at Physiological pH:
The net charge on gefitinib at physiological pH (7.4) is 0. For this reason, gefitinib is insoluble at pH 7 or above. The molecule precipitates out of solution between pH 4 to pH 6 corresponding to its two biologically relevant pKa values of 5.4 and 7.2. Gefitinib does not have an isoelectric pH (for biological purposes).
Drug Target Information: 2ITY, 2ITZ, 2ITO, 3UG2, 2ITN1; EGFR (receptor protein) tyrosine kinase (transferase) 2.7.10.1
Drug Target Structure Analysis: There is only one chain in the PDB file 2ITY. It is designated “Chain A”, comprising 300 amino acids. The chain in the PDB file (designated Chain A) is the 300 amino acid tyrosine kinase section of the larger protein Epidermal Growth Factor Receptor Tyrosine Kinase. The tyrosine kinase portion of EGFR is composed of two domains commonly referred to as the N-lobe (Domain 1) and C-lobe (Domain 2). Domain 1 (Red) is composed of one beta sheet and one alpha helix. Domain 2 (Blue) is much larger than Domain 1 and is comprised of two beta sheets and seven alpha helices (shown in blue).
The drug is positioned in between the two domains and regularly makes two separate Hydrogen Bonds, one to the N-terminal residue of Domain 2 and another the residue immediately adjacent to the C-terminal residue of Domain 1. EGFR Tyrosine Kinase has an active and an inactive conformation. Switching between these two involves the rearrangement of 4 main segments of the enzyme.These 4 segments include the alpha C-helix, the phosphate binding loop, the activation loop and the catalytic loop. These are shown in Figure 2 and 3. The target enzyme is part of a highly conserved and integral part of any Eukaryotic cell. The enzymatic (drug target) portion of the protein sits just inside the cell membrane. The adjacent portion of the protein spans the cell membrane and the majority of the protein sits on the cell surface acting as the Epidermal Growth Factor Receptor.
Drug Target Function Analysis and Drug Efficacy: Epidermal Growth Factor Receptor Tyrosine Kinase requires two Mg2+ ions as cofactors. These participate in salt bridges with the phosphate groups of the substrate molecule ATP. EGFR Tyrosine Kinase is a transferase (E.C.1). It catalyzes the transfer of a phosphate group from Adenosine-Tri-Phosphate to a Tyrosine residue of another protein. Gefitinib interacts with the active site of EGFR Tyrosine Kinase in several ways. It experiences differing levels of effectiveness with wild type kinase and various mutants (L858R, G719S, T790M) of its target enzyme (EGFR TK). Gefitinib interaction with EGFR TK is primarily defined by a hydrogen bond between the quinazoline nitrogen cluster of the drug and M793 of EGFR TK as well as hydrophobic interactions between EGFR TK’s hydrophobic pocket and the quinazoline and aniline rings of the drug (shown in figure 9).
These two interactions are present when Gefitinib binds to wild-type TK and to mutants L858R, and G719S. Gefitinib is most effective against mutation L858R. Bound to L858R Tyrosine Kinase, Gefitinib has a KD value of 2.4 nM (Eck, 2010) to 2.6 nM (Yun, 2007). This mutation forces the enzyme into the active state by disrupting the hydrophobic core of the active loop (Eck, 2010). The substituted arginine forces the hydrophobic pocket to disassemble and in its place a hydrogen bonding network is formed to stabilize the enzyme in its active state, accounting for the increased activity of the mutation. Also, a bridged hydrogen bond is formed between Cysteine 797 and Aspartate 800 of L858R and the classical amine of Gefitinib, shown in figure 11.
This extra hydrogen bond is only found between the L858R mutation and Gefitinib and it likely accounts for at least part of the high level of Gefitinib sensitivity in the L858R mutation. Another orientation of the aniline ring has been observed in L858R, however, that may account for a large portion of L858R sensitivity to Gefitinib, as well. In this second orientation of the aniline ring, the Chlorine is pointing downward, engaging in a halogen bond with D855. Since this orientation is seen only in L858R mutants, it is a second reasonable justification for L858R sensitivity to Gefitinib (Eck, 2010). Bound to wild type Tyrosine Kinase, Gefitinib has a KD value of 35 nM (Eck, 2010) to 53 nM (Yun, 2007). In the wild type active site, Gefitinib appears to hydrogen bond with M793 alone, which might account for the reduced affinity for the wild type enzyme compared to L858R. The strength of the binding affinity of Gefitinib for Tyrosine Kinase can be loosely correlated with the distance of the Hydrogen bond between M793 and the nitrogen cluster of Gefitinib. In other words, a hydrogen bond between M793 and the nitrogen cluster of the quinazoline ring that is around 2.66 angstroms correlates with a lower value and a higher affinity, as seen in the binding of Gefitinib to wild-type kinase and to mutant L858R. A hydrogen-bond of 2.91 angstroms, as seen in the mutant G719S, correlates with a much greater KD value, of about 123 nM (Yun 2007). The G719S mutation disrupts the beta sheet in Domain 1 of the enzyme directly adjacent to the P-Loop (Yoshikawa, 2012). The abnormally shaped P-Loop may reduce the ability of the enzyme to shield the ligand from the surrounding environment. C797 and D800 do not have the structured water present in G719S. Instead S719 and D800 seem to interact directly with the ligand via a possible hydrogen bonding with the ether and classical amine of Gefitinib, respectively, shown in figure 12.
The hydrogen bond interactions between S719, D800 and Gefitinib in G719S are much longer and weaker than the hydrogen bonds between C797, D800 and Gefitinib in L858R, shown in figure 11 and 12. G719S may have decreased binding affinity because S719 and D800 may be acting against the hydrogen bond between M793 and N1, essentially tugging at both ends of the ligand, and destabilizing Gefitinib in the active site. In L858R, the hydrogen bonds appear to reinforce one another in the crystal structure. This may explain the gap in binding affinity between the two mutants. The mutation T790M, like L858R, is quite sensitive to Gefitinib. T790M has a KD value of 4.6 (Eck, 2010), making it the second most sensitive point mutation to Gefitinib. Examination of its 3d structure shows that the Sulfur of the M790 residue is in good position to hydrogen bond with the N2 nitrogen of Gefitinib via structured. The mutation to methionine from threonine (sometimes called the “gatekeeper mutation”) does not interfere with the hydrophobic pocket of the active site and Gefitinib is able to interact with the hydrophobic pocket in a way very similar to that of L858R, shown in figure 9 (Eck, 2010).
The central quinazoline aromatic ring system and the aniline (chlorofluoro-benzene) ring of Gefitinib both interact with EGFR Tyrosine Kinase (EGFR TK) through hydrophobic interactions with EGFR TK’s sizeable hydrophobic pocket, shown in “yellow tint” space filling model in figure 9 (Yoshikawa, 2006). These hydrophobic interactions occur in both of the mutants mentioned (L858R and G719S) and also in the wild type enzyme.
Drug Action: Gefitinib is primarily used to treat Non-Small Cell Lung Cancer when other medications have failed and it is most effective on patients with the L858R mutation in Epidermal Growth Factor Receptor Tyrosine Kinase. This mutation causes the Tyrosine Kinase domain of Epidermal Growth Factor Receptor to become permanently active by destabilizing the inactive state and this eventually leads to cancer (06/13/2005. DrugBank.ca. Retrieved from http://www.drugbank.ca/drugs/DB00317) Gefitinib functions as a reversible competitive inhibitor of Tyrosine Kinase, in competition with ATP (Yun, 2007) The most dangerous side effect of taking Gefitinib is the development of Interstitial Lung Disease. It is thought that the development of this disease results from the successful suppression of Tyrosine Kinase by Gefitinib. This disrupts the signaling pathway which tells the cell to repair itself in the event of damage. Difficulty breathing may be an indication of the development of this disease. Other serious side effects of the drug are allergic reaction, and eye irritation. The most common side effects appear to be diarrhea, rash and acne. Less common side effects include loss of appetite, vomiting, nausea, asthenia, anorexia, and dry skin (10/2/2008. Rxlist.com. Retrieved from http://www.rxlist.com/iressa-side-effects-drug-center.htm). Gefitinib inhibits not only EGFR TK but many other Tyrosine Kinases in the cell (06/13/2005. DrugBank.ca. Retrieved from http://www.drugbank.ca/drugs/DB00317). This is because the kinases are structurally very similar from one to the next (Endicott, 2012). Gefitinib is a first generation EGFR Tyrosine Kinase Inhibitor, along with erlotinib (Naito, 2009). First generation inhibitors of Tyrosine Kinase are reversible by definition. The second generation of TK inhibitors form covalent bonds to the kinase and are thus irreversible (Kosaka, 2011). The second generation of TKI drugs are being designed to target all point mutations by binding the active site regardless of the combination of mutations. These drugs are targeting the C797 residue of Tyrosine Kinase. This will hopefully erase the issue of new mutations resisting TKI drugs over time, as has been the case (Kosaka, 2011). Gefitinib is an anti-cancer drug. It is not a cure for cancer; it only serves to reduce tumor growth in patients with NSCLC. There are a lot of improvements to be made on the drug. It is considered effective against the L858R mutation, but it proves ineffective against many of the other oncogenic mutants, especially G719S. Structures for many of the mutations in complex with gefitinib are not available yet. To date, gefitinib is only available in complex with wild type EGFR TK, L858R EGFR TK, G719S EGFR TK and the double mutant G719S/T790M EGFR TK. Some of the mutant structures are not available with any ligand at all (Eck, 2010).