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Van Der Steen et al. Cancer Drug Resist 2018;1:230-49 I http://dx.doi.org/10.20517/cdr.2018.13 Page 233

The residue L858 (exon 21) is part of a hydrophobic cluster on the A-loop. This cluster stabilizes the
inactive conformation of EGFR through its hydrophobic interactions. The L858R mutation disrupts this
hydrophobic interaction, which leads to destabilization of the inactive form and thus promotes the active
conformation of EGFR [3,11] . The L861Q mutation is very close to the L858 residue, and most likely disturbs
[8]
the inactive conformation in the same way .

Exon 19 deletion and exon 20 insertion take place in the C-helix. The exon 19 deletion in the loop of the
C-helix “pulls” the regulatory helix in the active conformation. In contrast, the exon 20 insertion on the
other end of the helix “pushes” the helix into its active conformation. Detailed crystal structures lack to
[3]
confirm this hypothesis .
The G719 residue (exon 18) is a less frequent point mutation occurring in EGFR. This Glycine is located on
the P-loop and is the first Gly in the GXGXXG motif. In the inactive form of EGFR, the P-loop contributes
to the hydrophobic interactions that favor the inactive location of the C-helix, together with the cluster of
L858. The G719 residue is necessary to cope with the torsion in the P-loop. Mutation to any other residue
leads to a displacement of the P-loop, disturbs the hydrophobic interactions and thus destabilizes the
[3]
inactive conformation of the receptor, resulting in receptor activation .
A study by Cho et al. showed that dimerization of EGFR is not always necessary for downstream
[27]
signaling. The wild-type EGFR receptor and the L858R mutant are dependent on dimerization for their
activation, whereas in case of the exon 19 deletion, the exon 20 insertion and the L858R/T790M double
mutant, dimerization is not necessary for EGFR-activation. This hypothesis is supported by studies with
cetuximab, a monoclonal antibody that prevents EGFR dimerization. Cetuximab has been shown to inhibit
EGFR-signaling most efficiently when the L858R mutation is present, whereas for the other mutations it
[27]
only has a modest effect .
First- and second-generation EGFR-TKIs
Several of the above mentioned mutations in EGFR render the tumor sensitive to EGFR-TKIs. Erlotinib
and gefitinib are the first generation of EGFR-TKIs that were FDA and EMA-approved. Both erlotinib
[28]
and gefitinib have anilinoquinazoline-based structures and serve as ATP-competitors . The efficacy of
[29]
[29]
both drugs is dependent on the type of sensitizing mutation . Jiang et al. observed a 100x increased
sensitivity of the Ba/F3 variant with the L858R mutation to gefitinib, compared to a 6x increased sensitivity
for the G719S mutant. This difference in sensitivity might be related to the increased affinity for ATP of
both mutants. In comparison to wild-type EGFR, the L858R mutant has a 50x increased ATP-affinity,
[29]
in contrast to a 10x increased affinity for the G719S mutant . These differences in affinity and response
might be related to the position of both mutants. As discussed above, the L858R mutation is located in the
A-loop, whereas the G719S mutation is located in the P-loop of the receptor.

Although there is a difference in affinity of erlotinib and gefitinib for the mutant and wild-type EGFR
receptors, the drugs will bind wild-type EGFR as well. This binding results in on-target side effects such as
[30]
skin rash or diarrhea . Regarding brain activity, the actual concentrations of erlotinib and gefitinib in the
central nervous system are too low to be active due to activity of PGP and BCRP drug transporters [31,32] .

Afatinib is a second-generation EGFR-TKI. The afatinib structure is also anilinequinazoline-based, and it
inhibits both EGFR and ErbB family members Her2, Her3 and Her4. Afatinib is also able to penetrate the
[33]
blood-brain-barrier (BBB) , but it showed moderate activity against brain metastasis. In contrast to the
[34]
first generation EGFR-TKIs, afatinib binds the receptor irreversibly at the C797 residue . This irreversible
binding of the receptor often leads to more severe on-target side effects like skin rash and diarrhea. A
meta-analysis on 3000 patients showed that afatinib has a limited effect on the L858R mutation, whereas it