and 5L6O). However, compound 43a inhibited EGFR more potently than EphB3.
The optimized analog 44 at 50 nM possessed a clean profile in a panel of 98 kinases
while fully abrogating EphB3 activity. No substantial inhibition of LKB1 also
possessing an F1 cysteine was observed. In this context, it is worth mentioning
that although cysteines in LBK1 and PINK1 are positioned at F1 (i.e., two residues
before the start of the αD helix), the hinge region of these kinases is shortened by one
amino acid (GK + 5 position vs. GK + 6 in EphB3). Consequently, these cysteine
residues adopt different orientations in the latter two kinases. A clickable analog of
compound 44 suggested cellular target engagement and limited off-target modification at concentrations below 100 nM. Noteworthily, inhibitor 44 was used as a
covalent probe for EphB1 in a chemical-genetics-based study where EphB1 was
engineered to feature an equivalent cysteine (G703C mutant). An analogous
approach was also applicable to the kinases FGFR4, ABL, and RAF [102].
Being among the few kinases known to harbor a cysteine residue in the F4
(αD + 6) position, the lipid kinase PI3Kα was addressed with inhibitor CNX-1351
(45, Fig. 17b) [103]. This compound potently hits PI3Kα (6.8 nM) with good
selectivity against some other PI3Ks. It caused prolonged inhibition of PI3Kα
signaling in cells and target engagement was demonstrated in vivo. Despite being
equipped with an enone warhead, inhibitor 45 possessed only low reactivity toward
GSH and several plasma proteins, showcasing that the β,β-dimethyl substitution
efficiently attenuates the enone’s intrinsic reactivity [25]. Covalent modification of
Cys862 was demonstrated by MS and X-ray crystallography (PDB: 3ZIM). Due to
the cysteine being located relatively far outside the ATP pocket, a long spacer was
required. Remarkably, the morpholine oxygen atom acts as the non-canonical hingebinding anchor of this compound.
2.5 Development of Inhibitors Targeting Cysteines around
the P-Loop and in the Roof Region
2.5.1 Inhibitors Targeting the R1 Subsite
ErbB3 (HER3), a pseudokinase which forms catalytically active heterodimers with
other ErbB kinases, is lacking the F2 cysteine residue common to the remaining
ErbB family members. However, this protein has recently been addressed via
Cys721, a cysteine located at the R1 subsite at the roof of the ATP binding cleft
[104]. R1-cysteines are only known in four other kinases (WNK1–4) [7]. In the
WNK family kinases, however, these moieties are located two positions further
C-terminally (β3 + 3 in ErbB3 vs. β3 + 5 in WNKs) making the positioning of
Cys271 unique. A non-reactive screening hit was developed to the initial lead
compound TX1-85-1 (46, Fig. 18), a covalent ErbB3 ligand with nanomolar
potency. Unfortunately, 46 was inefficient in suppressing the proliferation of
ErbB3-addicted cell lines and ErbB3-dependent downstream signaling at concentrations that would fully label the target protein. However, transforming 46 into
Covalent Kinase Inhibitors: An Overview
67
The optimized analog 44 at 50 nM possessed a clean profile in a panel of 98 kinases
while fully abrogating EphB3 activity. No substantial inhibition of LKB1 also
possessing an F1 cysteine was observed. In this context, it is worth mentioning
that although cysteines in LBK1 and PINK1 are positioned at F1 (i.e., two residues
before the start of the αD helix), the hinge region of these kinases is shortened by one
amino acid (GK + 5 position vs. GK + 6 in EphB3). Consequently, these cysteine
residues adopt different orientations in the latter two kinases. A clickable analog of
compound 44 suggested cellular target engagement and limited off-target modification at concentrations below 100 nM. Noteworthily, inhibitor 44 was used as a
covalent probe for EphB1 in a chemical-genetics-based study where EphB1 was
engineered to feature an equivalent cysteine (G703C mutant). An analogous
approach was also applicable to the kinases FGFR4, ABL, and RAF [102].
Being among the few kinases known to harbor a cysteine residue in the F4
(αD + 6) position, the lipid kinase PI3Kα was addressed with inhibitor CNX-1351
(45, Fig. 17b) [103]. This compound potently hits PI3Kα (6.8 nM) with good
selectivity against some other PI3Ks. It caused prolonged inhibition of PI3Kα
signaling in cells and target engagement was demonstrated in vivo. Despite being
equipped with an enone warhead, inhibitor 45 possessed only low reactivity toward
GSH and several plasma proteins, showcasing that the β,β-dimethyl substitution
efficiently attenuates the enone’s intrinsic reactivity [25]. Covalent modification of
Cys862 was demonstrated by MS and X-ray crystallography (PDB: 3ZIM). Due to
the cysteine being located relatively far outside the ATP pocket, a long spacer was
required. Remarkably, the morpholine oxygen atom acts as the non-canonical hingebinding anchor of this compound.
2.5 Development of Inhibitors Targeting Cysteines around
the P-Loop and in the Roof Region
2.5.1 Inhibitors Targeting the R1 Subsite
ErbB3 (HER3), a pseudokinase which forms catalytically active heterodimers with
other ErbB kinases, is lacking the F2 cysteine residue common to the remaining
ErbB family members. However, this protein has recently been addressed via
Cys721, a cysteine located at the R1 subsite at the roof of the ATP binding cleft
[104]. R1-cysteines are only known in four other kinases (WNK1–4) [7]. In the
WNK family kinases, however, these moieties are located two positions further
C-terminally (β3 + 3 in ErbB3 vs. β3 + 5 in WNKs) making the positioning of
Cys271 unique. A non-reactive screening hit was developed to the initial lead
compound TX1-85-1 (46, Fig. 18), a covalent ErbB3 ligand with nanomolar
potency. Unfortunately, 46 was inefficient in suppressing the proliferation of
ErbB3-addicted cell lines and ErbB3-dependent downstream signaling at concentrations that would fully label the target protein. However, transforming 46 into
Covalent Kinase Inhibitors: An Overview
67