isoforms (Aurora C, IC 50 ¼ 4 nM) but otherwise totally inactive (IC 50 > 10 μM) on
all other kinases tested. Our hypothesis to explain such high and rare level of
selectivity shared by compounds 4, 5, and other analogs made in the series relies
first on the fact that the αC-helix out conformation is not accessible to a broad
ensemble of kinases for ligands/inhibitors [26]. Key determinants which would drive
the occurrence of this inactive kinase conformation remain unclear but may be
related to the gatekeeper size and the packing forces developed along the hydrophobic spine [41]. In the case of compounds 3 and 5 in complex with Aurora A, the
benzimidazole core interacts with a glutamine residue (Gln185) localized in position
“
αC-helix
glutamate + 4,” a reported privileged residue/position. We analyzed among
the kinome the occurrence of a glutamine in this position and found it rare at 2%.
Compound (+)-5 was able to inhibit both Aurora A and Histone-H3 phosphorylation in HCT116 cell lines (IC 50 ¼ 70 and 10 nM, respectively), two relevant
biomarkers of Aurora A and B inhibition to monitor pharmacodynamic modulation
in vitro and in tumor models. Moreover, this compound displayed low nanomolar
anti-proliferative activities against a large panel of cell lines and exhibited no toxicity
on quiescent PBL cells providing evidence that targeting Aurora isoforms triggers
specific and lethal effects on cancer cell lines but no damage on non-proliferative
normal cells. Drug-likeness liabilities of compound (+)-5 (Cyp inhibition, exposure
in rats, hepatocyte clearance, and PDE3 inhibition) were then optimized to give rise
to SAR156497, a molecule which has demonstrated a narrow therapeutic window
when tested in a murine model of human colon adenocarcinoma xenograft [39].
3 Development of a Selective MET Kinase Inhibitor Active
Against Oncogenic Mutants
MET is a tyrosine kinase which is involved in embryonic development and wound
healing in normal cells. MET acts as a transmembrane receptor which is stimulated
by the hepatocyte growth factor (HGF) inducing in fine cell proliferation, migration,
and invasion [42]. Abnormal activation of the HGF-MET pathway has been frequently observed in human cancers in particular in solid tumors where MET protein
is usually overexpressed [43]. Moreover, MET gene has been found amplified in
5–12% of gastric carcinoma, 2–13% of lung cancers, and 4–12% of colon-rectal
carcinoma. Oncogenic mutations of the MET gene have been observed in multiple
human cancer types (hepatocellular, papillary renal cell and head and neck carcinoma, etc.) leading to protein structure changes and activation [44]. Five major
mutants of the kinase domain have been expressed in our group and tested for
catalytic activity and have exhibited a four- to five-time enhancement of catalytic
activity (Kcat) compared to the wild-type protein (internal data – Fig. 7a for mutation
locations). Whereas Y1230H, Y1235D, L1195V, and H1094Y are mutated residues
located at the vicinity of the ATP binding site (Figure 7b for the KMM representation), M1250T is remote to the ATP binding cleft and may influence catalytic
Achieving High Levels of Selectivity for Kinase Inhibitors
103
all other kinases tested. Our hypothesis to explain such high and rare level of
selectivity shared by compounds 4, 5, and other analogs made in the series relies
first on the fact that the αC-helix out conformation is not accessible to a broad
ensemble of kinases for ligands/inhibitors [26]. Key determinants which would drive
the occurrence of this inactive kinase conformation remain unclear but may be
related to the gatekeeper size and the packing forces developed along the hydrophobic spine [41]. In the case of compounds 3 and 5 in complex with Aurora A, the
benzimidazole core interacts with a glutamine residue (Gln185) localized in position
“
αC-helix
glutamate + 4,” a reported privileged residue/position. We analyzed among
the kinome the occurrence of a glutamine in this position and found it rare at 2%.
Compound (+)-5 was able to inhibit both Aurora A and Histone-H3 phosphorylation in HCT116 cell lines (IC 50 ¼ 70 and 10 nM, respectively), two relevant
biomarkers of Aurora A and B inhibition to monitor pharmacodynamic modulation
in vitro and in tumor models. Moreover, this compound displayed low nanomolar
anti-proliferative activities against a large panel of cell lines and exhibited no toxicity
on quiescent PBL cells providing evidence that targeting Aurora isoforms triggers
specific and lethal effects on cancer cell lines but no damage on non-proliferative
normal cells. Drug-likeness liabilities of compound (+)-5 (Cyp inhibition, exposure
in rats, hepatocyte clearance, and PDE3 inhibition) were then optimized to give rise
to SAR156497, a molecule which has demonstrated a narrow therapeutic window
when tested in a murine model of human colon adenocarcinoma xenograft [39].
3 Development of a Selective MET Kinase Inhibitor Active
Against Oncogenic Mutants
MET is a tyrosine kinase which is involved in embryonic development and wound
healing in normal cells. MET acts as a transmembrane receptor which is stimulated
by the hepatocyte growth factor (HGF) inducing in fine cell proliferation, migration,
and invasion [42]. Abnormal activation of the HGF-MET pathway has been frequently observed in human cancers in particular in solid tumors where MET protein
is usually overexpressed [43]. Moreover, MET gene has been found amplified in
5–12% of gastric carcinoma, 2–13% of lung cancers, and 4–12% of colon-rectal
carcinoma. Oncogenic mutations of the MET gene have been observed in multiple
human cancer types (hepatocellular, papillary renal cell and head and neck carcinoma, etc.) leading to protein structure changes and activation [44]. Five major
mutants of the kinase domain have been expressed in our group and tested for
catalytic activity and have exhibited a four- to five-time enhancement of catalytic
activity (Kcat) compared to the wild-type protein (internal data – Fig. 7a for mutation
locations). Whereas Y1230H, Y1235D, L1195V, and H1094Y are mutated residues
located at the vicinity of the ATP binding site (Figure 7b for the KMM representation), M1250T is remote to the ATP binding cleft and may influence catalytic
Achieving High Levels of Selectivity for Kinase Inhibitors
103