In vitro pharmacological characterization of PD 166285, a new nanomolar potent and broadly active protein tyrosine kinase inhibitor.
PD 166285, a novel protein tyrosine kinase inhibitor of a new structural class, the 6-aryl-pyrido[2,3-d]pyrimidines, was synthesized as the most potent and soluble analog of a series of small molecules originally identified by screening a compound library with assays that measured protein tyrosine kinase activity. PD 166285 was found to inhibit Src nonreceptor tyrosine kinase, fibroblast growth factor receptor-1, epidermal growth factor receptor and platelet-derived growth factor receptor beta subunit (PDGFR-beta), tyrosine kinases with half-maximal inhibitory potencies (IC50 values) of 8.4 +/- 2.3 nM (n = 6), 39.3 +/- 2.8 nM (n = 16), 87.5 +/- 13.7 nM (n = 6) and 98.3 +/- 7.9 nM (n = 16), respectively. PD 166285 also demonstrated inhibitory activity against mitogen-activated protein kinase (IC50 = 5 microM) and protein kinase C (IC50 = 22.7 microM). PD 166285 was further characterized as an ATP competitive inhibitor of Src nonreceptor tyrosine kinase, PDGFR-beta, fibroblast growth factor receptor-1 and epidermal growth factor receptor tyrosine kinases. In addition, PD 166285 inhibited PDGF- and EGF-stimulated receptor autophosphorylation in vascular smooth muscle cells (VSMCs) and A431 cells, respectively, and basic fibroblast growth factor-mediated tyrosine phosphorylation in Sf9 cells, with IC50 values of 6.5 nM, 1.6 microM and 97.3 nM, respectively, further establishing a tyrosine kinase mechanism of inhibition. The inhibition of PDGF receptor autophosphorylation in VSMCs by PD 166285 was long lasting and persisted for 4 days after a single 1-hr exposure followed by extensive washing. The PDGF-induced tyrosine phosphorylation of the 44- and 42-kDa mitogen-activated protein kinase isoforms was also blocked as a result of the inhibition of PDGF-stimulated receptor autophosphorylation by PD 166285 in VSMCs. The effects of PD 166285 were also demonstrated in functional assays of cell attachment, migration and proliferation, in which vascular cell adhesion to vitronectin, PDGF-directed chemotaxis and serum-stimulated cell growth were all potently inhibited with IC50 values of 80 yo 120 nM. Finally, PD 166285 uniquely demonstrated potent inhibition of phorbol ester-induced production of 92-kDa gelatinase A (MMP-9) in VSMC without affecting 72-kDa gelatinase B (MMP-2) as measured by gelatin zymography. These results highlight the biological characteristics of PD 166285 as a broadly active protein tyrosine kinase capable of potently inhibiting a number of kinase mediated cellular functions, including cell attachment, movement and replication. The potential therapeutic utility of this broadly acting inhibitor as an antiproliferative and antimigratory agent could extend to such diseases as cancer, atherosclerosis and restenosis, in which redundancies in protein kinase signaling pathways are known to exist.
Panek
R L
RL
Department of Vascular and Cardiac Diseases, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105, USA.
Lu
G H
GH
Klutchko
S R
SR
Batley
B L
BL
Dahring
T K
TK
Hamby
J M
JM
Hallak
H
H
Doherty
A M
AM
Keiser
J A
JA
D000818:Animals; D004247:DNA / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011728:Pyridones / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D011817:Rabbits; D051381:Rats; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D018411:Spodoptera; D014443:Tyrosine / Q000378:metabolism
D004791:Enzyme Inhibitors; C109647:PD 166285; D010982:Platelet-Derived Growth Factor; D011728:Pyridones; D011743:Pyrimidines; D014443:Tyrosine; D004247:DNA; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor