Bone-targeted Src SH2 inhibitors block Src cellular activity and osteoclast-mediated resorption.
Src, a nonreceptor tyrosine kinase, is an important regulator of osteoclast-mediated resorption. We have investigated whether compounds that bind to the Src SH2 domain inhibit Src activity in cells and decrease osteoclast-mediated resorption. Compounds were examined for binding to the Src SH2 domain in vitro using a fluorescence polarization binding assay. Experiments were carried out with compounds demonstrating in vitro binding activity (nmol/L range) to determine if they inhibit Src SH2 binding and Src function in cells, demonstrate blockade of Src signaling, and lack cellular toxicity. Cell-based assays included: (1) a mammalian two-hybrid assay; (2) morphological reversion and growth inhibition of cSrcY527F-transformed cells; and (3) inhibition of cortactin phosphorylation in csk-/- cells. The Src SH2 binding compounds inhibit Src activity in all three of these mechanism-based assays. The compounds described were synthesized to contain nonhydrolyzable phosphotyrosine mimics that bind to bone. These compounds were further tested and found to inhibit rabbit osteoclast-mediated resorption of dentine. These results indicate that compounds that bind to the Src SH2 domain can inhibit Src activity in cells and inhibit osteoclast-mediated resorption.
Violette
S M
SM
ARIAD Pharmaceuticals Inc., Cambridge, MA 02139, USA.
Guan
W
W
Bartlett
C
C
Smith
J A
JA
Bardelay
C
C
Antoine
E
E
Rickles
R J
RJ
Mandine
E
E
van Schravendijk
M R
MR
Adams
S E
SE
Lynch
B A
BA
Shakespeare
W C
WC
Yang
M
M
Jacobsen
V A
VA
Takeuchi
C S
CS
Macek
K J
KJ
Bohacek
R S
RS
Dalgarno
D C
DC
Weigele
M
M
Lesuisse
D
D
Sawyer
T K
TK
Baron
R
R
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001862:Bone Resorption / Q000378:metabolism*; D002461:Cell Line, Transformed; D003804:Dentin / Q000378:metabolism; D004164:Diphosphonates / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D008024:Ligands; D008322:Mammals; D051379:Mice; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D010024:Osteoporosis / Q000378:metabolism; D011817:Rabbits; D011869:Radioligand Assay; D051381:Rats; D014316:Tritium; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains / Q000502:physiology*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C427504:AP21946; C427505:AP22188; C413184:AP22408; C427503:AP22409; D000199:Actins; D004164:Diphosphonates; D004791:Enzyme Inhibitors; D008024:Ligands; D014316:Tritium; D019061:src-Family Kinases