Structure-based design of specific inhibitors of janus kinase 3 as apoptosis-inducing antileukemic agents

A novel homology model of the kinase domain of Janus kinase (JAK) 3 was use d for the structure-based design of dimethoxyquinazoline compounds with pot ent and specific inhibitory activity against JAK3. The active site of JAK3 in this homology model measures roughly 8 Angstrom x 11 Angstrom x 20 Angst rom, with a volume of similar to 530 Angstrom(3) available for inhibitor bi nding. Modeling studies indicated that 4-(phenyl)-amino-6,7-dimethoxyquinaz oline (parent compound WHI-258) would likely fit into the catalytic site of JAK3 and that derivatives of this compound that contain an OH group at the 4' position of the phenyl ring would more strongly bind to JAK3 because of added interactions with Asp-967, a key residue in the catalytic site of JA K3. These predictions were consistent with docking studies indicating that compounds containing a 4'-OH group, WHI-P131 [4-(4'-hydroxyphenyl)-amino-6, 7-dimethoxyquinazoline], WHI-P154 [4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7 -dimethoxyquinazoline], and WHI-P97 [4-(3',5'-dibromo-4'-hydroxylphenyl)-am ino-6,7-dimethoxyquinazoline], were likely to bind favorably to JAK3, with estimated K(i)s ranging from 0.6 to 2.3 mu M. These compounds inhibited JAK 3 in immune complex kinase assays in a dose-dependent fashion. In contrast, compounds lacking the 4'-OH group, WHI-P79 [4-(3'-bromophenyl)-amino-6,7-d imethoxyquinazoline], WHI-P111 [4-(3'-bromo-4'-methylphenyl)-amino-6,7-dime thoxyquinazoline], WHI-P112 [4-(2',5'dibromophenyl)-amino-6,7-dimethoxyquin azoline], WHI-P132 [4-(2'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline], and WHI-P258 [4-(phenyl)-amino-6,7-dimethoxyquinazoline], were predicted to bind less strongly, with estimated K(i)s ranging from 28 to 72 mu M. These compounds did not show any significant JAK3 inhibition in kinase assays, F urthermore, the lead dimethoxyquinazoline compound, WHI-P131, which showed potent JAK3-inhibitory activity (IC50 of 78 mu M), did not inhibit JAK1 and JAK2, the ZAP/SYK family tyrosine kinase SYK, the TEC family tyrosine kina se BTK, the SRC family tyrosine kinase LYN, or the receptor family tyrosine kinase insulin receptor kinase, even at concentrations as high as 350 mu M . WHI-P131 induced apoptosis in JAK3-expressing human leukemia cell lines N ALM-6 and LC1;19 but not in melanoma (M24-MET) or squamous carcinoma (SQ20B ) cells, Leukemia cells were not killed by dimethoxyquinazoline compounds t hat were inactive against JAK3, WHI-P131 inhibited the clonogenic growth of JAK3-positive leukemia cell lines DAUDI, RAMOS, LC1;19, NALM-6, MOLT-3, an d HL-60 (but not JAK3-negative BT-20 breast cancer, M24-MET melanoma, or SQ 20B squamous carcinoma cell lines) in a concentration-dependent fashion. Po tent and specific inhibitors of JAK3 such as WHI-P131 may provide the basis for the design of new treatment strategies against acute lymphoblastic leu kemia, the most common form of childhood cancer.