1. Design of allele-specific inhibitors to probe protein kinase signaling
Anthony C. Bishop, Kavita Shah, Yi Liu, Laurie Witucki, Chi-yun Kung, Kevan M. Shokat 
Current Biology 
Volume 8, Issue 5, Pages (February 1998)
DOI: /S (98)

2. Figure 1
The specificity problems associated with using small-molecule protein-kinase inhibitors to deconvolute cell signaling. Kinase catalytic domains (red ovals) are highly conserved. Thus, (a) the majority of potent inhibitors block the activity of closely related kinases and broadly down-regulate pathways mediated by kinase activity. (b) The selective protein kinase inhibition approach described here. A space-creating mutation is introduced into the ATP-binding site of the kinase of choice (Src). This mutation creates an active-site pocket in Src that can be uniquely recognized by the rationally designed small-molecule inhibitor. The inhibitor contains a bulky chemical group that makes it unable to bind wild-type protein kinases. Design of the complementary kinase–inhibitor pair allows for highly selective inhibition of the target kinase in the context of a whole cell. S indicates serine and Y indicates tyrosine.
Current Biology 1998 8, DOI: ( /S (98) )

3. Figure 2
(a) Structure of N-6 cyclopentyloxyadenosine (1). (b) Synthesis of pyrazolo[3,4-d]pyrimidine inhibitor analogues. Compound 2 was synthesized according to Hanefeld et al. [32]. (i) Acyl chloride (RCOCl; 10 equivalents), pyridine, 5°C, 1 h; then warm to 22°C, 11 h; (ii) LiAlH4 (3.0 equivalents), dry tetrahydrofuran under argon, 0°C, 30 min; then heat to reflux for 30 min.
Current Biology 1998 8, DOI: ( /S (98) )

4. Figure 3
(a) Chemical structures of quercetin (5) and AMPPNP (6). (b) Predicted binding orientation of 2 in Src family kinase active sites. The crystal structures of Hck bound to AMPPNP (red) and Hck bound to quercetin (blue) were superimposed according to the Hck protein backbone (white) [25]. The structure of 2 (yellow) was subsequently docked into the kinase active site by superimposing the pyrazolo[3,4-d]pyrimidine ring system of 2 onto the adenine ring of AMPPNP. (c) Predicted close contact between N-4 of 2 and the side chain of residue 338 in Src family kinases. Molecule 2 has been docked into the ATP-binding site of the Src family kinase Hck as in (b). The atoms of the Thr338 side chain and 2 are colored according to their elemental make-up (green, carbon; blue, nitrogen; red, oxygen; white, hydrogen) and the Hck backbone is shown in pink. The methyl hydrogens of the threonine side chain are not shown. Images were generated using the program InsightII.
Current Biology 1998 8, DOI: ( /S (98) )

5. Figure 4
Inhibitor analogue 3g does not inhibit tyrosine phosphorylation triggered by the active B-cell receptor. Murine spleen cells were incubated with 1.1% DMSO (lanes 1,2), 100 μM 3g in 1.1% DMSO (lane 3), or 100 μM 2 in 1.1% DMSO (lane 4). B-cell stimulation (lanes 2–4) was initiated by the addition of 10 μg/ml goat anti-mouse IgM. Cellular proteins were resolved by 10% SDS–PAGE, transferred to nitrocellulose, and immunoblotted with a monoclonal anti-phosphotyrosine antibody (4G10).
Current Biology 1998 8, DOI: ( /S (98) )

6. Figure 5
Inhibitor 3g blocks p36 phosphorylation in NIH3T3 fibroblasts transformed with I338G v-Src but not wild-type v-Src. Non-transformed NIH3T3 cells (lane 1), wild-type v-Src-transformed NIH3T3 cells (lanes 2,3), and I338G v-Src-transformed NIH3T3 cells (lanes 4,5) were incubated with 1.1% DMSO (lanes 1,2,4) or 100 μM 3g in 1.1% DMSO (lanes 3,5). After 12 h, the cells were lysed and protein phosphorylation levels determined as in Figure 4.
Current Biology 1998 8, DOI: ( /S (98) )

7. Figure 6
Fibroblasts transformed with I338G v-Src acquire a flattened morphology and regain actin stress fibers when they are incubated with 3g. (a,b) Non-transformed, (c,d,g,h) wild-type v-Src-transformed, and (e,f,i,j) I338G v-Src-transformed NIH3T3 fibroblasts were treated with either 1.1% DMSO (a–c,e,g,i) or 100 μM 3g in 1.1% DMSO (d,f,h,j). After 48 h, cells were photographed (a,c–f), stained with FITC–phalloidin, and visualized by fluorescence microscopy (b,g–j).
Current Biology 1998 8, DOI: ( /S (98) )