1. Volume 23, Issue 9, Pages 1103-1112 (September 2016)
Structural and Biochemical Basis for Intracellular Kinase Inhibition by Src-specific Peptidic Macrocycles 
Saadat U. Aleem, George Georghiou, Ralph E. Kleiner, Kip E. Guja, Barbara P. Craddock, Agatha Lyczek, Alix I. Chan, Miguel Garcia- Diaz, W. Todd Miller, David R. Liu, Markus A. Seeliger 
Cell Chemical Biology 
Volume 23, Issue 9, Pages (September 2016)
DOI: /j.chembiol
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2. Cell Chemical Biology 2016 23, 1103-1112DOI: (10. 1016/j. chembiol
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3. Figure 1
Chemical Structures of the Second-Generation Compounds MC4 and MC25 Described in This Work
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 2 X-Ray Cocrystal Structure of Src and MC25b
(A) MC25b binds to the active site of the kinase, located between the amino-terminal N lobe and the carboxy-terminal C lobe. Src is in the Cdk/Src-like inactive conformation, and the phosphate binding P loop (red), helix αC (orange), and the activation loop (blue) are highlighted.
(B) Details of the MC25b binding pocket (enlarged from A). The nitrophenylalanine (A group) occupies the adenine binding pocket, the fluorophenylalanine (B group) occupies a pocket underneath the β3-αC loop, and the cyclohexylalanine (C group) occupies a pocket near the peptide binding patch.
(C) Comparison of the structures of MC25b, MC1, and MC4b when complexed with the Src kinase domain. The macrocycle structures are shown from a perspective that fixes the kinase domains (not shown) in the same orientation.
(D) Interactions (dashed line) made between MC25b and Src kinase domain. The nitrophenyl group of MC25b (red) forms a salt bridge with the catalytic lysine (Lys295). The ornithine backbone of MC25b forms hydrogen bonds with Gln275, Cys277, and Gly279 of the P loop, Asn391 in the C lobe, and a water-mediated hydrogen bond with Arg388 in the activation loop of Src.
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5. Figure 3
The Effect of Ionic Strength on the Binding of Nitrophenylalanine and Pyrazine Macrocycles to Src Kinase
(A) Fluorescence anisotropy was used to measure the binding of the purified forms of the indicated Src kinase domains with a fluorescein-labeled macrocycle containing either a pyrazine (fluorescein-MC2) or nitrophenylalanine (fluorescein-MC9) group at the A position at 0 mM NaCl (low salt) or 500 mM NaCl (high salt). Experiments were performed in triplicate. Error bars represent mean values ± SD.
(B) KD values from (A) are plotted. Error bars represent mean values ± SD.
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6. Figure 4
A Bcr-Abl Activation-Loop Inhibitor-Resistance Mutation Modeled in Src(R419P) Results in a Loss of Potency for MC25b
(A) Inhibitory potency of macrocycles in biochemical kinase assay. Experiments were performed in triplicate, and data represent mean values ± SD.
(B) Destabilization of Src/Cdk-like inactive conformation results in loss of macrocycle potency. Src autophosphorylation (pTyr-416) or the L407G mutation cause a loss of potency for both MC4b and MC25b. Experiments were performed in triplicate, and data represent mean values ± SD.
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7. Figure 5
MC25a Inhibits Cell Migration in a Metastatic Breast Cancer Cell Line
(A) Dose-dependent inhibition of MDA-MB-231 cells by MC25a. MDA-MB-231 cells were treated for 24 hr with MC25a or DMSO. Following cell lysis and immunoprecipitation with Src antibody, radioactive kinase activity assays were performed, yielding an IC50 of 0.06 μM. Data represent mean values of four experiments ±SEM.
(B) Western blot of immunoprecipitated Src from MC25a-treated cells as input control for radioactive kinase assays in (A).
(C) Wound-closure assay of MDA-MB-231 cells. Following serum starvation for 12 hr, cells were treated with either 20 μM of MC25a, or DMSO vehicle control.
(D) Following introduction of a sterile wound, wound closure was quantified by measuring the free space unoccupied by cells at the indicated time points.
Data represent mean values ±SEM. *p < 0.05.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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