Methods and uses of bifunctional enzymatic clamp molecules
A technology of medicinal salts and compounds, applied in the field of treating these disorders
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Embodiment 1
[0214] Embodiment 1. Material source
[0215] Acetone and potassium carbonate were purchased from Sigma-Aldrich, catalog numbers #270725 and #209619, respectively.
[0216] EGFR "Inhibitor 3" was purchased from Calbiochem, cat. no. 324833.
[0217]
[0218] Mercaptan starting materials were purchased as follows:
[0219] 2-Mercaptoethanol (2-ME): SIGMA#M3148;
[0220] ·
[0221] EES5: Maybridge#SPB02607SC;
[0222] EES18: Maybridge#BTB12608SC;
[0223] EES18A: Oakwood product #60725-23-7;
[0224] EES18B: Enamine#T5878273;
[0225] EES18D: OtavaChemicals#7212920033;
[0226] EES18F: Maybridge#TL00159ZZ
Embodiment 2
[0227] Embodiment 2: the synthesis of compound
[0228]Several embodiments of the invention were synthesized by reacting the chloroacetamide group of a model tyrosine kinase inhibitor molecule with the thiol group of several mimic mutant compounds. Although not intended to limit the scope of the invention, these syntheses provide a means of producing these defined and broader embodiments.
[0229] In a separate synthesis, 2 mg of EES compound was mixed with 1 mg of Inhibitor 3 in 1 mL of anhydrous acetone (containing 15 mg of anhydrous potassium carbonate) and allowed to incubate at room temperature for 1 hour or overnight. The reaction mixture was loaded on a C4 reversed-phase Beckmann C18 semi-preparative HPLC column (P / N235328) for further purification. In each case, the identity of the target molecule was confirmed by the Mass Spectrometry Center, Department of Chemistry, University of Pennsylvania.
[0230] Compounds identified as C53, C300, C318, C318A, C318B, C318D ...
Embodiment 3
[0255] NE91 cells (mouse fibroblasts overexpressing wild-type human-type EGFR) were starved overnight with an exemplary compound that mimics the mutation (designated EEO3, see below; 0.1 μM; figure 2 2nd and 6th tracks), (0.1μM; figure 2 Tracks 3 and 7) or both ( figure 2 4th and 8th lanes) were incubated for 1 hour and harvested ( figure 2 lanes 1-4) or induced with EGF (50 ng / mL) for 15 min and harvested. Equal amounts of total protein were loaded on 10% SDS-PAGE gels, separated, transferred to PVDF membranes, and treated with pY99, pERK antibody (SantaCruz Biotechnology) or antibodies against specific phosphotyrosine of EGFR (pY845, pY1068, pY992) or AKT phosphorescence-serine (473; Cell Signaling Technology) probe. As controls, total EGFR and γ-tubulin levels were determined using 1005 (Santa Cruz Biotechnology) and GTU88 (Sigma) antibodies, respectively. The obtained data is shown in figure 2 middle.
[0256] Studies described in U.S. Patent Application Seria...
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