Inhibitors of anti-apoptotic proteins
A technology of solvates and compounds, applied in the field of chemical compounds of thiazolidine parts, can solve problems such as safety and incompetence
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Embodiment 1
[0093] Example 1. Protein expression and purification
[0094] recombinant full-length BCL-X L Generated from pET-19b (Novagen) plasmid construct containing the entire nucleotide sequence of BID fused to an N-terminal poly-His tag. Untagged proteins were expressed in E. coli BL21 in LB medium at 37°C and induced with 1 mM IPTG for a period of 3-4 hours. produce similarly 15 N-labeled protein, whose growth occurs when adding 0.5g / L 15 NH 4 Cl M9 medium. After cell lysis, soluble protein was purified by Hi-Trap chelating column (Amersham, Pharmacia) followed by ion exchange purification by MonoQ (Amersham, Pharmacia) column. Final BID samples were dialyzed against a buffer suitable for subsequent experiments.
Embodiment 2
[0095] Example 2. Molecular Modeling
[0096] Molecular modeling studies were performed using the software package Sybyl version 6.9 (TRIPOS) on several R12000 SGI Octane workstations. The docked structure of the compound was originally obtained by Gold. The molecular models of the compounds were energy minimized with MAXIMN2 (Sybyl). For each molecule, 20 solutions were generated and graded according to Goldscore. Solutions were finally graded by visual observation of linked compounds in deep hydrophobic grooves on the BCL-xL surface. Surface representations are generated by MOLCAD.
Embodiment 3
[0097] Example 3. NMR Spectrum
[0098] For all NMR experiments, BCL-xL was exchanged in 50 mM phosphate buffer, pH 7.5 and measured at 30°C. with 0.5mM 15 N-labeled protein samples to measure 2D of BCL-xL[ 15 N, 1 H]-HSQC spectrum. All experiments were performed with a 600 MHz Bruker Avance spectrometer equipped with either a TXI probe or a TCI cryoprobe. In all experiments, dephasing of the remaining water signal was obtained in the WATERGATE sequence. To examine the ability of test compounds to bind Bcl-xL, 25 μM protein samples were prepared and collected 1D in the absence and presence of test compounds 1 H NMR spectrum. Binding is easily detected in these simple experiments by observing the aliphatic region of the spectrum due to the altered chemical shift of the active-site methyl group of Ile, Leu, Thr, Val or Ala (region between 0.8 and 0.3 ppm ).
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