D-3-phosophoglycerate dehydrogenase allosteric inhibitor and application thereof
A technology based on medicinal salts and carboxylate groups, applied in medical preparations containing active ingredients, drug combinations, organic chemistry, etc., can solve problems such as no PHGDH inhibitors and no drug effects
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Embodiment 1
[0035] Embodiment 1, the discovery of PHGDH allosteric inhibitor
[0036] 1. Prediction of PHGDH allosteric sites
[0037] The allosteric sites of PHGDH (PDB code: 2G76) were predicted using the protein surface exploration program CAVITY. First, the program probes the surface of the protein by erasing the ball method to find potential binding sites on the protein surface; then, the program uses the empirical formula (CavityScore=(Volume-AdjustVolume) / (SurfaceArea-AdjustSurfaceArea)) to analyze the protein-binding small molecule ability to score. AdjustVolume and AdjustSurfaceArea are related to the hydrophobic area of residues and the number of hydrogen bond acceptors and donors in the predicted sites. by the maximum pK for known binding site-ligand binding pairs D Score and compare with known experimental pK D A good linear correlation was obtained by fitting the values. Therefore, the program can score the predicted binding site-ligand maximum pK according to the abov...
Embodiment 2
[0040] Embodiment 2, the synthesis of allosteric molecule
[0041] 1. Design of allosteric molecules
[0042] figure 2 Docking results of the designed allosteric molecules with PHGDH are shown. From figure 2 It can be seen from A that the compound (E)-4-(5-((2-(hydrazine carbonyl) hydrazono) methyl) furan-2-yl) benzoic acid (No. PKUMDL-PG-001) and PHGDH Interaction mode: The 2-phenylfuran aromatic ring occupies the hydrophobic cavity in the pocket, and the 4-position carboxyl oxygen in the benzene ring can form a hydrogen bond with the 59-position threonine; the NH in the thioformyl dihydrazide 2 And one of the NH groups can form a hydrogen bond with the 12-position aspartic acid. From figure 2 B It can be seen that the compound methyl (E)-4-(5-((2-(ethylcarbamoyl)hydrazono)methyl)thiophen-2-yl)benzoate (code PKUMDL- PG-021) and PHGDH interaction mode: 2-phenylthiophene aromatic ring occupies the hydrophobic cavity in the pocket, one of the NH groups in the thiosemica...
Embodiment 3
[0091] Example 3, Fluorescence kinetic method to determine the influence of allosteric molecules on PHGDH enzyme activity in vitro
[0092] The determination of PHGDH enzyme activity is realized by detecting the fluorescence emission spectrum of NADH at 456nm. First, PHGDH (final concentration 30 ng / μL) was incubated with HEPES buffer (25 mM, pH 7.1, 400 mM KCl), 5 μM PLP, 0.5 mM αKG, 150 μM NADH and PSAT1 (final concentration 30 ng / μL) in a 96-well plate for 10 minutes. Subsequently, 10 μL of DMSO (control group) or DMSO solution containing small molecules was added, and shaken and balanced at 25° C. at 550 rpm for 5 minutes. The final concentration (v / v) of DMSO was kept at 5% in the enzyme activity test system. Finally, Pser aqueous solution (final concentration 0.5mM) was added to start the reaction, and the consumption of NADH at 456nm was monitored over time with a UV-Vis microplate reader. Protein activity was assessed using the initial reaction rate within 30 s, wher...
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