Table 3.
Compound Analogs of C6
| ID | IUPAC Name and Structure | MW | ΔTma (°C) | Protection against Tryptic Proteolysisb | KM (PBG)c (μM) | Vmaxc (nmol/min/mg) |
|---|---|---|---|---|---|---|
| Control | – | – | – | – | 86 ± 5 | 61 ± 2 |
| C6-1 | 5-benzyl-2-hydroxy-3-nitrobenzaldehyde
|
257.24 | 6.6 | +/− | – | – |
| C6-2 | 4-[(2-chlorophenyl)methyl]-2-(hydroxymethyl)-6-nitrophenol
|
293.7 | 4.7 | +/− | – | – |
| C6-3 | (4-chloro-3-nitrophenyl)(phenyl)methanone
|
261.66 | 4.9 | +* | 84 ± 8 | 54 ± 2** |
The analogs of C6 were selected from the compound similarity search calculated using the Tanimoto coefficient. The Tanimoto coefficient is used to calculate the 2D similarity between sets of chemical structures.38 The effect of the analogs on both the ΔTm measured by DSF and on the limited tryptic proteolysis of HMBS was determined. Steady-state enzymatic kinetic parameters of HMBS in the presence of analogs were only determined for the analog that protected toward tryptic proteolysis (C6-3). *p < 0.05 and **p < 0.01 for significant difference compared with the values for the control sample (with 2% DMSO), calculated by unpaired two-tailed t test. Data are presented as mean ± SD. IUPAC, International Union of Pure and Applied Chemistry.
The thermal upshift values (ΔTm) monitored by DSF. The average compound concentration in DSF screening was 122 μM (2% DMSO).
+/−, ±2%; +, >4%.
Effect of the compounds on the enzyme kinetic parameters for HMBS activity, measured at fixed compound concentration (84 μM with 2% DMSO) and variable PBG (0–1 mM) at 37°C, and fitted to Michaelis-Menten kinetics.