Table 1.
Primary Hit Compounds
| Compound ID | ΔTma (°C) | Relative Activity, Standardb | Relative Activity, Pre-Incubated at 70°Cc | Protection against Tryptic Proteolysisd |
|---|---|---|---|---|
| Control | – | 1.00 | 1.00 | – |
| C4 | 1.6 | 1.04 ± 0.05 | 0.92 ± 0.07** | ND |
| C5e | 2.1 | 1.05 ± 0.02 | 1.04 ± 0.05 | +* |
| C6e | 1.6 | 0.98 ± 0.06 | 1.01 ± 0.06 | ++** |
| C8 | 4.5 | 1.01 ± 0.03 | 0.92 ± 0.05** | ND |
| C9 | 3.8 | 0.98 ± 0.03 | 1.04 ± 0.07 | +/− |
| C11e | 1.6 | 1.00 ± 0.03 | 1.01 ± 0.04 | + |
| C12 | 2.2 | 1.07 ± 0.05 | 0.94 ± 0.09 | +/− |
| C15 | 1.5 | 1.00 ± 0.02 | 0.91 ± 0.07* | ND |
| C16 | 2.0 | 1.04 ± 0.04 | 0.91 ± 0.03*** | ND |
| C17e | 2.3 | 1.09 ± 0.05 | 1.07 ± 0.04 | +* |
| C19 | 2.0 | 1.04 ± 0.02 | 0.95 ± 0.06 | +/− |
| C23 | 1.6 | 0.92 ± 0.05** | 0.96 ± 0.06* | ND |
| C24 | 2.3 | 1.08 ± 0.03 | 0.81 ± 0.06**** | ND |
The effect of the 13 primary hit compounds on the Tm of HMBS measured by DSF, enzymatic activity, and limited tryptic proteolysis of the enzyme. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 for significance compared with the DMSO control sample, calculated by unpaired two-tailed t test. Data are presented as mean ± SD.
The thermal upshift values (ΔTm) monitored by DSF. The average compound concentration in DSF screening was 122 μM (2% DMSO).
Activity assay performed at standard conditions, with 100 μM PBG at 37°C, 84 μM compound, and 2% DMSO, which was added in all controls.
Assay, including preincubation of HMBS with compound at 70°C, and subsequent standard activity assay, with 100 μM PBG at 37°C, 84 μM compound, and 2% DMSO.
+/−, ±2%; +, >4%; ++, 10% remaining full-length HMBS relative to DMSO; ND, not determined; see main text and Figure 1B for details.
Hit compounds selected for cell studies.