| Assay Method Information | |
| | Biochemical Characterization of Cif Inhibitors |
| Description: | The first step in characterizing the two compounds identified by high throughput screening was to verify that the inhibition was reproducible using fresh preparations. Chemical libraries are often stored for extended periods of time, which can lead to breakdown products contributing to the assay outcome. While the inhibition observed with a fresh tiratircol solution was consistent with the primary and secondary screening results, a fresh benserazide-HCl solution lost all observable inhibition. Additionally, it was noticed that benserazide-HCl solutions, either aqueous or in DMSO, acquired a red color over time, suggesting that the compound was susceptible to breakdown.After aging the freshly prepared benserazide-HCl solution for 3 months at room temperature, Cif inhibition was re-tested. Once more, there was no detectable inhibition. Cocrystallization of Cif protein with the red benserazide hydrochloride breakdown solution resulted in crystals with an enriched color over the well solution, indicating that the chromophore had some affinity for Cif protein. However, clear electron density was not obtainable for any additional compounds bound specifically to Cif. This observation could either be due to low occupancy of a molecule bound to Cif, or a non-specific interaction. 1D proton NMR revealed that a large number of additional peaks were present in the spectra of the breakdown sample. LC/MS analysis detected >100 additional compounds present after aging a benserazide-HCl solution in water. Due to the high diversity of products generated by benserazide-HCl breakdown, as well as the inability to repeat inhibition of Cif EH enzyme activity, this compound was not further investigated.After successfully recapitulating Cif inhibition with fresh tiratricol, the effect was confirmed with an independent substrate and assay. Using the adrenochrome reporter assay and epoxyhexane, an epoxide substrate previously shown to be hydrolyzed by Cif, a robust inhibition of Cif enzyme activity was observed.Subsequently, tiratricol inhibition was characterized kinetically, once again using the fluorogenic substrate CMNGC. The Ki was found to be 4.1±0.4 μM. Additionally, the Ki was independent of substrate concentration, indicating that tiratricol functioned via a non-competitive mechanism of inhibition. |
| Affinity data for this assay | |
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