To follow up on the prediction that the rigid ADEPs would bind ClpP more tightly, Robert Sauer and Karl Schmitz at MIT measured the capacity of the ADEP analogs and the parent compound to produce the "runaway garbage disposal" phenomenon in solutions containing the ClpP protein. The experiments showed that the modified ADEPs produced the effect at much lower concentrations, indicating a higher binding efficiency. The results implied that the modified molecules were about seven times better than the standard ones at binding to ClpP.
The final step was testing whether the rigid ADEPs were better at killing bacteria in a test tube. Those tests showed that, compared to published reports for standard ADEPs, the modified compounds were much more potent against three different dangerous bacteria 32 times more potent against S. aureus, 600 times more potent against E. faecalis, and 1,200 times more potent against S. pneumoniae.
Sello was a bit surprised by the dramatic increase in ADEP potency compared to the much more modest improvement in ClpP binding.
"We found that the most potent ADEP analog binds ClpP seven-fold better than the parent compound, yet it has 1,200-fold better antibacterial activity," Sello said. "We believe that some of the increase in potency may stem from the fact that the rigidified ADEPs bind ClpP more tightly and have an enhanced capacity to cross the cell membrane. The improved cell permeability of the ADEP analogs is consistent with reports in the literature that molecules with strong intramolecular hydrogen bonds are particularly good at
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