Characterization of Gain-of-Function Mutant Provides New Insights into ClpP Structure

ATP-dependent Clp protease (ClpP), a highly conserved serine protease in vast bacteria, could be converted into a noncontrollable enzyme capable of degrading mature proteins in the presence of acyldepsipeptides (ADEPs). Here, we design such a gain-of-function mutant of <i>Staphylococcus aureus</i> ClpP (<i>Sa</i>ClpP) capable of triggering the same level of dysfunctional activity that occurs upon ADEPs treatment. The <i>Sa</i>ClpPY63A mutant degrades FtsZ <i>in vivo</i> and inhibits staphylococcal growth. The crystal structure of <i>Sa</i>ClpPY63A indicates that Asn42 would be an important domino to fall for further activation of ClpP. Indeed, the <i>Sa</i>ClpPN42AY63A mutant demonstrates promoted self-activated proteolysis, which is a result of an enlarged entrance pore as observed in cryo-electron microscopy images. In addition, the expression of the engineered <i>clpP</i> allele phenocopies treatment with ADEPs; inhibition of cell division occurs as does showing sterilizing with rifampicin antibiotics. Collectively, we show that the gain-of-function <i>Sa</i>ClpPN42AY63A mutant becomes a fairly nonspecific protease and kills persisters by degrading over 500 proteins, thus providing new insights into the structure of the ClpP protease

Characterization of Gain-of-Function Mutant Provides New Insights into ClpP Structure

ATP-dependent Clp protease (ClpP), a highly conserved serine protease in vast bacteria, could be converted into a noncontrollable enzyme capable of degrading mature proteins in the presence of acyldepsipeptides (ADEPs). Here, we design such a gain-of-function mutant of <i>Staphylococcus aureus</i> ClpP (<i>Sa</i>ClpP) capable of triggering the same level of dysfunctional activity that occurs upon ADEPs treatment. The <i>Sa</i>ClpPY63A mutant degrades FtsZ <i>in vivo</i> and inhibits staphylococcal growth. The crystal structure of <i>Sa</i>ClpPY63A indicates that Asn42 would be an important domino to fall for further activation of ClpP. Indeed, the <i>Sa</i>ClpPN42AY63A mutant demonstrates promoted self-activated proteolysis, which is a result of an enlarged entrance pore as observed in cryo-electron microscopy images. In addition, the expression of the engineered <i>clpP</i> allele phenocopies treatment with ADEPs; inhibition of cell division occurs as does showing sterilizing with rifampicin antibiotics. Collectively, we show that the gain-of-function <i>Sa</i>ClpPN42AY63A mutant becomes a fairly nonspecific protease and kills persisters by degrading over 500 proteins, thus providing new insights into the structure of the ClpP protease

Discovery of Novel Small Molecule Inhibitors of Dengue Viral NS2B-NS3 Protease Using Virtual Screening and Scaffold Hopping

By virtual screening, compound <b>1</b> was found to be active against NS2B-NS3 protease (IC₅₀ = 13.12 ± 1.03 μM). Fourteen derivatives (<b>22</b>) of compound <b>1</b> were synthesized, leading to the discovery of four new inhibitors with biological activity. In order to expand the chemical diversity of the inhibitors, small-molecule-based scaffold hopping was performed on the basis of the common scaffold of compounds <b>1</b> and <b>22</b>. Twenty-one new compounds (<b>23</b>, <b>24</b>) containing quinoline (new scaffold) were designed and synthesized. Protease inhibition assays revealed that 12 compounds with the new scaffold are inhibitors of NS2B-NS3 protease. Taken together, 17 new compounds were discovered as NS2B-NS3 protease inhibitors with IC₅₀ values of 7.46 ± 1.15 to 48.59 ± 3.46 μM, and 8 compounds belonging to two different scaffolds are active to some extent against DENV based on luciferase reporter replicon-based assays. These novel chemical entities could serve as lead structures for discovering therapies against DENV