Virtual Screening Uncovers DspS Activators That Disperse <i>Pseudomonas aeruginosa</i> Biofilms

<i>Pseudomonas aeruginosa</i> is the predominant bacterium found in many chronic biofilm infections. Over the past few decades, biofilm-related infections have posed a significant challenge to medical practice due to the increasing emergence of multidrug resistance. <i>Cis</i>-2-decenoic acid (CDA), a small molecule found in <i>P. aeruginosa</i>, has been shown to disperse biofilms formed by various bacteria and to work in synergy with common antibiotics. Despite that, the binding mechanism between CDA and the predicted cyclases/histidine kinases associated sensory extracellular (CHASE) domain of sensor protein DspS remains unknown in the absence of a crystallized protein structure. Moreover, the therapeutic potential of CDA is limited by its susceptibility to oxidative degradation and isomerization. In this work, we propose a structural model for the DspS CHASE domain. The resulting model displays an overall topology reminiscent of the sensor protein PcrK in <i>Xanthomonas campestris</i>. Through molecular dynamics simulations, a stable potential binding site for CDA was further identified. Virtual screening against the predicted site of DspS CHASE using our developed pipeline discovered two promising compounds, compounds <b>2</b> and <b>9</b>, capable of dislodging 7-day <i>P. aeruginosa</i> biofilms at 50 μM without affecting bacterial growth. These compounds also enhanced the effects of ciprofloxacin against <i>P. aeruginosa</i>, reduced the survival of dispersed cells, and increased the expression of matrix-degrading enzyme genes <i>pelA</i>, <i>pslG</i>, and <i>eddA</i>. This study provides insights into CDA recognition by DspS and represents the first large-scale effort to uncover first-in-class DspS activators. At the same time, this work also underscores the effectiveness of a computational-aided drug discovery process in finding new activators, even without a known protein structure.