The development of novel inhibitors for the treatment of sepsis

<div>Sepsis is a potentially dangerous disease that is caused by the response of the body to</div><div>an infection. Its major cause is the invasion of microorganisms in the sterile regions of the</div><div>body. In septic patients, platelets are activated by the bacteria leading to haemostatic</div><div>complications including thrombocytopenia and associated bleeding complications. With</div><div>the widespread development of antibiotic resistance, we have explored the development</div><div>of inhibitors that prevent host colonization as well as inhibitors of platelet activation that</div><div>prevent the bacterial platelet interaction.</div><div><br></div><div><b>Aims: </b>The aim of this study was to test drugs that have the potential to aid the treatment of</div><div>sepsis. These drugs were previously identified as potentially targeting the FcgRIIa through</div><div>computational modelling. As FcgRIIa activation is necessary for both Gram-positive and</div><div>Gram-negative bacterial-induced platelet activation, this project was designed to</div><div>determine their efficacy ex vivo for E. coli and S. aureus. We further explored the</div><div>mechanism of action of a colonisation inhibitor allantodapsone.</div><div><br></div><div><b>Methods: </b>We used bacterial induced platelet aggregation assays in platelet-rich plasma (PRP) and</div><div>single loss of platelets in PRP, to assess approved drugs for activity as well as ADP</div><div>induced aggregation assays for specificity studies. We also tested a novel compound for</div><div>binding to the S. aureus surface protein ClfB using isothermal calorimetry.</div><div><br></div><div><b>Results: </b>46 drugs were identified as potential inhibitors of the platelet FcgRIIa. The testing of</div><div>porfimer, rifaximin, dactinomycin (actinomycin), rifampin, methotrexate and doxycycline</div><div>significantly inhibited S. aureus Newman as well as E. coli O157-induced platelet</div><div>compared to their vehicle control at 100 μM. Ritonavir and vinorelbine were only found to</div><div>inhibit E. coli O157-induced aggregation at this concentration. All other drugs did not show</div><div>a significant difference subsequent to treatment at a high concentration. Porfimer and</div><div>actinomycin were the most potent inhibiters with IC50’s of 11.4 μM and 37.7 μM, respectively for S. aureus and 11.6 μM and 36.5 μM, respectively for E. coli induced</div><div>platelet aggregation. Rifaximin, Rifampin and methotrexate had slightly lower IC50’s for E.</div><div>coli compared to S. aureus induced aggregations (E. coli: 64.8 μM, 79.5 μM, 66.8 μM; S.</div><div>aureus: 70 μM, 107.2 μM, 103.1 μM, respectively). Furthermore, doxycycline inhibited E.</div><div>coli induced aggregation with an IC50 of 64.6 μM. Porfimer and actinomycin inhibited ADP</div><div>induced platelet aggregation, suggesting that their mechanism is not specific.</div><div>Furthermore, porfimer led to a loss of single platelets in a single loss of platelets assay.</div><div>This suggests that porfimer has agonistic activity and is therefore not suitable for</div><div>repurposing.</div><div>We further explored the binding of allantodapsone to a S. aureus surface protein, ClfB.</div><div>The interaction of soluble allantodapsone with recombinant ClfB N123 domain was</div><div>explored using isothermal calorimetry. The allantodapsone was used at 150 μM in 10%</div><div>DMSO with a range of rClfB concentrations. 15μM and 22μM, demonstrated energy</div><div>release with each injection indicating binding, however the KD could not be reliably</div><div>calculated as the experiments did not reach saturation.</div><div><b><br></b></div><div><b>Conclusion: </b>Screening of 42 drugs identified four drugs that inhibited S. aureus and E. coli-induced</div><div>platelet aggregation ex vivo. These are methotrexate, doxycycline, rifampin, and rifaximin.</div><div>Doxycycline is toxic at the doses required for inhibition of the FcgRIIa and is therefore not</div><div>a suitable candidate. Rifaximin is not suitable for intravenous delivery and is not absorbed</div><div>in the gastrointestinal tract, and therefore is also not a viable candidate. Therefore,</div><div>methotrexate and rifampin should be explored further for repurposing.</div>