The development of novel inhibitors for the treatment of sepsis
Creators: name: Afnan Ali
Publisher: name: Royal College of Surgeons in Ireland
Year: 2021
Subjects: subject: 111504 Pharmaceutical Sciences
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine

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