The development of novel inhibitors for the treatment of sepsis

Sepsis is a potentially dangerous disease that is caused by the response of the body to

an infection. Its major cause is the invasion of microorganisms in the sterile regions of the

body. In septic patients, platelets are activated by the bacteria leading to haemostatic

complications including thrombocytopenia and associated bleeding complications. With

the widespread development of antibiotic resistance, we have explored the development

of inhibitors that prevent host colonization as well as inhibitors of platelet activation that

prevent the bacterial platelet interaction.

Aims: The aim of this study was to test drugs that have the potential to aid the treatment of

sepsis. These drugs were previously identified as potentially targeting the FcgRIIa through

computational modelling. As FcgRIIa activation is necessary for both Gram-positive and

Gram-negative bacterial-induced platelet activation, this project was designed to

determine their efficacy ex vivo for E. coli and S. aureus. We further explored the

mechanism of action of a colonisation inhibitor allantodapsone.

Methods: We used bacterial induced platelet aggregation assays in platelet-rich plasma (PRP) and

single loss of platelets in PRP, to assess approved drugs for activity as well as ADP

induced aggregation assays for specificity studies. We also tested a novel compound for

binding to the S. aureus surface protein ClfB using isothermal calorimetry.

Results: 46 drugs were identified as potential inhibitors of the platelet FcgRIIa. The testing of

porfimer, rifaximin, dactinomycin (actinomycin), rifampin, methotrexate and doxycycline

significantly inhibited S. aureus Newman as well as E. coli O157-induced platelet

compared to their vehicle control at 100 μM. Ritonavir and vinorelbine were only found to

inhibit E. coli O157-induced aggregation at this concentration. All other drugs did not show

a significant difference subsequent to treatment at a high concentration. Porfimer and

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

platelet 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 ADP

induced 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 for

repurposing.

We further explored the binding of allantodapsone to a S. aureus surface protein, ClfB.

The interaction of soluble allantodapsone with recombinant ClfB N123 domain was

explored using isothermal calorimetry. The allantodapsone was used at 150 μM in 10%

DMSO with a range of rClfB concentrations. 15μM and 22μM, demonstrated energy

release with each injection indicating binding, however the KD could not be reliably

calculated as the experiments did not reach saturation.

Conclusion: Screening of 42 drugs identified four drugs that inhibited S. aureus and E. coli-induced

platelet 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 not

a suitable candidate. Rifaximin is not suitable for intravenous delivery and is not absorbed

in the gastrointestinal tract, and therefore is also not a viable candidate. Therefore,

methotrexate and rifampin should be explored further for repurposing.