The development of novel inhibitors for the treatment of sepsis - Afnan Ali.pdf (8.21 MB)

The development of novel inhibitors for the treatment of sepsis

Download (8.21 MB)
thesis
posted on 25.01.2021, 09:32 by Afnan Ali
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.

History

First Supervisor

Dr. Marian Brennan

Comments

A thesis submitted for the degree of Master of Science from the Royal College of Surgeons in Ireland in 2020.

Published Citation

Afnan A, The development of novel inhibitors for the treatment of sepsis [MSc Thesis]. Dublin: Royal College of Surgeons in Ireland; 2021

Degree Name

Master of Science (MSc): Research

Date of award

31/05/2020

Programme

  • Master of Science (MSc): Research

Usage metrics

Theses and Dissertations

Exports