UC San Diego
Direct and Indirect Targeting of Amyloid Fibrils with Small Molecules and Polymeric Nanoparticles to Inhibit Disease Transmission and Progression
- Author(s): Sheik, Daniel
- Advisor(s): Yang, Jerry
- et al.
This dissertation focuses on targeting amyloid aggregates as a means to inhibit disease transmission, as with semen-derived enhancer of virus infection (SEVI), or disease progression, as with β amyloid (Aβ). The main focus has been to use polymeric nanoparticles to inhibit enhanced human immunodeficiency virus (HIV) infection in the presence of SEVI amyloid fibrils. By incorporating amyloid-binding benzothiazole anline (BTA) moieties into
polyacrylate scaffolds, we were able to bind SEVI fibrils and inhibit SEVI-mediated HIV infection at low concentrations. We formulated the amyloid-binding polymers into discreet spherical nanoparticles of comparable size to HIV virions (~150 nm) in order to analyze the effects of steric hindrance on SEVI-mediated HIV infection. The amyloid-binding nanoparticles we developed were shown to inhibit SEVI-mediated HIV infection better than any previously studied SEVI-neutralizing agent. Our results suggest that the improvement in activity against SEVI-mediated HIV infection is due to the increased size of the amyloid-binding nanoparticles relative to previously described small molecules and oligomers. From these results, we believe that we have introduced steric repulsion as a factor possibly as important as binding affinity in the inhibition of SEVI-mediated enhancement of HIV infection.
Further studies in the area of SEVI-HIV interactions have investigated denaturation of the secondary structure of SEVI fibrils to remove potential binding sites to HIV. Using hydrophobic acrylate-based polymers we were able to show that the β-sheet content of preformed SEVI fibrils was decreased by approximately half that of the native β-sheet content. Through our analyses, we were able to correlate the presence of hydrophobic β-sheets in SEVI fibrils with enhanced Thioflavin T (ThT) fluorescence after binding SEVI to fibrils as well as enhancement of HIV infection in the presence of SEVI. The relationship between HIV infection activity and hydrophobic β-sheet content suggests that HIV may bind to SEVI fibrils in hydrophobic pockets composed of β-sheets, similar to the mechanism of ThT binding. To apply this hydrophobic system in cellular HIV infection assays, the hydrophobic polymers were formulated into nanoparticles of controlled size and suspended in aqueous solution. The results of HIV infection assays suggest that we are able to denature the secondary structure of SEVI fibrils in solution and thereby inhibit SEVI-mediated HIV infection by over 60%. This is the first application of denaturation of SEVI fibrils as a mechanism to inhibit enhanced HIV infection to our knowledge.
Further research in the field of amyloid-related diseases has directed our focus to neurodegeneration and its relationship to amyloidogenic peptide aggregates. It has been previously determined that oligomers and aggregates of Aβ are toxic to neuronal cells, leading to neurodegeneration. Several neuronal receptors have been implicated in the clearance of such toxic amyloid aggregates. In an effort to inhibit Aβ toxicity in neurons we have designed small molecule analogs of agonists of these neuroprotective receptors. We have conducted a Structure-Activity Relationship (SAR) study to guide our synthetic efforts and have been able to develop fluorescently tagged agonists of two receptors, TrkB and SSTR4. We are seeking to quantify the binding affinities and activation of the receptors using these fluorescent binders. By targeting receptors implicated in neuroprotective processes we are seeking to prevent the progression of neurodegenerative diseases and introduce new treatments for these diseases.