Immune checkpoint blockade (ICB) using monoclonal antibodies against programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) is the treatment of choice for cancer immunotherapy. However, low tissue permeability, immunogenicity, immune-related adverse effects, and high cost could be possibly improved using alternative approaches. On the other hand, synthetic low-molecular-weight (LMW) PD-1/PD-L1 blockers have failed to progress beyond in vitro studies, mostly due to low binding affinity or poor pharmacological characteristics resulting from their limited solubility and/or stability. Here, we report the development of polymer-based anti-human PD-L1 antibody mimetics (α-hPD-L1 iBodies) by attaching the macrocyclic peptide WL12 to a N-(2-hydroxypropyl)methacrylamide copolymer. We characterized the binding properties of iBodies using surface plasmon resonance, enzyme-linked immunosorbent assay, flow cytometry, confocal microscopy, and a cellular ICB model. We found that the α-hPD-L1 iBodies specifically target human PD-L1 (hPD-L1) and block the PD-1/PD-L1 interaction in vitro, comparable to the atezolizumab, durvalumab, and avelumab licensed monoclonal antibodies targeting PD-L1. Our findings suggest that iBodies can be used as experimental tools to target hPD-L1 and could serve as a platform to potentiate the therapeutic effect of hPD-L1-targeting small molecules by improving their affinity and pharmacokinetic properties.
Aggregation of small neuronal protein α-synuclein (αSyn) in amyloid fibrils is considered to be one of the main causes of Parkinson's disease. Inhibition of this aggregation is a promising approach for disease treatment. Dozens of compounds able to inhibit αSyn fibrillization in solution were developed during the last decade. However, the applicability of most of them in the cellular environment was not established because of the absence of a suitable cell-based assay. In this work, we developed an assay for testing αSyn aggregation inhibitors in cells that is based on fluorescence resonance energy transfer (FRET) between labeled αSyn molecules in fibrils. The assay directly reports the amount of fibrillized αSyn and is more reliable than the assays based on cell viability. Moreover, we showed that cell viability decline does not always correlate with the amount of misfolded αSyn. The developed FRET-based assay does not interfere with the aggregation process and is suitable for high-throughput testing of αSyn aggregation inhibitors. Its application can sort out non-specific inhibitors and thus significantly facilitate the development of drugs for Parkinson`s disease.
- MeSH
- alfa-synuklein analýza antagonisté a inhibitory metabolismus MeSH
- benzodioxoly farmakologie MeSH
- elektroporace metody MeSH
- HeLa buňky MeSH
- intracelulární tekutina chemie účinky léků metabolismus MeSH
- lidé MeSH
- proteinové agregáty účinky léků fyziologie MeSH
- pyrazoly farmakologie MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- viabilita buněk účinky léků fyziologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
