Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.
- MeSH
- antifungální látky chemie metabolismus farmakologie MeSH
- aspartátové proteasy antagonisté a inhibitory genetika metabolismus MeSH
- Cryptococcus neoformans enzymologie MeSH
- fungální proteiny antagonisté a inhibitory genetika metabolismus MeSH
- HIV-proteasa chemie metabolismus MeSH
- HIV enzymologie MeSH
- houby účinky léků MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- preklinické hodnocení léčiv MeSH
- rekombinantní proteiny biosyntéza chemie izolace a purifikace MeSH
- simulace molekulární dynamiky MeSH
- substrátová specifita MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The polyhistidine tag (His-tag) is one of the most commonly used epitope tags in protein engineering. While His-tagged proteins can be detected reliably using immunological methods such as ELISA and Western blot, these methods are costly and time-intensive, necessitating more facile solutions for preliminary qualitative determination and concentration estimation. To this end, we present a rapid test strip assay based on iBody antibody mimetics that target the His-tag. We compare this strategy to commercial antibody-based assays and discuss the advantages and caveats of lateral flow assay design. Our test strip detected a panel of His-tagged proteins with different tag attachment strategies with a visual detection limit of 1 μM and densitometric detection limit of 0.5 μM. Due to its chemical nature, the presented assay exhibits wide reagent compatibility in comparison to antibody-based assays.
Glutamate carboxypeptidase II (GCPII) is a membrane protease overexpressed by prostate cancer cells and detected in the neovasculature of most solid tumors. Targeting GCPII with inhibitor-bearing nanoparticles can enable recognition, imaging, and delivery of treatments to cancer cells. Compared to methods based on antibodies and other large biomolecules, inhibitor-mediated targeting benefits from the low molecular weight of the inhibitor molecules, which are typically stable, easy-to-handle, and able to bind the enzyme with very high affinity. Although GCPII is established as a molecular target, comparing previously reported results is difficult due to the different methodological approaches used. In this work, we investigate the robustness and limitations of GCPII targeting with a diverse range of inhibitor-bearing nanoparticles (various structures, sizes, bionanointerfaces, conjugation chemistry, and surface densities of attached inhibitors). Polymer-coated nanodiamonds, virus-like particles based on bacteriophage Qβ and mouse polyomavirus, and polymeric poly(HPMA) nanoparticles with inhibitors attached by different means were synthesized and characterized. We evaluated their ability to bind GCPII and interact with cancer cells using surface plasmon resonance, inhibition assay, flow cytometry, and confocal microscopy. Regardless of the diversity of the investigated nanosystems, they all strongly interact with GCPII (most with low picomolar Ki values) and effectively target GCPII-expressing cells. The robustness of this approach was limited only by the quality of the nanoparticle bionanointerface, which must be properly designed by adding a sufficient density of hydrophilic protective polymers. We conclude that the targeting of cancer cells overexpressing GCPII is a viable approach transferable to a broad diversity of nanosystems.
- MeSH
- antigeny povrchové metabolismus MeSH
- antitumorózní látky aplikace a dávkování MeSH
- chemie farmaceutická MeSH
- glutamátkarboxypeptidasa II antagonisté a inhibitory metabolismus MeSH
- hydrofobní a hydrofilní interakce MeSH
- inhibitory enzymů aplikace a dávkování MeSH
- lidé MeSH
- ligandy MeSH
- nádorové buněčné linie MeSH
- nádory farmakoterapie patologie MeSH
- nanokonjugáty chemie MeSH
- rekombinantní proteiny metabolismus MeSH
- syntetická chemie okamžité shody MeSH
- thiazolidiny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
