Genome-wide forward genetic screening to identify receptors and proteins mediating nanoparticle uptake and intracellular processing.

Understanding how cells process nanoparticles is crucial to optimize nanomedicine efficacy. However, characterizing cellular pathways is challenging, especially if non-canonical mechanisms are involved. In this Article a genome-wide forward genetic screening based on insertional mutagenesis is applied to discover receptors and proteins involved in the intracellular accumulation (uptake and intracellular processing) of silica nanoparticles. The nanoparticles are covered by a human serum corona known to target the low-density lipoprotein receptor (LDLR). By sorting cells with reduced nanoparticle accumulation and deep sequencing after each sorting, 80 enriched genes are identified. We find that, as well as LDLR, the scavenger receptor SCARB1 also mediates nanoparticle accumulation. Additionally, heparan sulfate acts as a specific nanoparticle receptor, and its role varies depending on cell and nanoparticle type. Furthermore, some of the identified targets affect nanoparticle trafficking to the lysosomes. These results show the potential of genetic screening to characterize nanoparticle pathways. Additionally, they indicate that corona-coated nanoparticles are internalized via multiple receptors.

Induced protein degradation of histone deacetylases 3 (HDAC3) by proteolysis targeting chimera (PROTAC).

Histone deacetylases (HDACs) play important roles in inflammatory diseases like asthma and chronic obstructive pulmonary disease (COPD). Unravelling of and interfering with the functions of specific isoenzymes contributing to inflammation provides opportunities for drug development. Here we synthesize proteolysis targeting chimeras (PROTACs) for degradation of class I HDACs in which o-aminoanilide-based class I HDAC inhibitors are tethered to the cereblon ligand pomalidomide. One of these PROTACs, denoted HD-TAC7, showed promising degradation effects for HDAC3 with a DC50 value of 0.32 µM. In contrast to biochemical evidence using siRNA, HD-TAC7 showed a minimal effect on gene expression in LPS/IFNγ-stimulated RAW 264.7 macrophages. The lack of effect can be attributed to downregulation of the NF-κB subunit p65, which is a known side effect of pomalidomide treatment. Altogether, we describe a novel PROTAC that enables selective downregulation of HDAC3 levels, however we note that concomitant downregulation of the NF-κB subunit p65 can confound the biological outcome.

Targeting HDAC Complexes in Asthma and COPD.

Around three million patients die due to airway inflammatory diseases each year. The most notable of these diseases are asthma and chronic obstructive pulmonary disease (COPD). Therefore, new therapies are urgently needed. Promising targets are histone deacetylases (HDACs), since they regulate posttranslational protein acetylation. Over a thousand proteins are reversibly acetylated, and acetylation critically influences aberrant intracellular signaling pathways in asthma and COPD. The diverse set of selective and non-selective HDAC inhibitors used in pre-clinical models of airway inflammation show promising results, but several challenges still need to be overcome. One such challenge is the design of HDAC inhibitors with unique selectivity profiles, such as selectivity towards specific HDAC complexes. Novel strategies to disrupt HDAC complexes should be developed to validate HDACs further as targets for new anti-inflammatory pulmonary treatments.