Protein aggregation is a phenomenon that has attracted considerable attention within the pharmaceutical industry from both a developability standpoint (to ensure stability of protein formulations) and from a research perspective for neurodegenerative diseases. Experimental identification of aggregation behavior in proteins can be expensive; and hence, the development of accurate computational approaches is crucial. The existing methods for predicting protein aggregation rely mostly on the primary sequence and are typically trained on amyloid-like proteins. However, the training bias toward beta amyloid peptides may worsen prediction accuracy of such models when applied to larger protein systems. Here, we present a novel algorithm to identify aggregation-prone regions in proteins termed "AggScore" that is based entirely on three-dimensional structure input. The method uses the distribution of hydrophobic and electrostatic patches on the surface of the protein, factoring in the intensity and relative orientation of the respective surface patches into an aggregation propensity function that has been trained on a benchmark set of 31 adnectin proteins. AggScore can accurately identify aggregation-prone regions in several well-studied proteins and also reliably predict changes in aggregation behavior upon residue mutation. The method is agnostic to an amyloid-specific aggregation context and thus may be applied to globular proteins, small peptides and antibodies.

Purpose Initial studies indicate that the newly developed hCMEC/D3 cell line may prove to be a useful model for studying the physiology of the human blood-brain barrier (BBB) endothelium. The purpose of this study was to assess the mRNA expression of several ABC and SLC transporters, with an emphasis on the Proton-Coupled Oligopeptide Transporter Superfamily (POT) transporters in this immortalized BBB cell model. The transport kinetics of POT-substrates was also evaluated. Methods The hCMEC/D3 cell line was maintained in a modified EGM-2 medium in collagenated culture flasks and passaged every 3–4 days at approximately 85%–95% confluence. Messenger RNA (mRNA) expression of a variety of ABC and SLC transporters was evaluated using qRT-PCR arrays, while additional qRT-PCR primers were designed to assess the expression of POT members. The transport kinetics of mannitol and urea were utilized to quantitatively estimate the intercellular pore radius, while POT substrate transport was also determined to assess the suitability of the cell model from a drug screening perspective. Optimization of the cell line was attempted by culturing with on laminin and fibronectin enhanced collagen and in the presence of excess Ca2+. Results HCMEC/D3 cells express both hPHT1 and hPHT2, while little to no expression of either hPepT1 or hPepT2 was observed. The relative expression of other ABC and SLC transporters is discussed. While POT substrate transport does suggest suitability for BBB drug permeation screening, the relative intercellular pore radius was estimated at 19Å, significantly larger than that approximated in vivo. Culturing with extracellular matrix proteins did not alter mannitol permeability. Conclusion These studies characterized this relevant human hCMEC/D3 BBB cell line with respect to both the relative mRNA expression of various ABC and SLC transporters, and its potential utility as an in vitro screening tool for brain permeation. Additional studies are required to adequately determine the potential to establish an in vivo correlation.