Albumin domain mutants with enhanced Aß binding capacity identified by phage display analysis for application in various peripheral Aß elimination approaches of Alzheimer's disease treatment.

Deposition of amyloid protein, particularly Aß1-42 , is a major contributor to the onset of Alzheimer's disease (AD). However, almost no deposition of Aß in the peripheral tissues could be found. Human serum albumin (HSA), the most abundant protein in the blood, has been reported to inhibit amyloid formation through binding Aß, which is believed to play an important role in the peripheral clearance of Aß. We identified the Aß binding site on HSA and developed HSA mutants with high binding capacities for Aß using a phage display method. HSA fragment 187-385 (Domain II) was found to exhibit the highest binding capacity for Aß compared with the other two HSA fragments. To elucidate the sequence that forms the binding site for Aß on Domain II, a random screening of Domain II display phage biopanning was constructed. A number of mutants with higher Aß binding capacities than the wild type were identified. These mutants exhibited stronger scavenging abilities than the wild type, as revealed via in vitro equilibrium dialysis of Aß experiments. These findings provide useful basic data for developing a safer alternative therapy than Aß vaccines and for application in plasma exchange as well as extracorporeal dialysis.

Acute Kidney Injury Caused by Rhabdomyolysis Is Ameliorated by Serum Albumin-Based Supersulfide Donors through Antioxidative Pathways.

Oxidative stress is responsible for the onset and progression of various kinds of diseases including rhabdomyolysis-induced acute kidney injury (AKI). Antioxidants are, therefore, thought to aid in the recovery of illnesses linked to oxidative stress. Supersulfide species have been shown to have substantial antioxidative activity; however, due to their limited bioavailability, few supersulfide donors have had their actions evaluated in vivo. In this study, human serum albumin (HSA) and N-acetyl-L-cysteine polysulfides (NACSn), which have polysulfides in an oxidized form, were conjugated to create a supersulfide donor. HSA is chosen to be a carrier of NACSn because of its extended blood circulation and high level of biocompatibility. In contrast to a supersulfide donor containing reduced polysulfide in HSA, the NACSn-conjugated HSAs exhibited stronger antioxidant activity than HSA and free NACSn without being uptaken by the cells in vitro. The supersulfide donor reduced the levels of blood urea nitrogen and serum creatinine significantly in a mouse model of rhabdomyolysis-induced AKI. Supersulfide donors significantly reduced the expression of oxidative stress markers in the kidney. These results indicate that the developed supersulfide donor has the therapeutic effect on rhabdomyolysis-induced AKI.

Analysis of the Binding of Aripiprazole to Human Serum Albumin: The Importance of a Chloro-Group in the Chemical Structure.

Aripiprazole (ARP), a quinolinone derivative, is an atypical antipsychotic drug that is used in the treatment of schizophrenia. ARP has an extensive distribution and more than 99% of the ARP and dehydro-ARP, the main active metabolite, is bound to plasma proteins. However, information regarding the protein binding of ARP is limited. In this study, we report on a systematic study of the protein binding of ARP. The interaction of ARP and structurally related compounds with human serum albumin (HSA) was examined using equilibrium dialysis, circular dichroism (CD) spectroscopy, fluorescent probe displacement, and an X-ray crystallographic analysis. The binding affinities (nK) for ARP and its main metabolite, dehydro-ARP with HSA were found to be significantly higher than other structurally related compounds. The results of equilibrium dialysis experiments and CD spectral data indicated that the chloro-group linked to the phenylpiperazine ring in the ARP molecule plays a major role in the binding of these ligands to HSA. Furthermore, fluorescent probe displacement results indicated that ARP appears to bind at the site II pocket in subdomain III. A detailed CD spectral analysis suggests that the chloro-group linked to the phenylpiperazine ring may control the geometry of the ARP molecule when binding in the site II binding pocket. X-ray crystallographic analysis of the ARP-HSA complex revealed that the distance between the chlorine atom at the 3-positon of dichlorophenyl-piperazine on ARP and the sulfur atom of Cys392 in HSA was 3.4-3.6 Å. A similar halogen bond interaction has also been observed in the HSA structure complexed with diazepam, which also contains a chloro-group. Thus, the mechanism responsible for the binding of ARP to a protein elucidated here should be relevant for assessing the pharmacokinetics and pharmacodynamics of ARP in various clinical situations and for designing new drugs.