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Hiroshi Fukui, Hideto Kawaratani, Kosuke Kaji, Hiroaki Takaya, Hitoshi Yoshiji Department of Gastroenterology, Endocrinology and Metabolism, Nara Medical University, Nara, Japan Abstract: Among the various risky complications of liver cirrhosis, refractory ascites is associated with poor survival of cirrhotics and persistently worsens their quality of life (QOL). Major clinical guidelines worldwide define refractory ascites as ascites that cannot be managed by medical therapy either because of a lack of response to maximum doses of diuretics or because patients develop complications related to diuretic therapy that preclude the use of an effective dose of diuretics. Due to the difficulty in receiving a liver transplantation (LT), the ultimate solution for refractory ascites, most cirrhotic patients have selected the palliative therapy such as repeated serial paracentesis, transjugular intrahepatic portosystemic shunt, or peritoneovenous shunt to improve their QOL. During the past several decades, new interventions and methodologies, such as indwelling peritoneal catheter, peritoneal-urinary drainage, and cell-free and concentrated ascites reinfusion therapy, have been introduced. In addition, new medical treatments with vasoconstrictors or vasopressin V2 receptor antagonists have been proposed. Both the benefits and risks of these old and new modalities have been extensively studied in relation to the pathophysiological changes in ascites formation. Although the best solution for refractory ascites is to eliminate hepatic failure either by LT or by causal treatment, the selection of the best palliative therapy for individual patients is of utmost importance, aiming at achieving the longest possible, comfortable life. This review briefly summarizes the changing landscape of variable treatment modalities for cirrhotic patients with refractory ascites, aiming at clarifying their possibilities and limitations. Evolving issues with regard to the impact of gut-derived systemic and local infection on the clinical course of cirrhotic patients have paved the way for the development of a new gut microbiome-based therapeutics. Thus, it should be further investigated whether the early therapeutic approach to gut dysbiosis provides a better solution for the management of cirrhotic ascites. Keywords: pathophysiology, nonselective beta-blockers, V2 receptor antagonists, large-volume paracentesis, peritoneovenous shunt, transjugular intrahepatic portosystemic shunt, antibiotics

Background: It has been shown that expression of the potent angiogenic factor, vascular endothelial growth factor (VEGF), and its receptors, fit-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), increased during the development of [...]

Protein-protein interactions (PPIs) play a crucial role in most biological processes and are important targets in the development of therapeutic agents. However, small molecule drug discovery that targets PPIs remains very challenging. Targeting hot spot residues is considered the best option for inhibiting such interactions, but there are few examples of how knowledge of hot spots can be used in high throughput screening to find hit compounds. A substrate adaptor protein for a ubiquitin ligase complex, Kelch-like ECH-associated protein 1 (Keap1), negatively modulates the expression of genes involved in cellular protection against oxidative stress. Here, we focused on three arginine hot spot residues in the Keap1 substrate binding pocket (Arg380, Arg415, and Arg483), and screened the carboxylic acid library owned by Japan Tobacco Inc. for compounds that interact with the arginine residues in differential scanning fluorescence assays. Furthermore, we identified several small molecule compounds that specifically bind to the Keap1 Kelch domain hot spots by comparing binding to alanine mutant proteins (R380A, R415A, and R483A) with binding to the wild-type protein using surface plasmon resonance (SPR) screening. These compounds inhibited the protein-protein interaction between the Keap1 Kelch domain and the nuclear factor erythroid 2-related factor 2 (Nrf2) peptide, and the ubiquitination of Nrf2 catalyzed by the Cul3/RINGBox 1 E3 ligase. In addition, the binding mode of one compound (Compound 4) was determined by X-ray crystallography after validation of binding by isothermal titration calorimetry, native mass spectrometry, and nuclear magnetic resonance. Compound 4 had favorable thermodynamic properties, and noncovalently bound to Keap1 with a stoichiometry of 1:1. Our results suggest that Compound 4 could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions such as oxidative stress response, inflammation, and carcinogenesis. We believe that the use of a set of complementary biophysical techniques including the SPR assay with single alanine mutant of hot spots provides opportunities to identify hit compounds for developing inhibitors of PPIs.

Proteins fold into their most stable yet dynamic structures that enable their functions and exist in a balance between stability and instability. Protein folding and binding can be evaluated based on their thermodynamics determined using various methods. In this study, we analyzed the folding and binding thermodynamics of the C-terminal Src homology 2 domain (cSH2) of phosphoinositide 3-kinase and its Cys mutants, C656S, C659V, C670L, and C656S/C659V/C670L. The protein binds to the consensus sequence including phosphorylated Tyr, and the binding affinity to CD28-derived phosphopeptides decreased upon the Cys mutation: wild-type > C656S > C659V > C670L > C656S/C659V/C670L. The decreased affinity was mainly due to the increased binding enthalpy change. The thermal stability analysis showed the opposite tendency: wild-type < C656S < C659V < C670L < C656S/C659V/C670L, indicating a trade-off in protein architecture between stability and binding function. Based on the crystal structures of the cSH2 wild-type and C656S/C659V/C670L, the increased stability could be explained by the increased hydrophobic contacts. The wild-type protein would have evolved to have a native structure while maintaining the dynamics to express its binding function. Furthermore, a unique unfolding transition was observed for the cSH2 wild-type, possibly due to Cys oxidation, which was significantly changed upon the single mutation of Cys residues. We also demonstrated the validity of various methods for analyzing protein stability.