Lesinurad, a novel, oral compound for gout, acts to decrease serum uric acid through inhibition of urate transporters in the kidney.

BackgroundExcess body burden of uric acid promotes gout. Diminished renal clearance of uric acid causes hyperuricemia in most patients with gout, and the renal urate transporter (URAT)1 is important for regulation of serum uric acid (sUA) levels. The URAT1 inhibitors probenecid and benzbromarone are used as gout therapies; however, their use is limited by drug-drug interactions and off-target toxicity, respectively. Here, we define the mechanism of action of lesinurad (Zurampic®; RDEA594), a novel URAT1 inhibitor, recently approved in the USA and Europe for treatment of chronic gout.MethodssUA levels, fractional excretion of uric acid (FEUA), lesinurad plasma levels, and urinary excretion of lesinurad were measured in healthy volunteers treated with lesinurad. In addition, lesinurad, probenecid, and benzbromarone were compared in vitro for effects on urate transporters and the organic anion transporters (OAT)1 and OAT3, changes in mitochondrial membrane potential, and human peroxisome proliferator-activated receptor gamma (PPARγ) activity.ResultsAfter 6 hours, a single 200-mg dose of lesinurad elevated FEUA 3.6-fold (p < 0.001) and reduced sUA levels by 33 % (p < 0.001). At concentrations achieved in the clinic, lesinurad inhibited activity of URAT1 and OAT4 in vitro, did not inhibit GLUT9, and had no effect on ABCG2. Lesinurad also showed a low risk for mitochondrial toxicity and PPARγ induction compared to benzbromarone. Unlike probenecid, lesinurad did not inhibit OAT1 or OAT3 in the clinical setting.ConclusionThe pharmacodynamic effects and in vitro activity of lesinurad are consistent with inhibition of URAT1 and OAT4, major apical transporters for uric acid. Lesinurad also has a favorable selectivity and safety profile, consistent with an important role in sUA-lowering therapy for patients with gout

Determination of the phosphorylation level and deamidation susceptibility of equine beta-casein

beta-Casein was isolated from Haflinger mare’s milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine b-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare’s b-casein (25 514 6 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27–34) encoded by an exon sometimes out-spliced (25 511.40 Da). Hence, the beta-casein isolated from Haflinger mare’s milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi-phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2-DE, ranging from 4.74 to 5.30.Moreover, the equine beta-casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif 135Asn-Gly136. Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions

Characterization of isoforms of equine alpha-s1 casein: post-transcriptional variants and phosphorylation post-translational variants

 Characterization of isoforms of equine alpha-s1 casein: post-transcriptional variants and phosphorylation post-translational variants. 1. International Symposium on Minerals and Dairy Product

Caractérisation des isoformes de la caséine alpha-s1 équine : variants post-transcriptionnels et post-traductionnels

 Caractérisation des isoformes de la caséine alpha-s1 équine : variants post-transcriptionnels et post-traductionnels. Séminaire de l'Ecole doctorale sciences et ingénierie Ressources Procédés Produits Environnement RP2

Equine alpha S1-casein: Characterization of alternative splicing isoforms and determination of phosphorylation levels

αS1-Casein was isolated from Haflinger mare’s milk by hydrophobic interaction chromatography and displayed great micro-heterogeneity by 2-dimensional electrophoresis, probably because of a variable degree of phosphorylation and alternative splicing events. The aim of the present work was to investigate the complexity of the mare’s αS1-casein. The different isoforms present in milk were submitted to a double treatment of dephosphorylation, first by using alkaline phosphatase and then acid phosphatase to achieve complete dephosphorylation. The apoforms were then analyzed by electrospray ionization mass spectrometry. The results revealed the existence of a full-length protein and 7 variants resulting from posttranscriptional modifications; that is, exon skipping involving exon 7, exon 14, or both and use of a cryptic splice site encoding a glutamine residue. The determination of the different phosphorylation degrees of the native isoforms of αS1- casein was finally achieved by electrospray ionization mass spectrometry analysis after fractionation of the isoforms by ion-exchange chromatography. Thus, 36 different variants of equine αS1-casein were identified with several phosphate groups ranging from 2 to 6 or 8 depending on whether exon 7 was skipped. Key words: alternative splicing , cryptic splice site , equine αS1-casein , phosphorylatio