Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Structure of an Alkaline Pectate Lyase and Rational Engineering with Improved Thermo-Alkaline Stability for Efficient Ramie Degumming

Alkaline pectate lyases have biotechnological applications in plant fiber processing, such as ramie degumming. Previously, we characterized an alkaline pectate lyase from Bacillus clausii S10, named BacPelA, which showed potential for enzymatic ramie degumming because of its high cleavage activity toward methylated pectins in alkaline conditions. However, BacPelA displayed poor thermo-alkaline stability. Here, we report the 1.78 Å resolution crystal structure of BacPelA in apo form. The enzyme has the characteristic right-handed β-helix fold of members of the polysaccharide lyase 1 family and shows overall structural similarity to them, but it displays some differences in the details of the secondary structure and Ca2+-binding site. On the basis of the structure, 10 sites located in flexible regions and showing high B-factor and positive ΔTm values were selected for mutation, aiming to improve the thermo-alkaline stability of the enzyme. Following site-directed saturation mutagenesis and screening, mutants A238C, R150G, and R216H showed an increase in the T5015 value at pH 10.0 of 3.0 °C, 6.5 °C, and 7.0 °C, respectively, compared with the wild-type enzyme, interestingly accompanied by a 24.5%, 46.6%, and 61.9% increase in activity. The combined mutant R150G/R216H/A238C showed an 8.5 °C increase in the T5015 value at pH 10.0, and an 86.1% increase in the specific activity at 60 °C, with approximately doubled catalytic efficiency, compared with the wild-type enzyme. Moreover, this mutant retained 86.2% activity after incubation in ramie degumming conditions (4 h, 60 °C, pH 10.0), compared with only 3.4% for wild-type BacPelA. The combined mutant increased the weight loss of ramie fibers in degumming by 30.2% compared with wild-type BacPelA. This work provides a thermo-alkaline stable, highly active pectate lyase with great potential for application in the textile industry, and also illustrates an effective strategy for rational design and improvement of pectate lyases

Knockout of alanine racemase gene attenuates the pathogenicity of Aeromonas hydrophila

Abstract Background Aeromonas hydrophila is an opportunistic pathogen of poikilothermic and homoeothermic animals, including humans. In the present study, we described the role of Alanine racemase (alr-2) in the virulence of A. hydrophila using an alr-2 knockout mutant (A.H.Δalr). Results In mouse and common carp models, the survival of animals challenged with A.H.Δalr was significantly increased compared with the wild-type (WT), and the mutant was also impaired in its ability to replicate in the organs and blood of infected mice and fish. The A.H.Δalr significantly increased phagocytosis by macrophages of the mice and fish. These attenuation effects of alr-2 could be complemented by the addition of D-alanine to the A.H.Δalr strain. The histopathology results indicated that the extent of tissue injury in the WT-infected animals was more severe than in the A.H.Δalr-infected groups. The expression of 9 virulence genes was significantly down-regulated, and 3 outer membrane genes were significantly up-regulated in A.H.Δalr. Conclusions Our data suggest that alr-2 is essential for the virulence of A. hydrophila. Our findings suggested alanine racemase could be applied in the development of new antibiotics against A. hydrophila

Selection and characterization of alanine racemase inhibitors against Aeromonas hydrophila

Abstract Background Combining experimental and computational screening methods has been of keen interest in drug discovery. In the present study, we developed an efficient screening method that has been used to screen 2100 small-molecule compounds for alanine racemase Alr-2 inhibitors. Results We identified ten novel non-substrate Alr-2 inhibitors, of which patulin, homogentisic acid, and hydroquinone were active against Aeromonas hydrophila. The compounds were found to be capable of inhibiting Alr-2 to different extents with 50% inhibitory concentrations (IC50) ranging from 6.6 to 17.7 μM. These compounds inhibited the growth of A. hydrophila with minimal inhibitory concentrations (MICs) ranging from 20 to 120 μg/ml. These compounds have no activity on horseradish peroxidase and d-amino acid oxidase at a concentration of 50 μM. The MTT assay revealed that homogentisic acid and hydroquinone have minimal cytotoxicity against mammalian cells. The kinetic studies indicated a competitive inhibition of homogentisic acid against Alr-2 with an inhibition constant (K i) of 51.7 μM, while hydroquinone was a noncompetitive inhibitor with a K i of 212 μM. Molecular docking studies suggested that homogentisic acid binds to the active site of racemase, while hydroquinone lies near the active center of alanine racemase. Conclusions Our findings suggested that combining experimental and computational methods could be used for an efficient, large-scale screening of alanine racemase inhibitors against A. hydrophila that could be applied in the development of new antibiotics against A. hydrophila

Crystallization and preliminary X-ray study of alkaline alanine racemase from Bacillus pseudofirmus OF4

An alkaline alanine racemase from alkaliphilic B. pseudofirmus OF4 was expressed in E. coli and purified. Crystallization and preliminarily X-­ray crystallographic analysis were performed for the recombinant enzyme

Structural based sequence alignment of Alr_<i>Tt</i>, DadX_<i>Tt</i> and other three representative bacterial alanine racemases.

<p>Amino acid sequences of alanine racemase from a gram positive bacteria <i>Bacillus stearothermophilus</i> (Alr_<i>Bst</i>), a gram negative bacteria <i>Pseudomonas aeruginosa</i> (DadX_<i>pao</i>), and <i>Clostridium difficile</i> strain 630 (Alr_<i>Cd</i>) are aligned with Alr_<i>Tt</i> and DadX_<i>Tt</i> from <i>T</i>. <i>tengcongensis</i> MB4. Amino acids are numbered and secondary structures are labeled, strictly conserved amino acids are highlighted in yellow box. Amino acids form the substrate entryway are colored in blue (middle layer) and magenta (inner layer), key catalytic residues mediating the phosphate group and L-Ala binding are colored in red, residues necessary for hydrogen bonding interactions for PLP-binding are colored in green. Two key catalytic residues Lys40 and Tyr268’ are marked with a star. The hydrophobic patch (Pro225-Arg337) in Alr_<i>Tt</i> is indicated by a red box.</p

Comparison of the active site pocket of Alr_<i>Tt</i> with other three bacterial alanine racemases.

<p><b>(A)</b> The active site pocket of Alr_<i>Tt</i> (PDB 4Y2W), phosphate group, L-Ala and key amino acids that involved in substrate binding are shown in sticks, the hydrogen bonding interactions are indicated. Same view of the active site pocket of other three bacteria alanine racemase in complex with substrates are shown in same profile: <b>(B)</b> the PLP-D-Ala complex of Alr_<i>Bst</i> (PDB 1L6G), <b>(C)</b> PLP complex of Alr_<i>Cd</i> (PDB 4LUS), <b>(D)</b> PLP and DLY complex of DadX_<i>pao</i> (PDB 1RCQ).</p