Functional role of aspartate-31 and leucine-32 in Mycobacterium avium dihydrofolate reductase

Scope and Method of Study: Dihydrofolate reductase (DHFR: 1.5.1.3) has long been a drug target in antibacterial therapy. However, DHFR of Mycobacterium avium and other mycobacteria are naturally resistant to trimethoprim and other antituberculous drugs. Recent reports show that a new class of drugs: 2,4-diaminodeazapteridine (DMDPs) are showing increased selectivity for Mycobacterium avium. Better understanding of the binding sites of M. avium DHFR, will contribute towards developing better and more effective drugs. Based on sequence alignments and X-ray crystal structures of other DHFR's, aspartic acid 31 (D31) and leucine 32 (L32) were identified as functionally important residues in interactions with the substrate dihydrofolate and inhibitors, respectively. D31 and L32 of M. avium DHFR were modified by site-directed mutagenesis (GeneEditor, Promega). to D31A, D31E, D31Q, D31N, D31L,. L32A, L32F and L32D. Mutations were verified by full length gene sequencing. These mutants were then expressed in E. coli BL21(DE3)pLysS and the recombinant mutant protein purified using HisBind-Resin (Novangen). Functionality of the mutants was assessed in comparison with the recombinant wild type by a standard enzyme assay as well as by growth complementation. Kinetic parameters were determined and computed using the non-linear curve fit program Enzfitter (BioSoft, UK).Findings and Conclusions: All D31 mutations rendered the enzyme severely dysfunctional. Enzyme activity of the mutants D31E, D31Q and D31N were reduced by between 80 and 90%. Functionality of the mutants D31A and D31L were reduced by over 90% compared to the wild type.All D31 mutants show differences in kinetics compared to the wild type. Of the L32 mutants, only L32D reduced the enzyme's activity by two-thirds and showed differences in kinetic behavior compared to the wild type. L32F and L32A did not show selectivity for trimethoprim, while L32D did. The DMDP inhibitors were highly effective against the wild type. The mutants showed differences in selectivity to the DMDPs. The findings support the hypotheses that D31 plays a functional role with the substrate and L32 plays a functional role with inhibitors. All D31 mutations studies resulted in a dysfunctional enzyme, regardless of changes in side chain size or charge. Modification of L32 led to increased selectivity for trimethoprim, but decreased selectivity for the DMDPs

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie