ABSTRACT
Chronic kidney disease (CKD) has major morbidity and mortality for children and adults. While in adults CKD often is associated with diabetic complications, genetic variants can be the underlying cause in both populations. Beginning in 2016 with the emergence of more affordable next-generation sequencing (NGS) technologies, the Molecular Diagnostics Lab at Nemours Children's Hospital-Delaware developed the first clinically actionable pediatric NGS kidney panel comprised of 46 genes including APOL1. Apolipoprotein L1 (APOL1) associated nephropathy is reported along a spectrum of non-diabetic kidney disease. It is significantly associated with two "risk alleles" defined as G1 and G2 and typically found in individuals of African descent. In early 2020, as COVID-19 spread across the globe, reports of patients with kidney failure began to emerge. A collapsing glomerulopathy in Black patients with COVID-19 was found to be associated with the APOL1 predisposition of the known G1 and/or G2 risk variants. We identified genetic variants in 11 genes (NPHS1; NPHS2; LAMB2; WT1; COL4A4; COL4A5; COQ8B; CUBN; MEFV; PMM2; SMARCAL1) known to be associated with pediatric onset nephrotic syndrome, or detection of the high-risk haplotype of APOL1, in the majority (78%) of patients tested. These clinically actionable results guided medical care and improved patient outcomes.
ABSTRACT
Sequence validation of plasmid DNA is a crucial quality control step that must occur prior to adeno-associated virus (AAV) vector packaging through plasmid transfection. AAV cis-plasmids present unique challenges to sequence analysis, as they contain inverted terminal repeats and are prone to sequence rearrangements. An accurate and rapid next-generation sequencing approach has been established to analyze full-length sequences of AAV cis-plasmids within 3.5 days. Here, a step-by-step protocol is described that can reliably detect and identify the location and frequency of sequence variants commonly observed in AAV cis-plasmids.
Subject(s)
Dependovirus/chemistry , High-Throughput Nucleotide Sequencing/methods , Plasmids/chemistry , Sequence Analysis, DNA/methods , Animals , Dependovirus/genetics , High-Throughput Nucleotide Sequencing/standards , Humans , Plasmids/genetics , Reproducibility of Results , Sequence Analysis, DNA/standardsABSTRACT
Herein we describe the discovery of compounds that are competitive antagonists of the CP101-606 binding site within the NR2B subtype of the NMDA receptor. The compounds identified do not possess phenolic functional groups such as those in ifenprodil and related analogs. Initial identification of hits in this series focused on a basic, secondary amine side chain which led to good potency, but also presented a hERG liability. Further modifications led to examples of non-basic replacements which demonstrated much less liability in this regard. Finally, one compound in the series, 6a, was tested in the mouse forced swim depression assay and found to show activity (s.c. 60 mg/kg).
Subject(s)
Antidepressive Agents/chemical synthesis , Pyrazines/chemical synthesis , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Animals , Antidepressive Agents/chemistry , Antidepressive Agents/pharmacology , Binding Sites , Binding, Competitive , Dose-Response Relationship, Drug , Inhibitory Concentration 50 , Mice , Molecular Structure , Motor Activity/drug effects , Protein Binding/drug effects , Pyrazines/chemistry , Pyrazines/pharmacologyABSTRACT
Initial high throughput screening efforts identified highly potent and selective kappa opioid receptor antagonist 3 (κ IC(50)=77 nM; µ:κ and δ:κ IC(50) ratios>400) which lacked CNS exposure in vivo. Modification of this scaffold resulted in development of a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides showing potent and selectivity κ antagonism as well as good brain exposure. Analog 6c (κ IC(50)=20 nM; µ:κ=36, δ:κ=415) was also shown to reverse κ-agonist induced rat diuresis in vivo.
