d-Amino Acid Substitution of Peptide-Mediated NF-κB Suppression in mdx Mice Preserves Therapeutic Benefit in Skeletal Muscle, but Causes Kidney Toxicity

Abstract

In Duchenne muscular dystrophy (DMD) patients and the mdx mouse model of DMD, chronic activation of the classical nuclear factor-κB (NF-κB) pathway contributes to the pathogenesis that causes degeneration of muscle fibers, inflammation and fibrosis. Prior studies demonstrate that inhibition of inhibitor of κB kinase (IKK)-mediated NF-κB activation using L-isomer NF-κB essential modulator (NEMO)-binding domain (NBD) peptide-based approaches reduce muscle pathology in the mdx mouse. For our studies, the NBD peptide is synthesized as a fusion peptide with an eight-lysine (8K) protein transduction domain to facilitate intracellular delivery. We hypothesized that the D-isoform peptide could have a greater effect than the naturally occurring L-isoform peptide due to the longer persistence of the D-isoform peptide in vivo. In this study, we compared systemic treatment with low (1 mg/kg) and high (10 mg/kg) doses of L-and D-isomer 8K-wild-type-NBD peptide in mdx mice. Treatment with both L-or D-isoform 8K-wild-type-NBD peptide resulted in decreased activation of NF-κB and improved histology in skeletal muscle of the mdx mouse. However, we observed kidney toxicity (characterized by proteinuria), increased serum creatinine, activation of NF-κB and pathological changes in kidney cortex that were most severe with treatment with the D-isoform of 8K-wild-type-NBD peptide. The observed toxicity was also seen in normal mice.

Anti-Inflammatory and General Glucocorticoid Physiology in Skeletal Muscles Affected by Duchenne Muscular Dystrophy: Exploration of Steroid-Sparing Agents

“…In mdx myotubes or mice, several studies were performed with NF-κB inhibitors, such as NK-κB Essential MOdulator (NEMO)-Binding Domain (NBD), the antioxidant pyrrolidine dithiocarbamate (PDTC), the inhibitor of lipid peroxidation IRFI-042, and the free radical scavengers, N-acetylcysteine (NAC) and (ALA)/L-carnitine (L-Car) [ 31 , 37 , 38 , 39 , 40 , 41 , 42 ]. Unfortunately, NBD induced renal toxicity in mdx mice despite encouraging results showing decreased necrosis and increased regeneration in the diaphragm and hind limb muscles [ 37 , 43 , 44 ]. Administration of the NF-κB inhibitor PDTC showed increased strength and muscle regeneration along with decreased fatigue and muscle necrosis in mdx mice.…”

Roles of d-Amino Acids on the Bioactivity of Host Defense Peptides

“…Considering the importance of low off-target toxicity and the absence of dedicated mechanism study, further research into how and why toxicity against healthy mammalian cells by d -AA-containing HDPs occurs could be of vital importance in facilitating further clinical applications. A recent study from Reay et al [ 123 ] addressed this nascent and important area of research on d -AA-containing HDPs by investigating the in vivo toxicity of the d -isoform of the 8K-wild-type-NBD peptide (i.e. where NBD is an acronym for NEMO-binding domain and NEMO is an acronym for NF- κ B essential modulator).…”

“…where NBD is an acronym for NEMO-binding domain and NEMO is an acronym for NF- κ B essential modulator). At the onset, Reay et al [ 123 ] hypothesized that the d -isoform may potentially have higher therapeutic effect than their l -isoform owing to their longer persistence in vivo due to their inherent protease resistance. Their findings indicated that although the d -isoform afforded a similar level of bioactivity as the l -isoform but to their surprise the renal toxicity of the d -isoform was significantly higher and this was attributed to inherent toxicity of d -Ser as described previously by Krug et al [ 124 ].…”

Proteomic and cell biological profiling of the renal phenotype of the mdx-4cv mouse model of Duchenne muscular dystrophy

“…In analogy, we have used here kidney specimens from the established mdx-4cv mouse model of X-linked muscular dystrophy (Banks et al, 2010;Im et al, 1996;Tichy and Mourkioti, 2017). Genetic mdxtype mouse models have previously been used for studying cellular abnormalities in the kidney (Gusel'nikova et al, 2018) and the evaluation of kidney toxicity in relation to pharmacological applications (Reay et al, 2015) and experimental exon-skipping therapy (Zhang et al, 2015) to treat muscular dystrophy. Importantly, adult mdx mice were shown to exhibit reduced renal function using serum Cystatin C testing and dynamic computed tomography scanning with an angiographic agent (Wada et al, 2019).…”