Comparison of pharmaceutical properties and biological activities of prednisolone, deflazacort, and vamorolone in DMD disease models.

Duchenne muscular dystrophy (DMD) is a progressive disabling X-linked recessive disorder that causes gradual and irreversible loss of muscle, resulting in early death. The corticosteroids prednisone/prednisolone and deflazacort are used to treat DMD as the standard of care; however, only deflazacort is FDA approved for DMD. The novel atypical corticosteroid vamorolone is being investigated for treatment of DMD. We compared the pharmaceutical properties as well as the efficacy and safety of the three corticosteroids across multiple doses in the B10-mdx DMD mouse model. Pharmacokinetic studies in the mouse and evaluation of p-glycoprotein (P-gP) efflux in a cellular system demonstrated that vamorolone is not a strong P-gp substrate resulting in measurable central nervous system (CNS) exposure in the mouse. In contrast, deflazacort and prednisolone are strong P-gp substrates. All three corticosteroids showed efficacy, but also side effects at efficacious doses. After dosing mdx mice for two weeks, all three corticosteroids induced changes in gene expression in the liver and the muscle, but prednisolone and vamorolone induced more changes in the brain than did deflazacort. Both prednisolone and vamorolone induced depression-like behavior. All three corticosteroids reduced endogenous corticosterone levels, increased glucose levels, and reduced osteocalcin levels. Using micro-computed tomography, femur bone density was decreased, reaching significance with prednisolone. The results of these studies indicate that efficacious doses of vamorolone, are associated with similar side effects as seen with other corticosteroids. Further, because vamorolone is not a strong P-gp substrate, vamorolone distributes into the CNS increasing the potential CNS side-effects.

Modified Aminoglycosides Bind Nucleic Acids in High-Molecular-Weight Complexes.

Aminoglycosides represent a large group of antibiotics well known for their ability to target the bacterial ribosome. In studying 6"-substituted variants of the aminoglycoside tobramycin, we serendipitously found that compounds with C12 or C14 linear alkyl substituents potently inhibit reverse transcription in vitro. Initial observations suggested specific inhibition of reverse transcriptase. However, further analysis showed that these and related compounds bind nucleic acids with high affinity, forming high-molecular weight complexes. Stable complex formation is observed with DNA or RNA in single- or double-stranded form. Given the amphiphilic nature of these aminoglycoside derivatives, they likely form micelles and/or vesicles with surface-bound nucleic acids. Hence, these compounds may be useful tools to localize nucleic acids to surfaces or deliver nucleic acids to cells or organelles.

Roles of specific aminoglycoside-ribosome interactions in the inhibition of translation.

Aminoglycosides containing a 2-deoxystreptamine core (AGs) represent a large family of antibiotics that target the ribosome. These compounds promote miscoding, inhibit translocation, and inhibit ribosome recycling. AG binding to helix h44 of the small subunit induces rearrangement of A-site nucleotides A1492 and A1493, which promotes a key open-to-closed conformational change of the subunit and thereby increases miscoding. Mechanisms by which AGs inhibit translocation and recycling remain less clear. Structural studies have revealed a secondary AG binding site in H69 of the large subunit, and it has been proposed that interaction at this site is crucial for inhibition of translocation and recycling. Here, we analyze ribosomes with mutations targeting either or both AG binding sites. Assaying translocation, we find that ablation of the h44 site increases the IC50 values for AGs dramatically, while removal of the H69 site increases these values modestly. This suggests that the AG-h44 interaction is primarily responsible for inhibition, with H69 playing a minor role. Assaying recycling, we find that mutation of h44 has no effect on AG inhibition, consistent with a primary role for AG-H69 interaction. Collectively, these findings help clarify the roles of the two AG binding sites in mechanisms of inhibition by these compounds.

Epistasis analysis of 16S rRNA ram mutations helps define the conformational dynamics of the ribosome that influence decoding.

The ribosome actively participates in decoding, with a tRNA-dependent rearrangement of the 30S A site playing a key role. Ribosomal ambiguity (ram) mutations have mapped not only to the A site but also to the h12/S4/S5 region and intersubunit bridge B8, implicating other conformational changes such as 30S shoulder rotation and B8 disruption in the mechanism of decoding. Recent crystallographic data have revealed that mutation G299A in helix h12 allosterically promotes B8 disruption, raising the question of whether G299A and/or other ram mutations act mainly via B8. Here, we compared the effects of each of several ram mutations in the absence and presence of mutation h8Δ2, which effectively takes out bridge B8. The data obtained suggest that a subset of mutations including G299A act in part via B8 but predominantly through another mechanism. We also found that G299A in h12 and G347U in h14 each stabilize tRNA in the A site. Collectively, these data support a model in which rearrangement of the 30S A site, inward shoulder rotation, and bridge B8 disruption are loosely coupled events, all of which promote progression along the productive pathway toward peptide bond formation.

Modification of targets related to the Entner-Doudoroff/pentose phosphate pathway route for methyl-D-erythritol 4-phosphate-dependent carotenoid biosynthesis in Escherichia coli.

BACKGROUND: In engineered strains of Escherichia coli, bioconversion efficiency is determined by not only metabolic flux but also the turnover efficiency of relevant pathways. Methyl-D-erythritol 4-phosphate (MEP)-dependent carotenoid biosynthesis in E. coli requires efficient turnover of precursors and balanced flux among precursors, cofactors, and cellular energy. However, the imbalanced supply of glyceraldehyde 3-phosphate (G3P) and pyruvate precursors remains the major metabolic bottleneck. To address this problem, we manipulated various genetic targets related to the Entner-Doudoroff (ED)/pentose phosphate (PP) pathways. Systematic target modification was conducted to improve G3P and pyruvate use and rebalance the precursor and redox fluxes. RESULTS: Carotenoid production was improved to different degrees by modifying various targets in the Embden-Meyerhof-Parnas (EMP) and ED pathways, which directed metabolic flux from the EMP pathway towards the ED pathway. The improvements in yield were much greater when the MEP pathway was enhanced. The coordinated modification of ED and MEP pathway targets using gene expression enhancement and protein coupling strategies in the pgi deletion background further improved carotenoid synthesis. The fine-tuning of flux at the branch point between the ED and PP pathways was important for carotenoid biosynthesis. Deletion of pfkAB instead of pgi reduced the carotenoid yield. This suggested that anaplerotic flux of G3P and pyruvate might be necessary for carotenoid biosynthesis. Improved carotenoid yields were accompanied by increased biomass and decreased acetate overflow. Therefore, efficient use of G3P and pyruvate precursors resulted in a balance among carotenoid biosynthesis, cell growth, and by-product metabolism. CONCLUSIONS: An efficient and balanced MEP-dependent carotenoid bioconversion strategy involving both the ED and PP pathways was implemented by the coordinated modification of diverse central metabolic pathway targets. In this strategy, enhancement of the ED pathway for efficient G3P and pyruvate turnover was crucial for carotenoid production. The anaplerotic role of the PP pathway was important to supply precursors for the ED pathway. A balanced metabolic flux distribution among precursor supply, NADPH generation, and by-product pathways was established.