No abstract

New drugs are needed to enhance premature termination codon (PTC) suppression to treat the underlying cause of cystic fibrosis (CF) and other diseases caused by nonsense mutations. We tested new synthetic aminoglycoside derivatives expressly developed for PTC suppression in a series of complementary CF models. Using a dual-luciferase reporter system containing the four most prevalent CF transmembrane conductance regulator (CFTR) nonsense mutations (G542X, R553X, R1162X, and W1282X) within their local sequence contexts (the three codons on either side of the PTC), we found that NB124 promoted the most readthrough of G542X, R1162X, and W1282X PTCs. NB124 also restored full-length CFTR expression and chloride transport in Fischer rat thyroid cells stably transduced with a CFTR-G542XcDNA transgene, and was superior to gentamicin and other aminoglycosides tested. NB124 restored CFTR function to roughly 7% of wild-type activity in primary human bronchial epithelial (HBE) CF cells (G542X/ delF508), a highly relevant preclinical model with endogenous CFTR expression. Efficacy was further enhanced by addition of the CFTR potentiator, ivacaftor (VX-770), to airway cells expressing CFTR PTCs. NB124 treatment rescued CFTR function in a CF mouse model expressing a human CFTR-G542X transgene; efficacy was superior to gentamicin and exhibited favorable pharmacokinetic properties, suggesting that in vitro results translated to clinical benefit in vivo. NB124 was also less cytotoxic than gentamicin in a tissue-based model for ototoxicity. These results provide evidence that NB124 and other synthetic aminoglycosides provide a 10-fold improvement in therapeutic index over gentamicin and other first-generation aminoglycosides, providing a promising treatment for a wide array of CFTR nonsense mutations.

Polyamines are essential polycations present in all living cells. Polyamine levels are maintained from the diet and de novo synthesis, and their decline with age is associated with various pathologies. Here we show that polyamine levels oscillate in a daily manner. Both clock- and feeding-dependent mechanisms regulate the daily accumulation of key enzymes in polyamine biosynthesis through rhythmic binding of BMAL1:CLOCK to conserved DNA elements. In turn, polyamines control the circadian period in cultured cells and animals by regulating the interaction between the core clock repressors PER2 and CRY1. Importantly, we found that the decline in polyamine levels with age in mice is associated with a longer circadian period that can be reversed upon polyamine supplementation in the diet. Our findings suggest a crosstalk between circadian clocks and polyamine biosynthesis and open new possibilities for nutritional interventions against the decay in clock's function with age.

Edited by Linda SpremulliApoptosis is thought to play a critical role in several pathological processes, such as neurodegenerative diseases (i.e. Parkinson's and Alzheimer's diseases) and various cardiovascular diseases. Despite the fact that apoptotic mechanisms are well defined, there is still no substantial therapeutic strategy to stop or even slow this process. Thus, there is an unmet need for therapeutic agents that are able to block or slow apoptosis in neurodegenerative and cardiovascular diseases. The outer mitochondrial membrane protein voltage-dependent anion channel 1 (VDAC1) is a convergence point for a variety of cell survival and death signals, including apoptosis. Recently, we demonstrated that VDAC1 oligomerization is involved in mitochondrion-mediated apoptosis. Thus, VDAC1 oligomerization represents a prime target for agents designed to modulate apoptosis. Here, high-throughput compound screening and medicinal chemistry were employed to develop compounds that directly interact with VDAC1 and prevent VDAC1 oligomerization, concomitant with an inhibition of apoptosis as induced by various means and in various cell lines. The compounds protected against apoptosis-associated mitochondrial dysfunction, restoring dissipated mitochondrial membrane potential, and thus cell energy and metabolism, decreasing reactive oxidative species production, and preventing detachment of hexokinase bound to mitochondria and disruption of intracellular Ca 2؉ levels. Thus, this study describes novel drug candidates with a defined mechanism of action that involves inhibition of VDAC1 oligomerization, apoptosis, and mitochondrial dysfunction. The compounds VBIT-3 and VBIT-4 offer a therapeutic strategy for treating different diseases associated with enhanced apoptosis and point to VDAC1 as a promising target for therapeutic intervention.Mitochondria play crucial roles in cellular energy generation and metabolism, maintenance of the cell redox potential, calcium homeostasis, pH control and fatty acid oxidation, cell signaling, proliferation, differentiation, aging, and death (1). It is therefore not surprising that mitochondrial dysfunction is associated with various human diseases (1, 2).Located at the outer mitochondrial membrane (OMM), 2 the voltage-dependent anion channel (VDAC) serves as a mitochondrial gatekeeper. Three VDAC isoforms have been discovered (3), but only for VDAC1 is there a complete set of structural and functional information available. VDAC1 controls the metabolic and energy cross-talk between mitochondria and the rest of the cell, mediating the fluxes of ions, nucleotides, and other metabolites across the OMM (4 -7).

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania , affects millions of people worldwide. Aminoglycosides are mostly known as highly potent, broad-spectrum antibiotics that exert their antibacterial activity by selectively targeting the decoding A site of the bacterial ribosome, leading to aberrant protein synthesis. Recently, some aminoglycosides have been clinically approved and are currently used worldwide for the treatment of leishmaniasis; however the molecular details by which aminoglycosides induce their deleterious effect on Leishmaina is still rather obscure. Based on high conservation of the decoding site among all kingdoms, it is assumed that the putative binding site of these agents in Leishmania is the ribosomal A site. However, although recent X-ray crystal structures of the bacterial ribosome in complex with aminoglycosides shed light on the mechanism of aminoglycosides action as antibiotics, no such data are presently available regarding their binding site in Leishmania . We present crystal structures of two different aminoglycoside molecules bound to a model of the Leishmania ribosomal A site: Geneticin (G418), a potent aminoglycoside for the treatment of leishmaniasis at a 2.65-Å resolution, and Apramycin, shown to be a strong binder to the leishmanial ribosome lacking an antileishmanial activity at 1.4-Å resolution. The structural data, coupled with in vitro inhibition measurements on two strains of Leishmania , provide insight as to the source of the difference in inhibitory activity of different Aminoglycosides. The combined structural and physiological data sets the ground for rational design of new, and more specific, aminoglycoside derivatives as potential therapeutic agents against leishmaniasis.