The use of peptide as novel antibiotic development against the bacterial ribosome

International audienc

Selection of Peptides That Target the Aminoacyl-tRNA Site of Bacterial 16S Ribosomal RNA

International audienceFor almost five decades, antibiotics have been used successfully to control infectious diseases caused by bacterial pathogens. More recently, however, two-thirds of bacterial pathogens exhibit resistance and are continually evolving new resistance mechanisms against almost every clinically used antibiotic. Novel efforts are required for the development of new drugs or drug leads to combat these infectious diseases. A number of antibiotics target the bacterial aminoacyl-tRNA site (A site) of 16S rRNA (rRNA). Mutations in the A-site region are known to cause antibiotic resistance. In this study, a bacterial (Escherichia coli) A-site rRNA model was chosen as a target to screen for peptide binders. Two heptapeptides, HPVHHYQ and LPLTPLP, were selected through M13 phage display. Both peptides display selective binding to the A-site 16S rRNA with on-bead fluorescence assays. Dissociation constants (Kd's) of the amidated peptide HPVHHYQ-NH2 to various A-site RNA constructs were determined by using enzymatic footprinting, electrospray ionization mass spectrometry (ESI-MS), and isothermal titration calorimetry (ITC) under a variety of buffer and solution conditions. HPVHHYQ-NH2 exhibits moderate affinity for the A-site RNA, with an average Kd value of 16 microM. In addition, enzymatic footprinting assays and competition ESI-MS with a known A-site binder (paromomycin) revealed that peptide binding occurs near the asymmetric bulge at positions U1495 and G1494 and leads to increased exposure of residues A1492 and A1493

Inhibition of the interactions between eukaryotic initiation factors 4E and 4G impairs long-term associative memory consolidation but not reconsolidation

Considerable evidence indicates that the general blockade of protein synthesis prevents both the initial consolidation and the postretrieval reconsolidation of long-term memories. These findings come largely from studies of drugs that block ribosomal function, so as to globally interfere with both cap-dependent and -independent forms of translation. Here we show that intra-amygdala microinfusions of 4EGI-1, a small molecule inhibitor of cap-dependent translation that selectively disrupts the interaction between eukaryotic initiation factors (eIF) 4E and 4G, attenuates fear memory consolidation but not reconsolidation. Using a combination of behavioral and biochemical techniques, we provide both in vitro and in vivo evidence that the eIF4E–eIF4G complex is more stringently required for plasticity induced by initial learning than for that triggered by reactivation of an existing memory

PTEN recruitment controls synaptic and cognitive function in Alzheimer's models

Dyshomeostasis of amyloid-β peptide (Aβ) is responsible for synaptic malfunctions leading to cognitive deficits ranging from mild impairment to full-blown dementia in Alzheimer's disease. Aβ appears to skew synaptic plasticity events toward depression. We found that inhibition of PTEN, a lipid phosphatase that is essential to long-term depression, rescued normal synaptic function and cognition in cellular and animal models of Alzheimer's disease. Conversely, transgenic mice that overexpressed PTEN displayed synaptic depression that mimicked and occluded Aβ-induced depression. Mechanistically, Aβ triggers a PDZ-dependent recruitment of PTEN into the postsynaptic compartment. Using a PTEN knock-in mouse lacking the PDZ motif, and a cell-permeable interfering peptide, we found that this mechanism is crucial for Aβ-induced synaptic toxicity and cognitive dysfunction. Our results provide fundamental information on the molecular mechanisms of Aβ-induced synaptic malfunction and may offer new mechanism-based therapeutic targets to counteract downstream Aβ signaling.status: publishe

Biomimetic divalent ligands for the acute disruption of synaptic AMPAR stabilization

The interactions of the AMPA receptor (AMPAR) auxiliary subunit Stargazin with PDZ domain–containing scaffold proteins such as PSD-95 are critical for the synaptic stabilization of AMPARs. To investigate these interactions, we have developed biomimetic competing ligands that are assembled from two Stargazin-derived PSD-95/DLG/ZO-1 (PDZ) domain–binding motifs using 'click' chemistry. Characterization of the ligands in vitro and in a cellular FRET-based model revealed an enhanced affinity for the multiple PDZ domains of PSD-95 compared to monovalent peptides. In cultured neurons, the divalent ligands competed with transmembrane AMPAR regulatory protein (TARP) for the intracellular membrane-associated guanylate kinase resulting in increased lateral diffusion and endocytosis of surface AMPARs, while showing strong inhibition of synaptic AMPAR currents. This provides evidence for a model in which the TARP-containing AMPARs are stabilized at the synapse by engaging in multivalent interactions. In light of the prevalence of PDZ domain clusters, these new biomimetic chemical tools could find broad application for acutely perturbing multivalent complexes