Recombinant Expression and Stapling of a Novel Long-Acting GLP-1R Peptide Agonist.

Owing to their pleiotropic metabolic benefits, glucagon-like peptide-1 receptor (GLP-1R) agonists have been successfully utilized for treating metabolic diseases, such as type 2 diabetes and obesity. As part of our efforts in developing long-acting peptide therapeutics, we have previously reported a peptide engineering strategy that combines peptide side chain stapling with covalent integration of a serum protein-binding motif in a single step. Herein, we have used this strategy to develop a second generation extendin-4 analog rigidified with a symmetrical staple, which exhibits an excellent in vivo efficacy in an animal model of diabetes and obesity. To simplify the scale-up manufacturing of the lead GLP-1R agonist, a semisynthesis protocol was successfully developed, which involves recombinant expression of the linear peptide followed by attachment of a polyethylene glycol (PEG)-fatty acid staple in a subsequent chemical reaction step.

Redox-Inactive Peptide Disrupting Trx1-Ask1 Interaction for Selective Activation of Stress Signaling.

Thioredoxin 1 (Trx1) and glutaredoxin 1 (Grx1) are two ubiquitous redox enzymes that are central for redox homeostasis but also are implicated in many other processes, including stress sensing, inflammation, and apoptosis. In addition to their enzymatic redox activity, a growing body of evidence shows that Trx1 and Grx1 play regulatory roles via protein-protein interactions with specific proteins, including Ask1. The currently available inhibitors of Trx1 and Grx1 are thiol-reactive electrophiles or disulfides that may suffer from low selectivity because of their thiol reactivity. In this report, we used a phage peptide library to identify a 7-mer peptide, 2GTP1, that binds to both Trx1 and Grx1. We further showed that a cell-permeable derivative of 2GTP1, TAT-2GTP1, disrupts the Trx1-Ask1 interaction, which induces Ask1 phosphorylation with subsequent activation of JNK, stabilization of p53, and reduced viability of cancer cells. Notably, as opposed to a disulfide-derived Trx1 inhibitor (PX-12), TAT-2GTP1 was selective for activating the Ask1 pathway without affecting other stress signaling pathways, such as endoplasmic reticulum stress and AMPK activation. Overall, 2GTP1 will serve as a useful probe for investigating protein interactions of Trx1.

Post-transcriptional Modifications Modulate rRNA Structure and Ligand Interactions.

Post-transcriptional modifications play important roles in modulating the functions of RNA species. The presence of modifications in RNA may directly alter its interactions with binding partners or cause structural changes that indirectly affect ligand recognition. Given the rapidly growing list of modifications identified in noncoding and mRNAs associated with human disease, as well as the dynamic control over modifications involved in various physiological processes, it is imperative to understand RNA structural modulation by these modifications. Among the RNA species, rRNAs provide numerous examples of modification types located in differing sequence and structural contexts. In addition, the modified rRNA motifs participate in a wide variety of ligand interactions, including those with RNA, protein, and small molecules. In fact, several classes of antibiotics exert their effects on protein synthesis by binding to functionally important and highly modified regions of the rRNAs. These RNA regions often display conservation in sequence, secondary structure, tertiary interactions, and modifications, trademarks of ideal drug-targeting sites. Furthermore, ligand interactions with such regions often favor certain modification-induced conformational states of the RNA. Our laboratory has employed a combination of biophysical methods such as nuclear magnetic resonance spectroscopy (NMR), circular dichroism, and UV melting to study rRNA modifications in functionally important motifs, including helix 31 (h31) and helix h44 (h44) of the small subunit rRNA and helix 69 (H69) of the large subunit rRNA. The modified RNA oligonucleotides used in these studies were generated by solid-phase synthesis with a variety of phosphoramidite chemistries. The natural modifications were shown to impact thermal stability, dynamic behavior, and tertiary structures of the RNAs, with additive or cooperative effects occurring with multiple, clustered modifications. Taking advantage of the structural diversity offered by specific modifications in the chosen rRNA motifs, phage display was used to select peptides that bind with moderate (low micromolar) affinity and selectivity to modified h31, h44, and H69. Interactions between peptide ligands and RNAs were monitored by biophysical methods, including electrospray ionization mass spectrometry (ESI-MS), NMR, and surface plasmon resonance (SPR). The peptides compare well with natural compounds such as aminoglycosides in their binding affinities to the modified rRNA constructs. Some candidates were shown to exhibit specificity toward different modification states of the rRNA motifs. The selected peptides may be further optimized for improved RNA targeting or used in screening assays for new drug candidates. In this Account, we hope to stimulate interest in bioorganic and biophysical approaches, which may be used to deepen our understanding of other functionally important, naturally modified RNAs beyond the rRNAs.

Antioxidative activities of histidine containing caffeic acid-dipeptides.

Antioxidants have been utilized in both the food and cosmetics industries to neutralize the activities of reactive oxygen species (ROS) and free radicals. Histidine-containing peptides are powerful antioxidants that exist in nature. Additionally, hydroxycinnamic acid (HCA)-peptide conjugates exhibit a synergistically enhanced antioxidative activity. Thus, caffeic acid (CA), a natural antioxidant, was conjugated to histidine-containing dipeptides (His dipeptides) in order to develop better antioxidants. The antioxidative activities were measured using 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test and lipid peroxidation test with ferric thiocyanate method. Some of the CA-His dipeptides exhibited better radical scavenging activities than CA, and all of the CA-His dipeptides showed enhanced lipid peroxidation inhibitory activities. His dipeptide enhanced the antioxidative activity of CA, and the position of histidine also affected the antioxidative activity of the compounds. CA-proline-histidine amide (CA-Pro-His-NH(2)) exhibited the highest activity in both the free radical scavenging test and the lipid peroxidation inhibition test.

Kojic acid-amino acid conjugates as tyrosinase inhibitors.

Kojic acid (KA), a well known tyrosinase inhibitor, has insufficient inhibitory activity and stability. We modified KA with amino acids and screened their tyrosinase inhibitory activity. Among them, kojic acid-phenylalanine amide (KA-F-NH(2)) showed the strongest inhibitory activity, which was maintained for over 3 months at 50 degrees C, and acted as a noncompetitive inhibitor as determined by kinetic analysis. It also exhibited dopachrome reducing activity. We also propose a new tyrosinase inhibition mechanism based on the docking simulation data.

Synergistic antioxidative activities of hydroxycinnamoyl-peptides.

Antioxidants have become an important subject of study as an active ingredient for cosmetics and preservatives for food. We synthesized antioxidative peptide conjugates of hydroxycinnamic acids (HCAs) such as ferulic acid (FA), caffeic acid (CA), and sinapic acid (SA) by SPPS method. We measured their potential antioxidant properties by 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging test and lipid autoxidation inhibition test. When the antioxidative peptides, such as glutathione analogue (GS(Bzl)H) and carnosine (CAR), were conjugated to HCAs, their antioxidative activities were enhanced significantly. CA-peptides exhibited the highest free radical scavenging activity by the DPPH test, and showed good antioxidative activity in the lipid autoxidation test. FA- and SA-peptides showed excellent antioxidative activity in the lipid autoxidation test. Furthermore, we demonstrated a synergistic antioxidative activity of HCA-peptide conjugates by comparing their antioxidative activity with that of a simple mixture of HCAs and the antioxidant peptides.