Peptidream (Japan)

525 Background: Glypican-3 (GPC3) is a membrane-anchored oncofetal protein whose expression is largely absent in normal tissues. Significant upregulation of GPC3 protein has been observed in approximately 75% of hepatocellular carcinomas (HCC), and is associated with poor prognosis. The differential expression of GPC3 between tumor and normal tissues provides an opportunity for targeted radiopharmaceutical therapy (RPT) to treat HCC, a leading cause for cancer-related deaths worldwide. Methods: RAYZ-8009 comprises a novel macrocyclic peptide binder to GPC3, a linker, and a chelator that can be complexed with different radioisotopes. The affinity of peptide binders to GPC3 was determined by surface plasma resonance (SPR) and radioligand binding assays. Target-mediated cellular internalization was radiometrically measured at multiple time points. In vivo biodistribution, monotherapy and combination treatments with 177Lu or 225Ac were performed in HCC xenografts. Results: RAYZ-8009 showed high binding affinity (KD=0.7 nM) to human GPC3, with comparable affinity to GPC3 of human, mouse, canine and monkey origins, and no binding to other GPC family members. Potent cellular binding was confirmed in GPC3+ HepG2 cells, and was not affected by isotope switching. RAYZ-8009 achieved efficient internalization upon binding, with 42% internalized by 20 minutes in HepG2 cells. Biodistribution study of 177Lu-RAYZ-8009 showed sustained tumor uptake and fast renal clearance, with minimal or no uptake in other normal tissues. Exquisite tumor-specific uptake was also demonstrated in orthotopic HCC tumors with no uptake in surrounding normal liver tissue. Therapeutically, significant and durable anti-tumor effect and survival benefit were achieved with 177Lu- and 225Ac-labeled RAYZ-8009, as monotherapy or in combination with lenvatinib, in GPC3+ HCC xenografts. Conclusions: Preclinical in vitro and in vivo data demonstrate the potential of RAYZ-8009 as a theranostic agent for the treatment of patients with GPC3+ HCC.

An automated continuous-flow liquid-phase peptide synthesizer for preparing C-terminal free peptides was developed. This system includes aqueous workup, concentration, and in-line NIR monitoring.

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.

Advances in the design of permeable peptides and in the synthesis of large arrays of macrocyclic peptides with diverse amino acids have evolved on parallel but independent tracks. Less precedent combines their respective attributes, thereby limiting the potential to identify permeable peptide ligands for key targets. Herein, we present novel 6-, 7-, and 8-mer cyclic peptides (MW 774-1076 g·mol-1) with passive permeability and oral exposure that feature the amino acids and thioether ring-closing common to large array formats, including DNA- and RNA-templated synthesis. Each oral peptide herein, selected from virtual libraries of partially N-methylated peptides using in silico methods, reflects the subset consistent with low energy conformations, low desolvation penalties, and passive permeability. We envision that, by retaining the backbone N-methylation pattern and consequent bias toward permeability, one can generate large peptide arrays with sufficient side chain diversity to identify permeability-biased ligands to a variety of protein targets.

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.