Guanine nucleotide exchange factor DOCK11-binding peptide fused with a single chain antibody inhibits hepatitis B virus infection and replication.

Hepatitis B virus (HBV) infection is a major global health problem with no established cure. Dedicator of cytokinesis 11 (DOCK11), known as a guanine nucleotide exchange factor (GEF) for Cdc42, is reported to be essential for the maintenance of HBV. However, potential therapeutic strategies targeting DOCK11 have not yet been explored. We have previously developed an in vitro virus method as a more efficient tool for the analysis of proteomics and evolutionary protein engineering. In this study, using the in vitro virus method, we screened and identified a novel antiasialoglycoprotein receptor (ASGR) antibody, ASGR3-10M, and a DOCK11-binding peptide, DCS8-42A, for potential use in HBV infection. We further constructed a fusion protein (10M-D42AN) consisting of ASGR3-10M, DCS8-42A, a fusogenic peptide, and a nuclear localization signal to deliver the peptide inside hepatocytes. We show using immunofluorescence staining that 10M-D42AN was endocytosed into early endosomes and released into the cytoplasm and nucleus. Since DCS8-42A shares homology with activated cdc42-associated kinase 1 (Ack1), which promotes EGFR endocytosis required for HBV infection, we also found that 10M-D42AN inhibited endocytosis of EGFR and Ack1. Furthermore, we show 10M-D42AN suppressed the function of DOCK11 in the host DNA repair system required for covalently closed circular DNA synthesis and suppressed HBV proliferation in mice. In conclusion, this study realizes a novel hepatocyte-specific drug delivery system using an anti-ASGR antibody, a fusogenic peptide, and DOCK11-binding peptide to provide a novel treatment for HBV.

A novel phenylphthalimide derivative, pegylated TC11, improves pharmacokinetic properties and induces apoptosis of high-risk myeloma cells via G2/M cell-cycle arrest.

Despite the development of new drugs for multiple myeloma (MM), the prognosis of MM patients with high-risk cytogenetic abnormalities such as t (4; 14) and del17p remains poor. We reported that a novel phenylphthalimide derivative, TC11, induced apoptosis of MM cells in vitro and in vivo, and TC11 directly bound to α-tubulin and nucleophosmin-1 (NPM1). However, TC11 showed low water solubility and poor pharmacokinetic properties. Here we synthesized a water-soluble TC11-derivative, PEG(E)-TC11, in which HOEtO-TC11 is pegylated with PEG through an ester bond, and we examined its anti-myeloma activity. We observed that PEG(E)-TC11 and its hydrolyzed product, HOEtO-TC11, induced G2/M arrest and the apoptosis of MM cells. Intraperitoneal administration of PEG(E)-TC11 to xenografted mice revealed improved pharmacokinetic properties and significantly delayed tumor growth. TC11 and its derivatives did not bind to cereblon (CRBN), which is a responsible molecule for thalidomide-induced teratogenicity. These results suggest that PEG(E)-TC11 is a good candidate drug for treating high-risk MM.

Structural basis for inhibition of the MDM2:p53 interaction by an optimized MDM2-binding peptide selected with mRNA display.

The oncoprotein MDM2 binds to tumor suppressor protein p53 and inhibits its anticancer activity, which leads to promotion of tumor cell growth and tumor survival. Abrogation of the p53:MDM2 interaction reportedly results in reactivation of the p53 pathway and inhibition of tumor cell proliferation. We recently performed rigorous selection of MDM2-binding peptides by means of mRNA display and identified an optimal 12-mer peptide (PRFWEYWLRLME), named MDM2 Inhibitory Peptide (MIP), which shows higher affinity for MDM2 (and also its homolog, MDMX) and higher tumor cell proliferation suppression activity than known peptides. Here we determined the NMR solution structure of a MIP-MDM2 fusion protein to elucidate the structural basis of the tight binding of MIP to MDM2. A region spanning from Phe3 to Met11 of MIP forms a single α-helix, which is longer than those of the other MDM2-binding peptides. MIP shares a conserved Phe3-Trp7-Leu10 triad, whose side chains are oriented towards and fit into the hydrophobic pockets of MDM2. Additionally, hydrophobic surface patches that surround the hydrophobic pockets of MDM2 are covered by solvent-exposed MIP residues, Trp4, Tyr6, and Met11. Their hydrophobic interactions extend the interface of the two molecules and contribute to the strong binding. The potential MDM2 inhibition activity observed for MIP turned out to originate from its enlarged binding interface. The structural information obtained in the present study provides a road map for the rational design of strong inhibitors of MDM2:p53 binding.

MIP-2A is a novel target of an anilinoquinazoline derivative for inhibition of tumour cell proliferation.

We recently identified a novel anilinoquinazoline derivative, Q15, as a potent apoptosis inducer in a panel of human cancer cell lines and determined that Q15 targets hCAP-G2, a subunit of condensin II complex, leading to abnormal cell division. However, whether the defect in normal cell division directly results in cell death remains unclear. Here, we used an mRNA display method on a microfluidic chip to search for other Q15-binding proteins. We identified an additional Q15-binding protein, MIP-2A (MBP-1 interacting protein-2A), which has been reported to interact with MBP-1, a repressor of the c-Myc promoter. Our results indicate that Q15 inhibits the interaction between MIP-2A and MBP-1 as well as the expression of c-Myc protein, thereby inducing cell death. This study suggests that the simultaneous targeting of hCAP-G2 and MIP-2A is a promising strategy for the development of antitumor drugs as a treatment for intractable tumours.