Hiroshi Inooka

Solution conformation of endothelin determined by nuclear magnetic resonance and distance geometry

Endothelin; Vasoconstrictor peptide; Three‐dimensional structure; NMR, 1H; Distance geometry

A PEGylated analog of short-length Neuromedin U with potent anorectic and anti-obesity effects

Neuromedin U (NMU) is a neuropeptide known to regulate food intake and energy homeostasis that is widely distributed in the gastrointestinal tract, hypothalamus, and pituitary. A short form of NMU, porcine NMU-8 has potent agonist activity for the receptors NMUR1 and NMUR2; however, its short half-life precludes its effective use in vivo. To address this limitation, we designed and synthesized NMU-8 analogs modified by polyethylene glycol (PEG) with a molecular weight of 30kDa (PEG30k) via a variety of linkers (i.e., ω-amino- and ω-imino-carboxylic acid linker). Integrated evaluation of NMUR1 and NMUR2 binding affinities in vitro and anorectic activity in mice revealed that the introduction of a linker with a rigid ring group, e.g., 2-(piperazin-1-yl)acetic acid (PipAc), yielded a highly potent anorectic peptide, PEG30k-PipAc-NMU-8 (14), possessing improved receptor binding affinity. Subsequent optimization of the molecular weight of the PEG moiety led to the discovery of a PEG20k conjugate (15), which exhibited significant anti-obesity effect upon once-daily subcutaneous administration in diet-induced obese mice with 10% and 22% body weight loss at doses of 10 and 30nmol/kg, respectively. In addition, 15 reduced the weights of the liver and adipose tissue in a dose-dependent manner and improved the plasma biochemical parameters, e.g., insulin, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and total cholesterol. Thus, our results suggest that 15 (NMU-0002), which showed potent and long-lasting biological profiles in vivo, represents a candidate peptide for investigating the central and peripheral actions of NMU and its potential for clinical use.

PEGylated neuromedin U-8 shows long-lasting anorectic activity and anti-obesity effect in mice by peripheral administration

HIGHLIGHTSIntraperitoneal injection of neuromedin U‐23 and neuromedin S inhibited food intake.PEGylation gave neuromedin U‐8 a long‐lasting anorectic activity.Subcutaneous injection of PEGylated neuromedin U‐8 exerted an anti‐obesity effect. ABSTRACT Neuromedin U (NMU) is a neuropeptide found in the brain and gastrointestinal tract. The NMU system has been shown to regulate energy homeostasis by both a central and a peripheral mechanism. Peripheral administration of human NMU‐25 was recently shown to inhibit food intake in mice. We examined the possibility that other NMU‐related peptides exert an anorectic activity by intraperitoneal (i.p.) administration. We found that rat NMU‐23 and its structurally‐related peptide rat neuromedin S (NMS) significantly reduced food intake in lean mice, whereas NMU‐8, an active fragment of the octapeptide sequence conserved in porcine, human and mouse NMU, had no effect. When rat NMU‐23, NMU‐8, and rat NMS were covalently conjugated to polyethylene glycol (PEG) (PEGylation) at the N‐terminus of these peptides, PEGylated NMU‐8 showed the most long‐lasting and robust anorectic activity. The exploration of the linker between NMU‐8 and PEG using hetero‐bifunctional chemical cross‐linkers led to an identification of PEGylated NMU‐8 analogs with higher affinity for NMU receptors and with more potent anorectic activity in lean mice. The PEGylated NMU‐8 showed potent and robust anorectic activity and anti‐obesity effect in diet‐induced obesity (DIO) mice by once‐daily subcutaneous (s.c.) administration. These results suggest that PEGylated NMU‐8 has the therapeutic potential for treatment of obesity.

Discovery of a novel B-cell lymphoma 6 (BCL6)–corepressor interaction inhibitor by utilizing structure-based drug design

B-cell lymphoma 6 (BCL6) is a transcriptional repressor that can form complexes with corepressors via protein-protein interactions (PPIs). The complexes of BCL6 and corepressors play an important role in the formation of germinal centers (GCs), and differentiation and proliferation of lymphocytes. Therefore, BCL6-corepressor interaction inhibitors would be drug candidates for managing autoimmune diseases and cancer. Starting from high-throughput screening hits 1a and 2a, we identified a novel BCL6-corepressor interaction inhibitor 8c (cell-free enzyme-linked immunosorbent assay [ELISA] IC50=0.10µM, cell-based mammalian two-hybrid [M2H] assay IC50=0.72µM) by utilizing structure-based drug design (SBDD) based on an X-ray crystal structure of 1a bound to BCL6. Compound 8c also showed a good pharmacokinetic profile, which was acceptable for both in vitro and in vivo studies.

Differential effects of selective agonists of neuromedin U1 and U2 receptors in obese and diabetic mice

Neuromedin U (NmU) may be a novel target for obesity treatment owing to its anorectic and energy expenditure enhancing effects. Although two receptors, NMU1 and NMU2, are both responsible for the NmU‐mediated anti‐obesity effects, the receptor agonist with the most appropriate profiles for treating obesity and diabetes in terms of efficacy and safety is as yet unknown. Thus, we developed and evaluated novel NMU1/2 receptor‐selective agonists.

Identification of ligand-selective peptidic ActRIIB-antagonists using phage display technology

ActRIIB (activin receptor type-2B) is an activin receptor subtype constitutively expressed in the whole body, playing a role in cellular proliferation, differentiation, and metabolism. For its various physiological activities, ActRIIB interacts with activin and multiple other ligands including myostatin (MSTN), growth differentiation factor 11 (GDF11), and bone morphogenetic protein 9 (BMP9). Notably, the protein-protein interaction (PPI) between ActRIIB and MSTN negatively controls muscular development. Therefore, this PPI has been targeted for effective treatment of muscle degenerative diseases such as muscular dystrophy and sarcopenia. Here, we report the identification of ligand-selective peptidic ActRIIB-antagonists by phage display technology. Our peptides bound to the extracellular domain of ActRIIB, inhibited PPIs between ActRIIB expressed on the cell surface and its ligands, and subsequently suppressed activation of Smad that serves as the downstream signal of the ActRIIB pathway. Interestingly, these peptidic antagonists displayed different ligand selectivities; the AR2mini peptide inhibited multiple ligands (activin A, MSTN, GDF11, and BMP9), AR9 inhibited MSTN and GDF11, while AR8 selectively inhibited MSTN. This is the first report of artificial peptidic ActRIIB-antagonists possessing ligand-selectivity.