Junichi Sakamoto

A new class of non-thiazolidinedione, non-carboxylic-acid-based highly selective peroxisome proliferator-activated receptor (PPAR) γ agonists: design and synthesis of benzylpyrazole acylsulfonamides.

Herein, we describe the design, synthesis, and structure-activity relationships of novel benzylpyrazole acylsulfonamides as non-thiazolidinedione (TZD), non-carboxylic-acid-based peroxisome proliferator-activated receptor (PPAR) γ agonists. Docking model analysis of in-house weak agonist 2 bound to the reported PPARγ ligand binding domain suggested that modification of the carboxylic acid of 2 would help strengthen the interaction of 2 with the TZD pocket and afford non-carboxylic-acid-based agonists. In this study, we used an acylsulfonamide group as the ring-opening analog of TZD as an isosteric replacement of carboxylic acid moiety of 2; further, preliminary modification of the terminal alkyl chain on the sulfonyl group gave the lead compound 3c. Subsequent optimization of the resulting compound gave the potent agonists 25c, 30b, and 30c with high metabolic stability and significant antidiabetic activity. Further, we have described the difference in binding mode of the carboxylic-acid-based agonist 1 and acylsulfonamide 3d.

Discovery of a B-Cell Lymphoma 6 Protein–Protein Interaction Inhibitor by a Biophysics-Driven Fragment-Based Approach

B-cell lymphoma 6 (BCL6) is a transcriptional factor that expresses in lymphocytes and regulates the differentiation and proliferation of lymphocytes. Therefore, BCL6 is a therapeutic target for autoimmune diseases and cancer treatment. This report presents the discovery of BCL6-corepressor interaction inhibitors by using a biophysics-driven fragment-based approach. Using the surface plasmon resonance (SPR)-based fragment screening, we successfully identified fragment 1 (SPR KD = 1200 μM, ligand efficiency (LE) = 0.28), a competitive binder to the natural ligand BCoR peptide. Moreover, we elaborated 1 into the more potent compound 7 (SPR KD = 0.078 μM, LE = 0.37, cell-free protein-protein interaction (PPI) IC50 = 0.48 μM (ELISA), cellular PPI IC50 = 8.6 μM (M2H)) by a structure-based design and structural integration with a second high-throughput screening hit.

A Selective Bombesin Receptor Subtype 3 Agonist Promotes Weight Loss in Male Diet-Induced–Obese Rats With Circadian Rhythm Change

Bombesin receptor subtype 3 (BRS-3) is an orphan G protein-coupled receptor. Based on the obese phenotype of male BRS-3-deficient mice, BRS-3 has been considered an attractive target for obesity treatment. Here, we developed a selective BRS-3 agonist (compound-A) and evaluated its antiobesity effects. Compound-A showed anorectic effects and enhanced energy expenditure in diet-induced-obese (DIO)-F344 rats. Moreover, repeated oral administration of compound-A for 7 days resulted in a significant body weight reduction in DIO-F344 rats. We also evaluated compound-A for cardiovascular side effects using telemeterized Sprague-Dawley (SD) rats. Oral administration of compound-A resulted in transient blood pressure increases in SD rats. To investigate the underlying mechanisms of BRS-3 agonist effects, we focused on the suprachiasmatic nucleus (SCN), the main control center of circadian rhythms in the hypothalamus, also regulating sympathetic nervous system. Compound-A significantly increased the messenger RNA expression of Brs-3, c-fos, and circadian rhythm genes in SCN of DIO-F344 rats. Because SCN also controls the hypothalamic-pituitary-adrenal (HPA) axis, we evaluated the relationship between BRS-3 and the HPA axis. Oral administration of compound-A caused a significant increase of plasma corticosterone levels in DIO-F344 rats. On this basis, energy expenditure enhancement by compound-A may be due to a circadian rhythm change in central and peripheral tissues, enhancement of peripheral lipid metabolism, and stimulation of the sympathetic nervous system. Furthermore, the blood pressure increase by compound-A could be associated with sympathetic nervous system stimulation via SCN and elevation of plasma corticosterone levels through activation of the HPA axis.

Discovery of GPX4 inhibitory peptides from random peptide T7 phage display and subsequent structural analysis

The phospholipid hydroperoxidase glutathione peroxidase (GPX4) is an enzyme that reduces lipid hydroperoxides in lipid membranes. Recently, GPX4 has been investigated as a target molecule that induces iron-dependent cell death (ferroptosis) selectively in cancer cells that express mutant Ras. GPX4 inhibitors have the potential to become novel anti-cancer drugs. However, there are no druggable pockets for conventional small molecules on the molecular surface of GPX4. To generate GPX4 inhibitors, we examined the use of peptides as an alternative to small molecules. By screening peptide libraries displayed on T7 phages, and analyzing the X-ray crystal structures of the peptides, we successfully identified one peptide that binds to near Sec73 of catalytic site and two peptides that bind to another site on GPX4. To our knowledge, this is the first study reporting GPX4 inhibitory peptides and their structural information.

Structure-activity relationships and key structural feature of pyridyloxybenzene-acylsulfonamides as new, potent, and selective peroxisome proliferator-activated receptor (PPAR) γ Agonists.

In our search for a novel class of non-TZD, non-carboxylic acid peroxisome proliferator-activated receptor (PPAR) γ agonists, we explored alternative lipophilic templates to replace benzylpyrazole core of the previously reported agonist 1. Introduction of a pentylsulfonamide group into arylpropionic acids derived from previous in-house PPARγ ligands succeeded in the identification of 2-pyridyloxybenzene-acylsulfonamide 2 as a lead compound. Docking studies of compound 2 suggested that a substituent para to the central benzene ring should be incorporated to effectively fill the Y-shaped cavity of the PPARγ ligand-binding domain (LBD). This strategy led to significant improvement of PPARγ activity. Further optimization to balance in vitro activity and metabolic stability allowed the discovery of the potent, selective and orally efficacious PPARγ agonist 8f. Structure-activity relationship study as well as detailed analysis of the binding mode of 8f to the PPARγ-LBD revealed the essential structural features of this series of ligands.

Potent Body Weight-Lowering Effect of a Neuromedin U Receptor 2-selective PEGylated Peptide

Neuromedin U (NMU) is a neuropeptide that mediates a variety of physiological functions via its receptors, NMUR1 and NMUR2. Recently, there has been an increased focus on NMU as a promising treatment option for diabetes and obesity. A short form of NMU (NMU-8) has potent agonist activity for both receptors but is metabolically unstable. Therefore, we designed and synthesized NMU-8 analogues modified by polyethylene glycol (PEG; molecular weight, 20 kDa; PEG20k) via a linker. 3-(2-Naphthyl)alanine substitution at position 19 increased NMUR2 selectivity of NMU-8 analogues with retention of high agonist activity. Compound 37, an NMUR2-selective PEG20k analogue containing piperazin-1-ylacetyl linker, exhibited a potent body weight-lowering effect with concomitant inhibition of food intake in a dose-dependent manner (body weight loss of 12.4% at 30 nmol/kg) by once-daily repeated dosing for 2 weeks in mice with diet-induced 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.

Crystal Structure of a Human K-Ras G12D Mutant in Complex with GDP and the Cyclic Inhibitory Peptide KRpep-2d

The Ras proteins play roles in cell differentiation, proliferation, and survival. Aberrant signaling through Ras-mediated pathways in tumor cells occurs as a result of several types of mutational damage, which most frequently affects the amino acids G12, G13, and Q61. Recently, KRpep-2d was identified as a K-Ras(G12D) selective inhibitory peptide against the G12D mutant of K-Ras, which is a key member of the Ras protein family and an attractive cancer therapeutic target. In this study, the crystal structure of the human K-Ras(G12D) mutant was determined in complex with GDP and KRpep-2d at 1.25 Å resolution. This structure revealed that the peptide binds near Switch II and allosterically blocks protein-protein interactions with the guanine nucleotide exchange factor. This discovery of a unique binding pocket provides valuable information that will facilitate the design of direct Ras inhibitors.

Identification and Characterization of a New Series of Ghrelin O-Acyl Transferase Inhibitors:

Ghrelin O-acyl transferase (GOAT; MBOAT4) catalyzes O-acylation at serine-3 of des-acyl ghrelin. Acyl ghrelin is secreted by stomach X/A-like cells and plays a role in appetite and metabolism. Therefore, GOAT has been expected to be a novel antiobesity target because it is responsible for acyl ghrelin production. Here, we report homogeneous time-resolved fluorescence (HTRF) and enzyme-linked immunosorbent assay (ELISA) methods utilizing human GOAT-expressing microsomes as a novel high-throughput assay system for the discovery of hit compounds and optimization of lead compounds. Hit compounds exemplified by compound A (2-[(2,4-dichlorobenzyl)sulfanyl]-1,3-benzoxazole-5-carboxylic acid) were identified by high-throughput screening using the HTRF assay and confirmed to have GOAT inhibitory activity using the ELISA. Based on the hit compound information, the novel lead compound (compound B, (4-chloro-6-{[2-methyl-6-(trifluoromethyl)pyridin-3-yl]methoxy}-1-benzothiophen-3-yl)acetic acid) was synthesized and exhibited potent GOAT inhibition with oral bioavailability. Both the hit compound and lead compound showed octanoyl-CoA competitive inhibitory activity. Moreover, these two compounds decreased acyl ghrelin production in the stomach of mice after their oral administration. These novel findings demonstrate that GOAT is a druggable target, and its inhibitors are promising antiobesity drugs.

Discovery of an irreversible and cell-active BCL6 inhibitor selectively targeting Cys53 located at the protein-protein interaction interface

B-cell lymphoma 6 (BCL6) is the most frequently involved oncogene in diffuse large B-cell lymphomas (DLBCLs). BCL6 shows potent transcriptional repressor activity through interactions with its corepressors, such as BCL6 corepressor (BCOR). The inhibition of the protein-protein interaction (PPI) between BCL6 and its corepressors suppresses the growth of BCL6-dependent DLBCLs, thus making BCL6 an attractive drug target for lymphoma treatment. However, potent small-molecule PPI inhibitor identification remains challenging because of the lack of deep cavities at PPI interfaces. This article reports the discovery of a potent, cell-active small-molecule BCL6 inhibitor, BCL6-i (8), that operates through irreversible inhibition. First, we synthesized irreversible lead compound 4, which targets Cys53 in a cavity on the BCL6-BTB domain dimer by introducing an irreversible warhead to high-throughput screening hit compound 1. Further chemical optimization of 4 based on kinact/KI evaluation produced BCL6-i with a kinact/KI value of 1.9 × 104 M-1 s-1, corresponding to a 670-fold improvement in potency compared to that of 4. By exploiting the property of irreversible inhibition, engagement of BCL6-i to intracellular BCL6 was confirmed. BCL6-i showed intracellular PPI inhibitory activity between BCL6 and its corepressors, thus resulting in BCL6-dependent DLBCL cell growth inhibition. BCL6-i is a cell-active chemical probe with the most potent BCL6 inhibitory activity reported to date. The discovery process of BCL6-i illustrates the utility of irreversible inhibition for identifying potent chemical probes for intractable target proteins.