DeepCubist: Molecular Generator for Designing Peptidomimetics based on Complex three-dimensional scaffolds.

Mimicking bioactive conformations of peptide segments involved in the formation of protein-protein interfaces with small molecules is thought to represent a promising strategy for the design of protein-protein interaction (PPI) inhibitors. For compound design, the use of three-dimensional (3D) scaffolds rich in sp3-centers makes it possible to precisely mimic bioactive peptide conformations. Herein, we introduce DeepCubist, a molecular generator for designing peptidomimetics based on 3D scaffolds. Firstly, enumerated 3D scaffolds are superposed on a target peptide conformation to identify a preferred template structure for designing peptidomimetics. Secondly, heteroatoms and unsaturated bonds are introduced into the template via a deep generative model to produce candidate compounds. DeepCubist was applied to design peptidomimetics of exemplary peptide turn, helix, and loop structures in pharmaceutical targets engaging in PPIs.

A novel mRNA decay inhibitor abolishes pathophysiological cellular transition.

In cells, mRNA synthesis and decay are influenced by each other, and their balance is altered by either external or internal cues, resulting in changes in cell dynamics. We previously reported that it is important that an array of mRNAs that shape a phenotype are degraded before cellular transitions, such as cellular reprogramming and differentiation. In adipogenesis, the interaction between DDX6 and 4E-T had a definitive impact on the pathway in the processing body (PB). We screened a library of α-helix analogs with an alkaloid-like backbone to identify compounds that inhibit the binding between DDX6 and 4E-T proteins, which occurs between the α-helix of structured and internally disordered proteins. IAMC-00192 was identified as a lead compound. This compound directly inhibited the interaction between DDX6 and 4E-T. IAMC-00192 inhibited the temporal increase in PB formation that occurs during adipogenesis and epithelial-mesenchymal transition (EMT) and significantly suppressed these cellular transitions. In the EMT model, the half-life of preexisting mRNAs in PBs was extended twofold by the compound. The novel inhibitor of RNA decay not only represents a potentially useful tool to analyze in detail the pathological conditions affected by RNA decay and how it regulates the pathological state. The identification of this inhibitor may lead to the discovery of a first-in-class RNA decay inhibitor drug.

Development of Low-Molecular-Weight Compounds Targeting the Cancer-Associated KLF5 Transcription Factor.

Krüppel-like factor 5 (KLF5) is a potential target for anticancer drugs. However, as an intrinsically disordered protein (IDP) whose tertiary structure cannot be solved, innovative strategies are needed. We focused on its hydrophobic α-helix structure, defined as an induced helical motif (IHM), which is a possible interface for protein-protein interaction. Using mathematical analyses predicting the α-helix's structure and hydrophobicity, a 4-amino-acid site (V-A-I-F) was identified as an IHM. Low-molecular-weight compounds that mimic the main chain conformation of the α-helix with the four side chains of V-A-I-F were synthesized using bicyclic pyrazinooxadiazine-4,7-dione. These compounds selectively suppressed the proliferation and survival of cancer cells but not noncancer cells and decreased the protein but not mRNA levels of KLF5 in addition to reducing proteins of Wnt signaling. The compounds further suppressed transplanted colorectal cancer cells in vivo without side effects. Our approach appears promising for developing drugs against key IDPs.

Involvement of activator protein-1 family members in ß-catenin and p300 association on the genome of PANC-1 cells.

Wnt/ß-catenin is believed to regulate different sets of genes with different coactivators, cAMP response element-binding protein (CREB)-binding protein (CBP) or p300. However, the factors that determine which coactivators act on a particular promoter remain elusive. ICG-001 is a specific inhibitor for ß-catenin/CBP but not for ß-catenin/p300. By taking advantage of the action of ICG-001, we sought to investigate regulatory mechanisms underlying ß-catenin coactivator usage in human pancreatic carcinoma PANC-1 cells through combinatorial analysis of chromatin immunoprecipitation-sequencing and RNA-sequencing. CBP and p300 preferentially bound to regions with the TCF motif alone and with both the TCF and AP-1 motifs, respectively. ICG-001 increased ß-catenin binding to regions with both the TCF and AP-1 motifs, flanking the genes induced by ICG-001, concomitant with the increments of the p300 and AP-1 component c-JUN binding. Taken together, AP-1 possibly coordinates ß-catenin coactivator usage in PANC-1 cells. These results would further our understanding of the canonical Wnt/ß-catenin signaling divergence.

Comprehensive exploration of chemical space using trisubstituted carboranes.

A total of 42 trisubstituted carboranes categorised into five scaffolds were systematically designed and synthesized by exploiting the different reactivities of the twelve vertices of o-, m-, and p-carboranes to cover all directions in chemical space. Significant inhibitors of hypoxia inducible factor transcriptional activitay were mainly observed among scaffold V compounds (e.g., Vi-m, and Vo), whereas anti-rabies virus activity was observed among scaffold V (Va-h), scaffold II (IIb-g), and scaffold IV (IVb) compounds. The pharmacophore model predicted from compounds with scaffold V, which exhibited significant anti-rabies virus activity, agreed well with compounds IIb-g with scaffold II and compound IVb with scaffold IV. Normalized principal moment of inertia analysis indicated that carboranes with scaffolds I-V cover all regions in the chemical space. Furthermore, the first compounds shown to stimulate the proliferation of the rabies virus were found among scaffold V carboranes.

Synthesis of Three-Dimensional (Di)Azatricyclododecene Scaffold and Its Application to Peptidomimetics.

A novel sp3 carbon-rich tricyclic 3D scaffold-based peptide mimetic compound library was constructed to target protein-protein interactions. Tricyclic framework 7 was synthesized from 9-azabicyclo[3,3,1]nonan-3-one (11) via a gold(I)-catalyzed Conia-ene reaction. The electron-donating group on the pendant alkyne of cyclization precursor 12 b-e was the key to forming 6-endo-dig cyclized product 7 with complete regioselectivity. Using the synthetic strategy for regioselective construction of bridged tricyclic framework 7, a diazatricyclododecene 3D-scaffold 8 a, which enables the introduction of substituents into the scaffold to mimic amino acid side chains, was designed and synthesized. The peptide mimetics 21 a-u were synthesized via step-by-step installation of three substituents on diazatricyclododecene scaffold 8 a. Compounds 21 a-h were synthesized as α-helix peptide mimics of hydrophobic ZZxxZ and ZxxZZ sequences (Z=Leu or Phe) and subjected to cell-based assays: antiproliferative activity, HIF-1 transcriptional activity which is considered to affect cancer malignancy, and antiviral activity against rabies virus. Compound 21 a showed the strongest inhibitory activity of HIF-1 transcriptional activity (IC50 =4.1±0.8 µM), whereas compounds 21 a-g showed antiviral activity with IC50 values of 4.2-12.4 µM, suggesting that the 3D-scaffold 8 a has potential as a versatile peptide mimic skeleton.

E7386, a Selective Inhibitor of the Interaction between ß-Catenin and CBP, Exerts Antitumor Activity in Tumor Models with Activated Canonical Wnt Signaling.

The Wnt/ß-catenin signaling pathway plays crucial roles in embryonic development and the development of multiple types of cancer, and its aberrant activation provides cancer cells with escape mechanisms from immune checkpoint inhibitors. E7386, an orally active selective inhibitor of the interaction between ß-catenin and CREB binding protein, which is part of the Wnt/ß-catenin signaling pathway, disrupts the Wnt/ß-catenin signaling pathway in HEK293 and adenomatous polyposis coli (APC)-mutated human gastric cancer ECC10 cells. It also inhibited tumor growth in an ECC10 xenograft model and suppressed polyp formation in the intestinal tract of ApcMin /+ mice, in which mutation of Apc activates the Wnt/ß-catenin signaling pathway. E7386 demonstrated antitumor activity against mouse mammary tumors developed in mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice. Gene expression profiling using RNA sequencing data of MMTV-Wnt1 tumor tissue from mice treated with E7386 showed that E7386 downregulated genes in the hypoxia signaling pathway and immune responses related to the CCL2, and IHC analysis showed that E7386 induced infiltration of CD8+ cells into tumor tissues. Furthermore, E7386 showed synergistic antitumor activity against MMTV-Wnt1 tumor in combination with anti-PD-1 antibody. In conclusion, E7386 demonstrates clear antitumor activity via modulation of the Wnt/ß-catenin signaling pathway and alteration of the tumor and immune microenvironments, and its antitumor activity can be enhanced in combination with anti-PD-1 antibody. SIGNIFICANCE: These findings demonstrate that the novel anticancer agent, E7386, modulates Wnt/ß-catenin signaling, altering the tumor immune microenvironment and exhibiting synergistic antitumor activity in combination with anti-PD-1 antibody.

Design and synthesis of 14 and 15-membered macrocyclic scaffolds exhibiting inhibitory activities of hypoxia-inducible factor 1α.

Inspired by the privileged molecular skeletons of 14- and 15-membered antibiotics, we adopted a relatively unexplored synthetic approach that exploits alkaloidal macrocyclic scaffolds to generate modulators of protein-protein interactions (PPIs). As mimetics of hot-spot residues in the α-helices responsible for the transcriptional regulation, three hydrophobic sidechains were displayed on each of the four distinct macrocyclic scaffolds generating diversity of their spatial arrangements. Modular assembly of the building blocks followed by ring-closing olefin metathesis reaction and subsequent hydrogenation allowed concise and divergent synthesis of scaffolds 1-4. The 14-membered alkaloidal macrocycles 2-4 demonstrated similar inhibition of hypoxia-inducible factor (HIF)-1α transcriptional activities (IC50 between 8.7 and 10 µM), and 4 demonstrated the most potent inhibition of cell proliferation in vitro (IC50 = 12 µM against HTC116 colon cancer cell line). A docking model suggested that 4 could mimic the LLxxL motif in HIF-1α, in which the three sidechains are capable of matching the spatial arrangements of the protein hot-spot residues. Unlike most of the stapled peptides, the 14-membered alkaloidal scaffold has a similar size to the α-helix backbone and does not require additional atoms to induce α-helix mimetic structure. These experimental results underscore the potential of alkaloidal macrocyclic scaffolds featuring flexibly customizable skeletal, stereochemical, substitutional, and conformational properties for the development of non-peptidyl PPI modulators targeting α-helix-forming consensus sequences responsible for the transcriptional regulation.

Anti-tumor Activity of the Small Molecule Inhibitor PRI-724 Against ß-Catenin-activated Hepatocellular Carcinoma.

BACKGROUND/AIM: CBP is a transcriptional coactivator in the Wnt/ß-catenin pathway that is related to cell kinetics and differentiation. This study aimed to characterize ß-catenin-activated hepatocellular carcinoma (HCC) and evaluate the direct effects of PRI-724 (a selective inhibitor of Wnt/ß-catenin/CBP signaling) on HCC. MATERIALS AND METHODS: Immunohistochemistry for ß-catenin was performed in 199 HCC resected samples. Moreover, using cultured HCC cell lines, cell kinetics and its related proteins were analyzed after treatment of cells with C-82 (active form of PRI-724). RESULTS: Nuclear ß-catenin expression was found in 18% of HCC cases and the tumor sizes in these positive samples were larger. In HCC cell lines with a constitutively activated ß-catenin, C-82 inhibited cell proliferation. C-82 led to an increase in the percentage of cells in the G0/G1 phase of the cell cycle. The percentage of cells in the sub-G1 phase also increased. Moreover, C-82 treatment significantly decreased the expression of cell proliferating markers and increased the expression of apoptosis-related proteins. CONCLUSION: PRI-724(C-82) may be a novel drug for ß-catenin-activated HCC therapy.

ß-catenin signaling inhibitors ICG-001 and C-82 improve fibrosis in preclinical models of endometriosis.

Endometriosis exhibits unique characteristics, such as fibrosis, resistance to apoptosis, and promotion of cell proliferation; however, its pathophysiology is not fully understood. Recurrence rates after treatment are high, and the progression risk continues until menopause; hence, more effective therapy for endometriosis is needed. CREB-binding protein (CBP)/ß-catenin signaling inhibitors have demonstrated antifibrogenetic effects in liver, lung, and skin diseases. The present study evaluated the effects of two CBP/ß-catenin signaling inhibitors, ICG-001 and C-82, on the progression of endometriosis using endometriotic cyst stromal cells from the ovary and normal endometrial stromal cells from the uterus. ICG-001 was also evaluated in a mouse model. ICG-001 and C-82 inhibited cell proliferation, fibrogenesis, and cell migration, and promoted apoptosis in vitro. ICG-001 inhibited the growth of endometriotic lesions in the mouse model. CBP/ß-catenin signaling plays an important role in the pathophysiology of endometriosis. Inhibiting the CBP/ß-catenin signal can be a therapeutic target for endometriosis.

The novel inhibitor PRI-724 for Wnt/ß-catenin/CBP signaling ameliorates bleomycin-induced pulmonary fibrosis in mice.

Purpose/Aim of the Study: Wnt/ß-catenin signaling was reported to be activated in pulmonary fibrosis, and was focused on as a target for antifibrotic therapy. However, the mechanism how the inhibition of Wnt/ß-catenin signaling ameliorate pulmonary fibrosis has not been fully elucidated. The purpose of this study is to explore the target cells of Wnt/ß-catenin inhibition in pulmonary fibrosis and to examine the antifibrotic effect of the novel inhibitor PRI-724 specifically disrupting the interaction of ß-catenin and CBP. Materials and Methods: The effect of C-82, an active metabolite of PRI-724, on the expression of TGF-ß1 and α-smooth muscle actin (SMA) was examined on fibroblasts and macrophages. We also examined the effects of PRI-724 in mouse model of bleomycin-induced pulmonary fibrosis. Results: The activation and increased accumulation of ß-catenin in the canonical pathway were detected in lung fibroblasts as well as macrophages stimulated by Wnt3a using Western blotting. Treatment with C-82 reduced CBP protein and increased p300 protein binding to ß-catenin in the nucleus of lung fibroblasts. In addition, C-82 inhibited the expression of SMA in lung fibroblasts treated with TGF-ß, indicating the inhibition of myofibroblast differentiation. In the fibrotic lungs induced by bleomycin, ß-catenin was stained strongly in macrophages, but the staining of ß-catenin in alveolar epithelial cells and fibroblasts was weak. The administration of PRI-724 ameliorated pulmonary fibrosis induced by bleomycin in mice when administered with a late, but not an early, treatment schedule. Analysis of bronchoalveolar fluid (BALF) showed a decreased number of alveolar macrophages. In addition, the level of TGF-ß1 in BALF was decreased in mice treated with PRI-724. C-82 also inhibited the production of TGF-ß1 by alveolar macrophages. Conclusions: These results suggest that the ß-catenin/CBP inhibitor PRI-724 is a potent antifibrotic agent that acts by modulating the activity of macrophages in the lungs.

mS-11, a mimetic of the mSin3-binding helix in NRSF, ameliorates social interaction deficits in a prenatal valproic acid-induced autism mouse model.

Growing evidence suggests pivotal roles for epigenetic mechanisms in both animal models of and individuals with autism spectrum disorders (ASD). Neuron-restrictive silencer factor (NRSF) binds to neuron-restrictive silencing elements in neuronal genes and recruits co-repressors, such as mSin3, to epigenetically inhibit neuronal gene expression. Because dysregulation of NRSF is related to ASD, here we examined the effects of mS-11, a chemically optimized mimetic of the mSin3-binding helix in NRSF, on the behavioral and morphological abnormalities found in a mouse model of valproic acid (VPA)-induced ASD. Chronic treatment with mS-11 improved prenatal VPA-induced deficits in social interaction. Additionally, we found that NRSF mRNA expression was greater in the somatosensory cortex of VPA-exposed mice than of controls. Agreeing with these behavioral findings, mice that were prenatally exposed to VPA showed lower dendritic spine density in the somatosensory cortex, which was reversed by chronic treatment with mS-11. These findings suggest that mS-11 has the potential for improving ASD-related symptoms through inhibition of mSin3-NRSF binding.

Selective Inhibition of ß-Catenin/Co-Activator Cyclic AMP Response Element-Binding Protein-Dependent Signaling Prevents the Emergence of Hapten-Induced Atopic Dermatitis-Like Dermatitis.

BACKGROUND: The canonical Wnt/ß-catenin signaling pathway is a fundamental regulatory system involved in various biological events. ICG-001 selectively blocks the interaction of ß-catenin with its transcriptional co-activator cyclic AMP response element-binding protein (CBP). Recent studies have provided convincing evidence of the inhibitory effects of ICG-001 on Wnt-driven disease models, such as organ fibrosis, cancer, acute lymphoblastic leukemia, and asthma. However, the effects of ICG-001 in atopic dermatitis (AD) have not been investigated. OBJECTIVE: To investigate whether ß-catenin/CBP-dependent signaling was contributed in the pathogenesis of AD and ICG-001 could be a therapeutic agent for AD. METHODS: We examined the effects of ICG-001 in an AD-like murine model generated by repeated topical application of the hapten, oxazolone (Ox). ICG-001 or vehicle alone was injected intraperitoneally every day during the development of AD-like dermatitis arising from once-daily Ox treatment. RESULTS: Ox-induced AD-like dermatitis characterized by increases in transepidermal water loss, epidermal thickness, dermal thickness accompanied by increased myofibroblast and mast cell counts, and serum levels of thymic stromal lymphopoietin and thymus and activation-regulated chemokine, and decreases in stratum corneum hydration, were virtually normalized by the treatment with ICG-001. Elevated serum levels of periostin tended to be downregulated, without statistical significance. CONCLUSION: These results suggest that ß-catenin/CBP-dependent signaling might be involved in the pathogenesis of AD and could be a therapeutic target.

A mimetic of the mSin3-binding helix of NRSF/REST ameliorates abnormal pain behavior in chronic pain models.

The neuron-restrictive silencing factor NRSF/REST binds to neuron-restrictive silencing elements in neuronal genes and recruits corepressors such as mSin3 to inhibit epigenetically neuronal gene expression. Because dysregulation of NRSF/REST is related to neuropathic pain, here, we have designed compounds to target neuropathic pain based on the mSin3-binding helix structure of NRSF/REST and examined their ability to bind to mSin3 by NMR. One compound, mS-11, binds strongly to mSin3 with a binding mode similar to that of NRSF/REST. In a mouse model of neuropathic pain, mS-11 was found to ameliorate abnormal pain behavior and to reverse lost peripheral morphine analgesia. Furthermore, even in the less well epigenetically defined case of fibromyalgia, mS-11 ameliorated symptoms in a mouse model, suggesting that fibromyalgia is related to the dysfunction of NRSF/REST. Taken together, these findings show that the chemically optimized mimetic mS-11 can inhibit mSin3-NRSF/REST binding and successfully reverse lost peripheral and central morphine analgesia in mouse models of pain.

Establishment of an intermittent cold stress model using Tupaia belangeri and evaluation of compound C737 targeting neuron-restrictive silencer factor.

Previous studies have shown that intermittent cold stress (ICS) induces depression-like behaviors in mammals. Tupaia belangeri (the tree shrew) is the only experimental animal other than the chimpanzee that has been shown to be susceptible to infection by hepatitis B and C viruses. Moreover, full genome sequence analysis has revealed strong homology between host proteins in Tupaia and in humans and other primates. Tupaia neuromodulator receptor proteins are also known to have a high degree of homology with their corresponding primate proteins. Based on these similarities, we hypothesized that induction of ICS in Tupaia would provide a useful animal model of stress responses. We exposed young adult Tupaia to ICS and observed decreases in body temperature and body weight in both female and male Tupaia, suggesting that Tupaia are an appropriate animal model for ICS studies. We further examined the efficacy of a new small-molecule compound, C737, against the effects of ICS. C737 mimics the helical structure of neuron-restrictive silencer factor (NRSF/REST), which regulates a wide range of target genes involved in neuronal function and pain modulation. Treatment with C737 significantly reduced stress-induced weight loss in female Tupaia; these effects were stronger than those elicited by the antidepressant agomelatine. These results suggest that Tupaia represents a useful non-rodent ICS model. Our data also provide new insights into the function of NRSF/REST in stress-induced depression and other disorders with epigenetic influences or those with high prevalence in women.

Follow-up: Prospective compound design using the 'SAR Matrix' method and matrix-derived conditional probabilities of activity.

In a previous Method Article, we have presented the 'Structure-Activity Relationship (SAR) Matrix' (SARM) approach. The SARM methodology is designed to systematically extract structurally related compound series from screening or chemical optimization data and organize these series and associated SAR information in matrices reminiscent of R-group tables. SARM calculations also yield many virtual candidate compounds that form a "chemical space envelope" around related series. To further extend the SARM approach, different methods are developed to predict the activity of virtual compounds. In this follow-up contribution, we describe an activity prediction method that derives conditional probabilities of activity from SARMs and report representative results of first prospective applications of this approach.

Identification of new agonists of urotensin-II from a cyclic peptide library.

Urotensin-II (UT-II) is thought to be involved in the regulation of cardiovascular homeostasis and pathology. A head-to-tail cyclic hexapeptide library based on UT-II sequence was designed, synthesized, and evaluated by the activity on the UT-II receptor (GPR-14). A new synthetic sequence, WK[Xaa] (Xaa: amino acid with aromatic side chain), was identified as a characteristic minimum fragment activating hUT-II receptor instead of the WK[Y] sequence. Compound 1 showed an agonistic activity with an EC(50) value of 6.94 nM. The conformational investigation suggested that 1 did not have typical secondary structure in the message sequence. Structural analyses may enable us to investigate the active conformation of UT-II and lead to the identification of new ligands for GPR-14.

Deprotection of the indole (N(ind))-formyl (For) group on tryptophan employing a new reagent, N,N'-dimethylethylendiamine (DMEDA) in an aqueous solution.

The deprotection of the indole (N(ind))-formyl (For) group on Trp was achieved in a 95% yield using N,N'-dimethylethylendiamine (DMEDA) (1.5, 2.0, 3.0 eq) in water at room temperature. A new reagent was successfully applied to the deprotection of a model peptide, H-Phe-Trp(N(ind)-For)-Lys-Tyr-OH, to give H-Phe-Trp-Lys-Tyr-OH in a 91% yield.

Virtual screening leads to the discovery of an effective antagonist of lymphocyte function-associated antigen-1.

The binding of lymphocyte function-associated antigen-1 (LFA-1) to its ligand on endothelial cells, intercellular adhesion molecule-1 (ICAM-1), is a crucial step in the migration of leukocytes during the early stages of inflammation and is also involved in T-cell activation. In this paper, we report the identification of a series of novel antagonists of the LFA-1/ICAM-1 interaction using ligand-based virtual screening (VS), analogue design, and structure-activity relationship (SAR) analysis. Candidate compounds were evaluated in protein binding and cell adhesion assays. Experimental evaluation of only 25 candidates selected from a pool of approximately 2.5 million database compounds identified an initial hit that could be expanded and converted into a lead that effectively blocked the interaction between LFA-1 and ICAM-1.