Discovery of artificial VIPR2-antagonist peptides possessing receptor- and ligand-selectivity.

Vasoactive intestinal peptide receptor 2 (VIPR2, also known as VPAC2) is a class B G-protein coupled receptor (GPCR) and plays important roles in the physiology of central nervous system (CNS) by interaction with natural ligands; vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Because it has been reported that high-expression and/or overactivation of VIPR2 link to schizophrenic symptoms, VIPR2 antagonists could be good drug candidates for schizophrenia therapeutics. In this study, we discovered several artificial peptides that antagonize both human and rodent VIPR2 with selectivities against receptor subtypes VIPR1 (also known as VPAC1) and pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1). Of them, the representative 16-mer cyclic peptide VIpep-3 (Ac-CPPYLPRRLCTLLLRS-OH) exhibited strong binding affinity with KD value of 41 nM to extracellular domain of human VIPR2 in SPR analysis and showed potent antagonist activity with IC50 values of 47 nM (human), 180 nM (mouse), and 44 nM (rat) against VIP-VIPR2 signal in cell-based Ca influx assay. This is not only the first report on artificial VIPR2-selective antagonist peptides but also good example of the effective approach to discover novel antagonist against class B GPCR. Our peptides will contribute to study and development of the novel CNS drugs targeting to VIPR2.

Biochemical Properties of TAK-828F, a Potent and Selective Retinoid-Related Orphan Receptor Gamma t Inverse Agonist.

BACKGROUND/AIMS: Retinoid-related orphan receptor gamma t (RORγt) is a master regulator of T helper 17 cells that plays a pivotal role in the production of inflammatory cytokines including interleukin (IL)-17. Therefore, RORγt has attracted much attention as a target receptor for the treatment of inflammatory diseases including rheumatoid arthritis, multiple sclerosis, inflammatory bowel diseases, and psoriasis. This study aims to characterize TAK-828F, a potent and selective RORγt inverse agonist. METHODS: The biochemical properties of TAK-828F were evaluated using Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay, surface plasmon resonance (SPR) biosensor assay, cofactor recruitment assay, reporter assay, and IL-17 expression assay. RESULTS: TR-FRET binding assay and SPR biosensor assay revealed rapid, reversible, and high affinity binding of TAK-828F to RORγt. The cofactor recruitment assay showed that TAK-828F inhibited the recruitment of steroid receptor coactivator-1 to RORγt. Furthermore, TAK-828F inhibited the transcriptional activity of human and mouse RORγt with selectivity against human RORα and RORß. TAK-828F also suppressed IL-17 production in Jurkat cells, overexpressing human RORγt. CONCLUSION: These favorable properties will be of advantage in the evaluation of TAK-828F in clinical studies for inflammatory diseases. Furthermore, these findings demonstrate that TAK-828F could serve as a pharmacological tool for further studies of RORγt and inflammatory diseases.

Discovery of a Kelch-like ECH-associated protein 1-inhibitory tetrapeptide and its structural characterization.

Keap1 constitutively binds to the transcription factor Nrf2 to promote its degradation, resulting in negative modulation of genes involved in cellular protection against oxidative stress. Keap1 is increasingly recognized as an attractive target for treating diseases involving oxidative stress, including cancer, atherosclerosis, diabetes, arthritis, and neurodegeneration. We used phage-display peptide screening to identify a tetrapeptide showing moderate binding affinity, which inhibits the interaction between Nrf2 and Keap1. The tetrapeptide does not include an ETGE motif, which is a commonly found consensus sequence in known peptidic inhibitors. In addition to affinity parameters, IC50, KD, and thermodynamic parameters, the crystal structure of the complex was determined to elucidate the binding conformation. The binding interactions resemble those of known small-molecule inhibitors as opposed to those of substrates and peptidic inhibitors. Although the tetrapeptide's affinity is not very high, our results may help facilitate the designing of small-molecule inhibitors during lead generation in drug discovery.

Novel DOCK2-selective inhibitory peptide that suppresses B-cell line migration.

Dedicator of cytokinesis 2 (DOCK2) is a key molecule for lymphocyte activation and migration. DOCK2 interacts with Ras-related C3 botulinus toxin substrate 1 (Rac1, GTPase) and mediates the GDP-GTP exchange reaction, indicating that inhibitors against protein-protein interaction (PPI) between DOCK2 and Rac1 would be good drug candidates for treating immune-related disorders. Here, we report DOCK2-selective PPI inhibitory peptides discovered using random peptide T7 phage display technology. These peptides inhibited DOCK2 activity at nanomolar concentrations and were delivered to intracellular compartments by combination with cell-penetrating peptide (CPP). Consequently, one peptide, R4-DCpep-2(V2W/K4R/ox)-NH2 (Ac-RRRRCWARYHGYPWCRRRR-NH2), inhibited migration in human B lymphocyte MINO cell line at IC50 = 120 nM. To our knowledge, this is the first report of a DOCK2-selective peptide inhibitor; this study will contribute to the development of novel DOCK2-targeting immunosuppressive drugs.

Discovery of high-affinity BCL6-binding peptide and its structure-activity relationship.

B cell lymphoma 6 (BCL6) is a transcriptional repressor that interacts with its corepressors BcoR and SMRT. Since this protein-protein interaction (PPI) induces activation and differentiation of B lymphocytes, BCL6 has been an attractive drug target for potential autoimmune disease treatments. Here we report a novel BCL6 inhibitory peptide, F1324 (Ac-LWYTDIRMSWRVP-OH), which we discovered using phage display technology; we also discuss this peptide's structure-activity relationship (SAR). For BCL6(5-129) binding, KD and IC50 values of F1324 were 0.57 nM and 1 nM according to the results of an SPR analysis and cell-free ELISA assay, respectively. In contrast, BcoR(Arg498-514Pro) and SMRT(Leu1422-Arg1438) exhibited relatively weak micromole-order binding to BCL6. Furthermore, Fusion protein AcGFP-F1324 transiently expressed in HEK293T cells inhibited intracellular PPI in cell-based M2H assay. By examination of the truncation and fragmentation of F1324, the C-terminal sequence WRVP, which is similar to the BcoR(509-512) sequence WVVP, was identified as being critical for BCL6 binding. In addition, subsequent single-crystal X-ray diffraction analysis of F1324/BCL6(5-129) complex revealed that the high affinity of F1324 was caused by effective interaction of its side chains while its main chain structure was similar to that of BcoR(Arg498-514Pro). To our knowledge, F1324 is the strongest BCL6-binding peptide yet reported.

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.

K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.

Amino-acid mutations of Gly12 (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH2) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. KD and IC50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH2) that inhibited enzyme activity of K-Ras(G12D) with IC50 = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 µM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs.

Discovery of selective ATP-competitive eIF4A3 inhibitors.

Eukaryotic initiation factor 4A3 (eIF4A3), an ATP-dependent RNA helicase, is a core component of exon junction complex (EJC). EJC has a variety of roles in RNA metabolism such as translation, surveillance, and localization of spliced RNA. It is worthwhile to identify selective eIF4A3 inhibitors with a view to investigating the functions of eIF4A3 and EJC further to clarify the roles of the ATPase and helicase activities in cells. Our chemical optimization of hit compound 2 culminated in the discovery of ATP-competitive eIF4A3 inhibitor 18 with submicromolar ATPase inhibitory activity and excellent selectivity over other helicases. Hence, compound 18 could be a valuable chemical probe to elucidate the detailed functions of eIF4A3 and EJC.

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.

Proposal of an in vitro thrombus-growth model for evaluating anticoagulants.

The general anticoagulant evaluation requires high expense equipment, reagents, and space. Therefore, not all laboratories can perform research related to anticoagulant. In this study, we propose a novel simple method "in vitro thrombus-growth model" that can evaluate anticoagulant ability by measuring weight. The in vitro thrombus-growth model is prepared by creating a "growth-clot" with citrate plasma, calcium chloride, and thrombin, and then pouring new citrate plasma onto it. The prepared growth-clots were increased in volume in citrated human plasma, including surpluses calcium chloride, which was released slowly, leading to clot coagulation around the plasma. As a result of evaluating the anticoagulant ability of direct thrombin inhibitor using this in vitro thrombus-growth model, it was confirmed that clot growth was suppressed in a concentration-dependent manner. Therefore, this thrombus-growth model is useful as a primary anticoagulant test that can to discover compounds with anticoagulant activity perform in any laboratory.

Design and Synthesis of Novel Spiro Derivatives as Potent and Reversible Monoacylglycerol Lipase (MAGL) Inhibitors: Bioisosteric Transformation from 3-Oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl Moiety.

The therapeutic potential of monoacylglycerol lipase (MAGL) inhibitors in central nervous system-related diseases has attracted attention worldwide. However, the availability of reversible-type inhibitor is still limited to clarify the pharmacological effect. Herein, we report the discovery of novel spiro chemical series as potent and reversible MAGL inhibitors with a different binding mode to MAGL using Arg57 and His121. Starting from hit compound 1 and its co-crystal structure with MAGL, structure-based drug discovery (SBDD) approach enabled us to generate various spiro scaffolds like 2a (azetidine-lactam), 2b (cyclobutane-lactam), and 2d (cyclobutane-carbamate) as novel bioisosteres of 3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl moiety in 1 with higher lipophilic ligand efficiency (LLE). Optimization of the left hand side afforded 4f as a promising reversible MAGL inhibitor, which showed potent in vitro MAGL inhibitory activity (IC50 6.2 nM), good oral absorption, blood-brain barrier penetration, and significant pharmacodynamic changes (2-arachidonoylglycerol increase and arachidonic acid decrease) at 0.3-10 mg/kg, po. in mice.

Functional and physical interaction of yeast Mgs1 with PCNA: impact on RAD6-dependent DNA damage tolerance.

Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.

T-448, a specific inhibitor of LSD1 enzyme activity, improves learning function without causing thrombocytopenia in mice.

Dysregulation of histone H3 lysine 4 (H3K4) methylation has been implicated in the pathogenesis of several neurodevelopmental disorders. Targeting lysine-specific demethylase 1 (LSD1), an H3K4 demethylase, is therefore a promising approach to treat these disorders. However, LSD1 forms complexes with cofactors including growth factor independent 1B (GFI1B), a critical regulator of hematopoietic differentiation. Known tranylcypromine-based irreversible LSD1 inhibitors bind to coenzyme flavin adenine dinucleotide (FAD) and disrupt the LSD1-GFI1B complex, which is associated with hematotoxicity such as thrombocytopenia, representing a major hurdle in the development of LSD1 inhibitors as therapeutic agents. To discover LSD1 inhibitors with potent epigenetic modulation and lower risk of hematotoxicity, we screened small molecules that enhance H3K4 methylation by the inhibition of LSD1 enzyme activity in primary cultured rat neurons but have little impact on LSD1-GFI1B complex in human TF-1a erythroblasts. Here we report the discovery of a specific inhibitor of LSD1 enzyme activity, T-448 (3-((1S,2R)-2-(cyclobutylamino)cyclopropyl)-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzamide fumarate). T-448 has minimal impact on the LSD1-GFI1B complex and a superior hematological safety profile in mice via the generation of a compact formyl-FAD adduct. T-448 increased brain H3K4 methylation and partially restored learning function in mice with NMDA receptor hypofunction. T-448-type LSD1 inhibitors with improved safety profiles may provide unique therapeutic approaches for central nervous system disorders associated with epigenetic dysregulation.

Cholesterol Unbound RORγt Protein Enables a Sensitive Inverse Agonist Screening.

The retinoic acid-related orphan receptor gamma T (RORγt) plays an important role in Th17 cell proliferation and functionality. Thus, RORγt inverse agonists are thought to be potent therapeutic agents for Th17-mediated autoimmune diseases, such as rheumatoid arthritis, asthma, inflammatory bowel disease, and psoriasis. Although RORγt has constitutive activity, it is recognized that the receptor is physiologically regulated by various cholesterol derivatives. In this study, we sought to identify RORγt inverse agonists through a high-throughput screening campaign. To this end, we compared an apo-RORγt protein from Escherichia coli and a cholesterol-bound RORγt protein from insect cells. The IC50 of the known RORγt inverse agonist TO901317 was significantly lower for the apoprotein than for the cholesterol-bound RORγt. Through high-throughput screening using a fluorescence-based cholesterol binding assay with the apoprotein, we identified compound 1 as a novel cholesterol-competitive RORγt inverse agonist. Compound 1 inhibited the RORγt-TopFluor cholesterol interaction, coactivator recruitment, and transcriptional activity of RORγt. Cell-based reporter gene assay demonstrated that compound 1 showed higher potency by lipid depletion treatment. Collectively, our findings indicate that eliminating cholesterol from the RORγt protein is suitable for sensitive high-throughput screening to identify RORγt inverse 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.

LSD1 Inhibitor T-3775440 Inhibits SCLC Cell Proliferation by Disrupting LSD1 Interactions with SNAG Domain Proteins INSM1 and GFI1B.

T-3775440 is an irreversible inhibitor of the chromatin demethylase LSD1, which exerts antiproliferative effects by disrupting the interaction between LSD1 and GFI1B, a SNAG domain transcription factor, inducing leukemia cell transdifferentiation. Here, we describe the anticancer effects and mechanism of action of T-3775440 in small-cell lung cancer (SCLC). T-3775440 inhibited proliferation of SCLC cells in vitro and retarded SCLC tumor growth in vivo T-3775440 disrupted the interaction between LSD1 and the transcriptional repressor INSM1, thereby inhibiting expression of neuroendocrine-associated genes, such as ASCL1 INSM1 silencing phenocopied the effects of T-3775440 on gene expression and cell proliferation, consistent with the likelihood T-3775440 mediated its effects in SCLC by inhibiting INSM1. T-3775440 also inhibited proliferation of an SCLC cell line that overexpressed GFI1B, rather than INSM1, by disrupting the interaction between LSD1 and GFI1B. Taken together, our results argue that LSD1 plays an important role in neuroendocrine-associated transcription and cell proliferation of SCLC via interactions with the SNAG domain proteins INSM1 and GFI1B. Targeting these critical interactions with LSD1 inhibitors offers a novel rational strategy to therapeutically manage SCLC. Cancer Res; 77(17); 4652-62. ©2017 AACR.

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.

A pyridone derivative activates SERCA2a by attenuating the inhibitory effect of phospholamban.

The cardiac sarco/endoplasmic reticulum Ca2+-dependent ATPase 2a (SERCA2a) plays a central role in Ca2+ handling within cardiomyocytes and is negatively regulated by phospholamban (PLN), a sarcoplasmic reticulum (SR) membrane protein. The activation of SERCA2a, which has been reported to improve cardiac dysfunction in heart failure, is a potential therapeutic approach for heart failure. Therefore, we developed a novel small molecule, compound A and characterized it both in vitro and in vivo. Compound A activated the Ca2+-dependent ATPase activity of cardiac SR vesicles but not that of skeletal muscle SR vesicles that lack PLN. The surface plasmon resonance assay revealed a direct interaction between compound A and PLN, suggesting that the binding of compound A to PLN attenuates its inhibition of SERCA2a, resulting in SERCA2a activation. This was substantiated by inhibition of the compound A-mediated increase in Ca2+ levels within the SR of HL-1 cells by thapsigargin, a SERCA inhibitor. Compound A also increased the Ca2+ transients and contraction and relaxation of isolated adult rat cardiomyocytes. In isolated perfused rat hearts, the compound A enhanced systolic and diastolic functions. Further, an infusion of compound A (30mg/kg, i.v. bolus followed by 2mg/kg/min, i.v. infusion) significantly enhanced the diastolic function in anesthetized normal rats. These results indicate that compound A is a novel SERCA2a activator, which attenuates PLN inhibition and enhances the systolic and diastolic functions of the heart in vitro and in vivo. Therefore, compound A might be a novel therapeutic lead for heart failure.

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.

Identification of cytidine-5-triphosphate synthase1-selective inhibitory peptide from random peptide library displayed on T7 phage.

Cytidine triphosphate synthase 1 (CTPS1) is an enzyme expressed in activated lymphocytes that catalyzes the conversion of uridine triphosphate (UTP) to cytidine triphosphate (CTP) with ATP-dependent amination, using either L-glutamine or ammonia as the nitrogen source. Since CTP plays an important role in DNA/RNA synthesis, phospholipid synthesis, and protein sialyation, CTPS1-inhibition is expected to control lymphocyte proliferation and size expansion in inflammatory diseases. In contrast, CTPS2, an isozyme of CTPS1 possessing 74% amino acid sequence homology, is expressed in normal lymphocytes. Thus, CTPS1-selective inhibition is important to avoid undesirable side effects. Here, we report the discovery of CTpep-3: Ac-FRLGLLKAFRRLF-OH from random peptide libraries displayed on T7 phage, which exhibited CTPS1-selective binding with a KD value of 210nM in SPR analysis and CTPS1-selective inhibition with an IC50 value of 110nM in the enzyme assay. Furthermore, two fundamentally different approaches, enzyme inhibition assay and HDX-MS, provided the same conclusion that CTpep-3 acts by binding to the amidoligase (ALase) domain on CTPS1. To our knowledge, CTpep-3 is the first CTPS1-selective inhibitor.