Identification of α-Synuclein Proaggregator: Rapid Synthesis and Streamlining RT-QuIC Assays in Parkinson's Disease.

We report the discovery of two compounds, TKD150 and TKD152, that promote the aggregation of α-synuclein (aSN) using a real-time quaking-induced conversion (RT-QuIC) assay to detect abnormal aSN. By utilizing a Pd-catalyzed C-H arylation of benzoxazole with iodoarenes and implementing a planar conformation to the design, we successfully identified TKD150 and TKD152 as proaggregators for aSN. In comparison to a previously reported proaggregator, PA86, the two identified compounds were able to promote aggregation of aSN at twice the rate. Application of TKD150 and TKD152 to the RT-QuIC assay will shorten the inherent lag time and may allow wider use of this assay in clinical settings for the diagnosis of α-synucleinopathy-related diseases.

Identification of the first highly selective inhibitor of human lactate dehydrogenase B.

Lactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate and NADH to NAD+; it has two isoforms, LDHA and LDHB. LDHA is a promising target for cancer therapy, whereas LDHB is necessary for basal autophagy and cancer cell proliferation in oxidative and glycolytic cancer cells. To the best of our knowledge, selective inhibitors for LDHB have not yet been reported. Here, we developed a high-throughput mass spectrometry screening system using an LDHB enzyme assay by detecting NADH and NAD+. As a result, we identified a small-molecule LDHB selective inhibitor AXKO-0046, an indole derivative. This compound exhibited uncompetitive LDHB inhibition (EC50 = 42 nM). X-ray crystallography revealed that AXKO-0046 bound to the potential allosteric site away from the LDHB catalytic active site, suggesting that targeting the tetramerisation interface of the two dimers is critical for the enzymatic activity. AXKO-0046 and its derivatives can be used to validate LDHB-associated pathways in cancer metabolism.

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.

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.

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.

Discovery of Allosteric Inhibitors Targeting the Spliceosomal RNA Helicase Brr2.

Brr2 is an RNA helicase belonging to the Ski2-like subfamily and an essential component of spliceosome. Brr2 catalyzes an ATP-dependent unwinding of the U4/U6 RNA duplex, which is a critical step for spliceosomal activation. An HTS campaign using an RNA-dependent ATPase assay and initial SAR study identified two different Brr2 inhibitors, 3 and 12. Cocrystal structures revealed 3 binds to an unexpected allosteric site between the C-terminal and the N-terminal helicase cassettes, while 12 binds an RNA-binding site inside the N-terminal cassette. Selectivity profiling indicated the allosteric inhibitor 3 is more Brr2-selective than the RNA site binder 12. Chemical optimization of 3 using SBDD culminated in the discovery of the potent and selective Brr2 inhibitor 9 with helicase inhibitory activity. Our findings demonstrate an effective strategy to explore selective inhibitors for helicases, and 9 could be a promising starting point for exploring molecular probes to elucidate biological functions and the therapeutic relevance of Brr2.

Discovery of a novel prolyl-tRNA synthetase inhibitor and elucidation of its binding mode to the ATP site in complex with l-proline.

Prolyl-tRNA synthetase (PRS) is a member of the aminoacyl-tRNA synthetase family of enzymes and catalyzes the synthesis of prolyl-tRNAPro using ATP, l-proline, and tRNAPro as substrates. An ATP-dependent PRS inhibitor, halofuginone, was shown to suppress autoimmune responses, suggesting that the inhibition of PRS is a potential therapeutic approach for inflammatory diseases. Although a few PRS inhibitors have been derivatized from natural sources or substrate mimetics, small-molecule human PRS inhibitors have not been reported. In this study, we discovered a novel series of pyrazinamide PRS inhibitors from a compound library using pre-transfer editing activity of human PRS enzyme. Steady-state biochemical analysis on the inhibitory mode revealed its distinctive characteristics of inhibition with proline uncompetition and ATP competition. The binding activity of a representative compound was time-dependently potentiated by the presence of l-proline with Kd of 0.76 nM. Thermal shift assays demonstrated the stabilization of PRS in complex with l-proline and pyrazinamide PRS inhibitors. The binding mode of the PRS inhibitor to the ATP site of PRS enzyme was elucidated using the ternary complex crystal structure with l-proline. The results demonstrated the different inhibitory and binding mode of pyrazinamide PRS inhibitors from preceding halofuginone. Furthermore, the PRS inhibitor inhibited intracellular protein synthesis via a different mode than halofuginone. In conclusion, we have identified a novel drug-like PRS inhibitor with a distinctive binding mode. This inhibitor was effective in a cellular context. Thus, the series of PRS inhibitors are considered to be applicable to further development with differentiation from preceding halofuginone.

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.

Phase I study of TP300 in patients with advanced solid tumors with pharmacokinetic, pharmacogenetic and pharmacodynamic analyses.

BACKGROUND: A Phase I dose escalation first in man study assessed maximum tolerated dose (MTD), dose-limiting toxicity (DLT) and recommended Phase II dose of TP300, a water soluble prodrug of the Topo-1 inhibitor TP3076, and active metabolite, TP3011. METHODS: Eligible patients with refractory advanced solid tumors, adequate performance status, haematologic, renal, and hepatic function. TP300 was given as a 1-hour i.v. infusion 3-weekly and pharmacokinetic (PK) profiles of TP300, TP3076 and TP3011 were analysed. Polymorphisms in CYP2D6, AOX1 and UGT1A1 were studied and DNA strand-breaks measured in peripheral blood mononuclear cells (PBMCs). RESULTS: 32 patients received TP300 at 1, 2, 4, 6, 8, 10, 12 mg/m(2). MTD was 10 mg/m(2); DLTs at 12 (2/4 patients) and 10 mg/m(2) (3/12) included thrombocytopenia and febrile neutropenia; diarrhoea was uncommon. Six patients (five had received irinotecan), had stable disease for 1.5-5 months. TP3076 showed dose proportionality in AUC and Cmax from 1-10 mg/m(2). Genetic polymorphisms had no apparent influence on exposure. DNA strand-breaks were detected after TP300 infusion. CONCLUSIONS: TP300 had predictable hematologic toxicity, and diarrhoea was uncommon. AUC at MTD is substantially greater than for SN38. TP3076 and TP3011 are equi-potent with SN38, suggesting a PK advantage. TRIAL REGISTRATION: EU-CTR2006-001345-33.

Design and synthesis of novel allosteric MEK inhibitor CH4987655 as an orally available anticancer agent.

The MAP kinase pathway is one of the most important pathways involved in cell proliferation and differentiation, and its components are promising targets for antitumor drugs. Design and synthesis of a novel MEK inhibitor, based on the 3D-structural information of the target enzyme, and then multidimensional optimization including metabolic stability, physicochemical properties and safety profiles were effectively performed and led to the identification of a clinical candidate for an orally available potent MEK inhibitor, CH4987655, possessing a unique 3-oxo-oxazinane ring structure at the 5-position of the benzamide core structure. CH4987655 exhibits slow dissociation from the MEK enzyme, remarkable in vivo antitumor efficacy both in mono- and combination therapy, desirable metabolic stability, and insignificant MEK inhibition in mouse brain, implying few CNS-related side effects in human. An excellent PK profile and clear target inhibition in PBMC were demonstrated in a healthy volunteer clinical study.

A water soluble prodrug of a novel camptothecin analog is efficacious against breast cancer resistance protein-expressing tumor xenografts.

PURPOSE: Identification of a novel topoisomerase I inhibitor which shows superior efficacy and less individual variation than irinotecan hydrochloride (CPT-11). METHODS: A novel camptothecin analog that is effective against breast cancer resistance protein (BCRP)-positive cells was screened, and a water soluble prodrug was generated. Antitumor activity of the prodrug was examined in BCRP-positive and -negative xenografts both as a single agent and in combination with other anti-cancer drugs. RESULTS: A novel camptothecin analog, CH0793076, was discovered. Because CH0793076 was found to be highly lipophilic, a water soluble prodrug (TP300) was generated. TP300 is stable in an acidic solution but is rapidly converted to CH0793076 under physiological pH conditions such as in sera. This efficient prodrug activation would minimize interpatient differences in pharmacokinetic and toxicity profiles. Unlike CPT-11, TP300 does not exhibit cholinergic interaction or cause acute diarrhea at effective doses. In mouse xenograft models, TP300 showed antitumor activity against both BCRP-positive and -negative xenografts, whereas CPT-11 was less active against BCRP-positive xenografts. In addition, the effective dose range (MTD/ED(50)) for TP300 was wider than for CPT-11 and TP300 showed additive or synergistic antitumor effects in combination with other anti-cancer drugs such as capecitabine, oxaliplatin, cisplatin, bevacizumab and cetuximab. CONCLUSION: It is therefore expected that TP300 will provide an additional treatment option for patients who will undergo chemotherapy with camptothecins.

Design and synthesis of novel prodrugs of 2'-deoxy-2'-methylidenecytidine activated by membrane dipeptidase overexpressed in tumor tissues.

DNA microarray analysis comparing human tumor tissues with normal tissues including hematopoietic progenitor cells resulted in identification of membrane dipeptidase as a prodrug activation enzyme. Novel prodrugs of 2'-deoxy-2'-methylidenecytidine (DMDC) including compound 23 that are activated by membrane dipeptidase (MDP) preferentially in tumor tissue were designed and synthesized to generate the active drug, DMDC, after hydrolysis of the dipeptide bond followed by spontaneous cyclization of the promoiety.

Augmentation of the antitumor activity of capecitabine by a tumor selective dihydropyrimidine dehydrogenase inhibitor, RO0094889.

Capecitabine is an orally available fluoropyrimidine and is finally converted to 5-FU selectively in tumor tissues. In our study, we examined whether the antitumor activity of capecitabine is directly affected by a modulation of dihydropyrimidine dehydrogenase (DPD). The modulations were carried out by the overexpression of DPD in tumor cells and by tumor selective DPD inhibition. The DPD-overexpressing cells were obtained by transfection of human DPD cDNA into HCT116 human colorectal cancer cells. The HCT116 cells bearing DPD cDNA expressed about 13 times higher DPD activities than the parental HCT116 cells, and they became significantly less susceptible to capecitabine than the parental cells when transplanted into nude mice. Administration of RO0094889 that is converted to a DPD inhibitor 5-vinyluracil selectively in tumor tissues restored the antitumor activity of capecitabine against the tumor of the HCT116 cells carrying DPD cDNA and various tumors expressing DPD. As compared to 5-ethynyluracil or 5-vinyluracil, which inhibited DPD not only in tumor tissues but also in other non-cancerous tissues, the effective dose range of RO0094889 in augmenting the efficacy of capecitabine was much broader. These results indicate that the antitumor activity of capecitabine is directly affected by DPD activities in tumor tissues and therefore, the combination of capecitabine and a tumor selective DPD inhibitor, RO0094889, will be beneficial to patients who have tumors with high levels of DPD.

Design and synthesis of the tumor-activated prodrug of dihydropyrimidine dehydrogenase (DPD) inhibitor, RO0094889 for combination therapy with capecitabine.

A series of tumor-activated prodrugs of the inhibitors of dihydropyrimidine dehydrogenase (DPD), an enzyme catabolizing 5-fluorouracil (5-FU: 4g), has been designed and synthesized. RO0094889 (11c) is a prodrug of 5-vinyluracil (4c), a known DPD inhibitor, and was designed to generate 4c selectively in tumor tissues by sequential conversion of 11c by three enzymes: esterase, cytidine deaminase and thymidine phosphorylase, the latter two of which are known to be highly expressed in various tumor tissues. When capecitabine (1), a tumor-activated prodrug of 5-FU, was co-administered orally with 11c, 5-FU in tumor tissues was significantly increased with only a slight increase of 5-FU in plasma as compared with oral capecitabine alone.

A G protein-coupled receptor responsive to bile acids.

So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5'-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids.