Cryo-EM Structure of K+-Bound hERG Channel Complexed with the Blocker Astemizole.

The hERG channel is a voltage-gated potassium channel involved in cardiac repolarization. Off-target hERG inhibition by drugs has become a critical issue in the pharmaceutical industry. The three-dimensional structure of the hERG channel was recently reported at 3.8-Å resolution using cryogenic electron microscopy (cryo-EM). However, the drug inhibition mechanism remains unclear because of the scarce structural information regarding the drug- and potassium-bound hERG channels. In this study, we obtained the cryo-EM density map of potassium-bound hERG channel complexed with astemizole, a well-known hERG inhibitor that increases risk of potentially fatal arrhythmia, at 3.5-Å resolution. The structure suggested that astemizole inhibits potassium conduction by binding directly below the selectivity filter. Furthermore, we propose a possible binding model of astemizole to the hERG channel and provide insights into the unusual sensitivity of hERG to several drugs.

Discovery of novel serine palmitoyltransferase inhibitors as cancer therapeutic agents.

We pursued serine palmitoyltransferase (SPT) inhibitors as novel cancer therapeutic agents based on a correlation between SPT inhibition and growth suppression of cancer cells. High-throughput screening and medicinal chemistry efforts led to the identification of structurally diverse SPT inhibitors 4 and 5. Both compounds potently inhibited SPT enzyme and decreased intracellular ceramide content. In addition, they suppressed cell growth of human lung adenocarcinoma HCC4006 and acute promyelocytic leukemia PL-21, and displayed good pharmacokinetic profiles. Reduction of 3-ketodihydrosphingosine, the direct downstream product of SPT, was confirmed under in vivo settings after oral administration of compounds 4 and 5. Their anti-tumor efficacy was observed in a PL-21 xenograft mouse model. These results suggested that SPT inhibitors might have potential to be effective cancer therapeutics.

Discovery of [ cis-3-({(5 R)-5-[(7-Fluoro-1,1-dimethyl-2,3-dihydro-1 H-inden-5-yl)carbamoyl]-2-methoxy-7,8-dihydro-1,6-naphthyridin-6(5 H)-yl}carbonyl)cyclobutyl]acetic Acid (TAK-828F) as a Potent, Selective, and Orally Available Novel Retinoic Acid Receptor-Related Orphan Receptor γt Inverse Agonist.

A series of tetrahydronaphthyridine derivatives as novel RORγt inverse agonists were designed and synthesized. We reduced the lipophilicity of tetrahydroisoquinoline compound 1 by replacement of the trimethylsilyl group and SBDD-guided scaffold exchange, which successfully afforded compound 7 with a lower log  D value and tolerable in vitro activity. Consideration of LLE values in the subsequent optimization of the carboxylate tether led to the discovery of [ cis-3-({(5 R)-5-[(7-fluoro-1,1-dimethyl-2,3-dihydro-1 H-inden-5-yl)carbamoyl]-2-methoxy-7,8-dihydro-1,6-naphthyridin-6(5 H)-yl}carbonyl)cyclobutyl]acetic acid, TAK-828F (10), which showed potent RORγt inverse agonistic activity, excellent selectivity against other ROR isoforms and nuclear receptors, and a good pharmacokinetic profile. In animal studies, oral administration of compound 10 exhibited robust and dose-dependent inhibition of IL-17A cytokine expression in a mouse IL23-induced gene expression assay. Furthermore, development of clinical symptoms in a mouse experimental autoimmune encephalomyelitis model was significantly reduced. Compound 10 was selected as a clinical compound for the treatment of Th17-driven autoimmune diseases.

Identification of novel quinazolinedione derivatives as RORγt inverse agonist.

Novel small molecules were synthesized and evaluated as retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists for the treatment of inflammatory and autoimmune diseases. A hit compound, 1, was discovered by high-throughput screening of our compound library. The structure-activity relationship (SAR) study of compound 1 showed that the introduction of a chlorine group at the 3-position of 4-cyanophenyl moiety increased the potency and a 3-methylpentane-1,5-diamide linker is favorable for the activity. The carbazole moiety of 1 was also optimized; a quinazolinedione derivative 18i suppressed the increase of IL-17A mRNA level in the lymph node of a rat model of experimental autoimmune encephalomyelitis (EAE) upon oral administration. These results indicate that the novel quinazolinedione derivatives have great potential as orally available small-molecule RORγt inverse agonists for the treatment of Th17-driven autoimmune diseases. A U-shaped bioactive conformation of this chemotype with RORγt protein was also observed.

Farnesoid X receptor antagonist exacerbates dyslipidemia in mice.

BACKGROUND: The effects of farnesoid X receptor (FXR) antagonists on plasma lipid profile in mice have not been investigated thus far. The aim of this study was to investigate the antidyslipidemic effects of an FXR antagonist in dyslipidemic mice, and to clarify the mechanisms underlying the lipid modulatory effect. METHODS: Compound-T0 (1-100 mg/kg) was orally administered to C57BL/6J mice fed a Western-type diet or low-density lipoprotein receptor knockout (LDLR-/-) mice fed a Western-type diet for a week, and plasma lipid levels were investigated. Effects on lipid clearance, hepatic triglyceride secretion after Triton WR-1339 challenge, and intestinal lipid absorption were investigated after multiple dosing. RESULTS: Compound-T0 significantly increased plasma level of non-high-density lipoprotein cholesterol in both C57BL/6 and LDLR-/- mice; in addition, it significantly increased plasma triglyceride level in LDLR-/- mice. Compound-T0 failed to enhance the clearance of 3,3'-dioctadecylindocarbocyanine (DiI)-labeled LDL in C57BL/6J mice. Although compound-T0 did not affect triglyceride clearance and hepatic triglyceride secretion, it significantly increased intestinal [3H]cholesterol absorption in LDLR-/- mice. CONCLUSIONS: It was found that the FXR antagonist, compound-T0 exacerbated dyslipidemia in mice because it enhanced intestinal lipid absorption via acceleration of bile acid excretion.

Discovery of orally efficacious RORγt inverse agonists, part 1: Identification of novel phenylglycinamides as lead scaffolds.

A series of novel phenylglycinamides as retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists were discovered through optimization of a high-throughput screen hit 1. (R)-N-(2-((3,5-Difluoro-4-(trimethylsilyl)phenyl) amino)-1-(4-methoxyphenyl)-2-oxoethyl)-3-hydroxy-N-methylisoxazole-5-carboxamide (22) was identified as one of the best of these compounds. It displayed higher subtype selectivity and specificity over other nuclear receptors and demonstrated in vivo potency to suppress the transcriptional activity of RORγt in a mouse PD (pharmacodynamic) model upon oral administration.

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 Novel and Potent Stearoyl Coenzyme A Desaturase 1 (SCD1) Inhibitors as Anticancer Agents.

A lead compound A was identified previously as an stearoyl coenzyme A desaturase (SCD) inhibitor during research on potential treatments for obesity. This compound showed high SCD1 binding affinity, but a poor pharmacokinetic (PK) profile and limited chemical accessibility, making it suboptimal for use in anticancer research. To identify potent SCD1 inhibitors with more promising PK profiles, we newly designed a series of 'non-spiro' 4, 4-disubstituted piperidine derivatives based on molecular modeling studies. As a result, we discovered compound 1a, which retained moderate SCD1 binding affinity. Optimization around 1a was accelerated by analyzing Hansch-Fujita and Hammett constants to obtain 4-phenyl-4-(trifluoromethyl)piperidine derivative 1n. Fine-tuning of the azole moiety of 1n led to compound 1o (T-3764518), which retained nanomolar affinity and exhibited an excellent PK profile. Reflecting the good potency and PK profile, orally administrated compound 1o showed significant pharmacodynamic (PD) marker reduction (at 0.3mg/kg, bid) in HCT116 mouse xenograft model and tumor growth suppression (at 1mg/kg, bid) in 786-O mouse xenograft model. In conclusion, we identified a new series of SCD1 inhibitors, represented by compound 1o, which represents a promising new chemical tool suitable for the study of SCD1 biology as well as the potential development of novel anticancer therapies.

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.

Investigation of the structural requirements of K-Ras(G12D) selective inhibitory peptide KRpep-2d using alanine scans and cysteine bridging.

A structure-activity relationship study of a K-Ras(G12D) selective inhibitory cyclic peptide, KRpep-2d was performed. Alanine scanning of KRpep-2d focusing on the cyclic moiety showed that Leu7, Ile9, and Asp12 are the key elements for K-Ras(G12D) selective inhibition of KRpep-2d. The cysteine bridging was also examined to identify the stable analog of KRpep-2d under reductive conditions. As a result, the KRpep-2d analog (12) including mono-methylene bridging showed potent K-Ras(G12D) selective inhibition in both the presence and the absence of dithiothreitol. This means that mono-methylene bridging is an effective strategy to obtain a reduction-resistance analog of parent disulfide cyclic peptides. Peptide 12 inhibited proliferation of K-Ras(G12D)-driven cancer cells significantly. These results gave valuable information for further optimization of KRpep-2d to provide novel anti-cancer drug candidates targeting the K-Ras(G12D) mutant.

Studies of CDK 8/19 inhibitors: Discovery of novel and selective CDK8/19 dual inhibitors and elimination of their CYP3A4 time-dependent inhibition potential.

In this article, synthetic studies around a pyridylacrylamide-based hit compound (1), utilizing structure-based drug design guided by CDK8 docking models, is discussed. Modification of the pendant 4-fluorophenyl group to various heteroaromatic rings was conducted aiming an interaction with the proximal amino acids, and then replacement of the morpholine ring was targeted for decreasing potential of time-dependent CYP3A4 inhibition. These efforts led to the compound 4k, with enhanced CDK8 inhibitory activity and no apparent potential for time-dependent CYP3A4 inhibition (CDK8 IC50: 2.5nM; CYP3A4 TDI: 99% compound remaining). Compound 4k was found to possess a highly selective kinase inhibition profile, and also showed favorable pharmacokinetic profile. Oral administration of 4k (15mg/kg, bid. for 2weeks) suppressed tumor growth (T/C 29%) in an RPMI8226 mouse xenograft model.

Design and synthesis of selective CDK8/19 dual inhibitors: Discovery of 4,5-dihydrothieno[3',4':3,4]benzo[1,2-d]isothiazole derivatives.

To develop a novel series of CDK8/19 dual inhibitors, we employed structure-based drug design using docking models based on a library compound, 4,5-dihydroimidazolo[3',4':3,4]benzo[1,2-d]isothiazole 16 bound to CDK8. We designed various [5,6,5]-fused tricyclic scaffolds bearing a carboxamide group to maintain predicted interactions with the backbone CO and NH of Ala100 in the CDK8 kinase hinge region. We found that 4,5-dihydrothieno[3',4':3,4]benzo[1,2-d]isothiazole derivative 29a showed particularly potent enzymatic inhibitory activity in both CDK8/19 (CDK8 IC50: 0.76nM, CDK19 IC50: 1.7nM). To improve the physicochemical properties and kinase selectivity of this compound, we introduced a substituted 3-pyridyloxy group into the scaffold 8-position. The resulting optimized compound 52h showed excellent in vitro potency (CDK8 IC50: 0.46nM, CDK19 IC50: 0.99nM), physicochemical properties, and kinase selectivity (only 5 kinases showed <35% unbound fraction at 300nM. CDK19: 4.6%, CDK8: 8.3%, HASPIN: 23%, DYRK1B: 27%, HIP1: 32%). Based on a docking model of 52h bound to CDK8, we could explain the highly specific kinase activity profile found for this compound, based on the interaction of the pyridyl group of 52h interacting with Met174 of the CDK8 DMG activation loop. In vitro pharmacological evaluation of 52h revealed potent suppression of phosphorylated STAT1 in various cancer cells. The high oral bioavailability found for this compound enabled in vivo studies, in which we demonstrated a mechanism-based in vivo PD effect as well as tumor growth suppression in an RPMI8226 human hematopoietic and lymphoid xenograft model in mouse [T/C: -1% (2.5mg/kg, qd)].

Identification of PARP14 inhibitors using novel methods for detecting auto-ribosylation.

Poly(ADP-ribose) polymerases (PARPs) use nicotinamide adenine dinucleotide (NAD+) as a co-substrate to transfer ADP-ribose when it releases nicotinamide as the metabolized product. Enzymes of the PARP family play key roles in detecting and repairing DNA, modifying chromatin, regulating transcription, controlling energy metabolism, and inducing cell death. PARP14, the original member of the PARP family, has been reported to be associated with the development of inflammatory diseases and various cancer types, making it a potential therapeutic target. In this study, we purified the macrodomain-containing PARP14 enzyme and established an assay for detecting the auto-ribosylation activity of PARP14 using RapidFire high-throughput mass spectrometry and immunoradiometric assay using [3H]NAD+. Subsequently, we performed high-throughput screening using the assays and identified small-molecule hit compounds, which showed NAD+-competitive and PARP14-selective inhibitory activities. Co-crystal structures of PARP14 with certain hit compounds revealed that the inhibitors bind to the NAD+-binding site. Finally, we confirmed that the hit compounds interacted with intracellular PARP14 by a cell-based protein stabilization assay. Thus, we successfully identified primary candidate compounds for further investigation.