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.

Strictly regulated agonist-dependent activation of AMPA-R is the key characteristic of TAK-653 for robust synaptic responses and cognitive improvement.

Agonistic profiles of AMPA receptor (AMPA-R) potentiators may be associated with seizure risk and bell-shaped dose-response effects. Here, we report the pharmacological characteristics of a novel AMPA-R potentiator, TAK-653, which exhibits minimal agonistic properties. TAK-653 bound to the ligand binding domain of recombinant AMPA-R in a glutamate-dependent manner. TAK-653 strictly potentiated a glutamate-induced Ca2+ influx in hGluA1i-expressing CHO cells through structural interference at Ser743 in GluA1. In primary neurons, TAK-653 augmented AMPA-induced Ca2+ influx and AMPA-elicited currents via physiological AMPA-R with little agonistic effects. Interestingly, TAK-653 enhanced electrically evoked AMPA-R-mediated EPSPs more potently than AMPA (agonist) or LY451646 (AMPA-R potentiator with a prominent agonistic effect) in brain slices. Moreover, TAK-653 improved cognition for both working memory and recognition memory, while LY451646 did so only for recognition memory, and AMPA did not improve either. These data suggest that the facilitation of phasic AMPA-R activation by physiologically-released glutamate is the key to enhancing synaptic and cognitive functions, and nonselective activation of resting AMPA-Rs may negatively affect this process. Importantly, TAK-653 had a wide safety margin against convulsion; TAK-653 showed a 419-fold (plasma Cmax) and 1017-fold (AUC plasma) margin in rats. These findings provide insight into a therapeutically important aspect of AMPA-R potentiation.

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.

TAK-137, an AMPA-R potentiator with little agonistic effect, has a wide therapeutic window.

Activation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPA-R) is a promising strategy to treat psychiatric and neurological diseases if issues of bell-shaped response and narrow safety margin against seizure can be overcome. Here, we show that structural interference at Ser743 in AMPA-R is a key to lower the agonistic effect of AMPA-R potentiators containing dihydropyridothiadiazine 2,2-dioxides skeleton. With this structural insight, TAK-137, 9-(4-phenoxyphenyl)-3,4-dihydropyrido[2,1-c][1,2,4]thiadiazine 2,2-dioxide, was discovered as a novel AMPA-R potentiator with a lower agonistic effect than an AMPA-R potentiator LY451646 ((R)-N-(2-(4'-cyanobiphenyl-4-yl)propyl)propane-2-sulfonamide) in rat primary neurons. TAK-137 induced brain-derived neurotrophic factor in neurons in rodents and potently improved cognition in both rats and monkeys. Compared to LY451646, TAK-137 had a wider safety margin against seizure in rats. TAK-137 enhanced neural progenitor proliferation over a broader range of doses in rodents. Thus, TAK-137 is a promising AMPA-R potentiator with potent procognitive effects and lower risks of bell-shaped response and seizure. These data may open the door for the development of AMPA-R potentiators as therapeutic drugs for psychiatric and neurological diseases.

Design, Synthesis, and Evaluation of Piperazinyl Pyrrolidin-2-ones as a Novel Series of Reversible Monoacylglycerol Lipase Inhibitors.

Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyzes 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain. Because 2-AG and AA are endogenous biologically active ligands in the brain, inhibition of MAGL is an attractive therapeutic target for CNS disorders, particularly neurodegenerative diseases. In this study, we report the structure-based drug design of novel piperazinyl pyrrolidin-2-ones starting from our hit compounds 2a and 2b. By enhancing the interaction of the piperazinyl pyrrolidin-2-one core and its substituents with the MAGL enzyme via design modifications, we identified a potent and reversible MAGL inhibitor, compound ( R)-3t. Oral administration of compound ( R)-3t to mice decreased AA levels and elevated 2-AG levels in the brain.

HBT1, a Novel AMPA Receptor Potentiator with Lower Agonistic Effect, Avoided Bell-Shaped Response in In Vitro BDNF Production.

α-Amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor (AMPA-R) potentiators with brain-derived neurotrophic factor (BDNF)-induction potential could be promising as therapeutic drugs for neuropsychiatric and neurologic disorders. However, AMPA-R potentiators such as LY451646 have risks of narrow bell-shaped responses in pharmacological effects, including in vivo BDNF induction. Interestingly, LY451646 and LY451395, other AMPA-R potentiators, showed agonistic effects and exhibited bell-shaped responses in the BDNF production in primary neurons. We hypothesized that the agonistic property is related to the bell-shaped response and endeavored to discover novel AMPA-R potentiators with lower agonistic effects. LY451395 showed an agonistic effect in primary neurons, but not in a cell line expressing AMPA-Rs, in Ca2+ influx assays; thus, we used a Ca2+ influx assay in primary neurons and, from a chemical library, discovered two AMPA-R potentiators with lower agonistic effects: 2-(((5-methyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)acetyl)amino)-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxamide (HBT1) and (3S)-1-(4-tert-butylphenyl)-N-((1R)-2-(dimethylamino)-1-phenylethyl)-3-isobutyl-2-oxopyrrolidine-3-carboxamide (OXP1). In a patch-clamp study using primary neurons, HBT1 showed little agonistic effect, whereas both LY451395 and OXP1 showed remarkable agonistic effects. HBT1, but not OXP1, did not show remarkable bell-shaped response in BDNF production in primary neurons. HBT1 bound to the ligand-binding domain (LBD) of AMPA-R in a glutamate-dependent manner. The mode of HBT1 and LY451395 binding to a pocket in the LBD of AMPA-R differed: HBT1, but not LY451395, formed hydrogen bonds with S518 in the LBD. OXP1 may bind to a cryptic binding pocket on AMPA-R. Lower agonistic profile of HBT1 may associate with its lower risks of bell-shaped responses in BDNF production in primary neurons.

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.

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 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.

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.

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.

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 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.

Discovery of Potent and Selective Inhibitors of Phosphodiesterase 1 for the Treatment of Cognitive Impairment Associated with Neurodegenerative and Neuropsychiatric Diseases.

A diverse set of 3-aminopyrazolo[3,4-d]pyrimidinones was designed and synthesized. The structure-activity relationships of these polycyclic compounds as phosphodiesterase 1 (PDE1) inhibitors were studied along with their physicochemical and pharmacokinetic properties. Systematic optimizations of this novel scaffold culminated in the identification of a clinical candidate, (6aR,9aS)-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-3-(phenylamino)-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4-(2H)-one phosphate (ITI-214), which exhibited picomolar inhibitory potency for PDE1, demonstrated excellent selectivity against all other PDE families and showed good efficacy in vivo. Currently, this investigational new drug is in Phase I clinical development and being considered for the treatment of several indications including cognitive deficits associated with schizophrenia and Alzheimer's disease, movement disorders, attention deficit and hyperactivity disorders, and other central nervous system (CNS) and non-CNS disorders.

Discovery of 6-[5-(4-fluorophenyl)-3-methyl-pyrazol-4-yl]-benzoxazin-3-one derivatives as novel selective nonsteroidal mineralocorticoid receptor antagonists.

In the course of our study on selective nonsteroidal mineralocorticoid receptor (MR) antagonists, a series of novel benzoxazine derivatives possessing an azole ring as the core scaffold was designed for the purpose of attenuating the partial agonistic activity of the previously reported dihydropyrrol-2-one derivatives. Screening of alternative azole rings identified 1,3-dimethyl pyrazole 6a as a lead compound with reduced partial agonistic activity. Subsequent replacement of the 1-methyl group of the pyrazole ring with larger lipophilic side chains or polar side chains targeting Arg817 and Gln776 increased MR binding activity while maintaining the agonistic response at the lower level. Among these compounds, 6-[1-(2,2-difluoro-3-hydroxypropyl)-5-(4-fluorophenyl)-3-methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (37a) showed highly potent in vitro activity, high selectivity versus other steroid hormone receptors, and good pharmacokinetic profiles. Oral administration of 37a in deoxycorticosterone acetate-salt hypertensive rats showed a significant blood pressure-lowering effect with no signs of antiandrogenic effects.

Discovery of potent Mcl-1/Bcl-xL dual inhibitors by using a hybridization strategy based on structural analysis of target proteins.

Mcl-1 and Bcl-xL are crucial regulators of apoptosis, therefore dual inhibitors of both proteins could serve as promising new anticancer drugs. To design Mcl-1/Bcl-xL dual inhibitors, we performed structure-guided analyses of the corresponding selective Mcl-1 and Bcl-xL inhibitors. A cocrystal structure of a pyrazolo[1,5-a]pyridine derivative with Mcl-1 protein was successfully determined and revealed the protein-ligand binding mode. The key structure for Bcl-xL inhibition was further confirmed through the substructural analysis of ABT-263, a representative Bcl-xL/Bcl-2/Bcl-w inhibitor developed by Abbott Laboratories. On the basis of the structural data from this analysis, we designed hybrid compounds by tethering the Mcl-1 and Bcl-xL inhibitors together. The results of X-ray crystallographic analysis of hybrid compound 10 in complexes with both Mcl-1 and Bcl-xL demonstrated its binding mode with each protein. Following further optimization, compound 11 showed potent Mcl-1/Bcl-xL dual inhibitory activity (Mcl-1, IC50 = 0.088 µM; and Bcl-xL, IC50 = 0.0037 µM).

Design, synthesis, and structure-activity relationships of dihydrofuran-2-one and dihydropyrrol-2-one derivatives as novel benzoxazin-3-one-based mineralocorticoid receptor antagonists.

Dihydrofuran-2-one and dihydropyrrol-2-one derivatives were identified as novel, potent and selective mineralocorticoid receptor (MR) antagonists by the structure-based drug design approach utilizing the crystal structure of MR/compound complex. Introduction of lipophilic substituents directed toward the unfilled spaces of the MR and identification of a new scaffold, dihydropyrrol-2-one ring, led to potent in vitro activity. Among the synthesized compounds, dihydropyrrol-2-one 11i showed an excellent in vitro activity (MR binding IC50=43nM) and high selectivity over closely related steroid receptors such as the androgen receptor (AR), progesterone receptor (PR) and glucocorticoid receptor (GR) (>200-fold for AR and PR, 100-fold for GR).