In Vitro Pharmacological Profile of KW-6356, a Novel Adenosine A2A Receptor Antagonist/Inverse Agonist.

KW-6356 is a novel adenosine A2A (A2A) receptor antagonist/inverse agonist, and its efficacy as monotherapy in Parkinson's disease (PD) patients has been reported. Istradefylline is a first-generation A2A receptor antagonist approved for use as adjunct treatment to levodopa/decarboxylase inhibitor in adult PD patients experiencing "OFF" episodes. In this study, we investigated the in vitro pharmacological profile of KW-6356 as an A2A receptor antagonist/inverse agonist and the mode of antagonism and compared them with istradefylline. In addition, we determined cocrystal structures of A2A receptor in complex with KW-6356 and istradefylline to explore the structural basis of the antagonistic properties of KW-6356. Pharmacological studies have shown that KW-6356 is a potent and selective ligand for the A2A receptor (the -log of inhibition constant = 9.93 ± 0.01 for human receptor) with a very low dissociation rate from the receptor (the dissociation kinetic rate constant = 0.016 ± 0.006 minute-1 for human receptor). In particular, in vitro functional studies indicated that KW-6356 exhibits insurmountable antagonism and inverse agonism, whereas istradefylline exhibits surmountable antagonism. Crystallography of KW-6356- and istradefylline-bound A2A receptor have indicated that interactions with His2506.52 and Trp2466.48 are essential for the inverse agonism, whereas the interactions at both deep inside the orthosteric pocket and the pocket lid stabilizing the extracellular loop conformation may contribute to the insurmountable antagonism of KW-6356. These profiles may reflect important differences in vivo and help predict better clinical performance. SIGNIFICANCE STATEMENT: KW-6356 is a potent and selective adenosine A2A receptor antagonist/inverse agonist and exhibits insurmountable antagonism, whereas istradefylline, a first-generation adenosine A2A receptor antagonist, exhibits surmountable antagonism. Structural studies of adenosine A2A receptor in complex with KW-6356 and istradefylline explain the characteristic differences in the pharmacological properties of KW-6356 and istradefylline.

siRNA potency enhancement via chemical modifications of nucleotide bases at the 5'-end of the siRNA guide strand.

Small interfering RNAs (siRNAs) can be utilized not only as functional biological research tools but also as therapeutic agents. For the clinical use of siRNA as drugs, various chemical modifications have been used to improve the activity of siRNA drugs, and further chemical modifications are expected to improve the utility of siRNA therapeutics. As the 5' nucleobase of the guide strand affects the interaction between an siRNA and AGO2 and target cleavage activity, structural optimization of this specific position may be a useful strategy for improving siRNA activity. Here, using the in silico model of the complex between human AGO2 MID domain and nucleoside monophosphates, we screened and synthesized an original adenine-derived analog, 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), that fits better than the natural nucleotide bases into the MID domain of AGO2. Introduction of the 6-mCEPh-purine analog at the 5'-end of the siRNA guide strand significantly enhanced target knockdown activity in both cultured cell lines and in vivo animal models. Our findings can help expand strategies for rationally optimizing siRNA activity via chemical modifications of nucleotide bases.

Development of a novel class of peroxisome proliferator-activated receptor (PPAR) gamma ligands as an anticancer agent with a unique binding mode based on a non-thiazolidinedione scaffold.

We previously identified dibenzooxepine derivative 1 as a potent PPARγ ligand with a unique binding mode owing to its non-thiazolidinedione scaffold. However, while 1 showed remarkably potent MKN-45 gastric cancer cell aggregation activity, an indicator of cancer differentiation-inducing activity induced by PPARγ activation, we recognized that 1 was metabolically unstable. In the present study, we identified a metabolically soft spot, and successfully discovered 3-fluoro dibenzooxepine derivative 9 with better metabolic stability. Further optimization provided imidazo[1,2-a]pyridine derivative 17, which showed potent MKN-45 gastric cancer cell aggregation activity and excellent PK profiles compared with 9. Compound 17 exerted a growth inhibitory effect on AsPC-1/AG1 pancreatic tumor in mice. Furthermore, the decrease in the hematocrit (an indicator of localized edema, a serious adverse effect of PPARγ ligands) was tolerable even with oral administration at 200 mg/kg in healthy mice.

Crystal structure of the GH-46 subclass III chitosanase from Bacillus circulans MH-K1 in complex with chitotetraose.

BACKGROUND: Chitosanases (EC 3.2.1.132) hydrolyze chitosan which is a polymer of glucosamine (GlcN) linked by ß - 1,4 bonds, and show cleavage specificity against partially acetylated chitosan containing N-acetylglucosamine (GlcNAc) residues. Chitosanases' structural underpinnings for cleavage specificity and the conformational switch from open to closed structures are still a mystery. METHODS: The GH-46 subclass III chitosanase from Bacillus circulans MH-K1 (MH-K1 chitosanase), which also catalyzes the hydrolysis of GlcN-GlcNAc bonds in addition to GlcN-GlcN, has had its chitotetraose [(GlcN)4]-complexed crystal structure solved at 1.35 Å resolution. RESULTS: The MH-K1 chitosanase's (GlcN)4-bound structure has numerous structural similarities to other GH-46 chitosanases in terms of substrate binding and catalytic processes. However, subsite -1, which is absolutely specific for GlcN, seems to characterize the structure of a subclass III chitosanase due to its distinctive length and angle of a flexible loop. According to a comparison of the (GlcN)4-bound and apo-form structures, the particular binding of a GlcN residue at subsite -2 through Asp77 causes the backbone helix to kink, which causes the upper- and lower-domains to approach closely when binding a substrate. CONCLUSIONS: Although GH-46 chitosanases vary in the finer details of the subsites defining cleavage specificity, they share similar structural characteristics in substrate-binding, catalytic processes, and potentially in conformational change. GENERAL SIGNIFICANCE: The precise binding of a GlcN residue to the -2 subsite is essential for the conformational shift that occurs in all GH-46 chitosanases, as shown by the crystal structures of the apo- and substrate-bound forms of MH-K1 chitosanase.

Structural insight into an anti-BRIL Fab as a G-protein-coupled receptor crystallization chaperone.

Structure determination of G-protein-coupled receptors (GPCRs) is key for the successful development of efficient drugs targeting GPCRs. BRIL is a thermostabilized apocytochrome b562 (with M7W/H102I/R106L mutations) from Escherichia coli and is often used as a GPCR fusion protein for expression and crystallization. SRP2070Fab, an anti-BRIL antibody Fab fragment, has been reported to facilitate and enhance the crystallization of BRIL-fused GPCRs as a crystallization chaperone. This study was conducted to characterize the high-resolution crystal structure of the BRIL-SRP2070Fab complex. The structure of the BRIL-SRP2070Fab complex was determined at 2.1 Šresolution. This high-resolution structure elucidates the binding interaction between BRIL and SRP2070Fab. When binding to BRIL, SRP2070Fab recognizes conformational epitopes, not linear epitopes, on the surface of BRIL helices III and IV, thereby binding perpendicularly to the helices, which indicates stable binding. Additionally, the packing contacts of the BRIL-SRP2070Fab co-crystal are largely due to SRP2070Fab rather than BRIL. The accumulation of SRP2070Fab molecules by stacking is remarkable and is consistent with the finding that stacking of SRP2070Fab is predominant in known crystal structures of BRIL-fused GPCRs complexed with SRP2070Fab. These findings clarified the mechanism of SRP2070Fab as a crystallization chaperone. Moreover, these data will be useful in the structure-based drug design of membrane-protein drug targets.

The structure of L-amino-acid ligase from Bacillus licheniformis.

L-Amino-acid ligases (LALs) are enzymes which catalyze the formation of dipeptides by linking two L-amino acids. Although many dipeptides are known and expected to have medical and nutritional benefits, their practical use has been limited owing to their low availability and high expense. LALs are potentially desirable tools for the efficient production of dipeptides; however, the molecular basis of substrate recognition by LAL has not yet been sufficiently elucidated for the design of ideal LALs for the desired dipeptides. This report presents the crystal structure of the LAL BL00235 derived from Bacillus licheniformis NBRC 12200 determined at 1.9 Å resolution using the multi-wavelength anomalous dispersion method. The overall structure of BL00235 is fairly similar to that of YwfE, the only LAL with a known structure, but the structure around the catalytic site contains some significant differences. Detailed structural comparison of BL00235 with YwfE sheds some light on the molecular basis of the substrate specificities.

The dual pocket binding novel tankyrase inhibitor K-476 enhances the efficacy of immune checkpoint inhibitor by attracting CD8+ T cells to tumors.

The Wnt/ß-catenin pathway, which is associated with disease progression, is activated in many cancers. Tankyrase (TNKS) has received attention as a target molecule for Wnt/ß-catenin pathway inhibition. We identified K-476, a novel TNKS inhibitor, a dual pocket binder that binds to both the nicotinamide and ADP-ribose pockets. In a human colon cancer cell line, K-476 specifically and potently inhibited TNKS and led to stabilization of the Axin protein, resulting in Wnt/ß-catenin pathway suppression. Aberrant Wnt/ß-catenin pathway activation was recently reported as a possible mechanism of ineffectiveness in immune checkpoint inhibitor (ICI) treatment. Because the Wnt/ß-catenin pathway activation causes dendritic cell inactivation and suppresses chemokine production, resulting in a paucity of CD8+ T cells in tumor tissue, which is an important effector of ICIs. Thus, TNKS inhibitors may enhance the efficacy of ICIs. To examine whether K-476 enhances the antitumor effect of anti-PD-L1 antibodies, K-476 was administered orally with an anti-PD-L1 antibody to melanoma-bearing C57BL/6J mice. Although K-476 was ineffective as a monotherapy, it significantly enhanced the antitumor effect in combination with anti-PD-L1 antibody. In mice, intra-tumor infiltration of CD8+ T cells was increased by combination treatment. K-476 upregulated the chemokine expression (e.g., Ccl3 and Ccl4), which attracted CD8+ T cells. This was considered to contribute to the increased CD8+ T cells in the tumor microenvironment. Furthermore, while the potential gastrointestinal toxicity of TNKS inhibitors has been reported, it was not observed at effective doses. Thus, K-476 could be an attractive therapeutic option to enhance the efficacy of ICIs.

The discovery of a new antibody for BRIL-fused GPCR structure determination.

G-protein-coupled receptors (GPCRs)-the largest family of cell-surface membrane proteins-mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT1B and AT2R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them.

The novel potent TEAD inhibitor, K-975, inhibits YAP1/TAZ-TEAD protein-protein interactions and exerts an anti-tumor effect on malignant pleural mesothelioma.

The Hippo signaling pathway regulates cell fate and organ development. In the Hippo pathway, transcriptional enhanced associate domain (TEAD) which is a transcription factor is activated by forming a complex with yes-associated protein 1 (YAP1) or transcriptional coactivator with PDZ-binding motif (TAZ, also called WWTR1). Hyper-activation of YAP1/TAZ, leading to the activation of TEAD, has been reported in many cancers, including malignant pleural mesothelioma (MPM). Therefore, the YAP1/TAZ-TEAD complex is considered a novel therapeutic target for cancer treatment. However, few reports have described YAP1/TAZ-TEAD inhibitors, and their efficacy and selectivity are poor. In this study, we performed a high-throughput screening of a neurofibromin 2 (NF2)-deficient MPM cell line and a large tumor suppressor kinase 1/2 (LATS1/2)-deficient non-small-cell lung cancer cell line using a transcriptional reporter assay. After screening and optimization, K-975 was successfully identified as a potent inhibitor of YAP1/TAZ-TEAD signaling. X-ray crystallography revealed that K-975 was covalently bound to an internal cysteine residue located in the palmitate-binding pocket of TEAD. K-975 had a strong inhibitory effect against protein-protein interactions between YAP1/TAZ and TEAD in cell-free and cell-based assays. Furthermore, K-975 potently inhibited the proliferation of NF2-non-expressing MPM cell lines compared with NF2-expressing MPM cell lines. K-975 also suppressed tumor growth and provided significant survival benefit in MPM xenograft models. These findings indicate that K-975 is a strong and selective TEAD inhibitor with the potential to become an effective drug candidate for MPM therapy.

Development of Dihydrodibenzooxepine Peroxisome Proliferator-Activated Receptor (PPAR) Gamma Ligands of a Novel Binding Mode as Anticancer Agents: Effective Mimicry of Chiral Structures by Olefinic E/ Z-Isomers.

A novel class of PPARγ ligand 1 (EC50 = 197 nM) with a dibenzoazepin scaffold was identified through high-throughput screening campaign. To avoid the synthetically troublesome chiral center of 1, its conformational analysis using the MacroModel was conducted, focusing on conformational flip of the tricyclic ring and the conformational restriction by the methyl group at the chiral center. On the basis of this analysis, scaffold hopping of dibenzoazepine into dibenzo[ b, e]oxepine by replacing the chiral structures with the corresponding olefinic E/ Z isomers was performed. Consequently, dibenzo[ b, e]oxepine scaffold 9 was developed showing extremely potent PPARγ reporter activity (EC50 = 2.4 nM, efficacy = 9.5%) as well as differentiation-inducing activity against a gastric cancer cell line MKN-45 that was more potent than any other well-known PPARγ agonists in vitro (94% at 30 nM). The X-ray crystal structure analysis of 9 complexed with PPARγ showed that it had a unique binding mode to PPARγ ligand-binding domain that differed from that of any other PPARγ agonists identified thus far.

Bacillus circulans MH-K1 chitosanase: amino acid residues responsible for substrate binding.

To identify the amino acids responsible for the substrate binding of chitosanase from Bacillus circulans MH-K1 (MH-K1 chitosanase), Tyr148 and Lys218 of the chitosanase were mutated to serine and proline, respectively, and the mutated chitosanases were characterized. The enzymatic activities of Y148S and K218P were found to be 12.5% and 0.16% of the wild type, respectively. When the (GlcN)3 binding ability to the chitosanase was evaluated by fluorescence spectroscopy and thermal unfolding experiments, the binding abilities of both mutant enzymes were markedly reduced as compared with the wild type enzyme. The affinity of the enzyme for the trisaccharide decreased by 1.0 kcal/mol of binding free energy for Y148S, and 3.7 kcal/mol for K218P. The crystal structure of K218P revealed that Pro218 forms a cis-peptide bond and that the state of the flexible loop containing the 218th residue is considerably affected by the mutation. Thus, we conclude that the flexible loop containing Lys218 plays an important role in substrate binding, and that the role of Tyr148 is less critical, but still important, due to a stacking interaction or hydrogen bond.