Objective: Hippo signaling pathway is known to regulate organ development. In Hippo signaling pathway, YAP or TAZ works as a transcriptional co-activator and forms a transcriptional complex with TEAD. In several cancers, upstream factors in Hippo pathway are inactivated by genetic alterations. When the upstream factors are inactivated, TEAD is activated and forms a complex with YAP/TAZ resulting in enhancement of cell proliferation, drug resistance and so on. In the activation process, S-palmitoylation of TEAD is necessary for binding to YAP/TAZ. Malignant pleural mesothelioma (MPM) is one of cancer types which have genetic alterations in Hippo pathway genes. Although YAP/TAZ-TEAD inhibitor should be an ideal drug for MPM therapy, there are only a few reports about YAP/TAZ-TEAD inhibitor and the efficacy and selectivity are not sufficient. In this study, we succeeded to synthesize a small molecule TEAD inhibitor, K-975, and evaluated its mechanism of action and anti-tumor effect against MPM. Materials/methods: Inhibitory activity of K-975 on YAP/TAZ-TEAD protein-protein interaction (PPI) was evaluated in surface plasmon resonance (SPR) and co-immunoprecipitation assay. The effect of K-975 on palmitoylation status of TEAD was also evaluated. The three-dimensional structure of YAP-binding domain of TEAD1 in complex with K-975 was determined by X-ray crystallography. Anti-tumor effect of K-975 was evaluated by using MPM cell lines. Furthermore, using a derivative of K-975, 2 week-toxicity studies in rats and monkeys were performed. Results: K-975 inhibited YAP-TEAD and TAZ-TEAD PPI in NCI-H226 cells, a human MPM cell line. Also, K-975 inhibited palmitoylation of TEAD. The crystal structure revealed that K-975 directly bound to cysteine residue in YAP-binding domain of TEAD1. This cysteine residue is highly conserved in TEAD family and known as a site of S-palmitoylation. K-975 inhibited the cell proliferation of NCI-H226 with GI50 of about 20 nmol/L. K-975 also induced a change of gene expressions similar to that induced by YAP knockdown. In vivo experiments, K-975 strongly suppressed the tumor growth in several s.c. xenograft models and showed a significant survival benefit in an orthotopic xenograft model. However, 2 week-toxicity studies of a K-975 derivative with optimized bioavailability showed some pathological findings which suggested the renal toxicity. Conclusion: We synthesized a first-in-class drug which directly binds to TEAD protein and inhibits YAP/TAZ-TEAD PPI. K-975 showed a strong anti-tumor effect in pre-clinical MPM models. Although the renal toxicity might cause some difficulty in clinical use, we believe that a K-975 derivative has a possibility to become an effective drug candidate for MPM therapy. Citation Format: Ayumi Kaneda, Toshihiro Seike, Takeshi Uemori, Kensuke Myojo, Kensuke Aida, Tomohiro Danjo, Takahiro Nakajima, Daisuke Yamaguchi, Tomoko Hamada, Yoshiro Tsuji, Kaori Hamaguchi, Mai Yasunaga, Nobumasa Otsubo, Hideyuki Onodera, Yoichi Nishiya, Michihiko Suzuki, Junichi Saito, Toshihiko Ishii, Ryuichiro Nakai. Discovery of a first-in-class TEAD inhibitor which directly inhibits YAP/TAZ-TEAD protein-protein interaction and shows a potent anti-tumor effect in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3086.

Several studies have suggested that high blood pressure is associated with abnormalities in calcium metabolism, leading to increased calcium loss, secondary activation of the parathyroid gland, and increased removal of calcium from bone. [1][2][3][4][5][6][7] Consistently, studies in hypertensive rats have shown that hypercalciuria and ensuing hyperparathyroidism lead to reduced growth and detectable decrease in total bonemineral content later in life. 8,9) Recently, it was also shown that higher blood pressure in elderly white women is statistically associated with increased bone loss at the femoral neck, which may contribute to bone fracture. 10) Drugs used as primary choice for the treatment of hypertension include calcium channel blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor type1 (AT1) antagonists, diuretics, b-blockers, and a-blockers. 11) The first three drugs are especially widely used in Japan. The mechanism by which each drug lowers blood pressure differs. Calcium channel blockers primarily inhibit calcium influx through the L-type voltage-dependent calcium channel at the level of vascular smooth muscle, thereby disrupting the excitation contraction process. 12,13) Both ACE inhibitors and AT1 antagonists interfere with the renin-angiotensin system (RAS). The former inactivates the conversion of angiotensin I into angiotensin II (Ang II), which is a vasoconstrictive peptide, while the latter blocks the binding of Ang II to its receptor. 14,15) Osteoblasts are derived from mesenchymal stem cells and play a pivotal role in bone formation. During differentiation, they first express type I collagen, then alkaline phosphatase (ALP), and other bone matrix proteins and finally form mineralized bone. Osteoblasts express voltage-dependent calcium channels. 16) Various bone regulatory factors such as vitamin D 3 , [17][18][19] parathyroid hormone, 17,20,21) and prostaglandin E 2 17,22) cause a rise in intracellular calcium concentration ([Ca 2ϩ ] i ), at least part of which is decreased by dihydropyridine-type calcium channel blockers. These factors also promote or inhibit osteoblast differentiation. [23][24][25][26] Thus, it is suggested that signaling through the L-type calcium channel may be important for osteoblast functions. However, the fact that these factors give rise to multiple signals independent of calcium influx (i.e., transcription of specific genes, 27) cAMP production, 20,28) release of calcium ion from intracellular store 17) ) obscures the role of L-type calcium channel. Recently, Kosaka and Uchii found that a calcium channel blocker benidipine, but not amlodipine or nifedipine, increased ALP activity of osteoblastic cells isolated from neonatal mouse calvaria. 29) We further investigated the effects of benidipine on osteoblastic functions using osteoblastic cell line MC3T3-E1, 30) which is widely used in studies on various aspects of osteoblast differentiation since it expresses osteoblast markers and forms a mineralized extracellular matrix. 31,32) There we ...

During their differentiation, osteoblasts sequentially express type I collagen, alkaline phosphatase (ALP), and osteocalcin, and then undergo mineral deposition. Among dihydropyridine-type calcium channel blockers, only benidipine stimulated ALP activity of osteoblastic cells derived from neonatal mouse calvaria. To identify the molecular target of benidipine and elucidate the mechanism of action of the drug in osteoblasts, the mouse osteoblastic cell line MC3T3-E1 was used. Benidipine prompted ALP activity and ALP transcription induced by ascorbic acid, and mineral deposition by ascorbic acid and b-glycerophosphate. Benidipine, however, did not change collagen accumulation. MC3T3-E1 cells expressed the L-type Ca channel a1C subunit throughout the differentiation process, and Ca influx by potassium ions and Bay K 8644, an agonist, was strongly attenuated by benidipine. Each one of three structurally different classes of Ca channel blockers, nifedipine, verapamil, and diltiazem stimulated ALP activity, although at much higher concentrations of ca. 100 nM than benidipine, 1 pM. These results suggest that benidipine directly exerts its effect on osteoblasts and promotes osteoblast differentiation after the step of collagen accumulation by blocking the L-type Ca channel. Since benidipine blocked Ca influx more potently than the three other Ca channel blockers, the unique and potent osteoblast differentiating ability of benidipine may be due to its high affinity for Ca channel together with its high membrane retaining ability, as has been previously reported.

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In the second step of the two consecutive transesterifications of the self-splicing reaction of the group I intron, the conserved guanosine at the 3' terminus of the intron (omegaG) binds to the guanosine-binding site (GBS) in the intron. In the present study, we designed a 22-nt model RNA (GBS/omegaG) including the GBS and omegaG from the Tetrahymena group I intron, and determined the solution structure by NMR methods. In this structure, omegaG is recognized by the formation of a base triple with the G264 x C311 base pair, and this recognition is stabilized by the stacking interaction between omegaG and C262. The bulged structure at A263 causes a large helical twist angle (40 +/- 80) between the G264 x C311 and C262 x G312 base pairs. We named this type of binding pocket with a bulge and a large twist, formed on the major groove, a "Bulge-and-Twist" (BT) pocket. With another twist angle between the C262 x G312 and G413 x C313 base pairs (45 +/- 100), the axis of GBS/omegaG is kinked at the GBS region. This kinked axis superimposes well on that of the corresponding region in the structure model built on a 5.0 A resolution electron density map (Golden et al., Science, 1998, 282:345-358). This compact structure of the GBS is also consistent with previous biochemical studies on group I introns. The BT pockets are also found in the arginine-binding site of the HIV-TAR RNA, and within the 16S rRNA and the 23S rRNA.

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