CLK2

研发进展推介、博士后招聘联系公众号（备注：单位+姓名） 2024年3月18日，李志裕教授等人在Journal of Medicinal Chemistry 上发表了题为Structure-Guided Discovery of Potent and Selective CLK2 Inhibitors for the Treatment of Knee Osteoarthritis的文章，在这篇文章中，作者等人设计并合成了一系列高效的基于吲唑类化合物的CLK2抑制剂，其中化合物LQ23对CLK2的抑制活性高于先导化合物(IC50=1.4 nM)，具有较高的CLK2/CLK3选择性(>70倍)。此外，LQ23在体内外均有显著的抗骨关节炎作用，其作用机制可能是通过抑制CLK2/Wnt信号通路，降低炎症细胞因子，下调软骨降解酶，增加关节软骨。总体而言，这些数据支持LQ23作为膝关节内OA治疗的潜在候选者，利用其独特的作用机制进行靶向治疗。骨关节炎是一种典型的退行性疾病，传统上具有不同的病因，但具有相似的生物学、形态学和临床特征。在各种类型的骨性关节炎中，膝关节骨性关节炎的发病率尤其高。骨性关节炎的疼痛和残疾严重影响患者的生活质量，已成为当今社会严重的社会和经济负担之一。目前，骨性关节炎的治疗相对有限，主要集中在抗炎药物上。然而，消炎药并不能修复软骨。因此，对治疗疾病的骨性关节炎药物(DMOADs)的需求尚未得到满足。DMOADs不仅具有抗炎作用，还可通过促进软骨细胞再生和抑制软骨分解代谢来减轻软骨损伤。研究表明Wnt信号在骨性关节炎发病机制中的核心作用，许多与Wnt途径相关的新的生物学过程和靶点已被发现。在这些靶点中，酪氨酸和丝氨酸/苏氨酸激酶CLKs被报道通过调节细胞内信号来调节Wnt途径。基于CLK2在退行性疾病和肿瘤中的重要作用，研究机构相继设计了许多不同类型的CLK2抑制剂。目前，有五种药物正在进行临床试验，用于治疗退行性疾病和肿瘤。2018年，J.Kallen的团队报告了一系列有效的吲唑类CLK抑制剂的发现。确定了吲唑1是一种高效的吲唑CLK抑制剂，作为先导化合物。随后，作者等人引入了芳香族乙炔基团，合成了一系列新型的CLK2抑制剂。在这些抑制剂中，LQ23(CLK2，IC50=1.4 nM)表现出显著的CLK2抑制活性。并且LQ23对CLK1/2/4(CLK1，IC50=2.1 nM；CLK2，IC50=1.4 nM；CLK4，IC50=3.2 nM)显示出更高的抑制效力。此外，LQ23对CLK2的选择性高于CLK3(IC50>100 nm)。随后作者等人进一步证实LQ23对蛋白激酶的选择性，结果表明化合物LQ23是一种很好的选择性CLK2抑制剂。（图1）图1 (A)化合物LQ23对CLK2、CLK3和DYRK1A的抑制活性。(B) pSRSF4、pSRSF5和pSRSF6在二甲亚砜(DMSO)或Lorecivivint (30 nM)或LQ23(10、30和100 nM)处理1小时后的蛋白表达情况。(C) pSRSF4、pSRSF5和pSRSF6在转染靶向CLK2的siRNA的软骨细胞中的蛋白表达情况接下来，作者等人评估了LQ23在体外诱导软骨细胞早期分化的能力。据报道，转化生长因子β3可刺激骨髓间充质干细胞分化为高度特异的软骨细胞。结果表明LQ23可诱导骨髓间充质干细胞分化为软骨细胞样细胞(图2A)。此外，在单层条件下用LQ23处理14天的BMSCs表明软骨细胞正在分化，这是由成熟软骨细胞特异性蛋白(II型胶原，Col IIA)的存在支持的(图2B)。提示LQ23可促进BMSCs向软骨细胞分化。图2 LQ23诱导骨髓间充质干细胞的软骨细胞分化。(A)骨髓间质干细胞(用30 nM LQ23或DMSO或20 ng/mL TGF β3处理14天)用甲苯胺蓝和阿利新蓝染色。(B)骨髓间充质干细胞(用30 nM LQ23或DMSO或20 ng/mL TGF β3处理14天)用Col IIA染色，显示成熟软骨细胞除促进软骨再生外，骨性关节炎的长期疾病治疗还需要抑制软骨的降解。因此，作者等人还评估了LQ23对软骨细胞和骨髓间充质干细胞在病理生理条件下的分解代谢的影响。当肿瘤坏死因子TNF-α(20 ng/m L)和肿瘤抑素M(OM，10 ng/m L)处理时，软骨细胞和骨髓间充质干细胞MMP3、MMP13和ADAMTS5的基因表达上调(图3A，B)。LQ23(30 NM)可显著抑制细胞因子诱导的基质降解酶的表达，包括MMP3、MMP13和ADAMTS5(图3A，B)，其效力与Lorecivivint(30 nM)相似或提高。LQ23治疗组与模型组比较，NO释放量明显减少。提示LQ23在体外对软骨降解有抑制作用。图3 LQ23保护软骨细胞和骨髓间充质干细胞免受体外分解代谢破坏。细胞因子TNF-α (20 ng/mL)和Oncostatin M (10 ng/mL)刺激软骨细胞和骨髓间充质干细胞，DMSO或Lorecivivint (30 nM)或LQ23 (30 nM)处理72 h如图4所示，LQ23和Lorecivivint均能有效抑制肿瘤坏死因子TNF-α、IL-6和IL-β在TNF-α和OM刺激的软骨细胞中的表达。同时，LQ23的抑制作用与siCLK2一致。体外实验表明，LQ23具有与Lorecivivint相当的抗炎活性。随后，在TNF-α和OM刺激的软骨细胞中，观察了LQ23对STAT3的影响。当用TNF-α和OM处理软骨细胞时，Western Blot检测到STAT3的磷酸化水平显著升高(图5)，而LQ23则呈剂量依赖性地抑制STAT3的磷酸化。提示LQ23具有较强的体外抗炎作用。图4 LQ23抑制软骨细胞的炎症反应。用细胞因子TNF-α (20 ng/mL)和Oncostatin M (10 ng/mL)刺激软骨细胞，用DMSO或Lorecivivint (30 nM)或LQ23 (30 nM)处理48 h。用qRT-PCR检测细胞因子(TNF-α、IL-6和IL-1β)的基因表达图5 LQ23剂量依赖性地抑制TNF-α和OM刺激的软骨细胞中STAT3磷酸化。(A) Western blot检测TNF-α和OM刺激软骨细胞中磷酸化和总STAT3蛋白表达水平。(B)通过三次独立实验定量测定pSTAT3和STAT3的比例半月板损伤常见于膝关节骨性关节炎的MRI图像，可导致膝关节骨性关节炎。然而，修复半月板损伤的努力在很大程度上并不成功，也未能阻止导致膝关节骨性关节炎的退行性改变的进展。在大鼠炎性半月板损伤模型和大鼠前交叉韧带切断内侧半月板部分切除(ACLT+pMMx)模型上，采用生化和组织病理学方法观察LQ23对软骨再生和保护的影响。MIA可引起局部炎症、软骨退变和关节疼痛增加，或LQ23(1.5μg/kg，单次IA注射)可降低血浆中炎症细胞因子(TNF-α、IL-6和IL-1β)水平和软骨中分解代谢酶(MMP3、MMP13和ADAMTS5)水平。此外，与OA疾病严重程度相关的循环COMP水平在LQ23治疗的大鼠中自治疗后第11天起显著降低。LQ23在治疗后28天仍有效(图6A，B)。第28天苏木精-伊红(H&E)染色显示，LQ23治疗组关节形态明显改善。经LQ23治疗后，关节软骨表面光滑，细胞排列更规则，滑膜厚度减少，股骨与胫骨间隙增大(图6C)。而Lorecivivint或LQ23治疗显著降低了光镜下软骨检测评价标准OARSI评分51(图6D)，提示LQ23抑制炎症，保护软骨。图6 LQ23在碘乙酸钠(MIA)诱导的大鼠膝关节OA模型中抑制炎症，保护软骨，改善功能。注射mia的大鼠经IA注射给药后，分别给药整车或Lorecivivint (1.5μg/kg)或LQ23 (1.5μg/kg)。软骨和血浆分别于第11天和第28天采集综上所述， CLK2是开发DMOAD药物的一个与Wnt途径相关的有前景的靶点。作者团队发现了一系列新的化合物，确定了具有代表性的化合物LQ23，该化合物对CLK2具有较强的抑制活性，IC50为1.4 nM，且比CLK3具有更高的选择性。LQ23还可诱导BMSCs向软骨细胞分化。在体外，LQ23还能降低细胞因子诱导的NO生成和体内循环COMP水平，这表明LQ23可以预防OA病理条件下的基质分解。体内药代动力学数据和受试化合物在体外人体细胞中的有效性证明了LQ23的药物设计理论是合理的。这些发现强调了LQ23作为一种小分子潜在的DMOAD的前景，用于膝骨性关节炎的有效治疗。原文链接：https://doi.org/10.1021/acs.jmedchem.3c02092声明：发表/转载本文仅仅是出于传播信息的需要，并不意味着代表本公众号观点或证实其内容的真实性。据此内容作出的任何判断，后果自负。若有侵权，告知必删！长按关注本公众号   粉丝群/投稿/授权/广告等请联系公众号助手 觉得本文好看，请点这里↓

Biomarkers that could be targets for novel drugs to treat glioblastoma brain tumors have been identified by investigators at Georgetown Lombardi Comprehensive Cancer Center, providing hope for a cancer that is highly lethal. Currently, the drug most often used to treat glioblastoma, temozolomide, is uniquely able to cross the blood/brain barrier to attack the tumor but resistance develops rapidly, and many patients do not survive for more than a year after diagnosis. This new finding provides early evidence that there may be a benefit in targeting specific alterations in cancer cells with newer agents once a patients tumor becomes resistant to temozolomide. The finding appeared June 22, 2022, in Science Advances. "As a field, we have struggled to deal with the short-term effectiveness of temozolomide, as many of the drugs used successfully in other cancers are disappointing when they are subsequently tested in glioblastoma clinical trials. One way to deal with this problem is to learn enough about how we can target features that help drug-resistant glioblastoma survive," says Rebecca B. Riggins, PhD, Associate Professor and Associate Director of Education and Training at Georgetown Lombardi and co-corresponding author of the study. "We focused on the details of how temozolomide damages DNA to help radiation treatments work better. Our team found that temozolomide-resistant glioblastoma relies on a protein called CLK2, and that inhibiting the activity of CLK2 could cause widespread confusion, leading to cancer cell death." The targets the researchers identified were alterations in a key structural component of both DNA and RNA, specifically in guanine, one of the four bases that comprise DNA. Modifications to guanine can ultimately have an impact on CLK2, which has been implicated in the aggressiveness of the tumor. Beyond identifying vulnerable modifications, the researchers were able to identify drugs that help stabilize RNA and could potentially slow or stop the resistance that typically develops to temozolomide. Only about 5% of patients diagnosed with glioblastoma live for five years after diagnosis and median survival is a little over a year; survival rates havent changed much since the mid-1970s. Temozolomide (Temodar) has been the standard of care since 2005 in combination with surgery and radiation. Temozolomide's targeting of guanine also impacts structures that regulate key cancer-causing genes. If these cancer-causing genes, called oncogenes, could be kept turned off, the drug might have a longer period of activity. Some of this knowledge was gleaned from studies in a neurogenerative disease, Amyotrophic Lateral Sclerosis (ALS), commonly known as Lou Gehrigs disease. ALS has some features that are similar to glioblastoma, therefore they might also inform new strategies for glioblastoma treatment, the researchers theorized. "Some of the mechanisms underlying neurodegenerative diseases appear to be relevant to temozolomide resistance in glioblastoma," says Deanna M. Tiek, PhD, a F99/K00 fellow at the Northwestern University Feinberg School of Medicine and The Robert H. Lurie Comprehensive Cancer Center and co-corresponding author. Tiek was a PhD student in Riggins' lab when this research began. "This work demonstrates that inspiration and insight can come from places we might not have considered, and that it's so important to take a risk, do the experiment, and see if you were right or not." The investigators are now undertaking studies in small animal models, where they will test to see if the novel CLK2 inhibitor can efficiently enter the brain and shrink temozolomide-resistant glioblastoma. "We are also investigating whether other anti-cancer drugs that attack guanine and are commonly used in triple-negative breast cancer and colorectal cancer, for example, change RNA structures in a similar way, which could make CLK2 inhibition more effective in recurrent, drug-resistant forms of those cancers as well," concludes Riggins. Deanna M Tiek, Beril Erdogdu, Roham Razaghi, Lu Jin, Norah Sadowski, Carla Alamillo-Ferrer, J Robert Hogg, Bassem R Haddad, David H Drewry, Carrow I Wells, Julie E Pickett, Xiao Song, Anshika Goenka, Bo Hu, Samuel A Goldlust, William J Zuercher, Mihaela Pertea, Winston Timp, Shi-Yuan Cheng, Rebecca B. Riggins. Temozolomide-induced guanine mutations create exploitable vulnerabilities of guanine-rich DNA and RNA regions in drug-resistant gliomas. Science Advances, 2022. doi: 10.1126/sciadv.abn3471

KANAGAWA, Japan, June 7, 2022 /PRNewswire/ -- Chordia Therapeutics Inc. ("Chordia"), a biotech company engaged in the research and development of novel therapies for cancers, today announced that it has presented the interim results from the Phase 1 clinical trial of CTX-712, a selective pan-CDC-like kinase ("CLK") inhibitor discovered by Chordia, at the 2022 Annual Meeting of the American Society of Clinical Oncology (ASCO), which was held from June 3 to June 7. A Phase 1 clinical trial of CTX-712 in solid tumors and hematological malignancies demonstrated a clinically acceptable safety profile. As for antitumor efficacy, it was observed in multiple subjects, establishing an initial Proof of Concept (POC). Dose limiting toxicities (DLTs) observed included dehydration, decreased platelet count, and hypokalemia, and the maximum tolerated dose (MTD) for twice-weekly dosing was determined to be 140 mg. Additionally, two partial responses (PRs) and two complete responses (CRs) were observed in patients with ovarian cancer and acute myeloid leukemia, respectively. A dose-dependent increase in systemic exposure was observed in pharmacokinetics (PK) analysis, and a dose-dependent increase in exon skipping of RNAs set as pharmacodynamics (PD) markers confirmed mRNA splicing modification by CTX-712. Further studies are currently underway to determine the recommended Phase 2 dosing. "It is a great honor to be involved in the first-in-human clinical trial of a new anticancer drug candidate with a novel mechanism of action," said Dr. Noboru Yamamoto, Director of the Department of Experimental Therapeutics at the National Cancer Center Hospital and Principal Investigator of the study. "Although it is still in the early stages of clinical trials, we hope that CTX-712 will become an effective treatment for patients with advanced, relapsed, or refractory malignancies in the future." ASCO abstract number: 3080 Title: A First-in-Human Phase 1 Study of CTX-712 in Patients with Advanced, Relapsed or Refractory Malignant Tumors About CTX-712 CTX-712 is a first-in-class, orally available and selective small molecule inhibitor of CDC-like kinase (CLK), a key regulator of the RNA splicing process that plays an important role in cell growth. CTX-712 inhibits the growth of various human tumor cell lines in vitro, and in addition, exhibits antitumor activity in multiple xenograft mouse models in vivo. Details of Phase 1 Clinical Trial The Phase 1 clinical trial in Japan is investigating the tolerability, safety, and pharmacokinetics (PK) of CTX-712 in patients with advanced, relapsed, or refractory malignancies. For details of the study, please refer to JapicCTI-184188. About RNA Splicing The RNA immediately after transcription is called precursor messenger RNA which contains noncoding sequences (introns) as well as coding sequences (exons) that are needed to make proteins. RNA splicing is a process to remove the introns and connect the exons to form mature mRNA, which is then translated into protein. About CDC-like Kinases (CLKs) Kinase is a general term for enzymes that catalyze the transfer of phosphate groups in biological substances, such as ATP, over to target substances that are called substrates. Over 500 protein kinases are known in humans, of which the CLK family consists of four members – CLK1, CLK2, CLK3, and CLK4, and phosphorylates serine/arginine-rich (SR) proteins as substrates. About Serine/Arginine-rich Proteins (SR Proteins) SR protein is a general term for a group of proteins with serine (S) and arginine (R)-rich SR domains, of which about 10 types have been reported in humans. The serine in the SR domain is phosphorylated by CLK kinase. The phosphorylated SR proteins bind to the exon region of the precursor mRNA and facilitate the incorporation of the bound exon into the mature mRNA during splicing. About Exon Skipping When CTX-712 inhibits CLK kinases and the SR protein dephosphorylates, a splicing change called exon skipping occurs, in which the exons fail to be incorporated into the mature mRNA. About Chordia Therapeutics Chordia was established in November 2017 at Shonan Health Innovation Park ("Shonan iPark") in Fujisawa, Kanagawa Prefecture, as a biotech company engaged in the research and development of novel therapies for cancers, with the goal of researching and developing first-in-class anti-cancer drugs and creating innovative new drugs. In addition to its leading program for CTX-712, Chordia is engaged in the research of several developments in our pipeline, including CTX-439, a CDK12 inhibitor, which is expected to be effective in cancers with specific abnormalities, as well as GCN2 inhibitors. This press release is an English translation of a Japanese-language press release. The official language of this press release is Japanese, and the Japanese version takes precedence over the English version in terms of content and interpretation. SOURCE Chordia Therapeutics Inc.