Dordaviprone

– Phase 3 ACTION Study On-Track; First Interim Overall Survival Data Expected Third Quarter 2025 – – ONC206 Phase 1 PK and Safety Data Demonstrate Dose Proportional Exposure with No Dose Limiting Toxicity to Date – – Dordaviprone Filed for Provisional Determination with Therapeutic Goods Administration in Australia – – Conference Call at 8:30 a.m. ET Today – DURHAM, N.C., Aug. 13, 2024 (GLOBE NEWSWIRE) -- Chimerix (NASDAQ: CMRX), a biopharmaceutical company whose mission it is to develop medicines that meaningfully improve and extend the lives of patients facing deadly diseases, today reported financial results for the second quarter ended June 30, 2024 and provided an operational update. “We continued our strong execution of the Phase 3 ACTION study and expect the first interim overall survival (OS) data in the third quarter of 2025. As we approach this important milestone, we continue to strengthen our U.S. launch capabilities and readiness,” said Mike Andriole, Chief Executive Officer of Chimerix. “Additionally, we are making great strides advancing ONC206 through the remaining dose cohorts in two Phase 1 trials, which recently achieved dosing within an expected therapeutic range. As we escalate and intensify the dose within this range, we are encouraged by ongoing pharmacokinetic (PK) data that is in line with modeled expectations for delivering dose proportionate exposures for extended durations. Importantly, these exposures have not been associated with dose limiting toxicities thus far. We expect to complete enrollment in the ONC206 dose escalation trials by the end of this year.” “We have also made progress expanding the global reach for dordaviprone with the recent filing of a Provisional Determination application in Australia. This marks the second of three steps to potential Provisional Approval. Our team continues to be driven by the urgent need of patients focusing on indications where the unmet medical need remains frustratingly high. We will continue to drive our pipeline forward in order to accelerate access to patients in need of new treatment alternatives,” added Mr. Andriole. Dordaviprone (ONC201) Dordaviprone, a first-in-class imipridone, has the potential to be the first treatment approved for H3 K27M-mutant diffuse glioma. It is an oral small molecule that crosses the blood-brain barrier and selectively binds to the mitochondrial protease ClpP and the G-protein-coupled dopamine receptor D2 (DRD2). Dordaviprone's unique mechanism of action includes alterations of key epigenetic modifications such as reversal of H3 K27me3-loss (H3 K27 trimethyl loss), which is the hallmark of H3 K27M-mutant gliomas. The Company estimates that approximately 2,000 patients with diffuse glioma harbor the H3 K27M mutation in the United States and approximately 5,000 patients in the top seven markets globally. With no approved therapies specific to this patient population, the standard of care following upfront radiotherapy remains palliative in nature. Dordaviprone is being evaluated in the Phase 3 ACTION trial that is currently enrolling H3 K27M-mutant diffuse glioma patients at over 140 sites in 13 countries. Chimerix expects interim OS data in the third quarter of 2025. For more information on the ACTION trial, please visit clinicaltrials.gov Earlier this year, Chimerix initiated the evaluation process for dordaviprone to be considered for Provisional Registration in Australia. The Provisional Registration process is a three-step process which begins with a Pre-Submission Meeting evaluating current data, as well as other program features, including the status of pivotal studies. Chimerix recently initiated the second of three steps in the process, the filing of a Provisional Determination application. Should the TGA approve the Provisional Determination application, the final step is to apply for Provisional Registration. Should Chimerix proceed to the Provisional Registration step, it is expected that a filing could occur as early as year-end 2024 with possible commercial availability in 2026. ONC206 The imipridone ONC206 is a second generation ClpP agonist and DRD2 antagonist which also crosses the blood-brain barrier and is 10x more potent in vitro than dordaviprone. It has demonstrated monotherapy in vivo anti-cancer activity in central nervous system (CNS) tumor models, as well as in vivo solid tumors models outside of the CNS. The two Phase 1 dose escalation trials conducted in partnership with the Pacific Pediatric Neuro-Oncology Consortium (PNOC) and the National Institutes of Health (NIH) have enrolled over 75 pediatric and adult patients with unselected CNS tumors. The dose escalation studies have reached dose level 10 (of 11 planned levels) at 150mg twice per day for three consecutive days, with no dose limiting toxicity observed to date. The safety profile of ONC206 has been consistent across both pediatric and adult populations, with the majority of treatment-related adverse events being mild to moderate, including fatigue, lymphocyte count decrease and vomiting. No significant change in the overall safety profile has been reported to date as dosing has escalated and intensified in frequency from once per week to twice per day on three consecutive days per week. Completion of enrollment in the remaining two planned dose escalation cohorts is expected to occur in 2024. Assessment of objective responses in patients where a monotherapy treatment effect can be reliably evaluated is ongoing in dose cohorts at or above target exposure thresholds. The company expects to assess any objective responses in the first half of 2025, allowing sufficient time for response onset and confirmation in current and future dose cohorts. Additionally, ONC206 nonclinical studies remain ongoing to identify candidate oncology indications and biomarkers to inform future development plans. Second Quarter 2024 Financial Results Chimerix reported a net loss of $20.7 million, or $0.23 per basic and diluted share, for the second quarter of 2024. During the same period in 2023, Chimerix recorded a net loss of $18.6 million, or $0.21 per basic and diluted share. Research and development expenses increased to $18.4 million for the second quarter of 2024, compared to $16.9 million for the same period in 2023. General and administrative expenses increased to $4.5 million for the second quarter of 2024, compared to $4.4 million for the same period in 2023. Chimerix's balance sheet at June 30, 2024 included $171.5 million of capital available to fund operations, approximately 89.6 million outstanding shares of common stock and no outstanding debt. Upcoming Events The Company expects to participate in the 2024 Wedbush PacGrow Healthcare Conference occurring August 13-14, 2024 and the H.C. Wainwright 26th Annual Global Investment Conference taking place September 9-11,2024. Conference Call and Webcast Chimerix will host a conference call and live audio webcast to discuss second quarter 2024 financial results and provide a business update today at 8:30 a.m. ET. To access the live conference call, please dial 646-307-1963 (domestic) or 800-715-9871 (international) at least five minutes prior to the start time and refer to conference ID 5436125. A live audio webcast of the call will also be available on the Investors section of Chimerix’s website, www.chimerix.com. An archived webcast will be available on the Chimerix website approximately two hours after the event. About Chimerix Chimerix is a biopharmaceutical company with a mission to develop medicines that meaningfully improve and extend the lives of patients facing deadly diseases. The Company’s most advanced clinical-stage development program, dordaviprone (ONC201), is in development for H3 K27M-mutant diffuse glioma. The Company is conducting Phase 1 dose escalation studies of ONC206 to evaluate safety and PK data. Forward-Looking Statements This press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995 that are subject to risks and uncertainties that could cause actual results to differ materially from those projected. Forward-looking statements include those relating to, among other things, expectations regarding interim OS data from the ACTION study, plans for Provisional Registration in Australia, expectations regarding completion of enrollment and assessment of responses in the ONC206 dose escalation trials, and the characteristics and development of ONC206. Among the factors and risks that could cause actual results to differ materially from those indicated in the forward-looking statements are risks related to the ability to obtain and maintain accelerated approval; risks related to the timing, completion and outcome of the Phase 3 ACTION study of ONC201; risks associated with repeating positive results obtained in prior preclinical or clinical studies in future studies; risks related to the clinical development of ONC206; and additional risks set forth in the Company's filings with the Securities and Exchange Commission. These forward-looking statements represent the Company's judgment as of the date of this release. The Company disclaims, however, any intent or obligation to update these forward-looking statements. CONTACT: Will O’ConnorStern Investor Relations212-362-1200will@sternir.com CHIMERIX, INC.CONSOLIDATED BALANCE SHEETS(in thousands, except share and per share data)(unaudited) June 30, December 31, 2024 2023ASSETS Current assets: Cash and cash equivalents$15,658 $27,661 Short-term investments, available-for-sale 132,511 155,174 Accounts receivable 129 4 Prepaid expenses and other current assets 5,157 6,271 Total current assets 153,455 189,110 Long-term investments 23,315 21,657 Property and equipment, net of accumulated depreciation 276 224 Operating lease right-of-use assets 1,223 1,482 Other long-term assets 242 301 Total assets$178,511 $212,774 LIABILITIES AND STOCKHOLDERS' EQUITY Current liabilities: Accounts payable$4,163 $2,851 Accrued liabilities 17,939 15,592 Total current liabilities 22,102 18,443 Line of credit commitment fee - 125 Lease-related obligations 827 1,177 Total liabilities 22,929 19,745 Stockholders’ equity: Preferred stock, $0.001 par value, 10,000,000 shares authorized at June 30, 2024 and December 31, 2023; no shares issued and outstanding as of June 30, 2024 and December 31, 2023; no shares issued and outstanding as of June 30, 2024 and - - Common stock, $0.001 par value, 200,000,000 shares authorized at June 30, 2024 and December 31, 2023; 89,632,385 and 88,929,300 shares issued and outstanding as of June 30, 2024 and December 31, 2023, respectively 90 89 Additional paid-in capital 993,778 988,457 Accumulated other comprehensive (loss) gain, net (208) 7 Accumulated deficit (838,078) (795,524) Total stockholders’ equity 155,582 193,029 Total liabilities and stockholders’ equity$178,511 $212,774 CHIMERIX, INC.CONSOLIDATED STATEMENTS OF OPERATIONS AND COMPREHENSIVE LOSS(in thousands, except share and per share data)(unaudited) Three Months Ended June 30, Six Months Ended June 30, 2024 2023 2024 2023Revenues: Contract and grant revenue$129 $26 $129 $260 Licensing revenue - - - 49 Total revenues 129 26 129 309 Operating expenses: Research and development 18,428 16,926 37,272 35,748 General and administrative 4,533 4,448 10,079 10,127 Total operating expenses 22,961 21,374 47,351 45,875 Loss from operations (22,832) (21,348) (47,222) (45,566)Other income: Interest income and other, net 2,147 2,772 4,668 5,618 Net loss (20,685) (18,576) (42,554) (39,948)Other comprehensive loss: Unrealized loss on debt investments, net (30) (582) (215) (476) Comprehensive loss$(20,715) $(19,158) $(42,769) $(40,424)Per share information: Net loss, basic and diluted$(0.23) $(0.21) $(0.48) $(0.45) Weighted-average shares outstanding, basic and diluted 89,630,959 88,583,567 89,445,033 88,439,894

感谢关注转发，欢迎学术交流 回复药渡Cyber，领取文献原文 请点击此处链接观看CyberSAR系统详细使用教程 快看版 G蛋白偶联受体G2A是开发神经性疼痛、急性髓性白血病和炎症新疗法的有希望的靶点。但仍然缺乏有效的、选择性的、具有类药性的G2A激动剂来用作工具化合物或Hit。本文主要介绍了一种新的有效且选择性的G2A激动剂骨架的发现和构效关系研究。经过结构优化发现(3-(吡啶-3-基甲氧基)苯甲酰基)-D-苯丙氨酸（T-10418）比参考和天然配体9-HODE更高的效力，并且在G蛋白偶联受体中具有高选择性。凭借其良好的活性、高选择性、优异的溶解度和高代谢稳定性，T-10418可以作为研究G2A激活效果的药理学工具。 药渡CyberSAR系统在本研究中发挥了关键作用，提供了对GPR132靶点分子的深入解析。系统通过聚类结构视图和原始结构视图，展示了与靶点相关的活性分子，并以研发阶段时光轴的形式呈现了潜力Hit。此外，CyberSAR还提供了适应症和试验设计的可视化分析，帮助研发人员快速获取靶向结构信息，开拓研究思路。尽管CyberSAR在分子的初步开发中未被使用，但其在解析和优化药物分子方面显示出巨大的应用潜力。 图1.化合物T-10418发现和分子优化过程 正文版 G蛋白偶联受体GPR132，也称为G2A。1998年，Ludwig等人于2003年提出了质子敏感GPCR的概念，并将AGPCR类的四种孤儿受体：GPR4、TDAG8（GPR65）、OGR1（GPR68）和G2A归入该家族。质子感应GPCR的作用最初认为是感应局部pH值降低的组织损伤、炎症或肿瘤生长。然而，G2A的质子敏感性在文献中存在争议。在生理条件下，GPR4、OGR1和G2A普遍表达，其中 G2A在白细胞、中性粒细胞和巨噬细胞中表达最强。G2A在共表达瞬时受体电位香草酸1通道（TRPV1）的外周感觉神经元中表达。在这种环境中，G2A与发育密切相关通过蛋白激酶C（PKC）激活和随后的TRPV1致敏，奥沙利铂诱导的神经性疼痛（OINP）持续存在。因此，抑制G2A是减弱OINP的新方法。G2A也是促进乳腺癌转移的因素。由于G2A在过氧化物酶体增殖物激活受体γ激活时受到转录抑制，因此其抑制可能有助于罗格列酮的抗肿瘤作用，并表明抑制G2A可作为治疗乳腺癌的新策略。 G2A也被认为是脂质受体，溶血磷脂酰胆碱（LPC）是第一个激活剂。然而，LPC 作为G2A激动剂尚不明确。一项关于LPC激活G2A的研究被撤回，其他几项研究证实LPC缺乏G2A激动作用，一项研究甚至得出结论，LPC实际上是G2A 的弱拮抗剂（IC50>10μM）。源自亚油酸（羟基十八碳二烯酸）的氧化脂质，HODEs）或来自花生四烯酸（羟基-叶二十碳三烯酸，HETE）表现出弱至中度G2A激动作用的G2A配体，其中亚油酸代谢物9-HODE认为是最有效的内源性G2A激动剂（图1A）。值得注意的是，G2A的结构与其内源性激动剂形成复合物9-HODE最近用冷冻电子显微镜得到解析。天然细菌产物Commendamide以及N-棕榈酰甘氨酸和N-亚油酰甘氨酸也被发现可作为G2A激活剂，强化了G2A是信号脂质受体而不是质子传感器的概念。化合物SB583831（图1B）和SKF-95667（图1C）是有效的合成激动剂，SB-583831的EC50值在纳摩尔范围内。另一方面，化合物SB-583355（图1D）和替米沙坦类似物GSK1820795A（图1E）为有效的G2A拮抗剂。 G2A激活在造血、脓毒症和急性髓性白血病（AML）中具有潜力。抗癌药物ONC201和ONC212（图1F、G）在β-抑制蛋白募集过程中激活G2A的观察结果的支持其具有抗AML活性。ONC212还在AML中发挥显著的促凋亡作用，但在健康骨髓细胞中没有，并且减少了AML体内的生长，表明G2A激活可能是治疗AML的潜在治疗方法。 总而言之，目前的证据表明G2A激活和抑制对于开发新疗法的潜力。本文介绍了新型有效且选择性G2A激动剂骨架的发现和SAR研究。优化后的类似物在G2A激活功效上超过了内源性激动剂9-HODE，并且具有优异的溶解度和代谢稳定性，从而成为研究G2A的化合物工具。 G2A激动剂高通量筛选 为了发现新型G2A激动剂，在无偏高通量筛选（HTS）设置中筛选了Enamine库中的25,000种化合物。化合物均匀地来自文库的所有子集，并且优先考虑含有较多数量的含有羧酸的化合物的文库板，从而在筛选组中产生大约3,000种羧酸。这些化合物在基于功能细胞的测定中以50μM进行测试，检测肌醇单磷酸（IP-1）的积累作为人G2A激活的检测指标。与内源性激动剂9-HODE（EC50=7.5μM）比较效力。选择效力（EC50=1.74μM）和合成可及性方面最有希望的Hit（1，图2）进行进一步优化。 小鼠初级感觉神经元的Hit验证 为了验证效果，测试化合物1基于钙成像的TRPV1敏化实验。如前所述，G2A感觉神经元的激活以Gq-PKC依赖性方式导致TRPV1活性增强；因此，作者用两次相同的辣椒素刺激物（100nM，30s）和在第二次辣椒素刺激物之前预孵育的化合物1刺激小鼠初级感觉神经元两次。对钙反应幅度的分析[Δratio（F340/F380）] 表明，化合物1可以有效地对辣椒素诱导的TRPV1反应敏感，且与参考G2A激动剂9-HODE相当（图3A、B）。 首先，合成了来自HTS的原始外消旋产物（1）的两个对映体，从而发现(R)异构体（3命名为T-10418，EC50=0.82μM），其活性比(S)二异构体（2，EC50=31.1μM）强30倍以上。研究了具有高苄基和苯基残基的化合物（4-7），它们的活性明显低于具有苄基残基的Hit化合物，但在立体化学方面表现出相同的趋势。在化合物8-11中测试了一小部分芳香族取代基。结果表明，对位取代（8，EC50=1.96μM；9，EC50=3.34μM）似乎比间位（10，无活性）邻位取代（11，EC50=13.5μM）具有更好的耐受性。带有吡啶环的化合物（13和14）也仅显示出较弱的效力。苄基位置或羧基α位置的甲基取代基是可以耐受的（12和15），但不会提高活性。 接下来，分析了一系列具有或不具有杂原子的脂肪族取代基（16-32）。一般来说，除了化合物19（EC50=7.33μM）和22（EC50=6.87μM）外，该系列的化合物活性较弱（EC50>10μM）或无活性。如果化合物19为(S)-构型，则相应的对映体(R)的活性较低（20，EC50=64.8μM）。然而，化合物19在>20μM时仅表现出非常有限的效力，在这方面明显劣于对映体(R)，这与此前(R)-构型的观察结果一致。最后，带有二氢化茚取代的化合物33仅具有轻微活性。 考虑到第一批化合物（2-33）SAR结果，设计并合成了第二批化合物。第二批化合物的G2A活性如表2所示。 从之前的系列来看，外消旋化合物22是脂肪族取代苯环衍生物中最有前途的衍生物。此后，还合成并测试了其对映体。在这种情况下，与化合物19和20对一样，EC50=2.92μM的(S)-对映体（41）显示出比其(R)-对映体（40，EC50=40.6μM）更高的效力。化合物42和43均为R构型，并且在分子右侧的苯环上具有对位取代基。正如前一组化合物中所显示的那样，这些对位取代基是可以耐受的，但这些取代基并未带来 EC50改进，尽管43表现出最大活化作用。接下来，研究了吡啶环上氮原子位置不同的外消旋化合物。具有邻位氮的化合物48是有效的，显示EC50=2.87μM，而具有邻位氮的化合物49和具有苯环的50仅具有弱活性。最后，中心环中取代基的对位配置的化合物54导致效力丧失。SAR结果如图4所示。 为了将其用作参考工具化合物，还制备了所述拮抗剂SB-583355（56）。在 IP-One 测定中，随后研究了所描述的G2A拮抗剂56（SB-583355） 是否可以抵消化合物T-10418对G2A的激活（图5A）。化合物56以与参考激动剂9-HODE相比相同的剂量反应依赖性方式完全抑制了T-10418的激活。 直接结合G2A和β-Arrestin-2招募 通过LC-MS分析从稳定表达人G2A的CHO-K1细胞获得的膜蛋白中T-10418与G2A的直接结合。来自野生型CHO-K1的膜蛋白作为对照。尽管T-10418的亲和力可能在nM范围内，但在较高浓度的T-10418（支持信息图S3中显示的600nM）下，转染细胞与未转染细胞的膜蛋白之间存在微小但具有统计学意义的差异与G2A直接作用。为了进一步表征T-10418作为G2A配体，作者利用分裂荧光素酶互补测定报告β-arrestin2[βarr2；uniport ID P32121]在受体激活后募集至G2A，证实化合物在细胞中的直接与靶标作用（图5B）。 小鼠感觉神经元的靶标作用 T-10418在小鼠感觉神经元的TRPV1致敏试验中进行了测试；图6为100nM T-10418平均导致244% TRPV1致敏，相当于或超过用400nM 9-HODE处理后观察到的致敏。因此，T-10418比外消旋化合物1更具活性，其中1μM导致的TRPV1致敏作用与使用200nM(±)9-HODE观察到的结果相当（图3）。此外，两种激动剂T-10418和9-HODE的TRPV1敏化作用在与G2A拮抗剂56共同治疗后被完全消除，这为T-10418与靶标作用提供了额外的证据。 GPCR选择性 为了评估其作为化学工具的潜力，在测量受体与β-抑制蛋白的关联的PRESTO-Tango测定中测试了T-10418的GPCR选择性。该检测覆盖了300多个非嗅觉 GPCR。除了预期的G2A激活之外，T-10418仅对另一种受体GPR1（人类蛋白质序列）有活性，这表明GPCR具有出色的选择性（图7）。然而，应该指出的是，这种GPCR筛选有其局限性。PRESTO-Tango panel中的300多个GPCR包括大量孤儿受体，对于一些已经表征的受体，在该特定测定中尚未证明参考激动剂的激活作用。此外，不会观察到拮抗剂活性，因为通常只有当测试的化合物充当受体的激动剂时才会预期β-抑制蛋白的募集。此后，化合物T-10418也在Eurofins的SafetyScreen中进行了分析，显示整个组中的特异性结合抑制<25%（支持信息，图S4）。 GPCR1（GPR1；uniprot P46091）由凯莫瑞（一种136个氨基酸蛋白，也称为凯莫瑞-S157）以及凯莫瑞受体家族的其他两个GPCR激活：趋化因子样受体1（CMKLR1）和CC-基序趋化因子受体样2（CCRL2）。Chemerin-S157的C端九肽，称为Chemerin-9（C9），经鉴定能够激活CMKLR126以及GPR1。对于GPR1，仅可检测到抑制蛋白募集，而对于CMKLR1，在配体刺激后也可检测到G蛋白信号转导。因此，确定了T-10418对GPR1和CMKLR1处的内化和βarr2募集的影响。 C9与荧光染料TAMRA（四甲基罗丹明）的结合可以使其定位和细胞摄取可视化。用TAMRA-(EG)4-C9系列稀释液处理表达CMKLR1-eYFP或GPR1-eYFP的细胞，由于配体的结合和细胞摄取，导致TAMRA特异性红色荧光增加。然后通过30μM T-10418共处理来。尽管CMKLR1不受T-10418的影响（图8A），但与不含化合物的对照相比，稳定转染GPR1-eYFP的HEK293细胞的EC50发生了2倍的变化，表明T-10418对GPR1内化的影响较小（图8A、8B、C）。图8D显示了与用作阴性对照肽的TAMRA-(EG)4-scr2C9（乱序C9序列）相比，TAMRA-(EG)4-C9刺激的GPR1内化。 T-10418的调节在βarr2募集生物发光共振能量转移（BRET）测定中更强，该测定测量与βarr2的关联，作为受体配体依赖性激活的读数（图9）。T-10418仅表现出对GPR1的部分激动作用（图9E），这种对βarr2募集的有限刺激导致再次间接测试其效果。与CMKLR1相比，T-10418导致C9诱导的GPR1激活发生显著变化（图9A、B）。与不含化合物的对照相比，60μM浓度的T-10418减弱了 C9刺激的βarr2募集，C9的EC50变化了232倍。当没有内源配体应用于GPR1时，60μM T-10418的部分激动作用达到C9完全激活时观察到的最大βarr2募集的大约50%（图9B，w/o）。在对照组中，抑制蛋白3半最大招募需4nM C9。 最后，按照最近开发的方法进行置换结合测定28（图10）。证明T-10418无法置换Nluc-GPR1构建体上结合的TAMRA标记的C9，而C9显示完全置换，Ki为35.4nM。因此，T-10418根本不干扰C9与GPR1的结合，或者该化合物在正位C9位点处是低亲和力结合（激动剂），其亲和力太低而无法在该实验设置和浓度范围内显示效果。尽管在βarr2 NanoBRET测定中观察到对β-arr2招募的大量刺激（图9B、C），但T-10418介导的C9诱导的GPR1内化抑制（图8B）是有限的，这与GPR1的鉴定一致。PRESTO-Tango选择性（图7）。然而，在NanoBRETβ-arr2招募测定中，T-10418抵消了C9的影响[>EC50（C9）；9C]，因此，T-10418 vs C9对β-arr2招募具有调节作用。当使用T-10418作为药理学工具时，预计GPR1的显著调节仅在≥10μM时发生。对T-10418和GPR1相关化合物的进一步研究将有助于进一步表征其作用模式。 受这些观察结果的鼓舞，继续测试了T-10418与原型咪咪酮ONC201一起对AML细胞的影响。将AML细胞系Molm-13和ML-2与ONC201或T-10418单独孵育或与Bcl2抑制剂ABT-199（称为Venetoclax）联合孵育。ONC201和ABT-199之间的协同效应。72小时后，观察/检测到在ONC201存在的情况下，不存活的Molm-13和ML-2细胞明显减少，而T-10418不影响细胞扩增或活力，如图11所示。 此外，作者证实ONC201和ABT199联合治疗对细胞活力有更强的影响。细胞凋亡的诱导可以通过使用流式细胞术的AnnexinV/7-AAD测量实验验证。AML细胞对ONC201和T-10418敏感性的差异以及对T-10418对G2A选择性的研究结果表明，这些细胞系可能对G2A激活没有反应，并且ONC201诱导了独立于G2A的强抗白血病作用。 因此，作者质疑这些化合物是否激活了AML细胞系中的Gq通路，并在IP-One测定中对此进行了测试。为了跟进之前的实验，首先在表达G2A + GNA11的CHO-K1细胞上测试了ONC201和ONC212。与Nii等人的报告一致，ONC201（EC50=38.6μM）表现出有限的活性，并且低于ONC212（EC50=3.92μM）。为了测试不是由受体介导的任何反应，在仅过表达GNA1但不过表达G2A的CHO-K1细胞上进行了对照实验。当浓度≥30μM时，ONC201和ONC212均刺激IP-1显著增加。以前用9-HODE、T-10418或表1和表2中列出的任何衍生物都没有观察到这种情况。这表明ONC201和ONC212解决了CHO-K1细胞中可以激活Gq途径的一些其他靶标。然后继续在IP-One测定中测试Molm-13和ML-2细胞的反应。用9-HODE、T-10418或ONC212（在G2A上更活跃）治疗后，无法检测到Gq通路的任何激活（支持信息图S5）。因此，ONC201在AML细胞中的强烈作用很可能独立于G2A，这可能解释了T-10418无法抑制AML细胞存活。ONC201和ABT199有明显的加和作用，据Nii等人报道。然而，当ABT-199和T-10418联合使用时，没有检测到额外的抑制作用。 T-10418细胞毒性、水溶性、代谢稳定性和体内药代动力学研究 使用CellTiter-Glo作为代谢活性和细胞存活的总体测量方法，在肝细胞癌细胞（HepG2；DSMZ #ACC 180）中测试了化合物1（外消旋）和T-10418（对映体 R）的毒理学特征，在用任一化合物以高达100μM处理72小时后结果均不受影响（阈值≥90%）（支持信息图S6）。 为了评估T-10418是否也适合作为体内研究的化学工具，首先研究了其水溶性和代谢稳定性，这是药物良好吸收和低清除率的关键因素。在磷酸盐缓冲盐水（PBS）缓冲液中测定溶解度，并使用大鼠肝微粒体研究体外代谢半衰期（表3）。T-10418表现出非常高的代谢稳定性和大于3mM的优异水溶性。 由于T-10418具有高体外效力和优异的理化性质等有吸引力的特性，因此选择T-10418在雄性C57Bl/6N小鼠中进行PK研究。将化合物以1mg/kg静脉内（iv）施用并以10mg/kg皮下（sc）施用在PBS中。iv研究在15分钟、30分钟、1小时、2小时、4小时和8小时测量血浆浓度，皮下研究在30分钟、1小时、2小时、4小时、8小时和12小时测量血浆浓度。所得血浆浓度分布以及计算的PK参数显示在表4中。静脉注射途径在15分钟后产生最大血浆浓度，然后浓度呈指数下降，直到8小时的最后一次测量。另一方面，当应用T-10418时sc，在30分钟时达到峰值血浆浓度，因此，这又是记录的最早时间点。与静脉注射相比，皮下注射时的AUC接近10倍，这完全符合扫描静脉注射剂量之间的比率。总体而言，该化合物呈现出高血浆浓度和良好的PK特性，这使得T-10418成为G2A激动治疗潜力体内研究的合适候选者。 结论 孤儿GPCR G2A正在成为各种潜在治疗应用中有吸引力的靶标，但仍处于实验阶段通过药理学G2A激活来验证治疗效果正在进行中。在本文中发现并优化了一种新G2激动剂骨架。T-10418是其中最有效的G2A激活剂，并在GPCR家族中具有高选择性的特点。T-10418未能诱导白血病细胞系凋亡，表明G2A激动作用不会促进亚咪酮的抗白血病作用。此外，T-10418良好的理化性质和 PK 特性说明其适合作为体内化合物工具。因此，T-10418是研究 G2A 生物学和治疗潜力的非常有价值的工具。 文献来源 https://doi.org/10.1021/acs.jmedchem.3c02164 请点击此处链接观看CyberSAR系统详细使用教程 1.药渡CyberSAR结合药物设计思想，挖掘了文献及专利报道的活性的结构，通过CyberSAR以方便快速获得研发人员兴趣靶向结构，以供开拓思路，就Probable G-protein coupled receptor 132（Homo sapiens）举例如下： 2.在靶点界面选中“化学空间”选项标签下级联“聚类结构视图”选项卡，可以将CyberSAR平台收录的文献和专利具有关于GPR132相关实验测试活性的分子以“母核结构聚类”的形式展示。 3.在靶点界面选中“化学空间”选项标签下级联“原始结构视图”选项卡，可以将CyberSAR平台收录的文献具有关于GPR132相关实验测试活性的分子以“研发阶段时光轴”的形式展示。其中绿色字体高亮的“数据挖掘”即为潜力Hit。 登录方式 CyberSAR在电脑浏览器端登录网址： https://cyber.pharmacodia.com/，欢迎猛烈试用。 请点击此处链接观看CyberSAR系统详细使用教程 如需进一步沟通，请扫码添加微信联系药渡赵博士或药渡CyberSAR沟通群。