Zavondemstat

研究用工具药QC6352和已进入临床试验的药物zavondemstat，其抗癌效果并非来自它们被认为靶向的KDM4蛋白，而是来自一个意外的“兼职”靶点DHODH。 在抗癌药物研发领域，找到一个明确的分子靶点，往往意味着数年研究的方向、巨额资金的投向，以及临床试验中患者筛选的标准。然而，靶点搞错的情况并不罕见，而且后果可能相当严重。 日前，发表在国际杂志Nature Chemical Biology上题为“DHODH is the key target of the KDM4 inhibitor QC6352 in glioblastoma stem cells”的研究报告中，来自悉尼大学等机构的科学家们通过研究揭露了一个令人警醒的案例：两款备受关注的抗癌化合物：研究用工具药QC6352和已进入临床试验的药物zavondemstat，其抗癌效果并非来自它们被认为靶向的KDM4蛋白，而是来自一个意外的“兼职”靶点DHODH。 KDM4是一类组蛋白赖氨酸去甲基化酶，在多种癌症中频繁过表达，被认为能促进癌细胞生长和扩散，因此长期被视为有前景的治疗靶点。QC6352作为公认的“最佳级别”KDM4化学探针，在多种癌症模型中展现出强效抗肿瘤活性；其临床类似物zavondemstat也已推进到患者试验阶段，用于结直肠癌、胰腺癌和前列腺癌等。整个领域对这两个化合物的理解，都建立在“它们通过抑制KDM4发挥作用”这一前提之上。 但研究人员在尝试将这两种化合物“老药新用”于胶质母细胞瘤时发现了不对劲的地方；胶质母细胞瘤是最常见也最具侵袭性的脑癌，治疗手段有限，患者预后极差。研究人员使用患者来源的胶质母细胞瘤干细胞、肿瘤模型，并结合一系列遗传学、机制和分子实验，对QC6352的抗癌作用进行了系统追踪。 KDM4抑制剂QC6352在体外和体内均显示出强效的抗胶质母细胞瘤效果 关键线索来自对照实验，当研究人员测试其他KDM4抑制剂时，发现它们无法复制QC6352的抗癌效果。研究者Munoz对此打了个比方：“如果阻断KDM4是驱动这些效果的原因，我们本应看到其他抑制剂表现类似。但结果提示，QC6352走的是一条不同的路。”顺着这条线索，研究人员最终锁定了二氢乳清酸脱氢酶（DHODH）—嘧啶从头合成途径中的关键酶。癌细胞快速增殖需要大量合成DNA，而DHODH正是为DNA复制提供“砖块”的核心机器；研究者表示，DHODH就像一台生产DNA砖块的机器。关掉它，细胞就会缺砖，无法有效复制DNA并继续分裂。” 验证实验进一步夯实了这一结论，研究者指出，补充尿苷（可绕过DHODH依赖的嘧啶合成途径）能完全消除QC6352和zavondemstat的生长抑制作用；表达一种对抑制剂不敏感的DHODH突变体，同样可以逆转药效。而另一种正交KDM4抑制剂ML324，由于不具备DHODH抑制能力，无法重现QC6352的表型，这些结果一致表明，QC6352和zavondemstat实际上是KDM4–DHODH双重抑制剂，而在抗胶质母细胞瘤活性中，DHODH抑制才是主导因素。 这一发现的影响远不止于胶质母细胞瘤，多年来，全球众多研究者将QC6352作为“金标准”工具来研究KDM4的生物学功能，相关文献数量可观；zavondemstat进入临床试验，也是基于同样的机制假设。如果这些化合物的实际作用主要来自DHODH，那么部分已发表结论可能需要重新审视，临床试验的患者筛选和疗效解读也可能受到影响。Munoz指出，搞错药物机制会导致试验设计不佳、患者选择不当，以及多年研究聚焦在错误的靶点上。她强调，这不是历史上的孤立问题，而是当下仍在发生的事，需要更严格的研究规范，在药物进入临床测试前弄清楚它到底怎么工作。 当然，这项研究也带来了积极信息，一方面，DHODH抑制被确认为抗胶质母细胞瘤的潜在新方向，而目前已有多种靶向DHODH的药物正在其他癌症中接受研究，未来或有机会在脑癌患者中检验；另一方面，研究人员开发了不抑制DHODH的新型KDM4抑制剂，为今后准确研究KDM4的生物学功能提供了更干净的工具。从“认错靶点”到“将错就错”，这项研究给抗癌药物研发提了个醒：工具药未必如标签所写那样专一，临床试验的根基也可能比想象中脆弱。（生物谷Bioon.com） 参考文献： Sterling, J.R., Holtsmark, J.R., Joun, G.L. et al. DHODH is the key target of the KDM4 inhibitor QC6352 in glioblastoma stem cells. Nat Chem Biol (2026). doi：10.1038/s41589-026-02306-x

Scientists have uncovered that an important chemical ‘probe’ compound widely used to study cancer in the lab – and also a related compound that has entered clinical evaluation in cancer patients – do not in fact work in brain cancer cells via their long-believed mechanism of action. Instead, they act on an entirely different and unexpected molecular target – with important implications for research and treatment. The study, led by Professor Lenka Munoz at the University of Sydney Charles Perkins Centre and published in the journal Nature Chemical Biology, began with the observation that a chemical probe known as QC6352 – for many years regarded as the best small-molecule chemical inhibitor compound for a family of gene-regulating proteins called KDM4 enzymes – was identified as a strong hit in a screen to discover compounds that inhibit growth of hard-to-treat human brain cancer stem cells. A similar promising effect was also seen with zavondemstat – a closely-related compound, derived from QC6352, that has actually entered a clinical trial as a KDM4 inhibitor in cancer patients. Intrigued to have discovered a potential new treatment for lethal glioblastoma (GBM) brain tumours through KDM4 blockade, Professor Munoz next carried out what was expected to be a confirmatory experiment – testing an additional KDM4 inhibitor, structurally different from QC6352 and zavondemstat – to see if the promising finding could be reproduced with this ‘orthogonal control’ tool. Surprisingly, this second type of KDM4 inhibitor, ML324, was completely inactive against GBM cells, sowing the first seeds of doubt that the effectiveness of QC6352 and zavondemstat might not be achieved through KDM4 blockade – and setting in motion a complex follow-up programme which culminated in identifying the crucial target of the probe and drug in GBM cells. This turned out be a protein called dihydroorotate dehydrogenase (DHODH) which is involved in a totally different biochemical pathway in cells – namely creating the pyrimidine building blocks for making DNA and RNA. The follow-up detective work was carried out by an international team of chemical biology collaborators who worked closely together, first to definitively rule out KDMs as the effective target of QC6352 and zavondemstat in GBM stem cells and then to identify the critical target. Professor Paul Workman, Harrap Professor of Pharmacology and Therapeutics at The Institute of Cancer Research, London, was a senior member of the collaborative team and co-author on the paper. He helped to guide the investigation using his experience in developing chemical probes for potential drug targets in the Centre for Cancer Drug Discovery at The Institute of Cancer Research (ICR) and in his role as Director of the Chemical Probes Portal. Hosted at ICR, the Portal is a web-based resource that develops and promulgates best practice in the use of chemical probes for bioscience research The study was funded by the Tour de Cure Foundation and the National Health and Medical Research Council in Australia. The Chemical Probes Portal has received long-term funding from the Wellcome Trust. It is now supported through generous philanthropic funding from Diego Megia and Lola Monterola, philanthropists and founders of CRIS Cancer. Other teams involved came from the Goethe University, Frankfurt, Harvard University and Oxford University. Chemical probes are well-characterised small-molecule compounds, usually inhibitors, that are designed to modify the activity of a specific protein target. They are commonly used in biological research to investigate the function of specific proteins of interest in biological models, such as cancer cell lines, and to validate potential new targets for drug discovery. But to be effective for these purposes scientists need to be confident that the chemical probes they use are indeed acting primarily on their target of interest in the cell and not through ‘off-target’ effects on other proteins. Yet, in practice it can be very challenging to rule out unanticipated effects, even if the probe is counter-screened against tens or hundreds of other proteins – as was the case in the initial development of QC6352 and zavondemstat – therefore some off-targets may still easily be missed QC6352 has become a popular chemical probe in lab experiments to block the activity of KDM4 histone demethylases – enzymes that regulate gene activity and are thought to play a role in many cancers. Based on information in scientific publications, QC6352 was until now strongly recommended by the expert review-based Chemical Probes Portal as a best-in-class research probe for studying KDM4 biology – and is sold as such by multiple vendor companies and widely used in lab research. But the doubt about the QC6352’s KDM4 mechanism in GBM cells initially sown by the failure of the second, chemically distinct KDM4 inhibitor to replicate the effectiveness of QC6352 (and the clinical candidate zavondemstat) was then further strengthened by genetic evidence. Firstly, the team showed that knocking out KDM4 enzymes in GBM cells using CRISPR technology had no effect on GBM cell growth. And secondly, they demonstrated that GBM cells with KDM4 knocked out retained full sensitivity to QC6352. Thus, KDM4 was ruled out as the crucial target of QC6352 and zavondemstat, at least in brain cancer cells. The search was then on to identify the critical target for these compounds in GBM models. Image: Representation of the 3D X-ray crystal structure of QC6352 bound to an active site ‘pocket’ on the DHODH protein (DB912Y) based on data from Sterling et al Nature Chemical Biology publication. Courtesy of Dr Andreas Kraemer, University of Frankfurt. The surface of QC6352 is shown is pink with the chemical backbone visible inside. Although the team confirmed that QC6352 and zavondemstat are able to inhibit KDM4 enzyme in cell-free biochemical assays, by deploying a comprehensive range of state-of-the-art technologies they eventually discovered that both compounds inhibit DHODH about a thousand-fold more strongly than they block KDM4 – and established beyond reasonable doubt that DHODH is their critical target in GBM cells. The first clue to the crucial target came from the team’s global analysis by RNA sequencing of genes that are switched on or off in GBM cells treated with QC6352. Comparison with publicly-available ‘transcriptome’ data bases revealed the best match to be with known inhibitors of pyrimidine biosynthesis – suggesting the mechanism of action to involve a protein in this pathway. Seeking even greater clarity, the team then used mass spectrometry-based metabolomic profiling to look at the changes in the metabolite pattern of GBM cells treated with QC6352. This again pointed to a block in pyrimidine biosynthesis and in fact to DHODH as the key target – confirmed by demonstrating extremely potent inhibition by QC6352 in enzymatic assays using purified recombinant DHODH. Precise molecular details of how DHODH is so strongly inhibited came from the team solving the 3D X-ray crystal structure of QC6352 and zavondemstat bound to the active site of the DHODH protein. Not content with this already high degree of probability that DHODH is the effective functional target in GBM cells, the team carried out two ‘killer’ studies – known as ‘rescue’ experiments. In the first, they showed that GBM cells could be fully rescued (or protected) from the effects of QC6352 and zavondemstat by providing the metabolite uridine which bypasses and overcomes the blockade of DHODH. This is known as pharmacological or biochemical rescue. In the second killer experiment, the team performed a genetic rescue by inserting into GBM cells a form of DHODH that was mutated to make it resistant to inhibition by QC6352 and zavondemstat. Again, this strongly protected the brain cancer cells against the effects of the two compounds. Taken together, these studies, particularly the genetic knockout and pharmacological and genetic rescue experiments, provide a dossier of gold-standard evidence that firmly nails down DHODH as the crucial functional target of QC6352 and zavondemstat in GBM stem cells. Evidence supporting this mechanism was also obtained in human kidney cells and paediatric neuroblastoma cells, indicating potential for more general relevance beyond brain cancer cells. A clear learning point arising from this research is to consider DHODH as a possible off-target when developing and using KDM4 inhibitors as probes – and also when seeking the targets of compounds found to be active in cell-based phenotypic screens. Using more than one structurally distinct chemical inhibitor when studying the function of a biological target is highly recommended, but there are few high-quality, selective chemical probes available for KDM4. To help address this, the researchers used insights from their work to design new compounds that retain KDM4 inhibitory activity but avoid targeting DHODH. These provide a starting point for developing more selective, next-generation KDM4 chemical probes in the future. The use of state-of-the-art technologies, especially analysing the effects of KDM4 target knockdown and genetic rescue, were especially useful in identifying the critical mechanism of action of QC6352 and zavondemstat in brain cancer cells. The findings underline a general challenge in biomedical research, determining whether the effects of a chemical probe or drug are truly due to action on its nominal intended target – and the approaches described are generally applicable. In addition to cautioning against use of QC6352 as a chemical probe for KDM4 enzymes, the study could also have implications for the clinical development of zavondemstat, a close structural analogue of QC6352 that has entered early clinical trial for advanced cancers. The consequences of inhibiting DHODH would need to be considered in any future development of the drug. Lead author Professor Munzo, Professor of Molecular Cancer Pharmacology at the University of Sydney, said: “By uncovering the true mechanism of QC6352 and its clinical analogue zavondemstat, we’ve gained important new insights for both chemical biology and cancer drug discovery. Our work also provides a foundation for developing improved, more selective probes to study KDM4 enzymes and their role in cancer.” Professor Workman said: “Chemical probes are amazingly powerful and versatile tools for understanding fundamental biology and the molecular changes underpinning cancer and other diseases – but they must be used with great care, even when probes appear to be high-quality and target-selective. One should always be aware of potential limitations, especially off-target effects, and wherever possible design critical experiments to check for these. It’s case of ‘caveat emptor’ – buyer beware! “This study is a powerful example of using multiple approaches to check the properties and potential liabilities of chemical probes. Even widely used ones can have unexpected off-target activities, and understanding, mitigating or eliminating those is essential to draw robust conclusions about cancer biology and identify the best target opportunities for drug discovery.” In response to the new findings, the Chemical Probes Portal will be updating the entries on QC6352 and zavondemstat, highlighting the new data and including a health warning on the use of these compounds as chemical probes for KDM4. The Portal will also be commissioning additional new expert reviews. The Chemical Probes Portal hosted in the Centre for Cancer Drug Discovery is a free public online resource that helps scientists worldwide to identify and use high-quality chemical probes for bioscience research and drug discovery, providing expert recommendations and advice. It recently celebrated its 10th anniversary.