GRIA3

The pharmaceutical giant Lilly, valued at $700 billion, publicly disclosed a WO patent on March 14, 2024. This patent is for a crystalline salt form of an FGFR3 selective inhibitor, as shown in the figure below. It covers the crystalline salt form of a class of pyrimido[4,5-b]pyrazine small molecule compounds. Based on Lilly's strategic development around the FGFR3 target, one of the two compounds illustrated is very likely the structure of LOXO-435. The main difference between the structures of these two compounds is the presence or absence of an F substitution at the meta position of the pyridine segment. Lilly FGFR3 Inhibitor Crystalline PatentIn April 2019, Erdafitinib was approved, becoming the first FGFR selective compound approved by the FDA for second-line treatment of metastatic urothelial carcinoma. It is an effective orally administered pan-FGFR inhibitor, with IC50 values in the low nanomolar range for all members of the FGFR family (FGFR1 to FGFR4). Pemigatinib, developed by Incyte Pharmaceuticals and approved in 2020, is an oral, potent FGFR1-3 selective inhibitor and the first FDA-approved targeted therapy for cholangiocarcinoma. Infigratinib, developed by QED Therapeutic and indicated in clinical trials primarily for chemotherapy-refractory FGFR2 fusion-positive cholangiocarcinoma, is an oral, ATP-competitive, selective FGFR1-3 inhibitor. However, common treatment-related adverse events (TRAEs) for the three approved FGFR inhibitors mentioned above, as seen in clinical trials, include hyperphosphatemia, dry mouth, fatigue, skin changes, nail changes, and ocular disease. Approved FGFR inhibitorsThe development of pan-FGFR inhibitors as well as FGFR1-3 specific inhibitors has been successful; however, off-target side effects have informed the direction of subsequent development. For instance, FGFR1-mediated hyperphosphatemia is a dose-limiting toxicity of pan-FGFR inhibitors; cutaneous/ungual, ocular, and perioral toxicities mediated by FGFR2 lead to chronic intolerance of pan-FGFR inhibitors; domestic and international companies are dedicated to developing highly selective inhibitors targeting precise subtypes of FGFR2, FGFR3, FGFR4 and second-generation FGFR inhibitors that overcome resistance to existing FGFR inhibitors. The Selectivity of FGFR Inhibitors in PatentAccording to the description in patent WO2024054814A1, the crystalline salt form molecule protected by Eli Lilly has been previously disclosed in patents WO/2022/187443 and US2023/0095122A1, which have published the synthesis methods. Examples number 45 and 46 in WO/2022/187443 correspond to two isomers with pyridine carrying a fluoro substituent; example number 151 is the racemate of pyridine without a fluoro substituent. The patent only provides a general range for activity, indicating that selectivity is over 10-fold. Although the selectivity of the compound in the patent is not specified in exact multiples, more biological data are disclosed in the poster. At the 2021 AACR conference, Eli Lilly released preclinical data for LOXO-435, and the Figure below demonstrates that LOXO-435 has strong efficacy and high selectivity against FGFR3 and FGFR3 V555M kinase, while retaining certain activity against FGFR1 and FGFR2. The selectivity for FGFR1 and FGFR2 isoforms reached 361-fold and 66-fold, respectively. Cellular activity data shows that LOXO-435 has an IC50 of 15.1 nM against the FGFR3-TACC3 (RT112) mutant. The selectivity for UMUC-14 over DMS114 reached 374-fold. LOXO435 Enzymatic Activity Data and SelectivityThe design philosophy for the aforementioned FGFR3 selective inhibitor is speculated as follows: Based on the discovery of erdafitinib, initially, a compound with a pyrazole moiety integrated into a quinoline scaffold, Compound 1, was identified from a compound library of VEGFR2 inhibitors. This compound exhibited good selectivity towards FGFR3. By attaching an amino-substituted meta-dimethoxyphenyl ring to the right side of the benzene ring fragment in Compound 1, Compound 2 was obtained. The dimethoxyphenyl ring of Compound 2 occupies the hydrophobic pocket within the ATP-binding site through van der Waals interactions, with one of the methoxy groups forming a hydrogen bond with Asp641. Subsequent optimization of the flexible amino chain led to the development of Erdafitinib. Extending and further optimizing the pyrazole moiety resulted in the red key pharmacophore fragment shown in Figure 5, which ultimately led to the discovery of an FGFR3 selective inhibitor, specifically Compound 91 in the patent CN113490666A. Enzymatic activity assays demonstrated that FGFR3 had selectivity over FGFR1 and FGFR2 by factors of 142 and 9.5, respectively. In cellular activity assays, FGFR3's selectivity over FGFR1 was 22.7-fold. Retaining the pyrazole piperidine linker in the red fragment, introducing a cyano substitution, and derivatizing the methoxy substituent in the parent nucleus resulted in the potential PCC molecule, and substitutions at this position are also likely to enhance selectivity towards other FGFR subtypes. Searching for Eli Lilly's clinical molecule LOXO-435 in the Synapse database yields the following information. This is a public clinical disclosure. The primary objective is to learn more about the safety, side effects, and efficacy of LOXO-435. LOXO-435 can be used for the treatment of cancers of the urinary system cell lineage and other solid tumors with specific genetic alterations, known as FGFR3 gene mutations. This is an open-label, multi-center, Phase 1a/b study, and involves participants with advanced solid tumors that exhibit FGFR3 alterations, including metastatic urothelial carcinoma (UC). The study will be conducted in two phases: dose escalation and dose optimization (1a), and dose expansion (1b). Phase 1a will include up to two cohorts to evaluate the safety, tolerability, and pharmacokinetics of LOXO-435, in order to determine the recommended Phase 2 dose (RP2D) (or the optimal dose). Phase 1b will consist of four dose-expansion cohorts with participants pre-specified for FGFR3 alterations to evaluate the efficacy and safety of LOXO-435 at the RP2D. One of the cohorts will involve the combination treatment of LOXO-435 with Merck's Keytruda monoclonal antibody. FGFRs, comprised of four receptor subtypes (FGFR1 to FGFR4), consist of three distinct structural domains: the extracellular ligand-binding domain, single-pass transmembrane domain, and intracellular tyrosine kinase (TK) domain. Upon binding with fibroblast growth factor (FGF) ligands, the activated FGFR phosphorylates several downstream signaling proteins including PI3K-AKT, RAS-MAPK, and STAT. Therefore, FGFR plays a crucial role in numerous intracellular processes such as development, differentiation, survival, migration, and angiogenesis. However, dysregulation of FGFR can lead to constitutive activation, which is closely associated with the development of several cancers, including FGFR2 alterations in 10 - 16% of intrahepatic cholangiocarcinomas and FGFR3 alterations in 20% of advanced urothelial carcinomas, among others, with additional abnormal subtypes depicted in the classification below the text. We await the disclosure of clinical data for LOXO-435 by Eli Lilly, a selective inhibitor of FGFR3 that potentially avoids the dose-limiting toxicities—such as hyperphosphatemia and other chronic intolerances—associated with inhibiting FGFR1 and FGFR2. There is a significant unmet clinical need for treatments that can offer patients safer therapeutic options. It is hoped that Eli Lilly's potent and selective inhibitor will soon be available on the market, bringing a blessing to those afflicted. Reference: Loxo Oncology；Preclinical characterization of LOXO-435 (LOX-24350), a potent and highly isoform-selective FGFR3 inhibitor; AACR-NCI-EORTC VIRTUAL INTERNATIONAL CONFERENCE Date: October 7, 2021.

看过斯皮尔伯格经典科幻电影《少数派报告》的读者想必对其中“犯罪预测”的设定记忆深刻。然而，在现实中“犯罪”真的可以预测吗？ “犯罪预测”可能太过科幻，“暴力行为”也许真的可以预测！近日，南京大学医学院模式动物研究所石云教授团队在Nature子刊《Molecular Psychiatry》发表了题为《Dysfunction of AMPA receptor GluA3 is associated with aggressive behavior in human》的最新研究论文。研究人员通过人群SNP分析基因敲除小鼠直接证明了GRIA3基因和人类的攻击行为相关，揭示了人类攻击行为新的遗传因素，有助于解决暴力行为精神病药物的开发。对于大多数动物来说，攻击行为是一种具有积极意义的生物学行为。它可以帮助动物在种群中获得有利的支配地位，方便获取资源、领土和繁殖。但是，对于人类来说，攻击行为不仅具有生物学意义，还具有一定的社会意义。不恰当的攻击行为会造成严重损害社会，如暴力犯罪。因此，研究攻击行为具有十分重要的生物学和社会学意义。光遗传学工具和神经解剖学追踪技术的发展让研究人员能够从小鼠模型汇总研究攻击行为背后的神经回路。而大规模测序技术的发展使得研究人员可以从遗传角度对人类攻击行为相关的基因进行检测。其中内侧前额叶皮质（mPFC）病变已有研究会引起人类冲动和攻击行为。而人和小鼠X染色体上编码谷氨酸受体GluA3的GRIA3基因和人与小鼠的攻击行为相关。但是仍然缺乏直接证据来证明GRIA3基因的作用。Fig.1 Mutations in GluA3 serve as a marker for patients with aggressive outbursts.在这项研究中，4个有突发攻击行为的欧洲患者体内携带了两种罕见的GRIA3变异体，因此表现出强烈的攻击行为。其中3个患者的GRIA3基因突变导致GluA3谷氨酸受体发生G630R的氨基酸残基变化，另一个患者的基因突变导致了E787G的变化。通过与GRIA3基因敲除小鼠模型的对比，研究人员确定了GRIA3的功能障碍与攻击行为的直接关系。Fig.2 GluA3 variants disrupt ion channel function.紧接着，对这些突变体进行了功能分析。研究人员对多个GluA3突变体进行了克隆并转染至293T细胞，但是结果表明G630R突变是功能丧失性突变（LoF）。而对E787G突变的功能分析表明也是类似结果。为了分析这些功能丧失的原因，研究人员又通过WesternBlot检测表明功能的缺失是因为离子通道被破坏而不是缺乏表面运输蛋白。Fig.3 The SNP rs3216834 regulates GRIA3 gene expression.前述的研究表明，GluA3 LoF突变可能与人类的攻击行为有关。进一步地，研究人员又探究了遗传因素是否能够影响GluA3表达。因此，研究人员对人群GRIA3基因座中的单核苷酸多态性位点（SNP）进行了分析。结果表明在192个高频SNP中，rs3216834由于容易形成鸟苷酸四联体的单链DNA结构（在人群中大多数人（约78%）带有9个鸟苷酸（9G），少数人带有7G、8G、10G和11G），因此阻滞了mRNA的转录，进一步实验表明10G和11G会严重抑制GluA3的表达。为了进一步确定rs3216834和攻击行为相关的证据。研究人员对294位男性暴力犯罪服刑人员进行了SNP检测，结果发现其中9人携带rs3216834-10G，1人携带11G（3.4%）。而在937名正常受调查人员中仅有2人携带10G（0.2%）。这些结果再次证明rs3216834和攻击行为相关。Fig.4 Behavioral analysis of Gria3Y/-mice最后，研究人员利用GluA3敲除小鼠探究了GluA3功能缺失导致攻击性行为的神经环路机理。经过基因敲除后，小鼠内侧前额叶皮层的神经活动减低，在前额叶皮层将GluA3进行补偿后，显著缓解了小鼠的攻击行为。这些结果表明前额叶皮层异常的神经活动是攻击性行为的关键神经环路机制。综上，本文从临床数据到敲除小鼠模型直接证明了GRIA3突变引起的功能障碍能够导致攻击行为发生，而内侧前额叶皮层（mPFC）的兴奋性神经传递和神经元活动受损是攻击行为发生的主要机制。本文中的“主角”谷氨酸受体（GRIA3）被认为是与侵略相关的最佳候选基因之一。然而谷氨酸却是哺乳动物大脑中主要的兴奋性神经递质受体，在各种正常的神经生理过程中被激活。从这个角度出发，我们也能理解为什么GRIA3的突变会导致人类暴力行为的发生，这可能是由于神经系统的过度兴奋。也许，未来有一天，通过对GRIA3突变的检测，我们可以对暴力攻击型的精神类疾病进行预防和针对性治疗。参考文献：1.Peng, SX.et al. Dysfunction of AMPA receptor GluA3 is associated with aggressive behavior in human. Mol Psychiatry (2022).撰文 | 安小猪编辑 | 小耳朵上万科研人员交流基地，扫码加入“科研交流群”，找到组织，让科研更轻松！