一种新型 HCK 和 BTK 双抑制剂 Kin-8194 在 MYD88 突变 B 细胞淋巴瘤中显示出优于依鲁替尼的活性并克服了 BTKC481S 介导的依鲁替尼体外和体内耐药性,Blood

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一种新型 HCK 和 BTK 双抑制剂 Kin-8194 在 MYD88 突变 B 细胞淋巴瘤中显示出优于依鲁替尼的活性并克服了 BTKC481S 介导的依鲁替尼体外和体内耐药性
Blood ( IF 21.0 ) Pub Date : 2019-11-13 , DOI: 10.1182/blood-2019-130636
Guang Yang _{1,

2} , Jinhua Wang ₃ , Xia Liu ₄ , Manit Munshi ₄ , Jiaji G Chen ₄ , Amanda Kofides ₄ , Lian Xu ₄ , Nicholas Tsakmaklis ₄ , Maria Demos ₄ , Maria Luisa Guerrera ₄ , Gloria G Chan ₄ , Cristina Jimenez ₄ , Zachary R Hunter _{2,

4} , Christopher Patterson ₄ , Jorge J. Castillo _{2,

5} , Sara J Buhrlage ₃ , Nathanael Gray ₆ , Steven P Treon _{2,

4}

Affiliation

MYD88 中的激活突变通过包括 BTK 和 HCK 在内的多种途径促进恶性细胞的生长和存活。HCK 被突变的 MYD88 转录上调和激活，进而激活 BTK 本身以及 ERK 和 AKT。Ibrutinib 是一种与 BTKCys481 结合的共价抑制剂，在 MYD88 突变的 B 细胞恶性肿瘤（包括 WM、MZL、ABC DLBCL 和 PCNSL）中显示出活性。基于 BTKCys481 以及下游突变对依鲁替尼的耐药性越来越被认可。因此，我们寻求开发针对 HCK 的有效和选择性抑制剂。我们开发了 HCK 和 BTK 的非共价双重抑制剂 KIN-8194，经过筛选 > 220 个临床和临床前激酶抑制剂，并在合成 > 400 个类似物后进行了先导优化。KINOMEscan® 和活细胞靶标参与研究（KiNativ™ 分析和活细胞 ATP-生物素竞争测定）均证实了 HCK 和 BTK 双重抑制。在这些研究中，KIN-8194 显示出对 HCK (IC50<0.495nM) 和 BTK (IC50=0.915nM) 的强烈抑制，并在野生型和突变的 BTKCys481 WM 和 ABC DLBCL 细胞以及原代 WM 细胞中阻断了 pHCK 和 pBTK。重要的是，KIN-8194 显示选择性杀死 MYD88 突变的 WM 和 ABC DLBCL 细胞，并克服了 WM 和 ABC DLBCL 细胞对依鲁替尼的耐药性，这些细胞被设计用于表达突变的 BTKCys481。KIN-8194 在包括人类在内的多个物种中表现出优异的微粒体稳定性（T1/2=49.5 分钟）。小鼠的药代动力学研究显示出优异的生物利用度 (F=55%)、血清半衰期 (T1/2=15.1 小时) 和药物清除率 (CL=17. 4mL/min/kg) 适合每日一次口服给药。这些化合物表现出出色的体外安全性，包括对包括 hERG 在内的 100 种其他受体靶标没有观察到相关抑制，AMES 在高达 50 µM 时为阴性，而 Cyp 抑制研究显示可接受的抑制高达 10 µM。口服给药后的药效学研究表明，KIN-8194 阻断了植入 NOD SCID 小鼠的野生型和突变 BTKCys481 表达 TMD8 ABC DLBCL 细胞的 pHCK 和 pBTK。这些小鼠对高达 100 mg/kg 的连续给药耐受性良好。KIN-8194 处理的 NOD SCID 小鼠与野生型 (A) 或突变 (B) BTKCys481 TMD 细胞（N = 8/组）相比，在 50 mg/kg 时显示出优于载体对照或依鲁替尼处理的小鼠的肿瘤生长抑制和存活率. 在治疗 6 周的野生型 BTKCys481 TMD8 异种移植小鼠中，在另外 12 周的观察后，一半小鼠观察到肿瘤消除，随后没有生长，这与治愈一致。因此，我们描述了一种新型、高效的非共价 HCK 和 BTK 双重抑制剂，该抑制剂在小鼠中具有良好的耐受性，可选择性杀死 MYD88 突变的 WM 和 ABC DLBCL 细胞，并且可以克服突变的 BTKCys481 相关的依鲁替尼耐药性。披露猎人：杨森：咨询。Castillo:Beigene：咨询、研究经费；TG Therapeutics：研究经费；艾伯维：研究经费；杨森：咨询、研究经费；Pharmacyclics：咨询，研究经费。灰色：Gatekeeper、Syros、Petra、C4、B2S 和 Soltego。：股权；诺华、武田、Astellas、Taiho、Janssen、Kinogen、Voronoi、Her2llc、Deerfield 和赛诺菲：股权所有权、研究经费。Treon：Pharmacyclics：研究经费；BMS：研究经费；杨森：咨询。

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A Novel HCK and BTK Dual Inhibitor Kin-8194 Shows Superior Activity over Ibrutinib and Overcomes BTKC481S Mediated Ibrutinib Resistance in Vitro and In Vivo in MYD88 Mutated B-Cell Lymphomas

Activating mutations in MYD88 promote malignant cell growth and survival through multiple pathways that include BTK and HCK. HCK is transcriptionally upregulated and activated by mutated MYD88 and in turn activates BTK itself, as well as ERK and AKT. Ibrutinib is a covalent inhibitor that binds to BTKCys481 and shows activity in MYD88 mutated B-cell malignancies, including WM, MZL, ABC DLBCL, and PCNSL. Resistance to ibrutinib on the basis of BTKCys481 as well as downstream mutations is increasingly being recognized. We therefore sought to develop potent and selective inhibitors that target HCK. We developed a non-covalent dual inhibitor, KIN-8194, of HCK and BTK following a screen >220 clinical and preclinical kinase inhibitors, and lead optimization following synthesis >400 analogs. Dual HCK and BTK inhibition was confirmed by both KINOMEscan® and live-cell target engagement studies (KiNativ™ profiling and live cell ATP-biotin competition assay). KIN-8194 showed robust suppression of HCK (IC50<0.495nM) and BTK (IC50=0.915nM) in these studies, and blocked pHCK and pBTK in both wild-type and mutated BTKCys481 WM and ABC DLBCL cells, and primary WM cells. Importantly, KIN-8194 showed selective killing of MYD88 mutated WM and ABC DLBCL cells and overcame resistance to ibrutinib in WM and ABC DLBCL cells engineered to express mutated BTKCys481. KIN-8194 showed excellent microsomal stability across multiple species including human (T1/2=49.5 minutes). Pharmacokinetic studies in mice showed excellent bioavailability (F=55%), serum half-life (T1/2=15.1 hours) and drug clearance (CL=17.4mL/min/kg) amenable to once daily oral dosing. The compounds exhibited an excellent in vitro safety profiling including no relevant inhibition observed against a panel of 100 other receptor targets, including hERG, AMES was negative up to 50 µM, and Cyp inhibition studies showed acceptable inhibition up to 10 µM. Pharmacodynamic studies following oral administration showed that KIN-8194 blocked both pHCK and pBTK in wild-type and mutated BTKCys481 expressing TMD8 ABC DLBCL cells engrafted in NOD SCID mice. Continuous dosing up to 100 mg/kg was well tolerated in these mice. KIN-8194 treated NOD SCID mice xenografted with either wild-type (A) or mutated (B) BTKCys481 TMD cells (N=8/cohort) showed superior tumor growth suppression and survival over vehicle control or ibrutinib treated mice at 50 mg/kg. Among wild-type BTKCys481 TMD8 xenografted mice treated for 6 weeks, elimination of tumor was observed in half the mice with no subsequent growth following 12 additional weeks of observation, consistent with a cure. We therefore describe a novel, highly potent non-covalent dual HCK and BTK inhibitor that is well tolerated in mice, shows selective killing of MYD88 mutated WM and ABC DLBCL cells, and can overcome mutated BTKCys481 related ibrutinib resistance. Disclosures Hunter: Janssen: Consultancy. Castillo:Beigene: Consultancy, Research Funding; TG Therapeutics: Research Funding; Abbvie: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Treon:Pharmacyclics: Research Funding; BMS: Research Funding; Janssen: Consultancy.

更新日期：2019-11-13

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