CLPP

来源：药渡撰文：balabala     编辑：维他命靶向蛋白降解技术利用真核细胞内固有的两大蛋白降解系统——泛素-蛋白酶体系统（UPS）和溶酶体途径，实现了多种疾病相关蛋白的靶向降解，正引领着下一代小分子药物的发展方向。近年来，研究人员围绕上述问题积极探索，开发了多维度的靶向蛋白降解技术。本文将盘点从2023年至今的靶向蛋白降解的新型策略，以供读者参考。1基于纳米平台的靶向蛋白降解迄今为止，许多基于邻近效应的双功能分子已经被开发用于靶向调控生物大分子中，如靶向蛋白质泛素化/磷酸化、靶向RNA降解等等。它们在分别接近两个目标分子后将其拉近，从而形成复合物而发挥下游功能。但这些双功能分子往往是一价的，需要经过复杂耗时的筛选和优化。SM-PROTAC纳米平台2023年3月，北京大学深圳研究生院李子刚/尹丰团队开发了一种基于“分裂-混合”的纳米平台，并将其命名为split-and-mixPROTAC(SM-PROTAC)（图1），他们将能够自组装的核心单体分别与PROTAC中的两个组件（靶蛋白招募模块和E3连接酶招募模块）融合，以不同的比例将二者结合，再通过一些表征方法筛选二者的最佳比例，同时该纳米平台的多价性质也保证了目的蛋白的降解效果。之后，研究人员通过对一系列靶点的成功降解，证明了SM-PROTAC可以通过连接目标蛋白配体和E3连接酶配体实现快速的靶标蛋白降解以及配方优化。同时，该平台还具有极强的应用潜力，可以扩展到任何生物分子，包括DNA、RNA、蛋白质和其他生物分子的功能调控，如磷酸化/去磷酸化、泛素化、靶向降解、表观遗传学等。图1. 基于“分裂和混合”的SM-PROTAC模型示意图[1]LipoSM-PROTAC纳米自调节平台但是基于肽的SM-PROTAC的低药效，限制了其在生物医学领域的进一步发展。因此，迫切需要开发一种更有效的用于生物医学应用的PROTAC纳米平台。由于脂质体具有易于配制、自动组装和高负载运输的特性。研究人员将SM-PROTAC与脂质体结合，从而提出了一种基于脂质体的分合（Split-and-Mix）纳米自调节平台(LipoSM-PROTAC)（图2C），并对其进行了生物学表征和验证。研究发现，LipoSM-PROTAC系统具有叶酸选择性降解靶标蛋白的功能。且研究人员以雌激素受体蛋白（ERɑ）为模型，通过体外实验验证发现该体系具有显著降解ERɑ蛋白的效果。与基于肽的SM-PROTAC相比，LipoSM-PROTAC系统可以以更低的浓度达到治疗效果，并为临床转化提供了机会。总体而言，基于LipoSM的平台显示出更高的药物功效，这为PROTAC和其他生物分子调控提供了潜在的应用前景。图2. 基于脂质体的分合(Split-and-Mix)纳米自调节平台(LipoSM-PROTAC)模型示意图[2]2针对特定细胞器内蛋白的靶向蛋白降解目前，真核细胞中所有的靶向蛋白质稳定（TPD）都依赖于泛素蛋白酶体或溶酶体系统，因此对缺乏蛋白酶体和溶酶体的膜细胞器中的靶蛋白（如线粒体）无能为力。杭州医学研究所的方晓红团队设计并开发了一种线粒体蛋白酶靶向嵌合体（MtPTAC），用于实现线粒体基质中蛋白的靶向降解（图3）。MtPTAC是一种双功能小分子，一端与线粒体酪蛋白水解蛋白酶P（ClpP）结合，另一端与靶蛋白结合，MtPTAC激活了ClpP的水解酶活性，同时使目标蛋白与ClpP靠近，进而水解掉靶蛋白。研究人员以线粒体RNA聚合酶（POLRMT）为靶蛋白，在体内和体外均展示了MtPTAC强大的蛋白水解能力和抗肿瘤应用前景。这是第一个模块化设计的能够特异性水解线粒体内靶蛋白的TPD。图3. 线粒体蛋白酶靶向嵌合体(MtPTAC)模型示意图[3]3可逆的蛋白靶向降解嵌合体对于传统的PROTAC分子而言，其使用的过程中往往会表现出钩状效应，即当PROTAC分子的浓度达到某一特定值后，进一步增加其浓度反而会降低靶蛋白的降解效率，而这一效应也在一定程度上限制了PROTAC分子在活体和临床研究中的应用。基于这一背景，佛罗里达大学的Thomas Kodadek教授和来自康奈尔大学的Francis Barany教授团队提出了一种自组装蛋白降解靶向嵌合体（SAPTAC）的设计策略（图4），将PROTAC分子拆分为与靶蛋白和E3泛素连接酶结合的两部分（用于实现可逆耦合的“X和Y”），如果在平衡状态下，存在大量未连接的目标蛋白和E3连接酶配体，这些低分子量分子可以比稳定连接的PROTAC更有效地渗透细胞/组织。在渗透细胞后，目标蛋白和E3连接酶之间将形成共价可逆结构，引向关键的三元复合物，触发目标蛋白的多泛素化和随后的蛋白酶体介导的破坏，从而实现消除钩状效应。图4. 自组装蛋白降解靶向嵌合体(SAPTAC)模型示意图[4]4基于非传统降解途径的靶向蛋白质降解在哺乳动物受到多种刺激后，可激活早期基因（IEG），IEG介导生长因子、神经元和免疫刺激的转录反应，编码Fos、EGR和NR4A家族的转录因子，促进迟发反应基因（LRG）的转表达，介导细胞对刺激作出响应。而LRG具有细胞反应特异性，在对首次刺激的适应性反应中至关重要，IEG mRNA在首次刺激后的几分钟内积累，一旦翻译，它们的蛋白质会迅速降解，从而出现蛋白质瞬时爆发式表达，促使细胞对各种刺激产生适当的反应。尽管IEG的转录机制得到了很好的研究，但IEG蛋白的降解机制仍然是个谜。为了表征IEG蛋白的降解机制，Michael E. Greenberg和Stephen J. Elledge团队通过全局蛋白质稳定性分析（GPS）系统来测定IEG蛋白质的稳定性，并进行全基因组CRISPR-Cas9筛选，以寻找那些调节IEG蛋白稳定性的基因（图2）。他们发现，midnolin蛋白是哺乳动物中一种未被充分表征的蛋白质，它的过表达促进c-Fos、FosB、EGR1和NR4A1等多种IEG蛋白的蛋白酶体降解，抑制蛋白酶体的活性而不是抑制泛素化酶的活性可抑制IEG蛋白降解，表明IEG蛋白降解依赖于midnolin和蛋白酶体，而不是泛素化降解途径。midnolin-蛋白酶体途径可能代表了蛋白酶体绕过经典泛素化途径，以实现核蛋白选择性降解的一般机制。图5. midnolin-蛋白酶体途径独立于泛素化降解许多核蛋白[5]O-糖基化是真核生物中最普遍的糖基化形式。在多种类型的O-糖基化蛋白中，最突出的是黏蛋白（mucins），它主要由上皮细胞分泌，在一系列类型的肿瘤细胞中表达，并通过调节蛋白稳定性诱导各种致癌信号分子促进肿瘤进展。黏蛋白酶StcE是一种选择性切割黏蛋白的蛋白酶。StcE可以选择性地从生物样品中去除黏液结构域糖蛋白，并将其切割成片段，便于分析。StcE对黏蛋白表现出基于肽和聚糖的选择性。斯坦福大学的Carolyn R. Bertozzi教授团队通过纳米抗体将StcE酶靶向到癌细胞表面，并通过酶工程化改造降低StcE酶的酶活性，从而仅在癌细胞表面富集的情况下完成降解过程，避免误伤健康细胞的黏蛋白，具有肿瘤治疗的前景（图6）。图6. ColabFold (Methods)62预测的纳米体-黏液酶偶联物的结构[6]5通过分子内二价胶进行靶向降解IBG1是专利报道的一个类PROTAC的降解剂，其是由BET抑制剂JQ1和DCAF15的芳基磺酰胺配体E7820连接而成，能够在多个细胞系中诱导BRD4的高效降解。实验结果表明，IBG1诱导的BRD4降解依赖于蛋白酶体途径，但与DCAF15无关。英国邓迪大学的Ciulli团队与奥地利科学院CeMM分子医学研究中心的Winter团队通过研究IBG1分子对BRD4的靶向降解机制，揭示了IBG1是一种分子内二价胶水，并发现了一种新型的靶蛋白降解模式，即小分子同时顺式（cis）结合靶蛋白的两个相邻结构域，靶蛋白构象的变化使其粘连到E3连接酶上，与一般的PROTAC等反式（trans）蛋白-连接酶结合有着本质的区别。这种两个结构域的分子内二聚化修饰蛋白质表面并调节蛋白-蛋白相互作用的方法，是一种高效靶向蛋白质降解的新策略（图7），为药理学的发展提供了新的设计方向。图7. 传统的一价胶和二价PROTACs与分子内二价胶分子识别模式的示意图模型[7]参考文献：1.Targeted Biomolecule Regulation Platform: A Split-and-Mix PROTAC Approach2.Selective Protein of Interest Degradation through the Split-and-Mix Liposome Proteolysis Targeting Chimera Approach3.Mitochondrial Protease Targeting Chimeras for Mitochondrial Matrix Protein Degradation4.Reversible Assembly of Proteolysis Targeting Chimeras5.The midnolin-proteasome pathway catches proteins for ubiquitination-independent degradation6.Design of a mucin-selective protease for targeted degradation of cancer-associated mucins7.Targeted protein degradation via intramolecular bivalent glues*声明：本文仅是介绍医药疾病领域研究进展或简述研究概况或分享医药相关讯息，并非也不会进行治疗或诊断方案推荐，也不对相关投资构成任何建议。内容如有疏漏，欢迎沟通指出！利润飙升408.11%！微芯生物“两驾马车”驱动，研发之路高歌猛进这十款药物，今年有望获FDA批准！舒沃哲上市四个月卖9128 万，迪哲医药做对了什么？

弥漫性中线神经胶质瘤（DMG）是一种极其难治的脑瘤，属于最严重的4级胶质瘤，且主要发病于儿童，诊断后平均预期寿命仅为10个月，5年生存率不到1%。此前受到广泛报道的郑州女孩心蕊就是罹患这一疾病而过世。由于主要发病于患者三脑室、丘脑、脑干、脊髓等位置，手术治疗很难达成。放疗是目前的主要手段，化疗带来的获益似乎有限。然而即便如此，复发实际上也是不可避免的，近年来也没有其他系统性疗法被证明是有效的，仅有几项初步临床展现出了一线可能的希望。本文将对目前可能有希望的疗法进行简单总结。ONC-201ONC-201是Chimerix公司的一款药物，本身是一种多巴胺受体D2/D3抑制剂和线粒体酪蛋白水解蛋白酶P（CIpP）变构激动剂。因为多巴胺受体抑制剂的性质，最初是打算将用于精神相关疾病的，但研究人员发现这种小分子还可以穿透血脑屏障，因此他们就将其用于脑瘤开发。适应症也因此选择了脑瘤中占据不小比例的胶质母细胞瘤（GBM）作为适应症，但临床最终失败，并没能改善患者的无进展生存期（PFS）。好在天无绝人之路。在这项试验的两个GBM组中，四名偶然入选的H3K27M突变患者中有两名表现出多处病变的持续消退。后续的多项在H3K27M突变患者中的临床轶事和肿瘤消退也证明了其疗效。这就使得研究人员将注意力转移到H3K27M突变上。而刚好H3K27M突变DMG肿瘤细胞侵袭性更高，具有更差的预后。因此研究人员就将适应症开发集中在了H3K27M突变DMG上。而DMG上的临床开发也并没有辜负他们的期待，ONC201单药治疗H3K27M-DMG患者展现出相当有希望的疗效，在非复发患者中已经超过了化疗的金标准——ONC201治疗患者的诊断中位OS为21.7月，而外部化疗对照组仅为12个月。近期，发表于Journal of Clinical Oncology为ONC-201补充了在复发H3K27M-DMG患者的初步疗效，其机制此前也得到了阐述。ONC-201作为多巴胺受体抑制剂之外的额外作用，它可以过度激活ClpP，导致线粒体蛋白质组成分的选择性降解，并随后激活整合的应激反应和细胞凋亡，最终造成线粒体降解来实现靶向H3K27M突变蛋白质的方式诱导肿瘤细胞凋亡。GD2-CAR-TGD2-CAR-T同样是H3K27M突变DMG的可能解决方案，二硫根神经节苷脂（GD2）是一种二唾液酸神经节苷脂，主要在神经母细胞瘤、黑素瘤、骨肉瘤、肺癌和乳腺癌等肿瘤中高表达。通过调控MAPK、PI3K/Akt和FAK/paxillin等信号通路，能够促进肿瘤细胞增殖、迁移，维持肿瘤干性，以及促进化疗耐药等。而这种蛋白同时高表达于H3K27M突变的癌细胞，所以GD2-CAR-T也可以用于针对这种疾病，不过与ONC-201不同的是，这毕竟是一种CAR-T，脑内用药副作用和风险有些大。因此目前的临床主要用于儿童和青壮年脑桥和脊髓DMG患者，而丘脑或小脑肿瘤患者就会被排除入组，因为这些位置过于关键，具有极大风险。2022年披露的一项临床结果表明，4例患者中有3例表现出临床和影像学改善，患者神经细胞有所改善。该研究主要展现了临床转化过程中应对炎症等CAR-T副作用的相关处理方式。目前仍然在伦敦大学和斯坦福大学等几个机构中探索最佳剂量、途径和方案，并确定GD2-CAR T细胞疗法在H3K27M + DMG患者中的疗效。总结总的来说，目前较为有希望的治疗方案主要是ONC-201和GD2-CAR-T，其他治疗方案如溶瘤病毒，ADC，BBB穿梭肽，TILs，抗体等方式大多位于临床早期或临床前研究。在我国人口基数巨大的情况下，可以期待该领域药物的开发能够为患者带来更多机遇。参考来源：1.Venneti S, Kawakibi AR, Ji S, Waszak SM, Sweha SR, Mota M, Pun M, Deogharkar A, Chung C, Tarapore RS, Ramage S, Chi A, Wen PY, Arrillaga-Romany I, Batchelor TT, Butowski NA, Sumrall A, Shonka N, Harrison RA, de Groot J, Mehta M, Hall MD, Daghistani D, Cloughesy TF, Ellingson BM, Beccaria K, Varlet P, Kim MM, Umemura Y, Garton H, Franson A, Schwartz J, Jain R, Kachman M, Baum H, Burant CF, Mottl SL, Cartaxo RT, John V, Messinger D, Qin T, Peterson E, Sajjakulnukit P, Ravi K, Waugh A, Walling D, Ding Y, Xia Z, Schwendeman A, Hawes D, Yang F, Judkins AR, Wahl D, Lyssiotis CA, de la Nava D, Alonso MM, Eze A, Spitzer J, Schmidt SV, Duchatel RJ, Dun MD, Cain JE, Jiang L, Stopka SA, Baquer G, Regan MS, Filbin MG, Agar NYR, Zhao L, Kumar-Sinha C, Mody R, Chinnaiyan A, Kurokawa R, Pratt D, Yadav VN, Grill J, Kline C, Mueller S, Resnick A, Nazarian J, Allen JE, Odia Y, Gardner SL, Koschmann C. Clinical Efficacy of ONC201 in H3K27M-Mutant Diffuse Midline Gliomas Is Driven by Disruption of Integrated Metabolic and Epigenetic Pathways. Cancer Discov. 2023 Nov 1;13(11):2370-2393. doi: 10.1158/2159-8290.CD-23-0131.2.Isabel Arrillaga-Romany et al, ONC201 (Dordaviprone) in Recurrent H3 K27M–Mutant Diffuse Midline Glioma, Journal of Clinical Oncology (2024). DOI: 10.1200/JCO.23.011343.Miguel Llordes G, Medina Pérez VM, Curto Simón B, Castells-Yus I, Vázquez Sufuentes S, Schuhmacher AJ. Epidemiology, Diagnostic Strategies, and Therapeutic Advances in Diffuse Midline Glioma. J Clin Med. 2023 Aug 12;12(16):5261. doi: 10.3390/jcm12165261.

– ONC201 ACTION Study Progressing; Reiterate Interim OS Data Expected in 2025, Final OS Data Expected in 2026 – – Phase 2 ONC201 Data Published in Peer-Reviewed Journal of Clinical Oncology – – $204 Million in Cash and Equivalents at December 31, 2023 – – Conference Call at 8:30 a.m. ET Today – DURHAM, N.C., Feb. 29, 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 fourth quarter and full-year ended December 31, 2023 and provided an operational update. “Following strong clinical development in 2023, we remain very focused on advancing the ONC201 ACTION study, completing ONC206 dose escalation this year and strengthening our executive team as we prepare for potential commercialization of ONC201,” said Mike Andriole, Chief Executive Officer of Chimerix. “We are making good progress enrolling our global Phase 3 ACTION study and are excited about the prospect of having interim overall survival data next year. In addition, we are pleased to share that the ONC201 Phase 2 data was recently published in the Journal of Clinical Oncology which further elucidates key characteristics of response and detailed patient-level data.” “During the fourth quarter, we were delighted to strengthen our Board of Directors with the addition of Lisa Decker, Ph.D., as well as strengthen the management team with the promotion of Michelle LaSpaluto to Chief Financial Officer and the additions of Tom Riga as Chief Operating and Commercial Officer and Pablo Lee, MD, as Vice President of Medical Affairs. We are confident their collective expertise will be invaluable assets to Chimerix as we seek to maximize our future growth potential for patients and shareholders,” added Mr. Andriole. ONC201 Journal of Clinical Oncology Publication In February 2024, “ONC201 (dordaviprone) in Recurrent H3 K27M-mutant Diffuse Midline Glioma,” was published in the Journal of Clinical Oncology (JCO), a peer reviewed journal of the American Society of Clinical Oncology (ASCO). The manuscript reports in detail the results of 50 patients with recurrent H3 K27M-DMG treated with monotherapy ONC201 who were evaluable for objective response by Response Assessment in Neuro-Oncology (RANO) high grade glioma (HGG) criteria. ONC201 demonstrated a median overall survival (mOS) of 13.7 months (95% CI: 8.0 - 20.3), with an overall two-year rate of survival of 35% (95% CI: 21-49) from the start of ONC201 treatment post-recurrence. Chimerix previously conducted a natural disease history study (n=43) in the recurrent setting evaluating patients who did not receive ONC201 which showed a mOS of 5.1 months (95% CI: 3.9 - 7.7) with an overall two-year survival rate of 11% (95% CI:3.3-24.2).The top-line data from this JCO publication were previously disclosed by Chimerix. The journal can be accessed here. The Phase 3 ACTION trial is currently enrolling patients at over 130 sites in 13 countries. The trial enrolls patients shortly after completion of front-line radiation therapy that is the standard of care. The study is designed to enroll 450 patients randomized 1:1:1 to receive ONC201 at one of two dosing frequencies or placebo. Participants are randomized to receive 625mg of ONC201 once per week (the Phase 2 dosing regimen), 625mg twice per week on two consecutive days or placebo. The dose will be scaled by body weight for patients <52.5kg. For more information, please visit clinicaltrials.gov ONC206 ONC206 is a second generation ClpP agonist and DRD2 antagonist that has demonstrated monotherapy anti-cancer activity in pre-clinical models in primary CNS tumors and solid tumors outside of the CNS. Phase I dose escalation trials continue at the National Institutes of Health (NIH) and the Pacific Pediatric Neuro-Oncology Consortium (PNOC) in adult and pediatric CNS tumor patients, respectively. To date, ONC206 has been generally well tolerated with no dose limiting toxicities. The dose escalation trials are currently dosing at more frequent dose schedules, which are expected to increase the duration of therapeutic exposure. Chimerix expects to report preliminary safety and pharmacokinetic data from these trials beginning in mid-2024. Fourth Quarter 2023 Financial Results Chimerix’s balance sheet at December 31, 2023 included $204.5 million of capital available to fund operations, no debt, and approximately 88.9 million outstanding shares of common stock. Chimerix reported a net loss of $18.2 million, or $0.20 per basic and diluted share, for the fourth quarter of 2023, compared to a net loss of $21.0 million, or $0.24 per basic and diluted share for the fourth quarter of 2022. Research and development expenses decreased to $15.6 million for the three-month period ended December 31, 2023, compared to $19.3 million for the same period in 2022. This decrease was primarily driven by one-time costs associated with a reduction in force related to the TEMBEXA divestiture in the comparable 2022 period. General and administrative expenses decreased to $5.2 million for the fourth quarter of 2023, compared to $5.3 million for the same period in 2022. Full Year 2023 Financial Results Chimerix reported a net loss of $82.1 million, or $0.93 per basic and diluted share, for the year ended December 31, 2023. For the year ended December 31, 2022, Chimerix recorded net income of $172.2 million, or $1.97 per basic and $1.94 per diluted share. The decrease was primarily driven by the gain on sale of TEMBEXA to Emergent BioSolutions in 2022. Revenues for 2023 decreased to $0.3 million, compared to $33.8 million in 2022. The decrease was primarily related to deliveries under international TEMBEXA procurement agreements in the comparable 2022 period. Research and development expenses decreased to $68.8 million for the year ended December 31, 2023, compared to $71.6 million for the year ended December 31, 2022. General and administrative expenses increased to $24.6 million for the year ended December 31, 2023, compared to $22.1 million for the year ended December 31, 2022. Conference Call and Webcast Chimerix will host a conference call and live audio webcast to discuss fourth quarter and full-year 2023 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 6933453. 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, ONC201, is in development for H3 K27M-mutant glioma. 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, enrollment and timing of data for the Phase 3 ACTION study, the results of dose escalation trials of ONC206, and the impact of recent changes to the Board of Directors and management team. 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 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) December 31, December 31, 2023 2022 ASSETS Current assets: Cash and cash equivalents$27,661 $25,842 Short-term investments, available-for-sale 155,174 191,492 Accounts receivable 4 1,040 Prepaid expenses and other current assets 6,271 9,764 Total current assets 189,110 228,138 Long-term investments 21,657 48,626 Property and equipment, net of accumulated depreciation 224 227 Operating lease right-of-use assets 1,482 1,964 Other long-term assets 301 386 Total assets$212,774 $279,341 LIABILITIES AND STOCKHOLDERS' EQUITY Current liabilities: Accounts payable$2,851 $3,034 Accrued liabilities 15,592 17,381 Total current liabilities 18,443 20,415 Loan Fees 125 250 Lease-related obligations 1,177 1,819 Total liabilities 19,745 22,484 Stockholders’ equity: Preferred stock, $0.001 par value, 10,000,000 shares authorized at December 31, 2023 and 2022; no shares issued and outstanding as of December 31, 2023 and 2022 - - Common stock, $0.001 par value, 200,000,000 shares authorized at December 31, 2023 and 2022; 88,929,300 and 88,054,127 shares issued and outstanding as of December 31, 2023 and 2022, respectively 89 88 Additional paid-in capital 988,457 970,535 Accumulated other comprehensive gain (loss), net 7 (337) Accumulated deficit (795,524) (713,429) Total stockholders’ equity 193,029 256,857 Total liabilities and stockholders’ equity$212,774 $279,341 CHIMERIX, INC.CONSOLIDATED STATEMENTS OF OPERATIONS AND COMPREHENSIVE (LOSS) INCOME(in thousands, except share and per share data) Three Months Ended December 31, Years Ended December 31, 2023 2022 2023 2022 Revenues: Procurement revenue$- $- $- $31,971 Contract and grant revenue 4 439 275 942 Licensing revenue - - 49 536 Royalty revenue - 375 - 375 Total revenues 4 814 324 33,824 Cost of goods sold - - - 447 Gross Profit 4 814 324 33,377 Operating expenses: Research and development 15,644 19,281 68,788 71,631 General and administrative 5,170 5,347 24,601 22,132 Total operating expenses 20,814 24,628 93,389 93,763 Loss from operations (20,810) (23,814) (93,065) (60,386)Other income: Interest income and other, net 2,649 2,737 10,970 2,919 Gain on sale of business, net - - - 229,670 (Loss) income before income taxes (18,161) (21,077) (82,095) 172,203 Income tax expense - (117) - 36 Net (loss) income (18,161) (20,960) (82,095) 172,167 Other comprehensive income (loss): Unrealized income (loss) on investments, net 632 (300) 344 (316) Comprehensive (loss) income$(17,529) $(21,260) $(81,751) $171,851 Per share information: Net (loss) income, basic$(0.20) $(0.24) $(0.93) $1.97 Net (loss) income, diluted$(0.20) $(0.24) $(0.93) $1.94 Weighted-average shares outstanding, basic 88,910,300 88,049,138 88,604,026 87,555,110 Weighted-average shares outstanding, diluted 88,910,300 88,049,138 88,604,026 88,776,147