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Contribution of Noncovalent Recognition and Reactivity to the Optimization of Covalent Inhibitors: A Case Study on KRasG12C. 非共价识别和反应性对优化共价抑制剂的贡献:KRasG12C 案例研究。
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-11 DOI: 10.1021/acschembio.4c00217
Nikolett Péczka, Ivan Ranđelović, Zoltán Orgován, Noémi Csorba, Attila Egyed, László Petri, Péter Ábrányi-Balogh, Márton Gadanecz, András Perczel, József Tóvári, Gitta Schlosser, Tamás Takács, Levente M Mihalovits, György G Ferenczy, László Buday, György M Keserű

Covalent drugs might bear electrophiles to chemically modify their targets and have the potential to target previously undruggable proteins with high potency. Covalent binding of drug-size molecules includes a noncovalent recognition provided by secondary interactions and a chemical reaction leading to covalent complex formation. Optimization of their covalent mechanism of action should involve both types of interactions. Noncovalent and covalent binding steps can be characterized by an equilibrium dissociation constant (KI) and a reaction rate constant (kinact), respectively, and they are affected by both the warhead and the scaffold of the ligand. The relative contribution of these two steps was investigated on a prototypic drug target KRASG12C, an oncogenic mutant of KRAS. We used a synthetically more accessible nonchiral core derived from ARS-1620 that was equipped with four different warheads and a previously described KRAS-specific basic side chain. Combining these structural changes, we have synthesized novel covalent KRASG12C inhibitors and tested their binding and biological effect on KRASG12C by various biophysical and biochemical assays. These data allowed us to dissect the effect of scaffold and warhead on the noncovalent and covalent binding event. Our results revealed that the atropisomeric core of ARS-1620 is not indispensable for KRASG12C inhibition, the basic side chain has little effect on either binding step, and warheads affect the covalent reactivity but not the noncovalent binding. This type of analysis helps identify structural determinants of efficient covalent inhibition and may find use in the design of covalent agents.

共价药物可能带有亲电体,对其靶标进行化学修饰,并有可能以高强度靶向以前无法药物治疗的蛋白质。药物分子的共价结合包括由次级相互作用提供的非共价识别和导致共价复合物形成的化学反应。优化药物的共价作用机制应同时涉及这两种类型的相互作用。非共价和共价结合步骤可分别用平衡解离常数(KI)和反应速率常数(kinact)来表征,它们同时受到配体的弹头和支架的影响。我们以 KRAS 的致癌突变体 KRASG12C 为原型药物靶点,研究了这两个步骤的相对贡献。我们使用了源自 ARS-1620 的合成上更容易获得的非手性核心,该核心配备了四种不同的弹头和之前描述过的 KRAS 特异性基本侧链。结合这些结构变化,我们合成了新型共价 KRASG12C 抑制剂,并通过各种生物物理和生物化学实验测试了它们与 KRASG12C 的结合情况和生物效应。这些数据使我们得以剖析支架和弹头对非共价和共价结合事件的影响。我们的研究结果表明,ARS-1620 的异构体核心对 KRASG12C 的抑制作用并非不可或缺,基本侧链对两个结合步骤的影响都很小,而弹头会影响共价反应性,但不会影响非共价结合。这类分析有助于确定高效共价抑制的结构决定因素,并可用于设计共价制剂。
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引用次数: 0
Quantitative Measurement of Rate of Targeted Protein Degradation. 定量测量目标蛋白质降解率
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-09 DOI: 10.1021/acschembio.4c00262
Thomas L Lynch, Violeta L Marin, Ryan A McClure, Colin Phipps, Judith A Ronau, Milad Rouhimoghadam, Ashley M Adams, Soumya Kandi, Malerie L Wolke, Andrea G Shergalis, Gregory K Potts, Omprakash Nacham, Paul Richardson, Stephan J Kakavas, Gekleng Chhor, Gary J Jenkins, Kevin R Woller, Scott E Warder, Anil Vasudevan, Justin M Reitsma

Targeted protein degradation (TPD) is a therapeutic approach that leverages the cell's natural machinery to degrade targets instead of inhibiting them. This is accomplished by using mono- or bifunctional small molecules designed to induce the proximity of target proteins and E3 ubiquitin ligases, leading to ubiquitination and subsequent proteasome-dependent degradation of the target. One of the most significant attributes of the TPD approach is its proposed catalytic mechanism of action, which permits substoichiometric exposure to achieve the desired pharmacological effects. However, apart from one in vitro study, studies supporting the catalytic mechanism of degraders are largely inferred based on potency. A more comprehensive understanding of the degrader catalytic mechanism of action can help aspects of compound development. To address this knowledge gap, we developed a workflow for the quantitative measurement of the catalytic rate of degraders in cells. Comparing a selective and promiscuous BTK degrader, we demonstrate that both compounds function as efficient catalysts of BTK degradation, with the promiscuous degrader exhibiting faster rates due to its ability to induce more favorable ternary complexes. By leveraging computational modeling, we show that the catalytic rate is highly dynamic as the target is depleted from cells. Further investigation of the promiscuous kinase degrader revealed that the catalytic rate is a better predictor of optimal degrader activity toward a specific target compared to degradation magnitude alone. In summary, we present a versatile method for mapping the catalytic activity of any degrader for TPD in cells.

靶向蛋白质降解(TPD)是一种治疗方法,它利用细胞的天然机制来降解靶点,而不是抑制它们。具体方法是使用单功能或双功能小分子,诱导靶蛋白与 E3 泛素连接酶接近,导致泛素化,随后靶蛋白酶体依赖性降解。TPD 方法最重要的特性之一是它所提出的催化作用机制,它允许亚计量暴露,以达到所需的药理作用。然而,除了一项体外研究外,支持降解剂催化机制的研究大多是根据药效推断的。更全面地了解降解剂的催化作用机制有助于化合物的开发。为了填补这一知识空白,我们开发了一种定量测量细胞中降解剂催化率的工作流程。通过比较选择性 BTK 降解剂和杂合性 BTK 降解剂,我们证明这两种化合物都是高效的 BTK 降解催化剂,而杂合性降解剂由于能诱导出更有利的三元复合物而表现出更快的催化速率。通过利用计算建模,我们发现随着细胞中目标物的耗竭,催化速率也会发生很大的变化。对杂合激酶降解器的进一步研究表明,与降解幅度本身相比,催化率更能预测降解器对特定靶点的最佳活性。总之,我们提出了一种多功能方法,用于绘制细胞中任何降解剂对 TPD 的催化活性图。
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引用次数: 0
A Repurposed Drug Interferes with Nucleic Acid to Inhibit the Dual Activities of Coronavirus Nsp13. 一种可干扰核酸以抑制冠状病毒 Nsp13 双重活性的重塑药物。
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-09 DOI: 10.1021/acschembio.4c00244
Nathan Soper, Isabelle Yardumian, Eric Chen, Chao Yang, Samantha Ciervo, Aaron L Oom, Ludovic Desvignes, Mark J Mulligan, Yingkai Zhang, Tania J Lupoli

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlighted a critical need to discover more effective antivirals. While therapeutics for SARS-CoV-2 exist, its nonstructural protein 13 (Nsp13) remains a clinically untapped target. Nsp13 is a helicase responsible for unwinding double-stranded RNA during viral replication and is essential for propagation. Like other helicases, Nsp13 has two active sites: a nucleotide binding site that hydrolyzes nucleoside triphosphates (NTPs) and a nucleic acid binding channel that unwinds double-stranded RNA or DNA. Targeting viral helicases with small molecules, as well as the identification of ligand binding pockets, have been ongoing challenges, partly due to the flexible nature of these proteins. Here, we use a virtual screen to identify ligands of Nsp13 from a collection of clinically used drugs. We find that a known ion channel inhibitor, IOWH-032, inhibits the dual ATPase and helicase activities of SARS-CoV-2 Nsp13 at low micromolar concentrations. Kinetic and binding assays, along with computational and mutational analyses, indicate that IOWH-032 interacts with the RNA binding interface, leading to displacement of nucleic acid substrate, but not bound ATP. Evaluation of IOWH-032 with microbial helicases from other superfamilies reveals that it is selective for coronavirus Nsp13. Furthermore, it remains active against mutants representative of observed SARS-CoV-2 variants. Overall, this work provides a new inhibitor for Nsp13 and provides a rationale for a recent observation that IOWH-032 lowers SARS-CoV-2 viral loads in human cells, setting the stage for the discovery of other potent viral helicase modulators.

最近由严重急性呼吸系统综合征冠状病毒 2(SARS-CoV-2)引起的大流行凸显了发现更有效抗病毒药物的迫切需要。虽然已有针对 SARS-CoV-2 的疗法,但其非结构蛋白 13(Nsp13)仍是临床上尚未开发的靶点。Nsp13 是一种螺旋酶,负责在病毒复制过程中解开双链 RNA,对病毒的传播至关重要。与其他螺旋酶一样,Nsp13 也有两个活性位点:一个是核苷酸结合位点,用于水解三磷酸核苷(NTP);另一个是核酸结合通道,用于解开双链 RNA 或 DNA。用小分子靶向病毒螺旋酶以及鉴定配体结合位点一直是个难题,部分原因是这些蛋白的灵活性。在这里,我们利用虚拟筛选技术从一系列临床用药中找出了 Nsp13 的配体。我们发现,已知的离子通道抑制剂 IOWH-032 在低微摩尔浓度下可抑制 SARS-CoV-2 Nsp13 的 ATPase 和螺旋酶双重活性。动力学和结合试验以及计算和突变分析表明,IOWH-032 与 RNA 结合界面相互作用,导致核酸底物位移,但不导致结合 ATP 位移。将 IOWH-032 与其他超家族的微生物螺旋酶进行评估后发现,它对冠状病毒 Nsp13 具有选择性。此外,它对已观察到的 SARS-CoV-2 变异株的突变体仍有活性。总之,这项工作为 Nsp13 提供了一种新的抑制剂,并为最近观察到的 IOWH-032 降低人类细胞中 SARS-CoV-2 病毒载量提供了依据,为发现其他强效病毒螺旋酶调节剂创造了条件。
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引用次数: 0
Glutathione-Based Photoaffinity Probe Identifies Caffeine as a Positive Allosteric Modulator of the Calcium-Sensing Receptor. 基于谷胱甘肽的光亲和探针确定咖啡因是钙传感受体的正性异构调节剂
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-08 DOI: 10.1021/acschembio.4c00335
Nadee N J Matarage Don, Rayavarapu Padmavathi, Talan D Khasro, Md Rumman U Zaman, Hai-Feng Ji, Jeffrey L Ram, Young-Hoon Ahn

The calcium-sensing receptor (CaSR), abundantly expressed in the parathyroid gland and kidney, plays a central role in calcium homeostasis. In addition, CaSR exerts multimodal roles, including inflammation, muscle contraction, and bone remodeling, in other organs and tissues. The diverse functions of CaSR are mediated by many endogenous and exogenous ligands, including calcium, amino acids, glutathione, cinacalcet, and etelcalcetide, that have distinct binding sites in CaSR. However, strategies to evaluate ligand interactions with CaSR remain limited. Here, we developed a glutathione-based photoaffinity probe, DAZ-G, that analyzes ligand binding to CaSR. We showed that DAZ-G binds to the amino acid binding site in CaSR and acts as a positive allosteric modulator of CaSR. Oxidized and reduced glutathione and phenylalanine effectively compete with DAZ-G conjugation to CaSR, while calcium, cinacalcet, and etelcalcetide have cooperative effects. An unexpected finding was that caffeine effectively competes with DAZ-G's conjugation to CaSR and acts as a positive allosteric modulator of CaSR. The effective concentration of caffeine for CaSR activation (<10 μM) is easily attainable in plasma by ordinary caffeine consumption. Our report demonstrates the utility of a new chemical probe for CaSR and discovers a new protein target of caffeine, suggesting that caffeine consumption can modulate the diverse functions of CaSR.

钙传感受体(CaSR)在甲状旁腺和肾脏中大量表达,在钙平衡中发挥着核心作用。此外,CaSR 还在其他器官和组织中发挥多模式作用,包括炎症、肌肉收缩和骨骼重塑。CaSR 的多种功能由许多内源性和外源性配体介导,包括钙、氨基酸、谷胱甘肽、西那卡西酮和依替卡西酮,这些配体在 CaSR 中具有不同的结合位点。然而,评估配体与 CaSR 相互作用的策略仍然有限。在这里,我们开发了一种基于谷胱甘肽的光亲和探针 DAZ-G,用于分析配体与 CaSR 的结合。我们的研究表明,DAZ-G 可与 CaSR 的氨基酸结合位点结合,成为 CaSR 的正异构调节剂。氧化和还原的谷胱甘肽和苯丙氨酸能有效地竞争 DAZ-G 与 CaSR 的结合,而钙、西那卡西酮和依替卡西酮则具有协同作用。一个出乎意料的发现是,咖啡因能有效地竞争 DAZ-G 与 CaSR 的结合,并充当 CaSR 的正异构调节剂。咖啡因激活 CaSR 的有效浓度 (
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引用次数: 0
ALTering Cancer by Triggering Telomere Replication Stress through the Stabilization of Promoter G-Quadruplex in SMARCAL1. 通过稳定SMARCAL1的启动子G-四联体触发端粒复制应激,ALTering Cancer。
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-03 DOI: 10.1021/acschembio.4c00285
Suman Panda, Tanaya Roychowdhury, Anindya Dutta, Sourio Chakraborty, Tanya Das, Subhrangsu Chatterjee

Most of the human cancers are dependent on telomerase to extend the telomeres. But ∼10% of all cancers use a telomerase-independent, homologous recombination mediated pathway called alternative lengthening of telomeres (ALT). Due to the poor prognosis, ALT status is not being considered yet in the diagnosis of cancer. No such specific treatment is available to date for ALT positive cancers. ALT positive cancers are dependent on replication stress to deploy DNA repair pathways to the telomeres to execute homologous recombination mediated telomere extension. SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1) is associated with the ALT telomeres to resolve replication stress thus providing telomere stability. Thus, the dependency on replication stress regulatory factors like SMARCAL1 made it a suitable therapeutic target for the treatment of ALT positive cancers. In this study, we found a significant downregulation of SMARCAL1 expression by stabilizing the G-quadruplex (G4) motif found in the promoter of SMARCAL1 by potent G4 stabilizers, like TMPyP4 and BRACO-19. SMARCAL1 downregulation led toward the increased localization of PML (promyelocytic leukemia) bodies in ALT telomeres and triggered the formation of APBs (ALT-associated promyelocytic leukemia bodies) in ALT positive cell lines, increasing telomere replication stress and DNA damage at a genomic level. Induction of replication stress and hyper-recombinogenic phenotype in ALT positive cells mediated by G4 stabilizing molecules already highlighted their possible application as a new therapeutic window to target ALT positive tumors. In accordance with this, our study will also provide a valuable insight toward the development of G4-based ALT therapeutics targeting SMARCAL1.

大多数人类癌症都依赖端粒酶来延长端粒。但有10%的癌症使用一种不依赖端粒酶的同源重组介导的途径,即端粒替代性延长(ALT)。由于预后不佳,目前在诊断癌症时还没有考虑 ALT 状态。迄今为止,还没有针对 ALT 阳性癌症的特殊治疗方法。ALT阳性癌症依赖复制压力将DNA修复途径部署到端粒,以执行同源重组介导的端粒延长。SMARCAL1(与SWI/SNF相关的、基质相关的、依赖肌动蛋白的染色质调节器,类A亚家族1)与ALT端粒相关,可解决复制压力,从而提供端粒稳定性。因此,SMARCAL1等复制压力调节因子的依赖性使其成为治疗ALT阳性癌症的合适治疗靶点。在这项研究中,我们发现通过强效G4稳定剂(如TMPyP4和BRACO-19)稳定SMARCAL1启动子中的G-四叠体(G4)图案,可以显著下调SMARCAL1的表达。SMARCAL1 下调导致 PML(早幼粒细胞白血病)体在 ALT 端粒中的定位增加,并在 ALT 阳性细胞系中引发 APB(ALT 相关早幼粒细胞白血病体)的形成,从而在基因组水平上增加端粒复制压力和 DNA 损伤。由 G4 稳定分子介导的 ALT 阳性细胞复制压力和超重组表型的诱导已经突出表明,它们可能成为针对 ALT 阳性肿瘤的新的治疗窗口。因此,我们的研究也将为开发基于 G4 的、以 SMARCAL1 为靶点的 ALT 治疗药物提供有价值的见解。
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引用次数: 0
Confounding Factors in Targeted Degradation of Short-Lived Proteins. 短寿命蛋白质定向降解过程中的干扰因素
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-03 DOI: 10.1021/acschembio.4c00152
Vesna Vetma, Laura Casares Perez, Ján Eliaš, Andrea Stingu, Anju Kombara, Teresa Gmaschitz, Nina Braun, Tuncay Ciftci, Georg Dahmann, Emelyne Diers, Thomas Gerstberger, Peter Greb, Giorgia Kidd, Christiane Kofink, Ilaria Puoti, Valentina Spiteri, Nicole Trainor, Harald Weinstabl, Yvonne Westermaier, Claire Whitworth, Alessio Ciulli, William Farnaby, Kirsten McAulay, Aileen B Frost, Nicola Chessum, Manfred Koegl

Targeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but to what extent it influences target protein degradation has not been systematically explored. Using simple mathematical modeling of protein degradation, we find that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a degrader agent which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein-degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required to conclude that a novel agent works as a bona fide targeted protein degrader.

靶向蛋白质降解最近已成为药物发现的一种新选择。蛋白质的天然半衰期预计会影响降解剂的药效,但它在多大程度上影响靶蛋白降解还没有系统的研究。利用简单的蛋白质降解数学模型,我们发现目标蛋白质的天然半衰期对降解剂诱导的蛋白质降解水平有显著影响,这会给筛选工作带来巨大障碍。此外,我们还发现,在筛选短效蛋白质降解剂时,GSPT1 降解剂和一般细胞毒性化合物等阻碍蛋白质合成的药剂会被误认为是蛋白质降解剂。例如,在 GSPT1 降解和多柔比星等细胞毒性药物的作用下,MCL1 和 MDM2 等短寿命蛋白质会消失。这些发现对靶标选择以及得出新型制剂是真正的靶向蛋白降解剂这一结论所需的对照实验类型都有影响。
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引用次数: 0
HCV 5-Methylcytosine Enhances Viral RNA Replication through Interaction with m5C Reader YBX1. HCV 5-甲基胞嘧啶通过与 m5C 阅读器 YBX1 相互作用加强病毒 RNA 复制
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-02 DOI: 10.1021/acschembio.4c00322
Zhu-Li Li, Yan Xie, Yuke Xie, Hongliang Chen, Xiang Zhou, Min Liu, Xiao-Lian Zhang

Hepatitis C virus (HCV) is a positive-stranded RNA virus that mainly causes chronic hepatitis, cirrhosis and hepatocellular carcinoma. Recently we confirmed m5C modifications within NS5A gene of HCV RNA genome. However, the roles of the m5C modification and its interaction with host proteins in regulating HCV's life cycle, remain unexplored. Here, we demonstrate that HCV infection enhances the expression of the host m5C reader YBX1 through the transcription factor MAX. YBX1 acts as an m5C reader, recognizing the m5C-modified NS5A C7525 site in the HCV RNA genome and significantly enhancing HCV RNA stability. This m5C-modification is also required for YBX1 colocalization with lipid droplets and HCV Core protein. Moreover, YBX1 facilitates HCV RNA replication, as well as viral assembly/budding. The tryptophan residue at position 65 (W65) of YBX1 is critical for these functions. Knockout of YBX1 or the application of YBX1 inhibitor SU056 suppresses HCV RNA replication and viral protein translation. To our knowledge, this is the first report demonstrating that the interaction between host m5C reader YBX1 and HCV RNA m5C methylation facilitates viral replication. Therefore, hepatic-YBX1 knockdown holds promise as a potential host-directed strategy for HCV therapy.

丙型肝炎病毒(HCV)是一种正链 RNA 病毒,主要导致慢性肝炎、肝硬化和肝细胞癌。最近,我们证实了 HCV RNA 基因组 NS5A 基因中的 m5C 修饰。然而,m5C 修饰及其与宿主蛋白的相互作用在调控 HCV 生命周期中的作用仍有待探索。在这里,我们证明了 HCV 感染会通过转录因子 MAX 增强宿主 m5C 阅读器 YBX1 的表达。YBX1 可作为 m5C 阅读器,识别 HCV RNA 基因组中经 m5C 修饰的 NS5A C7525 位点,并显著增强 HCV RNA 的稳定性。这种 m5C 修饰也是 YBX1 与脂滴和 HCV 核心蛋白共定位所必需的。此外,YBX1 还有助于 HCV RNA 复制以及病毒组装/萌发。YBX1 第 65 位(W65)的色氨酸残基对这些功能至关重要。敲除 YBX1 或使用 YBX1 抑制剂 SU056 可抑制 HCV RNA 复制和病毒蛋白翻译。据我们所知,这是第一份证明宿主 m5C 阅读器 YBX1 与 HCV RNA m5C 甲基化之间相互作用促进病毒复制的报告。因此,肝脏-YBX1 基因敲除有望成为一种潜在的宿主导向 HCV 治疗策略。
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引用次数: 0
Engineered Branaplam Aptamers Exploit Structural Elements from Natural Riboswitches. 利用天然核糖开关的结构元素设计 Branaplam Aptamers。
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-02 DOI: 10.1021/acschembio.4c00358
Michael G Mohsen, Matthew K Midy, Aparaajita Balaji, Ronald R Breaker

Drug candidates that fail in clinical trials for efficacy reasons might still have favorable safety and bioavailability characteristics that could be exploited. A failed drug candidate could be repurposed if a receptor, such as an aptamer, were created that binds the compound with high specificity. Branaplam is a small molecule that was previously in development to treat spinal muscular atrophy and Huntington's disease. Here, we report the development of a small (48-nucleotide) RNA aptamer for branaplam with a dissociation constant of ∼150 nM. Starting with a combinatorial RNA pool integrating the secondary and tertiary structural scaffold of a Guanine-I riboswitch aptamer interspersed with regions of random sequence, in vitro selection yielded aptamer candidates for branaplam. Reselection and rational design were employed to improve binding of a representative branaplam aptamer candidate. A resulting variant retains the pseudoknot and two of the paired elements (P2 and P3) from the scaffold but lacks the enclosing paired element (P1) that is essential for the function of the natural Guanine-I riboswitch aptamer. A second combinatorial RNA pool based on the scaffold for TPP (thiamin pyrophosphate) riboswitches also yielded a candidate offering additional opportunities for branaplam aptamer development.

因疗效原因而在临床试验中失败的候选药物可能仍然具有良好的安全性和生物利用度特性,可以加以利用。如果能创造出一种能与化合物高度特异性结合的受体(如aptamer),那么失败的候选药物就能被重新利用。Branaplam 是一种小分子药物,以前曾用于治疗脊髓性肌萎缩症和亨廷顿氏症。在这里,我们报告了一种小的(48 个核苷酸)RNA 与 branaplam 的吻合器的开发情况,其解离常数为 ∼150 nM。从结合了鸟嘌呤-I核糖开关适配体的二级和三级结构支架以及随机序列区域的组合 RNA 池开始,体外筛选产生了 branaplam 的候选适配体。通过重新选择和合理设计,改进了具有代表性的 branaplam 候选灵敏配体的结合力。由此产生的一个变体保留了支架中的假结和两个成对元素(P2 和 P3),但缺少对天然 Guanine-I 核糖开关灵敏配体的功能至关重要的封闭成对元素(P1)。以 TPP(焦磷酸硫胺素)核糖开关支架为基础的第二个组合 RNA 池也产生了一个候选 RNA,为开发 branaplam aptamer 提供了更多机会。
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引用次数: 0
Tyrosine Sulfation Modulates the Binding Affinity of Chemokine-Targeting Nanobodies. 酪氨酸硫酸化改变了趋化因子靶向纳米抗体的结合亲和力
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-28 DOI: 10.1021/acschembio.4c00230
Joshua J Dilly, Alexandra L Morgan, Max J Bedding, Jason K K Low, Joel P Mackay, Anne C Conibear, Ram Prasad Bhusal, Martin J Stone, Charlotte Franck, Richard J Payne

Chemokines are an important family of small proteins integral to leukocyte recruitment during inflammation. Dysregulation of the chemokine-chemokine receptor axis is implicated in many diseases, and both chemokines and their cognate receptors have been the targets of therapeutic development. Analysis of the antigen-binding regions of chemokine-binding nanobodies revealed a sequence motif suggestive of tyrosine sulfation. Given the well-established importance of post-translational tyrosine sulfation of receptors for chemokine affinity, it was hypothesized that the sulfation of these nanobodies may contribute to chemokine binding and selectivity. Four nanobodies (16C1, 9F1, 11B1, and 11F2) were expressed using amber codon suppression to incorporate tyrosine sulfation. The sulfated variant of 16C1 demonstrated significantly improved chemokine binding compared to the non-sulfated counterpart, while the other nanobodies displayed equipotent or reduced affinity upon sulfation. The ability of tyrosine sulfation to modulate chemokine binding, both positively and negatively, could be leveraged for chemokine-targeted sulfo-nanobody therapeutics in the future.

趋化因子是炎症期间白细胞招募过程中不可或缺的重要小蛋白家族。许多疾病都与趋化因子-趋化因子受体轴的失调有关,趋化因子及其同源受体一直是治疗开发的目标。对趋化因子结合纳米抗体的抗原结合区进行分析,发现了一个暗示酪氨酸硫酸化的序列基序。鉴于已证实受体翻译后酪氨酸硫酸化对趋化因子亲和力的重要性,我们假设这些纳米抗体的硫酸化可能有助于趋化因子的结合和选择性。利用琥珀色密码子抑制技术表达了四种纳米抗体(16C1、9F1、11B1 和 11F2),以结合酪氨酸硫酸化。与未硫酸化的纳米抗体相比,硫酸化的 16C1 变体明显提高了趋化因子的结合力,而其他纳米抗体在硫酸化后亲和力相当或降低。酪氨酸硫酸化能正反两方面调节趋化因子的结合,未来可用于趋化因子靶向磺化纳米抗体疗法。
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引用次数: 0
A C-Degron Structure-Based Approach for the Development of Ligands Targeting the E3 Ligase TRIM7. 基于 C-egron 结构的配体靶向 E3 配体 TRIM7 的开发方法。
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-27 DOI: 10.1021/acschembio.4c00301
Christian J Muñoz Sosa, Christopher Lenz, Anton Hamann, Frederic Farges, Johannes Dopfer, Andreas Krämer, Veronika Cherkashyna, Andrey Tarnovskiy, Yurii S Moroz, Ewgenij Proschak, Václav Němec, Susanne Müller, Krishna Saxena, Stefan Knapp

TRIM7 is a ubiquitin E3 ligase with key regulatory functions, mediating viral infection, tumor biology, innate immunity, and cellular processes, such as autophagy and ferroptosis. It contains a PRYSPRY domain that specifically recognizes degron sequences containing C-terminal glutamine. Ligands that bind to the TRIM7 PRYSPRY domain may have applications in the treatment of viral infections, as modulators of inflammation, and in the design of a new class of PROTACs (PROteolysis TArgeting Chimeras) that mediate the selective degradation of therapeutically relevant proteins (POIs). Here, we developed an assay toolbox for the comprehensive evaluation of TRIM7 ligands. Using TRIM7 degron sequences together with a structure-based design, we developed the first series of peptidomimetic ligands with low micromolar affinity. The terminal carboxylate moiety was required for ligand activity but prevented cell penetration. A prodrug strategy using an ethyl ester resulted in enhanced permeability, which was evaluated using confocal imaging.

TRIM7 是一种泛素 E3 连接酶,具有关键的调控功能,可介导病毒感染、肿瘤生物学、先天免疫以及自噬和铁变态等细胞过程。它含有一个 PRYSPRY 结构域,能特异性识别含有 C 端谷氨酰胺的降解素序列。与 TRIM7 PRYSPRY 结构域结合的配体可能会应用于病毒感染的治疗、炎症的调节以及新型 PROTACs(PROteolysis TArgeting Chimeras)的设计,这种 PROTACs(PROteolysis TArgeting Chimeras)可介导治疗相关蛋白(POIs)的选择性降解。在这里,我们开发了一个用于全面评估 TRIM7 配体的检测工具箱。利用 TRIM7 降解子序列和基于结构的设计,我们开发出了第一个具有低微摩尔亲和力的拟肽配体系列。配体活性需要末端的羧酸盐分子,但它阻碍了细胞的穿透。使用乙酯的原药策略增强了渗透性,并使用共聚焦成像技术对渗透性进行了评估。
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