Pub Date : 2024-05-31DOI: 10.1021/acscentsci.4c00210
Jonathan A. Kephart, Daniel Y. Zhou, Jason Sandwisch, Nathalia Cajiao, Sebastian M. Krajewski, Paul Malinowski, Jiun-Haw Chu, Michael L. Neidig, Werner Kaminsky and Alexandra Velian*,
Directing groups guide substitution patterns in organic synthetic schemes, but little is known about pathways to control reactivity patterns, such as regioselectivity, in complex inorganic systems such as bioinorganic cofactors or extended surfaces. Interadsorbate effects are known to encode surface reactivity patterns in inorganic materials, modulating the location and binding strength of ligands. However, owing to limited experimental resolution into complex inorganic structures, there is little opportunity to resolve these effects on the atomic scale. Here, we utilize an atomically precise Fe/Co/Se nanocluster platform, [Fe3(L)2Co6Se8L′6]+ ([1(L)2]+; L = CNtBu, THF; L′ = Ph2PN(−)Tol), in which allosteric interadsorbate effects give rise to pronounced site-differentiation. Using a combination of spectroscopic techniques and single-crystal X-ray diffractometry, we discover that coordination of THF at the ligand-free Fe site in [1(CNtBu)2]+ sets off a domino effect wherein allosteric through-cluster interactions promote the regioselective dissociation of CNtBu at a neighboring Fe site. Computational analysis reveals that this active site correlation is a result of delocalized Fe···Se···Co···Se covalent interactions that intertwine edge sites on the same cluster face. This study provides an unprecedented atom-scale glimpse into how interfacial metal–support interactions mediate a collective and regiospecific path for substrate exchange across multiple active sites.
Akin to directing groups in organic chemistry, allostery directs reaction chemistry across multiple active sites at the surface of an inorganic cluster.
定向基团可引导有机合成方案中的取代模式,但人们对生物无机辅因子或扩展表面等复杂无机系统中控制反应模式(如区域选择性)的途径知之甚少。众所周知,吸附剂间效应可编码无机材料的表面反应模式,调节配体的位置和结合强度。然而,由于复杂无机结构的实验分辨率有限,几乎没有机会在原子尺度上解析这些效应。在这里,我们利用原子精确的铁/钴/硒纳米簇平台 [Fe3(L)2Co6Se8L′6]+ ([1(L)2]+;L = CNtBu,THF;L′ = Ph2PN(-)Tol),其中的异构吸附剂间效应引起了明显的位点差异。利用光谱技术和单晶 X 射线衍射测定法,我们发现 THF 在 [1(CNtBu)2]+ 中无配体的 Fe 位点上的配位引发了多米诺骨牌效应,在这种效应中,异构通簇相互作用促进了 CNtBu 在邻近 Fe 位点上的区域选择性解离。计算分析表明,这种活性位点相关性是Fe--Se--Co--Se共价相互作用的结果,这种相互作用使同一簇面上的边缘位点交织在一起。这项研究以前所未有的原子尺度揭示了界面金属-支持物相互作用是如何在多个活性位点之间介导集体和特定区域的底物交换路径的。
{"title":"Caught in the Act of Substitution: Interadsorbate Effects on an Atomically Precise Fe/Co/Se Nanocluster","authors":"Jonathan A. Kephart, Daniel Y. Zhou, Jason Sandwisch, Nathalia Cajiao, Sebastian M. Krajewski, Paul Malinowski, Jiun-Haw Chu, Michael L. Neidig, Werner Kaminsky and Alexandra Velian*, ","doi":"10.1021/acscentsci.4c00210","DOIUrl":"10.1021/acscentsci.4c00210","url":null,"abstract":"<p >Directing groups guide substitution patterns in organic synthetic schemes, but little is known about pathways to control reactivity patterns, such as regioselectivity, in complex inorganic systems such as bioinorganic cofactors or extended surfaces. Interadsorbate effects are known to encode surface reactivity patterns in inorganic materials, modulating the location and binding strength of ligands. However, owing to limited experimental resolution into complex inorganic structures, there is little opportunity to resolve these effects on the atomic scale. Here, we utilize an atomically precise Fe/Co/Se nanocluster platform, [Fe<sub>3</sub>(L)<sub>2</sub>Co<sub>6</sub>Se<sub>8</sub>L′<sub>6</sub>]<sup>+</sup> ([<b>1</b>(L)<sub>2</sub>]<sup>+</sup>; L = CN<sup><i>t</i></sup>Bu, THF; L′ = Ph<sub>2</sub>PN<sup>(−)</sup>Tol), in which allosteric interadsorbate effects give rise to pronounced site-differentiation. Using a combination of spectroscopic techniques and single-crystal X-ray diffractometry, we discover that coordination of THF at the ligand-free Fe site in [<b>1</b>(CN<sup><i>t</i></sup>Bu)<sub>2</sub>]<sup>+</sup> sets off a domino effect wherein allosteric through-cluster interactions promote the regioselective dissociation of CN<sup><i>t</i></sup>Bu at a neighboring Fe site. Computational analysis reveals that this active site correlation is a result of delocalized Fe···Se···Co···Se covalent interactions that intertwine edge sites on the same cluster face. This study provides an unprecedented atom-scale glimpse into how interfacial metal–support interactions mediate a collective and regiospecific path for substrate exchange across multiple active sites.</p><p >Akin to directing groups in organic chemistry, allostery directs reaction chemistry across multiple active sites at the surface of an inorganic cluster.</p>","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":12.7,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acscentsci.4c00210","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141191569","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-22DOI: 10.1021/acscentsci.4c00258
Tobias Vornholt, Mojmír Mutný, Gregor W. Schmidt, Christian Schellhaas, Ryo Tachibana, Sven Panke, Thomas R. Ward, Andreas Krause, Markus Jeschek
Tailored enzymes are crucial for the transition to a sustainable bioeconomy. However, enzyme engineering is laborious and failure-prone due to its reliance on serendipity. The efficiency and success rates of engineering campaigns may be improved by applying machine learning to map the sequence-activity landscape based on small experimental data sets. Yet, it often proves challenging to reliably model large sequence spaces while keeping the experimental effort tractable. To address this challenge, we present an integrated pipeline combining large-scale screening with active machine learning, which we applied to engineer an artificial metalloenzyme (ArM) catalyzing a new-to-nature hydroamination reaction. Combining lab automation and next-generation sequencing, we acquired sequence-activity data for several thousand ArM variants. We then used Gaussian process regression to model the activity landscape and guide further screening rounds. Critical characteristics of our pipeline include the cost-effective generation of information-rich data sets, the integration of an explorative round to improve the model’s performance, and the inclusion of experimental noise. Our approach led to an order-of-magnitude boost in the hit rate while making efficient use of experimental resources. Search strategies like this should find broad utility in enzyme engineering and accelerate the development of novel biocatalysts.
量身定制的酶对于向可持续生物经济过渡至关重要。然而,由于酶工程依赖于偶然性,因此既费力又容易失败。应用机器学习来绘制基于小型实验数据集的序列-活性图谱,可以提高工程活动的效率和成功率。然而,要在保持实验工作量可控的同时对大型序列空间进行可靠建模,往往具有挑战性。为了应对这一挑战,我们提出了一种将大规模筛选与主动机器学习相结合的集成管道,并将其应用于设计一种人工金属酶(ArM),以催化一种新的自然氢化反应。结合实验室自动化和新一代测序技术,我们获得了数千个 ArM 变体的序列-活性数据。然后,我们使用高斯过程回归法建立活性模型,并指导进一步的筛选工作。我们的方法的关键特点包括:经济高效地生成信息丰富的数据集、整合探索轮以提高模型的性能,以及包含实验噪音。我们的方法在有效利用实验资源的同时,还将命中率提高了一个数量级。像这样的搜索策略应该在酶工程中得到广泛应用,并加速新型生物催化剂的开发。
{"title":"Enhanced Sequence-Activity Mapping and Evolution of Artificial Metalloenzymes by Active Learning","authors":"Tobias Vornholt, Mojmír Mutný, Gregor W. Schmidt, Christian Schellhaas, Ryo Tachibana, Sven Panke, Thomas R. Ward, Andreas Krause, Markus Jeschek","doi":"10.1021/acscentsci.4c00258","DOIUrl":"https://doi.org/10.1021/acscentsci.4c00258","url":null,"abstract":"Tailored enzymes are crucial for the transition to a sustainable bioeconomy. However, enzyme engineering is laborious and failure-prone due to its reliance on serendipity. The efficiency and success rates of engineering campaigns may be improved by applying machine learning to map the sequence-activity landscape based on small experimental data sets. Yet, it often proves challenging to reliably model large sequence spaces while keeping the experimental effort tractable. To address this challenge, we present an integrated pipeline combining large-scale screening with active machine learning, which we applied to engineer an artificial metalloenzyme (ArM) catalyzing a new-to-nature hydroamination reaction. Combining lab automation and next-generation sequencing, we acquired sequence-activity data for several thousand ArM variants. We then used Gaussian process regression to model the activity landscape and guide further screening rounds. Critical characteristics of our pipeline include the cost-effective generation of information-rich data sets, the integration of an explorative round to improve the model’s performance, and the inclusion of experimental noise. Our approach led to an order-of-magnitude boost in the hit rate while making efficient use of experimental resources. Search strategies like this should find broad utility in enzyme engineering and accelerate the development of novel biocatalysts.","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":18.2,"publicationDate":"2024-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141146734","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-21DOI: 10.1021/acscentsci.4c00380
Lili Zhang, Mei Chen, Zhiqiang Wang, Minjuan Zhong, Hong Chen, Ting Li, Linlin Wang, Zhihui Zhao, Xiao-Bing Zhang, Guoliang Ke*, Yanlan Liu* and Weihong Tan*,
Coacervates formed by liquid–liquid phase separation emerge as important biomimetic models for studying the dynamic behaviors of membraneless organelles and synchronously motivating the creation of smart architectures with the regulation of cell fate. Despite continuous progress, it remains challenging to balance the trade-offs among structural stability, versatility, and molecular communication for regulation of cell fate and systemic investigation in a complex physiological system. Herein, we present a self-stabilizing and fastener-bound gain-of-function methodology to create a new type of synthetic DNA membraneless organelle (MO) with high stability and controlled bioactivity on the basis of DNA coacervates. Specifically, long single-strand DNA generated by rolling circle amplification (RCA) is selected as the scaffold that assembles into membraneless coacervates via phase separation. Intriguingly, the as-formed DNA MO can recruit RCA byproducts and other components to achieve self-stabilization, nanoscale condensation, and function encoding. As a proof of concept, photoactivatable DNA MO is constructed and successfully employed for time-dependent accumulation and spatiotemporal management of cancer in a mouse model. This study offers new, important insights into synthetic membraneless organelles for the basic understanding and manipulation of important life processes.
Coacervate membraneless organelles formed through liquid−liquid phase separation (LLPS) have been actively investigated as an emerging cargo delivery platform to harbor proteins, RNAs, and therapeutic molecules. However, a trade-off among structural stability, versatility, and molecular communication in physiological conditions hampers their application for systemic administration. Our study develops self-stabilizing and fastener-bound gain-of-function DNA membraneless organelles (MOs). Unlike traditional approaches to obtain functional MOs via complicated surface coating or hybridization, long single-strand DNA coacervates are used as scaffolds, enabling self-stabilization and function encoding by simply recruiting surrounding components during LLPS for systematic regulation of cell fate, without loss of communication properties.
液-液相分离形成的凝聚态成为研究无膜细胞器动态行为的重要生物仿生模型,同时也推动了智能结构的创造和细胞命运的调控。尽管取得了持续进展,但在复杂生理系统中平衡结构稳定性、多功能性和分子交流之间的权衡,以调控细胞命运和进行系统研究,仍然具有挑战性。在此,我们提出了一种自稳定和紧固件结合的功能增益方法,在DNA共凝胶的基础上创造出一种具有高稳定性和可控生物活性的新型合成DNA无膜细胞器(MO)。具体来说,该研究选择了由滚动圈扩增(RCA)产生的长单链DNA作为支架,通过相分离组装成无膜凝聚态。耐人寻味的是,形成后的 DNA MO 可以吸附 RCA 副产物和其他成分,从而实现自稳定、纳米级凝聚和功能编码。作为概念验证,我们构建了可光激活的 DNA MO,并在小鼠模型中成功用于随时间变化的积累和癌症的时空管理。这项研究为合成无膜细胞器提供了新的、重要的见解,有助于对重要生命过程的基本理解和操纵。通过液-液相分离(LLPS)形成的无膜细胞器作为一种新兴的货物运输平台,一直受到人们的积极研究,以容纳蛋白质、RNA 和治疗分子。然而,在生理条件下,结构稳定性、多功能性和分子交流之间的权衡阻碍了它们在全身给药中的应用。我们的研究开发了自稳定和紧固件结合的功能增益 DNA 无膜细胞器(MO)。与通过复杂的表面涂层或杂交获得功能性MOs的传统方法不同,我们采用了长单链DNA共凝胶作为支架,通过在LLPS过程中简单地招募周围成分来实现自稳定和功能编码,从而对细胞命运进行系统调控,而不会丧失通讯特性。
{"title":"Spatiotemporal Regulation of Cell Fate in Living Systems Using Photoactivatable Artificial DNA Membraneless Organelles","authors":"Lili Zhang, Mei Chen, Zhiqiang Wang, Minjuan Zhong, Hong Chen, Ting Li, Linlin Wang, Zhihui Zhao, Xiao-Bing Zhang, Guoliang Ke*, Yanlan Liu* and Weihong Tan*, ","doi":"10.1021/acscentsci.4c00380","DOIUrl":"10.1021/acscentsci.4c00380","url":null,"abstract":"<p >Coacervates formed by liquid–liquid phase separation emerge as important biomimetic models for studying the dynamic behaviors of membraneless organelles and synchronously motivating the creation of smart architectures with the regulation of cell fate. Despite continuous progress, it remains challenging to balance the trade-offs among structural stability, versatility, and molecular communication for regulation of cell fate and systemic investigation in a complex physiological system. Herein, we present a self-stabilizing and fastener-bound gain-of-function methodology to create a new type of synthetic DNA membraneless organelle (MO) with high stability and controlled bioactivity on the basis of DNA coacervates. Specifically, long single-strand DNA generated by rolling circle amplification (RCA) is selected as the scaffold that assembles into membraneless coacervates via phase separation. Intriguingly, the as-formed DNA MO can recruit RCA byproducts and other components to achieve self-stabilization, nanoscale condensation, and function encoding. As a proof of concept, photoactivatable DNA MO is constructed and successfully employed for time-dependent accumulation and spatiotemporal management of cancer in a mouse model. This study offers new, important insights into synthetic membraneless organelles for the basic understanding and manipulation of important life processes.</p><p >Coacervate membraneless organelles formed through liquid−liquid phase separation (LLPS) have been actively investigated as an emerging cargo delivery platform to harbor proteins, RNAs, and therapeutic molecules. However, a trade-off among structural stability, versatility, and molecular communication in physiological conditions hampers their application for systemic administration. Our study develops self-stabilizing and fastener-bound gain-of-function DNA membraneless organelles (MOs). Unlike traditional approaches to obtain functional MOs via complicated surface coating or hybridization, long single-strand DNA coacervates are used as scaffolds, enabling self-stabilization and function encoding by simply recruiting surrounding components during LLPS for systematic regulation of cell fate, without loss of communication properties.</p>","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":12.7,"publicationDate":"2024-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acscentsci.4c00380","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141117440","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-21DOI: 10.1021/acscentsci.4c00312
Selma Piranej, Luona Zhang, Alisina Bazrafshan, Mariana Marin, Gregory B. Melikian, Khalid Salaita
Assays that detect viral infections play a significant role in limiting the spread of diseases such as SARS-CoV-2. Here, we present Rolosense, a virus sensing platform that leverages the motion of 5 μm DNA-based motors on RNA fuel chips to transduce the presence of viruses. Motors and chips are modified with aptamers, which are designed for multivalent binding to viral targets and lead to stalling of motion. Therefore, the motors perform a “mechanical test” of the viral target and stall in the presence of whole virions, which represents a unique mechanism of transduction distinct from conventional assays. Rolosense can detect SARS-CoV-2 spiked in artificial saliva and exhaled breath condensate with a sensitivity of 103 copies/mL and discriminates among other respiratory viruses. The assay is modular and amenable to multiplexing, as demonstrated by our one-pot detection of influenza A and SARS-CoV-2. As a proof of concept, we show that readout can be achieved using a smartphone camera with a microscopic attachment in as little as 15 min without amplification reactions. Taken together, these results show that mechanical detection using Rolosense can be broadly applied to any viral target and has the potential to enable rapid, low-cost point-of-care screening of circulating viruses.
{"title":"Rolosense: Mechanical Detection of SARS-CoV-2 Using a DNA-Based Motor","authors":"Selma Piranej, Luona Zhang, Alisina Bazrafshan, Mariana Marin, Gregory B. Melikian, Khalid Salaita","doi":"10.1021/acscentsci.4c00312","DOIUrl":"https://doi.org/10.1021/acscentsci.4c00312","url":null,"abstract":"Assays that detect viral infections play a significant role in limiting the spread of diseases such as SARS-CoV-2. Here, we present Rolosense, a virus sensing platform that leverages the motion of 5 μm DNA-based motors on RNA fuel chips to transduce the presence of viruses. Motors and chips are modified with aptamers, which are designed for multivalent binding to viral targets and lead to stalling of motion. Therefore, the motors perform a “mechanical test” of the viral target and stall in the presence of whole virions, which represents a unique mechanism of transduction distinct from conventional assays. Rolosense can detect SARS-CoV-2 spiked in artificial saliva and exhaled breath condensate with a sensitivity of 10<sup>3</sup> copies/mL and discriminates among other respiratory viruses. The assay is modular and amenable to multiplexing, as demonstrated by our one-pot detection of influenza A and SARS-CoV-2. As a proof of concept, we show that readout can be achieved using a smartphone camera with a microscopic attachment in as little as 15 min without amplification reactions. Taken together, these results show that mechanical detection using Rolosense can be broadly applied to any viral target and has the potential to enable rapid, low-cost point-of-care screening of circulating viruses.","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":18.2,"publicationDate":"2024-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141146717","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-20DOI: 10.1021/acscentsci.3c01557
Chintan Soni, Noam Prywes, Matthew Hall, Malavika A. Nair, David F. Savage, Alanna Schepartz and Abhishek Chatterjee*,
Using directed evolution, aminoacyl-tRNA synthetases (aaRSs) have been engineered to incorporate numerous noncanonical amino acids (ncAAs). Until now, the selection of such novel aaRS mutants has relied on the expression of a selectable reporter protein. However, such translation-dependent selections are incompatible with exotic monomers that are suboptimal substrates for the ribosome. A two-step solution is needed to overcome this limitation: (A) engineering an aaRS to charge the exotic monomer, without ribosomal translation; (B) subsequent engineering of the ribosome to accept the resulting acyl-tRNA for translation. Here, we report a platform for aaRS engineering that directly selects tRNA-acylation without ribosomal translation (START). In START, each distinct aaRS mutant is correlated to a cognate tRNA containing a unique sequence barcode. Acylation by an active aaRS mutant protects the corresponding barcode-containing tRNAs from oxidative treatment designed to damage the 3′-terminus of the uncharged tRNAs. Sequencing of these surviving barcode-containing tRNAs is then used to reveal the identity of the aaRS mutants that acylated the correlated tRNA sequences. The efficacy of START was demonstrated by identifying novel mutants of the Methanomethylophilus alvus pyrrolysyl-tRNA synthetase from a naïve library that enables incorporation of ncAAs into proteins in living cells.
A novel approach to engineer aminoacyl-tRNA synthetases is reported that does not rely on translational readout. It will enable the selection of mutants for charging monomers that are poor substrates for the wild-type ribosome.
{"title":"A Translation-Independent Directed Evolution Strategy to Engineer Aminoacyl-tRNA Synthetases","authors":"Chintan Soni, Noam Prywes, Matthew Hall, Malavika A. Nair, David F. Savage, Alanna Schepartz and Abhishek Chatterjee*, ","doi":"10.1021/acscentsci.3c01557","DOIUrl":"10.1021/acscentsci.3c01557","url":null,"abstract":"<p >Using directed evolution, aminoacyl-tRNA synthetases (aaRSs) have been engineered to incorporate numerous noncanonical amino acids (ncAAs). Until now, the selection of such novel aaRS mutants has relied on the expression of a selectable reporter protein. However, such translation-dependent selections are incompatible with exotic monomers that are suboptimal substrates for the ribosome. A two-step solution is needed to overcome this limitation: (A) engineering an aaRS to charge the exotic monomer, without ribosomal translation; (B) subsequent engineering of the ribosome to accept the resulting acyl-tRNA for translation. Here, we report a platform for aaRS engineering that directly selects tRNA-acylation without ribosomal translation (START). In START, each distinct aaRS mutant is correlated to a cognate tRNA containing a unique sequence barcode. Acylation by an active aaRS mutant protects the corresponding barcode-containing tRNAs from oxidative treatment designed to damage the 3′-terminus of the uncharged tRNAs. Sequencing of these surviving barcode-containing tRNAs is then used to reveal the identity of the aaRS mutants that acylated the correlated tRNA sequences. The efficacy of START was demonstrated by identifying novel mutants of the <i>Methanomethylophilus alvus</i> pyrrolysyl-tRNA synthetase from a naïve library that enables incorporation of ncAAs into proteins in living cells.</p><p >A novel approach to engineer aminoacyl-tRNA synthetases is reported that does not rely on translational readout. It will enable the selection of mutants for charging monomers that are poor substrates for the wild-type ribosome.</p>","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":12.7,"publicationDate":"2024-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acscentsci.3c01557","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141146716","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-17DOI: 10.1021/acscentsci.4c00286
Melissa Lim, Thang Do Cong, Lauren M. Orr, Ethan S. Toriki, Andrew C. Kile, James W. Papatzimas, Elijah Lee, Yihan Lin, Daniel K. Nomura
Targeted protein degradation with monovalent molecular glue degraders is a powerful therapeutic modality for eliminating disease causing proteins. However, rational design of molecular glue degraders remains challenging. In this study, we sought to identify a transplantable and linker-less covalent handle that could be appended onto the exit vector of various protein-targeting ligands to induce the degradation of their respective targets. Using the BET family inhibitor JQ1 as a testbed, we synthesized and screened a series of covalent JQ1 analogs and identified a vinylsulfonyl piperazine handle that led to the potent and selective degradation of BRD4 in cells. Through chemoproteomic profiling, we identified DCAF16 as the E3 ligase responsible for BRD4 degradation─an E3 ligase substrate receptor that has been previously covalently targeted for molecular glue-based degradation of BRD4. Interestingly, we demonstrated that this covalent handle can be transplanted across a diverse array of protein-targeting ligands spanning many different protein classes to induce the degradation of CDK4, the androgen receptor, BTK, SMARCA2/4, and BCR-ABL/c-ABL. Our study reveals a DCAF16-based covalent degradative and linker-less chemical handle that can be attached to protein-targeting ligands to induce the degradation of several different classes of protein targets.
{"title":"DCAF16-Based Covalent Handle for the Rational Design of Monovalent Degraders","authors":"Melissa Lim, Thang Do Cong, Lauren M. Orr, Ethan S. Toriki, Andrew C. Kile, James W. Papatzimas, Elijah Lee, Yihan Lin, Daniel K. Nomura","doi":"10.1021/acscentsci.4c00286","DOIUrl":"https://doi.org/10.1021/acscentsci.4c00286","url":null,"abstract":"Targeted protein degradation with monovalent molecular glue degraders is a powerful therapeutic modality for eliminating disease causing proteins. However, rational design of molecular glue degraders remains challenging. In this study, we sought to identify a transplantable and linker-less covalent handle that could be appended onto the exit vector of various protein-targeting ligands to induce the degradation of their respective targets. Using the BET family inhibitor JQ1 as a testbed, we synthesized and screened a series of covalent JQ1 analogs and identified a vinylsulfonyl piperazine handle that led to the potent and selective degradation of BRD4 in cells. Through chemoproteomic profiling, we identified DCAF16 as the E3 ligase responsible for BRD4 degradation─an E3 ligase substrate receptor that has been previously covalently targeted for molecular glue-based degradation of BRD4. Interestingly, we demonstrated that this covalent handle can be transplanted across a diverse array of protein-targeting ligands spanning many different protein classes to induce the degradation of CDK4, the androgen receptor, BTK, SMARCA2/4, and BCR-ABL/c-ABL. Our study reveals a DCAF16-based covalent degradative and linker-less chemical handle that can be attached to protein-targeting ligands to induce the degradation of several different classes of protein targets.","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":18.2,"publicationDate":"2024-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141063298","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-16DOI: 10.1021/acscentsci.4c00073
Yunsheng Li, Xiaoyu Bai and Dan Yang*,
Mitochondria are essential organelles involved in various metabolic processes in eukaryotes. The imaging, targeting, and investigation of cell death mechanisms related to mitochondria have garnered significant interest. Small-molecule fluorescent probes have proven to be robust tools for utilizing light to advance the study of mitochondrial biology. In this study, we present the rational design of cationic Nile blue probes carrying a permanent positive charge for these purposes. The cationic Nile blue probes exhibit excellent mitochondrial permeability, unique solvatochromism, and resistance to oxidation. We observed weaker fluorescence in aqueous solutions compared to lipophilic solvents, thereby minimizing background fluorescence in the cytoplasm. Additionally, we achieved photoredox switching of the cationic Nile blue probes under mild conditions. This enabled us to demonstrate their application for the first time in single-molecule localization microscopy of mitochondria, allowing us to observe mitochondrial fission and fusion behaviors. Compared to conventional cyanine fluorophores, this class of dyes demonstrated prolonged resistance to photobleaching, likely due to their antioxidation properties. Furthermore, we extended the application of cationic Nile blue probes to the mitochondria-specific delivery of taxanes, facilitating the study of direct interactions between the drug and organelles. Our approach to triggering cell death without reliance on microtubule binding provides valuable insights into anticancer drug research and drug-resistance mechanisms.
Fluorescent cationic Nile blue probes were developed for SMLM imaging of mitochondria in living cells and the specific targeting of the antitumor drug paclitaxel into mitochondria.
{"title":"Development and Application of Cationic Nile Blue Probes in Live-Cell Super-Resolution Imaging and Specific Targeting to Mitochondria","authors":"Yunsheng Li, Xiaoyu Bai and Dan Yang*, ","doi":"10.1021/acscentsci.4c00073","DOIUrl":"10.1021/acscentsci.4c00073","url":null,"abstract":"<p >Mitochondria are essential organelles involved in various metabolic processes in eukaryotes. The imaging, targeting, and investigation of cell death mechanisms related to mitochondria have garnered significant interest. Small-molecule fluorescent probes have proven to be robust tools for utilizing light to advance the study of mitochondrial biology. In this study, we present the rational design of cationic Nile blue probes carrying a permanent positive charge for these purposes. The cationic Nile blue probes exhibit excellent mitochondrial permeability, unique solvatochromism, and resistance to oxidation. We observed weaker fluorescence in aqueous solutions compared to lipophilic solvents, thereby minimizing background fluorescence in the cytoplasm. Additionally, we achieved photoredox switching of the cationic Nile blue probes under mild conditions. This enabled us to demonstrate their application for the first time in single-molecule localization microscopy of mitochondria, allowing us to observe mitochondrial fission and fusion behaviors. Compared to conventional cyanine fluorophores, this class of dyes demonstrated prolonged resistance to photobleaching, likely due to their antioxidation properties. Furthermore, we extended the application of cationic Nile blue probes to the mitochondria-specific delivery of taxanes, facilitating the study of direct interactions between the drug and organelles. Our approach to triggering cell death without reliance on microtubule binding provides valuable insights into anticancer drug research and drug-resistance mechanisms.</p><p >Fluorescent cationic Nile blue probes were developed for SMLM imaging of mitochondria in living cells and the specific targeting of the antitumor drug paclitaxel into mitochondria.</p>","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":12.7,"publicationDate":"2024-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acscentsci.4c00073","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140970125","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-15DOI: 10.1021/acscentsci.4c00094
Yan Liang, Tiancen Bian, Komal Yadav, Qixin Zhou, Liejin Zhou, Rui Sun and Zuxiao Zhang*,
1,4-cis-Disubstituted cyclic compounds play a pivotal role in pharmaceutical development, offering enhanced potency and bioavailability. However, their stereoselective and modular synthesis remains a long-standing challenge. Here, we report an innovative strategy for accessing these structures via mild conditions employing cyclic 1,3-dienes/alkyl(aryl)halides and amines. This procedure exhibits a wide substrate scope that tolerates various functional groups. The utility of this method is demonstrated in the efficient synthesis of a TRPV6 inhibitor, CFTR modulator, and other bioactive molecules. Combined experimental and computational studies suggest that the hybrid palladium-catalyzed radical-polar crossover mechanism is crucial for achieving exceptional 1,4-syn-addition selectivity (dr > 20:1).
Via hybrid palladium catalysis, we pioneered a redox neutral 1,4-syn-addition to 1,3-cyclic dienes for constructing diverse cyclic compounds, unlocking quick access to bioactive molecules.
{"title":"Selective 1,4-syn-Addition to Cyclic 1,3-Dienes via Hybrid Palladium Catalysis","authors":"Yan Liang, Tiancen Bian, Komal Yadav, Qixin Zhou, Liejin Zhou, Rui Sun and Zuxiao Zhang*, ","doi":"10.1021/acscentsci.4c00094","DOIUrl":"10.1021/acscentsci.4c00094","url":null,"abstract":"<p >1,4-cis-Disubstituted cyclic compounds play a pivotal role in pharmaceutical development, offering enhanced potency and bioavailability. However, their stereoselective and modular synthesis remains a long-standing challenge. Here, we report an innovative strategy for accessing these structures via mild conditions employing cyclic 1,3-dienes/alkyl(aryl)halides and amines. This procedure exhibits a wide substrate scope that tolerates various functional groups. The utility of this method is demonstrated in the efficient synthesis of a TRPV6 inhibitor, CFTR modulator, and other bioactive molecules. Combined experimental and computational studies suggest that the hybrid palladium-catalyzed radical-polar crossover mechanism is crucial for achieving exceptional 1,4-syn-addition selectivity (dr > 20:1).</p><p >Via hybrid palladium catalysis, we pioneered a redox neutral 1,4-syn-addition to 1,3-cyclic dienes for constructing diverse cyclic compounds, unlocking quick access to bioactive molecules.</p>","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":12.7,"publicationDate":"2024-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acscentsci.4c00094","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140973924","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-14DOI: 10.1021/acscentsci.4c00071
Azmain Alamgir, Souvik Ghosal, Matthew P. DeLisa* and Christopher A. Alabi*,
Protein-based therapeutics comprise a rapidly growing subset of pharmaceuticals, but enabling their delivery into cells for intracellular applications has been a longstanding challenge. To overcome the delivery barrier, we explored a reversible, bioconjugation-based approach to modify the surface charge of protein cargos with an anionic “cloak” to facilitate electrostatic complexation and delivery with lipid nanoparticle (LNP) formulations. We demonstrate that the conjugation of lysine-reactive sulfonated compounds can allow for the delivery of various protein cargos using FDA-approved LNP formulations of the ionizable cationic lipid DLin-MC3-DMA (MC3). We apply this strategy to functionally deliver RNase A for cancer cell killing as well as a full-length antibody to inhibit oncogenic β-catenin signaling. Further, we show that LNPs encapsulating cloaked fluorescent proteins distribute to major organs in mice following systemic administration. Overall, our results point toward a generalizable platform that can be employed for intracellular delivery of a wide range of protein cargos.
A bioconjugation-based approach to remodel protein surface charges using novel anionic tags allows for the functional delivery of proteins using lipid-based nanoparticle carriers.
{"title":"Bioreversible Anionic Cloaking Enables Intracellular Protein Delivery with Ionizable Lipid Nanoparticles","authors":"Azmain Alamgir, Souvik Ghosal, Matthew P. DeLisa* and Christopher A. Alabi*, ","doi":"10.1021/acscentsci.4c00071","DOIUrl":"10.1021/acscentsci.4c00071","url":null,"abstract":"<p >Protein-based therapeutics comprise a rapidly growing subset of pharmaceuticals, but enabling their delivery into cells for intracellular applications has been a longstanding challenge. To overcome the delivery barrier, we explored a reversible, bioconjugation-based approach to modify the surface charge of protein cargos with an anionic “cloak” to facilitate electrostatic complexation and delivery with lipid nanoparticle (LNP) formulations. We demonstrate that the conjugation of lysine-reactive sulfonated compounds can allow for the delivery of various protein cargos using FDA-approved LNP formulations of the ionizable cationic lipid DLin-MC3-DMA (MC3). We apply this strategy to functionally deliver RNase A for cancer cell killing as well as a full-length antibody to inhibit oncogenic β-catenin signaling. Further, we show that LNPs encapsulating cloaked fluorescent proteins distribute to major organs in mice following systemic administration. Overall, our results point toward a generalizable platform that can be employed for intracellular delivery of a wide range of protein cargos.</p><p >A bioconjugation-based approach to remodel protein surface charges using novel anionic tags allows for the functional delivery of proteins using lipid-based nanoparticle carriers.</p>","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":12.7,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acscentsci.4c00071","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140930694","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}