R-3 Repository :: Browsing by Author "Xiao, Han"

Browsing by Author "Xiao, Han"

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Bone-Specific Enhancement of Antibody Therapy for Breast Cancer Metastasis to Bone
(American Chemical Society, 2022) Tian, Zeru; Yu, Chenfei; Zhang, Weijie; Wu, Kuan-Lin; Wang, Chenhang; Gupta, Ruchi; Xu, Zhan; Wu, Ling; Chen, Yuda; Zhang, Xiang H.-F.; Xiao, Han
Despite the rapid evolution of therapeutic antibodies, their clinical efficacy in the treatment of bone tumors is hampered due to the inadequate pharmacokinetics and poor bone tissue accessibility of these large macromolecules. Here, we show that engineering therapeutic antibodies with bone-homing peptide sequences dramatically enhances their concentrations in the bone metastatic niche, resulting in significantly reduced survival and progression of breast cancer bone metastases. To enhance the bone tumor-targeting ability of engineered antibodies, we introduced varying numbers of bone-homing peptides into permissive sites of the anti-HER2 antibody, trastuzumab. Compared to the unmodified antibody, the engineered antibodies have similar pharmacokinetics and in vitro cytotoxic activity, but exhibit improved bone tumor distribution in vivo. Accordingly, in xenograft models of breast cancer metastasis to bone sites, engineered antibodies with enhanced bone specificity exhibit increased inhibition of both initial bone metastases and secondary multiorgan metastases. Furthermore, this engineering strategy is also applied to prepare bone-targeting antibody–drug conjugates with enhanced therapeutic efficacy. These results demonstrate that adding bone-specific targeting to antibody therapy results in robust bone tumor delivery efficacy. This provides a powerful strategy to overcome the poor accessibility of antibodies to the bone tumors and the consequential resistance to the therapy.
Breaking Through the Current Obstacles of Non-Canonical Amino Acid Technology
(2022-08-26) Wu, Kuan-Lin; Xiao, Han
Genetic code expansion (GCE) technology has enabled more than 200 chemically and structurally diverse non-canonical amino acids (ncAAs) to be site-specifically introduced to proteins with high specificity and fidelity. This technology has allowed us to study biological processes including structure, catalysis, transport, and defense at a new level of molecular precision. Despite the massive success of GCE technology, there are still deficiencies that restrict its utility. First, high level of ncAA supplement is required for stop codon suppression, which is ineffective and eco-unfriendly. Second, there are only limited chemical functionalities that could be incorporated into proteins by GCE. Third, lots of reported ncAA only showed the genetic incorporation without demonstrating applications in solving actual biological questions. In this dissertation, we describe the above limitations of GCE technology and provide new insights and progressions. First, multiple “completely autonomous” species that possess the biosynthetic and translational machinery for making proteins that contain the 21st amino acid: O-methyltyrosine have been generated. We demonstrated that the endogenous biosynthesis of ncAA reaches higher intracellular ncAA concentration than attained through exogenous feeding, leading to greater genetic incorporation efficiency. Importantly, we showed that the limited bioavailability of exogenously fed ncAAs in multicellular systems can also be overcome by endogenous biosynthesis. The advantages of the autonomous system not only allow for enhanced efficiency of ncAA incorporation but also open new opportunities in multicellular systems for encoding ncAAs with poor bioavailability. Second, to expand the chemical toolbox of GCE technology, we designed and synthesized an isocyanide-containing ncAA: ε-N-2-isocyanoisobutyryl-lysine (NCibK). A mutant pyrrolysine tRNA synthetase HibK-1RS/tRNAPyl CUA pair that specifically recognize NCibK was identified. Several bioconjugation reactions at both small molecule and protein level have been demonstrated, showing the robustness of the methodology. The ease of synthesis, versatility of reactivity, and good compatibility of isocyanides make NCibK an attractive ncAA for future biological applications. Third, we demonstrate new applications for the reported ncAA 4-fluorophenyl lysine (FPheK) to resolve current limitations in cancer therapeutics and its production. A methodology to synthesize a bispecific small molecule - antibody conjugate from a commercially available antibody without protein engineering was developed. This methodology provides a powerful platform to generate bispecific agents by conjugating small-molecule-like ligands targeting different membrane markers to human antibodies, or to generate various kinds of antibody drug conjugates (ADC). In summary, the work in this dissertation has shown advances in every aspect of current obstacles in non-canonical amino acid technology.
Creation of Cells with Endogenous Noncanonical Amino Acids for Genetic Incorporation
(2022-04-14) Chen, Yuda; Xiao, Han
While most organisms utilize 20 canonical amino acid building blocks for protein synthesis, adding additional noncanonical amino acids (ncAAs) to the amino acid repertoire can greatly facilitate the investigation of protein structures and functions. Genetic incorporation of ncAAs requires 3 key components: a “blank” (normally non-coding) codon, a biorthogonal translational system and sufficient amount of intracellular ncAAs. Despite significant efforts focused on biorthogonal machinery and available codon for encoding ncAAs, current methodologies to reach sufficient amount of cellular ncAAs have largely relied on the exogenous feeding of chemically-synthesized ncAAs and successful uptake of these ncAAs by cells. The requirement of external addition of ncAAs limits the development of genetic code expansion in its efficiency as well as applicability in both academic and industrial settings. To solve this limitation, creation of completely autonomous cells with both biosynthetic pathway and genetic incorproation mechinery for ncAAs has been accomplished in this thesis. Before engineering cells with novel ncAA as its 21st amino acid, the reported bacterial cells with p-aminophenylalanine (pAF) as its 21st amino acid was optimized and its application to produce proteins with site-specific modifications was explored. Two bacterial cells with novel ncAAs (5-hydroxyltryptophan and 3,4-dihydroxylphenylalanine) as its 21st amino acid have been created by introducing external biosynthetic pathways reported in the literature and their corresponding genetic incorporation machineries. Comparing with classical genetic expansion technology, cells with those ncAAs as their 21st amino acid have been demonstrated with superior ability to detect oxidative stress and prepare site-specific conjugate of therapeutic proteins. Beyond the usage of reported biosynthetic way, a novel sulfotransferase was discovered from bioinformatics for biosynthesizing sulfotysoine (sTyr), which is inefficient to penetrate cell membrane. As an important protein post-translational modification, sTyr produced in situ could be genetically incorporated into proteins in both prokaryotic and eukaryotic cells with higher efficiency than classical genetic code expansion based on exogenous addition of sTyr. The cells with sTyr as its 21st amino acid was applied to produce thrombin inhibitors with enhanced affinity. As another component towards creation of cells with a 21st amino acid, the genetic incorporation machineries for two isocyanide-containing ncAAs were discovered and the genetic incorporation of this novel functional group was used for protein activation and site-specific conjugation.
Harnessing the power of antibodies to fight bone metastasis
(AAAS, 2021) Tian, Zeru; Wu, Ling; Yu, Chenfei; Chen, Yuda; Xu, Zhan; Bado, Igor; Loredo, Axel; Wang, Lushun; Wang, Hai; Wu, Kuan-Lin; Zhang, Weijie; Zhang, Xiang H.-F.; Xiao, Han
Antibody-based therapies have proved to be of great value in cancer treatment. Despite the clinical success of these biopharmaceuticals, reaching targets in the bone microenvironment has proved to be difficult due to the relatively low vascularization of bone tissue and the presence of physical barriers. Here, we have used an innovative bone-targeting (BonTarg) technology to generate a first-in-class bone-targeting antibody. Our strategy involves the use of pClick antibody conjugation technology to chemically couple the bone-targeting moiety bisphosphonate to therapeutic antibodies. Bisphosphonate modification of these antibodies results in the delivery of higher conjugate concentrations to the bone metastatic niche, relative to other tissues. In xenograft mice models, this strategy provides enhanced inhibition of bone metastases and multiorgan secondary metastases that arise from bone lesions. Specific delivery of therapeutic antibodies to the bone, therefore, represents a promising strategy for the treatment of bone metastatic cancers and other bone diseases. Precision modification of antibodies with bone-targeting moieties unleashes their potential for the treatment of bone metastases. Precision modification of antibodies with bone-targeting moieties unleashes their potential for the treatment of bone metastases.
Oxime as a general photocage for the design of visible light photo-activatable fluorophores
(Royal Society of Chemistry, 2021) Wang, Lushun; Wang, Shichao; Tang, Juan; Espinoza, Vanessa B.; Loredo, Axel; Tian, Zeru; Weisman, R. Bruce; Xiao, Han
Photoactivatable fluorophores have been widely used for tracking molecular and cellular dynamics with subdiffraction resolution. In this work, we have prepared a series of photoactivatable probes using the oxime moiety as a new class of photolabile caging group in which the photoactivation process is mediated by a highly efficient photodeoximation reaction. Incorporation of the oxime caging group into fluorophores results in loss of fluorescence. Upon light irradiation in the presence of air, the oxime-caged fluorophores are oxidized to their carbonyl derivatives, restoring strong fluorophore fluorescence. To demonstrate the utility of these oxime-caged fluorophores, we have created probes that target different organelles for live-cell confocal imaging. We also carried out photoactivated localization microscopy (PALM) imaging under physiological conditions using low-power light activation in the absence of cytotoxic additives. Our studies show that oximes represent a new class of visible-light photocages that can be widely used for cellular imaging, sensing, and photo-controlled molecular release.
Protein target highlights in CASP15: Analysis of models by structure providers
(Wiley, 2023) Alexander, Leila T.; Durairaj, Janani; Kryshtafovych, Andriy; Abriata, Luciano A.; Bayo, Yusupha; Bhabha, Gira; Breyton, Cécile; Caulton, Simon G.; Chen, James; Degroux, Séraphine; Ekiert, Damian C.; Erlandsen, Benedikte S.; Freddolino, Peter L.; Gilzer, Dominic; Greening, Chris; Grimes, Jonathan M.; Grinter, Rhys; Gurusaran, Manickam; Hartmann, Marcus D.; Hitchman, Charlie J.; Keown, Jeremy R.; Kropp, Ashleigh; Kursula, Petri; Lovering, Andrew L.; Lemaitre, Bruno; Lia, Andrea; Liu, Shiheng; Logotheti, Maria; Lu, Shuze; Markússon, Sigurbjörn; Miller, Mitchell D.; Minasov, George; Niemann, Hartmut H.; Opazo, Felipe; Phillips Jr, George N.; Davies, Owen R.; Rommelaere, Samuel; Rosas-Lemus, Monica; Roversi, Pietro; Satchell, Karla; Smith, Nathan; Wilson, Mark A.; Wu, Kuan-Lin; Xia, Xian; Xiao, Han; Zhang, Wenhua; Zhou, Z. Hong; Fidelis, Krzysztof; Topf, Maya; Moult, John; Schwede, Torsten
We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.
Single-atom replacement as a general approach towards visible-light/near-infrared heavy-atom-free photosensitizers for photodynamic therapy
(Royal Society of Chemistry, 2020) Tang, Juan; Wang, Lushun; Loredo, Axel; Cole, Carson C.; Xiao, Han
Photodynamic therapy has become an emerging strategy for the treatment of cancer. This technology relies on the development of photosensitizers (PSs) that convert molecular oxygen to cytotoxic reactive oxygen species upon exposure to light. In this study, we have developed a facile and general strategy for obtaining visible light/near-infrared-absorbing PSs by performing a simple sulfur-for-oxygen replacement within existing fluorophores. Thionation of carbonyl groups within existing fluorophore cores leads to an improvement of the singlet oxygen quantum yield and molar absorption coefficient at longer wavelengths (deep to 600–800 nm). Additionally, these thio-based PSs lack dark cytotoxicity but exhibit significant phototoxicity against monolayer cancer cells and 3D multicellular tumor spheroids with IC50 in the micromolar range. To achieve tumor-specific delivery, we have conjugated these thio-based PSs to an antibody and demonstrated their tumor-specific therapeutic activity.
Synthesis and application of caged-fluorescent probes
(2022-04-20) Loredo Pineda, Axel Alfredo; Xiao, Han
This dissertation describes the design, synthesis, and applications of photoactivatable fluorescent probes in the visible light region. Visible light-activated fluorescent probes are highly desirable due to the spatiotemporal control and widely available light sources. The use of photoactivatable probes in biological systems is optimal as the activation method is straightforward and non-invasive. A single atom substitution in fluorescent dyes has been demonstrated to efficiently generate lightactivatable fluorogenic cages. This methodology has been developed for use in superresolution imaging, photodynamic therapy, and inorganic mercury detection. The development of fluorogenic tetrazine probes allows the design of probes that are not limited to a single fluorophore core; it was then demonstrated that visible light could cleave the tetrazine moiety in fluorophore-conjugated dyes. The introduction of 3- methyilthio tetrazines improved turn-on efficiency and redshifted the wavelength of dissociation. The organization of this thesis is as follows: Chapter 1 will introduce photoremovable protecting groups ranging from UV to far red. The applications of caged groups are discussed in the fields of synthesis and biology. Chapter 2 describes the use of s-tetrazines as photocages. Light-activated fluorescence affords a powerful tool for monitoring subcellular structures and dynamics with enhanced temporal and spatial control of the fluorescence signal. Here, we demonstrate a general and straightforward strategy for using a tetrazine phototrigger to design photoactivatable fluorophores that emit across the visible spectrum. Tetrazine is known to efficiently quench the fluorescence of various fluorophores via a mechanism referred to as through-bond energy transfer. Upon light irradiation, restricted tetrazine moieties undergo a photolysis reaction that generates two nitriles and molecular nitrogen, thus restoring the fluorescence of fluorophores. Significantly, we find that this strategy can be successfully translated and generalized to a wide range of fluorophore scaffolds. Based on these results, we have used this mechanism to design photoactivatable fluorophores targeting cellular organelles and proteins. Compared to widely used phototriggers (e.g., o-nitrobenzyl and nitrophenethyl groups), this study affords a new photoactivation mechanism, in which the quencher is photodecomposed to restore the fluorescence upon light irradiation. Because of the exclusive use of tetrazine as a photoquencher in the design of fluorogenic probes, we anticipate that our current study will significantly facilitate the development of novel photoactivatable fluorophores. Chapter 3 provides further insight in the development of tetrazine photocages. The photolysis rates of carbon and sulfur tetrazines were obtained, showing a redshift in the photolysis wavelength when sulfur substitution is incorporated. This strategy was further implemented in through bond energy transfer tetrazine cassettes, with a one-pot methodology to convert carboxylic esters into 3-methylthio tetrazines. Higher turn-on ratios were obtained compared to 3-methyl capped tetrazine fluorophore counterparts. This strategy allowed the use of green light to unmask BODIPY based dyes. Chapter 4 will introduce the design and synthesis of a single-atom fluorescence switch. Applications of this methodology in cell imaging and photodynamic therapy are discussed. Chapter 5 of this thesis will detail the application of thionated fluorophores in the detection of inorganic mercury. By performing a single-atom replacement within common fluorophores, we have developed a facile and general strategy to prepare a broad-spectrum class of colorimetric and fluorogenic probes for the selective detection of mercury ions in aqueous environments. Thionation of carbonyl groups from existing fluorophore cores results in a great reduction of fluorescence. In the presence of mercury ions, the resulting thiocaged probes are efficiently desulfurized to their oxo derivatives, leading to pronounced changes in chromogenic and fluorogenic signals. Because these probes exhibit high selectivity, excellent sensitivity, good membrane-permeability, and rapid responses towards mercury ions, they are suitable for visualization of mercury in both aqueous and intracellular environments.
Synthesis of precision antibody conjugates using proximity-induced chemistry
(Ivyspring, 2021) Cao, Yu J.; Yu, Chenfei; Wu, Kuan-Lin; Wang, Xuechun; Liu, Dong; Tian, Zeru; Zhao, Lijun; Qi, Xuexiu; Loredo, Axel; Chung, Anna; Xiao, Han
Rationale: Therapeutic antibody conjugates allow for the specific delivery of cytotoxic agents or immune cells to tumors, thus enhancing the antitumor activity of these agents and minimizing adverse systemic effects. Most current antibody conjugates are prepared by nonspecific modification of antibody cysteine or lysine residues, inevitably resulting in the generation of heterogeneous conjugates with limited therapeutic efficacies. Traditional strategies to prepare homogeneous antibody conjugates require antibody engineering or chemical/enzymatic treatments, processes that often affect antibody folding and stability, as well as yield and cost. Developing a simple and cost-effective way to precisely couple functional payloads to native antibodies is of great importance. Methods: We describe a simple proximity-induced antibody conjugation method (pClick) that enables the synthesis of homogeneous antibody conjugates from native antibodies without requiring additional antibody engineering or post-synthesis treatments. A proximity-activated crosslinker is introduced into a chemically synthesized affinity peptide modified with a bioorthogonal handle. Upon binding to a specific antibody site, the affinity peptide covalently attaches to the antibody via spontaneous crosslinking, yielding an antibody molecule ready for bioorthogonal conjugation with payloads. Results: We have prepared well-defined antibody-drug conjugates and bispecific small molecule-antibody conjugates using pClick technology. The resulting conjugates exhibit excellent in vitro cytotoxic activity against cancer cells and, in the case of bispecific conjugates, superb antitumor activity in mouse xenograft models. Conclusions: Our pClick technology enables efficient, simple, and site-specific conjugation of various moieties to the existing native antibodies. This technology does not require antibody engineering or additional UV/chemical/enzymatic treatments, therefore providing a general, convenient strategy for developing novel antibody conjugates.
Unleashing the potential of noncanonical amino acid biosynthesis to create cells with precision tyrosine sulfation
(Springer Nature, 2022) Chen, Yuda; Jin, Shikai; Zhang, Mengxi; Hu, Yu; Wu, Kuan-Lin; Chung, Anna; Wang, Shichao; Tian, Zeru; Wang, Yixian; Wolynes, Peter G.; Xiao, Han; Center for Theoretical Biological Physics
Despite the great promise of genetic code expansion technology to modulate structures and functions of proteins, external addition of ncAAs is required in most cases and it often limits the utility of genetic code expansion technology, especially to noncanonical amino acids (ncAAs) with poor membrane internalization. Here, we report the creation of autonomous cells, both prokaryotic and eukaryotic, with the ability to biosynthesize and genetically encode sulfotyrosine (sTyr), an important protein post-translational modification with low membrane permeability. These engineered cells can produce site-specifically sulfated proteins at a higher yield than cells fed exogenously with the highest level of sTyr reported in the literature. We use these autonomous cells to prepare highly potent thrombin inhibitors with site-specific sulfation. By enhancing ncAA incorporation efficiency, this added ability of cells to biosynthesize ncAAs and genetically incorporate them into proteins greatly extends the utility of genetic code expansion methods.
Visible light-activated dyes and methods of use thereof
(2024-02-27) Xiao, Han; Tang, Juan; Rice University; United States Patent and Trademark Office
Disclosed herein are photoactivable fluorophores comprising one or more thiocarbonyl groups as well as conjugates and compositions thereof. The present disclosure also provides methods of preparing photoactivatable fluorophores as well as methods of imaging using the photoactivatable fluorophores, conjugates, and compositions of the present disclosure.
Xanthone-based solvatochromic fluorophores for quantifying micropolarity of protein aggregates
(Royal Society of Chemistry, 2022) Wang, Lushun; Hsiung, Chia-Heng; Liu, Xiaojing; Wang, Shichao; Loredo, Axel; Zhang, Xin; Xiao, Han
Proper three-dimensional structures are essential for maintaining the functionality of proteins and for avoiding pathological consequences of improper folding. Misfolding and aggregation of proteins have been both associated with neurodegenerative disease. Therefore, a variety of fluorogenic tools that respond to both polarity and viscosity have been developed to detect protein aggregation. However, the rational design of highly sensitive fluorophores that respond solely to polarity has remained elusive. In this work, we demonstrate that electron-withdrawing heteroatoms with (d–p)–π* conjugation can stabilize lowest unoccupied molecular orbital (LUMO) energy levels and promote bathochromic shifts. Guided by computational analyses, we have devised a novel series of xanthone-based solvatochromic fluorophores that have rarely been systematically studied. The resulting probes exhibit superior sensitivity to polarity but are insensitive to viscosity. As proof of concept, we have synthesized protein targeting probes for live-cell confocal imaging intended to quantify the polarity of misfolded and aggregated proteins. Interestingly, our results reveal several layers of protein aggregates in a way that we had not anticipated. First, microenvironments with reduced polarity were validated in the misfolding and aggregation of folded globular proteins. Second, granular aggregates of AgHalo displayed a less polar environment than aggregates formed by folded globular protein represented by Htt–polyQ. Third, our studies reveal that granular protein aggregates formed in response to different types of stressors exhibit significant polarity differences. These results show that the solvatochromic fluorophores solely responsive to polarity represent a new class of indicators that can be widely used for detecting protein aggregation in live cells, thus paving the way for elucidating cellular mechanisms of protein aggregation as well as therapeutic approaches to managing intracellular aggregates.
ZipA Uses a Two-Pronged FtsZ-Binding Mechanism Necessary for Cell Division
(American Society for Microbiology, 2021) Cameron, Todd A.; Vega, Daniel E.; Yu, Chenfei; Xiao, Han; Margolin, William
In most bacteria, cell division is centrally organized by the FtsZ protein, which assembles into dynamic filaments at the division site along the cell membrane that interact with other key cell division proteins. In gammaproteobacteria such as Escherichia coli, FtsZ filaments are anchored to the cell membrane by two essential proteins, FtsA and ZipA. Canonically, this interaction was believed to be mediated solely by the FtsZ C-terminal peptide (CTP) domain that interacts with these and several other regulatory proteins. However, we now provide evidence of a second interaction between FtsZ and ZipA. Using site-specific photoactivated cross-linking, we identified a noncanonical FtsZ-binding site on ZipA on the opposite side from the FtsZ CTP-binding pocket. Cross-linking at this site was unaffected by the truncation of the FtsZ linker and CTP domains, indicating that this noncanonical site must interact directly with the globular core domain of FtsZ. Mutations introduced into either the canonical or noncanonical binding sites on ZipA disrupted photo-cross-linking with FtsZ and normal ZipA function in cell division, suggesting that both binding modes are important for normal cell growth and division. One mutation at the noncanonical face was also found to suppress defects of several other canonical and noncanonical site mutations in ZipA, suggesting there is some interdependence between the two sites. Taken together, these results suggest that ZipA employs a two-pronged FtsZ-binding mechanism.

Browsing by Author "Xiao, Han"

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