Covalent Proteins as Targeted Radionuclide Therapies Enhance Antitumor Effects.

Molecularly targeted radionuclide therapies (TRTs) struggle with balancing efficacy and safety, as current strategies to increase tumor absorption often alter drug pharmacokinetics to prolong circulation and normal tissue irradiation. Here we report the first covalent protein TRT, which, through reacting with the target irreversibly, increases radioactive dose to the tumor without altering the drug's pharmacokinetic profile or normal tissue biodistribution. Through genetic code expansion, we engineered a latent bioreactive amino acid into a nanobody, which binds to its target protein and forms a covalent linkage via the proximity-enabled reactivity, cross-linking the target irreversibly in vitro, on cancer cells, and on tumors in vivo. The radiolabeled covalent nanobody markedly increases radioisotope levels in tumors and extends tumor residence time while maintaining rapid systemic clearance. Furthermore, the covalent nanobody conjugated to the α-emitter actinium-225 inhibits tumor growth more effectively than the noncovalent nanobody without causing tissue toxicity. Shifting the protein-based TRT from noncovalent to covalent mode, this chemical strategy improves tumor responses to TRTs and can be readily scaled to diverse protein radiopharmaceuticals engaging broad tumor targets.

Advanced flow cytometry for biomedical applications.

Flow cytometry (FC) is a versatile tool with excellent capabilities to detect and measure multiple characteristics of a population of cells or particles. Notable advancements in in vivo photoacoustic FC, coherent Raman FC, microfluidic FC, and so on, have been achieved in the last two decades, which endows FC with new functions and expands its applications in basic research and clinical practice. Advanced FC broadens the tools available to researchers to conduct research involving cancer detection, microbiology (COVID-19, HIV, bacteria, etc.), and nucleic acid analysis. This review presents an overall picture of advanced flow cytometers and provides not only a clear understanding of their mechanisms but also new insights into their practical applications. We identify the latest trends in this area and aim to raise awareness of advanced techniques of FC. We hope this review expands the applications of FC and accelerates its clinical translation.

Genetically encoded chemical crosslinking of carbohydrate.

Protein-carbohydrate interactions play important roles in various biological processes, such as organism development, cancer metastasis, pathogen infection and immune response, but they remain challenging to study and exploit due to their low binding affinity and non-covalent nature. Here we site-specifically engineered covalent linkages between proteins and carbohydrates under biocompatible conditions. We show that sulfonyl fluoride reacts with glycans via a proximity-enabled reactivity, and to harness this a bioreactive unnatural amino acid (SFY) that contains sulfonyl fluoride was genetically encoded into proteins. SFY-incorporated Siglec-7 crosslinked with its sialoglycan ligand specifically in vitro and on the surface of cancer cells. Through irreversible cloaking of sialoglycan at the cancer cell surface, SFY-incorporated Siglec-7 enhanced the killing of cancer cells by natural killer cells. Genetically encoding the chemical crosslinking of proteins to carbohydrates (GECX-sugar) offers a solution to address the low affinity and weak strength of protein-sugar interactions.

Prodrugs of Persulfide and Sulfide: Is There a Pharmacological Difference between the Two in the Context of Rapid Exchanges among Various Sulfur Species In Vivo?

Sulfide and persulfide are chemically different and one might expect persulfide to be more effective in mediating sulfur signaling because persulfide can directly modify protein cysteine residue. However, rapid scrambling, and interconversions occur among sulfur species. Then there is the question of whether the chemical reactivity differences between sulfide and persulfide would translate into pharmacological differences. Utilizing a delivery system to generate pure hydrogen sulfide (H2 S), hydrogen persulfide (H2 S2 ), and N-acetyl-l-cysteine persulfide (N-CysSSH), we examined the activities of sulfide and persulfide in vitro and in vivo. Persulfide prodrugs exhibited increased activities compared to the H2 S prodrug. In particular, the H2 S2 prodrug offers much-elevated analgesic effects compared to the H2 S prodrug in vivo. Persulfide prodrugs also possess a reduced level of toxicity compared to the H2 S prodrug in vivo, indicating persulfide might represent a better therapeutic paradigm than H2 S.

A Genetically Encoded Fluorosulfonyloxybenzoyl-l-lysine for Expansive Covalent Bonding of Proteins via SuFEx Chemistry.

Genetically introducing novel chemical bonds into proteins provides innovative avenues for biochemical research, protein engineering, and biotherapeutic applications. Recently, latent bioreactive unnatural amino acids (Uaas) have been incorporated into proteins to covalently target natural residues through proximity-enabled reactivity. Aryl fluorosulfate is particularly attractive due to its exceptional biocompatibility and multitargeting capability via sulfur(VI) fluoride exchange (SuFEx) reaction. Thus far, fluorosulfate-l-tyrosine (FSY) is the only aryl fluorosulfate-containing Uaa that has been genetically encoded. FSY has a relatively rigid and short side chain, which restricts the diversity of proteins targetable and the scope of applications. Here we designed and genetically encoded a new latent bioreactive Uaa, fluorosulfonyloxybenzoyl-l-lysine (FSK), in E. coli and mammalian cells. Due to its long and flexible aryl fluorosulfate-containing side chain, FSK was particularly useful in covalently linking protein sites that are unreachable with FSY, both intra- and intermolecularly, in vitro and in live cells. In addition, we created covalent nanobodies that irreversibly bound to epidermal growth factor receptors (EGFR) on cells, with FSK and FSY targeting distinct positions on EGFR to counter potential mutational resistance. Moreover, we established the use of FSK and FSY for genetically encoded chemical cross-linking to capture elusive enzyme-substrate interactions in live cells, allowing us to target residues aside from Cys and to cross-link at the binding periphery. FSK complements FSY to expand target diversity and versatility. Together, they provide a powerful, genetically encoded, latent bioreactive SuFEx system for creating covalent bonds in diverse proteins in vitro and in vivo, which will be widely useful for biological research and applications.

Mild cognitive impairment understanding: an empirical study by data-driven approach.

BACKGROUND: Cognitive decline has emerged as a significant threat to both public health and personal welfare, and mild cognitive decline/impairment (MCI) can further develop into Dementia/Alzheimer's disease. While treatment of Dementia/Alzheimer's disease can be expensive and ineffective sometimes, the prevention of MCI by identifying modifiable risk factors is a complementary and effective strategy. RESULTS: In this study, based on the data collected by Centers for Disease Control and Prevention (CDC) through the nationwide telephone survey, we apply a data-driven approach to re-exam the previously founded risk factors and discover new risk factors. We found that depression, physical health, cigarette usage, education level, and sleep time play an important role in cognitive decline, which is consistent with the previous discovery. Besides that, the first time, we point out that other factors such as arthritis, pulmonary disease, stroke, asthma, marital status also contribute to MCI risk, which is less exploited previously. We also incorporate some machine learning and deep learning algorithms to weigh the importance of various factors contributed to MCI and predicted cognitive declined. CONCLUSION: By incorporating the data-driven approach, we can determine that risk factors significantly correlated with diseases. These correlations could also be expanded to another medical diagnosis besides MCI.

Click and Release: A High-Content Bioorthogonal Prodrug with Multiple Outputs.

A high-content bioorthogonal prodrug with multiple outputs using the "click, cyclize, and release" concept is described. The proof of concept is established by the co-delivery of a gasotransmitter carbon monoxide, an anticancer drug floxuridine, and an in situ generated fluorescent reporter molecule for real-time monitoring of the prodrug activation. Bioorthogonal prodrugs as such are invaluable tools for the co-delivery of other drug payloads for multimodal therapy.

Enrichment-triggered prodrug activation demonstrated through mitochondria-targeted delivery of doxorubicin and carbon monoxide.

Controlled activation is a critical component in prodrug development. Here we report a concentration-sensitive platform approach for bioorthogonal prodrug activation by taking advantage of reaction kinetics. Using two 'click and release' systems, we demonstrate enrichment and prodrug activation specifically in mitochondria to demonstrate the principle of the approach. In both cases, the payload (doxorubicin or carbon monoxide) was released inside the mitochondrial matrix following the enrichment-initiated click reaction. Furthermore, mitochondria-targeted delivery yielded substantial augmentation of functional biological and therapeutic effects in vitro and in vivo when compared to controls, which did not result in enrichment. This method is thus a platform for targeted drug delivery that is amenable to conjugation with a variety of molecules and is not limited to cell-surface delivery. Taken together, these two 'click and release' pairs clearly demonstrate the concept of enrichment-triggered drug release and the critical feasibility of treating clinically relevant diseases such as acute liver injury and cancer.

Biomarker-Based Metabolic Labeling for Redirected and Enhanced Immune Response.

Installation of an antibody-recruiting moiety on the surface of disease-relevant cells can lead to the selective destruction of targets by the immune system. Such an approach can be an alternative strategy to traditional chemotherapeutics in cancer therapy and possibly other diseases. Herein we describe the development of a new strategy to selectively label targets with an antibody-recruiting moiety through its covalent and stable installation, complementing existing methods of employing reversible binding. This is achieved through selective delivery of 1,3,4- O-acetyl- N-azidoacetylmannosamine (Ac3ManNAz) to folate receptor-overexpressing cells using an Ac3ManNAz-folate conjugate via a cleavable linker. As such, Ac3ManNAz is converted to cell surface glycan bearing an azido group, which serves as an anchor to introduce l-rhamnose (Rha), a hapten, via a click reaction with aza-dibenzocyclooctyne (DBCO)-Rha. We tested this method in several cell lines including KB, HEK-293, and MCF7 and were able to demonstrate the following: 1) Rha can be selectively installed to the folate receptor overexpressing cell surface and 2) the Rha installed on the target surface can recruit anti-rhamnose (anti-Rha) antibodies, leading to the destruction of target cells via complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP).

An esterase-activated click and release approach to metal-free CO-prodrugs.

One major challenge in the development of CO as a therapeutic agent is its controllable delivery in a pharmaceutically acceptable form. Herein, we describe for the first time a general chemical strategy to esterase-sensitive organic CO-prodrugs.