Arginine Accelerates Sulfur Fluoride Exchange and Phosphorus Fluoride Exchange Reactions between Proteins.

Sulfur fluoride exchange (SuFEx) and phosphorus fluoride exchange (PFEx) click chemistries are advancing research across multiple disciplines. By genetically incorporating latent bioreactive unnatural amino acids (Uaas), these chemistries have been integrated into proteins, enabling precise covalent linkages with biological macromolecules and paving the way for new applications. However, their suboptimal reaction rates in proteins limit effectiveness, and traditional catalytic methods for small molecules are often incompatible with biological systems or in vivo applications. We demonstrated that introducing an arginine adjacent to the latent bioreactive Uaa significantly boosts SuFEx and PFEx reaction rates between proteins. This method is effective across various Uaas, target residues, and protein environments. Notably, it also enables efficient SuFEx reactions in acidic conditions, common in certain cellular compartments and tumor microenvironments, which typically hinder SuFEx reactions. Furthermore, we developed the first covalent cell engager that substantially enhances natural killer cell activation through improved covalent interaction facilitated by arginine. These findings provide mechanistic insights and offer a biocompatible strategy to harness these robust chemistries for advancing biological research and developing new biotherapeutics.

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.

Novel Hydrogen Sulfide (H2S)-Releasing BW-HS-101 and Its Non-H2S Releasing Derivative in Modulation of Microscopic and Molecular Parameters of Gastric Mucosal Barrier.

Hydrogen sulfide (H2S) is an endogenously produced molecule with anti-inflammatory and cytoprotective properties. We aimed to investigate for the first time if a novel, esterase-sensitive H2S-prodrug, BW-HS-101 with the ability to release H2S in a controllable manner, prevents gastric mucosa against acetylsalicylic acid-induced gastropathy on microscopic and molecular levels. Wistar rats were pretreated intragastrically with vehicle, BW-HS-101 (0.5-50 µmol/kg) or its analogue without the ability to release H2S, BW-iHS-101 prior to ASA administration (125 mg/kg, intragastrically). BW-HS-101 was administered alone or in combination with nitroarginine (L-NNA, 20 mg/kg, intraperitoneally) or zinc protoporphyrin IX (10 mg/kg, intraperitoneally). Gastroprotective effects of BW-HS-101 were additionally evaluated against necrotic damage induced by intragastrical administration of 75% ethanol. Gastric mucosal damage was assessed microscopically, and gastric blood flow was determined by laser flowmetry. Gastric mucosal DNA oxidation and PGE2 concentration were assessed by ELISA. Serum and/or gastric protein concentrations of IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-13, VEGF, GM-CSF, IFN-γ, TNF-α, and EGF were determined by a microbeads/fluorescent-based multiplex assay. Changes in gastric mucosal iNOS, HMOX-1, SOCS3, IL1-R1, IL1-R2, TNF-R2, COX-1, and COX-2 mRNA were assessed by real-time PCR. BW-HS-101 or BW-iHS-101 applied at a dose of 50 µmol/kg protected gastric mucosa against ASA-induced gastric damage and prevented a decrease in the gastric blood flow level. H2S prodrug decreased DNA oxidation, systemic and gastric mucosal inflammation with accompanied upregulation of SOCS3, and EGF and HMOX-1 expression. Pharmacological inhibition of nitric oxide (NO) synthase but not carbon monoxide (CO)/heme oxygenase (HMOX) activity by L-NNA or ZnPP, respectively, reversed the gastroprotective effect of BW-HS-101. BW-HS-101 also protected against ethanol-induced gastric injury formation. We conclude that BW-HS-101, due to its ability to release H2S in a controllable manner, prevents gastric mucosa against drugs-induced gastropathy, inflammation and DNA oxidation, and upregulate gastric microcirculation. Gastroprotective effects of this H2S prodrug involves endogenous NO but not CO activity and could be mediated by cytoprotective and anti-inflammatory SOCS3 and EGF pathways.

Making smart drugs smarter: The importance of linker chemistry in targeted drug delivery.

Smart drugs, such as antibody-drug conjugates, for targeted therapy rely on the ability to deliver a warhead to the desired location and to achieve activation at the same site. Thus, designing a smart drug often requires proper linker chemistry for tethering the warhead with a vehicle in such a way that either allows the active drug to retain its potency while being tethered or ensures release and thus activation at the desired location. Recent years have seen much progress in the design of new linker activation strategies. Herein, we review the recent development of chemical strategies used to link the warhead with a delivery vehicle for preferential cleavage at the desired sites.

Genetically encoding thyronine for fluorescent detection of peroxynitrite.

Peroxynitrite is a highly reactive oxidant effecting cell signaling and cell death. Here we report a fluorescent protein probe to selectively detect peroxynitrite. A novel unnatural amino acid, thyronine (Thy), was genetically encoded in E. coli and mammalian cells by evolving an orthogonal tRNAPyl/ThyRS pair. Incorporation of Thy into the chromophore of sfGFP or cpsGFP afforded a virtually non-fluorescent reporter. Upon treatment with peroxynitrite, Thy was converted into tyrosine via O-dearylation, regenerating GFP fluorescence in a time- and concentration-dependent manner. Genetically encoded thyronine may also be valuable for other redox applications.

Click and release: bioorthogonal approaches to "on-demand" activation of prodrugs.

Prodrug approaches represent an excellent solution to certain pharmaceutical issues commonly encountered in the drug discovery and development process. Along this line, the chemistry needed for the bio-reversible derivatization of drug functional groups for on-demand release is critical. In recent years, "click and release" approaches have shown great promise in the design of prodrugs because of their bioorthogonality and controlled bond-cleavage, which help ensure prodrug stability during circulation and ready cleavage at the desired site of action. This review highlights recent developments of this research field and discusses issues yet to be addressed.

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.

Toward Hydrogen Sulfide Based Therapeutics: Critical Drug Delivery and Developability Issues.

Hydrogen sulfide (H2 S), together with nitric oxide (NO) and carbon monoxide (CO), belongs to the gasotransmitter family and plays important roles in mammals as a signaling molecule. Many studies have also shown the various therapeutic effects of H2 S, which include protection against myocardial ischemia injury, cytoprotection against oxidative stress, mediation of neurotransmission, inhibition of insulin signaling, regulation of inflammation, inhibition of the hypoxia-inducible pathway, and dilation of blood vessels. One major challenge in the development of H2 S-based therapeutics is its delivery. In this manuscript, we assess the various drug delivery strategies in the context of being used research tools and eventual developability as therapeutic agents.

Toward Direct Protein S-Persulfidation: A Prodrug Approach That Directly Delivers Hydrogen Persulfide.

A general strategy of delivering hydrogen persulfide (H2S2) is described herein. Esterase- and phosphatase-sensitive H2S2 prodrugs with tunable release rates have been synthesized. Their utility is validated in examining protein S-persulfidation. With this unique approach of directly delivering H2S2, our findings reaffirmed that S-persulfidation leads to decreased activity of glyceraldehyde 3-phosphate dehydrogenase. This new approach complements available prodrugs/donors that directly deliver a single species, including hydrogen sulfide, perthiol, and COS, and will be very useful as part of the toolbox for delineating the mechanisms of sulfur signaling.

An Esterase-Sensitive Prodrug Approach for Controllable Delivery of Persulfide Species.

A strategy to deliver a well-defined persulfide species in a biological medium is described. Under near physiological conditions, the persulfide prodrug can be activated by an esterase to generate a "hydroxymethyl persulfide" intermediate, which rapidly collapses to form a defined persulfide. Such persulfide prodrugs can be used either as chemical tools to study persulfide chemistry and biology or for future development as H2 S-based therapeutic reagents. Using the persulfide prodrugs developed in this study, the reactivity between S-methyl methanethiosulfonate (MMTS) with persulfide was unambiguously demonstrated. Furthermore, a representative prodrug exhibited potent cardioprotective effects in a murine model of myocardial ischemia-reperfusion (MI/R) injury with a bell shape therapeutic profile.

Comparative analysis of Cu (I)-catalyzed alkyne-azide cycloaddition (CuAAC) and strain-promoted alkyne-azide cycloaddition (SPAAC) in O-GlcNAc proteomics.

O-linked ß-N-acetylglucosamine (O-GlcNAc) is emerging as an essential protein post-translational modification in a range of organisms. It is involved in various cellular processes such as nutrient sensing, protein degradation, gene expression, and is associated with many human diseases. Despite its importance, identifying O-GlcNAcylated proteins is a major challenge in proteomics. Here, using peracetylated N-azidoacetylglucosamine (Ac4 GlcNAz) as a bioorthogonal chemical handle, we described a gel-based mass spectrometry method for the identification of proteins with O-GlcNAc modification in A549 cells. In addition, we made a labeling efficiency comparison between two modes of azide-alkyne bioorthogonal reactions in click chemistry: copper-catalyzed azide-alkyne cycloaddition (CuAAC) with Biotin-Diazo-Alkyne and stain-promoted azide-alkyne cycloaddition (SPAAC) with Biotin-DIBO-Alkyne. After conjugation with click chemistry in vitro and enrichment via streptavidin resin, proteins with O-GlcNAc modification were separated by SDS-PAGE and identified with mass spectrometry. Proteomics data analysis revealed that 229 putative O-GlcNAc modified proteins were identified with Biotin-Diazo-Alkyne conjugated sample and 188 proteins with Biotin-DIBO-Alkyne conjugated sample, among which 114 proteins were overlapping. Interestingly, 74 proteins identified from Biotin-Diazo-Alkyne conjugates and 46 verified proteins from Biotin-DIBO-Alkyne conjugates could be found in the O-GlcNAc modified proteins database dbOGAP (http://cbsb.lombardi.georgetown.edu/hulab/OGAP.html). These results suggested that CuAAC with Biotin-Diazo-Alkyne represented a more powerful method in proteomics with higher protein identification and better accuracy compared to SPAAC. The proteomics credibility was also confirmed by the molecular function and cell component gene ontology (GO). Together, the method we reported here combining metabolic labeling, click chemistry, affinity-based enrichment, SDS-PAGE separation, and mass spectrometry, would be adaptable for other post-translationally modified proteins in proteomics.

Esterase-Sensitive Prodrugs with Tunable Release Rates and Direct Generation of Hydrogen Sulfide.

Prodrugs that release hydrogen sulfide upon esterase-mediated cleavage of an ester group followed by lactonization are described herein. By modifying the ester group and thus its susceptibility to esterase, and structural features critical to the lactonization rate, H2 S release rates can be tuned. Such prodrugs directly release hydrogen sulfide without the involvement of perthiol species, which are commonly encountered with existing H2 S donors. Additionally, such prodrugs can easily be conjugated to another non-steroidal anti-inflammatory agent, leading to easy synthesis of hybrid prodrugs. As a biological validation of the H2 S prodrugs, the anti-inflammatory effects of one such prodrug were examined by studying its ability to inhibit LPS-induced TNF-α production in RAW 264.7 cells. This type of H2 S prodrugs shows great potential as both research tools and therapeutic agents.

Stereoselective Synthesis of 1-Methyl-3',4',5',6'-tetrahydrospiro[indoline-3,2'-pyran]-2-one Derivatives via Prins Cyclization.

A novel spirocyclization has been developed for the construction of functionalized spirooxindole pyran via Lewis acid promoted Prins cyclization. The reaction proceeds through formation of a single diastereoisomer with high stereoselectivity. This approach can be used to construct biologically important substituted spirooxindole as well as fluorinated pyran scaffolds.

Genetically Enabling Phosphorus Fluoride Exchange Click Chemistry in Proteins.

Phosphorus Fluoride Exchange (PFEx), recently debuted in small molecules, represents the forefront of click chemistry. To explore PFEx's potential in biological settings, we developed amino acids PFY and PFK featuring phosphoramidofluoridates and incorporated them into proteins through genetic code expansion. PFY/PFK selectively reacted with nearby His, Tyr, Lys, or Cys in proteins, both in vitro and in living cells, demonstrating that proximity enabled PFEx reactivity without external reagents. The reaction with His showed unique pH-dependent properties and created thermally sensitive linkages. Additionally, Na2SiO3 enhanced PFEx reactions with Tyr and Cys. PFEx, by generating defined covalent P-N/O linkages, extends the utility of phosphorus linkages in proteins, aligning with nature's use of phosphate connectors in other biomolecules. More versatile and durable than SuFEx, PFEx in proteins expands the latent bioreactive arsenal for covalent protein engineering and will facilitate the broad application of this potent click chemistry in biological and biomedical fields.

Prodrugs of sulfide and persulfide species: Implications in their different pharmacological activities.

Reactive sulfur species (RSS), such as H2S, hydrogen polysulfide (H2Sn, n ≥ 2), and hydropersulfides (RSSnH, n ≥ 1), are known to mediate diverse signaling pathways and possess a plethora of exciting therapeutic opportunities. Historically, due to the rapid inter-conversion among those species in vivo, the biological differences of distinct sulfur species were often overlooked. These species were considered to enrich the global sulfur pool in almost an equal fashion. However, advancement in this field has revealed that sulfur species at different oxidation states result in different pharmacological effects including scavenging reactive oxygen species (ROS), activating ion channels, and exhibiting analgesic effects. Here, we summarize recent advances in studying the biological and pharmacological differences of distinct sulfur species; discuss this phenomenon from the view of chemical properties and sulfur signaling pathways; and lay out a roadmap to transforming such new knowledge into general principles in developing sulfur-based therapeutics.

The proximity-enabled sulfur fluoride exchange reaction in the protein context.

The proximity-enabled sulfur(vi) fluoride exchange (SuFEx) reaction generates specific covalent linkages between proteins in cells and in vivo, which opens innovative avenues for studying elusive protein-protein interactions and developing potent covalent protein drugs. To exploit the power and expand the applications of covalent proteins, covalent linkage formation between proteins is the critical step, for which fundamental kinetic and essential properties remain unexplored. Herein, we systematically studied SuFEx kinetics in different proteins and conditions. In contrast to in small molecules, SuFEx in interacting proteins conformed with a two-step mechanism involving noncovalent binding, followed by covalent bond formation, exhibiting nonlinear rate dependence on protein concentration. The protein SuFEx rate consistently changed with protein binding affinity as well as chemical reactivity of the functional group and was impacted by target residue identity and solution pH. In addition, kinetic analyses of nanobody SR4 binding with SARS-CoV-2 spike protein revealed that viral target mutations did not abolish covalent binding but decreased the SuFEx rate with affinity decrease. Moreover, off-target cross-linking of a SuFEx-capable nanobody in human serum was not detected, and the SuFEx-generated protein linkage was stable at cellular acidic pHs, suggesting SuFEx suitability for in vivo usage. These results advanced our understanding of SuFEx reactivity and kinetics in proteins, which is invaluable for ongoing exploration of SuFEx-enabled covalent proteins for basic biological research and creative biotherapeutics.

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.

Genetically encoded chemical crosslinking of RNA in vivo.

Protein-RNA interactions regulate RNA fate and function, and defects can lead to various disorders. Such interactions have mainly been studied by nucleoside-based UV crosslinking methods, which lack broad in vivo compatibility and the ability to resolve specific amino acids. In this study we genetically encoded latent bioreactive unnatural amino acids into proteins to react with bound RNA by proximity-enabled reactivity and demonstrated genetically encoded chemical crosslinking of proteins with target RNA (GECX-RNA) in vivo. Applying GECX-RNA to the RNA chaperone Hfq in Escherichia coli identified target RNAs with amino acid specificity. Combining GECX-RNA with immunoprecipitation and high-throughput sequencing of an N6-methyladenosine reader protein in mammalian cells allowed the in vivo identification of unknown N6-methyladenosine on RNA with single-nucleotide resolution throughout the transcriptome. GECX-RNA thus affords resolution at the nucleotide and amino acid level for interrogating protein-RNA interactions in vivo. It also enables the precise engineering of covalent linkages between a protein and RNA, which will inspire innovative solutions for RNA-related research and therapeutics.

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.