The Fe‐N‐C Nanozyme with Both Accelerated and Inhibited Biocatalytic Activities Capable of Accessing Drug–Drug Interactions

Emerging as a cost‐effective and robust enzyme mimic, nanozymes have drawn increasing attention with broad applications ranging from cancer therapy to biosensing. Developing nanozymes with both accelerated and inhibited biocatalytic properties in a biological context is intriguing to peruse more advanced functions of natural enzymes, but remains challenging, because most nanozymes are lack of enzyme‐like molecular structures. By re‐visiting and engineering the well‐known Fe‐N‐C electrocatalyst that has a heme‐like Fe‐N_x active sites, herein, it is reported that Fe‐N‐C could not only catalyze drug metabolization but also had inhibition behaviors similar to cytochrome P450 (CYP), endowing it a potential replacement of CYP for preliminary evaluation of massive potential chemicals, drug dosing guide, and outcome prediction. In addition, in contrast to electrocatalysts, the highly graphitic framework of Fe‐N‐C may not be obligatory for a competitive CYP‐like activity.     

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, H₂S release rates can be tuned. Such prodrugs directly release hydrogen sulfide without the involvement of perthiol species, which are commonly encountered with existing H₂S 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 H₂S 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 H₂S prodrugs shows great potential as both research tools and therapeutic agents.     

The feasibility of enzyme targeted activation for amino acid/dipeptide monoester prodrugs of floxuridine; cathepsin D as a potential targeted enzyme

The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5'-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0-105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5'-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5'-O-L-phenylalanyl-L-tyrosylfloxuridine and 5'-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enzymes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5'-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.     

Lysosome‐Targeting Amplifiers of Reactive Oxygen Species as Anticancer Prodrugs

Cancer cells produce elevated levels of reactive oxygen species, which has been used to design cancer specific prodrugs. Their activation relies on at least a bimolecular process, in which a prodrug reacts with ROS. However, at low micromolar concentrations of the prodrugs and ROS, the activation is usually inefficient. Herein, we propose and validate a potentially general approach for solving this intrinsic problem of ROS‐dependent prodrugs. In particular, known prodrug 4‐(N ‐ferrocenyl‐N ‐benzylaminocarbonyloxymethyl)phenylboronic acid pinacol ester was converted into its lysosome‐specific analogue. Since lysosomes contain a higher concentration of active ROS than the cytoplasm, activation of the prodrug was facilitated with respect to the parent compound. Moreover, it was found to exhibit high anticancer activity in a variety of cancer cell lines (IC₅₀=3.5–7.2 μm ) and in vivo (40 mg kg⁻¹, NK/Ly murine model) but remained weakly toxic towards non‐malignant cells (IC₅₀=15–30 μm ).     

Advances in Single‐Atom Nanozymes Research†

Nanozymes with intrinsic enzyme‐like properties have attracted significant interest owing to their capability to address the limitations of traditional enzymes such as fragility, high cost and difficult mass production. However, the currently reported nanozymes are generally less active than natural enzymes. In recent years, with the rapid development of nanoscience and nanotechnology, single‐atom nanozymes (SAzymes) with well‐defined electronic and geometric structures have shown a promise to serve as direct surrogates of traditional enzymes by mimicking the highly evolved catalytic center of natural enzymes. In this review, we will introduce the enzymatic characteristics and recent advances of SAzymes, and summarize their significant applications from in vitro detection to in vivo monitoring and therapy.     

When Nanozymes Meet Single‐Atom Catalysis

Nanomaterials with enzyme‐like activities, coined nanozymes, have been researched widely as they offer unparalleled advantages in terms of low cost, superior activity, and high stability. The complex structure and composition of nanozymes has led to extensive investigation of their catalytic sites at an atomic scale, and to an in‐depth understanding of the biocatalysis occurring. Single‐atom catalysts (SACs), characterized by atomically dispersed active sites, have provided opportunities for mimicking metalloprotease and for bridging the gap between natural enzymes and nanozymes. In this Minireview, we illustrate the unique properties of nanozymes and we discuss recent advances in the synthesis, characterization, and applications of SACs. Subsequently, we outline the impressive progress made in single‐atom nanozymes and we discuss their applications in sensing, degradation of organic pollutants, and in therapeutic roles. Finally, we present the major challenges and opportunities remaining for a successful marriage of nanozymes and SACs.     

Liver‐Targeted Small‐Molecule Inhibitors of Proprotein Convertase Subtilisin/Kexin Type 9 Synthesis

Targeting of the human ribosome is an unprecedented therapeutic modality with a genome‐wide selectivity challenge. A liver‐targeted drug candidate is described that inhibits ribosomal synthesis of PCSK9, a lipid regulator considered undruggable by small molecules. Key to the concept was the identification of pharmacologically active zwitterions designed to be retained in the liver. Oral delivery of the poorly permeable zwitterions was achieved by prodrugs susceptible to cleavage by carboxylesterase 1. The synthesis of select tetrazole prodrugs was crucial. A cell‐free in vitro translation assay containing human cell lysate and purified target mRNA fused to a reporter was used to identify active zwitterions. In vivo PCSK9 lowering by oral dosing of the candidate prodrug and quantification of the drug fraction delivered to the liver utilizing an oral positron emission tomography ¹⁸F‐isotopologue validated our liver‐targeting approach.     

Self‐immolative dendrimers as novel drug delivery platforms

Self‐immolative dendrimers were recently developed and introduced as a potential platform for a single‐triggered multi‐prodrug. These unique structural dendrimers can release all of their tail units through domino‐like chain fragmentation, which is initiated by a single cleavage at the dendrimer core. The incorporation of drug molecules as the tail units and an enzyme substrate as the trigger generates a multi‐prodrug unit that is activated with a single enzymatic cleavage. We have demonstrated several examples of self‐immolative dendritic prodrug systems and have shown significant advantages with respect to the appropriate monomeric prodrug. We anticipate that single‐triggered, dendritic prodrugs will be exploited to further improve selective chemotherapeutic approaches in cancer therapy. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 1569–1578, 2006     

Design of Block Copolymer Micellar Aggregates for Co‐Delivery of Enzyme and Anticancer Prodrug

Traditional enzyme–prodrug therapy (EPT) is a two‐step strategy, which has many serious deficiencies, so having a one‐step EPT treatment becomes a problem of immediate interest. This study aims to achieve an effective co‐delivery of horseradish peroxidase (HRP) as a kind of enzyme for prodrug activation and ethyl 3‐indoleacetate (EIA) as anticancer prodrug. A ternary block copolymer PEG‐PAsp(AED)‐CA consisting of poly(ethylene glycol) (PEG), reduction‐sensitive poly (N ‐(2,2′‐dithiobis(ethylamine)) aspartamide) PAsp(AED), and cholic acid (CA) was synthesized and assembled into spherical micelles which encapsulated EIA in its hydrophobic core and HRP in a reduction‐sensitive interlayer. TEM photographs show that the polymer micelle is around 40 nm, and the cell survival rate test shows that the EIA/HRP polymer micelle is highly lethal to human lung adenocarcinoma cells. Thus, co‐delivery of EIA and HRP demonstrates great potential in cancer therapy, offering a structurally simple and highly tunable platform for the synchronous delivery of enzymes and prodrugs in EPT.     

Direct Real‐Time Monitoring of Prodrug Activation by Chemiluminescence

The majority of theranostic prodrugs reported so far relay information through a fluorogenic response generated upon release of the active chemotherapeutic agent. A chemiluminescence detection mode offers significant advantages over fluorescence, mainly due to the superior signal‐to‐noise ratio of chemiluminescence. Here we report the design and synthesis of the first theranostic prodrug monitored by a chemiluminescence diagnostic mode. As a representative model, we prepared a prodrug from the chemotherapeutic monomethyl auristatin E, which was modified for activation by β‐galactosidase. The activation of the prodrug in the presence of β‐galactosidase is accompanied by emission of a green photon. Light emission intensities, which increase with increasing concentration of the prodrug, were linearly correlated with a decrease in the viability of a human cell line that stably expresses β‐galactosidase. We obtained sharp intravital chemiluminescent images of endogenous enzymatic activity in β‐galactosidase‐overexpressing tumor‐bearing mice. The exceptional sensitivity achieved with the chemiluminescence diagnostic mode should allow the exploitation of theranostic prodrugs for personalized cancer treatment.     

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 H₂S‐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.     

NIR‐Light‐Activated Combination Therapy with a Precise Ratio of Photosensitizer and Prodrug Using a Host–Guest Strategy

The co‐delivery of photosensitizers with prodrugs sensitive to reactive oxygen species (ROS) for light‐triggered ROS generation and cascaded prodrug activation has drawn tremendous attention. However, the absence of a feasible method to deliver the two components at a precise ratio has impaired the application potential. Herein, we report an efficient method to produce a nanosized platform for the delivery of an optimized ratio of the two components by the means of host–guest strategy for maximizing the combination therapy efficacy of cancer treatment. The key features of this host–guest strategy for the combination therapy are that the ratio between photosensitizer and ROS‐sensitive prodrug can be easily tuned, near‐infrared (NIR) irradiation can sensitize the photosensitizer and activate the paclitaxel prodrug for its release, and the accumulation process can be tracked by NIR imaging to maximize the efficacy of photodynamic and chemotherapy.     

Hypoxia‐Activated Prodrugs of PERK Inhibitors

Tumour hypoxia plays an important role in tumour progression and resistance to therapy. Under hypoxia unfolded proteins accumulate in the endoplasmic reticulum (ER) and this stress is relieved through the protein kinase R‐like ER kinase (PERK) signalling arm of the unfolded protein response (UPR). Targeting the UPR through PERK kinase inhibitors provides tumour growth inhibition, but also elicits on‐mechanism normal tissue toxicity. Hypoxia presents a target for tumour‐selective drug delivery using hypoxia‐activated prodrugs. We designed and prepared hypoxia‐activated prodrugs of modified PERK inhibitors using a 2‐nitroimidazole bioreductive trigger. The new inhibitors retained PERK kinase inhibitory activity and the corresponding prodrugs were strongly deactivated. The prodrugs were able to undergo fragmentation following radiolytic reduction, or bioreduction in HCT116 cells, to release their effectors, albeit inefficiently. We examined the effects of the prodrugs on PERK signalling in hypoxic HCT116 cells. This study has identified a 2‐substituted nitroimidazole carbamate prodrug with potential to deliver PERK inhibitors in a hypoxia‐selective manner.     

Cyclic‐Disulfide‐Based Prodrugs for Cytosol‐Specific Drug Delivery

The cytosolic conversion of therapeutically relevant nucleosides into bioactive triphosphates is often hampered by the inefficiency of the first kinase‐mediated step. Nucleoside monophosphate prodrugs can be used to bypass this limitation. Herein we describe a novel cyclic‐disulfide class of nucleoside monophosphate prodrugs with a cytosol‐specific, reductive release trigger. The key event, a charge‐dissipating reduction‐triggered cyclodeesterification leads to robust cytosolic production of the cyclic 3′,5′‐monophosphate for downstream enzymatic processing. The antiviral competence of the platform was demonstrated with an O‐benzyl‐1,2‐dithiane‐4,5‐diol ester of 2′‐C‐methyluridine‐3′,5′‐phosphate. Both in vitro and in vivo comparison with the clinically efficacious ProTide prodrug of 2′‐deoxy‐2′‐α‐fluoro‐β‐C‐methyluridine is provided. The cytosolic specificity of the release allows for a wide range of potential applications, from tissue‐targeted drug delivery to intracellular imaging.     

Targeting Cancer with PCPA‐Drug Conjugates: LSD1 Inhibition‐Triggered Release of 4‐Hydroxytamoxifen

Targeting cancer with small molecule prodrugs should help overcome problems associated with conventional cancer‐targeting methods. Herein, we focused on lysine‐specific demethylase 1 (LSD1) to trigger the controlled release of anticancer drugs in cancer cells, where LSD1 is highly expressed. Conjugates of the LSD1 inhibitor trans‐2‐phenylcyclopropylamine (PCPA) were used as novel prodrugs to selectively release anticancer drugs by LSD1 inhibition. As PCPA‐drug conjugate (PDC) prototypes, we designed PCPA‐tamoxifen conjugates 1 a and 1 b , which released 4‐hydroxytamoxifen in the presence of LSD1 in vitro. Furthermore, 1 a and 1 b inhibited the growth of breast cancer cells by the simultaneous inhibition of LSD1 and the estrogen receptor without exhibiting cytotoxicity toward normal cells. These results demonstrate that PDCs provide a useful prodrug method that may facilitate the selective release of drugs in cancer cells.     

Unveiling the Intrinsic Catalytic Activities of Single‐Gold‐Nanoparticle‐Based Enzyme Mimetics

Gold nanoparticles (AuNPs) have been demonstrated to serve as effective nanomaterial‐based enzyme mimetics (nanozymes) for a number of enzymatic reactions under mild conditions. The intrinsic glucose oxidase and peroxidase activities of single AuNPs and Ag–Au nanohybrids, respectively, were investigated by single NP collision electrochemical measurements. A significantly high turnover number of nanozymes was obtained from individual catalytic events compared with the results from the classical, ensemble‐averaged measurements. The unusual enhancement of catalytic activity of single nanozymes is believed to originate from the high accessible surface area of monodispersed NPs and the high activities of carbon‐supported NPs during single‐particle collision at a carbon ultramicroelectrode. This work introduces a new method for the precise characterization of the intrinsic catalytic activities of nanozymes, giving further insights to the design of high‐efficiency nanomaterial catalysts.     

A Designed 5‐Fluorouracil‐Based Bridged Silsesquioxane as an Autonomous Acid‐Triggered Drug‐Delivery System

Two new prodrugs, bearing two and three 5‐fluorouracil (5‐FU) units, respectively, have been synthesized and were shown to efficiently treat human breast cancer cells. In addition to 5‐FU, they were intended to form complexes through H‐bonds to an organo‐bridged silane prior to hydrolysis‐condensation through sol–gel processes to construct acid‐responsive bridged silsesquioxanes (BS). Whereas 5‐FU itself and the prodrug bearing two 5‐FU units completely leached out from the corresponding materials, the prodrug bearing three 5‐FU units was successfully maintained in the resulting BS. Solid‐state NMR (²⁹Si and ¹³C) spectroscopy show that the organic fragments of the organo‐bridged silane are retained in the hybrid through covalent bonding and the ¹H NMR spectroscopic analysis provides evidence for the hydrogen‐bonding interactions between the prodrug bearing three 5‐FU units and the triazine‐based hybrid matrix. The complex in the BS is not affected under neutral medium and operates under acidic conditions even under pH as high as 5 to deliver the drug as demonstrated by HPLC analysis and confirmed by FTIR and ¹³C NMR spectroscopic studies. Such functional BS are promising materials as carriers to avoid the side effects of the anticancer drug 5‐FU thanks to a controlled and targeted drug delivery.     

Activation of Prodrugs by NIR‐Triggered Release of Exogenous Enzymes for Locoregional Chemo‐photothermal Therapy

Enzymes have been used to direct the conversion of prodrugs in cancer therapy. However, non‐specific distribution of endogenous enzymes seriously hinders their bioapplications. Herein, we developed a near‐infrared‐triggered locoregional chemo‐photothermal therapy based on the exogenous enzyme delivery and remolded tumor mivroenvironment. The catalytic efficiency of enzymes was enhanced by the hyperthermia, and the therapeutic efficacy of photothermal therapy (PTT) was improved owing to the inhibition of heat shock protein 90 by chemotherapeutics. The locoregional chemo‐phototherapy achieved a one‐time successful cure in 4T1 tumor‐bearing mice model. Thus, a mutually reinforcing feedback loop between PTT and chemotherapy can be initiated by the irradiation, which holds a promising future in cancer therapy.     

Drug Delivery by an Enzyme‐Mediated Cyclization of a Lipid Prodrug with Unique Bilayer‐Formation Properties

Special delivery : Liposomal drug‐delivery systems in which prodrugs are activated specifically by disease‐associated enzymes have great potential for the treatment of severe diseases, such as cancer. A new type of phospholipid‐based prodrug has the ability to form stable small unilamellar vesicles (see picture). Activation of the prodrug vesicles by the enzyme sPLA₂ initiates a cyclization reaction, which leads to the release of the drug.

     

Bacteriophage Tail‐Tube Assembly Studied by Proton‐Detected 4D Solid‐State NMR

Obtaining unambiguous resonance assignments remains a major bottleneck in solid‐state NMR studies of protein structure and dynamics. Particularly for supramolecular assemblies with large subunits (>150 residues), the analysis of crowded spectral data presents a challenge, even if three‐dimensional (3D) spectra are used. Here, we present a proton‐detected 4D solid‐state NMR assignment procedure that is tailored for large assemblies. The key to recording 4D spectra with three indirect carbon or nitrogen dimensions with their inherently large chemical shift dispersion lies in the use of sparse non‐uniform sampling (as low as 2 %). As a proof of principle, we acquired 4D (H)COCANH, (H)CACONH, and (H)CBCANH spectra of the 20 kDa bacteriophage tail‐tube protein gp17.1 in a total time of two and a half weeks. These spectra were sufficient to obtain complete resonance assignments in a straightforward manner without use of previous solution NMR data.