Trimethyl Lock Research Articles

Trimethyl Lock Based Tools for Drug Delivery and Cell Imaging - Synthesis and Properties.

Trimethyl lock (TML) systems have become increasingly important in medicinal and bioorganic chemistry, particularly for their roles in the targeted delivery of therapeutic agents and as integral components in fluorogenic probes for cellular imaging. The simplicity and efficiency of their synthesis have established TML systems as versatile platforms for the controlled release of active molecules under particular physiological conditions. This review consolidates recent advancements in the application of TML systems, with a focus on their use in drug delivery, cellular imaging, and other areas where precise molecular release is crucial. Additionally, we discuss the synthetic strategies employed to construct TML-based conjugates, underscoring their potential to enhance the specificity and efficacy of bioactive compounds in various biomedical applications.

A chiral trimethyl lock based on the vicinal disubstituent effect: prolonged release of camptothecin into cancer cells.

Synthesis and in vitro testing of a prodrug designed for the controlled delivery of the anticancer drug camptothecin within pancreatic cancer cells are reported. Our study reveals a non-conventional pharmacokinetic release characterized by an exponential pattern before reaching the half-life (t1/2) and a linear pattern thereafter. The release mechanism was triggered either by hydrolytic enzymes and/or by the acid microenvironment of cancer cells.

Trimethyl Lock Quinone-Based Organic Molecular Probes for NQO1 Sensing and Imaging

NAD(P)H: quinone oxidoreductase isozyme 1 (NQO1) is a flavoenzyme and involved in protection against oxidative stress and the regulation of metabolic functions, which is strongly implicated in neurodegenerative diseases and carcinogenic processes. Furthermore, NQO1 is also involved in the modes of action of redox-active drugs (e.g., antimalarials). Determining the activity and localization of NQO1 in living organisms is of great importance for early disease diagnosis and therapy. As a promising and convenient biosensing technique, trimethyl lock quinone-based organic molecular probes have been well established for the specific detection and imaging of NQO1 in living cells and in vivo. In this review, the recent progress of NQO1 probes based on organic small molecules is summarized from the perspectives of molecular design strategies, sensing mechanisms and bioimaging applications. We also elucidate the potential limitations and prospects of current NQO1 probes to further promote the development of versatile imaging tools for NQO1-related biomedical investigation.

Fatty acids as molecular carriers in cleavable antifungal conjugates

Conjugates composed of C2-18 fatty acid (FA) residues as a molecular carrier and 5-fluorocytosine (5-FC) as an active agent, released upon the action of intracellular esterases on the ester bond between FA and “trimethyl lock” intramolecular linker, demonstrate good in vitro activity against human pathogenic yeasts of Candida spp. The minimal inhibitory concentrations (MIC) values for the most active conjugates containing caprylic (C8), capric (C10), lauric (C12), or myristic (C14) acid residues were in the 2–64 μg mL−1 range, except for these against the least susceptible Candida krusei. The least active conjugates containing C2, C16, or C18 FA were slowly hydrolyzed by esterase and probably poorly taken up by Candida cells, as found for their analogs containing a fluorescent label, Nap-NH2 instead of 5-FC.

Tetrazine-induced activation of a trimethyl lock as a click-to-release system for protected doxorubicin.

We herein report a novel chemically triggered click-to-release system, that combines the trimethyl lock (TML) lactonization with the bioorthogonal inverse electron demand Diels-Alder (IEDDA) reaction of a vinyl ether and a tetrazine. Kinetic studies were carried out on a vinyl phenol model system with six tetrazines using NMR and UV/Vis spectroscopy, revealing that within the three step sequence the IEDDA reaction was rate-limiting. The reaction rates were enhanced by increasing the electrophilicity of the tetrazine, while balancing reactivity and stability of the tetrazines. The anticancer drug doxorubicin was conjugated to a vinyl-modified TML. Its subsequent liberation from vinyl-TML was triggered by dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate and followed quantitatively by NMR, thereby providing a proof-of-concept for the tetrazine/TML click-to-release system. In addition the applicability of the reaction under physiolgoical conditions could be shown.

Design and synthesis of a new near-infrared and large Stokes shift fluorescence probe for NAD(P)H:quinone oxidoreductase 1 detection in living systems

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a kind of anti-oxidant enzyme and widely over-expressed in many cancers. Accurate detection of its activity may be applied for early screening certain cancers. In this work, a new near-infrared and large Stokes shift fluorescence probe (HHW-1) based on chloro-substituted dicyanoisophorone was designed for NQO1 detection. HHW-1 was constructed through trimethyl lock quinone to pull-push the optical tuable hydroxyl group of chloro-substituted dicyanoisophorone fluorophore. HHW-1 was applied to detect NQO1 with “off-on” fluorescent signal and large Stokes shift (150 nm) in a time-dependent manner, exhibiting a high selectivity and low detection limit (5.6 ng/mL). Moreover, HHW-1 possessed good optical performance to track the change of NQO1 activity in cells caused by dicoumarin (Dic). What’ more, HHW-1 was also applied to track NQO1 activity in tumor bearing mice. All taken together, HHW-1 was an excellent indicator for NQO1 detection in living bio-systems, which may be used for early tumor diagnose.

Structure-activity relationship study of a series of nucleoside derivatives bearing sulfonamide scaffold as potent and selective PRMT5 inhibitors

Protein arginine methyltransferase 5 (PRMT5) is a promising target for the treatment of malignant tumors. The discovery of nucleoside-derived inhibitors against PRMT5 with novel scaffold has been challenging. Herein, we report our effort on the design and synthesis of nucleoside derivatives bearing sulfonamide scaffold as potent PRMT5 inhibitors. The representative compound 23n was identified as a potent and selective PRMT5 inhibitor with an IC50 value of 8 nM. Molecular docking study demonstrated the binding mode of compound 23n and illustrated its inhibitory activity to PRMT5. The Trimethyl Lock prodrug strategy was used to afford prodrug 36 with lower polarity which could rapidly release the active compound 23n after entering the tumor cells. Cell-based assays revealed that the prodrug 36 restrained the proliferation of Z-138 and MOLM-13 cells and suppressed methylation of PRMT5 substrate more potently than 23n. Additionally, both compound 23n and 36 exerted antiproliferative effects against Z-138 cells mainly by inducing apoptosis effectively rather than arresting cell cycle. Thus, compounds 23n and 36 represent a series of potent PRMT5 inhibitor with novel scaffold.

Development of camptothecin nano-prodrugs based on trimethyl lock groups toward selective drug release in cancer cells

Selective drug release at the tumor site contributes to the reduction of side effects caused by anticancer treatments. To achieve a selective drug release system in cancer cells, we designed a new prodrug in which the anticancer drug camptothecin (CPT) is linked to a trimethyl lock (TML) group by an ester bond. Evaluation of in vitro cytostatic activity and drug release kinetics of the CPT-TML nano-prodrugs revealed that the ester of CPT-TML was stable against hydrolysis due to the steric hindrance around the ester derived from the tertiary hydroxy group. In contrast, CPT was smoothly released via sterically hindered ester cleavage by lactonization of the activated TML group. This finding will help in the development of nano-prodrugs with high selectivity at tumor site.

Development of Camptothecin Nano-Prodrugs Based on Trimethyl Lock Groups Toward Selective Drug Release in Cancer Cells

Development of Camptothecin Nano-Prodrugs Based on Trimethyl Lock Groups Toward Selective Drug Release in Cancer Cells

Masked Amino Trimethyl Lock (H2N‐TML) Systems: New Molecular Entities for the Development of Turn‐On Fluorophores and Their Application in Hydrogen Sulfide (H2S) Imaging in Human Cells

Masked trimethyl lock (TML) systems as molecular moieties enabling the bioresponsive release of compounds or dyes in a controlled temporal and spatial manner have been widely applied for the development of drug conjugates, prodrugs or molecular imaging tools. Herein, we report the development of a novel amino trimethyl lock (H2N‐TML) system as an auto‐immolative molecular entity for the release of fluorophores. We designed Cou‐TML‐N3 and MURh‐TML‐N3 , two azide‐masked turn‐on fluorophores. The latter was demonstrated to selectively release fluorescent MURh in the presence of physiological concentrations of the redox‐signaling molecule H2S in vitro and was successfully applied to image H2S in human cells

Convenient and ultrasensitive detection of live Salmonella using ratiometric electrochemical molecular substrates

Salmonella contamination is a major concern in food and public health safety, and carrying out episodic monitoring of Salmonella contamination in food and water bodies is essential for safeguarding public health and the economy. Therefore, there is an urgent need to develop an easy-to-operate Salmonella-targeting point-of-care detection platform. To this end, we designed two activity-based latent ratiometric electrochemical molecular substrates, denoted as Sal-CAF and Sal-NBAF, specifically for achieving easy, rapid, and selective profiling of Salmonella esterase (a Salmonella biomarker) under physiological conditions. The octyl esters of the substrates were cleaved by the esterase and triggered the trimethyl lock to eject the electron-rich aminoferrocene derivatives (CAF and NBAF), and the corresponding electrochemical signals were tracked at the negative region (−0.08 V vs Ag/AgCl) of the voltammetric spectrum. The Sal-CAF substrate was used to determine the concentration of Salmonella in a wide dynamic range (1.03 × 105–1.1 × 1010 CFU mL−1) with a low detection limit of 39.27 × 103 CFU mL−1. The developed probes were tested against various bacteria but were only activated by live Salmonella. Furthermore, the Sal-CAF probe was used directly in quantifying spiked live Salmonella spiked in milk samples and also used to effectively monitor and quantify Salmonella production in real-time. These achievements indicated the Sal-CAF probe to be a promising platform for point-of-care Salmonella analysis.

Development of a Redox-Sensitive Spermine Prodrug for the Potential Treatment of Snyder Robinson Syndrome.

Snyder Robinson Syndrome (SRS) is a rare disease associated with a defective spermine synthase gene and low intracellular spermine levels. In this study, a spermine replacement therapy was developed using a spermine prodrug that enters cells via the polyamine transport system. The prodrug was comprised of three components: a redox-sensitive quinone "trigger", a "trimethyl lock (TML)" aryl "release mechanism", and spermine. The presence of spermine in the design facilitated uptake by the polyamine transport system. The quinone-TML motifs provided a redox-sensitive agent, which upon intracellular reduction generated a hydroquinone, which underwent intramolecular cyclization to release free spermine and a lactone byproduct. Rewardingly, most SRS fibroblasts treated with the prodrug revealed a significant increase in intracellular spermine. Administering the spermine prodrug through feeding in a Drosophila model of SRS showed significant beneficial effects. In summary, a spermine prodrug is developed and provides a lead compound for future spermine replacement therapy experiments.

Esterase-Sensitive Prodrugs of a Potent Bisubstrate Inhibitor of Nicotinamide N-Methyltransferase (NNMT) Display Cellular Activity.

A recently discovered bisubstrate inhibitor of Nicotinamide N-methyltransferase (NNMT) was found to be highly potent in biochemical assays with a single digit nanomolar IC50 value but lacking in cellular activity. We, here, report a prodrug strategy designed to translate the observed potent biochemical inhibitory activity of this inhibitor into strong cellular activity. This prodrug strategy relies on the temporary protection of the amine and carboxylic acid moieties of the highly polar amino acid side chain present in the bisubstrate inhibitor. The modification of the carboxylic acid into a range of esters in the absence or presence of a trimethyl-lock (TML) amine protecting group yielded a range of candidate prodrugs. Based on the stability in an aqueous buffer, and the confirmed esterase-dependent conversion to the parent compound, the isopropyl ester was selected as the preferred acid prodrug. The isopropyl ester and isopropyl ester-TML prodrugs exhibit improved cell permeability, which also translates to significantly enhanced cellular activity as established using assays designed to measure the enzymatic activity of NNMT in live cells.

Molecular Umbrella as a Nanocarrier for Antifungals.

A molecular umbrella composed of two O-sulfated cholic acid residues was applied for the construction of conjugates with cispentacin, containing a “trimethyl lock” (TML) or o-dithiobenzylcarbamoyl moiety as a cleavable linker. Three out of five conjugates demonstrated antifungal in vitro activity against C. albicans and C. glabrata but not against C. krusei, with MIC90 values in the 0.22–0.99 mM range and were not hemolytic. Antifungal activity of the most active conjugate 24c, containing the TML–pimelate linker, was comparable to that of intact cispentacin. A structural analogue of 24c, containing the Nap-NH2 fluorescent probe, was accumulated in Candida cells, and TML-containing conjugates were cleaved in cell-free extract of C. albicans cells. These results suggest that a molecular umbrella can be successfully applied as a nanocarrier for the construction of cleavable antifungal conjugates.

Luminogenic D-Luciferin Derivatives as OATP1B1 and 1B3 Substrates in No-wash Assays.

The human hepatic organic ion transporting polypeptides OATP1B1 and -1B3 are uptake transporters that influence the disposition of several small molecule drugs and perpetrate certain adverse drug-drug interactions. To predict these invivo effects, invitro systems are used to screen new drug entities as potential transporter substrates or inhibitors. To simplify such studies, we synthesized luminogenic derivatives of the OATP1B1 and -1B3 substrate D-luciferin to test as probe substrates in a rapid, no-wash optical approach for substrate and inhibitor identification and characterization. Each derivative is a pro-luciferin containing a self-immolating trimethyl lock quinone linker that is sensitive to intracellular reducing environments that cause the release of free luciferin in proportion to the amount of probe taken up by the transporter. A subsequent luciferin-limited luciferase reaction produces light in proportion to transporter activity. We tested the derivatives in HEK293 cells that overexpress OATP1B1 or OATP1B3 by transient transfection or viral transduction. Derivatives were identified that showed OATP-dependent uptake that was time and concentration dependent, saturable and sensitive to inhibition by known OATP1B1 and -1B3 substrates and inhibitors. These luminogenic transporter probes enabled an add-only multi-well plate protocol suitable for automation and high throughput screening.

Developing Push-Pull Hydroxylphenylpolyenylpyridinium Chromophores as Ratiometric Two-Photon Fluorescent Probes for Cellular and Intravital Imaging of Mitochondrial NQO1.

This work highlights the use of push-pull hydroxylphenylpolyenylpyridinium fluorophores coupled with trimethyl lock quinone to engineer the ratiometric two-photon probes for cellular and intravital imaging of mitochondrial NAD(P)H:quinone oxidoreductase 1 (NQO1), a critical antioxidant enzyme responsible for detoxifying quinones. As a typical representative, QBMP showed favorable binding with NQO1 with a Michaelis constant of 12.74 μM and exhibited a suite of superior properties, including rapid response (4 min), large Stokes shift (162 nm), ultralow detection limit (0.9 nM), favorable two-photon cross section for the released fluorophore (70.5 GM), and deep tissue penetration (225 μm) in fixed brain tissues. More importantly, this probe was successfully applied for distinguishing different NQO1-expressing cancer and normal cells, revealing decreased NQO1 activity in a cellular Parkinson's disease model, screening NQO1 inducers as neuroprotective agents, and imaging of NQO1 in live mouse brain.

Trimethyl Lock Quinone‐Functionalized Coumarin for Real‐time Monitoring of NQO1 Activity in the Live Cells

We developed trimethyl lock quinone‐functionalized coumarin (probe 1) as a small fluorescent probe for the imaging of NQO1 activity in the live cells. NQO1 is highly expressed in most cancer cells and it is considered as a potential biomarker of NQO1‐related cancer. Probe 1 showed the high selectivity and rapid fluorescence response to the NQO1 activity. In addition, the probe is biocompatible and easily penetrates living cells. It was found that probe 1 provided relatively strong fluorescence in NQO1‐positive A549 cells, unlike NQO1‐negative NIH‐3T3 and MDA‐MB‐231 cells. Therefore, we demonstrated that probe 1 can detect NQO1 activity in real time in living cells and it can also distinguish different levels of NQO1 activity in various pathological cells.

Prodrug Strategies to Improve the Solubility of the HCV NS5A Inhibitor Pibrentasvir (ABT-530).

A research program to discover solubilizing prodrugs of the HCV NS5A inhibitor pibrentasvir (PIB) identified phosphomethyl analog 2 and trimethyl-lock (TML) prodrug 9. The prodrug moiety is attached to a benzimidazole nitrogen atom via an oxymethyl linkage to allow for rapid and complete release of the drug for absorption following phosphate removal by intestinal alkaline phosphatase. These prodrugs have good hydrolytic stability properties and improved solubility compared to PIB, both in aqueous buffer (pH 7) and FESSIF (pH 5). TML prodrug 9 provided superior in vivo performance, delivering high plasma concentrations of PIB in PK studies conducted in mice, dogs, and monkeys. The improved dissolution properties of these phosphate prodrugs provide them the potential to simplify drug dosage forms for PIB-containing HCV therapy.

Effect of the chemical structure on the drug release from brinzolamide based nano eye-drops

Commercially available eye-drops scarcely penetrate the eye because of the cornea, which is located at the surface of the eye and function of barrier to keep out foreign matter. To enhance the penetration ratio of eye-drop, we have proposed nanometer-sized eye-drops called “nano eye-drops.” A series of prodrug compounds derived from brinzolamide, a glaucoma therapeutic agent, connected with a trimethyl lock (TML) group were synthesized and the nano eye-drops of the resulting prodrugs were successfully fabricated by the reprecipitation method. We found that the drug release of nano eye-drops critically depended on the chemical structure of the prodrugs comprising the nano eye-drops. As a result, the timing of the ocular hypotensive effect was controlled by altering the chemical structure of brinzolamide prodrug. These results provide insight into drug design for effective controlled release.

Fluorogenic sensing of mitochondrial electron transferring dehydrogenase using a trimethyl lock quinone-linked hemicyanine

A trimethyl lock quinone (TLQ)-linked hemicyanine (probe 1) is developed for imaging mitochondrial dehydrogenase activity in the live human cells. In aqueous environment, the system underwent a fluorogenic reduction of TLQ moiety upon incubation with human recombinant NQO1, providing a fluorescence Off-On with a concomitant visual color change from yellow to orange. In contrast to other TLQ-based fluorescent probes, probe 1 is localized to the mitochondria of living cells where little NQO1 exist. And, it can provide a fluorescence turn-on response which was selectively modulated by antimycin A and myxothiazol, a potent inhibitor of the complex III in the electron transfer chain and showed poor response to dicoumarol, a potent inhibitor of NQO1. Likely, probe 1 acts as a fluorogenic chemosensor for the mitochondrial electron transferring reaction in the live cells.