The long-term goal of our research is to develop the working tools and methodologies that will form the foundation of “Therapeutic In Vivo Synthetic Chemistry”. The main benefit of this approach is that synthetic transformations can be directly performed at target regions within the body to generate molecules that elicit localized biological effects. This method should largely circumvent off-target binding and instability issues associated with current drug administration techniques. In these years, we have engaged this topic through two different approaches. The first is through the usage of glycosylated artificial metalloenzymes, where the primary aim is to exploit the chemoselectivity of embedded, non-natural transition metal catalysts for the synthesis/release of bioactive molecules. The second approach is rather centered on discovering chemical probes with novel and selective reactivity to biological metabolites naturally overexpressed in cancer cells. Once developed, the objective is then to adapt them for synthesizing diagnostic probes or anticancer drugs. Overall, we have been dedicated to developing the initial working proofs-of-concept and then to spearheading investigations toward their utility for therapeutic and diagnostic applications.
1. In vivo metal-catalyzed reactions with glycosylated artificial metalloenzymes
We developed the albumin-based artificial metalloenzymes, ArM, with gold (Au) and ruthenium (Ru) complexes, of which metals are efficiently protected inside the hydrophobic pocket of albumin, hence various transition metal-catalyzed transformation could be now possible in cells, mice, or even plants. These catalysts show quite high catalytic activity in the presence of 20 mM of glutathione and even in the whole blood (Angew. Chem. Int. Ed. 2017, Nature Catal. 2019, Nature Commun. 2019, Angew. Chem. Int. Ed. 2021). Furthermore, by conjugating the glycans on the albumin surface as a targeting vector, we successfully carried the artificial metalloenzymes to the cancer regions in mice through “glycan pattern recognition”, and synthesized the anti-cancer drugs as the true meaning of “catalytically” in mice, to disturb the cancer onset and growth. Our molecular technique is quite powerful; Just a single intravenous injection of the glycosylated artificial metalloenzyme and substrates (starting compounds) led to efficient metal-catalyzed drug synthesis leading to efficient cancer treatment.
Namely, we represented research on selective cell tagging (SeCT) therapy in vivo via the glycosylated ArM-Au-1 (Fig. 1A, Sci. Adv. 2021). The concept of SeCT therapy is based on a strategy of preferentially tagging (conjugating) specific cells with a biological small molecule. In contrast to traditional chemotherapy that directly eliminates cancer cells using highly cytotoxic drugs, the principal benefit of SeCT therapy allows cancer cells to be tagged using non-toxic chemical moieties that can either disrupt cellular function (ex/ inhibitors of adhesion) or elicit immunological responses (ex/ antigens). Subsequent functional impairment or related biological responses can indirectly lead to cancer cell death without significantly harming surrounding tissue. As depicted in Figure 1A, we showed that individual HeLa cancer cells in living mice could be tagged in vivo with cyclic-Arg-Gly-Asp (cRGD) moieties for integrin-blocking, leading to disrupted cell adhesion and compromised successfully seeding onto the extracellular matrix. The mice populations that received just one dosage of the SeCT labeling reagents via intraperitoneal injection showed a significant delay in tumor onset by 4 weeks (Fig. 1A), resulting in an improvement in overall survival rates over a period of 81 days.
Following the same concept of the SeCT therapy, we developed a cancer therapy based on targeted cell surface tagging with proapoptotic peptide 1 (Ac-GGKLFG-X; X = a benzyl fluoride moiety) that induces apoptosis when attached to the cell surface (Fig. 1B, Chem. Sci. 2021). Using the Ru-catalyzed alkylation, the proapoptotic peptide 1 showed excellent therapeutic effects in vivo. In particular, co-treatment with the proapoptotic peptide and the cRGD-coated ArM-Ru-1 significantly and synergistically inhibited tumor growth and prolonged survival rate of tumor-bearing mice after only a single injection (Fig. 1B).
Except for the above samples, we also successfully carried out cancer treatment through localized in vivo drug synthesis (Nature Commun. 2022, Angew. Chem. Int. Ed. 2022). As depicted in Fig. 1C, we investigated the design and optimization of synthetic prodrugs that can be robustly transformed in vivo to reach therapeutically relevant levels. To do this, retrosynthetic prodrug design led to the identification of combretastatin-based prodrugs, which form highly active cytostatic agents via sequential ring-closing metathesis and aromatization (Fig. 1C). In vivo activation by intravenously administered the glycosylated ArM-Ru-2 was also found to induce a significant reduction of implanted tumor growth in mice.
Figure 1. Therapeutic in vivo synthetic chemistry exemplified by (A), (B) in vivo drug-tagging therapy (SeCT) and (C) in vivo drug synthesis.
Since our technology is well targeting, non-invasive, without risk of immunogenicity, non-toxicity, and high efficiency of in vivo drug synthesis, we must point out that our technology could be a possible method to apply to the patients for disease treatment in a hospital, as we indeed have been actively investigating with the companies.
2. In vivo chemical probes with novel reactivity to cancer-related metabolite
Although endogenous metabolites are known to be essential for the biochemical process, the studies on small biogenic molecules (e.g., acrolein, polyamine, formaldehyde, CO, NO) are not gaining enough attention. We have been mainly working on utilizing small biomolecules, especially acrolein, as endogenous reagents for in vivo synthetic chemistry and using the reaction outcome for therapeutic and diagnostic applications (Fig. 2A). Acrolein, the most reactive a,b-unsaturated aldehyde, is generated inside the human body through exogenous and endogenous factors. The high accumulation of acrolein in the human body is often linked pathologically with several oxidative stress-related diseases, including Alzheimer’s disease and cancer. We found that the [3+2] cycloaddition between aryl azide 3 and acrolein, which proceeds without a catalyst to give a triazoline derivative 4 that rearranges into a diazo compound 5 as the major product (Fig. 2B, ACS Sens. 2016). We observed that the reaction rate of a bulky azide 3b with acrolein is about ten times faster than the rate observed for that of simple phenyl azide 3a (Chem. Sci. 2021). Subsequently, by attaching TAMRA fluorophore to the phenyl azides, the click-to-sense (CTS) probe 6 was developed to selectively detect the endogenous acrolein (Fig. 2C). Namely, when the CTS probe 6 encounters the intracellular acrolein, the [3+2] cycloaddition proceeds to give a diazo compound derivative, which further reacts with the various biomolecules to anchor the TAMRA fluorophore via covalent attachment (Fig. 2C, Pathway-1). By utilizing the method, we discovered that acrolein is generally overproduced in cancer cells, but is negligible in healthy cells (Adv. Sci. 2018, 2019).
We demonstrated the feasibility of using the CTS probe 6 to detect acrolein in the breast cancer tissues resected from the patients. The probe 6 can discriminate, in a clear-cut manner, cancer from the normal breast gland and sensitively visualize cancer morphology and localization in the resection stump for ONE minute (Eur. J. Surg. Oncol. 2022). The CTS probe is now under clinical trials for intraoperative breast cancer diagnosis in hospitals.
Figure 2. (A) Utilization of small biogenic molecules for Therapeutic In Vivo Synthetic Chemistry. (B) [3+2] Cycloaddition of aryl azide with acrolein. In-cell reaction with endogenous acrolein leading to (C) intraoperative breast cancer diagnosis, (D) α-radiotherapy, and (E) targeted chemotherapy.
Subsequently, we developed 211Astatine-labeled 2,6-diisopropylphenyl azide (ADIPA) 7, a simple targeted a-therapeutic molecule that can react effectively with endogenous acrolein to covalently bind the [211At] in the cancerous cells through the similar mechanism to CTS probe (Fig. 2D, Pathway-2). When just 70 kBq of ADIPA 7 was intratumorally or intravenously administered into the cancer mouse model, cancer growth was significantly inhibited without side effects (Chem. Sci. 2023). Since acrolein is generally overproduced in most cancer cells, our synthetic a-therapeutic approach could be generally used for any cancer treatment.
We have also applied the acrolein-based in vivo [3+2] cycloaddition reaction in cencerous cells to the targeted chemotherapy (Chem. Sci. 2021). We designed mitomycin C-based azidobenzylcarbamate (MMC-ABC) prodrug 8 to target acrolein in A549 (human lung adenocarcinoma epithelial)-derived cancer xenograft models (Fig. 2E). When the MMC-ABC 8 was administered to the mouse and reached the tumor area, it reacted with the endogeneous acrolein to selectively release the active drug molecule (Fig 2E, Pathway-3). The method successfully treats the cancer without inducing any side effects caused by the off-target reaction at the other regions. Such innovative prodrug strategy, on the basis of in vivo chemistry with acrolein in cancerous cells, could be considered for clinical application.
Overall, through our vision of “Therapeutic In Vivo Synthetic Chemistry”, the avenue exists for the reinvestigation of promising drug candidates that were previously deemed not viable due to off-target binding and instability problems. This is because these issues may possibly be circumvented by synthesizing their bioactive counterparts in vivo rather than administer them directly. If successful, our research could represent a pioneering new approach to drug delivery with significantly implications to not only pharmaceutical sciences, but the life sciences as a whole.