Expert interviews

We had an interview with Professor Zhao Ma, a researcher at the School of Pharmaceutical Sciences at Shandong University, who specializes in the research of subcellular structures. We spoke with Professor Ma regarding the contemporary management of malignant diseases. Professor Ma stated that secondary brain tumors are challenging to treat due to their potential to metastasize. We subsequently inquired of Professor Ma on his research on bioluminescence imaging technology, which holds significant promise for malignancy therapy. Professor Ma pointed out that bioluminescence imaging technology can monitor tumor cell proliferation and metastasis, thereby serving as a means to evaluate the efficacy of drug candidates. Furthermore, in gene therapy, this technique can be employed to ascertain the expression of genes at the tumor location. We questioned with Professor Ma regarding the benefits of contemporary bioluminescence imaging technologies in tumor treatment. Professor Ma identified five distinct advantages of bioluminescence imaging technique. Bioluminescence imaging technology is non-invasive, allowing for the acquisition of information about organisms with minimal harm. Secondly, the bioluminescence imaging technology is robust and capable of long-term real-time monitoring. Third, bioluminescence imaging method has excellent sensitivity and necessitates less signal input than fluorescence, thereby diminishing the impact of tissue interference on signal attenuation. Fourth, bioluminescence imaging technology facilitates high-throughput screening, enabling researchers to evaluate medications through rapid signal variations. Fifth, due to the presence of many bioluminescent enzymes in organisms, each emitting different wavelengths, bioluminescence imaging technology can facilitate multi-labeling to investigate the interactions among multiple biological reaction processes.

We subsequently had an interview with Professor Xiaohan Ye, who specializes in organic synthesis research at the School of Pharmaceutical Sciences, Shandong University. Professor Ye informed us about the present limitations of bioluminescence imaging technology, including the short wavelength of light production and restricted penetration. Professor Ye stated that advancements in bioluminescence imaging technologies will significantly enhance the treatment of malignant diseases. This motivated us to consider that enhancing bioluminescence technology could more effectively leverage its advantages.

We corresponded with Professor Minyong Li, Vice Dean of the School of Pharmaceutical Sciences at Shandong University and Editor-in-Chief of the prestigious journal Medicinal Research Reviews. Professor Li possesses extensive and enduring expertise in bioluminescence and visualization-based drug discovery. He significantly advanced the exploration of bioluminescence and offered extensive professional guidance.

Implementation

1. Idea hypothesis

Following three weeks of brainstorming, we came up with several possible solutions to the shortcomings of firefly luciferin.

Subsequently, we shared our ideas and inquiries with Professor Ye, an expert in organic synthesis at Shandong University. Professor Ye identified multiple choices for us. The initial option is to substitute groups on the molecular structure of luciferin. The alternative is to incorporate a conjugated rigid ring to extend its emission wavelength. We must identify a method to enhance the intensity and stability of the bioluminescence. A third option is the incorporation of hydrophilic groups that enhance the solubility of luciferin in water.

Upon examining the crystal structure of firefly luciferin and luciferase, we identified a substantial binding pocket adjacent to the hydroxyl or amino group. Consequently, we incorporated the cycloamino group at this location to enhance the affinity for luciferase. Furthermore, we propose that the incorporation of cycloamino groups may enhance the lipid-water partition coefficient and the permeability of the blood-brain barrier. It may improve the wavelength and duration of bioluminescence in organisms.

2. Chemical synthesis

After communicating with Professor Ye, we decided to carry out the cyclization reaction to achieve the purpose of introducing cycloamine groups. Finally,we obtain cycloaminoluciferin 5-cyL, 6-cyL, 7-cyL, and 8-cyL.

3. Inspection

After obtaining the compound, we decided to evaluate the biological activity. All animal studies were approved by the Ethics Committee of School of Pharmaceutical Sciences, Shandong University, and were conducted in compliance with European guidelines for the care and use of laboratory animals.

When we measured the substrate concentration dependence and ATP concentration dependence of the new substrate bioluminescence intensity at the enzyme level with the live animal imager, we found that the compound 8-cyL performed poorly, so we decided to use only the compounds 5-cyL, 6-cyL and 7-cyL in the following studies.

According to the imaging study of xenograft tumor model in nude mice, 7-cyL, a novel substrate with reasonable activity, was selected for the following experiments.

We then tested the animal penetration and bioluminescence time of 7-cyL, demonstrating the effectiveness of 7-cyL. At the same time, we performed cytotoxicity tests and used 7-cyL to evaluate the treatment and efficacy of mice with tumor cells, which demonstrated the safety of 7-cyL.

entrepreneurship

The ultimate goal of our research is to solve real social problems, to promote new developments in the field of medicine, and to contribute to human health problems.

The main advantages of our new substrate 7-cyL over D-luciferin and aminoluciferin are its high brain imaging ratio and enhanced blood-brain barrier permeability, which indicates that this substrate may have high application value in brain imaging in vivo. The bioluminescence intensity of compound 7-cyl is ten times than that of D-luciferin at the same dose, and the bioluminescence duration is 7 hours. In brain tumor imaging, 7-cyL exhibited a bioluminescence intensity more than 30 times higher than D-luciferin. This will make the continuous tracing of brain tumor’s pathogenesis possible. Additionally, this technology will make a great contribution in the field of target medicine selection. In the field of antitumor pharmacodynamics, the method of utilizing living bioluminescent imaging has higher sensitivity than conventional approaches. While it is not possible to detect tumor through conventional approaches, this technology can already detect fairly strong signals. Because the detection of this technology is merely restricted to living cells and dead tumor cells are not able to be detected, whereas through conventional approaches it is impossible to discriminate normal tumor cells and dead tumor cells, therefore this technology can discover drug therapeutic effect earlier than the conventional approaches and is more sensitive than conventional approaches. In contemporary times, the medicines that have already applied this technology to do research about their antitumor effects and successfully appeared in the market include Sunitinib and Topotecan.

Disadvantages: This technique requires unaffordable imaging systems. To apply this technique, the luciferase gene should be expressed in organisms. Based on the consideration of biological safety, it cannot be applied in human body so far.