Lead drug innovation and optimization: focussing on the use of cutting-edge screening technology, developing, identifying, and continuously incubating potential lead drugs to help more young scientists achieve successful biotechnology enterprises.
1. High-throughput screening technology: In recent years, the development of high-throughput screening technology has brought revolutionary changes to drug discovery, allowing researchers to test tens of thousands of compounds in a relatively short time, thus accelerating the discovery of lead drugs.
2. In-depth understanding of Hippo pathway: The scientific research community has gradually deepened its understanding of Hippo signaling pathway and recognizes its key role in many cancers, especially in the occurrence and progression of tumors caused by pathway dysregulation
3. Bioinformatics and artificial intelligence: With the development of big data, bioinformatics and artificial intelligence, researchers can conduct in-depth analysis of a large amount of data to identify and optimize potential compounds.
4. Demand for cancer treatment: The global incidence of cancer continues to rise, leading to an increase in the demand for effective treatment methods. Especially for those cancers whose traditional therapies are not effective, new treatments are needed.
5. The trend of target-oriented drugs: In recent years, the trend in the pharmaceutical industry is to develop target-oriented drugs, which have an effect on specific biomarkers or pathways, providing more accurate and less side-effect treatment options.
6. External innovation strategies of pharmaceutical companies: Many large pharmaceutical companies turn to external partners, such as biotechnology startups, to obtain new drug candidates. This provides an opportunity for start-ups to cooperate with large enterprises and further commercialize their discoveries.
7. Cognition of patients and doctors: As the public's awareness of the role of Hippo pathway in cancer increases, patients and doctors may be more inclined to try new therapies for this pathway.
The lead drug innovation incubator is based on the current advanced high-throughput screening technology, the in-depth understanding of the Hippo pathway in the scientific research community, and the continuous demand for new cancer treatments in the market.
Through cutting-edge technology and in-depth biological research, we explore and accelerate the development of lead drugs for cancers related to Hippo pathway dysregulations, providing patients with more accurate and effective treatment options to improve their quality of life and life expectancy.
We have established an open R&D platform to encourage young scientists to put forward innovative ideas and projects. This can be through internal innovation laboratories, cooperative research projects, or incubator spaces for young scientists. Encourage young scientists to participate in drug screening and incubation programs, and provide flexible cooperation models, such as equity incentives and project dividends. And provide training and development plans specially designed for young scientists, such as seminars, workshops, mentorship, etc., to ensure that they can grow rapidly and bring value to the company.
To achieve the above goals, the company will work on the following focuses.
1. Advanced screening platform: use high-throughput screening technology, bioinformatics, and artificial intelligence technology to provide technical support for new drug discovery.
2. Compound library: Establish and maintain a library containing multiple compounds, so that the screening process has enough candidate targets.
3. Intellectual property management: Ensure that all potential lead drugs are applied for and protected in a timely manner, so as to lay the foundation for subsequent authorization and cooperation.
4. Lead drug optimization: After determining that a compound has therapeutic potential, further structural optimization and pharmacodynamics research are carried out to make it more suitable for subsequent clinical development.
5. Legal and commercial policy research: establish strategic cooperation with large pharmaceutical companies or biotechnology companies to further develop and commercialize these lead drugs through authorized cooperation. In the agreement with partners, set up a milestone payment and final drug sales commission mechanism to ensure that the company can get continuous returns from successful drug development.
We bind “Donor” and “Acceptor” beads with MST2 and STRN3 in our experiment, separately. Donor beads contain a photosensitizing agent (phthalocyanine) that, when irradiated at 680 nm, excites ambient oxygen to a singlet state. Excitation of each Donor bead generates approximately 60,000 oxygen singlets per second, resulting in a glowing of receptor beads. We used the public platform of the School of Life Sciences to perform the high throughput screening. The library of small molecules screened included TargetMol's Natural Compound Library, Approved Drug Screening Library, and Inhibitor Library, totaling 3106 molecules. MST2 and STRN3 binding tightness were determined by simultaneously measuring the fluorescence intensity of each well in a 384-well plate. The first two columns and the last two columns of the plate are negative and positive controls, respectively. For the negative control, we added only magnetic beads without protein, so the light intensity produced will be very low. And the light intensity produced by the positive control will be very high. Therefore, we can use this method to calculate the separation rate of MST2 and STRN3:
1.Set the number except the first and last two columns equal to x
2.Average the negative and positive controls respectively and label them as AN and AP
3.Put data into the equation: {1- (x-AN) / AP}×100 (a.u.)
After the calculation, our group found CX6258 (A potent, kinase-selective inhibitor of pan-Pim kinase that has passed the preclinical studies and clinical trials, and their biological activities and safety have been verified) which has the highest separation rate for doing the rest of the experiment.
Functional validation of small molecule drug candidates using pull-down experiments. Through the interview with Dr. Song during human practice, we learned that although the AlphaScreen technology has the advantages of a wide range of applications, including small molecules to large complexes, high speed, and high sensitivity, its main limitations should not be ignored. These include the sensitivity of the reaction system to bright light or prolonged room light; the trapping of singlet oxygen molecules by some compounds can reduce the optical signal; and the bleaching effect of donor beads makes single-pass signal detection preferable. To verify the screening results in the previous step, we performed pull-down experiments with CX6258 to clarify the separation of MST2-STRN3. GST pull-down is a method to study protein interactions in vitro, and the basic principle is as follows: assuming that MST2 and STRN3 may have interactions, we will fuse MST2 with a GST tag. Then, GST-MST2, HST-STRN3, and Sephrose4B beads (which can specifically bind GST) are incubated for a certain period of time, the unbound proteins are washed sufficiently, the beads are boiled for SDS-PAGE electrophoresis, and finally radiolabeling is performed, and the corresponding bands of GST-MST2 and HST-STRN3 are seen, indicating that the two proteins are pulled down due to the interaction. If there is no interaction, only one band corresponds to GST-MST2.
The results showed that lane 1 was used to exclude the possibility of the GST tag interacting with STRN3. Lanes 4-7, as the INPUT of each component, served as a control and to make the results more convincing. Lanes 2 and 3 were used to test whether CX6258 could disrupt the MST2-STRN3 interaction. Lane 2, due to the addition of CX6258 in the sample, showed only one band in the SDS-PAGE (only GST-MST2 was pulled down), while lane 3 showed two bands (indicating that GST-MST2 and HST-STRN3 were pulled down, respectively), indicating that CX6258 has the ability to disrupt the MST2-STRN3 interaction.
A series of hit compounds can be obtained based on the AlphaScreen high-throughput screening system. To verify the reliability of this high-throughput screening system, we selected one of the candidates for the following in vitro pull-down and point mutation experiments to further confirm the accuracy of the screening results.
Virtual docking predicts sites of CX6258 interaction with MST2. From further human practice on the issue of future development of the drug, we learned that the current application of our drug in the treatment of hippo-related cancers belongs to the stage of lead compounds, and the subsequent chemical modifications need to be continuously carried out to achieve better efficacy and lower side effects. These optimizations are based on the understanding of the binding site of CX6258 with the target protein MST2 and the crystal structure of the binding site. Therefore, we used virtual docking prototypes to predict and validate the binding sites of both.
Autodock Lab is a computer software widely used for molecular docking research of drug molecules. Its principle is based on the theories of molecular mechanics and computational chemistry, used to predict the binding ability and binding mode between compounds. By simulating intermolecular interactions, Autodock can provide important information about molecular binding and drug development for drug designers. We imported the protein sequence information of MST2 and the molecular structure information of CX6258 into the software, and obtained the following prediction result graph, showing that the binding sites are L34, E36, and V42 (steps for using Autodock virtual docking are shown in the protocol).
The binding site predicted by virtual docking was mutagenized by PCR to determine the mode of action of CX6258. Based on the pET28a-GST-MST2 1-308 plasmid, we designed the primer sequences corresponding to the three amino acids to be mutated, i.e. GCA instead of CTT for L34A, GCA instead of GAA for E36A, and GCA instead of GTA for V42A, and then performed overlap PCR.
We performed protein purification of mutant MST2 (MST2 3 mutant). The experimental design was similar to the previous purification of MST2 and STRN3. The clear single band in lane 7 shows that the GST-MST2 3 mutant protein was successfully expressed and purified.
After the MST2 3 mutant protein was purified, we tested the role of CX6258 between MST2-STRN3 and MST2 3 mutant-STRN3. The results of lanes 1-4 show that after the binding site of CX6258 to MST2 is mutated, CX6258 no longer affects the interaction between MST2 and STRN3 (two bands in both lanes 1 and 2); the unmutated MST2 protein can still be affected by CX6258 and separated from STRN3 (only one distinct band in lane 3). Lanes 5 and 6 were used to exclude the effect of non-specific binding of GST-MST2 to HST tag or GST tag to HST-STRN3 on the results. Lanes 7-11 were used as the INPUT of each component and were used as a control to make the results more convincing. Overall, this result validates that the sites where CX6258 interacts with MST2 are L34, E36, and V42, elucidating the mechanism by which this small molecule is able to disrupt the MST2-STRN3 interaction. In addition, the elucidation of this mechanism will increase the commercial and translational value of the project results.
We chose a cancer cell called AGS to explore the effect of CX3. We prepared three different concentrations of CX, to investigate which concentration is the most effective. The three concentrations are 0 nmol/L, 25 nmol/L, and 50 nmol/L. We add the CX into about 300 AGS cells and leave them in the cell chamber. After 24 hours, we used Crystal Violet Staining Solution to dye the cells and count the number of the cells. Crystal Violet Staining Solution is a dye often used in tissue or cell staining to stain the nucleus a deep purple color. When it dissolves, it can be ingested by living cells, and it can color DNA, proteins, and fats. Quantitative analysis can evaluate cell growth and reproduction. As Figure 14 shows, the left one is 0 nmol/L, the middle one is 25 nmol/L and the right one is 50 nmol/L. It is obvious that there are more stained cells in the left one and in the right one there are fewer stained cells. In addition, we quantified the inhibitory effect of CX on AGS gastric cancer cell lines using histograms. Overall, the results showed that the higher concentration of CX in the concentration range of 0-50 nmol/L had a greater effect on reducing cancer cells, demonstrating that CX has the potential to inhibit tumor development.
Due to the short growth cycle and high fecundity of Drosophila and the high homology of the hippo pathway to humans, we have chosen the Drosophila model of colorectal cancer as the experimental animal for our project4. We will use two types of Drosophila, the experimental group Esgts>Yki/Esgts>GFP and the control group Esgts>ctrl/Esgts>GFP. Specific overexpression of Yki in Drosophila mesenteric stem cells promotes mesenteric stem cell proliferation, which thickens the colon and produces a colorectal tumor-like phenotype that can lead to Drosophila colorectal cancer. The Esg-GFP is specifically overexpressed in midgut stem cells. We fed 3-day-old adult Drosophila CX6258 for 7 days at 30°C. After dissection, the midgut was fixed and stained with p-H3 and Dapi to represent growing midgut stem cells and nuclei, respectively.
We found that the inhibition of midgut stem cell growth by CX6258 was significant in both experimental and control groups. In the experimental group, the proliferation of colon stem cells was abnormally active due to the overexpression of Yki (similar to colorectal carcinogenesis), and the application of CX6258 resulted in a nearly 3-fold decrease in the number of colon stem cells compared to the untreated group. These results suggest that CX6258 inhibits the proliferation of colon stem cells, and the inhibitory effect is even more pronounced in the colorectal cancer model.
Overall, the reliability of the AlphaScreen high-throughput drug screening system was confirmed by further experimental validation of the screening results, which is expected to provide us with more valuable hit compounds.
In the early stage of the company's development, it is planned to sell some hit compound-related patents with good results in animal experiments to support the company's preparatory expenses, and the remaining ones with a higher market value in the future will be further developed, researched and modified by our company.
Strengths:
Technology leadership: With a unique high-throughput drug screening technology, lead drugs can be quickly found.
Intellectual Property: Through the screening and incubation process, a large amount of intellectual property may be accumulated, which are potential assets.
Incubation mode: Attracting young scientists to join, may form a dynamic and innovative thinking research and development team.
Cooperation mode: Based on the intellectual property rights authorization and cooperation of new drugs, there is a low risk of research and development.
Weaknesses :
Funding pressures at the start-up stage: Initial investments in high-throughput screening and research and development can be considerable.
Low brand awareness: As a new company, market and industry recognition may not be enough.
Resource constraints: There may be limitations in resources, funding, and equipment compared to large pharmaceutical companies.
Opportunities:
Market demand: hippo pathway dysregulation-related cancer treatment is a hot spot, and the market demand for new drugs.
Partnerships and investments: This may attract partnerships and investments from pharmaceutical giants or venture capital firms.
Intellectual property transfer: For some non-core projects, it is possible for the company to earn revenue through technology transfer.
Technological advances: New technologies, such as artificial intelligence, have opened up new opportunities for drug screening and research and development.
Threats:
Competition is fierce: There are many large companies and research institutes in the pharmaceutical industry that are conducting related research.
Fast technology iteration: If you do not continue to invest in research and development, it may soon be replaced by new technologies or methods.
Regulatory pressure: The development and launch of new drugs involves intense regulatory scrutiny that can lead to project delays or failure.
Intellectual property disputes: During the research and development process, you may face intellectual property conflicts with other companies or research institutions.
To sum up, the following links are crucial for start-ups to succeed:
Continuous innovation and optimization of core technologies: As a drug screening and incubation company, the leadership and innovation ability of core technologies are the basis for its continued competitiveness. Continuous technology development and optimization to ensure that the company is always at the forefront of the industry is essential for success.
Strengthen the intellectual property strategy: Intellectual property is not only the core asset of the company, but also the guarantee to ensure that the technology and products are not copied and infringed. It is important to ensure that the innovations generated by the company are protected by the law and that these intellectual property rights are effectively managed and used.
Incubation and profit distribution policy: Attract and retain top talent in the industry to build an efficient, united, and passionate team. This is the key to ensuring technological leadership and product innovation.
Strategic Partnerships: Strategic partnerships with major pharmaceutical companies, research institutions, and venture capitalists can help companies access resources, capital, and market opportunities to accelerate the commercialization of their products.
Accurately target the first batch of target drugs: accurately evaluate the market demand and competition, optimize the development difficulty of lead drugs, and determine the first round of screening and research and development direction.
Strict quality control and compliance: Ensuring the accuracy, repeatability and reliability of drug screening, as well as strict compliance with relevant laws and regulations and industry standards, is the key to gaining the trust of industry and consumers.
Clear market positioning and effective promotion strategy: determine the target market, understand customer needs, and develop effective marketing strategies to ensure that the company's technology and products can be widely recognized and applied.
In the founding stage of a start-up company, the structure and role of the team are usually more concise and flexible. The following is a possible team structure:
Chief Executive Officer (CEO) :
Key responsibilities: Determine the overall strategy and direction of the company, lead the entire team, be responsible for external relations, partnerships and financing activities.
Chief Scientific Officer/Technology Officer (CSO/CTO) :
Main responsibilities: Responsible for the company's research and development activities, technology strategy and innovation, feasibility assessment, ensuring technology leadership and product quality.
R&D Team:
Drug screener
Molecular biologist
Chemist/Pharmaceutical chemist
Data scientist
Intellectual Property and Compliance Department:
Main responsibilities: Responsible for patent application, maintenance and management, ensuring that the company's research and development results are legally protected, and responsible for compliance with relevant regulations and standards.
Department of Business Development and Strategic Cooperation:
Main responsibilities: Seek and establish partnerships with other companies or research institutions to promote the commercialization of the company's technology and products. If early stage venture capital companies have rich experience and industry resources in the biomedical industry, it is recommended that early stage investors provide support for this part.
Seed round funding: The goal is to raise $500,000 to $1,000,000 from angel investors, early-stage venture capital firms or other strategic investors in related fields.
Series A funding: Plans to raise $3,000,000 to $5,000,000 after 18 to 24 months or after certain key technology or business milestones have been reached, primarily from venture capital institutions.
R&D expenditure:
Purchase and maintain high throughput screening equipment.
Buy or rent lab space.
Purchase lab supplies, chemicals and other necessary equipment.
An external service or contract research organization (CRO) that pays for experiments and research.
Personnel salary:
Pay salaries and benefits for key positions in the core team and start-up stage.
The money raised will likely be used to add research and development, sales and administrative positions.
Intellectual Property Protection:
Pay for patent applications, patent maintenance and other intellectual property services.
Market and Business Development:
Develop marketing strategy, brand building and initial product promotion.
Establish and maintain relationships with potential partners.
Operating expenses:
Daily administrative and operating costs, including rent, public utilities, software subscriptions, etc.
Initial training and team building activities.
Emergencies and unpredictable costs:
Set a portion of your budget for unpredictable expenses or contingencies that may arise.
The company's core revenue is technology transfer and drug sales share. We generate revenue through patent transfer for drugs with less risk and predictable market size. For some technologies with large market size, in early research, and not yet in clinical trials, revenue is obtained through sales sharing, which generally accounts for 2-3% of sales revenue.
| the first fiscal year | the second fiscal year | the third fiscal year | the 6th fiscal year | |
| Technology transfer | $500,000 | $1,000,000 | $1,500,000 | $3,500,000 |
| Drug sales share | $6,000,000 | |||
| R&D investment | $300,000 | $300,000 | $500,000 | $1,000,000 |
| Operation support | $100,000 | $120,000 | $150,000 | $250,000 |
| Research team Project revenue share | $150,000 | $300,000 | $450,000 | $1,230,000 |
| Market promotion | $50,000 | $60,000 | $80,000 | $100,000 |
| Legal and policy services | $50,000 | $100,000 | $120,000 | $200,000 |
| Annual income | $500,000 | $1,000,000 | $1,500,000 | $4,100,000 |
| Profit before tax | -$150,000 | $120,000 | $200,000 | $1,320,000 |