Exploring Mithramycin as an Antitumor Compound

Introduction

Malignant tumour growths have been an issue of major concern universally. Following the detrimental effects that it presents with on the victim, it needs a different approach for effecting curbing of the disease. Conventional medicine, which for long has been considered as the preferable approaches to dealing with cancers, has also presented with various challenges such as being costly13. Lately, chemotherapy is the major approach to the treatment of cancer. However, various antitumor compounds that are natural products or their derivatives produced by microorganisms can be considered for the address of tumour growths in the body16. One of the antitumor compounds that can be considered for the treatment of cancer is Mithramycin (MTM) 5. The product is an antineoplastic antibiotic that is produced by the microorganism Streptomyces argillaceous ATCC12956. It is a DNA or a RNA polymerase inhibitor besides being a DNA-binding transcriptional inhibitor. It thus facilitates tumour necrosis factors alpha and Fas-ligand induced apoptosis. MTM muddles to GC-rich structures that are in the minor groove of DNA and depends on MG2+5. This study will focus on providing a discussion of the strategies that can be use in then screening and the evaluation besides production of anti-tumoral drug such as mithramycin. For those involved in researching similar topics, such as healthcare dissertation help, understanding these antitumor compounds' potential is crucial for advancing treatment methods.

Screening Programmes

Screening programmes and bioassays is an important step in the development of antitumor drugs. In the preliminary assay of anticancer activity, MTT/MTS in vitro cell proliferation assay is one of the most common screening programs6. MTT and MTS in vitro assays are not only reliable but also convenient methods of assessing the preliminary anticancer activity. In the MTT assay, it involves the bioreduction of (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide by dehydrogenase. The process takes place inside of the living cells and forms a colourized formazan dye6. In the MTS bioassay, the same process takes place but 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt and an electron coupling reagent (phenazine ethosulfate) PES are used5.

The MTT bioassay needs solubilising agents that are important in the dissolution of formazan that is insoluble. MTS assay produces a hydrophilic formazan product and makes the assay relatively simpler. Viable cells are measured using calorimetry and works on the principle that the mitochondrial dehydrogenase reduces the colourless tetrazolium salt into a coloured aqueous soluble formazan product through the action of viable cells at a temperature of 37oC6. In addition, there is a direct proportionality between the quantity of the coloured product and the number of living cells in the culture. This is so since MTT/MTS reagent can only be reduced to formazan through the cells that are metabolically active. The assay is of much importance as it characterizes the prospective anticancer agents. The assay can also be undertaken routinely and easily in a laboratory with minimum involvement of intellectual property6. The MTT and the MTS assays examine the toxicity to the specific cell that is being investigated. Cytotoxicity is thus screened for against murine or human cancer cell lines besides normal cell line like the peripheral blood lymphocytes. In addition, the bioassays allows for the determination of the selectivity index of MTM that is under investigation for cancer cells over normal cells. Qualitative observations are important in the confirmation of MTS/MTT results6. This is usually done under the microscope of the cell morphology prior to and after the assay. Through this, potential modes of action of the product can be determined. Besides, it enables in deciding on which further analysis to perform and the choices are usually inclusive of caspase activation, assessing stage of the cell cycle arrest as well as microtubule steadying or subversion6.

MTM can also be taken through the NCI60 screen test, which would in vitro anticancer drug screening. The screening involves a panel of 60 distinct human tumour cell lines from various cancer types6. Some of the examples can be leukaemia or cancers of major organs of the body. The screen tests the degree of growth as well as the inhibition and cytotoxicity of a compound against each of the 60 cell lines. It uses a computer program in the assessment of the pattern of response across the distinct cell lines that are used. This also highlights a potential mechanism of action besides identifying the unique modes of the compound used in its activity against cancerous cells6. The assay only screens pure compounds thus the molecular structure of MTM is submitted that undergoes review before given consideration for testing.

In vitro inhibition assays are used in the determination of activity against particular molecular or cellular target for the confirmation of the mechanism of action of compound as well as investigating the selectivity towards distinct targets besides any off-target effects of the substance under assessment6. The in vitro inhibition assays are undertaken against purified cell enzymes or cell-free extracts that are rich in the enzyme target of interest. One of then assays that can be undertaken for MTM is the protein kinase inhibition since protein kinase have been reported to be the second most significant target for anticancer drug. These assays are based on the detection of the extent to which the potential drug compound can inhibit the activity of an enzyme6.

Production

Mithramycin (MTM) is of the aureolic acid family with anti-tumour activity and is produced by its host cell organism Streptomyces argillaceous. The microorganism is grown in plates at 30oC9. Protoplast transformation is usually based on standard pre-set procedures. The organism is grown on R5 solid medium plates to allow for the regeneration of protoplast. Spores of distinct strains or clones are grown in TSB medium at 30oC and 250rpm for 24 hours9. The seed culture is then used in the inoculation of Erlenmeyer flasks which with 50ml of R5A medium10. At different times, samples are then removed and are centrifuged, filtered, extracted before being analyzed by HPLC9.

Generation of Mutants

MTM is a bioactive compound that is glycosylated by oligosaccharide chains containing sugars that are dominantly derived from glucose-1-phosphate. The sugars in MTM have been reported to be responsible for its bioactivity besides taking part in the various interactions with the cell target. The sugar profile of the compound can thus be changed to generate mutants that are more active and with heightened pharmacological activity. This process of generating MTM mutants can take distinct approaches such as chemical synthesis, chemoenzymatic approach, or combinational biosynthesis10. Generating the mutants of MTM with is undertaken with novel saccharide profiles and the process first involves the transformation of the producer strain with distinct plasmids that directs the biosynthesis of different DOHS. These plasmids and genes are then used for various purposes9. The plasmids that are introduced contain DOH biosynthesis clusters to Streptomyces argillaceous through the synthesis of existing DOHs or the synthesis of novel DOHs that are produced in the recombinant strains10.

Four distinct glycosyltransferases are involved in the synthesis of mithramycin and the mutants can have new sugars besides the sugars situated at distinct positions of the disaccharide and trisaccharide chains10. Two types of compounds are usually expected. These include the tetracyclic compounds and tricyclic compounds with low and high activity respectively10. The absorption spectrum of both compounds is useful differentiating them as they have different absorption maxima of 230, 278, 317, and 411 nm for compounds that are like MTM and 232, 282, 329, and 424 nm for those that are premithramycin like10. After the sugar plasmids are introduced in the mithramycin producer, transformants are then cultivated on R5 A medium alongside thiostrepton and cultures that are removed from ethyl acetate10. HPLC and HPLC-MS is used in the analysis of the organic extracts leading to the classification of plasmids in distinct groups because of plasmids that do not alter the glycosylation profile, plasmids that induce the formulation of premithramycin-like compounds, and the plasmids that induce the formation of mithramycin like compounds15. Some of the compounds can undergo further purification and categorization. From NMR and MR structure analyses, some of the peaks that can be noted respond to compounds such as demycarosyl-mithramycin, dideolivosyl-6-β-d-amicetosyl-mithramycin, demycarosyl-3D-β-d-digitoxosyl-mithramycin, and deoliosyl-demycarosyl-3C-β-d-boivinosyl-mithramycin10.

Some of the mutants of MTM contain one DOH and their biosynthesis is mostly encoded by the sugar plasmids that are used9. Demycarosyl-3D-β-d-digitoxosyl-mithramycin and deoliosyl-demycarosyl-3C-β-d-boivinosyl-mithramycin usually contain D-amicetose and a D-digitoxose respectively15. In addition, demycarosyl-3D-β-d-digitoxosyl-mithramycin when produced by Streptomyces argillaceous produces DOH that is synthesised by the concerted action of enzymes that are encoded by the sugar plasmid and enzymes from the mithramycin sugars biosynthetic machinery9. The host produces 4-ketoreductase, which leads to the formation of D-amicetose through the MtmC involved in D-olivose that is attached at position C of mithramycin9. The advantage of combinatorial biosynthesis approach of sugar biosynthesis of genes is that it is a potent mechanism to the generation of mutant mithramycins with sugars being substituted at distinct positions of the bioactive compound10.

Industrial Production

Production of mithramycin to meet industrial demand needs scale-up processes. The polyketide antitumor compound is industrial produced by Streptomyces argillaceous with a tricyclic aglycone and two aliphatic side chains besides a trisaccharide and a disaccharide chain14. Its biosynthesis in the laboratory begins from the condensation of the malonyl-CoA units. This concentrates a carbon chain that undergoes change to a tetracyclic intermediary. It is then glycosylated by five deoxysugars that originate from glucose-1-phosphate. Subsequently, oxidation and reduction takes place thus rendering the final compound14. For industrial demand to be met, the production needs to be scaled up. In doing so, the predecessor source of malonyl-CoA or glucose-1-phosphate in Streptomyces argillaceous is increased to enhance the production of MTM14.

The pool of malonyl-CoA is hightened through the increased expression of the S.coelicolor phosphoglucomutase gene pgm. On the other hand, glucose-1-phosphate can be increased through the overexpression of the acetyl-CoA carboxylase ovmGIH genes from oviedomycin biosynthesis gene cluster in Streptomyces argillaceus14. These strategies enable an upsurge in the intracellular pool of glucose-1-phospahate and malonyl-CoA respectively. Also, the pool of glucose-1-phospahate and malonyl-CoA can be improved through the inactivation via cloning of S. argillaceous ADP-glucose pyrophosphorylase gene glgCa and the acyl-CoA:diacylglycerol acyltransferase gene aftAa. These enhancements would result in a heightened production of MTM. A combination of these strategies in the lab would enable industrial production as the highest production levels would be met in such a case14. Also, ten strategies have also been reported to be applicable for the increased production of MTM mutants that have better pharmacological properties such as demycarosyl-mithramycin, demycarosyl-3D-β-D-digitoxosyl-mithramycin, mithramycin SK and mithramycin SDK14.

Clinical Evaluation and Approval

After the identification of a product with potential antitumor activity, clinical trials are usually crucial before the regulatory approves them for usage. Normally, the clinical trials for regulatory approval of products such as MTM are needed to demonstrate the clinical benefits of the compound in a target population12. Clinical development of MTM would proceed through orderly clinical studies, which commences from the evaluation of both the safety as well as the pharmacokinetics of the product8. The recommended dosage and the administration of the compound should then be determined. After that, the efficacy and its safety are assessed in an exploratory manner12.

The clinical benefits of MTM should then be compared with the conventional standard medications that are already in existence8. Also, the uncertainties and relevance of external data are given consideration in relation to MTM that is being experimented on and the conditions that it would be used to treat. The clinical evaluation should aim at showing overall survival. The guideline on anticancer medicinal products focuses on cytotoxic compounds such as MTM8. Besides, the effect on the quality of life and overall safety benefits that is associated with new cancer medicines such as MTM12. After the regulatory bodies evaluate these, the drug can be approved. The cost of the critical evaluation is $50 000. Evaluation is done through structure-based design that gives insights into the development and optimization. It will be evaluated on both males and females aged 40 to 50 years as they suffer from cancer more relative to other age groups. The group is of black American ethnic group. The evaluation validates the usage of the antitumor product.

Conclusion

Mithramycin has been identified as one of the products with potential anticancer activity thus can be exploited. It is a polyketide antitumor compound from the Streptomyces argillaceous. Developing the compound alongside other anticancer products is ultimately crucial to the treatment of malignant tumours which has a become an issue of global concern. MTM is a secondary bioactive metabolite and is amongst the chemicals used in the curbing of various kinds of tumours. The study has noted that screening of this compound through MTT and MTS bioassays determines the potential anticancer activity of the product as a preliminary step. Genetic mutation can then be applied in the enhancement of the anticancer activity of the product by producing its derivatives with better pharmacological activity. In addition, the laboratory production can be scaled up to meet industrial production demand. Before the product is approved, clinical evaluation by regulatory body for its safety and efficacy is key. Discovery programmes are important in detecting anticancer activities of microorganism to curb the detrimental effects of cancer on the global population.

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Bibliography

Baltz (2017) Gifted microbes for genome mining and natural product discovery. J Ind Microbiol Biotechnol. 44(4-5):573-588. doi: 10.1007/s10295-016-1815-x.

Baltz (2017) Molecular beacons to identify gifted microbes for genome mining. J Antibiot (Tokyo). 70(5):639- 646. doi: 10.1038/ja.2017.1.

Baltz (2019). Natural product drug discovery in the genomic era: realities, conjectures, misconceptions, and opportunities. J Ind Microbiol Biotechnol. 46(3-4):281-299. doi: 10.1007/s10295- 018-2115-4.

Doroghazi et al (2014) A roadmap for natural product discovery based on large-scale genomics and metabolomics. Nat Chem Biol. 10(11):963-8. doi:10.1038/nchembio.1659.

Hamed et al (2018). Antimicrobial, antidermatophytic, and cytotoxic activities from Streptomyces sp. MER4 isolated from Egyptian local environment. Bulletin of the National Research Centre. 42, 22 - 28. https://doi.org/10.1186/s42269-018-0022-5.

McCauley, J., Zivanovic, A., & Skropeta, D. (2013). Bioassays for anticancer activities. In Metabolomics Tools for Natural Product Discovery (pp. 191-205). Humana Press, Totowa, NJ.

Noomnual et al (2016). Streptanoate, a new anticancer butanoate from Streptomyces sp. DC3. The Journal of Antibiotics (2016) 69, 124–127.

Penson, D. F. (2017). Re: Assessment of Overall Survival, Quality of Life, and Safety Benefits Associated with New Cancer Medicines. The Journal of urology, 197(5), 1220-1221.

Pérez, M., Baig, I., Braña, A. F., Salas, J. A., Rohr, J., & Méndez, C. (2008). Generation of new derivatives of the antitumor antibiotic mithramycin by altering the glycosylation pattern through combinatorial biosynthesis. Chembiochem: a European journal of chemical biology, 9(14), 2295.

Remsing, L. L., González, A. M., Nur-e-Alam, M., Fernández-Lozano, M. J., Braña, A. F., Rix, U., ... & Rohr, J. (2003). Mithramycin SK, A Novel Antitumor Drug with Improved Therapeutic Index, Mithramycin SA, and Demycarosyl-mithramycin SK: Three New Products Generated in the Mithramycin Producer Streptomyces a rgillaceus through Combinatorial Biosynthesis. Journal of the American Chemical Society, 125(19), 5745-5753.

Rigali et al (2018) Cracking the regulatory code of biosynthetic gene clusters as a strategy for natural product discovery. Biochem Pharmacol. 153:24-34. doi: 10.1016/j.bcp.2018.01.007.

Salas-Vega, S., Iliopoulos, O., & Mossialos, E. (2017). Assessment of overall survival, quality of life, and safety benefits associated with new cancer medicines. JAMA oncology, 3(3), 382-390.

Wright (2014) Something old, something new: revisiting natural products in antibiotic drug discovery. Can J Microbiol. 60(3):147-54. doi: 10.1139/cjm-2014-0063.

Zabala, D., Braña, A. F., Flórez, A. B., Salas, J. A., & Méndez, C. (2013). Engineering precursor metabolite pools for increasing production of antitumor mithramycins in Streptomyces argillaceus. Metabolic engineering, 20, 187-197.

Zeng et al (2020) High-Throughput Screening Technology in Industrial Biotechnology. Trends Biotechnol. 38(8):888-906. DOI: 10.1016/j.tibtech.2020.01.001

Zhang et al (2017) Using natural products for drug discovery: the impact of the genomics era. Expert Opin Drug Discov. 12(5):475-487. doi: 10.1080/17460441.2017.1303478.