Background
Why improve existing cancer therapies?
Cancer is amongst the largest killers in the world, with over 9.9 million people succumbing to its throes in the year 2021. The most widely accepted treatment for this is chemotherapy, which can pose deleterious effects to not only malignant, but also healthy cells of the body, along with reduction of the immune response due to its non-specificity and low efficacy. There is an immediate need to introduce targeted therapeutic drugs in order to combat more aggressive cancers. Realizing this tremendous potential for improved forms of targeted cancer therapy, our team resolved to work on a potential cancer therapy involving therapeutic drugs in the form of enzyme-prodrug therapy.
About cytosine deaminase and its enzymatic function
The enzyme cytosine deaminase is found in several prokaryotes and a few eukaryotes. Our project adopts the deamination of 5-fluorocytosine (5FC) as the inactive drug into the active 5- fluorouracil (5FU) by employing yeast cytosine deaminase (yCD). 5FU is an antimetabolite interfering with the synthesis of thymine by altering the shape of thymidylate synthase, causing errors in DNA replication and preventing tumor cell proliferation. Targeted delivery of this prodrug to the tumor regions can lead to apoptosis of the cells to produce anti-cancer effects. The direct administration of 5FU may induce several side effects such as increased risk of getting infections, bleeding, and breathlessness. Since 5FC is not deaminated in mammalian cells and is well absorbed, 5FC and CD delivery has little intrinsic toxicity and poses several advantages over direct 5FU delivery.
The enzyme CD is known to sustain an optimum pH of 7.0-7.4. Owing to the low pH conditions found in microtumor environments, the wild type enzyme needs to be mutated to stably function under slightly acidic conditions. Thus, we aim to engineer mutant CD strains with improved stability in the acidic pH range resembling the microtumor environment. We have chosen cytosine deaminase from Saccharomyces cerevisiae (yCD) for optimization over its bacterial counterpart (bCD) due to its improved efficacy and sensitivity towards 5-FC.
What is directed evolution?
Directed evolution is a method which aims to mimic the process of natural selection to attain a specific predefined-goal. Mutations are induced in a given protein and nucleic acid, followed by assaying proteins and selecting the best mutant for the required function. This requires the structure of the protein to be studied in detail, in order to better understand the regions to be mutated. The broad steps involved in performing directed evolution are the generation of multiple mutant libraries, followed by screening for the best mutants. A superior mutant may be produced from the best mutants through genetic recombination.
Owing to the refinements and advancements that directed evolution has brought to the field of synthetic biology, our team concluded that it was the best method to facilitate achieving our goal of creating a pH-stable mutant of cytosine deaminase.
