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Inside the lab:  testing anti-cancer drug candidates

Drug concentration: thinking small

The very first phase of the drug screening process involves invitro tests. Unlike invivo tests, which are carried out on animals and humans, invitro tests are about working on cells cultures. The concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century. Cells are grown and maintained at an appropriate temperature and gas in a cell incubator. In our case, cancer cells are cultivated in order to be treated with the drug candidate and further observed.

A scientist working on drug screening is interesting in two things: first, to know whether the drug candidate is effective against cancer cells, and secondly, to understand the way the drug is killing cancer cells.

Since drugs are natural or artificial- origin chemicals, they trigger certain chemical reactions in the cell, which eventually leads to its death or results in “cell cycle arrest”. “Cell cycle arrest” means preventing the cell from replicating. Cancer cells are known to replicate uncontrollably, dividing beyond normal limits .So drugs are actually supposed to stop these cells growing in number and invading and destroying other tissues.

However, drugs are not intelligent beings who can distinguish between healthy and cancer cells.  Drugs do harm all living cells. This is the reason why scientists focus on small numbers: the lesser the needed amount of a drug in order to be efficient, the better for the living organism. Now you understand why chemotherapy has so many side effects on humans!…

IC50 represents the concentration of a drug that is required for killing 50% of the cancer cells in vitro. Bearing in mind the drugs’ toxic potential, researchers select for further studies only those drugs who prove to have small IC50s concentrations.

Main Target: cancer cells

These small IC50 drugs become the subject of new investigations in order to understand the way they chemically affect the cell. This means identifying the “drug target”. The “drug target” may be theoretically any kind of cell compound, but the majority of today’s drugs are aimed at targeting proteins.

The interactions between proteins are important for the majority of biological functions. For example, proteins mediate outside-in the cell signals. Also, a protein may interact briefly with another protein just to modify it. This is the reason why more and more protein-targeted drugs are developed.

In order to be able to fully explain the drug’s mechanism, scientists are interested in narrowing their search to specific kinds of protein to protein interactions trying to find out exactly how that particular drug works.

In order to achieve this, scientists are carrying out controlled lab experiments or assays, testing or measuring the activity of a drug in an organic sample .These assays are aimed at saving time by identifying major protein categories the drug might have an effect upon.

One such example is the kinase assay.The kinase assay is aimed at deciding whether the drug candidate works as a “kinase inhibitor”.

The kinase enzymes – tiny, but important

Kinases are a very important class of enzymes found in the cell. Kinases are very important compounds that regulate many aspects such as cell growth, cell movement, cell death, metabolism, cell cycle, cell differentiation, etc.

When kinase enzymes are not working right, the healthy cell develops abnormalities and eventually, transforms itself into a cancer cell. So an anti-cancer drug’s “mission” would be to inhibit (turn off) the “broken” kinases in order to stop the cancer cells from proliferating.

Neverthless, kinases are not the only potential drug targets. Actually, one of the major challenges of today’s drug discovery is precisely the explosion of potential “protein drug targets”. The more scientists learned about protein interactions and the more druggable proteins they discovered (up to 3000 now!), the more difficult it became for them to test whether a drug has any effect on these growing number of compounds.

However, kinase inhibitors remain the largest and fastest growing category of drugs in development . Kinase inhibitors currently consume 30% or approximately US$12 billion in research each year.

Exterminating cancer cells

Basically, what a kinase enzyme does is to add a phosphate onto a molecule, typically a protein. This process is called phosphorylation. Before getting too technical, it is important to say that phosphorylation activates or deactivates many proteins, causing or preventing the mechanisms of diseases such as cancer and diabetes.

One such example of the regulatory role that phosphorylation plays is the p53 tumor suppressor protein. This protein’s name tells it all.  Activation of the p53 protein leads to cell cycle arrest (preventing the cell from further replicating), or apoptotic cell death.

Now if you wonder what apoptotic cell death means, this is actually a very simple and fascinating notion: cell’s suicide. Whenever a cell is damaged, her biological “program” tells her to commit suicide, for the sake of the whole organism. A cell will commit apoptosis whenever a biochemical event such as loss of cell membrane, nuclear fragmentation or chromosomal DNA fragmentation, will irreversibly affect her.

Of course, one may argue that a drug who induces cell suicide actually commits “crime”, but apoptosis is a much desired result. Apoptosis is actually a good way of getting rid of cancer cells or any other forms of cell death, such as necrosis, that are known to be dangerous for the living organism.

Cancer research, a long way to go…

To sum up, most of the anti-cancer drugs developed nowadays are very likely to target proteins. At the moment, scientists are focusing on understanding all the chemical reactions that are affecting different classes of proteins which exist inside the cell.

Understanding the way the drug works is an essential step of the drug screening process. Knowing a certain drug is effective against cancer cells is an incomplete information. The drug’s mechanism also needs to be deciphered.

This is the reason why invitro tests are a primary, essential phase of the drug development process.

Nevertheless, cancer research is a long way to go…

Irem Durmaz and Ebru Bilget, young researchers @Bilkent University ,preparing the kinase assay.

Irem Durmaz, performing the kinase assay

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