Hello Everyone,
We are attempting to develop a targeted therapy drug and would like to know what the likelihood is of the targeted molecule gaining resistance against our drug. Is there any assay that will confirm this or at least provide any level of insight?
Thank You
Please feel free to ask questions if you need more information.
Hi Aswini,
It has been a decade since the P-glycoprotein (Pgp) gene, which is associated with a form of MDR caused by reduced drug accumulation, was cloned. Thus, this would seem to be an appropriate time to evaluate our understanding of this form of MDR. The two MDR genes identified in humans to date (the MDR-associated protein [MRP] and Pgp genes) are structurally similar and both are members of the ATP-binding cassette (ABC) transporter family. Although the physiological role of MRP is not yet understood, one Pgp gene (mdr1) plays an important role in the blood-tissue barrier and the other (mdr2/3) is involved in phospholipid transport in the liver. A variety of compounds (chemosensitizing agents) can interfere with Pgp and MRP function; such agents may improve the efficacy of conventional therapy when used in combination with such regimens. Determining the roles cellular MDR mechanisms play in patients’ response to chemotherapy is a major challenge. Using Pgp and MRP as molecular markers to detect MDR tumor cells is technically demanding, and solid tumors in particular contain heterogeneous cell populations. Since MDR requires Pgp or MRP gene expression, clinically relevant gene expression thresholds need to be established; sequential samples from individual patients are valuable for correlating MDR gene expression with the clinical course of disease. Studies in leukemias, myelomas, and some childhood cancers show that Pgp expression correlates with poor response to chemotherapy. Such agents may inactivate Pgp in tumor cells or affect Pgp function in normal cells, resulting in altered pharmacokinetics. It would be interesting to determine whether patients who fail treatment in the presence of chemosensitizing agents acquire other MDR mechanisms. The ABC transporter superfamily in prokaryotes and eukaryotes is involved in the transport of substrates ranging from ions to large proteins. Of the 15 or more ABC transporter genes characterized in human cells, two (Pgp and MRP) cause MDR. Therefore, it would be relevant to determine the number of such genes present in the human genome; however, extrapolating from the number of ABC transporter genes in bacteria, the human gene probably contains a minimum of 200 ABC transporter superfamily members. Thus, tumor cells can potentially use many ABC transporters to mount resistance to known and future therapeutic agents. The challenge will be to determine which ABC transporters are clinically relevant.
Rapid diagnosis can be done with the help of reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (QPCR) assay to detect the drug-resistance of related genes in cancer cell lines. The expression of some drug-resistance genes was compared with the sensitivity and resistance-acquired cancer cell lines to anti-cancer drugs by Northern blot analysis and PCR assay. The resistance cell lines exhibited an enhanced expression of multi-drug resistance (MDR-1), thymidylate synthase (TS), c-fos and DNA polymerase beta genes. Then these genes that expressed mRNA were quantitated using RT-PCR. The expression of the genes was dependent on their sensitivity (IC50) to anti-cancer drugs. Additionally, the QPCR assay has been developed as a rapid method for the expression of drug-resistance genes and applied to the PCR products amplified by the RT-PCR. Thus the QPCR assay for the expression of genes will allow rapid detection of the drug-resistance to chemotherapy in human cancers.
Source: Pubmed
In antibacterial drug discovery, it is common practice to measure the frequency of resistance (FOR) to a drug. A large number of bacterial cells are exposed to a drug concentration that is sufficient to kill them (or at least prevent them from growing). Resistant cells will appear as colonies. The number of colonies that grow in the treated cells divided by the number of colonies that grow in the untreated cells is the FOR.
A similar approach to getting resistant cells can be used with cultured mammalian cells by growing the cells on gradually increasing drug concentrations.
Having thus selected resistant cells, they should be grown in the absence of the drug for a while, then re-exposed to the drug to see if they retain the resistance. If they do, the next step is to sequence the target gene to see if there are any mutations.
Sometimes, resistance is not due to target mutations. It can also be caused by increased target expression, or mutations in other genes, for example. Increased efflux pump expression (like P-glycoprotein) is often a cause of resistance.
If there are mutations in the target gene, the next step is to introduce the mutations into the drug-sensitive cells by site-directed mutagenesis to make sure that the mutation really causes resistance.
Dear Aswini,
The following links describe methods for assessing drug resistance:
1- Assays for Antiretroviral Resistance
http://hivinsite.ucsf.edu/InSite?page=kb-02-02-03
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776203/
http://jac.oxfordjournals.org/content/55/6/817.full.pdf
2-Rapid determination of anti-tuberculosis drug resistance from whole-genome sequences
https://genomemedicine.biomedcentral.com/articles/10.1186/s13073-015-0164-0
Hoping this will be helpful,
Rafik
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