I am working finding the drug targets of certain drugs whose MOA (mechanism of action) is not known. Will whole genome sequence comparison of the sensitve vs resistant bacterial strain yeild any meaningful data in terms of finding drug targets?
How to validate the difference in genomes found if any, as the specific drug target?
Can a gene knock-out approach be helpful?
Comparing whole genome might be a mammoth task and simple sequencing errors might lead to misleading results . There is an alternate way :
1) Find out how sensitive the background strain is to the new drug you are using.
2) At what rate the resistant mutants arise in a certain population of sensitive strain.
3) find out the highest concentration of drug of your interest that will allow only the growth of your resistant strain.
4) Make a genomic library of resistant strain and transform it into sensitive strain and grow them on medium containing highest concentration of Drug that you had determined before. The colonies that arise will contain the plasmid containing a gene of interest that could have a role in drug resistance.
Drug resistance can arise in a gene coding for target protein of drug, there are certain mechanisms that can cause random mutations in that gene which can lead to structural modifications of the target protein which further makes the strain resistant. And in this case you can find the mode of action of your drug by studying the activity of genes that you from the screening that is mentioned above.
Gene knock-out will be good, provided the number of genes to be considered for the knock-out study is validated. A genome comparison will provide a quicker and easier means of analysis. If the genomes represent closest homologs even a minor variation play a major role functionally. Genes corresponding to those segments can be then used for knock-out study.
I'm afraid you will find out a lot of differences by genomic comparison. further narrowing down the genes for KO is an issue.
But if the resistant strain originates from the sensitive strain and this is an experimental design of the researcher, the genomic differences will be predominately formed by this selection for resistance.
Comparing whole genome might be a mammoth task and simple sequencing errors might lead to misleading results . There is an alternate way :
1) Find out how sensitive the background strain is to the new drug you are using.
2) At what rate the resistant mutants arise in a certain population of sensitive strain.
3) find out the highest concentration of drug of your interest that will allow only the growth of your resistant strain.
4) Make a genomic library of resistant strain and transform it into sensitive strain and grow them on medium containing highest concentration of Drug that you had determined before. The colonies that arise will contain the plasmid containing a gene of interest that could have a role in drug resistance.
Drug resistance can arise in a gene coding for target protein of drug, there are certain mechanisms that can cause random mutations in that gene which can lead to structural modifications of the target protein which further makes the strain resistant. And in this case you can find the mode of action of your drug by studying the activity of genes that you from the screening that is mentioned above.
Whole genome comparison would not be a mammoth task and with NGS a high depth of coverage can be achieved to eliminate sequencing errors to identify putative targets . These could then be tested using the method described by Prashant, but using a more directed approach and only cloning those identified putative targets.
Thanks Prashant Desai for your answer. I have few molecules synthesized in our lab based on CADD. They are based on docking studies on a particular protein target. But there is considerable difference in terms of potency wrt enzyme and whole cell assay. I have hypothesized that the high potency might be due to some off/secondary targets. I am planning to identify those off/secondary targets by comparing the whole genomic transcriptional profile of bacteria in presence and absence of the molecule. Is the approach right??
Kindly advice.
In general bacterial may also develop resistance to your drug due to acquiring enzymes that hydrolyse/effflux your drug through plasmid (episome). This possibility should also be taken in to consideration before going for whole genome comparison approaches.
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