Help for an Organic Chemist
Posted 18 January 2010 - 12:08 PM
I am an organic chemistry graduate student and my work involves the synthesis of molecules as potential antibiotics. As such I've gone through and learned the techniques necessary to culture and test my compounds on a variety of bacteria.
We have made a compound that we feel very confident should work yet the initial results suggest it doesn't. One of our assumptions is that the cell wall of the bacteria is not permeable to the compound we've designed (which I realize doesn't help us from a pharmaceutical perspective, but we want to learn some science behind the bacteria too).
So I have heard in passing that one way to test for the cell wall permeability issue would be to hit the bacteria with a sub-lethal dose of a cell wall disruptor (like any of the penicillin class that it may be susceptible to). Is this actually a viable experiment? At a non-lethal concentration does the bacteria's cell wall really become like swiss cheese so that our compound may pass through? And if this is true, are there any guidelines to the concentration of cell wall disruptor to add or do you go with the highest concentration that doesn't kill the bacteria?
Thanks for helping the novice in advance.
Posted 18 January 2010 - 06:29 PM
Posted 19 January 2010 - 09:15 AM
Posted 19 January 2010 - 11:29 AM
Penicillin and related antibiotics don't 'disrupt' the cell wall or make it more permeable. Rather it inhibits cell wall synthesis and assembly.
Thank you for the reply I appreciate it.
I agree with the fact that the penicillin's serve as cell wall synthesis inhibitors, however, this is only true at lethal concenrations of the drug. Only when the concentration is high enough do you have complete cell wall synthesis inhibition.
What I am curious about however is what the effect is at sub-lethal concentrations. My thought process behind the "leakyness" is the following. The penicillin's act to bind to the enzymes which catalyze the cross-linking of the peptidoglycan oligimers and I believe I have heard that a properly constructed peptidoglycan wall will allow small molecules about 2 nm in size to permeate through. So im thinking that at sub-lethal concentrations, the antibiotic is still going to be active in its ability to bind to the proteins which drive the cross-linking, just not enough to cause a lack of survival for the cell. I'd imagine however that the compound won't be an all or none response, and despite not killing the cell, it has stopped the cross-linking of the oligimers in a whole bunch of places and therefore you now have area's where the "hole" which molecules can pass through will be larger due to there being missing peptidoglycan cross-links which would have taken up that space.
It would be analagous of trying to push a golf-ball through a quilt in my mind. The quilt is made of many many small strings which together only allow super small particles like dust to pass through. If I now take a pair of scissors (penicillin) I could cut a path for the golf ball. Sure, I could make 10,000 cutes (high conc) and totally demolish the quilt, but I could also make 10 cuts to form a hole large enough for the ball to fit.
So I don't know if my though process makes sense at all I felt I was rambling a bit. I guess where my questions in the logic come in deal with how the cell responds to this attack by a sub-lethal penicillin concentration (lets assume there are no beta-lactamases). Can the PBP's be upregulated to combat the presence of the drug? Can the cell repair any cross-links which have not been made thus plugging up any holes that might have been made?
Or is my biology just way too far off all together? Thank you again and hopefully my post makes sense!
Posted 19 January 2010 - 01:17 PM
Posted 19 January 2010 - 03:52 PM
Posted 02 February 2010 - 02:52 PM
You might try an aminoglycoside, perhaps neomycin or kanamycin, as they not only inhibit bacterial protein synthesis, but also cause the "formation of transient holes in the cell wall and disruption of the normal permeability of the cell wall" (see here). Using a compound like this, or perhaps a non-ionic detergent (like Tween) or a cationic detergent (like hexadecyltrimethylammonium bromide, benzalkonium chloride, or SDS), or a natural substance (like bile salts or bile extract), you might be able to affect permeability of the membrane.
Thank you for the reply. I did some reading on the aminoglycosides and I am going to use this approach. I'll let you know if it works out and again I appreciate the recommendation!