Cholera and other bacterial-based diarrheal diseases continue to kill many people around the world every year. It is risky to use antibiotics against these bacteria, because killing the bacteria can lead to a large amount of toxin release into the patient.
Scientists at the University of Texas Southwestern Medical Center have developed an alternative approach to combating these diseases. They have fabricated peptoid-coated microparticles that chemically bind to cholera toxin, preventing the toxin from damaging intestinal cells.
What is a peptoid molecule?
Both peptoid and peptide molecules are short chains of amino acids (the building blocks of proteins). Peptoid molecules are an entirely synthetic variant of peptide molecules.
The chemical difference between the two is in the location of the side chains which extend from the molecule's backbone. The side chains extend from the nitrogen atoms of the backbone in peptoid molecules, and from the "alpha-carbon" atom in peptide molecules.
Finding the ideal peptoid molecule.
The scientists first needed to discover a peptoid molecule that tightly binds to cholera toxin. For this purpose, they turned to combinatorial chemistry.
In this context, combinatorial chemistry refers to the chemical synthesis of a large number of molecules, each unique yet structurally related, and rapidly screening them for the desired effect. In this case, the desired effect was tight binding to cholera toxin.
Combinatorial chemistry may be unfairly disparaged as testing zillions of possibilities at random, hoping to find a positive result. However, in reality, rapidly screening large numbers of candidate molecules, in a systematic fashion, using small quantities of molecules, saves a great deal of time and provides vast data sets, from which relevant chemical trends may be elucidated.
These scientists screened 100,000 peptoid molecules, chemically bound to microparticles, for efficacy towards chemical binding to cholera toxin. The peptoid-coated microparticles were washed with cholera toxin, which had been chemically modified with biotin (vitamin B7).
The scientists then washed the microparticles with fluorescent streptavidin proteins (which chemically bind to biotin, and thus the peptoid molecules). In this manner, the scientists were able to visualize, with a microscope, which microparticles possessed peptoid molecules that chemically bind to cholera toxin.
The scientists manually isolated positive microparticles, rinsed the peptoid molecules off of them, and subjected them to a further screening procedure to isolate the desired peptoid molecules. In this manner, the scientists found two peptoid molecules that form tight chemical bonds with cholera toxin.
Strength of chemical binding.
It was important for the scientists to determine the strength of the chemical bond between their peptoid molecules and cholera toxin. For pharmaceutical purposes, one classification method is the minimum concentration of toxin needed for detectable chemical binding.
In this case, binding at 60 nanomolar concentrations was detected. This is 3.6 × 1016 cholera toxin molecules per liter of solution, which believe it or not is a very small concentration (remember that molecules are very small; proteins are considered to be very large molecules, and on average even they are only five billionths of one meter in diameter).
Protecting cells from cholera toxin.
The scientists then set out to prove that the peptoid-coated microparticles protects intestinal cells from cholera toxin. They utilized an assay based on an electrical current.
When a single layer of their cells is exposed to cholera toxin, an electrical current (toxicity) is observed. In contrast, in the presence of peptoid-coated microparticles, the electrical current is dramatically reduced, suggesting protection against cholera toxin.
Future directions.
Based on these laboratory results, the scientists intend to test the efficacy of their peptoid-coated microparticles in the intestines of living animals; if all goes well, in humans. In the absence of unforseen toxicity, this development holds much promise for combating not only cholera, but also other pathogenic bacteria that effect toxicity by releasing toxic molecules into their surroundings.
for more information:
Simpson, L. S.; Burdine, L.; Dutta, A. K.;
Feranchak, A. P.; Kodadek, T.
Selective toxin sequestrants for the treatment of
bacterial infections.
J. Am. Chem. Soc. 2009, 131, 5760-5762.