Scientists at the B23 beamline of the Diamond Light Source have used synchrotron light to make an important discovery about a common cancer therapy. Bleomycin is used to shrink a variety of tumours, but little is known about how this drug interacts with proteins in the bloodstream. The beamline scientists used synchrotron-grade circular dichroism to study how bleomycin interacts with two common blood proteins, one of which is normally elevated in people with cancer. Reporting in the International Journal of Molecular Sciences, they found that the drug bound more firmly to the protein elevated in cancer patients, suggesting there may be less of the free form available to elicit its therapeutic effect. On closer analysis, the team discovered that one of two variants of bleomycin binds more strongly to this protein than the other. They caution that the ratio of these two variants may need to be adjusted to improve the therapeutic benefit of this drug.
While screening compounds produced by bacteria in the 1960s, scientists made a serendipitous discovery. They stumbled upon a molecule called bleomycin with anticancer properties. Since then, this life-saving drug has been used to treat a variety of tumours from squamous cell carcinomas to lymphomas. The drug works by chopping up DNA in cancer cells, and this DNA-drug interaction has been characterised in the past. However, when bleomycin enters the bloodstream, it may interact with plasma proteins and less is known about how this impacts the drug’s effectiveness. Bleomycin shows promising outcomes when tested on cancer cells grown in the lab, but the serum extracts used in lab cultures have a different mix of proteins to the sera of cancer patients, so it’s worth exploring whether plasma proteins in patients could sequester the drug and reduce its effectiveness.
Beamline scientists at B23 were determined to explore this overlooked issue. Led by Rohanah Hussain, they harnessed ultraviolet light from synchrotron radiation to explore how the drug binds plasma proteins using a technique called circular dichroism.
Circular dichroism is the differential absorption of left- and right-circularly polarised ultraviolet light passing through a liquid solution containing biomolecules, in this case proteins with drugs The CD measurement is displayed as a curve (spectrum) of which shape reflects the architecture on how the protein in solution is folded in helical, ribbon, turn and unordered segments. Drug binding to protein can affect such a folding that is used to identify and quantify drug binding interaction, in this case bleomycin with the two major blood proteins. Another unique experiment carried out at B23 beamline is the use of the powerful synchrotron beamlight to irradiate multiple of times the protein-drug mixtures for photostability assessment, which varies depending upon the strength of the drug binding interactions.
The high photon flux available at the B23 beamline (Diamond Light Source) generated by synchrotron radiation is sufficient for disrupting the folding of biological macromolecules in a time scale of minutes to hours, providing a useful tool for accelerated photo-stability studies.
First, the team assessed whether Blenoxane®, a commercial preparation of bleomycin, could bind to two common plasma proteins: one was human serum albumin (HSA), an abundant serum protein that facilitates the delivery of drugs around the body through the bloodstream. The other was α1-acid glycoprotein (AGP), a protein produced by the liver in response to inflammation that is found in cancer patients at ten times the normal level.
To explore binding interactions with these two proteins, the team examined the circular dichroism curve for each protein across a spectrum of ultraviolet light, and then they observed whether addition of Blenoxane® altered the protein curve. Sizeable differences were observed with AGP, suggesting the drug binds and induced marked changes to the protein’s shape, but the curve didn’t shift for HSA. This doesn’t indicate that the drug doesn’t bind HSA, only that it doesn’t alter its shape upon interaction. The team adapted their circular dichroism experiments to confirm that Blenoxane® did bind to HSA by heating the sample to unfold (denature) the proteins’ architecture and then observing spectral changes with and without the drug present.
Read more on Diamond Light Source website
Image: Rohanah Hussain is a Senior Beamline Scientist, working on the B23 beamline at Diamond