Bleomycin: cancer drug with a hidden flaw

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.

Hussain explained:

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

Picking up good vibrations – of proteins – at CHESS

A new method for analyzing protein crystals – developed by Cornell researchers and given a funky two-part name – could open up applications for new drug discovery and other areas of biotechnology and biochemistry.

The development, outlined in a paper published March 3 in Nature Communications, provides researchers with the tools to interpret the once-discarded data from X-ray crystallography experiments – an essential method used to study the structures of proteins. This work, which builds on a study released in 2020, could lead to a better understanding of a protein’s movement, structure and overall function.

Protein crystallography produces bright spots, known as Bragg peaks, from the crystals, providing high-resolution information about the shape and structure of a protein. This process also captures blurry images – patterns and clouds related to the movement and vibrations of the proteins – hidden in the background of the Bragg peaks.

These background images are typically discarded, with priority given to the bright Bragg peak imagery that is more easily analyzed.

“We know that this pattern is related to the motion of the atoms of the protein, but we haven’t been able to use that information,” said lead author Steve Meisburger, Ph.D. ’14, a former postdoctoral researcher in the lab of Nozomi Ando, M.S. ’04, Ph.D. ’09, associate professor of chemistry and chemical biology in the College of Arts and Sciences. “The information is there, but we didn’t know how to use it.  Now we do.”

Meisburger worked closely with Ando to develop the robust workflow to decode the weak background signals from crystallography experiments called diffuse scattering. This allows researchers to analyze the total scattering from crystals, which depends on both the protein’s structure and the subtle blur of its movements.

Their two-part method – which the team dubbed GOODVIBES and DISCOBALL – simultaneously provides a high-resolution structure of the protein and information on its correlated atomic movements.

GOODVIBES analyzes the X-ray data by separating the movements – subtle vibrations – of the protein from other proteins that might be moving around it. DISCOBALL independently validates these movements for certain proteins directly from the data, allowing researchers to trust the results from GOODVIBES and understand what the protein might be doing.

Read more on CHESS website

Image: Meisburger, Case, & Ando (2020) Nat Commun 11, 1271

Undermining the foundations of bacterial resistance

Scientists from the University of Guelph have used the Canadian Light Source (CLS) at the University of Saskatchewan to better understand how several infectious bacteria, including E. coli., build a protective sugar-based barrier that helps cloak their cells.

Published in the Journal of Biological Chemistry, the Guelph research provides the very early steps toward new treatments for E. coli and a whole range of bacteria. Their particular focus is on strains of E. coli that cause urinary tract and bloodstream infections, particularly those that are antibiotic resistant.

The research is looking to understand the enzyme that many infectious bacteria use to build the foundations of their protective capsule. The capsule helps shield the bacterium from attack by the human immune system and exists in many clinically distinct variants.

Making vaccines or drugs that targets the capsule itself directly is impractical as such treatments would target only a few bacteria. Instead, the Guelph team is focused on a key enzyme that builds the capsule foundation. This foundation could serve as a common point of attack, allowing a single treatment for several key pathogens infecting humans and livestock.

“We are interested in the machinery that builds the bacterium’s protective layer,” said Dr. Chris Whitfield, Professor Emeritus in the Department of Molecular and Cellular Biology. “By understanding and targeting the machinery, we can render the pathogen unable to survive in the host”.

Read more on the Canadian Light Source website

Image : Matthew Kimber, Chris Whitfield, and enzyme

Using light to switch drugs on and off

Scientists at the Paul Scherrer Institute PSI have used the Swiss X-ray free-electron laser SwissFEL and the Swiss Light Source SLS to make a film that could give a decisive boost to developing a new type of drug. They made the advance in the field of so-called photopharmacology, a discipline that develops active substances which can be specifically activated or deactivated with the help of light. The study is being published today in the journal Nature Communications.

Photopharmacology is a new field of medicine that is predicted to have a great future. It could help to treat diseases such as cancer even more effectively than before. Photopharmacological drugs are fitted with a molecular photoswitch. The substance is activated by a pulse of light, but only once it has reached the region of the body where it is meant to act. And after it has done its job, it can be switched off again by another pulse of light.

This could limit potential side effects and reduce the development of drug resistance – to antibiotics, for example.

Licht-switchable drugs

To make conventional drugs sensitive to light, a switch is built into them. In their study, the scientists led by the principal authors Maximilian Wranik and Jörg Standfuss used the active molecule combretastatin A-4, which is currently being tested in clinical trials as an anti-cancer drug. It binds to a protein called tubulin, which forms the microtubules that make up the basic structure of the cells in the body, and also drive cell division. Combretastatin A-4, or “CA4” for short, destabilises these microtubules, thereby curbing the uncontrolled division of cancer cells, i.e. it slows down the growth of tumours.

In the modified CA4 molecule, a bridge consisting of two nitrogen atoms is added, which makes it particularly photoactive. In the inactive state, the so-called azo bridge stretches the molecular components to which it is attached to form an elongated chain. The pulse of light bends the bond, bringing the ends of the chain closer together – like a muscle contracting to bend a joint. Crucially, in its elongated form, the molecule does not fit inside the binding pockets of the tubulin – depressions on the surface of the protein where the molecule can dock in order to exert its effect. However, when the molecule is bent, it fits perfectly – like a key in a lock. Molecules like this, which fit into corresponding binding pockets, are also called ligands.

Read more on the PSI website

Image: Jörg Standfuss (left) and Maximilian Wranik in front of the experimental station Alvra of the Swiss X-ray free-electron laser SwissFEL, where the photopharmacological studies were carried out. In the long term, the aim is to develop drugs that can be switched on and off by light.

Credit: Paul Scherrer Institute/Markus Fischer