Cancer enzyme targets: Neutrons and glaucoma drug

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  • Published: Feb 15, 2018
  • Author: David Bradley
  • Channels: X-ray Spectrometry
thumbnail image: Cancer enzyme targets: Neutrons and glaucoma drug

Enzyme targets

This image shows the active site of hCA II. The active site is flanked by hydrophilic (violet) and hydrophobic (green) binding pockets that can be used to design specific drugs targeting cancer-associated hCAs. Five clinical drugs are shown superimposed in the hCA II active site, based on room-temperature neutron structures. Credit: (ORNL/Andrey Kovalevsky)

Medicinal chemists and drug designers have focused on human carbonic anhydrases (hCAs) as potential targets for many years as they play a wide range of roles in the cell. Now, a neutron crystallographic analysis of various drugs used to treat the eye condition glaucoma and their enzyme target could point the way to novel pharmaceuticals for targeting aggressive cancers more effectively because of the link with hCAs. hCAs are zinc metalloenzymes that interconvert carbon dioxide and bicarbonate and so are critical to many biological processes.

Andrey Kovalevsky, Mayank Aggarwal, Hector Velazquez, Matthew Cuneo, Kevin Weiss, and Jeremy Smith of Oak Ridge National Laboratory, Tennessee, USA, and Matthew Blakeley of the Institute Laue-Langevin, Zoë Fisher of the European Spallation Source and Lund University, and Robert McKenna of the University of Florida, used neutron macromolecular crystallography. They investigated the different states of three glaucoma drugs as they interact with their target enzyme, human carbonic anhydrase II (hCA II). Each drug is a sulfonamide inhibitor that binds to the zinc metal centre in the enzyme.

"Our goal was to observe differences in the presentation of three clinically used glaucoma drugs while they are bound to the hCA II enzyme," explains Kovalevsky, an instrument scientist at ORNL and a senior on the paper. "By looking at how well these drugs target hCA II in protonated, neutral and deprotonated states, we hoped to obtain insights that would make it possible to improve these medicines so they can better target enzymes linked to cancer."

Protonation state

Changing the protonation state of the drugs from neutral, positive or negative charge to another state could be used to modulate its ability to recognize and bind to its target enzyme, or other protein, and so boost its efficacy. Indeed, writing in the journal Structure, the team explains how they found that temperature, pH, and electrical charge of all the three glaucoma drugs could affect their ability to target and bind with the hCA II enzyme.

"This discovery was really a proof of principle for us," adds McKenna, also a senior co-author. Moreover, new information about the hydrogen-bonding networks that make up the active site within hCA II could be used to develop novel drugs for cancer treatment given that the family of hCA enzymes contains similar proteins, such as hCA IX and XII, which are, for instance, closely associated with aggressive breast cancers, such as triple negative breast cancer.

"If we can understand binding at the atomic level, we can redesign drugs and turn them into stronger and more selective 'magnets' that will be attracted to cancer-associated enzymes," Kovalevsky explains."Such drugs would be much more effective at killing cancer cells while leaving healthy cells unhurt." This would reduce putative side effects for patients significantly, he suggests. In earlier work, scientists have commonly used X-ray crystallography for their structural studies of hCA enzymes. However, these studies have lacked the requisite atomic information on drug binding because X-ray techniques cannot reproduce the details of the hydrogen atoms abundant in proteins and enzymes. Neutrons, by contrast are much more sensitive to lighter elements, so they provide much more detailed information on the location of hydrogen atoms.

Neutrons trump X-rays

Revealing details of the position of hydrogens atoms, hydrogen bonds and, critically, protonation, in the enzyme-substrate complex is well within the capabilities of neutron crystallography.

"When you use neutron diffraction you don't have radiation damage, so you can do your study at room temperature," McKenna adds. "In addition, freezing crystals may alter the drug and enzyme, introducing a false view into the study, while room temperature studies more closely resemble the environment the drug will be used in."

Related Links

Struct 2018, online: "To Be or Not to Be Protonated: Atomic Details of Human Carbonic Anhydrase-Clinical Drug Complexes by Neutron Crystallography and Simulation"

Article by David Bradley

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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