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European Biopharmaceutical Review
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For several decades, X-ray crystallography – in which the scattering of
X-rays by the electron clouds of the atoms in a crystalline sample
allows the atomic and molecular structure to be determined – has been
routinely used by the pharmaceutical industry to determine how their
drugs interact with their protein targets. Nevertheless, a limitation of
the technique is that hydrogen atoms (with only one electron) are
virtually invisible in X-ray analyses, leaving scientists to speculate
on their positions despite their importance in drug binding through
hydrogen bonding. This is where neutrons come into their own. Rather
than being scattered by electron clouds, neutrons are scattered by the
atomic nuclei in a crystalline structure, allowing the positions of all
atoms to be determined – including lighter elements such as hydrogen.
Neutron crystallography is therefore particularly suited for use in drug
discovery and development.
Neutron Crystallography
This
potential has been acutely demonstrated in a recent study of
interactions between a common clinical inhibitor and HIV-1 protease – an
enzyme essential for the replication of the virus, allowing it to break
polypeptide chains and generate proteins for viral maturation and the
production of new infectious virions.
After 20 years of analysis
with X-rays, results published earlier this year in the Journal of
Medicinal Chemistry, which demonstrated the use of neutrons, provided a
more detailed picture than ever before of how an antiviral drug used to
inhibit the enzyme actually works. The team were able to identify the
position of every hydrogen atom involved in the system, and discover
which were involved in the binding of the inhibitor to the enzyme
through hydrogen bonding. Critically, the analysis indicated which
hydrogen bonds were the weakest and how the binding – and, hence, the
drug’s performance – could be improved.
This research represents
a major step forward for a technique whose application in the field of
pharmaceutical R&D has been held back in the past due to various
technical challenges. These included the volume of sample crystals that
needed to be grown, the length of time it took for the results to be
collected, as well as the lack of dedicated instrumentation available
around the world with which scientists could carry out their studies.
During
this latest study, the neutron crystallography experiments were carried
out on the quasi-Laue neutron diffractometer (LADI-III) instrument at
the Institut Laue-Langevin (ILL), where developments in the last decade
have addressed the technical challenges. Using the LADI-III, crystals as
small as 0.05mm3 can now be analysed – making many more studies
feasible.
Developments on instruments such as LADI-III are
helping establish a reputation for neutron science as a complement to
X-ray studies during various stages of the drug development process. In
recent years, neutron scattering techniques at the ILL alone have been
the source of a number of major new insights.
Reducing Side-Effects
Earlier
this year, scientists from ILL and King’s College London looked at
another drug prescribed to HIV sufferers – this time a common
antibiotic, Amphotericin B (AmB). This protects against fungal
infections – a potentially deadly affliction to those whose immune
systems are significantly compromised fighting HIV or going through
chemotherapy. While a highly effective drug, the researchers wanted to
examine why increased dosages of AmB – prescribed in recent years in response to antibiotic resistance – has started to cause kidney failure and other life-threatening complications.
Using
neutron analysis of model membrane systems, the team discovered that at
low doses, AmB opens up holes in fungal cell walls – allowing cell
material to leak out or harmful material to get in, which kills the
cell; however, it is unable to penetrate all the way through the human
cell membranes. In the last 20 years, increases in dosage are producing
full penetration of both types of membranes, resulting in the damage to
healthy tissue observed in trials. By beginning to gain a true picture
of how AmB works, scientists can now aim to reduce its side-effects or
utilise its therapeutic properties in new, less harmful, alternative
drugs.
Nanomedicine
It is not only improvements
to existing drugs that are being considered – whole new areas of drug
development are being opened up. Earlier this year, researchers from the
University of Illinois and the Australian Nuclear Science and
Technology Organization used ILL’s neutron-scattering instruments to
investigate, on a molecular level, the physical changes undergone by
cell membranes as they come into contact with gold nanoparticles.
Gold nanoparticles have been identified by major pharmaceutical companies as a potential future drug delivery agent
for the treatment of cancer, based on their ability to target and
penetrate cells to transport drugs directly inside the infected tissue.
By targeting cancerous cells, this area of research – called
‘nanomedicine’ – aims to reduce or even eliminate the need for surgery.
However,
at present we do not understand in any detail the interaction
mechanisms between nanoparticles and the cell’s outer barrier – the cell
membrane. Without this, it is impossible to determine how dangerous
they are and whether their ability to penetrate and destroy cells can
ever be harnessed for good, such as in the fi ght against cancer.
The
first results from a new project at ILL to better understand these
interactions were published earlier this year in Langmuir and looked at
how the surface charge of the membranes affects the interaction. Using
neutron-scattering techniques, the team showed that depending on a
positive or negative surface charge, the interaction with the gold fl
akes could produce a protective shield effect, or the complete
penetration and destabilisation of the membrane. These are vital
insights for the development of future nano-treatments.
Shedding New Light
With
the case for the use of neutrons starting to build, there is the
potential to revisit our understanding of even common drugs, as was the
case in the latest study on the HIV inhibitor amprenavir (APV), which
has been used clinically for over a decade. In this latest study, its
binding with the HIV-1 protease enzyme was analysed by an international
team of scientists from Georgia State University, Purdue University and
Oak Ridge National Laboratory in the US, and Harwell Oxford in the UK.
Despite
its decade of use, it was only following the fi rst analysis with
neutrons that APV ’s true binding behaviour was revealed, painting a
rather different picture to that inferred from the X-ray studies, which
had overplayed the importance of several hydrogen bonds. In fact, the
team found only two strong direct hydrogen bonds between the drug and
the HIV enzyme.
The findings present drug designers with a set
of new sites to try and improve the drug’s chemistry to signifi cantly
strengthen the binding, increasing the effectiveness of this and other
inhibitors, and reducing the necessary dosages.
Suggestions of
areas for improvement include replacing weaker watermediated hydrogen
bonds with stronger direct hydrogen bonds to increase the enthalpy of
binding. This could be achieved by changing certain functional groups of
the drug in order to expel water molecules present in the active site,
which at the same time improves the entropy of binding.
Another
approach would be to increase the strength of the two direct hydrogen
bonds by the creation of what is known as a ‘low-barrier hydrogen bond’,
in which the proton is equidistant between the donor and acceptor
atoms. This can be achieved by lowering the ionisation constant of the
hydroxyl group of the drug to make it similar to that of the amino acids
it binds to, via the introduction of a strong electronegative atom such
as fluorine.
Drug Resistance
The findings in
this latest paper may also help address one of the biggest issues in
combating HIV infection – drug resistance. Evolution of the virus over
time produces enzyme variants with weakened binding affi nity to the
inhibitor – a process that is actually sped up by the introduction of
the drugs themselves. One way around this would be to improve the
binding of the inhibitor with the main-chain atoms of HIV-1 protease
rather than its side-chain atoms, as the main-chain atoms of the enzyme
cannot mutate.
Before this latest study, it was thought that the
potential for advances in this area were limited, because the hydrogen
bond interactions with the mainchain atoms were already very strong.
However, this has been shown not to be the case, creating a new avenue
for the development of HIV pharmaceuticals much less affected by virus
evolution and resistance.
Future Design
X-ray
crystallography has played a crucial role in structure-guided drug
design for over two decades. While its value to researchers in this fi
eld will continue for many years, combining the use of X-rays with
neutrons increases the clarity of how drugs interact with their protein
targets, providing the pharma industry with a powerful new tool to
improve the performance of their products. It is clear that the future
of drug design will feature a combination of the two crystallographic
techniques that can provide patients with newer, more effective
medicines to not only battle HIV infection, but for a whole host of
other diseases as well.
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