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Structural Biology in the Pharmaceutical Industry
by Roman Hillig, Bayer Schering Pharma AG, Berlin
Over the last 15 years protein crystallography has become a standard method in the
drug discovery process so that today there are interesting research areas and job
opportunities for structural biologists in the pharmaceutical industry. Despite this,
PhD students in Structural Biology often do not have a clear picture of how work in
pharma research is organized, and even the nomenclature of the drug discovery
process is often not very clear: What is the difference between “hits”, “leads” and
“drugs”? And is every protein implicated in a disease automatically a “drugable
target”? I would therefore like to introduce the different steps of the drug discovery
process and give an overview on the contributions of Structural Biology to the various
steps.
A typical project starts with the identification of a promising target protein, either
based on in-house research or on data from the public domain. During the
subsequent target validation phase, experiments such as siRNA knock-down
studies or overexpression studies are carried out to verify that inhibition of the
putative target indeed results in the expected effects in cellular assays (e.g. slowing
down the proliferation rate of cancer cell lines, while not affecting non-tumor cell
lines). Even at this early stage, long before a decision has been made to clone,
express and crystallize this protein, Structural Biology can already contribute: For
early target candidates, we check whether there are structures of the protein itself or
related proteins in the PDB and, if so, analyze carefully whether the structure
features a pocket suitable for binding of small molecule inhibitors. This assessment is
then one of the criteria based on which target candidates will be prioritized to arrive at
a balanced target portfolio.
If a protein is accepted as a validated target, the lead generation phase is initiated:
In a first step, low molecular weight compounds with at least some inhibitory effect on
the target (so-called initial hits) are identified (hit identification phase). This is
usually done via high-throughput screening (HTS) where all compounds in the
large proprietary compound library of a pharma company (often more than 1 million
compounds) are tested in a biochemical or cellular assay with the target protein. The
best inhibitors identified in this screening campaign show often still only weak affinity
to the target, but nevertheless represent the starting points for the subsequent
optimization steps. In the hit-to-lead process, the hits are further characterized for
their affinity to the target, selectivity against undesired related proteins, possible
toxicity, pharmacokinetic properties (such as solubility or metabolic stability) and their
chemical optimization potential. Usually hits fall into different groups (termed
‘clusters’) which share a common chemical scaffold. At the end of a successful hitto-lead phase, i.e. if compounds have indeed been identified which meet pre-defined
criteria in terms of affinity to target, selectivity, and chemical optimization potential,
the best compound of the most promising cluster is selected as the lead structure.
This forms the starting point of the lead optimization phase where often hundreds
or even thousands of variants of the lead molecule are synthesized and tested. The
aim is to identify improved compounds with even better affinity to the target, higher
selectivity and improved pharmacokinetic properties. The end point of a successful
lead optimization process is a development candidate, which still have to pass
through further tests and, ultimately, clinical studies on patients and approval by the
health authorities before it is an approved drug and can be marketed and used in
therapy.
Historically, the main contribution of protein crystallography was in the generation of
crystal structures of the target protein in complex with lead molecules during the lead
optimization process. This helped to speed up lead optimization by providing the
medicinal chemists with the view of the 3D binding mode. However, due to the
massive progress in protein production (multi-parallel cloning, expression and
purification) and crystallization (robot-based screening of thousands of crystallization
conditions, in parallel for several constructs, with and without ligands) over the last
few years, the time needed for the determination of a new crystal structure has
decreased from many years to often only several months. This now enables protein
crystallography to provide crystal structures not only in the lead optimization phase
but already in the hit-to-lead phase. Here, information on the 3D binding mode of the
various clusters is often even more valuable as it allows a better-informed decision
on which cluster to nominate as a lead.
Over the last 10 years, fragment screening or fragment-based drug discovery (FBDD)
has emerged as a new method for hit identification and an alternative to the
traditional high-throughput screening: Here, libraries of one or two thousand smaller
molecules, so called fragments (molecular weight 150 – 300 Da), are screened for
binding to the target protein, using either protein NMR methods, surface plasmon
resonance or high concentration screening. In a second step, the structural biologists
try to solve co-crystal structures of the target in complex with these fragment hits.
With the 3D binding mode known, even initially very weak-binding fragments can
represent excellent starting points for the subsequent hit-to-lead and lead
optimization process. Based on the crystal structures, the medicinal chemists,
computational chemists and proteins crystallographers can discuss and decide
together how to extend the fragment most efficiently to quickly fill adjacent
subpockets and arrive at more potent inhibitors. Protein crystallography has thus
developed into an established tool in hit finding, hit cluster prioritization and lead
optimization.
In every day life, work in pharma is not very different from work as a crystallographer
in academia. We try to design the right constructs, purify the protein most effectively
and search for crystallization conditions just as our colleagues in academia do – with
the additional requirement that the structures have to be delivered fast enough in the
drug discover process to make a meaningful contribution. What makes Structural
Biology in the pharmaceutical industry particularly attractive to me is that all projects
are directly connected to human diseases and to developing drugs against them.
And that every structure is eagerly awaited by the computational and medicinal
chemists in the project team, who will immediately act on it and base their next
synthetic efforts on the protein-ligand interactions seen in our structures.
Figure: A small molecule inhibitor bound to the ATP binding pocket of protein kinase
MK2, a target for inflammation (Hillig et al., J.Mol.Biol. 369, 735-45, 2007). (A)
Overall structure in surface representation, (B) Stereo representation of the active
site residues coordination the inhibitor. Carbon atoms of protein residues shown in
green, of the inhibitor in yellow, and sulphur atom co-crystallized sulphate ion in
orange. 2Fo-Fc density map contoured at 1σ shown in blue. Hydrogen bonds shown
as dotted lines.