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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.