X-ray structures of Aldose Reductase and AKR1B10 with the lead inhibitor JF0064

Human enzymes Aldose Reductase (AR or AKR1B1) and AKR1B10 are of biomedical interest because of their involvement in secondary diabetic complications and inflammatory disorders (AR) and in several type of cancers, e.g. hepatocellular carcinoma and smoking related lung cancer (AKR1B10). They belong to the Aldo-keto reductase family (AKRs) which present a highly conserved (α/β)8 barrel folding. Their substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable external loops. JF0064 (2,2',3,3',5,5',6,6'-octafluoro-4,4'-biphenyldiol) has been identified in a series of polyhalogenated compounds as a lead inhibitor of both enzymes. The determination of the X-ray structure of the AR:NADP+:JF0064 complex at ultrahigh-resolution (0.85 Å) allows to observe JF0064 interacting with the catalytic residue Tyr48 through a negatively charged hydroxyl group of the inhibitor (i.e., the acidic phenol). The non-competitive inhibition pattern observed for JF0064 with both enzymes suggests that this acidic hydroxyl group will also be present in the case of AKR1B10. The X-ray structure of the AKR1B10:NADP+:JF0064 complex (achieved with the combination of surface lysine methylation and the introduction of K125R/V301L mutations) and the comparison between both structures unveils some important hints for a structure-based drug design optimization.

Crystallographic fragment screening: challenges, opportunities, lessons learned

Fragment-based approaches are now routinely applied for lead development in pharmaceutical drug research. Usually, a small but well selected library of low molecular weight compounds is pre-screened by biochemical or biophysical methods such as surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) or thermal shift assay; often followed for promising hit candidates by X-ray crystallography. We designed a small fragment library consisting of 364 compounds that is not strictly compliant to the otherwise often followed Astex rule of three for fragment library composition.[1] Thereafter, our library was validated on the pepsin-like aspartyl protease endothiapepsin, which serves as a model system for proteins that are involved in serious diseases such as malaria (plasmepsins), hypertension (renin) and Alzheimer's disease (ß-secretase) and therefore, is a valid target for further drug development. Due to the small size of fragments, they frequently exhibit only low affinity to the applied target protein and thus are often hard to detect in any screening approach, reflected in little overlap between different screening methods. After initial screening, we decided to validate the entire library by X-ray crystallography, which requires a steady supply of crystals, reproducible soaking conditions and a reliable setup at a synchrotron source, such as HZB BESSY II BL14.1 [2], preferably with some automation in initial data processing and refinement. A total hit rate greater than 10% was obtained, which will be compared to results from other screening methods. The resulting crystal structures will be discussed and provide an ideal basis for further lead development.

Fragment-screening at the BESSY II MX-beamlines of the Helmholtz-Zentrum Berlin

"Within the last decade, the fragment-based screening approach has been matured to a reliable and powerful instrument of pharmaceutical drug discovery. The success of fragment screening strongly depends on the quality of the chosen fragment library (100-200 Da), the quality of the target protein diffraction as well as the possibility to use high throughput methods for the screen application. A thorough crystallographic analysis of many protein-fragment complex structures and their binding modes has the perspective to result in the development of new potential lead structures and to map the interaction landscape of protein surfaces. Recently we started the development of a dedicated experimental facility for high throughput fragment screening at the BESSY II storage ring. The in house data processing pipeline ""XDSAPP"" [1] has been developed to speed up the data evaluation of large amounts of diffraction data. We have assembled a fragment library of 96 compounds and have validated this library against two target proteins. These first results suggest that our library is capable of identifying binding partners at a hit rate of close to 10%. This library together with a fully automated beam line [2] will be made accessible to users, thus enabling fragment-screening experiments on a much broader basis. "