Identification and Visualization of Kinase-Specific Subpockets

Abstract

The identification and design of selective compounds is important for the reduction of unwanted side effects as well as for the development of tool compounds for target validation studies. This is, in particular, true for therapeutically important protein families that possess conserved folds and have numerous members such as kinases. To support the design of selective kinase inhibitors, we developed a novel approach that allows identification of specificity determining subpockets between closely related kinases solely based on their three-dimensional structures. To account for the intrinsic flexibility of the proteins, multiple X-ray structures of the target protein of interest as well as of unwanted off-target(s) are taken into account. The binding pockets of these protein structures are calculated and fused to a combined target and off-target pocket, respectively. Subsequently, shape differences between these two combined pockets are identified via fusion rules. The approach provides a user-friendly visualization of target-specific areas in a binding pocket which should be explored when designing selective compounds. Furthermore, the approach can be easily combined with in silico alanine mutation studies to identify selectivity determining residues. The potential impact of the approach is demonstrated in four retrospective experiments on closely related kinases, i.e., p38α vs Erk2, PAK1 vs PAK4, ITK vs AurA, and BRAF vs VEGFR2. Overall, the presented approach does not require any profiling data for training purposes, provides an intuitive visualization of a large number of protein structures at once, and could also be applied to other target classes.

A benchmark driven guide to binding site comparison: An exhaustive evaluation using tailor-made data sets (ProSPECCTs)

“…Consequently, the application of these tools might offer an opportunity to correlate ligand affinities with binding site similarities, e.g. for protein kinases [68,77,78]. A clear differentiation between minor dissimilarities is an essential necessity for such analyses and the abovementioned tools might be helpful to reliably rank binding sites according to their similarities.…”

Assessment of Flexible Shape Complementarity: New Opportunities to Explain and Induce Selectivity in Ligands of Protein Tyrosine Phosphatase 1B

“…The tool allows identification of selectivity relevant binding site areas and conformations, especially for cases where interaction feature patterns are highly similar and therefore of limited use to increase selectivity. A similar approach has been pursued before for kinases by comparing binding site grids of crystal structures from targets and off‐targets . Our tool, however, focuses on the detection of selective conformations and therefore cases where information from crystal structures does not reveal exploitable differences.…”

“…A similar approach has been pursued before for kinases by comparing binding site grids of crystal structures from targets and off-targets. [5] Our tool, however, focuses on the detection of selective conformations and therefore cases where information from crystal structures does not reveal exploitable differences. It further facilitates exploitation of the discovered differences for virtual screening of appropriate ligands favoring the selectivity relevant protein conformations, following the principle suggested by Huggins et al to lock the protein in a selective conformation with the help of a ligand.…”

A Comprehensive Structural Overview of p38α Mitogen‐Activated Protein Kinase in Complex with ATP‐Site and Non‐ATP‐Site Binders

“…[13] Through the gate area, variousp ockets becomea ccessible, which, altogether,c ompose the back cleft. The most important pocket in this area is the allosteric site (AS), ah ydrophobic region createdbymovement of the DFG motif and surrounded by Met78,V al83, Ile141, and His148.…”

“…The small size of the side chain of Thr106 createsalarge cavity known as hydrophobic region I( HR-I), surroundedb y Ala51, Lys53, Leu75, Ile84, Leu104, Thr106, and Leu167; [12] the properf illing of this target-specific pocket is knowna so ne of the most important selectivity-drivingf eatures for p38a MAPK inhibitors. [13] Through the gate area, variousp ockets becomea ccessible, which, altogether,c ompose the back cleft. The most important pocket in this area is the allosteric site (AS), ah ydrophobic region createdbymovement of the DFG motif and surrounded by Met78,V al83, Ile141, and His148.…”