In December 2016 I attended the SCI Protein-Protein Interaction symposium. Armed with Cresset’s powerful ligand centric molecular modeling suite Forge, and an embryonic version of our new structure-based design application, Flare, I was keen to see what could usefully be done with PPI’s.
Prof. Richard Baylis (University of Leeds, UK) presented new data on the interaction of N-MYC with Aurora A. N-MYC is a disordered multi-domain protein with a host of interaction partners. Dysregulation of N-MYC has been linked to a range of cancers. N-MYC is short lived in-vivo and its usual fate is to be ubiquitinylated and degraded. Binding with Aurora A protects N-MYC from this process allowing its various tumorogenic affects to persist. The Baylis group provided the first x-ray evidence showing how N-MYC interacts at an allosteric site of Aurora A which stabilises an active conformation of the Kinase (figure 1).
Figure 1: Aurora A kinase with N-MYC – light green (left), and detail of the N-MYC short helical domain 74-89 (right).
An alternative computational strategy, which occurred to Cresset at the time, was to employ a structure-based approach; to furnish molecular designs that could directly prevent this protein-protein interaction. For this purpose, an initial analysis of the surface interaction, including both electrostatic and lipophilic hot-spots, would be vital.
During the talk, I used Flare to quickly download the relevant PDB file (5G1X) and to load the protein coordinates directly into the application. An automated protein prep protocol (build-model) was used to refine the pdb structure before generating the surface interaction maps, using Cressets proprietary XED force field (figure 2).
Figure 2: (A) Positive protein electrostatic isopotential surface of Aurora (left), negative protein electrostatic isopotential surface (center), and neutral isopotential surface with some key residues of N-MYC (right).
Figure 3: Negative protein electrostatic isopotential surface of N-MYC short helical region (left), and positive protein electrostatic isopotential surface of the same (right).
We can exploit this information to generate chemical starting points, once each important set of residues is identified and mapped. Thus, from the 3D shape and detailed electrostatic information we can conduct de-novo design experiments to furnish ideas for synthesis, or use virtual screening (Blaze) to search for commercial compounds to purchase and test.
Since the distance between the two main hot spot regions was not ideal (27 Ang. Val61 to Trp77) and although linking them might have been possible using a fragment linking or growing technique e.g., using Spark (Using Cresset’s Spark to grow and link distant fragment hits with sensible chemistry), we chose to tackle them independently with a de-novo design technique. I used the key residues Pro75, Trp77, Glu80, Met81 and Glu84 from the short helical domain as a molecular reference. We used this reference to score our molecular ideas against, and to optimize them via iterative ‘molecular design > alignment > scoring’ cycles in Forge. This powerful technique scores 3D shape, electrostatics and protein steric clashes whilst simultaneously calculating and/or filtering in-silico physiochemical properties. This method as described is limited only by the imagination of the user. In conjunction with Spark as the idea generator however, the limit is set only by the availability of appropriate fragments in the Spark databases – which is a substantial resource.
Later, when we returned, we also ran a virtual screening test on this system using the Blaze demo server. Results of this quick virtual screen against a sub-set of the ChEMBL database are shown below (figure 4).
Figure 4: Forge ‘tile view‘ of example diverse 2D output results of the virtual screen using the Blaze Demo server against a sub-set of ChEMBL (left) and 3D alignments of two of these (pink and green sticks) against the reference N-MYC peptide (blue lines) bound to Aurora A (Forge screenshot).
A powerful combination of cutting edge ligand and structure-based modeling
Figure 5: Flare screenshot of the structure of an initial idea (left) superimposed on N-MYC hot spot residues, plus its calculated properties, and (right) a space filling model of a further example with superior properties, improved fit, better synthetic tractability and … an IP position.Although this is only a thought experiment (until the point at which any of these molecular designs are synthesized and tested) this illustrates how the powerful combination of both ligand centric and structure-based techniques in Flare, Forge, and perhaps also Spark, could be used to generate specific ideas that address the types of challenges presented by PPI’s or fragment enabled drug discovery projects. This is not untypical, in terms of a portfolio of tasks we might suggest to Cresset Discovery Services clients.
Download and evaluation of Flare, Forge, Spark and Blaze, or contact us to find out how Cresset Discovery Services can enhance your project with insightful and creative delivery of powerful molecular modeling.