Drug repurposing via target hopping

 Drug repositioning: similar sites are likely to bind the same ligand

Repurposing tested small molecules drugs for new indications or new mechanism of action is an appealing strategy. MED-SuMo can be used in cases where a drug is co-cristalized with its target. If a similar binding site can be found in the Protein Data Bank [1] (or any macromolecule structure database), it is likely that this drug would also bind to this similar target. This target hopping case is on one hand probably rare but, on another hand, could be a strong rational evidence to highlight a possible off-target and eventually a possible undesired side effect. During lead discovery for a new target, finding cross-reactivity to a target for which there are already leads, enables the fast discovery of new leads via target-hopping. With the potential of short-circuiting the lead discovery process on a genomic scale, target hopping is an important chemogenomic application of structural informatics.

 MED-SuMo is used in Site vs Binding Site database to find in the PDB the most similar sites to B-RAF/sorafenib

This case study is about an example of repurposing sorafenib from B-RAF to others protein kinases. The B-RAF-sorafenib complex 3D structure is available in the PDB (code 1UWH [2]) and is used as the input of MED-SuMo to query the PDB binding site database. The database is redundant in terms of unique site or pocket but very interestingly for our application, it is exhaustive in term of kinase conformations and bound ligands.

 

Fig1: The input of MED-SuMo: 58 Surface Chemical Features (rendered as colored ball and stick) in the 6 Å vicinity of sorafenib in the B-RAF-sorafeninb complex (PDB code 1UWH). The backbone of B-RAF is shown in grey (only the secondary structure is shown) ; the DFG-out ligand, sorafenib is rendered as sticks.

 

 
 


The top results are shown in the screenshot of the result table: top hits are mostly DFG-out protein kinases followed almost exclusively by  protein kinases. MED-Sumo identifies the most similar protein kinases binding sites (about 1/5 of all protein kinases), together with a few ATP binding proteins. The results are very different from a sequence based search tool where all protein kinases would be found first.
 
 
A simple measure of similarity between the site hit and the query site is obtained by normalizing the score of the hit to the maximum possible score (site query towards itself)).

Relative MED-SuMo score (%) = Hit score / Query score * 100

In this case study, we show that target hopping is likely to occur above a relative MED-SuMo score of 60%: the first hit which is not a DFG-out kinase is a DFG-in protein kinase: 3C4C ranked as the 33th hit. The relative score is 54% which makes sense as the DFG-in and the DFG-out binding mode share the hinge and the gatekeeper region which is about 50% of the pocket.

 Validation of the prediction of sorafenib repurposing

B-RAF and C-Kit have a common ligand, sorafenib (BAY439006), a drug on the market since 2006, which is an oral inhibitor of C-RAF, wild-type B-RAF, mutant V599E BRAF, vascular endothelial growth factor receptor VEGFR2, VEGFR3, FLT-3, platelet-derived growth factor receptor, p38, and C-KIT among other kinases [3]. More recently, sorafenib was shown to inhibit several protein kinases [4]: B-RAF: 540 nM, P38α: 370 nM, VEGFR2: 59 nM, LCK: 2700 nM, ABL1: 680 nM, C-KIT: 31 nM, Tie2: 2100 nM and others. The 3D binding site similarity between B-RAF and C-KIT had been highlighted previously by Debe et al. [5]. The authors pointed out the fact that the cross-reactivity of B-RAF and C-KIT can be rationalized by the 3D similarity of the binding site and not by sequence alignments because 1/6 of kinases are more similar to B-RAF than C-KIT. In the MED-SuMo results (Tab1), C-KIT is one of the best ranked (PDB code 1T46 [6]): 9th hit and 4th protein kinase after B-RAF, P38α, VEGFR2, LCK and ABL1. Interestingly, MED-SuMo points out, as top ranked hits, targets which are experimentally validated. This application is case where the concept that similar targets (sequence and conformation) can bind the same ligand with a similar binding mode.

Target (PDB code) Human B-RAF (1UWH) P38alpha (2BAK) VEGFR2 (3BE2) LCK (2OG8) C-KIT (1T46) ABL1 (3CS9) chicken SRC (2OIQ) Tie2 (2OSC)
Hit rank 1 3 4 6 9 10 28 48
Relative MED-SuMo Score 100% 80% 76% 75% 74% 73% 60% 44%

 Tab1: Results extracted from the whole result table: only the 1st occurence of each protein kinase is reported here with its name and its PDB code. The hit rank refers to a comparison towards the whole PDB binding sites. The relative MED-SuMo score is defined above in the text.

Tie2 is found among the hits with at a relative MED-SuMo score of 44% (below the 60% cutoff). Even if the conformation of 2OSC is DFG-out, like the query, the differences in the binding site (ATP pocket) are detected and a score significantly below 60% is found. This hit could be seen as a false negative if hte results are analysed with the cutoff value without further molecular modeling.

Though no experimental data are available to our knowledge, sorafenib is predicted to inhibit chicken SRC. This is likely to occur on a structural basis because the 2OIQ structure is DFG-out and contains imatinib which has the same binding mode as sorafenib. Interestingly, sorafenib is reported to be a low affinity binder to human SRC [6]. Further work would be needed to check if the boarderline relative score of 60% found with chicken SRC is a prediction of a lower experimental affinity.

 Conclusion

Starting from a B-RAF/sorafenib complex available in the PDB, MED-SuMo found, as top ranked, 5 protein kinases which are known experimentally to bind sorafenib: VEGFR2, P38α, ABL, C-KIT and LCK. MED-SuMo provides true positives and no false positives. False negatives could occur for two reasons: (1) lack of a similar structure in the PDB (in this case study, a DFG-out structure is needed) (2) sorafenib binds to others protein kinase with another binding mode. MED-SuMo is best suited to detect off-targets, though not all off targets can be detected. Interestingly, the detected off-targets with a high relative score higher than 60% are very likely to be real off-targets in vitro.
The complex of sorafenib with those 5 targets can be easily exported for further molecular modeling/scoring.

 References

[1] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne: “The Protein Data Bank” (2000) Nucleic Acids Research, 28 pp. 235-242.

[2] Wan, P.T., Garnett, M.J., Roe, S.M., Lee, S., Niculescu-Duvaz, D., Good, V.M., Jones, C.M., Marshall, C.J., Springer, C.J., Barford, D., Marais, R. “Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF” 2004 Cell 116: 855-867

[3] Ahmad T, Eisen T. “Kinase inhibition with BAY 43-9006 in renal cell carcinoma” (2004) Clin Cancer Res. 10(18 Pt 2):6388S-92S.

[4] Karaman MW, Herrgard S, Treiber DK, Gallant P, Atteridge CE, Campbell BT, Chan KW, Ciceri P, Davis MI, Edeen PT, Faraoni R, Floyd M, Hunt JP, Lockhart DJ, Milanov ZV, Morrison MJ, Pallares G, Patel HK, Pritchard S, Wodicka LM, Zarrinkar PP. A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol. 2008 Jan;26(1):127-32.

[5] Debe D.A., Hambly K. P., Danzer J.F. “Structural informatics: chemogenomics in silico” in Chemogenomics, knowledge-based approaches to Drug Discovery (2006) edited by Edgar Jacoby (Novartis Institutes for Biomedical Research, Switzerland)

[6] Mol, C.D., Dougan, D.R., Schneider, T.R., Skene, R.J., Kraus, M.L., Scheibe, D.N., Snell, G.P., Zou, H., Sang, B.C., Wilson, K.P. “Structural basis for the autoinhibition and STI-571 inhibition of c-Kit tyrosine kinase.” (2004) J.Biol.Chem. 279: 31655-31663

 


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