ally a consequence of its fast off-rate. Structural requirements for SCH772984 slow-off rates Based on the binding mode of SCH772984 with ERK1/2, the complex structures with the off-target haspin and JNK1 and available structural data on other kinases that are inhibited by SCH772984 including CLK131, GAK 32, JAK233, TNIK and TTK34, we sought to identify the structural requirements facilitating this novel binding mode as well as the mechanisms leading to slow off-rates. Comparison of 14 available crystal structures, the four structures determined here as well as structure-based sequence alignments revealed conservation of key residues lining the ERK1/2-SCH772984 interface. While the residues within the type-I adenine mimetic pocket were quite conserved, we hypothesized that the SCH772984 binding mode in ERK1/2 depends on a strong structural rearrangement of the 313348-27-5 web P-loop as well as an aromatic stacking interaction between Y64 located in helix C. 11 of the 16 off-targets harbour an aromatic residue at the tip of the P-loop that may facilitate stabilizing the distorted P-loop conformation necessary for the opening of the P-loop pocket. Furthermore, only two kinases, CLK1 and CLK2, contain also an aromatic residue in an analogues position to ERK1/2 C tyrosine that would allow a stacking interaction with SCH772984. To assist our structural comparison, we analysed the binding kinetics of SCH772984 on the off-targets CLK1, CLK2, JNK2, CSNK2A1/A2, DRAK1 and TTK. Interestingly, BLI binding data revealed that all studied off-targets exhibited fast association and dissociation rates. Thus, for the use of SCH772984 in cellular systems and in vivo the unique slow off-rate for ERK1/2 will additionally increase inhibitor efficacy and selectivity. Importance of aromatic stacking interactions Based on our structural comparison we hypothesized that two aromatic tyrosine residues located within the P-loop and C could be the keys for stabilization of the binding mode of SCH772984 in ERK1/2 and its slow binding kinetics. To test this hypothesis, we mutated the P-loop and C tyrosine residues to the corresponding residues present in offtargets that all showed fast off-rates, and studied the binding kinetics of SCH771984 with these mutants. Interestingly, all four single-site mutants tested retained slow association and dissociation kinetics, albeit with reduced binding affinities. Also the double mutants Y36A/Y64H and Y36Q/Y64H still showed slow binding kinetics suggesting that H64 can compensate the loss of the C tyrosine through a similar Nat Chem Biol. Author manuscript; available in PMC 2015 December 22. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Chaikuad et al. Page 8 stacking interaction with the inhibitor. In support of this notion, the other double mutants with the substitutions of Y64 to lysine or aspartate strongly reduced inhibitor off-rates. The mutagenesis study suggested that both tyrosine PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19812126 residues are required for tight binding and slow inhibitor binding kinetics in agreement with our prediction based on the analysed crystal structures. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Since we also 
observed slow binding kinetics of the type-I VTX-11e, we used the generated mutants to investigate if the slow binding kinetics of this inhibitor is also affected by these substitutions. Surprisingly, the two tested single mutants significantly reduced inhibitor off-rates. In particular, the st