For example, the critical His1201 from the D-loop of TNKS1is not conserved in other PARP proteins; the a3 helix N-terminal to the D-loop is slightly shorter in tankyrases due to the insertion of a prolineand deletion of two amino acids, resulting in a narrower induced pocket. Therefore, one is likely to achieve broader selectivity over PARP family members with compounds that bind to the induced pocket. For example, the selectivity of XAV939 for TNKS1 over PARP2 is only 10 fold whereas the selectivity of 2 is greater than 143 fold. The TNKS1/2 complex structure and molecular modeling analysis suggest a number of distinct routes to further optimize tankyrase inhibitors that bind to the induced pocket. Preliminary metabolic stability studies indicated enzymatic cleavage of the amide bond to be the primary clearance mechanism for IWRs. It is clear from our crystal structure that the amide quinoline 
of 2 can be replaced by other more stable moieties that maintain the same hydrogen bonding and stacking interactions. Modifications of the central phenyl group may also generate compounds with more favorable binding geometries. Quantum mechanical calculations suggest that the,60u dihedral between the phenyl and amide Fingolimod supply observed in the crystal structure of 2 results in an intrinsic reduction of potency by approximately 25-fold. The pyrrolidine dione group also does not appear optimal for tankyrase binding. One of the two carbonyl oxygens is not involved in hydrogen bonding or any other interaction with the protein and thus could be replaced. In addition, it is also conceivable that the norbornyl group does not interact optimally with the Tyr1213, Tyr1224, and Ile1228 of TNKS1. Furthermore, since the induced pocket is adjacent to the nicotinamide pocket which is unoccupied and unhindered, it may be possible to extend the induced pocket binding tankyrase inhibitors such as 2 into the nicotinamide pocket to gain additional interactions, resulting in even greater potency while maintaining good selectivity due to the specificity of the induced pocket. IWR compounds may have activity for proteins other than PARP family members; thus, minimizing potential side effects from the off-target interactions is essential for further development of tankyrase inhibitors derived from IWRs. Future studies such as chemical proteomics screens need to be carried out to identify potential unintended INCB28060 targets of these inhibitors. We note that induced pockets have been observed for other enzymes such as protein kinases. An allosteric binding pocket was reported for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase and the formation of this pocket requires a large conformation change. Improving interactions in this allosteric pocket and establishing additional interactions in the adjacent ATP pocket enhanced the affinity of the inhibitors by 12,000 fold. Imatinib, developed to treat chronic myelogenous leukemiaand gastrointestinal stromal tumor, binds to similar sites in the human Abl and Kit kinases and shows excellent efficacy and specificity for Abl and Kit. Interestingly, imatinib was found to inhibit stronglya non-kinase target, the oxidoreductase NQO2, from a screen carried out to identify off-target proteins. Vemurafenib, developed for the treatment of metastatic melanoma caused by the BRAFV600E mutation, also binds to an induced pocket created by an outward shift of the aC helix. In summary, the present structure reveals a novel binding mode for tankyrase inhibitors and, in conjunction with molecular modeling analysis, provides insights into the molecular basis for the key interactions between IWRs and tankyrases.