It is of interest to point out that all structural information was obtained using cocrystallization experiments, which demonstrates that, with suitable primary screening options, the crystallographic follow-up of a fragment-based screening campaign is not necessarily reliant upon the availability of a soakable crystal system. Although we allowed a slightly higher molecular weight cut-off than the 250 Da nowadays typically used in the design of a fragment library, interestingly six out of the nine crystallographically BI-D1870 confirmed fragment hits have a molecular weight below 200 Da and one has a molecular weight of 244.3 Da. With respective molecular weights of 253.3 and 261.3 Da, the other two fragment hits, compounds 13 and 19, are only marginally larger and well below the higher molecular weight cut-off of our fragment library. Importantly, all hits bind to the CHK2 hinge region, including compound 19 from the category of thermal shift hits and AlphaScreenTM inactives. This hit category should include any second site binders and therefore our findings confirm the adenine subpocket as the dominant fragment-binding site. Furthermore, we have shown that, in addition to the interactions with the hinge, these fragments exploit several of the interaction hot-spots used by advanced CHK2 inhibitors, but do so in different ways. Because no fragments were found to bind in other subpockets of the CHK2 ATP binding site, further development into potent lead molecules through fragment linking is not an option. However, since none of the CHK2 fragment hits is exemplified as a hinge-binding scaffold in the previously reported CHK2 inhibitors, they could be developed by merging them with existing CHK2 inhibitors. Furthermore, in keeping with the majority of advanced fragment-based kinase inhibitors, such as the B-raf inhibitor PLX4032, the PKB/Akt inhibitor AZD5363, and the Aurora Janus kinase 2 inhibitor AT9283, optimization using a fragment evolution/growing strategy would be the most promising way to develop our CHK2 fragment hit matter into potent lead molecules with favorable physicochemical properties. Moreover, the crystal structures of compound 13 and its followup compound 22 access a hydrophobic area above the hinge not previously explored in rational CHK2 inhibitor design. We speculate that this pocket could be exploited to enhance both potency and selectivity of CHK2 inhibitors. However, although compounds 13 and 22 have good ligand efficiencies, the usefulness of this pocket in CHK2 inhibitor design will need to be further investigated, starting from more potent but nonselective CHK2 inhibitors. Together the similar binding mode observed for JNK3 inhibitors, and the fact that many kinases have a phenylalanine or tyrosine residue in the gatekeeper +2 position, suggest that the area above the hinge could also be important in the design of selective ATP-competitive inhibitors for other kinases with a small gatekeeper +2 residue. Head and neck cancer is one of the cancers with a rising incidence over past 10 years while its survival rate has not been significantly improved. More than 90% of head and neck cancers are squamous cell carcinoma, arising in the lining epithelium of the oral cavity, larynx, NVP-BEZ235 purchase pharynx, and nasopharynx. HNSCC is classified as a complex molecular disease, which develops from dysfunctions of multiple interrelated pathways. Moreover, HNSCC has been shown to arise through an accumulation of genetic alterations and there is a need for better understanding of the mechanisms or pathways in responding to the proliferation and apoptosis of HNSCC.