vFLIP-IKK g Blocker

1. vFLIP-IKKg Blocker Edith Chan WIBR

2. Work Study • The study focuses on using X-ray crystal structures, biophysical screening and structure based design to identify blockers of the vFLIP-IKKg and p22-cFLIP-IKKg interaction, conducting lead optimisation and identifying a development candidate. • My immediate actions are • Understanding of the interaction of the vFLIP-IKKg complex using X-ray crystal structure • Selection of compounds mimicking the IKKg interaction of the complex

3. vFLIP C-terminus N-terminus IKKg E B A D X-ray crystal structure • Our collaborators at BBK have solved the structure between vFLIP-IKKg (3cl3). • Full length IKKg is 419aa long mulitdomain protein • Both proteins are truncated • ks-vFLIP (aa1-178) [188aa] • IKKg (aa150-272) [419aa] • The X-ray structure comprised of a dimer of two ks-vFLIP-IKKg complex. • The two vFLIP molecules come together solely through interactions between the two IKKg chains.

4. Protein-Protein Interactions • Each of the IKKg helix is interacting with a copy of the vFLIP via two adjacent vertical clefts (Cleft1 and Cleft2) • Cleft 1 involved more interactions between the complex, the hottest spot seems to be around Phe238 (of IKKg)

5. Cleft1 Cleft2 Where are the main Interactions? • SURFNET, which locates the all available clefts on a protein surface, also indentifies Cleft1 and Cleft2. However, they are smaller than expected. • In Cleft1, mainly F238, D242, and K246 from IKKg interact with vFLIP, with F238 reaching the deepest pocket. • In Cleft2, Q236 and E240 are pointing into the pocket. Interaction would be optimized.

6. Cleft1 Interactions FQEYDNHIK • Residues involved in this region are • F238 – all hydrophobic interaction, enclosed by F53, F79, L80, P54, and A57. • D242 and K246 – enclosed by H83,T87,Y90,S89 • K246 has a H-bond with C=O of M88 • D242 has a H-bond to H83 • Mutation study showed that A57L has impaired the forming of the complex. P54G shows a reduction in affinity while Y90L retains affinity. • D242R mutant has rendered IKKg largely incapable of forming a complex. Pro54G Tyr90F Ala57L

7. a-helix in Protein-Protein Interaction • There are many other biological systems that involved the protein recognition of a a-helix, such as bacterial autotransporter NaIP, Gp41, smMLCK/CaM, HDM2/p53, Bcl-2 family, Estrogen receptor-CoA, Tachyknin receptors,… • Many of them are studied extensively. • Let’s us look at the HDM2/p53 system. • HDM2 – Human double minute 2 protein/p53 is related in cancer therapy. • The complex has protein-protein interaction, with p53 adopting helical conformation.

8. p53 vs IKKg • The backbone of p53 and IKKg peptides overlap well. • 2 of the residues (F and D) that interact with protein also overlap well. • The left picture shows an overlap between p53 and a small molecule inhibitor P53 RFMDYWEAL IKKg LFQEYDNHIK

9. 1t4e MW = 580 HA = 4 HD = 2 cLogP = 6.41 1ttv MW = 454 HA = 3 HD = 1 cLogP = 8.3

10. Known Helix Mimeticsspecial groups • Some known helix mimetic and if any similar compounds are in our 30K screening database. Terphenyl 1 hit, no rgroup Trispyridylamide No hits Terephthaiamide About 10 hits but without the phenol

11. Known Helix MimeticSmall molecules - Common Chalcones Pyrrole system ~20 hit, 0 hit Plenty in commercial database Aryl sulphonamides 10 hits Isoindolinones 4 hit

12. Known Helix MimeticSmall molecules – Custom made 1,4-benzodiazepine-2,5-diones (BZD) No hit Trisubstituted imidazoles 0 hit Nutlins no hits