vFLIP-IKK g Blocker

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vFLIP-IKK Blocker

Edith Chan

WIBR

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Work Study

• The study focuses on using X-ray crystal structures, biophysical screening and structure based design to identify blockers of the vFLIP-IKK and p22-cFLIP-IKK interaction, conducting lead optimisation and identifying a development candidate.

• My immediate actions are

– Understanding of the interaction of the vFLIP-IKKcomplex using X-ray crystal structure

– Selection of compounds mimicking the IKK interaction of the complex

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X-ray crystal structure• Our collaborators at BBK have

solved the structure between vFLIP-IKK (3cl3).

• Full length IKK 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-IKKcomplex.

• The two vFLIP molecules come together solely through interactions between the two IKK chains.

A B

D

E

IKK

vFLIP

N-terminus

C-terminus

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Protein-Protein Interactions

• Each of the IKK 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 IKK)

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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 IKK interact with vFLIP, with F238 reaching the deepest pocket.

• In Cleft2, Q236 and E240 are pointing into the pocket. Interaction would be optimized.

Cleft1Cleft2

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Cleft1 Interactions• 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 IKK largely incapable of forming a complex.

Ala57L

Pro54G

Tyr90F

FQEYDNHIK

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-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.

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p53 vs IKK• 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 RFMDYWEALIKK LFQEYDNHIK

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N

N

O

OI

Cl

O

O

Cl

1t4e

N

NCl

Cl

O

O

1ttvMW = 454HA = 3HD = 1cLogP = 8.3

MW = 580HA = 4 HD = 2cLogP = 6.41

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Known Helix Mimeticsspecial groups

• Some known helix mimetic and if any similar compounds are in our 30K screening database.

R1

R2

R3

X

Y

Terphenyl1 hit, no rgroup

ONH

N O

O

R1O

O

R3

R4

R2

TerephthaiamideAbout 10 hits but without the phenol

N

NH

O

N

O

N

OR1

R2

N OH

N

COOMe

OR3

TrispyridylamideNo hits

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Known Helix MimeticSmall molecules - Common

OH

H

R4

R3

R1

R2

ChalconesPyrrole system~20 hit, 0 hitPlenty in commercial database

Aryl sulphonamides10 hits

OH

H R

N

S

S

OO

N

R

R

Isoindolinones4 hit

N R1

O

OR2

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Known Helix MimeticSmall molecules – Custom made

N

N

R

R

R

Trisubstituted imidazoles0 hit

Nutlinsno hits

N

N

R

R

NO N R2

O

R3

1,4-benzodiazepine-2,5-diones (BZD)No hit

N

N

O

O

R1

R2

R4

O

O

R3