This paper describes research into designing α/β-peptide foldamers that mimic the sequence and binding properties of the pro-apoptotic BH3 domains. The researchers took the primary sequence of the Puma BH3 domain and replaced some amino acids with β-amino acids to create a series of ααβαααβ foldamers. Foldamer 4 displayed nanomolar affinity for the anti-apoptotic proteins Bcl-xL and Mcl-1 similar to the natural Puma BH3 domain. Further experiments showed foldamers 4 and 5 bound directly but did not fully adopt an α-helical structure like the natural domain. The results demonstrate the potential of sequence-based

1. Sequence-based Design Of α/β-peptide
Foldamers That Mimic BH3 Domains
W. SETH HORNE, MELISSA D. BOERSMA, MATTHEW
A. WINDSOR, AND SAMUEL H. GELLMAN
Paper Discussion – Emerging Topics in Chemical Biology
A. Reddy & J. Ingram – Leeds University – 19/10/12

2. The Bcl-2 Protein Family
Pro-apoptotic Anti-apoptotic
• Bak • Bcl-2
• Bad • Bcl-xL
• Puma • Mcl-1

3. Foldamers
• Foldamer: A synthetic molecule that has the ability
to adopt the folded (secondary/tertiary) structures
typical of proteins.
• Foldamers have been considered good candidates for
binding to protein surfaces involved in protein-protein
interactions.

4. Foldamer Synthesis
• Structure-based drug design:
Looks first for structural homology first, and then
adding biological activity.
• Sequence based design:
Looks first for homology in the amino acid sequence,
and then modifies the structure to suit other
requirements.

5. α-Amino Acids vs. β-Amino Acids
• When R1 = side chain and R2 = H  β3-amino acid
• When R1 = H and R2 = side chain  β2-amino acid

6. Sequence Based Design - ααβαααβ
• Took the Puma BH3 domain primary sequence,
(1) NH2 - R - R - E - Y - Q - A - N - L - D - D - A - M - R - R - L - Q - A - G - I - E - R - A - W - Q - E - E - Ac
• Replaced some α-amino acids with β-amino acids
(1) NH2 - R - R - E - Y - Q - A - N - L - D - D - A - M - R - R - L - Q - A - G - I - E - R - A - W - Q - E - E - Ac
(2) NH2 - R - R - E - Y - Q - A - N - L - D - D - A - M - R - R - L - Q - A - G - I - E - R - A - W - Q - E - E - Ac
(3) NH2 - R - R - E - Y - Q - A - N - L - D - D - A - M - R - R - L - Q - A - G - I - E - R - A - W - Q - E - E - Ac
(4) NH2 - R - R - E - Y - Q - A - N - L - D - D - A - M - R - R - L - Q - A - G - I - E - R - A - W - Q - E - E - Ac
(5) NH2 - R - R - E - Y - Q - A - N - L - D - D - A - M - R - R - L - Q - A - G - I - E - R - A - W - Q - E - E - Ac
(6) NH2 - R - R - E - Y - Q - A - N - L - D - D - A - M - R - R - L - Q - A - G - I - E - R - A - W - Q - E - E - Ac
(7) NH2 - R - R - E - Y - Q - A - N - L - D - D - A - M - R - R - L - Q - A - G - I - E - R - A - W - Q - E - E - Ac
(8) NH2 - R - R - E - Y - Q - A - N - L - D - D - A - M - R - R - L - Q - A - G - I - E - R - A - W - Q - E - E - Ac

7. α-Helix Structures
R
D
Q Q
A W M 1 2 3
R I
E E
4 5
Q A L
A L
D R
E
R
R E
G
6 7 8
N A
Y

8. Testing the Puma BH3 Analogues
• Tests performed
• Fluorescence Polarisation Competition Assay
• Direct Binding Fluorescence Polarisation
• Proteolytic Stability Assay
• Circular Dichroism

9. Competitive Binding – FP Assay
The binding affinity of the
helices for anti apoptotic
Bcl-xL and Mcl-1 was
assessed.
This was done by calculating
the Ki in a competitive FP
assay where a fluorescently
labelled Bak BH3 domain
was used as the tracer.
Compound 4 displayed
nanomolar affinity for both
Bcl-xL and Mcl-1.

10. Direct Binding FP Assay
• To support the previous results, a direct binding FP
assay was done to measure Kd for the puma BH3
domain and α/β peptides 4 and 5.
• This involved the replacement of an N-acetyl group
with a fluorophore.
• The Ki and Kd values were reported as consistent.

11. Secondary Structure Comparison - CD
• To understand binding
better, they
investigated the
secondary structure of
4, 5, 7 and the wild-
type, 1 - with circular
dichroism.
• Found that all four have
similar chiral index, BUT
do not hold a helical
structure for most of the
time.
http://upload.wikimedia.org/wikipedia/commons/thumb/d/d1/Circular_dichroism.png/622px-Circular_dichroism.png

12. Future Research Opportunities
• Within the paper
• Discover the cause for the difference in binding constant
between the different ααβαααβ analogues.
• Conduct further backbone and/or sequence modification.
• α- and β-amino acids are commercially available, proteins
can be formed robotically – this can be used to rapidly
synthesise sequences.

13. Future Research: Gellman Group

14. Fin.
• Questions?