Remodeling of gp-41-C34 Peptide Leads to Highly Effective Inhibitors of the Fusion of HIV-1

with target cells

Promising anti-1 peptide developed

Nina PollakPanos AthanasopoulosFrits Hulshof

HIV Life Cycle

HIV Fusion Mechanism

1) gp120 binds to CD4 receptor. Chemokine binding domain of gp120 is exposed and binds Co-receptors (CCR5 , CXCR4).

2) Conformational change is induced: hydrophobic gp41 is exposed and the N-terminal fusion peptide of the gp41 penetrates the cell membrane

3) Packing of C-terminal and and N-terminal helical regions brings in close proximity the virus and the cell, causing fusion of the cell with the virus

Mimicking peptides could prevent fusion

Cynthetic C peptides

• DP178 : Resistant HIV-1 strains• C34: less soluble than DP178, reduced

susceptibility to the evolution of resistant viruses• SC34 1a: Nle is introduced: Solubility is

increased 1000-fold over that of C34

Concepts of amino acid substitutions

-Maintain residues critical for interaction (a, d, e)

-Replace solvent accesible residues (b,c, g, f) with Lys and Glu

improve α-helicity with salt bridges

improve solubility with charged aa

Solid phase synthesis

• Suitable for short peptides

• Expensive

• Purification

SC34 1a results

• SC34 1000 fold better soluble than C34

• Anti HIV activity 10 times higher in MAGI assay

• 7 fold decrease in NL4-3 strain

Further modifications

• Eight i+4 intrahelical salt bridges

• Change Lys and Glu to improve stability by dipole

• Three possible i+3 Lys-Glu intrahelical interactions

SC34EK 2• MAGI analyses showed:

– anti HIV-1 activity of SC34EK 2 peptide higher than that of 1

• Conclusion:– conserved residues in the solvent-accessible

face of SC34 peptides changeable– replacements have to interfer with α-helix

formation

SC35EK 3

• 5 repeats of Z-EE-ZZ-KK– Z: residues interacting with inner strand– E and K: residues stabilizing α-helix

conformation

• MAGI analyses showed:– anti HIV-1 activity comparable to SC34EK 2

Circular dichroism (CD) analyses• linearly polarized light

– polarized in a certain direction (A)

• circularly polarized light– used by CD– the electric field vector has a constant length, but rotates about its

propagation direction (C)

ellipticaly polarized light, nearly linear (B)

Circular dichroism (CD) analyses 2

• CD measures differences in the absorption of left-handed versus right-handed polarized light.

• The differences arise due to structural asymmetry.

• Helicity increases in order 1a < 2a < 3 (absence of N36)

• CD-spectra of equimolar mixtures of N36 and SC peptides are similar

• Conclusion:– Intrahelical saltbridges increase the helicity of SC

peptides

Stability of N36/SC peptide complexes

• changes in [θ]222 as function of temperature

• melting temperature (Tm) N36/SC34 peptide complexes higher than N36/C34 complex

• Conclusion– N36/SC34 are more stable through intrahelical saltbridges.

Conclusions

• Conserved residues in the solvent-accesible face of SC34 peptides changeable, but replacements have to interfer with α-helix formation.

• Intrahelical saltbridges increase the helicity of SC peptides.

• N36/SC34 are more stable through intrahelical saltbridges.

• The stability of the N36/SC peptide complex correlates to some extent with its anti-HIV activity.

Ultracentrifugation sedimentation• N36/SC peptides form six-helix complex• each complex consists of three N36 and three SC

petides

• Conclusion:– The stability of the N36/SC peptide complex correlates to

some extent with its anti-HIV activity.

Discussion

• Gp41 a good target for anti HIV therapy?

• Find a more cost effective way to make the peptides?

• Imune response?

• How can it be that there is no resistance to the shorter variant SC29EK?

Antimicrob Agents Chemother. 2008 Dec 29.

SC29EK, a peptide fusion inhibitor with enhanced {alpha}-helicity, inhibits replication of human immunodeficiency virus type 1 mutants resistant to enfuvirtide.Naito T, Izumi K, Kodama E, Sakagami Y, Kajiwara K, Nishikawa H, Watanabe K, Sarafianos SG, Oishi S, Fujii N, Matsuoka M.

• Enfuvirtide (T-20)– peptide-based drug– targets the step of HIV fusion– suppresses replication of HIV-1 strains

(wild-type or multi-drug resistant to reverse transcriptase and/or protease inhibitors)– BUT HIV-1 variants with T-20 RESISTENCE have emerged

• Development of a novel HIV fusion inhibitor, SC34EK– SC34EK inhibits replication of T-20-resistant HIV-1s as well as wild-type HIV-1

• In this study introduction of SC29EK– shorter variant of SC34EK– SC29EK blocks replication of T-20-resistant HIV-1s– antiviral activity even at high serum concentrations (up to 50%) maintained – Circular dichroism analysis revealed that the α-helicity of SC29EK was well maintained– parental C29 showed moderate and reduced inhibition of wild-type and T-20-resistant HIV-1s,

α-helicity was lower.

• Conclusion:– α-helicity in a peptide-based fusion inhibitor is a key factor for activity– this enables the design of short peptide inhibitors with improved pharmacological properties