Download SC34EK 2

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 Pollak
Panos Athanasopoulos
Frits 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 Nterminal 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