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Promising anti-1 peptide developed

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.

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Promising anti-1 peptide developed

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  1. 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

  2. HIV Life Cycle

  3. HIV Fusion Mechanism • gp120 binds to CD4 receptor. Chemokine binding domain of gp120 is exposed and binds Co-receptors (CCR5, CXCR4). • Conformational change is induced: hydrophobic gp41 is exposed and the N-terminal fusion peptide of the gp41 penetrates the cell membrane • 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

  4. Mimicking peptides could prevent fusion

  5. 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

  6. 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

  7. Solid phase synthesis • Suitable for short peptides • Expensive • Purification

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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)

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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?

  18. 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

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