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 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 SC 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 (T m ) 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 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