Interaction Analysis of HIV-1 BG5O5 SOSIP. 664 Trimer with Peptide Triazoles
Kriti Acharya1, Adel Ahmed1, Francesca Moraca2 Cameron Abrams2 and Irwin Chaiken1
1Department
of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia PA 19102 and
2School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA 19104
Abstract
HIV-1 Entry Inhibition By PT
CD4IgG2 competition SPR assay of
Monomeric gp120 by UM15.
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• Rationale
• BG5O5.SOSIP.664 is the only stabilized trimer identified so far
that is amenable to crystallographic analysis
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Increasing UM15
concentration
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• Stabilizing Sequence Modifications in BG5O5 SOSIP
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• A501C and T605C : gp120-gp41Ecto disulfide bond.
• I559P in gp41Ecto : Trimer stabilizing ;gp41-gp41 association
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PT Synthesis: Solid Phase Peptide Synthesis used Fmoc standard chemistry
on a microwave peptide synthesizer.
UM15: 6 Amino acid peptide with a Triazole moiety
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IC50 190nM
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IC50 plot of inhibition of monomeric binding
to CD4IgG2 by UM15.
Representative sensorgrams showing dose
dependent inhibition of monomeric gp120
binding to CD4IgG2 by UM15
• ΔMPER : Improve homogeneity and solubility
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UM15 concentration (nM)
Time (seconds)
Inhibition of SOSIP.664 binding to CD4 by UM15
CD4IgG2 competition SPR assay of
BG5O5.SOSIP.664 trimer by UM15.
UM15 Binding site on HIV-1 BG5O5.SOSIP.664
120
Docking Studies
• PT can bind (in silico) to SOSIP
gp120 at a site similar to that
observed with monomeric gp120.
• Interactions with the critical
residues (T257, S375, D474,
W112) identified for PTs
• MD simulation shows the high
stability of UM15 within the
binding site.
UM15 inhibition of SOSIP binding to CD4IgG2
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IC50 280 nM
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0
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10
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UM15 concentration (nM)
Representative sensorgrams showing dose
dependent inhibition of SOSIP binding to
CD4IgG2 by UM15
PT Inhibitor UM15
% gp120 binding to immobilized CD4
CD4IgG2 gp120 UM15 competition
Response Units
Human Immuno deficiency Virus Type 1 (HIV-1) is a global health problem
with over 33 million people infected worldwide and over 2.7 million new
infections annually. Host cell infection by HIV-1 is mediated by cell receptor
(CD4) interactions with the envelope glycoproteins consisting of a trimeric
complex (Env) of gp120 and gp41. Env thus presents itself as an attractive
target to attack the virus directly in order to block the cascade events that lead
to host cell infection. Peptide Triazoles (PT) are a class of entry inhibitors
developed in our lab that put gp120 in an inactive state and suppress the
binding of CD4 and co-receptor CCR5 leading to complete inhibition of viral
entry. However, most of our past biophysical investigations on the interaction
mechanism of PT involved monomeric gp120 protein, while the target on the
virus is the trimeric spike protein complex. Therefore, here we sought to
determine the inhibition mechanism of HIV mediated viral entry using the HIV
Env trimeric protein BG505.SOSIP.664. To do this we utilized a 6 amino acid
peptide (UM15) with a proline conjugated ferrocenyl-triazole residue.
BG505.SOSIP.664 binding to UM15 was studied using SPR competition assay.
In the presence of increasing concentrations of UM15, there was a significant
reduction in SOSIP binding to CD4. Strikingly, calculated IC50 value of
280nM was observed, indicating a highly specific interaction between the
trimeric Env protein and UM15. To further elucidate the binding mode of PT
onto the trimeric Env protein, docking studies were done with the
crystallographically derived BG5O5.SOSIP.664 structure (PDB: 4NCO).
Docking results showed that UM15 can bind (in silico) to trimeric protein in a
two cavity region overlapping the CD4 site, similar to that of monomeric
gp120. These results provide a framework for optimization of PT entry
inhibitor as well as understanding the mechanism of PT binding to HIV
envelope protein.
Method validation using monomeric gp120
HIV-1 BG5O5.SOSIP.664
IC50 plot showing inhibition of SOSIP binding
to CD4IgG2 by UM15.
• Lack of competition with the negative control inactive scrambled UM15 (UM15S)
validates the specificity of peptide triazole binding to BG5O5.SOSIP.664 trimer.
Binding Assay Configuration using SPR
Conclusions and Future Directions
Approach
• UM15 peptide binds to BG5O5.SOSIP.664 trimer and inhibits binding to CD4IgG2. This result is
critical in opening the way to using SOSIP to investigate structural mechanism of PT mode of action.
• Allows study of conformational effects on PT binding to SOSIP trimer via different antibody
competition experiments.
• SOSIP-PT complexes represent potential templates for co-crystallization studies for understanding PT
binding to trimeric protein.
• Deciphering the PT mode of action will enable further optimization of novel peptide-based lead
molecules as HIV-1 entry inhibitors.
• Comparing PT interaction with other forms of Env trimers (such as Env (-)ΔCT) is also being pursued.
Acknowledgements
•
We would like to thank Dr. John Moore, Dr. Al Cupo and Dr. PJ Klasse of Weill Cornell
Medical College for SOSIP constructs