Anti-HIV dendrimeric peptides

Qitao Yu, Ling Li and James P. Tam Department of Biochemistry, The Scripps Research Institute in Florida,

Jupiter, FL 33458, USA Introduction Novel dendrimeric peptides were developed in our laboratory as new architectural peptides to facilitate the peptide-based drug design [1]. Previously, we reported a tetrapeptide RLYR to be a motif that contributes to the antimicrobial activity of a cascade-type peptide dendrimer. Our studies have demonstrated that dendrimeric peptides containing several copies of this motif on their surface were found to possess broad antimicrobial activity [1]. Here we report the development of a branch-type of RLYR-containing dendrimeric peptide that displays potent HIV-1 inhibition. Comparing with a control α-peptide consisting of tandemly repeating RLYR, this dendrimer is 50-fold more resistant to proteinase K than the corresponding linear α-peptide. Our results show that the unusual architectural design of dendrimeric peptide allows us to integrate multiple copies of functional motifs in parallel while improving stability and simplifying the synthetic process. Results and Discussion Our prototypic design of a branched dendrimeric peptide contains an α-peptide backbone α-(Lys)4 and four pendant branches of RLYR tethered to the ε-amines of α-(Lys)4. This is based on our original design of the cascade-type (RLYR)4K2K which we now rearranged the backbone to an α-peptide and the architecture to a branch-type peptide dendrimer. For synthetic expedience, the N-terminal α-amine is capped with a Fmoc protecting group. For comparison, we also prepared a 16-residue of linear peptide containing four tandemly repeating RLYR (Figure 1).

The α-dendrimer was prepared by a combination of Boc- and Fmoc-chemistries, with the α-peptide backbone being assembled by the Fmoc chemistry and the side-chain RLYR peptides by the Boc chemistry (Figure 2).

To determine effect of peptide architecture on proteolytic stability, we compared the rates of proteinase K digestion on both linear and branched peptide. Using 0.05mg/ml proteinase K, the half life for linear peptide (RLYR)4 was <2 min whereas half life for α-dendrimer was 150 min, a 75 fold increases in resistance to proteinase K digestion comparing the corresponding linear peptide.

Fig. 1. Schematic representations of linear and α-dendrimeric peptides.

Arg-Leu-Tyr-Arg-Arg-Leu-Tyr-Arg-Arg-Leu-Tyr-Arg-Arg-Leu-Tyr-Arg-

Linear peptide (RLYR)4

Lys Lys

Arg-Leu-Tyr-Arg

Lys Lys NH2

α-Dendrimer

Arg-Leu-Tyr-Arg

Arg-Leu-Tyr-Arg

Arg-Leu-Tyr-Arg

Arg-Leu-Tyr-Arg ε

α

S. Del Valle et al. (eds.), Peptides for Youth: The Proceedings of the 20th American Peptide Symposium, 539DOI: 10.1007/978-0-387-73657-0_236, © Springer Science+Business Media, LLC 2009

The effect of our prototype dendrimer on HIV -1 was examined by Magi assay. Briefly, HIV-1 NL4-3 viral supernatant was harvested from transient transfection of 293T. The reporter cell line, P4-R5 magi cells were seeded 5x103 cells per well in 96 well plates. 24 hours later, cell media were replaced with viral supernatant containing serial diluted dendrimers. 48 hours later, the β-galactosidase activity was assayed using β-glo kit (Promega, Madison, WI).

Our result shown that dendrimer inhibits the infection of HIV NL4-3 with IC50 at ~0.15 µM level. However, α-dendrimer failed to inhibit VSV-G pseudo-typed NL4-3 luciferase in the same assay system, indicating that α-dendrimer inhibited NL4-3 replication at the entry level.

We found the prototype dendrimer has no harmful effect of on HIV-1 susceptible cells. Cell toxicity study was performed using three cell lines, P4R5 Magi cell, MT4 and SupT 1 cells, which are often used for the study of HIV-1 replication. Under our experimental conditions, no toxicity was observed with all three cell lines using concentration up to 1µM by MTT assay (Invitrogen, Carlsbad, CA) and Cell glo kit (Promega, Modison, WI).

In conclusions, our prototypic dendrimeric peptide displays potent anti-HIV activity, low cell toxicity, and enhanced stability. While the mechanism of their anti-HIV activity is still being investigated, the novel architectural design and easy preparation (requiring fewer chemical steps for synthesis) of dendrimers offer strong potential for developing “druggable” peptides. Acknowledgments The work was in part supported by US public Health Service NIH Grant EB001986 . P4R5 cells and plasmids, NL4-3 Luc were obtained from Dr. Nathaniel Landau through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. References 1. Tam, J. P., Lu, Y-A. and Yang J-L. Eur. J. Biochem. 269, 923-932 (2002).

H2N

O

Peptide chain assembly2. Boc chemistry

+

1. Remove Fmoc

Peptide = RLYR

OHBoc-NH

Fmoc-NH

O

NH2NH

-NH

O

-NH

O

-NH

ONH

-NH

NH-NH

O

OHNH

Fmoc-NH

O

Fmoc-NH

O

Fmoc-NH

ONH

Fmoc-NH

NHFmoc-NH

Peptide

Peptide Peptide

Peptide

Peptide

3. Cleavage

Fmoc chemistry coupling four times

Fig. 2.Synthetic scheme of α-dendrimer.

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