Development of a Functional Backbone Cyclic Mimetic of the HIV-1 Tat Arginine-rich Motif

Abstract

We have used the backbone cyclic proteinomimetics approach to develop peptides that functionally mimic the arginine-rich motif (ARM) of the HIV-1 Tat protein. This consensus sequence serves both as a nuclear localization signal (NLS) and as an RNA binding domain. Based on the NMR structure of Tat, we have designed and synthesized a backbone cyclic ARM mimetic peptide library. The peptides were screened for their ability to mediate nuclear import of the corresponding BSA conjugates in permeabilized cells. One peptide, designated "Tat11," displayed active NLS properties. Nuclear import of Tat11-BSA was found to proceed by the same distinct pathway used by the Tat-NLS and not by the common importin ␣ pathway, which is used by the SV40-NLS. Most of the Tat-derived backbone cyclic peptides display selective inhibitory activity as demonstrated by the inhibition of the nuclear import mediated by the Tat-NLS and not by the SV40-NLS. The Tat-ARM-derived peptides, including Tat-11, also inhibited binding of the HIV-1 Rev-ARM to its corresponding RNA element (Rev response element) with inhibition constants of 5 nM. Here we have shown for the first time (a) a functional mimetic of a protein sequence, which activates a nuclear import receptor and (b) a mimetic of a protein sequence with a dual functionality. Tat11 is a lead compound which can potentially inhibit the HIV-1 life cycle by a dual mechanism: inhibition of nuclear import and of RNA binding.Proteinomimetics are small molecules that can mimic the structure and/or function of active sites within proteins (1). They are useful for detailed study of protein folding, structure, and function. The major potential application of proteinomimetics is, however, therapeutic; such molecules can be used to block protein-protein and/or protein-nucleic acid interactions, thus interfering with undesired biological processes (2-4). Being relatively small, proteinomimetics often solve acute problems associated with the use of proteins as drugs, such as antigenicity, low bioavailability, high cost, and rapid enzymatic degradation.Two properties of the parent protein must be retained when designing proteinomimetics: (i) the bioactive conformation of the desired active site and (ii) a certain degree of conformational flexibility to allow induced fit. Linear peptides are not optimal candidates to mimic proteins, because they equilibrate between multiple conformations and thus adaptation of the bioactive conformation is at an entropic cost. Introduction of conformational constraints into the peptide is thus needed to generate a proteinomimetic. Being relatively small and conformationally constrained, cyclic peptides are excellent candidates to serve as proteinomimetics. Backbone cyclization of peptides is the method of cyclization developed and used in our lab. It results in peptides with improved selectivity, enhanced metabolic stability, and high bioavailability (5-9). To select the most active backbone cyclic (BC) 1 peptide based on a given sequence, we have developed the "cycloscan" ...

Sequence and structure space of RNA‐binding peptides

“…Cyclic versions of an HIV-1 Tat arginine-rich peptide were generated utilizing methylene linkers of different lengths, and one peptide, screened initially for nuclear localization, was able to bind HIV-1 TAR but also bound RRE RNA, perhaps as a result of the cyclic constraint. 37 In another study, a disulfide-linked cyclic BIV Tat ␤-hairpin peptide bound BIV TAR with 3-fold higher affinity than the noncyclized version, 38 consistent with the notion that prestabilizing peptide structure can enhance binding affinity, as described above for ␣-helical peptides.…”

Targeting RNA with Small Molecules

“…For example, backbone-cyclized peptides designed to mimic the Arg-rich motif of the HIV-1 Tat protein have been found to be potent RRE binders. [62] Eilatin-containing octahedral Ru II complexes have also been identified by the assay to be a) The proposed secondary structure of the 66-nucleotide RRE core of HIV-1 (the actual RRE is larger than 300 nucleotides long). The red nucleotides represent the high affinity Rev recognition element.…”

Peptides Containing Cyclin/Cdk-Nuclear Localization Signal Motifs Derived from Viral Initiator Proteins Bind to DNA When Unphosphorylated

“…Moreover, peptides that bind to single-stranded DNA have been reported (see, for example, references 7 and 69). In addition, a peptide mimetic of the human immunodeficiency virus type 1 Tat NLS is known to bind to RNA (21). Interest in these molecules reflects, in part, their potential use in gene therapy (37,40).…”