Biana Zalis Biana Zalis

Simultaneous Recognition of HIVSimultaneous Recognition of HIV--1 TAR 1 TAR RNA Bulge and Loop Sequences by Cyclic RNA Bulge and Loop Sequences by Cyclic

Peptide Mimics of Tat ProteinPeptide Mimics of Tat ProteinAmy Davidson, Thomas C. Amy Davidson, Thomas C. LeeperLeeper, , ZafiriaZafiria AthanassiouAthanassiou, , KrystynaKrystyna PatoraPatora--KomisarskaKomisarska, Jonathan , Jonathan

KarnKarn, John A. Robinson and Gabriele , John A. Robinson and Gabriele VaraniVarani

Introduction: Introduction: HIVHIV--1 Retrovirus1 Retrovirus

HIV is a lentivirus (retrovirus family) that causes acquired immunodeficiency syndrome (AIDS), a condition in humans in which the immune system begins to fail.From its discovery in 1981 to 2006, AIDS killed more than 25 million people.

HIV is different in structure from other retroviruses. It is roughly spherical with a diameter of about 120 nm (smaller than a red blood cell, yet large for a virus). It is composed of two copies of positive single-stranded RNAthat codes for the virus's nine genes enclosed by a conical capsid composed of 2,000 copies of the viral protein p24.

Tat Genes and LTRsTat Genes and LTRs

Tat genes are regulatory genes for proteins that control the ability of HIV to infect cells, produce new copies of virus (replicate), or cause disease. Tat proteins are transcriptional transactivators for the LTR (long terminal repeat ) promoter. LTRs are sequences of DNA that repeat hundreds or thousands of times. They are used by viruses to insert their genetic sequences into the host genomes and to control production of new viruses.

Computer model of Tat protein that helps HIV to replicate.

TatTat--TAR Interaction TAR Interaction

HIV transactivation response (TAR) RNA is required for the transactivation of LTRs and for virus replication. The TAR acts as a binding site for the Tat protein and this interaction stimulates the activity of the LTR promoter.

Interactions of Lys51 and Arg55 with G17 attract the U10-G17 stem bases into a Watson-Crick conformation.

Detailed view of Arg55 interactions with bases U13 and G16 in the TAR loop region.

Overall view of Tat binding to the stem-loop region of TAR. Bases of the loop section are labeled as well as residues of Tat that form tight interactions with TAR.

Example: Cyclin T1–Tat and TAR complex

MotivationMotivationThe most common medical treatment is by antiretroviral agents such as nucleoside analogue reverse transcriptase inhibitors (NARTIs or NRTIs), protease inhibitors or a non-nucleoside reverse transcriptase inhibitors (NNRTIs).Combinations of antiretrovirals are subject to positive and negative synergies, which limits the number of useful combinations.Other issues further limit some people's treatment options from antiretroviral drug combinations, including their complicated dosing schedules and often severe side-effects.Another problem is that HIV infection becomes resistant to antiretroviral-drugs, treatment becomes more complicated and prognosis may deteriorate.

MotivationMotivationAntiviral drugs are a class of medication used specifically for treating viral infections. Specific antivirals are used for specific viruses. Unlike most antibiotics, antiviral drugs do not destroy their target pathogen; instead they inhibit their development. The Tat-TAR complex is an attractive target for developing new antivirals because the interaction between Tat and TAR is unique to the virus.During the last 15 years scientists have tried to synthesize and to evaluate small-molecule and peptidic inhibitors of the Tat-TAR interaction.However, no one succeeded and inhibiting the Tat-TAR interaction has proven challenging.

Development of Cyclic Peptide Mimics of Development of Cyclic Peptide Mimics of BIV Tat ProteinBIV Tat ProteinFirst the scientists focused on mimics of BIV (bovine immunodeficiency virus) Tat protein to discover inhibitors of the Tat-TAR interactions in it.They used the available high-resolution structure of BIV complex (3-5).

All heavy atoms are shown. Adenines are black; guanines are blue; uracyls are yellow; cytosines are red; and the BIV-2 peptide is green and red (d-Pro-l-Pro template) (5 )

They recently (2004, 2007) published that it is possible to mimic BIV Tat protein by using cyclic peptides that bind to BIV TAR with nM affinity (6, 7).

BIVBIV--2 Cyclic Peptide2 Cyclic Peptide

Average solution NMR structure of BIV-2

One of good β-hairpin (intramolecular base pairing ) structured Tat-TAR inhibitors, called BIV-2. It binds to TAR with a KD ≈ 150 nM .

The structure of BIV-2 was investigated in aqueous solution by 1H NMR spectroscopy.

BIV2 appeared as a single species (>95%) in solution, with a strong Val12 C(α)−Hto d-Pro13 C(δ)−HNOE in NOESY spectra typical for a trans peptide bond. Many NH−NH NOE connectivities were seen, including as the strongest that between the Lys7 and Arg8peptide NHs. Many other cross-strand NOE connectivitiesprovided compelling prima facia evidence for a stable β-hairpin structure.

Development of ConformationallyConstrained Mimics of HIV-1 Tat

BIV Tat protein and TAR RNA are highly homologous to HIV-1 Tat and TAR.Given the similarities in sequence and secondary structures between BIV and HIV Tat and TAR, they reasoned that the same family of constrained cyclic peptides may also be able to act as HIV Tat inhibitors. So they based the design of HIV-1 Tat mimics on the structure of the Tat-TAR interaction inhibitors in BIV, that they discovered earlier (6, 7).

Identification of Identification of nMnM Inhibitors of the HIVInhibitors of the HIV--1 1 TatTat--TAR Interaction.TAR Interaction.The method was to vary the amino acids on the peptide surface, to predict to face the TAR RNA to optimize binding in HIV and to combine multiple individual amino acid changes.Peptide binding to TAR was measured by EMSAs. A presents EMSA data for one of the peptides, L-22 (Kd=30 nM) that also efficiently inhibits formation of the ADP-1-TAR complex (B).

Binding of cyclic peptides to HIV-1 TAR and inhibition of the Tat-TAR complex by EMSA. (A) Binding of L-22 peptide (all concentrations are in M) to HIV TAR (1 nM); the buffer contains a 10,000-fold excess of tRNA to ensure that the observed binding is specific. (B) Inhibition of the complex between the ADP-1 peptide (150 nM) and HIV TAR (1 nM) by L-22, in the presence of 10,000-fold excess of tRNA.

ADP-1 is a 37-mer linear peptide containing the entire RNA-binding activity of Tat protein

In remarkable contrast to linear peptides of similar length, binding to TAR could not be disrupted by a 10,000-fold excess of tRNA present in the binding buffer.

Identification of Identification of nMnM Inhibitors of the HIVInhibitors of the HIV--1 1 TatTat--TAR Interaction.TAR Interaction.Among the roughly 100 peptides investigated, 3 sequences stand outfor their potency. L-22, L-50, and L-51 all have activity in the low nM range.

The interaction we observe is also specific: some of these peptides discriminate even between HIV-1 and BIV TAR, although their similarities in sequence and secondary structures . The selectivity of L-51 toward HIV-1 TAR is remarkable given the similarities between the 2 RNAs and the origin of these peptides as structural mimics of BIV Tat.

Identification of Identification of nMnM Inhibitors of the HIVInhibitors of the HIV--1 1 TatTat--TAR Interaction.TAR Interaction.

Furthermore, the results indicate that small changes in peptide sequence lead to significant changes in activity even for RNAs as closely related as the TAR RNAs from BIV and HIV-1. This finding is again in marked contrast to previous linear peptidic and peptidomimetic inhibitors of HIV-1 Tat-TAR, where changes in sequence generally led to only modest changes in activity and specificity against unrelated RNAs.

Structure of the TARStructure of the TAR––LL--22 Complex22 ComplexTo understand the molecular basis for the activity of this class of Tat mimics, and to further improve their characteristics, we determined the structure of the L-22–TAR complex.L-22 peptide was selected for structure determination because of the very large number of intermolecular NOEs observed in NOESY spectra in the complex with TAR.The structure of the complex relied on 143 intermolecular NOEs to accurately define the relative position of the RNA and the peptide.

The orientation of the peptide in the RNA major groove is identical to what was observed in the complex between BIV TAR and a related peptide.

Structure of the TARStructure of the TAR––LL--22 Complex22 Complex

The RNA apical loop remains somewhat disordered as also observed by relaxation studies. Remarkably, most arginineguanidinium NH2 protons were assigned from the combined analysis of filtered and conventional NOESY spectra; presumably, strong interactions with TAR slowed down bond rotation and exchange with solvent sufficiently to allow their observation. As a consequence, at least 1 intermolecular NOE was visible from the guanidinium group for every arginine residue, facilitating their precise positioning in the structure. Superposition of 10 lowest-energy structures: guaninosines in

purple, cytosines in black, adenosines in red, and uracils in gray

Structure of the TARStructure of the TAR––LL--22 Complex22 Complex

The L-22 peptide binds TAR in the major groove of the upper RNA helix (nucleotides 26–29 and 36–39) with the D-Pro-L-Pro template facing down toward the lower helix. This orientation is analogous to what has been observed in the structure of the precursor peptide BIV-2 bound to BIV TAR RNA. This observation was important to the design of the HIV-directed library of peptides and it was reassuring to observe it in the final structure.

The peptide does not simply stick into the major groove, but organizes the bulge and loop residues to partially close around it, creating a much deeper binding pocket than seen in other structures of TAR.

The Cyclic Peptide Makes Intimate Contacts The Cyclic Peptide Makes Intimate Contacts with the Apical Loop of TAR, Burying It from with the Apical Loop of TAR, Burying It from Solvent into an Unusually Deep Binding GrooveSolvent into an Unusually Deep Binding Groove

A very surprising feature is the position of loop residue A35. It is flipped out from the RNA as in other structures of TAR, but it is also drawn down in an unprecedented way toward the UCU bulge through an interaction with the guanidinium group of Arg-11, which resides near the D-Pro-L-Pro end of the peptide.In doing so, A35 draws other loop residues to partially close around the peptide so as to create a much deeper binding pocket than observed in any other structure of TAR.

The Cyclic Peptide Makes Intimate Contacts The Cyclic Peptide Makes Intimate Contacts with the Apical Loop of TAR, Burying It from with the Apical Loop of TAR, Burying It from Solvent into an Unusually Deep Binding GrooveSolvent into an Unusually Deep Binding Groove

Other interactions with the apical loop appear to be of less importance, and indeed the remainder of the loop remains locally unstructured

A35,G33 and G34 close over the strand of the peptide between Arg-8 and Ile-12. As a consequence of the interaction between A35 and Arg-11, even Val-2, which was postulated to remain solvent exposed when the peptides were designed, is partially protected from solvent by interacting with the minor groove face of A35.

Hydrophobic And Polar InteractionsHydrophobic And Polar Interactions

Hydrophobic interactions observed in the HIV-1 TAR-L22 complex, stabilize a base triple and dock the peptide in the RNA groove. Amino hydrogen atoms are protected from solvent exchange and observable in NOESY experiments.

(A) The guanidinium group of Arg-1 contacts the RNA backbone between G21 and A22 and is within hydrogen-bonding distance of the A22 phosphate

(B) The Arg-5 guanidinium group stacks on top of the U23 base to provide a cation– interaction that stabilizes the base triple (hydrogen bonds are shown by dotted lines), while the aliphatic portion of the Lys-6 side chain lies over Arg-5, packing its guanidinium group against U23

(C) Arg-5 is also adjacent to the base of G28, forming hydrogen bonds with the O6 and N7 of G28

Discussion:Discussion:

The Tat-TAR complex has long been considered an attractive target for the discovery of novel antivirals because of its central role in promoting infection by up-regulating HIV transcription.Stable β-hairpin cyclic peptides bind specifically to HIV-1 TAR RNA with high affinity.Discovery of a family of structurally constrained -hairpin cyclic peptide mimics of HIV-1 Tat protein that bind to TAR RNA with nM affinity and have greatly improved specificity compared with previous small-molecule or peptidic ligands for TAR.The most surprising result was the observation that the peptide simultaneously organizes the bulge and loop residues to partially close around it, creating a much deeper binding pocketthan seen in all previous structures of TAR.

Discussion:Discussion:These cyclic peptides are potent inhibitors of viral replication with activity only 10-fold lower than the non-nucleoside reverse transcriptase inhibitor neviparine in primary T lymphocytes.These cyclic peptides are broad-spectrum HIV inhibitors and are active against a wide range of viral isolates from each of the HIV-1 clades without any cytotoxicity to at least 1 mM; their constrained cyclic structure makes them stable to proteolysis. Finally, mechanistic studies show that the peptides do not inhibit HIV interaction with its receptor or viral entry, off-target activity that stunted the development of several linear peptide and small-molecule inhibitors of the Tat-TAR interaction.Hopefully, these cyclic peptides in this class will provide new antiviral drug leads active against a wide range of viral strains, including strains that are resistant to the current range of drugs.

Thank you for attentionThank you for attention

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