An ultra-high affinity ligand of HIV-1 TAR reveals the RNA structure recognized by P-TEFb

Abstract

The HIV-1 trans-activator protein Tat binds the trans-activation response element (TAR) to facilitate recruitment of the super elongation complex (SEC) to enhance transcription of the integrated pro-viral genome. The Tat–TAR interaction is critical for viral replication and the emergence of the virus from the latent state, therefore, inhibiting this interaction has long been pursued to discover new anti-viral or latency reversal agents. However, discovering active compounds that directly target RNA with high affinity and selectivity remains a significant challenge; limiting pre-clinical development. Here, we report the rational design of a macrocyclic peptide mimic of the arginine rich motif of Tat, which binds to TAR with low pM affinity and 100-fold selectivity against closely homologous RNAs. Despite these unprecedented binding properties, the new ligand (JB181) only moderately inhibits Tat-dependent reactivation in cells and recruitment of positive transcription elongation factor (P-TEFb) to TAR. The NMR structure of the JB181–TAR complex revealed that the ligand induces a structure in the TAR loop that closely mimics the P-TEFb/Tat1:57/AFF4/TAR complex. These results strongly suggest that high-affinity ligands which bind the UCU bulge are not likely to inhibit recruitment of the SEC and suggest that targeting of the TAR loop will be an essential feature of effective Tat inhibitors.

Face-time with TAR: Portraits of an HIV-1 RNA with diverse modes of effector recognition relevant for drug discovery

“…5, C and D) to produce a number of cyclic peptides (82,137,138) that culminated recently in an "ultrahigh affinity" cyclic peptidomimetic. This inhibitor, JB181, binds HIV-1 TAR with an unprecedented K D (63). NMR solution analysis revealed that JB181 recognizes TAR in the s1b major groove and bulge (Fig.…”

“…H, close-up view of HIV-1 TAR recognition in the central UCU bulge by TBP6.7. Arg-47 engages TAR at the ASM, similar to F. I, global view of HIV-1 TAR recognition by the cyclic peptide JB181 (PDB entry 6d2u) (63). The S c value was calculated from the lowest energy NMR structure.…”

“…For these reasons, TAR is a highvalue drug target whose inhibition could potentially disrupt viral transcription in chronic as well as latent infections. However, no such inhibitors are clinically available, and TAR has resisted the development of therapeutics, despite success in the identification of compounds that target the RNA with specificity and affinity (62)(63)(64).…”

“…Additional structural improvements have been attained through engineered RNA constructs to promote crystal contacts or by exploiting structurally well-characterized proteins, such as U1A, as a starting platform for labbased evolution and structural studies. These developments have led to a series of new structures including: exciting TAR-Tat and TAR-Tat-SEC complexes (74 -76), a co-crystal structure of TAR bound to a lab-evolved protein from the Wedekind lab (77), and an ultrahigh-affinity cyclic peptide bound to TAR (63). Here, we put these novel discoveries into perspective by considering prior characterization of TAR apo-and boundstate conformations.…”

Design and Synthesis of WM5 Analogues as HIV-1 TAR RNA Binders

“…During the reviewing of this work, two interesting papers were published that could help the design of an effective TAR binder. In particular, Schulze-Gahmen and Hurley solved the crystal structure of the TAR in complex with Tat and the SEC core (CycT1/CDK9/AFF4) [43], while Varani and co-workers reported the NMR structure of TAR RNA in complex with a ultra-potent macrocyclic peptide that mimics the Arginine Rich Motif (ARM) of Tat [44], which however only weakly inhibit the Tat-mediated transcription.…”