Introduction to Tat-TAR inhibitors

1. Broad and potent inhibition of both HIV-1 reverse transcription and mRNA transcription by a Tat peptidomimetic binding to the Tat-transactivating RNA sequence Annette Ratcliff, Mathew Lalonde, Zafiria Athanassiou, Michael Lobritz, Krystyna Patora, Ricardo L.A. Dias, Mudit Tyagi, Julian Wong, Kerstin Moehle, Keith Olszens, Jonathan Karn, John A. Robinson, Gabriele Varani, and Eric J. Arts 1 Case Western Reserve University,Cleveland, USA 2 University of Washington, Seattle, USA 3 University of Zurich, Zurich, Switzerland

2. Introduction to Tat-TAR inhibitors • Cooperative binding of Tat and P-TEFb to TAR activates the CDK9 kinase of P-TEFb that phosphorylates RNAP II and the repressive NELF factors, leading to greatly enhanced RNAP II processivity • These considerations have led to the synthesis and valuation of numerous small-molecule and peptidic inhibitors of the Tat-TAR interaction during the last 15 years, e.g. aminoglycosides and arg rich peptides (Hua et al. Biochem 1999; Puglisi et al. Science 1992; Hamy et al., 1997 PNAS) • However, none of these molecules had sufficient potency or selectivity to progress into preclinical

3. Tat peptidomimenic binding to TAR Davidson et al., PNAS 2009

4. Cellular uptake of fluorescein labelled L50 • Inhibition of HIV-1? Inhibition of NL4-3 in U87.CD4.CXCR4 cells Inhibition in human PBMCs

5. Breath of L50 inhibition of HIV-1 Consensus TAR RNA sequence Consensus Tat protein sequence

6. Mechanism of L50 inhibition of HIV-1 L50 X X + pLTR-luc pNL4-3 X X L50 X X + pCMV-luc pNL4-3 No drug L50 (250 uM) Minimal inhibition of NL4-3 virus production from 293T cells Very weak inhibition of pLTR-luc but no statistical difference in pCMV-luc

7. Mechanism of L50 inhibition of HIV-1 Infect U87.CD4.CXCR4 cells with virus produced in 293T cells in the presence of L50 L50 L50 X L50 L50 L50 p<0.001 p<0.001 No drug L50 (250 uM) Complete inhibition of carryover virus replication in the U87.CD4.CXCR4 cells

8. Infectivity of recovered virus Infect U87.CD4.CXCR4 cells with virus produced in 293T cells in the presence of L50 Pellet and wash virus produced from 293T cells in the presence of L50 Infect U87.CD4.CXCR4 cells with equal virus quantities (by p24) ??? L50 L50 L50 L50 No inhibition of the virus produced in the presence of L50 OR cell-free virus treated with L50 then pelleted and washed

9. Time-of-drug inhibition study to determine the HIV-1 replication step of L50 inhibition

10. Two-phase decay of HIV-1 replication during L50 inhibition

11. Summary and Questions • Conformationally constrained cyclic peptide mimics can of HIV-1 Tat can effectively enter cells and inhibit HIV-1 at high nM concentrations. Potency of L50 in PBMC nears the antiviral activity of nevirapine. • This inhibition is broad and extends to primary HIV-1 of different subtypes and of both phenotypes (CXCR4- and CCR5-tropic). • Inhibition of HIV-1 transcription is weak and the majority of antiviral activity involves inhibition of reverse transcription. There is no inhibition of virus entry. • L50 inhibition occurs earlier than 3TC or nevirapine in the reverse transcription cycle. Inhibtion of (-) strand strong stop DNA and initiation from the tRNALys,3 appears to be the primary target.

12. Acknowledgements Case Western Reserve University,Cleveland, USA Annette Ratcliff Mathew Lalonde Michael Lobritz Mudit Tyagi Julian Wong Jonathan Karn University of Washington, Seattle, USA Zafiria Athanassiou Gabriele Varani University of Zurich, Zurich, Switzerland Krystyna Patora Ricardo L.A. Dias John A. Robinson NAID, NIH for funding