Broad and potent inhibition of both HIV-1 Broad and potent inhibition of both HIV-1 reverse transcription and mRNA transcription reverse transcription and mRNA transcription

by a Tat peptidomimetic binding to the Tat-by a Tat peptidomimetic binding to the Tat-transactivating RNA sequencetransactivating 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, USA3 University of Zurich, Zurich, Switzerland

Introduction to Tat-TAR inhibitorsIntroduction 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

Tat peptidomimenic binding to TARTat peptidomimenic binding to TAR

Davidson et al., PNAS 2009

Inhibition of HIV-1?Inhibition of HIV-1? Cellular uptake of fluoresceinlabelled L50

Inhibition of NL4-3 in U87.CD4.CXCR4 cells

Inhibition in human PBMCs

Breath of L50 inhibition of HIV-1Breath of L50 inhibition of HIV-1

Consensus TAR RNA sequence

Consensus Tat protein sequence

Mechanism of L50 inhibition of HIV-1Mechanism of L50 inhibition of HIV-1

pNL4-3 pLTR-luc+

pNL4-3 pCMV-luc+

L50

X

XX

X

L50

X

X

Very weak inhibition of pLTR-luc but no statistical difference in pCMV-luc

No drugL50 (250 uM)

Minimal inhibition of NL4-3 virus production from 293T cells

Mechanism of L50 inhibition of HIV-1Mechanism of L50 inhibition of HIV-1

No drugL50 (250 uM)

Complete inhibition of carryover virus replication in the U87.CD4.CXCR4 cells

Infect U87.CD4.CXCR4 cells with virus produced in 293T cells in the presence of L50

L50

L50

L50

L50

L50 X

p<0.001 p<0.001

Infectivity of recovered virusInfectivity of recovered virus

No inhibition of the virus produced in the presence of L50 OR cell-free virus treated with L50 then pelleted and washed

Infect U87.CD4.CXCR4 cells with virus produced in 293T cells in the presence of L50

L50L50

L50

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)

???

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

Two-phase decay of HIV-1 replication during Two-phase decay of HIV-1 replication during L50 inhibitionL50 inhibition

Summary and QuestionsSummary 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.

AcknowledgementsAcknowledgementsCase Western Reserve University,Cleveland, USA

Annette RatcliffMathew LalondeMichael LobritzMudit TyagiJulian WongJonathan Karn

University of Washington, Seattle, USA

Zafiria AthanassiouGabriele Varani

University of Zurich, Zurich, Switzerland

Krystyna PatoraRicardo L.A. DiasJohn A. Robinson

NAID, NIH for funding