Mechanism of action of the HIV-1 integrase inhibitory peptide LEDGF 361-370.

The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.

[1]  Alejandra Borjabad,et al.  Testing antiretroviral drug efficacy in conventional mice infected with chimeric HIV-1 , 2007, AIDS.

[2]  A. Friedler,et al.  Peptides Derived from HIV-1 Integrase that Bind Rev Stimulate Viral Genome Integration , 2009, PloS one.

[3]  J. Levy.  Pathogenesis of human immunodeficiency virus infection , 1989, Microbiological reviews.

[4]  Joseph Rosenbluh,et al.  Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium , 2007, Proceedings of the National Academy of Sciences.

[5]  K. Wüthrich.  NMR of proteins and nucleic acids , 1988 .

[6]  Maria Vanegas,et al.  Identification of the LEDGF/p75 HIV-1 integrase-interaction domain and NLS reveals NLS-independent chromatin tethering , 2005, Journal of Cell Science.

[7]  M. Kozal,et al.  The hunt for HIV-1 integrase inhibitors. , 2006, AIDS patient care and STDs.

[8]  A. Engelman,et al.  Structural basis for the recognition between HIV-1 integrase and transcriptional coactivator p75. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Ad Bax,et al.  MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy , 1985 .

[10]  J Desmyter,et al.  Mechanism of inhibitory effect of dextran sulfate and heparin on replication of human immunodeficiency virus in vitro. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[11]  A. Brooks,et al.  A mouse model for study of systemic HIV-1 infection, antiviral immune responses, and neuroinvasiveness. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[12]  W. Greene,et al.  Slipping through the door: HIV entry into the nucleus. , 2002, Microbes and infection.

[13]  Robert Craigie,et al.  HIV-1 DNA integration: Mechanism of viral DNA cleavage and DNA strand transfer , 1991, Cell.

[14]  Richard R. Ernst,et al.  Investigation of exchange processes by two‐dimensional NMR spectroscopy , 1979 .

[15]  Pamela A. Silver,et al.  Identification of an Evolutionarily Conserved Domain in Human Lens Epithelium-derived Growth Factor/Transcriptional Co-activator p75 (LEDGF/p75) That Binds HIV-1 Integrase* , 2004, Journal of Biological Chemistry.

[16]  O. Keppler,et al.  HIV-susceptible transgenic rats allow rapid preclinical testing of antiviral compounds targeting virus entry or reverse transcription , 2007, Proceedings of the National Academy of Sciences.

[17]  A. Engelman,et al.  Structure-based mutagenesis of the integrase-LEDGF/p75 interface uncouples a strict correlation between in vitro protein binding and HIV-1 fitness. , 2007, Virology.

[18]  H. Nagata,et al.  Active domains of salivary statherin on apatitic surfaces for binding to Fusobacterium nucleatum cells. , 2004, Microbiology.

[19]  A. Engelman,et al.  Solution structure of the HIV-1 integrase-binding domain in LEDGF/p75 , 2005, Nature Structural &Molecular Biology.

[20]  A. Friedler,et al.  Peptides derived from HIV‐1 Rev inhibit HIV‐1 integrase in a shiftide mechanism , 2008, Biopolymers.

[21]  E. Woolf,et al.  Determination of the HIV integrase inhibitor, MK-0518 (raltegravir), in human plasma using 96-well liquid-liquid extraction and HPLC-MS/MS. , 2007, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[22]  M S Baig,et al.  Homology modeling and docking studies of Comamonas testosteroni B-356 biphenyl-2,3-dioxygenase involved in degradation of polychlorinated biphenyls. , 2010, International journal of biological macromolecules.

[23]  Myriam Witvrouw,et al.  New Class of HIV Integrase Inhibitors that Block Viral Replication in Cell Culture , 2002, Current Biology.

[24]  A. Friedler,et al.  Correlation between shiftide activity and HIV-1 integrase inhibition by a peptide selected from a combinatorial library. , 2008, Journal of molecular biology.

[25]  A. Friedler,et al.  Interaction between HIV-1 Rev and Integrase Proteins , 2007, Journal of Biological Chemistry.

[26]  D. Volsky,et al.  Vaccine-induced protection from infection of mice by chimeric human immunodeficiency virus type 1, EcoHIV/NL4-3. , 2007, Vaccine.

[27]  Wagner,et al.  Gradient-Selected NOESY-A Fourfold Reduction of the Measurement Time for the NOESY Experiment , 1996, Journal of magnetic resonance. Series A.

[28]  C. Ehresmann,et al.  Inhibition of the initiation of HIV-1 reverse transcription by 3'-azido-3'-deoxythymidine. Comparison with elongation. , 2000, The Journal of biological chemistry.

[29]  A. Friedler,et al.  Peptide inhibitors of HIV-1 integrase: from mechanistic studies to improved lead compounds. , 2009, Bioorganic & medicinal chemistry.

[30]  V. Saudek,et al.  Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions , 1992, Journal of biomolecular NMR.