J Clin Invest. 2005 Jan;115(1):76-85.
Identification of cellular deoxyhypusine synthase as a novel target for antiretroviral therapy.
Hauber I1, Bevec D, Heukeshoven J, Krätzer F, Horn F, Choidas A, Harrer T, Hauber J.
Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany.
The introduction of highly active antiretroviral therapy (HAART) has significantly decreased morbidity and mortality among patients infected with HIV-1. However, HIV-1 can acquire resistance against all currently available antiretroviral drugs targeting viral reverse transcriptase, protease, and gp41. Moreover, in a growing number of patients, the development of multidrug-resistant viruses compromises HAART efficacy and limits therapeutic options. Therefore, it is an ongoing task to develop new drugs and to identify new targets for antiretroviral therapy. Here, we identified the guanylhydrazone CNI-1493 as an efficient inhibitor of human deoxyhypusine synthase (DHS). By inhibiting DHS, this compound suppresses hypusine formation and, thereby, activation of eukaryotic initiation factor 5A (eIF-5A), a cellular cofactor of the HIV-1 Rev regulatory protein. We demonstrate that inhibition of DHS by CNI-1493 or RNA interference efficiently suppressed the retroviral replication cycle in cell culture and primary cells. We show that CNI-1493 inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and viral strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Moreover, no measurable drug-induced adverse effects on cell cycle transition, apoptosis, and general cytotoxicity were observed. Therefore, human DHS represents a novel and promising drug target for the development of advanced antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.
10.1172/JCI21949
Inhibition of hypusine formation in eIF-5A by interfering with DHS activity. (A) Schematic representation of the pathway of eIF-5A biosynthesis. Hypusine is required for the biological activity of the protein. The conversion of a specific lysine residue (Lys50) into the unusual amino acid hypusine occurs in 2 posttranslational reactions that are mediated by the enzymes DHS and deoxyhypusine hydroxylase (DHH; see text for details). DHS is a target of experimental low–molecular weight drugs, such as the multivalent guanylhydrazone CNI-1493. (B) CNI-1493 inhibits hypusine formation in eIF-5A in vivo. Jurkat cells were metabolically labeled using either [14C]putrescine or [35S]cysteine in the presence of either DMSO (solvent control, lane 1) or CNI-1493 (1.0 μM, lane 2; 0.5 μM, lane 3). Equal amounts of the various radiolabeled cellular extracts were then subjected to eIF-5A–specific immunoprecipitation analysis and analyzed by SDS-PAGE and autoradiography. Molecular mass standards are indicated in kDa. (C) Two-dimensional electrophoresis of total proteins from PM1 cells that were metabolically labeled using [14C]spermidine in the presence of either DMSO (upper panel) or CNI-1493 (1.0 μM; lower panel). Protein separation was performed, using equal amounts of the radiolabeled extracts, by isoelectric focusing in the first dimension on an immobilized linear pH gradient (IPG) from pH 4.0 to 7.0, which was followed by SDS-PAGE separation in the second dimension. Only molecular mass of less than 25.0 kDa is shown in autoradiographs. No radioactive protein spots were detected elsewhere. Molecular mass standards are indicated in kDa on the right.
Identification of cellular deoxyhypusine synthase as a novel target for antiretroviral therapy
J Clin Invest. 2005 Jan 3;115(1):76-85.
Inhibition of macrophage-tropic (M-tropic) and T cell–tropic (T-tropic) HIV-1. PM1 cells were infected with HIV-1 BaL or NL4/3. Infected cells were cultured in the presence of the indicated concentrations of CNI-1493, or with DMSO alone (control). The culture medium was changed at days 3, 6, and 9. p24 antigen levels and cell viability were determined at days 6 and 12. The percentage of inhibition of virus replication as compared with replication in the control culture without drug, which was arbitrarily set at 100%, is shown in the upper panels. MTT assays (Roche Diagnostics GmbH) from the same cultures are shown in the lower panels.
Inhibition of clinical HIV-1 isolates. PBMCs from HIV-1–infected patients were cultured in the presence of CNI-1493. The culture medium was changed every 3 days. Every week, each culture was split 1:1, and fresh feeder PBMCs from 4 healthy donors, treated with PHA-P (6 μg/ml phytohemagglutinin) and PB (1 μg/μl polybrene) and preincubated in CNI-1493 for 4 days, were added along with recombinant IL-2. At day 14, cell counts, cell viability, and p24 antigen and/or bDNA levels were determined (cell counts ranged from 0.9 × 106 to 1.1 × 106 cells per milliliter; in all cultures tested, at least 80% of the cells were viable). The percentage of inhibition of virus replication as compared with replication in the respective untreated patient cells is shown.
Kinetics of HIV-1 inhibition by CNI-1493 and long-term drug exposure. (A) PM1 cells were infected with HIV-1 BaL and cultured for 12 days in the presence of CNI-1493 at a concentration of 0.5 μM. Subsequently, the cells were split in 2 equal halves (Cell-split). One of these cultures was further incubated in culture medium containing 0.5 μM CNI-1493 for another 12 days. The other culture was washed with medium and incubated for 12 days in the presence of DMSO (solvent control). The culture medium was changed at days 2, 5, 7, 10, and 12 before the drug was removed, and at days 2, 5, 7, and 9 after cell splitting. The viability of the cells, cell counts, and p24 antigen levels were determined at the days indicated. (B) HIV-1 BaL–infected PM1 cells were cultured for 12 weeks in the presence of 125 nM CNI-1493. Subsequently, the virus-containing supernatant was used for de novo infection of another batch of PM1 cells, and culturing of these cells, as well as of cells infected with wild-type (WT) HIV-1 BaL, continued for an additional 16 days in the presence of 1.0 μM CNI-1493 or DMSO (solvent control). Inhibition of virus replication (in percent; black bars) and drug toxicity (as measured by MTT assay; gray bars) at days 8, 12, and 16 are indicated.
Inhibition of DHS by RNAi. For DHS knock-down, HLCD4-CAT cells, which express the CD4 surface molecule, were repeatedly transfected with either human DHS–specific (DHS) or control scramble II duplex (SDII) siRNAs. (A) RNA was isolated 1 day after the final transfection, followed by first-strand cDNA synthesis. Gene silencing was monitored by DHS-specific PCR. A GAPDH-specific reaction served as internal control. (B) siRNA-treated cells were metabolically labeled using [14C]spermidine, and total cell proteins were analyzed by 2-dimensional electrophoresis and subsequent autoradiography as described in the legend to Figure . (C and D) The respective cell cultures were infected with HIV-1 NL4/3 1 day after the final transfection. At day 3 after infection, p24 antigen levels (C, black bars), cell viability (MTT assay; C, gray bars), and integrated proviral DNA (D) were analyzed. NC, negative control reaction.
CNI-1493 negatively affects HIV-1 Rev trans-activation. (A) Inhibition of cytoplasmic accumulation of incompletely spliced viral mRNAs. PM1 cells were infected with HIV-1 BaL and subsequently cultured in the presence of either CNI-1493 (0.8 μM) or DMSO. Cytoplasmic (C) or total (T) RNA was isolated at day 7 after infection and subjected to HIV-1 mRNA–specific or U6 snRNA–specific Northern analysis. (B) Inhibition of Rev-dependent virus production. HeLa cells were cultured for 7 days in 1.0 μM CNI-1493 or DMSO and subsequently cotransfected with a rev-defective proviral DNA, a Rev-expressing vector, or the respective parental plasmid (Rev-deficient control) and a construct expressing constitutively secreted alkaline phosphatase (SEAP). The transfected cell monolayers were further maintained in 1.0 μM CNI-1493 or DMSO and analyzed at 60 hours after transfection for levels of p24 antigen. The respective values were adjusted for transfection efficiency by determination of the level of SEAP (upper panel). In addition, total cell protein extracts were prepared and subjected to p55Gag-specific or α-tubulin–specific (protein loading control) Western analysis (lower panel). (C) Analysis of Rev trans-activation. HeLa cells were cultured in CNI-1493 or DMSO as before and subsequently cotransfected with the Rev-responsive reporter plasmid pCMVgagLucRRE, a Rev-expressing vector, or the respective parental plasmid (Rev-deficient control) and the constitutive internal control vector pBC12/CMV/βGal. (D) Analysis of Tat trans-activation. HeLa cells were cultured in CNI-1493 or DMSO as before and subsequently cotransfected with the Tat-responsive reporter plasmid pBC12/HIV/CAT, a Tat-expressing vector, or the respective parental plasmid (Tat-deficient control) and the constitutive internal control vector pBC12/CMV/βGal.
Inhibition of HAART-resistant HIV-1 isolates. PM1 cells were incubated for 6 days in the presence of 1.0 μM CNI-1493 or DMSO and then infected with various types of antiretroviral drug–resistant viruses. Results of the phenotypic drug sensitivity assay (red areas in the virograms; for details see text) are shown. The resistance-conferring mutations in the various viral genomes (genotypes), as determined by DNA sequencing, are summarized in the supplemental data section. (A) Infected cells were incubated in CNI-1493 at concentrations of 1.0 μM and 0.5 μM, or DMSO (control). Inhibition of virus replication (in percent) and drug toxicity (as measured by MTT assay) at days 6 and 12 are indicated in the right panels. (B) Three-week time course of inhibition of an "omni-resistant" virus isolate (FE9) by CNI-1493. p24 antigen level (p24Ag/CNI-1493 and p24Ag/DMSO control) and cell viability (MTT/CNI-1493 and MTT/DMSO control) were determined at days 6, 9, 12, 16, and 20 after infection (graphs). PI, protease inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; AZT, zidovudine; DDC, zalcitabine; ddI, didanosine; D4T, stavudine; 3TC, lamivudine.
Anti-HIV Agents/chemistry
Anti-HIV Agents/pharmacology*
Drug Resistance, Multiple
Gene Expression Regulation, Viral/drug effects
Gene Products, rev/metabolism
HIV Infections/enzymology*
HIV Infections/virology
HIV-1/drug effects
HIV-1/physiology
Hydrazones/chemistry
Hydrazones/pharmacology
Lysine/analogs & derivatives*
Lysine/metabolism
Macrophages/drug effects
Macrophages/virology
Oxidoreductases Acting on CH-NH Group Donors/antagonists & inhibitors*
Oxidoreductases Acting on CH-NH Group Donors/genetics
Oxidoreductases Acting on CH-NH Group Donors/metabolism
T-Lymphocytes/virology
Transcriptional Activation/drug effects
Virus Replication/drug effects
rev Gene Products, Human Immunodeficiency Virus
Gene Products, rev
Hydrazones
hypusine
semapimod
Oxidoreductases Acting on CH-NH Group Donors
deoxyhypusine synthase
American Society for Clinical Investigation
Highlights from Nature Reviews Drug Discovery - Nature Publishing Group
Highlights from Nature Reviews Microbiology - Nature Publishing Group
Palliative Care of Patients with HIV - HIV InSite
HIV/AIDS Medicines - MedlinePlus Health Information
L-Lysine - Hazardous Substances Data Bank