Abstracts Vol. 1 [International Conference on AIDS (11th: 1996: Vancouver, Canada)]

Mo.A.1093 - Mo.A.1098

Monday, July 8, 1996

Mo.A. 1093
DISCOVERY OF CYANOVIRIN-N,A NOVEL HIV-INACTIVATING PROTEIN FROM
NOSTOC ELLIPSOSPORUM THAT TARGETS VIRAL GP120
Boyd, MichaelR, Gustafson K, McMahon J, Shoemaker R. Laboratory of Drug Discovery
Research and Development, National Cancer Institute, Frederick, MD
Issue: There is an urgent international priority for discovery and development of femalecontrollable mrneasures against HIV transmission.The viral-gp 120/cellular-CD4 interaction
remains an especially attractive target both for developmental therapeutics and for prophylaxis against HIV infection.
Project: As a part of the U.S. National Cancer Institute (NCI)'s program to discover new
AIDS drug development leads, more than 80,000 extracts of diverse terrestrial plants,
marine plants and animals, and microorganisms have been screened to date for anti-HIV
activity Our research has focused upon the isolation and detailed chemical and biological
characterization of individual active constituents from selected extracts found positive in the
NCI screen. We disclose here for the first time a pertinent new discovery
Results: Cyanovirin-N (CV-N), a unique I I kDa antiviral protein, has been isolated and
identified fiorn cultures of the cyanobacterium (blue-green alga), Nostoc ellipsosporum. A
DNA sequence corresponding to the chemically deduced protein sequence has been synthesized and expressed in Escherischir coli; the recombinant protein product (r-CV-N) is
indistinguishable from natural CV-N. Low nanomolar concentrations of CV-N prevent the in
vitro replication and cytopathicity of primate retroviruses, including SIV and diverse laboratory strains and clinical isolates of HIV- I and HIV-2. CV-N mediates these antiviral effects
through apparently conserved interactions with the viral envelope glycoprotein that differ
from gp I 20 interactions with either the cellular receptor derivative, sCD4, or with antibodies directed against viral gp I 20 neutralizing determinants.
Lessons Learned: The novel gp I 20-targeting properties of CV-N are of interest for possible
broad-spectrum virucidal and therapeutic applications against HIV The NCI Decision
Network Committee has recently selected -N for high-priority preclinical development and
further investigation of topical and other ex vivo prophylactic applications.
Michael R. Boyd, M.D., Ph.D., NCI-FCRDC, Bldg. 1052, Rm 12 I, Frederick, MD 21702-1201
USA Telephone: 30 I1-846-539 I FAX [phone]
Mo.A. 1094
EFFECT OF THE TAT INHIBITOR, 7-CHLORO-5-(2-PYRRL)-3H- I,4-BENZODIAZEPIN2-(H)-ONE, IN IN VITRO RECONSTITUTED AND CELL-BASED TRANSACTIVATION
SYSTEMS
Tomassini oanne*, Blau C*, ByrnesV*, Flores O**, Graham P*, Hazuda D*,Yang L**.
*Merck Research Laboratories, W. Point. Pa. and **Tularik, Inc., San Francisco, California
Objective: The benzodiazepine compound, 7-chloro-S-(2-pyrrl)-3H- I,4-benzodiazepin-2 -(H)-one, was previously identified as an inhibitor ofTAT-activated HIV transcription and of
HIV replication in acute and chronically infected cells (I). Although the mechanism of action
of this inhibitor is not completely understood, studies have indicated that the compound
inhibits TAt-induced initiation of transcription from the HIV LTR, presumably by antagonizing
the effect of cooperative cellular factors (2,3).To better understand the inhibitory role of
this compound, we have further evaluated its activity in several TAT transactivation systems.
Method: The benzodiazepine inhibitor was evaluated in an in vitro transactivation system
reconstituted with recombinant TAT protein and in cell-based transactivation systems
induced by endogenously expressed TAT and/or exogenous recombinant TAT protein.
Results:The compound was found to inhibit transactivation in cell-based assays in which
TAT was endogenously expressed. but had no effect in in vitro reconstituted or cell-based
transactivation assays induced by exogenous recombinant TAT protein. Furthermore, the
amount of inhibition observed in cell-based, endogenous transactivation correlated with a
decrease in the level of TAT protein present.
Conclusion: Our studies indicate the benzodiazepine compound does not directly inhibit
TAT activation of transcription from the HIV LTR, as evidenced by a lack of effect in transactivation systerns induced by recornmbinant TAT protein.This result is in contrast to previous
reports in which the compound inhibited transactivation in both endogenous and exogenous TAT transactivation systems. Further investigation is required to discern these differences. I. Hsu, M.-C, et al., PNAS 90:6395-6399, 1993.2. Braddock, M. et al.,J.Virol. 68:25-33,
1994. 3. Cupelli, L. and Hsu, M.-C, J.Virol. 69:2640-2643, 1995.
Joainne Tornassini, Merck Research Laboratories, Bldg. 16-206, Sumneytown Pike,West Point,
PA 19486 USA Telephone: [phone] Fax: [phone]
Mo.A. 1095
SYNTHESIS AND BIOLOGICAL EVALUATION OF CYCLIC PHOSPHOLIPID
ANALOGUES TARGETING HIV- I
Ishaq, K 5.*, McLean, J.W*, Morris-Natschke, S.L.*, lyer N.**Kucera, Louis 5.**. *The
University of North Carolina, Chapel Hill, NC; **Wake Forest University Medical Center,
Winston-Salem, NC, U.S.A.
Objectives: The major objectives are to synthesize and evaluate membrane interactive
cyclic phospholipid analogues and to conjugate them with AZT to enhance anti-HIV- I
selectivity
Methods: We have synthesized conformationally constrained phospholipid analogues with
heteroalkyl chains and a phosphocholine moiety incorporated into cyclohexane rings.
Biological evaluations were performed using 3H-TdR incorporation into total DNA to measure cytotoxicity and a syncytial plaque assay for anti-HIV- I activity in CEM-SS cells.TC50
values for cytotoxicity and EC50 values for plaque inhibition were determined using a computer progran.
Results: The cis and trans isomers of 3-(hexadecylthio) cyclohexyl phosphocholine gave an
TC50 of 15.7 and I 1.5 pM, respectively for cytotoxicity and EC50 of 0.36 and 0.84 pM,
respectively for anti-HIV- I activity Also, we synthesized the cyclopentane-AZT and cyclohexane-AZT conjugates.The cyclopentane-AZT conjugate had an TC50 of 29.8 pM for
cytotoxicity and an EC50 of 0.05 pM for anti-HIV- I activity Experiments are in progress to
evaluate the cyclohexane-AZT conjugate.
Conclusions: The selectivity (TC50/EC50) of the cis isomer (44) was about three-fold better than the trans isomer (14) of the cyclohexyl phosphocholine.The cyclopentane-AZT
conjugate had a selectivity of 596. Further modifcation of conformationally constrained
phospholipids and their AZT conjugates are in progress to increase anti-HIV- I selectivity

L.S. Kucera.Wake Feorest University Medical Center, Medical Center Blvd.,Winston-Salem,
North Carolina, U.S.A., 2 7157-1064 Telephorie: 910-716-4875 Fax [phone] email:
[email protected]
Mo.A. 1096
HIV-I LOAD IS DECREASED IN CHRONIC INFECTED ACH-2 CELL LINE BY
SUCCESSIVE IN VITRO HYPERTHERMIATREATMENT
Lee Moon H*, Schick PM**, Hsu MYK*, Ly H*. *Harbor-UCLA Medical Center:
Torrance,CA; **Westside Research Foundation, Culver City CA; USA.
Objective: Extracorporeal whole body hyperthermia is cuirrently in clinical trials for treating
AIDS patients in the USA. Since efficacy of this treatment is not well established, we studied the
effects of hyperthermia treatment (42~C) in the HIV- I chronically infected T cell line (ACH-2).
Methods: Uninfected parent A3.0 I and infected ACH-2 T lymphocytes (5 x 105/ml 0.0 IM
phosphate buffered saline, pH 7.4) were heated in a 42"C water bath for I hr and then
placed in culture. Cells incubated at 37'C served as untreated controls. A series of two
heat treatments, 4 days apart, was also investigated. The cell % viability and number was
determined by trypan blue exclusion counting in a hemacytometer HIV- I production was
measured by p24 antigen EIA testing of the culture medium.
Results: Heat treated ACH-2 showed a lower cell % viability and number (49% and 0.8 x
105/ml) compared to heat treated A3.01 (69% and 1.4 x 10-/ml). In successive heat treat
ments of ACH-2 cells, although the first resulted in a iower cell % viablity and number it
induced a higher level of HIV p24 (2025 pg/ml) compared to untreated cells (1 313 pg/ml).
However, the second heat treatment further reduced cell % viability and number, and
resulted in lower HIV I p24 (333 pg/ml) compared to untreated ACH-2 cells (1020 pg/ml).
When the successive heat treatments were increased to 2 hr, this produced an even
greater reduction of cells and HIV- I p24 (83 pg/ml).
Conclusions: In vitro hyperthermia treatment shows selective killing of HIV- I chronic infect
edT lymphocytes that results in lowering the viral load.These in vitro studies provide a scientific basis for treatment protocols in the current hyperthermia clinical trials of AIDS
patients.
M.H. Lee, HarborUCLA Medical Center, 1124 W Carson St., Bldg E-6 Torrance, CA 90502.
USA Telephone:[phone] Fax:[phone] email:[email protected]
Mo.A.1097
OPEN-LABEL SORIVUDINE (BV-araU) FOR THE TREATMENT OF VARICELLA
ZOSTER VIRUS (VZV) INFECTIONS IN PEDIATRIC AIDS PATIENTS WHO FAILED
OR WERE INTOLERANT OF INTRAVENOUS ACYCLOVIR
Oshana, Scott*, Brennan-Rowe N*, Denisky G*,Thomis J*, DeHertogh D*, Srnaldone L*,
Freifeld A**. *Bristol-Myers Squibb, Wallingford, CT US: **National Institute of Health,
Bethesda, MD US
Objective: To determine efficacy and safety of oral sorivudine (BV-araU)i in pediatric AIDS
patients with cutaneous, visceral or ocular VZV disease who had failed or were intolerant of
intravenous acyclovir.
Methods: Patients were evaluated at entry during treatment and at post-treatrment.The
oral sonvudine dose approximated I mg/kg/day: In patient,- with active VZV disease response
was assessed as follows: resolved (complete healing of lesions followed by therapy discon
tinuation), suppression (Healing of lesions with cortinued therapy), improved (some lesions
persist), no change, progression of lesions despite therapy and unable to determine. In
those who entered for suppression of VZV disease. time to recurrence was the endpoint.
Results: Thirteen pediatric AIDS patients, aged 2 to I 4 years. were enrolled:; I0 were
female, 3 male. Median CD4 count at entry was 3 cells/mmn3.The dose of oral sorivudine
ranged from 5 to 30 mg/day and treatment duration ranged from 1I0 days to I I months.
Of the 6 patients (5 cutaneous, I myelitis) treated for active VZV disease, I had resolution,
3 had improvement, I had progression of disease and I was unable to determine. Of the 7
patients treated for suppression of VZV disease, timre to recurrence was > I0 months in I,
>4 months in I, >1I month in 2 > 10 days in 2 and I was free ofVZV but was discontinued on day 55 because of elevated transaminases. Seven patients had sorivudine therapy
interrupted due to laboratory abnormalities, 2 because of neutropenia (I only had normal
tests at baseline) and 5 because of abnormal transaminases (I had normal tests at baseline). One patient died < 30 days and 2 other deaths occrred > 30 days after the last
dose of sorivudine. All deaths were attributed to complica'ions of HIV disease.
Conclusions: Oral sorivudine appears to be of beneft in pediatric patients with advanced
AIDS and VZV disease who had failed or were intolerant of intravenous acyclovir
Scott Oshana - 5 Research Parkway Wallingford, CT 06492 - US
Telephone: [phone] FAX: [phone]
Mo.A. 1098
THE COMPROMISED MEMBRANE:A TARGET FOR HIV DRUG DESIGN
Paul Gatti, Allyson Haislip M, Douglas Plymrale R, Alla Makutonina, Bongkun Choi, Robert
Garry F. Tulane University School of Medicine, New Orleans, LA. USA
Objective: HIV infection alters permeability of the plasma membrane to ions and other
small molecules.The oblective of this study was to identify molecules that can selectively
enter HIV infected cells and inhibit progeny virus expression.
Methods: CD4+ T-Iymphoblastoid cells (RH9) and peripheral blood mononuclear cells
(PBMC) were infected with HIV- I, then incubated in medium containing hygromycin B. an
aminoglycoside protein synthesis inhibitor that is normally inpermeable to mammalian cells
at pM concentrations. HIV induced cytopathic effects were quantitated by photomicroscopy
and HIV production was measured by antigen capture EIA. Protein synthesis was measured
by 35S methionine-cysteine (trans-label) radiolabeling.
Results: HIV specihc cytopathic effects were dramatically reduced in the presence of pM
concentrations of hygromycin B. Hygromycin B also inhibited HIV production in a dosedependent manner during acute infection. Selective inhibition of protein synthesis by
hygromycin B was demonstrated in H9 cells and PBMC acutely infected by HIV as corn
pared to mock infected cells. Hygromycin B also reduced -IV production in RH9 cells persistently infected with HIV by selectively inhibiting protein synthesis.
Conclusions: These results demonstrate HIV alters the plasma membrane permeability of
infected cells to hygromycin B, rendering them more sensitive to this translational inhibitor