Final Program and Oral Abstracts [International Conference on AIDS (8th: 1992: Amsterdam, Netherlands)]

ThA 1503-ThA 1507

TRACK A: BASIC SCIENCE

ThA 1503 NOVEL, LOW MOLECULAR WEIGHT HIV-1 PROTEASE INHIBITORS,
Vaillancourt Marc*,** Vanasse, B*., Cohen, E.** & Sauv6, G.*,
* Institut Armand-Frappier, Laval, Qu6bec, Canada H7N 4Z3.
** Universit de Montrdal, Montreal, Qu6bec, Canada, H3C 3J7
Objective: The catastrophic dimensions of
the HIV-AIDS pandemic has spurred a worldwide quest for new therapeutic agents.
One such therapeutic approach is the use of inhibitors of HIV-protease. Previous
studies has shown that if the protease is catalytically defective and/or inhibited, viral
maturation is blocked, and therefore infection is arrested. With this in mind, we
report here novel inhibitors of HIV-protease.
Methods: The inhibitors were synthesized in a one-step chemical synthesis. These
inhibitors are non-peptidic, low molecular weight (<500 g/mol), difunctionalized
enols of simple amino acids. Kinetics and inhibitory constants (Ki) were determined
by incubating the enzyme with various amount of substrate and inhibitor, and the rate
was measured by High Performance Liquid Chromatography.
Results: The results of our research will be discussed in terms of structure-activityrelationship between the inhibitors and the HIV-protease. These inhibitors showed
mid-nanomolar inhibitory constant (Ki = 425 nM) against HIV-1 protease.
Preliminary biological results using infected MT4 cells will also be discussed.
Conclusion: The inhibition shown by our inhibitors is, to our knowledge, the best
reported anti HIV-protease activity for molecules that does not contain at least one
peptide linkage.
Vaillancourt Marc, Institut Armand-Frappier, Universitd du Qudbec, 531 boul
des Prairies, Laval, Qu6bec, Canada H7N 4Z3.
Telephone: [phone] ext.422, FAX: [phone]
ThA 1 5 IN VITRO ISOLATION AND PARTIAL CHARACTERIZATION OF HIV VARIANTS WITH
ThA 1 5 REDUCED SENSITIVITY TO C-2 SYMMETRICAL INHIBITORS OF HIV-1 PROTEASE.
Otto Michael., J.; Garber,S.; Winslow,D.; Cheng,Y-S.E.; Reid,C.D.;
Aldrich,P; Jadhav,P.K.; The DuPont Merck Pharmaceutical Company,
Glenolden, PA USA
Objectives: Recent reports have shown that the inhibition of HIV protease results in the
inhibition of infectious virus production in vitro. HIV protease, therefore, appears to be
an attractive target for anti-HIV therapy. Since development of resistance is a serious
concern with any new antiviral, we attempted to isolate variants of HIV with altered
sensitivity to a class of protease inibitors described as C-2 symmetrical diols. A
representative of these is shown below as the target compound.
Methods: H-9 cells were infected with HIV-1(Rf) in the presence of a suboptimal
concentration (0.lug/ml) of the target compound. The virus was allowed to replicate over
five passages with fresh H-9 cells and compound. The compound concentration was increased
to 0.3ug/ml for three passages. Virus which was able to replicate at 5.0ug/ml (five times
the IC90) was plaque purified, passaged in 5ug/ml compound and reisolated by endpoint
dilution. Virus isolates were tested for sensitivity to the target and related compounds
by yield reduction assays.
Results: Five isolates with altered sensitivity were obtained. Each isolate replicated as
well as the parental HIV-1(Rf) in H-9 and MT-2 cells. The concentration of the compound
sufficient to inhibit virus replication by 90% (IC90) ranged from 6.5 - 8.2ug/ml for the
five isolates versus 0.9ug/ml for the parental. Similar differences in sensitivity were
observed with related diol inhibitors of protease. The isolates, however, had no significant reduction in sensitivity to RO 31-8959 (Roche protease inhibitor) or to AZT or DDC.
Conclusions: Variants of HIV-1(Rf) can be obtained in vitro with modestly reduced
sensitivity to certain inhibitors of HIV-1 protease. No isolates were obtained which were
completely insensitive to these compounds. In addition the variants showed no cross
resistance to a structurally different protease inhibitor, RO 31-8959..Ph
0/ O H OH -fO H
L.N N o o..,NN No
H 0O OH H 0
Michael J. Otto, Ph.D. The DuPont Merck Pharmaceutical Company
500 S. Ridgeway Avenue, Glenolden, PA 19036 USA
Telephone [phone] FAX No. [phone]
ThA 1507 DISCOVERY OF POTENT, ORALLY BIOAVAILABLE INHIBITORS OF HIV
PROTEASE. Norbeck. Daniel; Kempf, D.; Marsh, K.; Paul, D.; Knigge,
M.; Vasavanonda, S; Clement, J.; Kohlbrenner, W.; Sham, H.;
Wideburg, N.; Plattner, J. Abbott Laboratories, Abbott Park, IL.
Objectives: The production of infectious HIV requires specific processing of the
gag and gag-pol polyproteins by a virally encoded aspartic protease. Inhibitors of
the HIV protease therefore have attracted intense interest as potential drugs for
the treatment of AIDS, but clinical evaluation of these agents has been hampered by
their extremely poor oral bioavailability. We have overcome this limitation by the
systematic chemical modification of C2 symmetric transition state analogues.
Methods: Inhibition of HIV-1 protease was determined using the fluorogenic
substrate DABCYL-S-Q-D-Y-P-I-V-Q-EDANS. In vitro anti-HIV activity was determined
by a CPE reduction assay in MT4 cells, or, in the case of patient isolates, by a p24
antigen assay in primary PBLs. Plasma samples were analyzed by HPLC.
Results: Non-peptidic lead compounds were identified which exhibited Ki's against
the purified protease between <1-5 nM and ED50's against the replication of HIV-13B
in MT4 cells between 1-6 pM. One of these compounds, A-80164, achieved peak plasma
levels of 22 pLM following a 50 mg/kg dose in rat. Chemical elaboration of this lead
gave rise to an increasingly potent and growing class of orally bioavailable
compounds. A-80987, with a Ki of 0.25 nM, inhibited the replication of laboratory
strains of HIV-1 in MT4 cells at an average ED50 of 0.13 [IM. A-80987 exhibited
comparable activity against virus obtained directly from AIDS and ARC patients,
against HIV-2, and against AZT resistant strains. Following a 10 mg/kg oral dose,
peak plasma levels of A-80987 in rat, monkey and dog ranged from 0.99 to 4.21 NM.
The oral bioavailability of A-80987 ranged from 13 to 26%, while the terminal
elimination half-life of a 5 mg/kg IV dose ranged from 1.5 to 2.0 hours.
Conclusions: The HIV protease inhibitor A-80987 possesses potent antiretroviral
activity in vitro and good oral bioavailability in three species. This compound may
be useful in the treatment of persons infected with HIV.
Norbeck, Daniel, D-47D, Abbott Laboratories, Abbott Park, IL,
60064, USA, Telephone: [phone], FAX: [phone].
Th66

ThA 1504 SHUT OFF OF HIV REPLICATION IN CHRONICALLY-INFECTED
MACROPHAGES BY AN INHIBITOR OF HIV PROTEASE
PGrno Carlo-F*; Bergamini A*; Capozzi M*; Milanese M*; Thaisrivongs S**; Zon
G***; Rocchi G*; Calib R*. *Univ. Rome Tor Vergata, Italy, **Upjohn Co.,
Kalamazoo, MI, USA, ***ABI, Foster City, CA, USA.
Objectives: Assess the efficacy of inhibitors of late stages of HIV replication in chronically-infected
macrophages (HIV-M/M) (the major reservoir of the virus).
Methods: Mature macrophages (M/M) were infected in vitro with HIV, and exposed at various time points
to the protease inhibitor U-75875 (Upjohn) or the antisense anti-rev 28 mer (AS). Results (assessed by
HIV-p24 assay, immunofluorescence (IF), and virus titration) were compared with C8166 T-cell line and
chronically- infected H9 (HIV-H9).
Results: U-75875 inhibits HIV replication in de-novo infected M/M (ID50:0.7.M) and in C8166 T-cells
(ID50:0.031.M). AS also potently inhibits HIV in M/M (ID50:0.07.M) and in C8166 (ID50:0.061tM). >99.9%
inhibition of HIV replication was achieved by 10LM U-75875 in HIV-M/M (ID50:3p1M). Moreover, no
virus production was detected up to 30 days after drug treatment IDo5 of U-75875 in HIV-H9 was 0.24tM.
No cell toxicity was detected at any drug concentration tested. Virus titration shows that 10pM U- 75875
induces >5 logs decrease of virus production in HIV-M/M. AS substantially inhibits HIV replication in M/M
if added 5 days after virus challenge (91% inhibition by 25j1M AS), while its activity substantially decreases
in HIV-M/M and in HIV-H9 (<25% HIV inhibition by 25MM AS). Similar pattern of virus antigen expression
(by IF) compared to control HIV-M/M was detected in U-75875-exposed HIV-M/M, even in the absence
of production of infectious particles. Ten gtM AZT and 1,000 U/ml alpha-interferon (used as control drugs)
inhibited HIV replication in de-novo infected M/M, while both had no effect in HIV-M/M and in HIV-H9.
Conclusions: U-75875 is the most potent inhibitor of HIV replication in HIV-M/M tested in our models.
No decrease in antigenicity of HIV-M/M treated with U-75875 was observed, thus it is conceivable that a
partially restored immune system may contribute to the destruction of infected but non-producing HIVM/M. (Supported by Grants of National Research Council and Ministry of Health, Italy).
Carlo-Federico Perno, MD. Dept of Experimental Medicine,
University of Rome Tor Vergata, Via Orazio Raimondo, Rome, Italy
Ph. No. 0039-6-72592118 - Fax No. 0039-6-7234793
ThA 1506 HIV-1 (RF) VARIANTS WITH A SINGLE AMINO ACID SUBSTITUTION
AT THE PROTEASE (VAL82 TO ALA) HAVE A REDUCED SENSITIVITY
TO C-2 SYMMETRICAL INHIBITORS OF HIV-1 PROTEASE.
Patterson. Catherine E.; Cheng, Y.-S. E.; Buckery, R.; Otto, M. J.; and
Winslow, D. L. The DuPont Merck Pharmaceutical Company, Wilmington,
DE 19880-0328 USA
Objectives: We have isolated HIV-1 (Rf) variants that have a reduced susceptibility to several C-2
symmetrical inhibitors of the HIV protease. Molecular cloning of the viral protease gene and
characterization of its sequence is our first attempt toward an understanding of the resistant
properties of these viral variants.
Methods: DNA samples extracted from infected cells were used as templates for polymerase chain
reactions (PCR) to amplify the N-terminal coding sequence of the pol gene (nt 1621-2067). The
amplified DNAs were sequenced either directly from PCR-amplified samples or after molecular
clonings. Cloned DNAs, which carry both protease and its N-terminal flanking sequences, were
inserted into pET3AM for the expression of HIV-protease. Both autoproteolytic and transcleavage activities of the expressed protease were measured as previously reported.
Results and Conclusion: A single-point mutation of "T" to "C" at nt 2015 was found in each of the
five resistant isolates, whereas, a "T" was present in this position of the sensitive (HIV-1 Rf)
isolate. This mutation results in a change from a valine codon (GTC) to an alanine codon (GCC).
The mutant Ala82-protease is able to participate in the initial autoproteolytic cleavage that liberates
the mature protease from the pol polyprotein. The yield of virus containing the Ala82 mutation in
productive infection is comparable to that of wild type. These observations suggest that the
Ala82-protease has a substantial level of protease activity.
Patterson, Catherine E., The DuPont Merck Pharmaceutical Company,
Wilmington, DE, 19880-0328, USA, Telephone: [phone]

NOTES