Infectious Disease Research Department,
Serquest/Southern Research Institute, Frederick, Maryland
217011; Biochemistry Department,
Southern Research Institute, Birmingham, Alabama
352052; and Developmental
Therapeutics Program, Division of Cancer Treatment, Diagnosis and
Centers, National Cancer Institute, Bethesda, Maryland
208923
Received 30 October 1998/Returned for modification 8 March
1999/Accepted 13 May 1999
(+)-Calanolide A (NSC 650886) has previously been reported to be a
unique and specific nonnucleoside inhibitor of the reverse transcriptase (RT) of human immunodeficiency virus (HIV) type 1 (HIV-1)
(M. J. Currens et al., J. Pharmacol. Exp. Ther., 279:645-651, 1996). Two isomers of calanolide A, (
)-calanolide B (NSC 661122; costatolide) and ()-dihydrocalanolide B (NSC 661123;
dihydrocostatolide), possess antiviral properties similar to those of
calanolide A. Each of these three compounds possesses the phenotypic
properties ascribed to the pharmacologic class of nonnucleoside RT
inhibitors (NNRTIs). The calanolide analogs, however, exhibit 10-fold
enhanced antiviral activity against drug-resistant viruses that bear
the most prevalent NNRTI resistance that is engendered by amino acid change Y181C in the RT. Further enhancement of activity is observed with RTs that possess the Y181C change together with mutations that
yield resistance to AZT. In addition, enzymatic inhibition assays have
demonstrated that the compounds inhibit RT through a mechanism that
affects both the Km for dTTP and the
Vmax, i.e., mixed-type inhibition. In fresh
human cells, costatolide and dihydrocostatolide are highly effective
inhibitors of low-passage clinical virus strains, including those
representative of the various HIV-1 clade strains, syncytium-inducing
and non-syncytium-inducing isolates, and T-tropic and monocyte-tropic
isolates. Similar to calanolide A, decreased activities of the two
isomers were observed against viruses and RTs with amino acid changes
at residues L100, K103, T139, and Y188 in the RT, although costatolide
exhibited a smaller loss of activity against many of these
NNRTI-resistant isolates. Comparison of cross-resistance data obtained
with a panel of NNRTI-resistant virus strains suggests that each of the
three stereoisomers may interact differently with the RT, despite their
high degree of structural similarity. Selection of viruses resistant to
each of the three compounds in a variety of cell lines yielded viruses with T139I, L100I, Y188H, or L187F amino acid changes in the RT. Similarly, a variety of resistant virus strains with different amino
acid changes were selected in cell culture when the calanolide analogs
were used in combination with other active anti-HIV agents, including
nucleoside and nonnucleoside RT and protease inhibitors. In assays with
combinations of anti-HIV agents, costatolide exhibited synergy with
these anti-HIV agents. The calanolide isomers represent a novel and
distinct subgroup of the NNRTI family, and these data suggest that a
compound of the calanolide A series, such as costatolide, should be
evaluated further for therapeutic use in combination with other
anti-HIV agents.
 |
INTRODUCTION |
The structurally diverse class of
nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) includes
compounds which are among the most potent anti-human immunodeficiency
virus (anti-HIV) agents identified (for a review, see references
17 and 18). The therapeutic
utility of these anti-HIV compounds, however, is severely compromised
by the rapid appearance of drug-resistant virus isolates in patients
(26). Similarly, growth of HIV in cell culture in the
presence of the NNRTIs rapidly selects for drug-resistant viruses
(26). The high specificity of the interaction of these
compounds at the hydrophobic nonnucleoside binding site on the HIV-1 RT
results in the ability of single amino acid changes in the NNRTI
binding pocket to reduce or eliminate the inhibitory activity of the
compound (14, 15, 20, 29). Amino acid changes in the RT
which affect drug sensitivity include A98G, L100I, K101E, K103N,
V106A, V108I, E138K, T139I, Y181C, Y188C, G190A, F227L, and P236L
(26).
The effective use of NNRTIs in patients is dependent on the definition
of appropriate combinations of agents which will prevent or retard the
selection of drug-resistant viruses or which will result in the
selection of drug-resistant virus isolates in which mutation of
critical amino acid residues renders the RT less fit to support virus
reproduction. NNRTIs may also be useful as part of a combination
anti-HIV strategy with a highly potent NNRTI and additional anti-HIV
type 1 (anti-HIV-1) agents in therapy-naive patients. The potential for
the therapeutic use of NNRTIs in patients has recently been reviewed
(17, 18). Results of clinical trials with nevirapine as a
component of a three-drug regimen in patients have highlighted the
possible benefits of the development of additional novel or more potent
NNRTIs (13). Although the use of NNRTIs alone is not
warranted, these compounds may be used in other ways, for example, as
topical microbicides for the prevention of the sexual transmission of
HIV, for postexposure prophylaxis, or as a first-line therapeutic
option for the treatment of patients without the elimination of future
therapy options.
It has become increasingly apparent that the HIV-1-specific inhibitor
class of compounds is quite diverse (2, 3, 19). This
diversity may allow the identification of therapeutically beneficial
combinations of anti-HIV compounds, including the NNRTIs. A variety of
structurally distinct NNRTIs have been identified through the efforts
of the National Cancer Institute's (NCI's) high-capacity anti-HIV
drug screening program (1, 6-11, 23, 24). Evaluation of the
activity of calanolide A against RT and NNRTI-resistant viruses, as
well as detailed evaluation of the kinetics of RT inhibition, has
suggested that calanolide A represents a novel new class of
HIV-1-specific inhibitor (7, 16, 22, 23). Like other members
of the NNRTI family of inhibitors, calanolide A exhibits potent
anti-HIV activity in established and fresh human cells and
synergistically inhibits HIV when it is used in combination with
nucleoside anti-HIV compounds. NNRTI-resistant virus isolates were
determined to possess a unique profile of sensitivity to the compound,
and the compound predominantly selected for drug-resistant virus
isolates with a previously unknown mutation at amino acid residue 139 (T139I) (7). These data, as well as those presented here,
indicate that the calanolide class of NNRTIs may interact in a
mechanistically different fashion with the HIV-1 RT and therefore may
be useful inhibitors when used in combination with other anti-HIV agents, including other NNRTIs.
| |
MATERIALS AND METHODS |
Cells and viruses.
The established human cells,
laboratory-derived virus isolates (including drug-resistant virus
isolates), and low-passage clinical virus isolates used in these
evaluations have previously been described in detail (11,
12). These cells were maintained in RPMI 1640 medium supplemented
with 10% fetal bovine serum, 2 mM glutamine, penicillin (100 U/ml),
and streptomycin (100 µg/ml). Fresh human cells were obtained from
the American Red Cross (Baltimore, Md.).
Materials.
(+)-Calanolide A, ()-calanolide B
(costatolide), and ()-dihydrocalanolide B (dihydrocostatolide) (NSC
654086, NSC 661122, and NSC 661123, respectively) were obtained from
the Developmental Therapeutics Program, Division of Cancer Treatment,
Diagnosis and Centers, NCI. The structures of each of these molecules
are provided in Fig. 1. Crystalline stock
materials were stored at 70°C and were solubilized in 100%
dimethyl sulfoxide. All stocks were diluted at least 400-fold prior to
performance of drug susceptibility assays. All of the compounds used in
these studies were obtained from NCI, including zidovudine (AZT; NSC
602670), didanosine (ddI), zalcitabine (ddC), lamivudine (3TC),
stavudine (d4T), nevirapine, E-EBTU (8), diphenylsulfone
(25), UC10 and UC781 (5), KNI272, and resobene
(21).
2',5'-Bis-O--(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"-oxathiole-2", 2"-dioxide-
-D-pentofuranosyl (TSAO) and
-APA were obtained from Jan Balzarini and Janssen Pharmaceuticals,
respectively. Enzyme-linked immunosorbent assay plates were purchased
from Coulter Immunotech (Hialeah, Fla.). Materials required for the
performance of RT inhibition assays and anti-HIV assays and for the
growth and maintenance of established and fresh human cells have been
described previously (4, 11).
Antiviral and cross-resistance assays.
The inhibitory
activities of the compounds against HIV were evaluated as described
previously (11) by microtiter anti-HIV assays with CEM-SS
cells or fresh human peripheral blood mononuclear cells (PBMCs); these
assays quantify the ability of a compound to inhibit HIV-induced cell
killing or HIV replication. Quantification was performed by the
tetrazolium dye XTT assay (CEM-SS, 174×CEM, MT2, and AA5 cell-based
assays), which is metabolized to a colored formazan product by viable
cells, RT assay (U937- and PBMC-based assays), and/or p24 enzyme-linked
immunosorbent assay (monocyte-macrophage assays). Antiviral and
toxicity data are reported as the quantity of drug required to inhibit
virus-induced cell killing or virus production by 50%
(EC50) and the quantity of drug required to reduce cell
viability by 50% (IC50).
Anti-HIV assays with drug combinations.
Analysis of drug
combinations was performed with CEM-SS cells acutely infected with the
IIIB strain of HIV-1 as described previously (11) by the
anti-HIV assay methodology described above. Statistical evaluations
were performed with MacSynergy II software (28). The results
of the assays with drug combinations are presented three dimensionally
for each combination concentration, yielding a surface of activity
extending above (synergy) or below (antagonism) the plane of additive
interaction. The volume of the surface is calculated and expressed as a
synergy volume (square micromolar percent) calculated at the 95%
confidence interval (28). For these studies, synergy is
defined as drug combinations with synergy volumes of greater than 50 µM2%. Slightly synergistic and highly synergistic
anti-HIV activities have been defined as synergy volumes of 50 to 100 and >100 µM2%, respectively. Synergy volumes of between
0 and 50 µM2% are considered additive, and synergy
volumes of less than 0 µM2% are considered antagonistic.
Selection of drug-resistant strains.
Resistant virus
isolates were selected in cell culture by serial passage of the IIIB
strain of HIV-1 in CEM-SS, MT2, H9, or AA5 cells in the presence of
increasing concentrations of antiviral compound. Identical selections
were performed with the clinical isolate ROJO in fresh human peripheral
blood cells. The initial selection was performed with two times the
EC50 of the compound as determined by the microtiter
anti-HIV assay. With successive passages the drug concentration was
increased twofold to enhance the selective pressure on the virus. The
final drug concentrations used in the selection of viruses resistant to
calanolide A, costatolide, and dihydrocostatolide were 2.7, 5.4, and
5.4 µM, respectively. Upon selection of a drug-resistant virus
isolate, cross-resistance testing was performed by the methods
described above for the performance of antiviral assays. Resistance has
been defined in this study as a greater than fivefold increase in the
EC50 compared to the concentration of the compound with
activity against the wild-type (IIIB) isolate. Two drug-resistant
strains were selected exactly as described above by beginning with two
times the EC50 of each drug and increasing the drug
concentration twofold at each passage.
Analysis of RT mutations.
Resistance-engendering mutations
were identified by the direct sequencing of PCR products amplified from
the RT region of proviral DNA obtained from acutely infected CEM-SS
cells. A PCR-amplified product was prepared from the first 750 bp of
the RT gene with the A'-NE1' primer set. Single-stranded, biotinylated
DNA was purified from this product with avidin-conjugated
supraparamagnetic beads (Dynabeads, M280; Dynal). Direct sequencing
with dideoxynucleotide chain termination was performed with each of the
appropriate G, A, C, and T dideoxy sequencing mixes with the Sequenase
T7 polymerase kit (United States Biochemicals) with 7-deaza-dGTP to
resolve compression artifacts, -33P, and five sets of
overlapping primers obtained with a primer analysis software package
(Oligo 4.04; National Bioscience, Inc.). Evaluation of the resulting
sequencing gels was accomplished with Millipore's automated gel
scanning system. RT sequences from drug-resistant isolates were aligned
with the parental wild-type HIV-IIIIB and HXB2 RTs with
Millipore and Bioimage's software run on a Sun Microsystems Sparc 10 Station microcomputer.
RT inhibition assays.
Analysis of the drug sensitivity of RT
containing defined amino acid substitutions was performed as described
previously (4). Evaluation of the activities of the
compounds with homopolymer and heteropolymer templates was performed as
described previously (7). For the Ki
studies, HIV-1 RT activity was measured in 50-µl reaction mixtures
containing 50 mM Tris (pH 8.0), 50 mM KCl, 10 mM MgCl2, 4 mM 2-mercaptoethanol, 3% glycerol, 1 mg of bovine serum albumin per
ml, 6.6 µg of primed 16S rRNA from Escherichia coli per
ml, 10 µM dATP, 10 µM dCTP, 10 µM dGTP, and various
concentrations of [3H]dTTP (27). Purified
recombinant HIV-1BH10 RT was used for these experiments
(30). The Km for dTTP was 1.67 µM,
and for the rRNA template it was 0.66 µg/ml.
| |
RESULTS |
Range of activity and mechanisms of action of calanolide
isomers.
The activities of calanolide A, costatolide, and
dihydrocostatolide were evaluated with established and fresh human
cells infected with both laboratory-derived and clinical strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV). Nevirapine (an
NNRTI) and AZT (a nucleoside RT inhibitor [NRT]) were used as
positive anti-HIV control compounds. These data are summarized in Table
1. Each of the compounds was determined
to be active against HIV-1 in the T-cell lines CEM-SS, H9, and MT2, in
the B-cell line AA5, in the monocytic line U937, and in the
T-cell-B-cell hybrid line 174×CEM. The activities (EC50)
of the compounds against HIV-1 ranged from 0.08 to 0.5, 0.06 to 1.4, and 0.1 to 0.8 µM for calanolide A, costatolide, and
dihydrocostatolide, respectively. None of the compounds was found to
have activity against HIV-2 or SIV. AZT and nevirapine exhibited the
expected levels of activity in each of the cell lines.
Each of the compounds was also evaluated in fresh human peripheral
blood leukocytes and monocytes-macrophages infected with a variety of
low-passage clinical virus isolates (Table 1). The compounds were
determined to be equally active against clinical virus strains,
including viruses representative of the various HIV-1 clades (clades A
through F) found worldwide, syncytium-inducing and
non-syncytium-inducing viruses, and T-tropic and monocyte-macrophage tropic viruses. The toxicity of each of the compounds was determined in
parallel in each cell line. The toxic concentrations
(IC50s) of each of the three compounds were reached at
approximately 10 to 20 µM for all cell lines, yielding a therapeutic
index (which is equal to IC50/EC50) of
approximately 100 to 200 for each of the compounds.
Mechanistic assays indicated that each of the calanolide analogs
inhibited RT when they were evaluated in a biochemical RT inhibition
assay with either a homopolymeric poly(rC)-oligo(dG) or a
heteropolymeric rRNA template-primer assay system. The compounds did
not inhibit virus attachment, integrase, protease, or cell-cell fusion
(data not shown). Also, the compounds did not inhibit late-stage virus
reproduction events on the basis of their inability to suppress virus
production in chronically infected cells (data not shown). Limited
pretreatment assays demonstrated that compound had to be continuously
present in order to be effective.
Evaluation of the abilities of the compounds to inhibit the enzymatic
activity of purified RT was performed in order to define the modes of
action of the calanolide analogs. When the rRNA template was present at
saturating concentrations and the dTTP was present at unsaturating
concentrations, costatolide and dihydrocostatolide inhibited the enzyme
with IC50s of 0.003 to 0.01 µM. Inhibition could be
reversed by increasing the dTTP concentration in the assay 10-fold
(IC50s, >1 µM) at either saturating or nonsaturating concentrations of template. Consistent with the results reported by
Currens et al. (16) for calanolide A, costatolide and
dihydrocostatolide inhibited HIV-1 RT by a complex mechanism. At
concentrations of between 0.02 and 0.15 µM, the inhibition
approximates a mixed type of inhibition in which the inhibitor affects
both the Km for dTTP and the
Vmax of the enzyme (data not shown). In addition to this complexity, our data for the highest concentration tested (0.2 µM) suggest that there is a second mechanism of inhibition for both
compounds. Considering that NNRTIs bind in a pocket of the enzyme
outside of the catalytic site, the complexity in the mechanism that the
data demonstrate is not surprising. These data provide inhibition
constants (Ki) for costatolide and
dihydrocostatolide of 0.06 and 0.03 µM, respectively.
Interaction of calanolide isomers with other anti-HIV agents.
Anti-HIV assays were performed with each of the calanolide analogs in
combination with a variety of anti-HIV agents including the NRTIs, AZT,
ddC, ddI, 3TC, and d4T, the NNRTIs UC10, UC781, and diarylsulfone, the
protease inhibitors ritonavir, indinavir, nelfinavir, and saquinavir,
and the surface-active attachment inhibitor resobene. A summary of the
data obtained with combinations of anti-HIV agents from the MacSynergy
II evaluations is presented in Table 2.
Data obtained from the MacSynergy II evaluations are presented in
synergy volume units (square micromolar percent) at the 95% confidence
interval as described above. Each of the compounds exhibited a
synergistic interaction with the NRTIs, with synergy volumes ranging
from approximately 100 to over 500 µM2%. The
interactions of the calanolides in combination with ritonavir and
saquinavir were also determined to be synergistic. Additive interactions were obtained with the compounds in combination with other
NNRTIs (with the exception of UC781), the attachment inhibitor resobene, and the protease inhibitors indinavir and nelfinavir. Costatolide in combination with the nucleoside analogs generally exhibited the highest levels of synergy. Calanolide A was less synergistic than costatolide, and dihydrocostatolide exhibited lower
levels of synergy than calanolide A. Antagonistic anti-HIV drug
interactions or synergistic toxicity was not observed with any of the
drug combinations evaluated.
Sensitivities of calanolide isomers to NNRTI-resistant
viruses.
The three calanolide stereoisomers and the positive
control compounds nevirapine and AZT were evaluated for their antiviral activities against viruses selected in cell culture for resistance to a
variety of NNRTIs (Table 3). Virus
isolates containing an L100I, K103N, or Y188H amino acid change in the
RT were resistant to the antiviral effects of the compounds. Calanolide
A also had lower levels of activity when it was used to challenge
viruses that possess either the V108I or the T139I amino acid change. Costatolide and dihydrocostatolide were not adversely affected by the
V108I change, and costatolide remained partially active against viruses
with the T139I amino acid change in the RT.
A striking enhanced activity of each of these compounds against mutant
virus with the Y181C amino acid change was observed. This enhanced
anti-HIV activity was absent against a mutant containing both the Y181C
and the K103N changes. However, viruses with these changes remained
sensitive to the calanolide analogs, whereas viruses with the K103N
amino acid change alone were completely resistant to the compounds.
Calanolide A remained active against viruses that possessed the M184I
and P236L amino acid changes, while slight but reproducible lower
levels of activity were observed with costatolide and
dihydrocostatolide when they were used to challenge these isolates.
Confirmation of the results of these assays was obtained by evaluating
the activity of each compound against viruses or purified RT with
single amino acid changes introduced by site-directed mutagenesis. As
noted above, viruses with the L100I, K101E, K103N, T139I, and Y188C
amino acid changes were resistant to the calanolide analogs (Table
4). The calanolide A-specific amino acid
change T139I resulted in a 4- to 20-fold loss of activity with
calanolide A and dihydrocostatolide but had a much greater effect on
costatolide (60-fold loss of activity). Enhanced sensitivity was
detected with the Y181C amino acid change in the RT, and further
enhancement (two- to threefold) was observed upon introduction of AZT
resistance-engendering mutations into the RT with the Y181C mutation.
As expected, each of the compounds was inactive against purified HIV-2
RT.
Selection and characterization of drug-resistant virus
isolates.
Viruses resistant to the calanolide isomers were
selected in CEM-SS cells infected with the IIIB strain of HIV-1 after a
short time of culture in the presence of increasing concentrations of each compound (four to six passages) (Table
5). Selection with calanolide A yielded a
virus isolate with the T139I amino acid change in the RT. This virus
was >100-fold more resistant than the wild type to calanolide was but
less resistant to costatolide and dihydrocostatolide. Selection with
costatolide yielded a virus that possessed both T139I and L100I amino
acid changes and that was highly cross-resistant to each of the
calanolide analogs. A virus strain selected with dihydrocostatolide
contains the L100I amino acid change. This virus was >100-fold more
resistant than the wild type to costatolide and dihydrocostatolide but
was less cross-resistant to calanolide A.
To examine the influence of the cell type in selecting resistant virus,
costatolide was used to treat a variety of different established human
cell lines and fresh human cells infected with HIV-1IIIB. A
variety of amino acid changes were observed in viruses isolated from
different cell types. In CEM-SS cells, the resistant isolate contained
both the T139 and L100I amino acid changes. In both MT2 and AA5 cells,
costatolide selected for virus that possessed the Y188H amino acid
change. In H9 cells, the resistant virus isolate possessed an L187F
amino acid change. Each of these substitutions was determined to yield
high-level resistance to costatolide (>27-fold greater resistance than
the wild type). Fresh human cells infected with a low-passage clinical
strain (ROJO) were also used to select for a costatolide-resistant
isolate by the methodology identical to that described above. In this experiment the virus selected contained the L100I amino acid change.
Finally, calanolide A and costatolide were used in combination with a
second NNRTI in strategies for the isolation of drug-resistant virus.
The compounds chosen for these assays with drug combinations were
selected on the basis of their ability to effectively inhibit the
replication of calanolide A-resistant viruses, as well as on the basis
of the ability of calanolide A to inhibit drug-resistant viruses
selected by the second compound. The compounds 3TC, diphenylsulfone, E-BPTU, -APA, UC10, TSAO, and diarylsulfone were used in these studies. In each drug combination, a virus strain with a mutation that
conferred resistance to both compounds was obtained in cell culture
(Table 6). These amino acid changes
differed from the changes selected by either compound when it was used
alone in these in vitro selection assays. These viruses possessed
changes in the amino acid sequence of the RT which included L100I,
K101E, V108I, K103N, V106I, and Y188H. On the basis of a direct
comparison of the number of passages required to select for a
drug-resistant virus, the NNRTIs most capable of suppressing virus
reproduction appeared to be a calanolide analog (which was active
against Y181C mutants) in combination with agents that were inhibitory
to viruses with the L100I amino acid change (-APA and
diarylsulfone). With these combinations, selection for viruses with
either K103N or Y188H amino acid changes conferred resistance to both
antiviral agents after approximately 13 to 14 passages in cell culture.
| |
DISCUSSION |
The stereoisomers (+)-calanolide A, ()-calanolide B
(costatolide), and ()-dihydrocalanolide B (dihydrocostatolide) were determined to be highly effective nonnucleoside inhibitors of RT.
Although less potent than many members of the NNRTI class, several
unique features regarding the anti-HIV activities of calanolide compounds have been defined, and these features suggest that the calanolides represent a novel and potentially useful subclass of the
NNRTI family of inhibitors. The major difference between the calanolide
isomers and other NNRTIs involves the activities of the compounds
against viruses that possess the Y181C amino acid change in the RT. In
most cases, the isomers exhibited approximately 10-fold enhanced
activity against these strains. This activity appears to be a result of
a general structural feature of the molecule since ()-calanolide A,
which failed to inhibit wild-type HIV-1, was determined to be active,
albeit at a higher concentration, against the Y181C-containing viruses
(EC50 = 1.5 µM) (4a). This enhanced level
of activity is more pronounced when the Y181C change is present in the
background of an AZT-resistant virus. In addition, although the K103N
amino acid change resulted in the loss of activity of the compounds,
introduction of the K103N change into the Y181C-possessing RT yielded a
virus which remained sensitive to the antiviral activities of all three
compounds. This suggests that the conformational change in the RT
pocket associated with the K103N amino acid change is compensated for
by the Y181C change. Enzymatic data and studies with chimeric HIV-1 and
HIV-2 RT have suggested that calanolide A may bind to two sites on the
RT (16, 22). One of the proposed sites is within the
hydrophobic NNRTI binding pocket, while the second site was determined
to be outside the pocket. Our kinetic data confirm that each of the
calanolides has a complex pattern of interaction with the RT. Our data
are consistent with those reported for calanolide A by Currens et al.
(16).
Sensitivity testing of the calanolide isomers also suggests that fine
specificity differences exist in the interaction of the three isomers
with the RT. For example, although only slightly resistant, costatolide
appears to be reproducibly affected by amino acid changes M184I-M184V
and P236L. Anti-HIV assays with drug combinations also suggest that
differences may exist between the isomers in regard to their
interactions with other compounds for the inhibition of HIV. Of the
three stereoisomers, costatolide exhibited the greatest level of
synergy with each of the nucleoside analogs. Unlike the majority of the
NNRTIs, each of the calanolide analogs was determined to have
synergistic activity with the NNRTI UC781. Most importantly, for
further clinical development of these compounds, all of the anti-HIV
assays performed with combinations of the calanolide isomers failed to
detect either synergistic toxicity or antagonism with respect to virus reproduction.
The selection of drug-resistant isolates in cell culture yielded
important information regarding the interaction of the isomers with the
RT and with other anti-HIV agents. Calanolide A selected for viruses
with a previously unidentified T139I amino acid change in the RT
(7). Costatolide, the only one of the three compounds which
remained active against viruses with the T139I change, selected for a
resistant isolate with both the T139I and L100I amino acid changes or
with the L100I change alone. The calanolide A-resistant virus with the
T139I change was not cross-resistant to any of the other NNRTIs
evaluated. Introduction of the L100I amino acid change, however,
yielded a virus that was highly cross-resistant to a variety of NNRTIs
with the exception of -APA, which remained completely active against
the strain. In addition, the virus with the L100I change exhibited only
10-fold reductions in sensitivity to UC781 and TSAO.
Our drug efficacy, drug combination, and viral resistance data suggest
that costatolide may be superior to calanolide A as an inhibitor of
HIV. Thus, costatolide was used to select for additional resistant
strains in different cell lines in order to define the range of
mutations which might be expected to appear in patient isolates during
therapy with this compound. In addition to the diagnostic T139I change,
a variety of amino acid changes were observed, including L100I, Y188H,
and L187F. Viruses with these changes likely represent subpopulations
of virus which exist in the wild-type virus pool with growth advantage
in the particular cell line used for selection.
In order to further explore the interaction of costatolide with other
NNRTIs in the context of resistant virus selection, compounds were
chosen for use in combination with the calanolide isomers on the basis
of their ability to inhibit the replication of viruses resistant to the
isomers. Resistant virus strains were selected with each of these drug
combinations. The amino acid changes observed in each resistant virus
were different from those selected with the individual compounds.
Selection of calanolide-resistant viruses by using a drug-resistant
strain as the starting material rapidly yielded drug-resistant virus
populations. Use of an AZT-resistant strain (strain G910-6) yielded
viruses with K103N and K122E amino acid changes upon selection with
costatolide and dihydrocostatolide in MT2 cells. The most difficult
selections to perform on the basis of a direct comparison of the number
of passages required to obtain a resistant strain included the
calanolide isomers with compounds that inhibited viruses with L100I
amino acid changes. In these cases, the number of passages required to
select for a resistant strain were approximately double the number of
passages required for other combinations of agents (11 to 14 passages
versus 5 to 6 passages).
Our results demonstrate that the calanolide isomers can be clearly
distinguished from other members of the NNRTI class, and thus, that the
compounds may be useful in combination with other anti-HIV agents.
Nonetheless, resistance to a single calanolide isomer or an isomer in
combination with a second active compound was easily achieved in cell
culture. Thus, the use of two NNRTIs may be ineffective in patients
unless sufficiently high concentrations of the compounds are maintained
to suppress the ability of the virus to replicate. The use of two
NNRTIs in combination will require the addition of a third anti-HIV
compound with a different mechanism of anti-HIV action in order to be
effective. The possibility of using two NNRTIs in a cocktail with a
third NRTI may be clinically useful from the viewpoint of preserving
the possibility of using more potent future therapies with the protease inhibitors.
This work was supported by contract NO1-CM-37818 to the
Southern Research Institute from NCI.
We gratefully acknowledge Larry Ross and Cathi Pyle for technical
laboratory support, Barbara Toyer and Michelle Wenzel for drug
preparation support, and Diana Markle for assistance with the
preparation of the manuscript.
| 1.
|
Bader, J. P.,
J. B. McMahon,
R. J. Schultz,
V. L. Narayanan,
J. B. Pierce,
W. A. Harrison,
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Abstract
Introduction
