Taking advantage of a standard assay on mouse LM cells (murine
fibroblast-like cells), we found that several diaminic carbonates, a
new class of organic compounds synthesized in our laboratories, were
able to inhibit human tumor necrosis factor 
(huTNF)-induced cytotoxicity in a dose-dependent manner. Structure-function
relationship studies indicated precise structural requirements for
compounds being active as huTNF inhibitors. ITF1779, one of the most
active compounds in inhibiting huTNF-induced cytotoxicity, was
selected for further studies. In vitro experiments showed
that ITF1779 inhibited not only huTNF-induced cytotoxicity on LM
cells but also another response of the same cells,
interleukin-1-induced interleukin-6 production. Receptor-binding
studies performed under nonequilibrium conditions and morphologic
evidence of vacuole formation in cells treated with high concentrations
of ITF1779 showed that the effects were strikingly similar to those of
chloroquine, a lysosomotropic agent. Consistent with a mechanism of
action of diaminic carbonates closely matching that of chloroquine are some structural similarities between the two classes of compounds, in
particular their both being diprotic weak bases. Moreover, ITF1779 was
shown to be active in vivo because it afforded protection against lipopolysaccharide-induced shock in mice, a systemic
inflammatory response crucially dependent on tumor necrosis factor
production.
 |
Introduction |
Tumor necrosis factor
(TNF)
is an inflammatory cytokine that plays a pivotal role in the host
defense against pathogens (Echtenacher et al., 1990
; Nakano
et al., 1990; Smith et al., 1990; Williams
et al., 1990). However, in case of chronic or acute systemic
or localized overproduction, TNF has been shown to lead, in
conjunction with other mediators of the inflammatory response, to a
large number of pathologic conditions. Sepsis syndrome, cachexia, cerebral malaria and rheumatoid arthritis are but a few examples in
which TNF has been shown to play a significant pathogenic role (Grau
et al., 1987; Oliff et al., 1987; Tracey et
al., 1986; Brennan et al., 1992). These findings have
fostered significant efforts to discover drugs able to inhibit TNF
in the hope of their having therapeutic application in inflammatory
disease states shown to be crucially dependent on TNF
overproduction. Research activity along these lines led to the
identification of several compounds able to inhibit, with different
degrees of selectivity, either the production (Moreira et
al., 1993; Han et al., 1990; Lee et al.,
1994) or the activity of TNF (Alzani et al., 1993; Tracey
et al., 1987). Results from clinical trials with
anti-huTNF monoclonal antibodies support the idea that approaches
aimed at blocking huTNF can lead to significant therapeutic
successes (Chikanza and Fernandes, 1996).
In the present report, we describe a class of diaminic carbonates that
were synthesized in our laboratories and some members of which were
found to inhibit huTNF-induced cytotoxicity in the course of a
screening program that had been established to find compounds with
anti-huTNF activity. A compound (ITF1779) that scored as one of the
most potent in the huTNF cytotoxicity assay was investigated in
detail. Results showed that its inhibitory effect was not specific for
huTNF but rather extended to at least one other response that has
been investigated in the same cell line, IL-1-induced IL-6 production.
Moreover, experiments performed to elucidate its mechanism of action
allowed us to conclude that it had effects closely matching those of
chloroquine (fig. 1), a lysosomotropic
agent (Mackenzie, 1983) that bears some structural resemblance to
diaminic carbonates in being a hydrophobic diamine. In in
vivo experiments, ITF1779 was shown to afford significant protection from lethality against LPS-induced shock in mice, a systemic
inflammatory response crucially dependent on TNF production.
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Fig. 1.
Structural requirements for diaminic carbonates
being optimally active in inhibiting huTNF-induced cytotoxicity.
Chloroquine and azaspirane (SK&F-106615) formulas.
|
|
| |
Materials and Methods |
Diaminic carbonates.
Diaminic carbonates were prepared using
a new methodology for the synthesis of both symmetrical and
asymmetrical compounds starting from the corresponding aminoalcohols
(Bertolini, G. et al., manuscript in preparation). All
compounds were identified by 1H nuclear magnetic resonance
(NMR), 13C-NMR, mass spectrometry and infrared
spectroscopy, and the purity of each batch was determined by gas
chromatography and elemental analysis. Diaminic carbonates are
hygroscopic, stable oil or low-melting solid (e.g., ITF1779
melting point = 95-98°C) and are stable for at least 30 days at
room temperature in aqueous solution.
Materials.
The following drugs and chemicals were used in
this study: huTNF (WOC, Vaduz, Liechtenstein), iodinated huTNF
(125I-huTNF) (930 µCi/µmol; DuPont NEN, Boston, MA)
and MTT, EGTA, AcD and CHX (Sigma Chemical Co., St. Louis, MO).
Cytotoxicity assays.
LM cells (murine fibroblast-like
cells
ATCC CCL1.2; 1.5 × 104/well) were cultured
overnight in 96-well plates (Costar, Cambridge, MA). After 24 h,
huTNF was added either alone or in combination with the indicated
diaminic carbonates. The plates were then incubated for 48 h at
37°C. Thereafter, cells were pulsed with 40 µl of MTT solution (5 mg/ml in PBS) for 4 h at 37°C; medium was then discarded,
dimethylsulfoxide (200 µl) was added and the O.D. was read at 570 nm.
Percent cytotoxicity was calculated as 100 
(O.D. of LM cells
incubated with huTNF/O.D. of LM cells alone) × 100; percent
inhibition of huTNF-induced cytotoxicity was calculated as 100 [(% cytotoxicity in the presence of the indicated diaminic carbonates/% cytotoxicity with huTNF alone) × 100]. The indicated results of these as well as of all other in vitro assays
reported here were obtained in representative experiments that were
performed at least twice with similar results.
Binding assays.
LM cells (1.5 × 105
cells/well) were cultured overnight in 24-well plates (Costar Italia,
Milan, Italy); the medium was then discarded and the cells incubated
with 125I-huTNF in the absence or presence of a 100-fold
excess of unlabeled huTNF or diaminic carbonates ITF1779 or ITF1493.
Assays were performed at 4°C or 37°C and for different incubation
times, as indicated. Cells were then washed three times with PBS 0.1%
BSA, and cell-bound radioactivity was recovered by solubilization of cells with 1 N NaOH and measured in a 
-counter (LKB-Pharmacia, Uppsala, Sweden). Internalized 125I-huTNF was measured
after treating cells with acidic buffer (0.1 M glycine, pH 3) for 5 min
at 4°C and was defined as the fraction that remained cell-associated
after this treatment. To measure degradation of
125I-huTNF, we treated cell-dissociated
125I-huTNF with 10% TCA and, after centrifugation,
determined the radioactivity of the TCA-soluble and insoluble
fractions. Scatchard plot analyses were performed with the aid of data
analysis program (EBDA, Elsevier Science Publishers, Amsterdam, The
Netherlands).
IL-1-induced IL-6 production.
LM cells (3 × 104 cells/well of 96-well plates) were incubated for
24 h or 48 h with mouse IL-1 (10 ng/ml; Genzyme,
Cambridge, MA) in the absence or presence of ITF1779 or ITF1493. At the
end of the incubation times, supernatants were harvested and IL-6 titers measured by means of a sandwich ELISA (Mouse IL-6 ELISA Kit,
Genzyme) according to the manufacturer's instructions.
LPS-induced shock.
BALB/c female mice (8-12 weeks old,
Charles River, Calco, Italy) were injected i.p. with Salmonella
enteritidis LPS (5 mg/kg, Sigma). Immediately afterwards, some
animals were treated s.c. with the indicated doses of ITF1779.
Dexamethasone (Sigma) was used as a positive control and was
administered i.p. 30 min before LPS at the dose of 30 mg/kg. Survival
was monitored for 7 days. Statistical analysis of survival was
performed by the log-rank test, using the computer software Graph Pad
Prism 2.01 (Intuitive Software for Science, San Diego, CA).
| |
Results |
Screening of diaminic carbonates for inhibition of huTNF-induced
cytotoxicity.
As part of a screening program that had been
established in our laboratories to find compounds with anti-huTNF
activity, we tested diaminic carbonates, a class of organic compounds
synthesized in our laboratories. The first members of this class were
originally synthesized for the salification of the polyanionic molecule
heparin with positively charged counterion molecules in order to
facilitate the passage of heparin through mucous membranes by
neutralizing the negative charges that it carries (Andriuoli et
al., 1990). Several diaminic carbonates were found to be
significantly active in inhibiting huTNF-induced cytotoxicity on LM
cells, the screening assay that had been selected to find active
compounds. We chose this assay to identify compounds with
huTNF-inhibitory activity because of the ease of its execution and
because of the possibility of obtaining a strictly quantitative
measurement of TNF activity (Ruff and Gifford, 1981). Table
1 shows the general structural formula of
diaminic carbonates and the individual formulas of some active and some
inactive compounds of this class. ITF1779, the most active compound
that had emerged during the first screening, and ITF1493, an inactive
compound, were selected for the studies reported here. On the basis of
the knowledge of the structure-activity relationship that we acquired
by screening the first panel of diaminic carbonates, we synthesized and
tested a second panel. Altogether, screening of these compounds enabled
us to establish precise structural requirements for diaminic carbonates
being active as huTNF inhibitors. These are summarized in figure 1.
ITF1779: In vitro activities and mechanism of
action.
Figure 2 shows the effect of
coincubating different doses of huTNF with different doses of
ITF1779. ITF1779 inhibited in a dose-dependent manner the cytotoxic
activity of huTNF on LM cells. The effect was inversely proportional
to the dose of huTNF.
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Fig. 2.
Inhibition of huTNF-induced cytotoxicity on LM
cells by ITF1779. LM cells were incubated with huTNF alone ( ) or
with huTNF and the following doses of ITF1779: ( ), 2 µM; ( ),
6 µM; ( ), 20 µM; ( ), 60 µM. After 48 h cytotoxicity
was measured as described in "Materials and Methods."
|
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Subsequent experiments were performed to clarify the mechanism of
action of ITF1779. In a first experiment, ITF1779 was added to LM cells
at the same time as, or at different times after, huTNF. As shown in
figure 3, the inhibitory effect of
ITF1779 was still detectable, though reduced, when it was added 4 h after huTNF. At later times it was essentially undetectable.
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Fig. 3.
Inhibition of huTNF-induced cytotoxicity on LM
cells by ITF1779 added at different times after huTNF. LM cells were
incubated with huTNF (3 ng/ml) either alone or with huTNF and
ITF1779 (50 µM) added at the same time as huTNF or at different
times after huTNF. Then, 48 h after the addition of huTNF,
cytotoxicity was measured and the percent inhibition of
huTNF-induced cytotoxicity was determined as described in
"Materials and Methods."
|
|
To determine whether ITF1779 could directly interfere with the
interaction between huTNF and TNFR, we tested the ability of ITF1779
to inhibit the binding of huTNF to LM cells under steady-state
conditions (4°C). This assay measures the binding of huTNF to only
one (p55 TNFR) of the two TNFR because huTNF does not interact with
the second TNFR (p75 TNFR) expressed on mouse cells (Lewis et
al., 1991). In parallel experiments, we also tested the effect of
ITF1779 on a soluble, recombinant form of the human p55 TNFR. Both
experiments yielded negative results at all doses tested (data not
shown).
These results, which showed that ITF1779 did not interfere with the
ligand-receptor interaction, prompted us to check the specificity of
the observed inhibitory effect on huTNF-induced cytotoxicity. For
this purpose we studied another biological response of LM cells
unrelated to the first: IL-1-induced IL-6 production (Akira et
al., 1993). In this experiment we also included ITF1493, which had
scored negative in the cytotoxicity assay (see table 1). Table
2 shows that ITF1779, but not ITF1493,
inhibited this cellular response in a dose-dependent manner. This
inhibition was not due to either additive or synergistic toxic effects
of ITF1779 and IL-1 on LM cells (data not shown). This result allowed us to conclude that the inhibitory effect was not specific for huTNF
but extended to at least one other LM cell response.
Further binding experiments performed with 125I-huTNF on
LM cells at 37°C were helpful in shedding light on the mechanism
underlying the previously described inhibitory effects of diaminic
carbonates on LM cells. At 37°C, huTNF induces TNFR aggregation
(Grazioli et al., 1994; Pennica et al., 1992) and
subsequent internalization (Tsujimoto et al., 1985).
Experiments performed at this temperature therefore make it possible to
investigate the fate of huTNF subsequent to the initial interaction
with its specific receptors.
Initially, we found that at 37°C ITF1779 induced a significant
increase of the binding of 125I-huTNF to LM cells (table
3) that was seemingly at odds with the
observed inhibition of the biological response. This increase was not
observed in the presence of ITF1493. The increase was not due to
enhanced background binding by ITF1779, because coincubation of
125I-huTNF, unlabeled huTNF and ITF1779 yielded
values of bound cpm similar to those obtained in the absence of ITF1779
(data not shown). By means of experiments measuring the amount of
internalized 125I-huTNF as determined after cell
washings carried out with acidic (pH 3) buffer, we could rule out the
increase of 125I-huTNF binding being due to enhanced
internalization of the ligand (table 4).
ITF1779 did not alter the ratio between cell surface-bound and
internalized 125I-huTNF: 20% to 30% of it remained
cell surface-bound, and 70% to 80% was internalized in either the
absence or the presence of ITF1779 or ITF1493.
Scatchard plots from binding experiments performed at 37°C in the
absence or presence of 60 µM ITF1779 or ITF1493 indicated that in the
presence of ITF1779, there was an approximately 4-fold increase in the
number of binding events/cell over the 4-h period considered without
any changes in the affinity constants (table 5). No significant effect was observed in
the presence of ITF1493.
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TABLE 5
Scatchard plots of binding of 125I-huTNF, in the absence or
presence of ITF1779 or ITF1493, to LM cells at 37°Ca
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These results prompted us to investigate whether enhanced binding of
125I-huTNF might have been due to increased receptor
synthesis or to the unmasking of cryptic plasma membrane TNFR. Both
possibilities were excluded by subsequent experiments.
Thus, even in conditions where synthesis of TNFR was blocked through
the addition of CHX or AcD, ITF1779 increased the binding of
125I-huTNF to LM cells (table
6). On the other hand, no such
enhancement was observed for the binding of 125I-huTNF
to purified LM cell plasma membranes; this result excluded the
possibility that ITF1779 acted by unmasking cryptic plasma membrane
TNFR (data not shown).
At this point we were left with the possibility that ITF1779 interfered
with steps subsequent to the internalization of TNF, in particular
lysosomal degradation (Tsujimoto et al., 1985). For this
purpose, we set up experiments including, as positive control,
chloroquine, a lysosomotropic agent (Mackenzie, 1983) known to
interfere with the intracellular handling of internalized ligands in
general and of TNF in particular (Tsujimoto et al., 1985). Thus, LM cells were incubated at 37°C in the absence or presence of ITF1779, ITF1493 or chloroquine. After removal of medium
and two washing steps, cells were incubated with
125I-huTNF for 1, 6 or 24 h at 37°C, and at the
end of each time period, we measured cell surface-bound (fig.
4A), internalized (fig. 4B) and
cell-dissociated, degraded 125I-huTNF (fig. 4C).
Preliminary experiments had shown that ITF1779 still inhibited
huTNF-induced cytotoxicity under these conditions (data not shown).
As can be seen, in contrast to LM cells incubated with medium or with
ITF1493, samples treated with ITF1779 or chloroquine did not release
degraded 125I-huTNF but rather, over time, accumulated
progressively increasing amounts of 125I-huTNF. These
latter experiments suggested that ITF1779 acted through a mechanism
very similar to that of chloroquine. Further substantiating such
similarity was morphologic evidence of vacuole formation and swelling
of LM cells treated with 
100 µM ITF1779. This was not observed at
lower concentrations. Moreover, vacuole formation was not observed at
any dose of ITF1493 tested (data not shown).
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Fig. 4.
Effect of ITF1779 on accumulation and degradation of
125I-huTNF. LM cells were incubated overnight at 37°C
in the absence () or presence of 60 µM ITF1779 (), 60 µM
ITF1493 () or 60 µM chloroquine (). After two washing steps,
125I-huTNF (6 × 10 10 M) was added
for the indicated times in the absence or presence of excess, unlabeled
huTNF. Surface-bound (panel A), internalized (panel B) and degraded
(panel C) 125I-huTNF was then measured as described in
"Materials and Methods." Values are indicated as percent of total
specifically bound 125I-huTNF.
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ITF1779: In vivo protection from LPS-induced
shock.
On the basis of the results obtained in
vitro, we investigated potential in vivo effects of
ITF1779. Preliminary, acute toxicity studies showed an LD50
of >400 mg/kg for ITF1779 administered s.c. Thus we selected, for the
in vivo studies, doses of ITF1779 that allowed indefinite
survival of the animals without overt signs of side effects (3 mg/kg
and 10 mg/kg). ITF1779 was studied for its capacity to afford
protection from LPS-induced shock in mice, a systemic inflammatory
response crucially dependent on TNF production (Bone, 1993; Mohler
et al., 1994; Tracey et al., 1987; Williams and
Summers, 1994). Indeed, as figure 5
shows, ITF1779 at 10 mg/kg significantly increased the survival of
treated animals (P < .001 vs. LPS alone). At 3 mg/kg
the percentage of long-term survivors was almost twice that in the LPS
group, although the difference did not attain statistical significance.
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Fig. 5.
Effect of ITF1779 on survival in LPS-induced shock.
BALB/c mice (20-30 animals/group) were treated i.p. with LPS and,
immediately thereafter after, s.c. with ITF1779 or with saline.
Dexamethasone was administered i.p. 30 min before LPS treatment.
Results were obtained in three separate experiments. Statistical
analysis was performed by the log-rank test: Dexamethasone
vs. LPS, P < .001; ITF1779 3 mg/kg vs. LPS,
not significant; ITF1779 10 mg/kg vs. LPS, P < .001.
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Discussion |
Diaminic carbonates are a class of organic compounds characterized
by the presence of two hydrophobic amines and by the insertion, between
the two aminic groups, of a both chemical and enzymatically labile
site, the carbonate diester (Nassar et al., 1992).
Some of these compounds were found to inhibit, in a dose-dependent
manner and with an IC50 in the micromolar range,
huTNF-induced cytotoxicity on LM cells. Because some of these
compounds (such as ITF1779) contain two stereogenic carbon atoms, they
are actually a mixture of three stereoisomers. In the case of ITF1779,
the independent stereoselective synthesis of the three stereoisomers showed that the enantiomerically pure compounds were as active as the
mixture (data not shown).
Initial experiments indicated that ITF1779 had to be added either at
the same time or shortly after huTNF in order to inhibit the
measured biological response. Moreover, inhibition of huTNF cytotoxicity was not a specific effect, because also IL-1-induced IL-6
production by the same cells was inhibited by ITF1779 but not by
ITF1493, a compound of the same class that is inactive in inhibiting
huTNF cytotoxicity. Further experiments aimed at demonstrating
possible effects of active diaminic carbonates on ligand binding and
post-binding events indicated that ITF1779 was essentially inactive in
inhibiting the specific binding of 125I-huTNF to LM
cells under steady-state conditions but that it interfered with events
subsequent to initial binding of the ligand and leading to progressive
intracellular accumulation of 125I-huTNF without
releasing degraded, TCA-soluble 125I-huTNF. These
results were very similar to those obtained in the same experiments
with chloroquine, a lysosomotropic agent (Mackenzie, 1983; De Duve
et al., 1974). Moreover, at high concentrations of ITF1779,
we observed extensive vacuole formation and swelling of LM cells,
another phenomenon that closely matched the reported effects of
chloroquine (Estes et al., 1987; Lie and Schofield, 1973)
and that has been ascribed to sequestration of large quantities of
cellular membrane within lysosomal vesicles (Mackenzie, 1983; De Duve
et al., 1974). Given that chloroquine and, more generally, 4-aminoquinolines bear some structural similarities to the diaminic carbonates described here (both are diprotic weak bases), it is reasonable to assume that, like chloroquine (Mackenzie, 1983), diaminic
carbonates become trapped within lysosomes, which causes the local pH
level to exceed the optimal range for acid hydrolases to perform their
digestive functions. This would then lead to inhibition of
intracellular degradation of huTNF, as described in the present
experiments. On the other hand, many cellular activities have been
shown to depend on a functional lysosomal system (Mackenzie, 1983). It
is therefore not surprising that interfering with it has resulted in
the consistent, reversible inhibition of basal cell work and
responsiveness to external stimuli (Hurvitz and Hirschhorn, 1965;
Goldstein et al., 1975), effects similar to the inhibition,
described here, of huTNF-induced cytotoxicity of LM cells and of
IL-1-induced IL-6 production by the same cells.
Notwithstanding the lack of specificity of active diaminic carbonates
as regards the inducing stimulus (huTNF or IL-1), during the
screening of the compounds, we observed precise structural requirements
for their being active as huTNF inhibitors (table 1). In summary, it
was found that 1) the diaminic system is crucial for the measured
activity because all monoaminic compounds tested (such as the alcohols
formed by hydrolysis of the carbonate diester) were inactive; 2) the
presence of the carbonate group is not relevant for activity but
represents a point in which the molecule can be degraded both
chemically and enzymatically, thus avoiding accumulation of the
compound, particularly during long-term treatment; 3) the length of the
carbon chain is crucial for good activity (ITF1779 vs.
ITF2083); 4) the nature of the alkyl chain on nitrogen atoms is very
important for activity, and the best results were obtained in the case
of linear, medium-sized alkyl chains such as n-propyl, n-butyl
and n-pentyl (ITF1779 vs. ITF2109); 5) the presence of branching near the carbonate group is very important for activity (ITF1779 vs. ITF2002). We observed higher activity for those
compounds that have a small alkyl group such as methyl
(e.g., ITF1779), ethyl and n-propyl near the carbonate.
In vivo studies performed with ITF1779 suggest that active
diaminic carbonates can be therapeutically useful. In fact, ITF1779 afforded protection from LPS-induced shock, a systemic inflammatory response the pathogenesis of which is crucially dependent on TNF production (Bone, 1993; Mohler et al., 1994; Tracey et
al., 1987; Williams and Summers, 1994). More recent, preliminary
results (F. Leoni, G. Bertolini, F. Di Pierro, unpublished
observations) in other in vivo models of inflammatory
responses confirm the therapeutic efficacy of ITF1779.
The present results could also be relevant for other classes of
molecules of pharmacological interest that bear some structural similarities to the diaminic carbonates reported here. The previously mentioned chloroquine is, together with hydroxychloroquine, a therapeutically useful agent in some conditions of chronic
inflammation, such as rheumatoid arthritis and systemic lupus
erythematosus (Fowler et al., 1984; Ten Wolde et
al., 1996; O'Dell et al., 1996). As previously
mentioned, chloroquine (fig. 1) and, more generally, molecules of the
4-aminoquinoline class of compounds have a general formula that, in
some essential features, resembles that of diaminic carbonates. Indeed,
although chloroquine contains a heterocyclic moiety, this compound can
also be considered a hydrophobic diamine like our diaminic carbonates.
The same is true of azaspiranes (fig. 1), a recently described new
class of compounds some members of which have been shown to possess
remarkable therapeutic activity in animal models of rheumatoid
arthritis (Badger and Swift, 1993; Badger and Wright, 1995).
Azaspiranes and diaminic carbonates are very similar; both are alkylic
hydrophobic diamines. It has been suggested that the anti-inflammatory
effects of active azaspiranes are due to induction of suppressor cells
(Badger and Swift, 1993). On the basis of the present results, it may
be worth while to reinvestigate the mechanism of action of these
interesting molecules. If the observations on diaminic carbonates
reported in the present paper can indeed be extended to these classes
of molecules, then it might be tempting to speculate that their
mechanism of action depends largely on a similar structural backbone
that allows them to act as lysosomotropic, diprotic weak bases.
However, although this structural motif might be necessary to endow
them with biological activity, it is clearly not sufficient for this
purpose, as shown by our structure-function relationship studies with
diaminic carbonates. The molecular bases that might help explain why
the presence of discrete, chemical substituents is necessary to the
described effects are at present unclear. Preliminary results (F. Leoni, G. Bertolini, F. DiPierro, unpublished observations) suggest
that these structural requirements vary according to the target cell that is investigated. Further elucidation of these issues will be
helpful in optimizing the therapeutic potential of diaminic carbonates,
4-aminoquinolines and, perhaps, other classes of molecules that may
work through a similar mechanism of action.
We thank Dr. Pietro Ghezzi for helpful suggestions and
discussions and Daniela Bretto for technical support.
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Abstract
Introduction
