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CUTTING EDGE |
Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We now report the discovery and characterization of (R)-5-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione (BIRT 377),2 a low m.w., nonionic compound that prevents LFA-1 from binding to ICAM-1. In addition to representing the first reported small molecule antagonist of this cell-adhesion process, BIRT 377 is unique from binding antagonists of other integrins in that it does not seem to possess any obvious structural features derived from known protein epitopes (for a recent review, see Ref. 14). Understanding of the mechanism by which BIRT 377 functions, therefore, could have important implications for the study of other integrin-ligand interactions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LFA-1, soluble ICAM-1 (sICAM-1), and Mac-1 were purified as previously reported (Refs. 15, 16, 17 , respectively). Abs were obtained from the following sources: IB4 from Ancell (Bayport, MN); MHM 23 and MHM 24 from Biomedia (Foster City, CA); CLB-LFA 1/2 and mAb 38 from RDI (Flanders, NJ); and TS 2/4, TS 1/22, and TS 1/18 from the American Type Culture Collection (Manassas, VA). KIM 185 and KIM 127 were a kind gift of Dr. Martyn Robinson (Celltech, Berkshire, U.K.). R 3.1, R 3.3, and R 7.1 were generated internally and have been previously reported (Refs. 18, 19, 20 , respectively). Small molecules 1a and 1b (BIRT 377) were synthesized via standard techniques from amino acid precursors and 3,5-dichlorophenylisocyanate.
LFA-1/ICAM-1 binding assay
LFA-1 binding to ICAM-1 was monitored by first incubating 50
µl of sICAM-1 (40 µg/ml in Dulbecco’s PBS with calcium and
magnesium, adjusted to 2.5 mM MgCl2 and 0.1 mM
PMSF), in a 96-well plate (Nunc, Naperville, IL) for 30 min at room
temperature. Plates were then blocked with 200 µl of 2% (w/v) BSA in
diluting buffer at 37°C for 30 min. Blocking solution was removed
from wells, test compounds were diluted into diluting buffer, and then
50 µl was added to the wells followed by the addition of 
25 ng of
LFA-1 (50 µl) at 37°C. After 1 h, wells were washed four times
with 200 µl of PBS. The bound LFA-1 was detected via the addition of
100 µl of a rabbit anti-CD11a (cytoplasmic tail) polyclonal Ab in
a 1:200 dilution with PBS and 1% BSA and it was allowed to incubate
for 30 min at 37°C. Wells were washed four times with 200 µl of PBS
and the bound polyclonal Ab was detected by the addition of a 1:4000
dilution of 50 µl of HRP conjugated to goat anti-rabbit Ig
(Zymed, South San Francisco, CA). This reagent was allowed to incubate
for 20 min at 37°C, wells were washed as above, and 200 µl of the
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)
substrate for the HRP was added to each well to develop a quantitative
colorimetric signal proportional to the amount of LFA-1 bound to
sICAM-1. sICAM-1 (70 µg/ml) was used as a positive control for
inhibition of the LFA-1/ICAM-1 interaction. A dose-response curve was
obtained for all test compounds.
Cellular assays
SKW3 cell assays (Fig. 2
, upper panels) were
performed as previously reported (21). Staphylococcal
enterotoxin B (SEB)-induced (Fig. 2, lower left panel)
and PMA/ionomycin-induced (Fig. 2, lower right panel)
IL-2 production assays were performed in the following manner. Purified
human PBMCs suspended in 10 ml of complete medium were counted and
plated in a 96-well microtiter format at 2 x
105 cells/well. Compounds and DMSO controls were
added to the wells using 1:10 serial dilutions. The final concentration
of DMSO was proportional to the concentration of compound and was a
maximum of 0.1%. SEB final concentration of 2 µg/ml), or PMA (final
concentration of 10 ng/ml) and ionomycin (final concentration of 1
µg/ml) were added to the wells and the total volume was adjusted to
200 µl with complete medium. The plates were incubated at 37°C and
5% CO2 overnight. IL-2 production was assayed
using a human IL-2 ELISA kit (R&D Systems, Minneapolis, MN). Anti-LFA-1
(R3.1, 10 µg/ml) is shown in the first column as a mechanistic
control. Data represent the mean of three replicate wells ±
the SE.
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BIRT 377 was evaluated in a competitive assay for its ability to
block the binding of various anti-LFA-1 mAbs to immobilized,
micellar LFA-1. LFA-1 (50 µl) was immobilized onto a microtiter plate
by adsorption at 3 µg/ml in the assay buffer (dPBS (Life
Technologies, Rockville, MD) + 2 mM MgCl2) for
1 h at room temperature. Nonspecific sites were then blocked for
30 min with 200 µl of 2% BSA in buffer. Purified mAb (50 µl at 10
µg/ml), and 50 µl of BIRT 377 or its enantiomer (5 µM in assay
buffer) were then added and allowed to bind for 1 h at 37°C. The
plate was then washed four times with assay buffer (200 µl) and 50
µl of a goat anti-mouse/HRP conjugate (Zymed) was added at a
1/4000 dilution in 1% BSA in buffer. After 20 min at 37°C, the plate
was washed and 200 µl of ABTS reagent was added to visualize binding.
After 15 min, absorbance was measured at 405 nm. Bars (see Fig. 3)
represent the mean of duplicate wells ± the SE.
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Female BALB/c mice (eight per group) obtained from The Jackson Laboratory (Bar Harbor, ME) (7- to 8-wk-old, weighing between 20 and 23 g), were dosed by oral gavage with BIRT 377 or vehicle control. One hour after dosing, each animal was challenged with 5 µg SEB (i.p., 0.2 ml from a 0.025 mg/ml stock; Sigma, St. Louis, MO). Blood was obtained from metophane-anesthetized mice 3 h after challenge, and plasma IL-2 levels were measured using a mouse IL-2 ELISA kit (R&D Systems). Data represent the mean of 15–16 mice ± SE for the BIRT 377 study and the mean of 8 mice ± SE for the anti-LFA-1 and enantiomer studies. Statistics were performed using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify compounds that directly antagonize the target protein-protein interaction, a high throughput screen was established which measured the binding of purified LFA-1 to plate-immobilized sICAM-1. This molecular assay had significantly increased sensitivity compared with a cell-based homotypic aggregation assay (22). Presumably, antagonists of the molecular assay must only prevent monomeric LFA-1/ICAM-1 interactions, whereas the cellular assay must overcome the high avidity of the multivalent presentation of LFA-1 on leukocytes interacting with the multivalent presentation of ICAM-1 on target cells. Consequently, the molecular assay allows for the detection of antagonists with a wider range of potencies, including weak antagonists that might be amenable to productive chemical modification.
Examination of a proprietary chemical collection through the high
throughput screen produced an initial hit (Fig. 1a) that inhibited the
association LFA-1 and ICAM-1 with a Kd
of 3.5 ± 1.0 µM. Structure-activity relationship studies were
performed around this hit and they successfully increased the potency
of the molecule resulting in BIRT 377 (Fig. 1b, m.w. = 442),
a compound with a Kd of 25.8 ±
6.3 nM. BIRT 377 is a single stereoisomer and is 35-fold more potent
than its enantiomer, indicating the occurrence of a true binding event
as opposed to inhibition of the assay via nonspecific detergent
effects. Furthermore, the binding of LFA-1 to ICAM-1 was restored upon
removal of BIRT 377 from the system, demonstrating that the molecule
binds reversibly and does not covalently modify either protein.
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, upper left
panel; IC50 = 2.6 ± 0.5 µM) but not
the ß1 integrin (very late Ag-4,
VLA-4)-mediated binding of SKW3 cells to fibronectin (Fig. 2, upper right panel; IC50 > 250 µM).
The SKW3 cell assays confirm that BIRT 377 is not acutely toxic to the
cell nor does it exert its action nonspecifically by inhibiting
cellular metabolism or by disrupting cytoskeletal integrity. Binding site studies
To date, several small molecules designed to antagonize the
binding of other integrins (e.g., gpIIbIIIa, VLA-4,
4ß7) have been derived
from peptide epitopes known through mutagenesis studies to be critical
for binding (23, 24, 25). BIRT 377 was the optimized product
of a screening hit and, hence, nothing was known originally about the
location of its binding site. Furthermore, the lipophilic chemical
nature of BIRT 377 separated it from other integrin antagonists that
were derived from highly charged peptide leads and suggested the
possibility that BIRT 377 might be acting via allosteric means. Binding
site identification studies were initiated to resolve these issues.
The LFA-1/ICAM-1 complex comprises the following three protein chains:
CD54 (ICAM-1), CD18 (ß2 integrin common chain),
and CD11a (LFA-1 -chain). Several experiments were undertaken to
determine which protein(s) contained the binding site for BIRT
377.
Analysis of the shape of the dose-response curve in the protein-protein
binding assay (for an explanation of this technique, see Ref.
26), as well as results from a competitive radioligand
binding assay (data not shown), provided preliminary evidence that BIRT
377 binds to LFA-1 and not to ICAM-1. To solidify this finding and to
determine whether the binding site for BIRT 377 was formed from one or
both subunits of LFA-1, an ELISA was established which measured the
ability of the antagonist to prevent the binding of a series of mAbs
with known specificity for either the -chain (CD11a) or ß-chain
(CD18) chain (Fig. 3). BIRT 377 (5 µM)
inhibited the binding of two of the seven anti-CD11a Abs but did
not inhibit the binding of any of the seven anti-CD18 Abs, despite
the fact that most of the Abs inhibit LFA-1-mediated adhesion. These
data, in conjunction with the data showing that BIRT 377 does not
inhibit Mac-1/ICAM-1 binding, provide strong evidence that the binding
site of BIRT 377 lies solely on the CD11a chain.
Knowledge of the epitopes on CD11a that recognize each of the Abs in
this study provides further information relevant to the binding of BIRT
377. The CD11a subunit of LFA-1 is proposed to have a ß-propeller
unit interrupted by an 200-amino acid "inserted-" or
"I-"domain (27). Mutagenesis (28) and
binding studies (29, 30) indicate that the I-domain
contains residues critical to support LFA/ICAM interactions. Most Abs
directed against CD11a that also block LFA-1-dependent cellular
adhesion appear to bind to one of a number of epitopes on the I-domain
(29, 30). Of the Abs used in this study, TS 2/4 binds to
the ß-propeller region and does not substantially inhibit cell
aggregation (28). The blocking Abs MHM 24, TS 1/22, and
mAb 38, which are not affected by BIRT 377, have previously been
reported to bind to I-domain (28, 29, 30). The two Abs that
are prevented from binding to LFA-1 by the addition of BIRT 377 (R 3.1
and R 7.1) block the homotypic aggregation of JY-cells and also bind to
purified I-domain (K. Last-Barney and R. Rothlein, unpublished
results). Thus, it appears that BIRT 377 affects the presentation of
some, but not all, I-domain-based epitopes, providing possible
mechanistic insight into how the compound antagonizes ICAM binding.
However, it cannot be ascertained from the current study whether this
inhibition is a result of the direct binding of BIRT 377 to the
I-domain or if it is a consequence of a remote allosteric event.
In vitro and in vivo profile
BIRT 377 blocks several in vitro assays that are known to be LFA-1
dependent. For example, the compound inhibits the phorbol ester
(PMA)-induced homotypic aggregation of JY cells (22) with
an IC50 of 0.24 ± 0.13 µM (data not
shown). To confirm that the inhibition of leukocyte function is due to
the interference with cell adhesion events and not due to peripheral
inhibition of other activation factors, the production of IL-2 from
stimulated leukocytes was measured under LFA-1-dependent and
LFA-1-independent conditions (Fig. 2, lower panels). BIRT
377 is effective in inhibiting the LFA-1-dependent production of IL-2
by human peripheral blood lymphocytes that have been stimulated with
superantigen (SEB; IC50 = 0.85 ± 0.03
µM). However, the compound does not inhibit IL-2 production in the
same cells stimulated with PMA and ionomycin, conditions that induce
activation while bypassing the need for LFA-1-mediated interactions.
Similar results were observed with mouse splenocytes (data not shown),
indicating that BIRT 377 is also active against murine LFA-1.
Low m.w. heterocyclic compounds have a potential advantage over protein
therapeutics in that, generally, they can be formulated more easily for
oral dosing. To assess in vivo efficacy and oral bioavailability, BIRT
377 was tested in a mouse model that measures the inhibition of
SEB-induced production of IL-2. The data in Fig. 4 demonstrate that the compound does
inhibit in this model in a dose-dependent manner and that 25 and 50
mg/kg oral doses of BIRT 377 are efficacious with the higher dose
reducing the level of serum IL-2 by 64% (p
< 0.001). At the higher dose, this compound inhibits the assay on the
same level as an anti-LFA-1 Ab (M17.2, 200 µg i.p.; 66%
inhibition vs saline control, p < 0.01; data not
shown). The production of IL-2, however, is not affected by the
nonbinding enantiomer of BIRT 377 (50 mg/kg per os; -7% inhibition vs
oil control (not significant); data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell adhesion plays a critical role in immunological function. There is strong evidence from the immunosuppressive effects of anti-cell adhesion Abs and anti-sense oligonucleotides that the inhibition of LFA-1 and ICAM-1 may well lead to therapeutically useful agents. The data presented herein demonstrate that a small, noncharged molecule can also block this large protein-protein interaction and, because this molecule inhibits leukocyte function both in vitro and in vivo, the prospects for developing an orally bioavailable anti-LFA-1 therapy are improved.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: BIRT 377, (R)-5-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione; sICAM-1, soluble ICAM-1; ABTS, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); SEB, staphylococcal enterotoxin B.
Received for publication July 26, 1999. Accepted for publication September 14, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7-mediated lymphoid cell adhesion to MAdCAM-1. Bioorg. Med. Chem. Lett. 8:1601.[Medline]
-subunits into a ß-propeller domain. Proc. Natl. Acad. Sci. USA 94:3162.This article has been cited by other articles:
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