Antitumor Steroids
447
Zn
++
ion located within the receptor
has been proposed to explain this ex-
tremely high binding afFnity; hydropho-
bic
interaction
with
a
tryptophane
or
phenylalanine has been advocated as an
alternative hypothesis. The 12
β
-site ac-
commodates small nonpolar groups as
methyl; otherwise, C
12
has been scarcely
investigated.
3.1.4
D-ring
C
14
can accept small, nonpolar groups at
the
α
-or
β
-sites without appreciable loss
of binding afFnity. C
15
is intolerant of
small hydrophobic substituents (methyl)
or replacement with oxygen. C
16
α
ac-
commodates large nonpolar groups (e.g.
iodine), but C
16
β
is sterically less ac-
cessible, and even fluorine substitution
decreases the binding afFnity substan-
tially. ER accepts small nonpolar groups
in
C
17
α
or
groups
in
which
the
ex-
tended bulk should be directed in an
endo manner, for example,
Z
CH
=
CHI
binds more effectively than the corre-
sponding
E
-isomer. Spatial tolerances of
this area are precise, because even an
ethyl group at this position results in
signiFcant loss of binding afFnity; this
effect may be secondary to alteration of
rotation of the 17
β
-hydroxyl group. The
17
β
-hydroxyl group acts as a H-bond ac-
ceptor from His524. There may be a lysine
and two cysteine residues (one positioned
on each face of the steroid) in the vicinity
of C
15
-C
16
-C
17
, based on the reactivity of
E
2
derivatives bearing covalently labeling
groups. The 18-methyl group is not a sig-
niFcant contributor to binding, indicating
no productive contact with the receptor at
this site.
Data summarized here deFne localiza-
tions for grafting of a cytotoxic compound
onto estradiol.
1. Positions 7
α
and 11
β
appear espe-
cially appropriate for large substituents
without provoking a dramatic loss of
binding afFnity. In fact, this has been
proved by linkage of a porphyrin in posi-
tion 11
β
through a side chain. Linkage
on 7
α
or 11
β
would localize the sub-
stituent within the same region of the
receptor: the steroid may indeed rotate
around a virtual symmetry axis between
C
3
and C
17
.Hence
,7
α
and 11
β
seem
quite equivalent for grafting a side chain
bearing a cytotoxic compound. ±lexibil-
ity of such chains does not appear of
prime importance as revealed by the
study of mobile [(CH
2
)
n
]o
rinflex
ib
le
[(C±
2
)
n
]spacers
.
2. Substitution in 17
α
may also be recom-
mended, provided that a short spacer
is introduced between E
2
and the cyto-
toxic compound to maintain an effec-
tive binding afFnity. Ethynyl and vinyl
groups are especially accurate since
they give an effective separation with-
out excessive conformational flexibility.
Directing these substituents away from
the
β
-face of the steroid (side of the
17 OH) is required to limit steric inter-
ference with the binding pocket.
3.2
Nucleocytoplasmic Shuttle of Estrogen
Receptors
Nuclear receptors are ‘‘translocating bind-
ing proteins,’’ which continuously move
between the cytoplasm and the cell nu-
cleus (shuttling mechanism). The nuclear
localization, however, always dominates
especially under agonist stimulation (a
dynamic event resulting from the con-
tinuous active transport of the receptor
into the nucleus, slightly counterbalanced
by its diffusion into the cytoplasm). Re-
ceptor detection in the cytosol of extracts
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