Chemistry Reference
In-Depth Information
laboratories. Details of the organic chemistry of epothilones and their analogs will
not be discussed. Likewise, the impressive advances in the elucidation of epothilone
biosynthesis and the development of heterologous expression systems is largely
outside of the scope of this chapter,
42
except for a few selected modified analogs
produced in heterologous expression systems. (For a recent review on the biosynth-
esis of epothilones see Ref. 42). These systems will be covered in Section 1.3.
1.2
BIOLOGICAL EFFECTS OF Epo B
1.2.1
In Vitro Activity
The basic biology and pharmacology of Epo B (as the most potent and most widely
studied natural epothilone) have been summarized in several previous review
articles.
15,23,37,39,43,44
As indicated in Section 1.1, the biological effects of the com-
pound are based on its ability to bind to microtubules and alter the intrinsic stability
and dynamic properties of these supramolecular structures. In cell-free in vitro sys-
tems, this is demonstrated by the prevention of Ca
2
þ
- or cold-induced depolymer-
ization of preformed microtubule polymers
19
as well as by the promotion of tubulin
polymerization (to form microtubule-like polymers) in the absence of either micro-
tubule-associated proteins (MAPs) and/or guanosine triphosphate (GTP), at tem-
peratures significantly below 37
C, and in the presence of Ca
2
þ
.
11,19
The latter
phenomenon, that is, the induction of tubulin polymerization, is frequently used
as a biochemical readout for the assessment of the interaction of microtubule-
stabilizing agents with tubulin in a quantitative fashion. Epo B is a more efficient
tubulin-polymerizing agent than paclitaxel, which in turn polymerizes tubulin with
about the same potency as Epo A. (e.g., EC
50
values for the polymerization of
microtubule protein by Epo A, Epo B, and paclitaxel have been determined as
1.12, 0.67, and 1.88 mM, respectively).
45
However, it should be noted that the
exact magnitude of tubulin-polymerizing effects in vitro (absolute and even relative
polymerization rates, extent of tubulin polymer formation) strongly depends on the
assay conditions employed (e.g., biological source and purity of tubulin, concentra-
tion of microtubule-stabilizing buffer components, and reaction temperature).
45
Epothilones can displace [
3
H]-paclitaxel from microtubules with efficiencies simi-
lar or superior to those of unlabeled paclitaxel or docetaxel.
11,19
Inhibition of pacli-
taxel binding occurs in a competitive fashion [with apparent K
i
values of 1.4 mM
(Epo A) and 0.7 mM (Epo B)], which thus suggests that the microtubule binding
sites of paclitaxel and Epo A and B are largely overlapping or even identical (vide
infra). More recently, the binding constants of Epo A and B to stabilized microtu-
bules in vitro have been determined as 2.93
10
7
M
1
(Epo A) and 6.08
10
8
M
1
(37
C) with a fluorescence-based displacement assay.
46
In line with its effects on tubulin polymerization in vitro (i.e., in an excellular
context), the prevention of cold-induced depolymerization of microtubules by
epothilones has also been demonstrated in cells.
37
Microtubule stabilization in
intact cells (as well as cancer cell growth inhibition, vide infra), however, is
observed at strikingly lower concentrations than those required for the induction
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