From Plant to Patient: Thapsigargin, a Tool for Understanding …
95
(1), two or three H
+ and an ATP are bound to the pump. The pump exchanges the H
+
with two cytosolic Ca
2+ ions and is transformed to the 2Ca
2+ E1 ATP state (2). After
phosphorylation of Asp-351 the high energy [2Ca
2+ ]E1P ADP (3) occluded state is
formed. ADP is exchanged with ATP to give a low energy phosphorylated occluded
intermediate [2Ca
2+ ]E2P ATP state (4). The two calcium ions are exchanged with
luminal protons after the opening of a channel to the lumen to give nH
+ E2P-ATP,
in which n equals 2 or 3 (5). After closing the luminal channel, the occluded state
[nH
+ ]E2P ATP (6) is formed, in which no channel to either the lumen or the cytosol
is open. The cycle is concluded by dephosphorylation to give nH
+ E2ATP. The ratelimiting steps are the E1P to E2P [190] and the E2 to E1 transformations (Fig. 17)
[191]. A slightly deviating model has been suggested [192].
Among the known SERCA inhibitors thapsigargin is by far the most potent [184,
193–195]. Thapsigargin (1) locks SERCA in an E2 stage. The X-ray structures of
the thapsigargin-SERCA complex with and without nucleotide have been solved,
enabling analysis of the topography of the binding site [196].
12.4 Thapsigargin and Other P-type Pumps
The P-type pumps are a major family consisting of a large number of different
pumps [117]. All of these are characterized by the presence of an aspartate moiety,
which is phosphorylated by ATP to enable the pump to transport typically an ion
against a concentration gradient. Some pumps in addition, are flipases, establishing
different concentrations of lipids in the inner and outer plasma membrane. Thapsigargin (1) also shows an affinity for the SPCA pump [76]. The SPCA pump transports
Ca
2+ /Mn
2+ into the Golgi compartment. The affinity of 1 for the SPCA is about 1,000
times lower (7.7 μM) than for the SERCAs (0.2–13 nM). Structure-activity relationship studies have revealed that 1 binds to SPCA differently from the way it binds
to SERCA [76]. Even though there are structural similarities between all the P-type
ATPases, 1 has a poor to almost vanishingly small activity against mammalian Ptype ATPases like PMCA and the Na
+ /K
+ ATPases and some Ca
2+ -ATPases from
nematodes [104, 150, 197, 198].
12.5 Thapsigargin Prodrug Design, Preparation,
and Preclinical Evaluation
The potency of thapsigargin (1) as a cytotoxin made this compound an interesting
choice as a potential cancer chemotherapeutic agent. Thapsigargin (1) is extremely
cytotoxic and kills cells in all stages, in contrast to many chemotherapeutics, which
are targeted against cells in the proliferative states [108, 199–204]. The preparation
of thapsigargin prodrugs, however, is not straightforward. First, the absence of amino
95
(1), two or three H
+ and an ATP are bound to the pump. The pump exchanges the H
+
with two cytosolic Ca
2+ ions and is transformed to the 2Ca
2+ E1 ATP state (2). After
phosphorylation of Asp-351 the high energy [2Ca
2+ ]E1P ADP (3) occluded state is
formed. ADP is exchanged with ATP to give a low energy phosphorylated occluded
intermediate [2Ca
2+ ]E2P ATP state (4). The two calcium ions are exchanged with
luminal protons after the opening of a channel to the lumen to give nH
+ E2P-ATP,
in which n equals 2 or 3 (5). After closing the luminal channel, the occluded state
[nH
+ ]E2P ATP (6) is formed, in which no channel to either the lumen or the cytosol
is open. The cycle is concluded by dephosphorylation to give nH
+ E2ATP. The ratelimiting steps are the E1P to E2P [190] and the E2 to E1 transformations (Fig. 17)
[191]. A slightly deviating model has been suggested [192].
Among the known SERCA inhibitors thapsigargin is by far the most potent [184,
193–195]. Thapsigargin (1) locks SERCA in an E2 stage. The X-ray structures of
the thapsigargin-SERCA complex with and without nucleotide have been solved,
enabling analysis of the topography of the binding site [196].
12.4 Thapsigargin and Other P-type Pumps
The P-type pumps are a major family consisting of a large number of different
pumps [117]. All of these are characterized by the presence of an aspartate moiety,
which is phosphorylated by ATP to enable the pump to transport typically an ion
against a concentration gradient. Some pumps in addition, are flipases, establishing
different concentrations of lipids in the inner and outer plasma membrane. Thapsigargin (1) also shows an affinity for the SPCA pump [76]. The SPCA pump transports
Ca
2+ /Mn
2+ into the Golgi compartment. The affinity of 1 for the SPCA is about 1,000
times lower (7.7 μM) than for the SERCAs (0.2–13 nM). Structure-activity relationship studies have revealed that 1 binds to SPCA differently from the way it binds
to SERCA [76]. Even though there are structural similarities between all the P-type
ATPases, 1 has a poor to almost vanishingly small activity against mammalian Ptype ATPases like PMCA and the Na
+ /K
+ ATPases and some Ca
2+ -ATPases from
nematodes [104, 150, 197, 198].
12.5 Thapsigargin Prodrug Design, Preparation,
and Preclinical Evaluation
The potency of thapsigargin (1) as a cytotoxin made this compound an interesting
choice as a potential cancer chemotherapeutic agent. Thapsigargin (1) is extremely
cytotoxic and kills cells in all stages, in contrast to many chemotherapeutics, which
are targeted against cells in the proliferative states [108, 199–204]. The preparation
of thapsigargin prodrugs, however, is not straightforward. First, the absence of amino