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Figure 2. Inhibition of sporozoite infection of hepatocytes by parent TSP (1) and
derivatives 9 and 10.
Briefly, Plasmodium berghei sporozoites were added to the human
hepatoma cell line, HepG2, in the presence of different compounds.
Sporozoite infectivity was determined by quantifying the number
of intracellular liver stages that developed 40 h post-infection.
As reported earlier, parent TSP inhibited the sporozoite infec-
Figure 3. Inhibition of sporozoite infection of hepatocytes by biotin–linker–TSPs 13
and 14.
regardless of linker used the large compounds simply lack suffi-
cient cellular penetration.
tion of hepatocytes by over 90% at a concentration of 2 lM. Desm-
ethyl derivative 9 and ethylamino-substituted derivative 10 also
show near complete inhibition of sporozoite infectivity at a con-
centration of 10 lM (Fig. 2). This data indicates that structural var-
iation at the secondary amine of the piperidine ring is possible and
does not result in significant loss of activity. Moreover, this data
suggests that future modification of the piperidine ring may yield
more potent derivatives as well.
It was shown in previous work studying anticoccidial activity of
TSP and TSP analogs that N-desmethyl TSP or TSP derivatives hav-
ing a basic amine were potent inhibitors of parasite cGMP-depen-
dent protein kinase in vitro, but greatly reduced activity was
observed for these compounds with cells.6 Thus the reduced ability
of compounds 9 and 10 to inhibit infection of hepatocytes as com-
pared to parent TSP (Fig. 2) may also be due to decreased cellular
penetration. These combined results are consistent with the target
protein being intracellular. Decrease or loss of potency for the com-
pounds in this study is likely due to their decreased permeability
into the parasite. In any case, the two biotin-containing com-
pounds were judged to have insufficient activity to move forward
with affinity purification of the unknown protein target(s).
In summary, derivatives of TSP (1) were synthesized in order to
assess whether structural variation at the piperidinyl nitrogen
would give analogs that maintain the ability to inhibit sporozoite
invasion, and thus are amenable to biotinylation and subsequent
pull-down of TSP-binding sporozoite protein(s). It was found that
structural variation at the piperidinyl nitrogen is tolerated. How-
ever, until the target protein can be identified and activity with
the target determined directly, it is unclear if structural modifica-
tions to TSP such as with compounds 9 and 10 here result in re-
duced activity due to reduced target binding or due to reduced
cellular penetration. Thus until the target of TSP can be identified
and isolated, further structure–function studies to find more po-
tent TSP-based inhibitors of sporozoite infectivity will be con-
founded by both target binding and cell penetration affecting
activity. In addition, this work demonstrates that addition of large,
biotin-containing substituents to the piperidine nitrogen is not
likely a viable strategy to identify the cellular targets of TSP; bio-
tin–linker–TSP conjugates 13 and 14 were inactive. Although it is
possible the biotin conjugates might bind the protein target in cell
lysate, where the barrier of cellular penetration is removed. Given
the importance of identifying the target protein(s) of TSP in spor-
ozoites, alternative methods involving the synthesis of bi-func-
tional TSPs are being pursued. These TSPs will be designed to
include a photoreactive aryl moiety to irreversibly bind the TSP
to its target protein(s), and a small bioorthogonal reactive substitu-
ent on the piperidinyl ring, such as a propargyl group that is capa-
ble of coupling to an azide-linked biotin via click chemistry.
Having shown that changing substitution of the piperidinyl
nitrogen from methyl to an ethylamine linker group did not result
in a loss of activity, the synthesis of biotin-containing derivatives of
TSP was undertaken. Compound 10 was coupled with two different
biotin containing conjugates, NHS esters 11 and 12 (Scheme 3), to
give the TSP–linker–biotin conjugates 13 and 14, respectively.
The depth of a putative binding pocket for TSP in the protein
target(s) is unknown. Thus, the two linkers chosen to separate
the biotin moiety from the TSP core are quite long. This approach
was taken in order to avoid a situation where the linker was too
short for the TSP core to interact with its target protein(s) while
the biotin moiety simultaneously interacts with an affinity purifi-
cation column. Furthermore, the LC–LC linker (11) was chosen be-
cause the hydrophobicity of the long alkyl chains are thought to
increase membrane permeability.10 The PEG4 linker (12) was cho-
sen to impart extra water solubility, as well as to decrease mem-
brane permeability.11 It was hypothesized that the hydrophobic
TSP–linker–biotin compound (13) would display greater activity
than that of the more hydrophilic TSP–linker–biotin compound
(14) if the drug target(s) are intracellular. Thus, these two linking
strategies were chosen in an attempt to gather information about
the cellular location of the protein target(s) and to ensure that
the TSP–linker–biotin conjugates would be able to interact with
both the target protein(s) and avidin/streptavidin column in
chorus.
TSP–linker–biotin compounds 13 and 14 each showed essen-
tially no inhibition of sporozoite infectivity of hepatocytes at either
2 or 10 lM concentrations (Fig. 3). It is possible this loss of activity
is due to a loss of binding affinity for the protein target of TSP that
is responsible for observed activity. The large biotin–linker moie-
ties may impede the ability of compounds 13 and 14 to interact
efficiently with the binding site of the target protein(s). However,
it is also possible that the protein target(s) are intracellular, and
Towle, Tyrell
