Biomedical Engineering Reference
In-Depth Information
In another study, MAL3-101 was used as a probe to validate a screening system for
identifying chemical species that are active against the causative parasite of human
African trypanosomiasis,
Trypanosoma brucei
[69]. The transport of variant surface
glycoproteins (VSGs) to the parasite's plasma membrane by means of the ER is
critical for the parasite's survival in the host bloodstream. In contrast to mammalian
systems, import into the protozoan ER is posttranslational and dependent on chap-
erones in the cytosol. It was hypothesized that MAL3-101 would inhibit the import
of VSGs into the ER by inhibiting the protozoan Hsp70/Hsp40. A model system for
the protozoan ER utilizing
T. brucei
microsomes and cytosol showed importation
of 80% of the VSG protein VSG_117 posttranslationally across the membrane into
the interior of microsomes. Import of the protein into the microsomes was shown by
treatment of the system with proteinase K. The imported VSG_117 (i.e., 80% of the
protein) was protected from proteinase K digestion by the microsomal membrane.
However, in a separate experiment where MAL3-101 was included in the system,
only 13% of the VSG protein was protected from digestion, indicating that the com-
pound blocked uptake of VSG_117 into the microsomes. When assessed for activity
against living
T. brucei
, MAL3-101 exhibited dose-dependent trypanocidal activity
with an IC
50
value of 1.5
M, confirming both the activity of MAL3-101 as a try-
panocidal agent and validating the assay as useful for identifying new trypanocidal
compounds.
Another inhibitor of Hsp70, MAL2-11B, a precursor to MAL3-101, was also
identified as a useful probe. In addition to inhibiting TAg-stimulated ATPase activity,
MAL2-11B inhibited both endogenous Hsp70 ATPase activity and TAg's ATPase
activity [22,70]. In subsequent studies exploring its anti-TAg properties, MAL2-11B
was found to inhibit DNA replication in polyomavirus SV40 and to reduce the growth
of the human BK virus in kidney cells without cytotoxic effects toward the host cells
[70,71].
The identification of Hsp70 modulators from the UPCMLD library allowed for
the rapid diversification of the structural space around the known Hsp70 inhibitor
deoxyspergualin and facile access to second- and third-generation libraries. The
structure-activity insight from these studies has provided a new class of Hsp70
probes with higher potency for the study of Hsp70-related functions [22].
18.4.2 Agonist of the Acetylcholine Muscarinic M
1
Receptor: Probes with
Higher Selectivity
The five muscarinic receptors M
1
to M
5
are G-protein-coupled receptors activated by
the neurotransmitter acetylcholine [72]. Among these subtypes, the M
1
receptor sub-
type, which is localized in the central nervous system (CNS), has attracted attention
as a target for new treatments of Alzheimer's disease and schizophrenia, due to its role
in cognitive processing and short-term memory [73]. However, conservation of the
acetylcholine binding site, the orthosteric site, in all five of the muscarinic receptor
subtypes has made the development of M
1
subtype-specific agonists difficult.
In overcoming this obstacle, Lebois et al. employed a functional high-throughput
screen and a subsequent DOS optimization to prepare M
1
-selective agonists that
