Biomedical Engineering Reference
In-Depth Information
probes that target novel mechanisms relevant to human diseases. In the study, a
DOS strategy was applied to construct a library and to prepare a secondary, focused
sublibrary for lead optimization. Target validation confirmed SL209 as a useful probe
for studying the assembly and disassembly of the HCV viral particle.
18.5 DEVELOPING PROBES FOR THERAPEUTICALLY DESIRABLE
PHENOTYPES
Screening DOS libraries for therapeutically desired phenotypes has emerged as a
powerful method for discovering novel lead compounds and for understanding disease
biology. In addition to the representative studies presented below, several important
examples of this approach are discussed in Chapter 17.
18.5.1
Inhibitors of Glucose Transport
The reliance of cancer cells on glycolysis in preference to oxidative phosphorylation
(OXYPHOS) for ATP synthesis, even in the presence of oxygen, is a well-known
phenomenon [86]. Consequently, molecules that interfere with glucose metabolism
or transport are of interest as probes in the study of neoplastic disorders [87]. A range
of molecules are known to inhibit various steps of glycolysis, such as 2-deoxyglucose,
arsenate compounds, 3-bromopyruvate, and iodoacetate [88]. An alternative approach
to perturbing glycolysis is to modify the activity of glucose transporters (GLUT pro-
teins) that facilitate the passive transport of glucose across cell membranes [89].
The natural product cytochalasin B is a well-studied inhibitor of transmembrane
glucose transport [90]. However, cytochalasin B also depolymerizes actin, com-
plicating its use as a probe in studying complex cellular systems [24]. Therefore,
glucose transport inhibitors that do not affect other important cellular processes are
desired as probes for studying the relationship between glycolysis and neoplastic
disorders.
Ulanovskaya et al. employed a pairwise chemical genetic screen to identify
molecules that modulate glucose metabolism based on the two available pathways
for ATP synthesis in cells: glycolysis and OXYPHOS [24]. When one pathway
is shut down (e.g., by a small-molecule inhibitor), a cell will rely on the other
pathway for ATP synthesis, and cellular ATP production becomes sensitized to
inhibitors of the alternative pathway. This approach was validated by studying several
pairs of molecules, where one molecule inhibits glycolysis or glucose transport (2-
deoxyglucose, sodium iodoacetate, or cytochalasin B) and the other molecule inhibits
OXYPHOS (e.g., antimycin A). An endogenous ATP level-dependent luciferase
reporter assay was developed to measure the effect of a compound or a pair of
compounds on ATP levels, and the assay was used to screen a library of 955 struc-
turally diverse compounds against antimycin A-treated A549 cells to identify new
inhibitors of glycolysis and glucose transport. Two compounds (Figure 18.7) that
reduced ATP levels were identified. Neither compound reduced ATP levels apprecia-
bly in the absence of antimycin A, and both compounds exhibited ATP suppression
