Recognizing and exploiting differences between RNAi and small-molecule inhibitors

Abstract

The biology of RNA interference has greatly facilitated analysis of loss-of-function phenotypes, but correlating these phenotypes with small-molecule inhibition profiles is not always straightforward. We examine the rationale of comparing RNA interference to pharmacological intervention in chemical biology.

Figure 1

Modes of action for inhibition of protein activity. (a) Inhibition of protein expression by siRNA. (b) Inhibition of protein activity by small molecules.

Figure 2

Alternative modes of Aurora B blockade. (a) Schematic showing that in unperturbed cells (i), the CPC comprising Aurora B, INCENP, survivin and borealin localizes to centromeres and phosphorylates substrates. Exposure to small-molecule inhibitors (ii) prevents substrate phosphorylation but leaves the CPC intact. Aurora B RNAi (iii) or high overexpression of dominant negative (DN) mutants (iv) not only prevents substrate phosphorylation but also mislocalizes and destabilizes remaining CPC components. Low overexpression of dominant negative mutants (v) leaves the CPC intact but potently inhibits substrate phosphorylation. (b) Immunofluorescence images of human cells exposed to a small-molecule Aurora B inhibitor (i) or Aurora B siRNAs (ii), or transfected with a dominant negative mutant (iii), and then stained to detect Aurora B (green) and DNA (red). Small-molecule-mediated inhibition allows correct localization of Aurora B, whereas RNAi greatly reduces Aurora B levels and the kinase mutant mislocalizes the CPC. Reproduced from ref. . Copyright 2003 Rockefeller University Press.

Figure 3

Differential effects of siRNA and small-molecule inhibitors against p110β. (a) Activation of growth factor receptor signals through an adaptor protein such as IRS1 to bring PI(3)K to the membrane and to relieve basal inhibition of p110α by p85. Catalytically active p110α then phosphorylates phosphoinositide-4,5-bisphosphate (PIP₂), generating the lipid second messenger phosphoinositide-3,4,5-trisphosphate (PIP₃). PIP₃ in turn recruits Akt to the membrane, where it is phosphorylated and regulates a broad range of substrates that promote growth, proliferation and survival. (b) The role of p110β in responding to growth factor receptors and in activating growth and survival is less central than that of p110α. Small-molecule inhibitors of p110β do not affect signaling through p110α and therefore do not appreciably affect growth or survival. (c) In contrast, siRNA against p110β leads to growth arrest, possibly by freeing up monomeric p85, which can sequester IRS1 in a nonsignaling cytosolic protein complex, thereby abrogating signaling through p110α.