Small Molecule Inhibitors as Biological Probes

There are many approaches to experimentally eliminate the function of individual proteins. These include genetic knockouts, introduction of dominant negative protein forms, antisense or RNA interference-mediated knockdown of expression, and the use of specific chemical inhibitors. Drug-like small molecules offer a number of advantages over other methods, although thus far the number of proteins for which such inhibitors exist is relatively small.

Small-molecule inhibitors are particularly useful for studying highly dynamic biological systems, such as the cytoskeletal network in cells (Peterson and Mitchison 2002). Small-molecule inhibitors can take effect on a timescale of seconds, thereby offering exquisite temporal control over protein inhibition. This rapid means of protein inactivation can allow for more informative perturbation experiments, as biologically important events can occur on a sub-second timescale. By contrast, genetic knockouts, dominant negative protein forms, and RNA interference-mediated protein downregulation typically require substantially greater time to take effect, opening the possibility of functional compensation through, for example, upregulated expression of other functionally redundant proteins. Thus, chemical inhibitors can be used to reveal the unique consequences of acute loss of protein function.

An additional strength of small molecules as research tools is that they are frequently effective in multiple cell types and species. Chemical compounds developed using one experimental system can often be applied to others, whereas, for example, genetic knockouts are inherently species specific. Furthermore, genes required for organismal viability can require the cumbersome generation of conditional knockouts for functional analysis. The use of temperature-sensitive alleles can mitigate this problem, but in some cases temperature shifts alone can independently introduce perturbations (Gasch et al. 2000). RNA interference reagents are also typically species specific due to nucleotide differences in genes from even closely related organisms. Thus, the "portability" of small molecules renders them more broadly useful to study diverse biological systems.

In addition, small-molecule inhibitors may allow for inhibition of a subset of a protein's functions, thus providing a finer scalpel for dissecting protein function. Whereas genetic knock-out of protein expression or knock-down by RNA interference eliminates all protein function, chemical inhibitors could, for example, inhibit catalytic activity while preserving target scaffolding functions (Knight and Shokat 2007). Similarly, inhibiting protein function through the use of so-called "dominant negative" forms of proteins, which often act by titrating interacting partners away from the corresponding endogenous, wild-type protein, can produce off-target effects that may be avoided by small molecules that bind and inhibit their targets directly.

For these reasons, small-molecule inhibitors offer a powerful and complementary approach for investigating protein function that is distinct from genetic, RNAi-dependent approaches and the use of dominant negative protein forms.

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