Abstract
DEAD-box proteins are an essential class of enzymes involved in all stages of RNA metabolism. The study of DEAD-box proteins is challenging in a native setting since they are structurally similar, often essential and display dosage sensitivity. Pharmacological inhibition would be an ideal tool to probe the function of these enzymes. In this work, we describe a chemical genetic strategy for the specific inactivation of individual DEAD-box proteins with small molecule inhibitors using covalent complementarity. We identify a residue of low conservation within the P-loop of the nucleotide-binding site of DEAD-box proteins and show that it can be mutated to cysteine without a substantial loss of enzyme function to generate electrophile-sensitive mutants. We then present a series of small molecules that rapidly and specifically bind and inhibit electrophile-sensitive DEAD-box proteins with high selectivity over the wild-type enzyme. Thus, this approach can be used to systematically generate small molecule-sensitive alleles of DEAD-box proteins, allowing for pharmacological inhibition and functional characterization of members of this enzyme family.
Highlights
Small molecule inhibitors are powerful tools for the study of cellular enzymatic processes due to their rapid onset of inhibition, which prevents cellular compensation and their ability to be administered at varying doses, allowing for partial as well as complete loss-of-function phenotypes
adenosine-5 -monophosphate (AMP)-acrylate reduces both the endpoint (Figure 5E) and the rate of duplex unwinding by DDX3ES, in contrast to AMP, which largely showed endpoint depression of duplex unwinding by DDX3WT [27]. To confirm that these results are not confined to one ES mutant DEAD-box protein, we show that duplex unwinding by ES Dbp2 (S161C) is reduced by AMP-acrylate, while Dbp2WT is unaffected (Supplementary Figure S4A and B)
We have developed a strategy for the chemical genetic inhibition of DEAD-box proteins through covalent complementarity
Summary
Small molecule inhibitors are powerful tools for the study of cellular enzymatic processes due to their rapid onset of inhibition, which prevents cellular compensation and their ability to be administered at varying doses, allowing for partial as well as complete loss-of-function phenotypes. As compared to the adenosine triphosphate (ATP)-binding site of kinases, the development of small molecules targeting the nucleotide-binding pocket of adenosine triphosphatases (ATPases) has been proven challenging. ATPcompetitive inhibitors of the AAA+ ATPase p97/VCP and structurally related family members have been discovered [1,2], a generalizable small molecule scaffold with high affinity for the ATPase nucleotide-binding pocket has not yet been identified. This is likely due to the reliance on electrostatic interactions for high-affinity binding with its native substrate (ATP). It is difficult to develop potent small molecule inhibitors of most ATPases, including the DEAD-box proteins
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