Small molecule inhibitors of dynein: how selective is EHNA?

Recent work in my lab has used siRNA-mediated depletion to intefere with dynein motor function in intact cells. We are now developing other ways of blocking dynein function that could act more quickly to provide rapid readouts in live cell imaging assays. It would also be nice if this inhibition was reversible. There are of course many ways in which this might work – we are trying a “knock-sideways” approach as exploited by Scottie Robinson to explore adaptor complex function. What I describe could of course easily be done using a small molecule inhibitor and if one does a quick search then it is evident that two “dynein inhibitors” are available: vanadate and EHNA. My exploration of the true specificity of EHNA was driven by its seemingly increasing naming as a “selective dynein inhibitor”. For examples of this see this recent paper (which incidentally I really liked and have even recently evaluated for F1000 – which I will post soon): Kaplan and Reiner (2011) Journal of Cell Science http://jcs.biologists.org/content/124/23/3989.

The use of vanadate as a dynein inhibitor was first described in 1978 by Kobayashi et al: http://www.sciencedirect.com/science/article/pii/0006291X78912792

It seems pretty clear to me at least that vanadate is not going to be selective in this context. Our knowledge of its use as a protein phosphatase inhibitor means that applying this to live cells is going to have all sorts of implications for protein phosphorylation. I would of course be interested in any contrary opinions.

So what about EHNA? EHNA, like vanadate, was first described as a dynein inhibitor based on work on flagellar movement in sperm:

Penningroth et al. (1982) Dynein ATPase is inhibited selectively in vitro by erythro-9-[3-2-(hydroxynonyl)]adenine. Biochem Biophys Res Comm. http://www.sciencedirect.com/science/article/pii/0006291X82919647

It does indeed appear to have some selectivity for the dynein motor and, at least in vitro, is a good tool to probe dynein function relative to other ATP-dependent motors such as myosins. However, it also causes significant morphological and functional changes to the actin cytoskeleton – see Schliwa et al (1984) erythro-9-[3-(2-Hydroxynonyl)]adenine is an effective inhibitor of cell motility and actin assembly.   http://www.pnas.org/content/81/19/6044.full.pdf.

Both vanadate and EHNA were used by Beckerle and Porter in their paper in Nature in 1983 (http://www.nature.com/nature/journal/v295/n5851/abs/295701a0.html) showing that dynein was required for motility of pigment granules in squirrelfish erythropores. Where this paper excels is that is shows very clearly that the effect of EHNA on granule motility is not due to inhibition of another EHNA target, protein carboxyl-methylase. This was done by microinjecting cells with S-adenosyl homocysteine. This had previously been shown to block monocyte chemotaxis (perhaps explaining some of the effects on the actin cytoskeleton in the PNAS paper from Schliwa et al above) again showing some pretty clear evidence for selectivity.

There are plenty of other papers that have used EHNA and shown some very clear dynein-dependent functions are perturbed. I am not citing these here – its a blog, not a review (but Google “EHNA dynein” for examples).

So I guess my conclusion here is that EHNA might indeed be a useful tool to study dynein activity in cells. My concern is really over the use of the label “selective inhibitor of dynein” when applied to EHNA. I would urge anyone using this in the hope of exploring dynein function in any way to read the Schliwa paper mentioned above (http://www.pnas.org/content/81/19/6044.full.pdf).

I guess one simple solution is to get some and for us to compare this to some of our own experiments. Maybe I’ll post some info on this in the future. In the meantime, if anyone has any experience with EHNA in particular, or comments to make on its use and/or the use of such “selective inhibitors” in general I would welcome them.

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