Imagine viewing the plasma membrane of a cell from an interior perspective, with a front-row seat to the process by which substances enter the cell. Over in one corner, the plasma membrane begins to bulge inward, forming a sphere stretching toward you with a droplet-shaped neck on the opposite side that remains connected to the cell surface. This sphere, a clathrin-coated vesicle, attracts long protein molecules that self-assemble head-to-tail into structures that coil around the neck of the droplet, pinching it off until it separates from the plasma membrane and becomes wholly incorporated into the cell’s interior.

This process is called clathrin-mediated endocytosis, and the elongated protein that acts as a molecular tourniquet to cleave off each vesicle is called dynamin. Its action is powered by hydrolysis of GTP to GDP. And until recently, measuring this GTPase activity with high sensitivity was impossible.

Dynamin InhibitionBut on September 28, 2017, a research team from the University of Texas Southwestern Medical Center aided by Bellbrook Labs scientist Meera Kumar published an article in PLoS ONE detailing an optimized, highly sensitive fluorescence-based assay that can detect dynamin’s basal GTPase activity under conditions compatible with high-throughput screening (HTS). 1

The fluorescence polarization assay (Transcreener® GDP FP) uses an antibody that selectively binds and is highly specific for GDP over GTP. As the GTPase activity of dynamin releases GDP, these GDP molecules displace the fluorescence tracer from the antibody, resulting in low fluorescence polarization. Measuring decreased polarization relative to a standard GDP curve thus permits quantification of GTPase activity. Furthermore, conducting assays in absence or presence of potential dynamin-inhibiting compounds enables a highly sensitive, selective, and high-throughput screen.

GDP Assay for Dynamin InhibitorsGiven the biological roles of dynamin and clathrin-coated endocytosis, the power of such a screen is significant. Dynamin is involved in a host of disease states including cancers,2,3 epilepsy, hereditary neuropathies such as Charcot-Marie-Tooth disease and centronuclear myopathy,4 and epilepsy.4 Given the role of endocytosis in synaptic signaling, dynamin inhibitors with antipsychotic activity have been described. 5 And researchers in Australia are currently conducting a drug discovery program focused on modulation of the GTPase activity of dynamin to treat epilepsy (an estimated $4.5B global market) and other central nervous system indications, hoping to help patients with few options for managing refractory epilepsy.6

Previous screens for small molecule inhibitors of dynamin’s GTPase activity detected released phosphate using a malachite green colorimetric assay, but this approach does not have sufficient sensitivity to detect basal GTPase activity of dynamin.1 As a result, two previously identified inhibitors, Dynasore and Dyngo-4a, turned out to not inhibit basal GTPase activity—even at relatively high concentrations.1

A screen of basal GTPase activity of unassembled recombinant Dyn1 on a library of 8,000 small molecules did allow researchers to identify 42 preliminary hits and 4 confirmed hits, one of which was characterized further and had an impressive IC50 value of 0.6 μM for inhibition of basal GTPase activity and 6 μM for inhibition of lipid nanotubule assembly-stimulated GTPase activity.1

Unfortunately, the scaffold of this compound proved to be unsuitable for further refinement as a drug.1 However, as a proof-of-concept effort, the screen is suitably sensitive and selective for use in identifying additional lead compounds and chemical scaffolds.

Since dynamin GTPases are involved in epilepsy and other devastating neurological diseases for which few therapeutic options exist, the possibility of identifying new inhibitors holds promise for patients and clinicians alike.

-Robyn M. Perrin, PhD

[1] Mohanakrishnan A, Tran TVM, Kumar M, Chen H, Posner BA, Schmid SL. 2017. A highly-sensitive high throughput assay for dynamin’s basal GTPase activity. PLoS One. 12(9):e0185639.

[2] Chircop M, Perera S, Mariana A, Lau H, Ma MP, Gilbert J, Jones NC, Gordon CP, Young KA, Morokoff A, Sakoff J, O’Brien TJ, McCluskey A, Robinson PJ. 2011. Inhibition of dynamin by dynole 34-2 induces cell death following cytokinesis failure in cancer cells. Mol Cancer Ther. 10(9):1553-62.

[3] Meng J. 2017. Distinct functions of dynamin isoforms in tumorigenesis and their potential as therapeutic targets in cancer. Oncotarget. 8(25):41701-41716.

[4] Ferguson SM, Camilli PD. 2012. Dynamin, a membrane remodelling GTPase. Nat Rev Mol Cell Biol. 13(2): 75–88.

[5] Daniel JA, Chau N, Abdel-Hamid MK, Hu L, von Kleist L, Whiting A, Krishnan S, Maamary P, Joseph SR, Simpson F, Haucke V, McCluskey A, Robinson PJ. 2015. Phenothiazine-derived antipsychotic drugs inhibit dynamin and clathrin-mediated endocytosis. Traffic. 16(6):635-54.

[6] Bio-Link Australia, http://bio-link.com/therapeutics/

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