Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that power transport of cellular cargos towards microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally-constrained isosteres. We identified dynapyrazoles, inhibitors more potent than ciliobrevins in vitro, and find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles block only microtubule-stimulated activity. Single-digit micromolar concentrations of dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Together, our studies suggest that chemical structure-based analyses can lead to inhibitors with distinct modes of inhibition and improved properties.
- Tarun M Kapoor
- Tarun M Kapoor
- Rand M Miller
- Andrew P Carter
- Jonathan Baruch Steinman
- Vladimir I Gelfand
- James K Chen
- Andrew P Carter
- Maxence V Nachury
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Wilfred A van der Donk, University of Illinois at Urbana-Champaign, United States
© 2017, Steinman et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.
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