Non-nucleoside Inhibitors of Zika Virus RNA-Dependent RNA Polymerase.

Zika virus (ZIKV) remains a potentially significant public health concern because it can cause teratogenic effects, such as microcephaly in newborns and neurological disease, like Guillain-Barré syndrome. Together with efforts to develop a vaccine, the discovery of antiviral molecules is important to control ZIKV infections and to prevent its most severe symptoms. Here, we report the development of small nonnucleoside inhibitors (NNIs) of ZIKV RNA-dependent RNA polymerase (RdRp) activity. These NNIs target an allosteric pocket (N pocket) located next to a putative hinge region between the thumb and the palm subdomains that was originally described for dengue virus (DENV) RdRp. We first tested the activity of DENV RdRp N-pocket inhibitors against ZIKV RdRp, introduced chemical modifications into these molecules, and assessed their potency using both enzymatic and cell-based assays. The most potent compound had a 50% inhibitory concentration value of 7.3 µM and inhibited ZIKV replication in a cell-based assay with a 50% effective concentration value of 24.3 µM. Importantly, we report four high-resolution crystal structures detailing how these NNIs insert into the N pocket of ZIKV RdRp. Our observations point to subtle differences in the size, shape, chemical environment, and hydration of the N pocket from ZIKV RdRp from those of the N pocket from DENV RdRp that are crucial for the design of improved antiviral inhibitors with activity against ZIKV.IMPORTANCE Zika virus belongs to the Flavivirus genus, which comprises several important human pathogens. There is currently neither an approved vaccine nor antiviral drugs available to prevent infection by ZIKV. The nonstructural protein 5 (NS5) polymerase, which is responsible for replicating the viral RNA genome, represents one of the most promising targets for antiviral drug development. Starting from compounds recently developed against dengue virus NS5, we designed and synthesized inhibitors targeting Zika virus NS5. We show that these novel compounds inhibit viral replication by targeting the polymerase activity. High-resolution X-ray crystallographic structures of protein-inhibitor complexes demonstrated specific binding to an allosteric site within the polymerase, called the N pocket. This work paves the way for the future structure-based design of potent compounds specifically targeting ZIKV RNA polymerase activity.

Ensa controls S-phase length by modulating Treslin levels.

The Greatwall/Ensa/PP2A-B55 pathway is essential for controlling mitotic substrate phosphorylation and mitotic entry. Here, we investigate the effect of the knockdown of the Gwl substrate, Ensa, in human cells. Unexpectedly, Ensa knockdown promotes a dramatic extension of S phase associated with a lowered density of replication forks. Notably, Ensa depletion results in a decrease of Treslin levels, a pivotal protein for the firing of replication origins. Accordingly, the extended S phase in Ensa-depleted cells is completely rescued by the overexpression of Treslin. Our data herein reveal a new mechanism by which normal cells regulate S-phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway.The Greatwall/Ensa/PP2A-B55 pathway controls mitotic substrate phosphorylation and mitotic entry. Here the authors show that cells regulate S phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway.

The substrate of Greatwall kinase, Arpp19, controls mitosis by inhibiting protein phosphatase 2A.

Initiation and maintenance of mitosis require the activation of protein kinase cyclin B-Cdc2 and the inhibition of protein phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall (Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition. We identified cyclic adenosine monophosphate-regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts.