Characterization of the Zika virus two-component NS2B-NS3 protease and structure-assisted identification of allosteric small-molecule antagonists.

The recent re-emergence of Zika virus (ZIKV)1, a member of the Flaviviridae family, has become a global emergency. Currently, there are no effective methods of preventing or treating ZIKV infection, which causes severe neuroimmunopathology and is particularly harmful to the developing fetuses of infected pregnant women. However, the pathology induced by ZIKV is unique among flaviviruses, and knowledge of the biology of other family members cannot easily be extrapolated to ZIKV. Thus, structure-function studies of ZIKV proteins are urgently needed to facilitate the development of effective preventative and therapeutic agents. Like other flaviviruses, ZIKV expresses an NS2B-NS3 protease, which consists of the NS2B cofactor and the NS3 protease domain and is essential for cleavage of the ZIKV polyprotein precursor and generation of fully functional viral proteins. Here, we report the enzymatic characterization of ZIKV protease, and we identify structural scaffolds for allosteric small-molecule inhibitors of this protease. Molecular modeling of the protease-inhibitor complexes suggests that these compounds bind to the druggable cavity in the NS2B-NS3 protease interface and affect productive interactions of the protease domain with its cofactor. The most potent compound demonstrated efficient inhibition of ZIKV propagation in vitro in human fetal neural progenitor cells and in vivo in SJL mice. The inhibitory scaffolds could be further developed into valuable research reagents and, ultimately, provide a roadmap for the selection of efficient inhibitors of ZIKV infection.

Cardiac Glycosides Activate the Tumor Suppressor and Viral Restriction Factor Promyelocytic Leukemia Protein (PML).

Cardiac glycosides (CGs), inhibitors of Na+/K+-ATPase (NKA), used clinically to treat heart failure, have garnered recent attention as potential anti-cancer and anti-viral agents. A high-throughput phenotypic screen designed to identify modulators of promyelocytic leukemia protein (PML) nuclear body (NB) formation revealed the CG gitoxigenin as a potent activator of PML. We demonstrate that multiple structurally distinct CGs activate the formation of PML NBs and induce PML protein SUMOylation in an NKA-dependent fashion. CG effects on PML occur at the post-transcriptional level, mechanistically distinct from previously described PML activators and are mediated through signaling events downstream of NKA. Curiously, genomic deletion of PML in human cancer cells failed to abrogate the cytotoxic effects of CGs and other apoptotic stimuli such as ceramide and arsenic trioxide that were previously shown to function through PML in mice. These findings suggest that alternative pathways can compensate for PML loss to mediate apoptosis in response to CGs and other apoptotic stimuli.

Radiation Sensitivity and Tumor Susceptibility in ATM Phospho-Mutant ATF2 Mice.

The transcription factor ATF2 was previously shown to be an ATM substrate. Upon phosphorylation by ATM, ATF2 exhibits a transcription-independent function in the DNA damage response through localization to DNA repair foci and control of cell cycle arrest. To assess the physiological significance of this phosphorylation, we generated ATF2 mutant mice in which the ATM phosphoacceptor sites (S472/S480) were mutated (ATF2(KI)). ATF2(KI) mice are more sensitive to ionizing radiation (IR) than wild-type (ATF2 (WT)) mice: following IR, ATF2(KI) mice exhibited higher levels of apoptosis in the intestinal crypt cells and impaired hepatic steatosis. Molecular analysis identified impaired activation of the cell cycle regulatory protein p21(Cip/Waf1) in cells and tissues of IR-treated ATF2(KI) mice, which was p53 independent. Analysis of tumor development in p53(KO) crossed with ATF2(KI) mice indicated a marked decrease in amount of time required for tumor development. Further, when subjected to two-stage skin carcinogenesis process, ATF2(KI) mice developed skin tumors faster and with higher incidence, which also progressed to the more malignant carcinomas, compared with the control mice. Using 3 mouse models, we establish the importance of ATF2 phosphorylation by ATM in the acute cellular response to DNA damage and maintenance of genomic stability.

A role for ATF2 in regulating MITF and melanoma development.

The transcription factor ATF2 has been shown to attenuate melanoma susceptibility to apoptosis and to promote its ability to form tumors in xenograft models. To directly assess ATF2's role in melanoma development, we crossed a mouse melanoma model (Nras(Q61K)::Ink4a⁻/⁻) with mice expressing a transcriptionally inactive form of ATF2 in melanocytes. In contrast to 7/21 of the Nras(Q61K)::Ink4a⁻/⁻ mice, only 1/21 mice expressing mutant ATF2 in melanocytes developed melanoma. Gene expression profiling identified higher MITF expression in primary melanocytes expressing transcriptionally inactive ATF2. MITF downregulation by ATF2 was confirmed in the skin of Atf2⁻/⁻ mice, in primary human melanocytes, and in 50% of human melanoma cell lines. Inhibition of MITF transcription by MITF was shown to be mediated by ATF2-JunB-dependent suppression of SOX10 transcription. Remarkably, oncogenic BRAF (V600E)-dependent focus formation of melanocytes on soft agar was inhibited by ATF2 knockdown and partially rescued upon shMITF co-expression. On melanoma tissue microarrays, a high nuclear ATF2 to MITF ratio in primary specimens was associated with metastatic disease and poor prognosis. Our findings establish the importance of transcriptionally active ATF2 in melanoma development through fine-tuning of MITF expression.

c-Jun regulates phosphoinositide-dependent kinase 1 transcription: implication for Akt and protein kinase C activities and melanoma tumorigenesis.

Mutations in N-RAS and B-RAF, which commonly occur in melanomas, result in constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) signaling. Active ERK increases expression and activity of the c-Jun transcription factor, linking ERK and Jun N-terminal kinase (JNK) cascades. Here, we show that c-Jun regulates transcription of phosphoinositide-dependent kinase 1 (PDK1) with a concomitant impact on Akt and protein kinase C (PKC) activity and related substrates. Inhibition of c-Jun reduces PDK1 expression and attenuates Akt and PKC activity, which can be restored by exogenous PDK1. c-Jun regulation of PDK1 in melanoma contributes to growth rate and the ability to form tumors in mice. Correspondingly, increased levels of c-Jun in melanoma cell lines coincide with up-regulation of PDK1 and phosphorylation of PKC and Akt. The identification of c-Jun as a transcriptional regulator of PDK1 expression highlights key mechanisms underlying c-Jun oncogenic activity, and provides new insight into the nature of up-regulated Akt and PKC in melanoma.

Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma tumorigenesis.

The E3 ubiquitin ligase Siah2 has been implicated in the regulation of the hypoxia response, as well as in the control of Ras, JNK/p38/NF-kappaB signaling pathways. Both Ras/mitogen-activated protein kinase (MAPK) and hypoxia pathways are important for melanoma development and progression, pointing to the possible use of Siah2 as target for treatment of this tumor type. In the present study, we have established a high-throughput electro-chemiluninescent-based assay in order to screen and identify inhibitors of Siah2 ubiquitin ligase activity. Of 1840 compounds screened, we identified and characterized menadione (MEN) as a specific inhibitor of Siah2 ligase activity. MEN attenuated Siah2 self-ubiquitination, and increased expression of its substrates PHD3 and Sprouty2, with concomitant decrease in levels of HIF-1alpha and pERK, the respective downstream effectors. MEN treatment no longer affected PHD3 or Sprouty2 in Siah-KO cells, pointing to its Siah-dependent effects. Further, MEN inhibition of Siah2 was not attenuated by free radical scavenger, suggesting it is ROS-independent. Significantly, growth of xenograft melanoma tumors was inhibited following the administration of MEN or its derivative. These findings reveal an efficient platform for the identification of Siah inhibitors while identifying and characterizing MEN as Siah inhibitor that attenuates hypoxia and MAPK signaling, and inhibits melanoma tumorigenesis.

BI-69A11-mediated inhibition of AKT leads to effective regression of xenograft melanoma.

The AKT/PKB pathway plays a central role in tumor development and progression and is often up-regulated in different tumor types, including melanomas. We have recently reported on the in silico approach to identify putative inhibitors for AKT/PKB. Of the reported hits, we selected BI-69A11, a compound which was shown to inhibit AKT activity in in vitro kinase assays. Analysis of BI-69A11 was performed in melanoma cells, a tumor type that commonly exhibits up-regulation of AKT. Treatment of the UACC903 human melanoma cells, harboring the PTEN mutation, with BI-69A11 caused efficient inhibition of AKT S473 phosphorylation with concomitant inhibition of AKT phosphorylation of PRAS40. Treatment of melanoma cells with BI-69A11 also reduced AKT protein expression, which coincided with inhibition of AKT association with HSP-90. BI-69A11 treatment not only caused cell death of melanoma, but also prostate tumor cell lines. Notably, the effect of BI-69A11 on cell death was more pronounced in cells that express an active form of AKT. Significantly, intra-peritoneal injection of BI-69A11 caused effective regression of melanoma tumor xenografts, which coincided with elevated levels of cell death. These findings identify BI-69A11 as a potent inhibitor of AKT that is capable of eliciting effective regression of xenograft melanoma tumors.