Allosteric Modulation of the YAP/TAZ-TEAD Interaction by Palmitoylation and Small-Molecule Inhibitors.

The Hippo signaling pathway is a highly conserved signaling network that plays a central role in regulating cellular growth, proliferation, and organ size. This pathway consists of a kinase cascade that integrates various upstream signals to control the activation or inactivation of YAP/TAZ proteins. Phosphorylated YAP/TAZ is sequestered in the cytoplasm; however, when the Hippo pathway is deactivated, it translocates into the nucleus, where it associates with TEAD transcription factors. This partnership is instrumental in regulating the transcription of progrowth and antiapoptotic genes. Thus, in many cancers, aberrantly hyperactivated YAP/TAZ promotes oncogenesis by contributing to cancer cell proliferation, metastasis, and therapy resistance. Because YAP and TAZ exert their oncogenic effects by binding with TEAD, it is critical to understand this key interaction to develop cancer therapeutics. Previous research has indicated that TEAD undergoes autopalmitoylation at a conserved cysteine, and small molecules that inhibit TEAD palmitoylation disrupt effective YAP/TAZ binding. However, how exactly palmitoylation contributes to YAP/TAZ-TEAD interactions and how the TEAD palmitoylation inhibitors disrupt this interaction remains unknown. Utilizing molecular dynamics simulations, our investigation not only provides detailed atomistic insight into the YAP/TAZ-TEAD dynamics but also unveils that the inhibitor studied influences the binding of YAP and TAZ to TEAD in distinct manners. This discovery has significant implications for the design and deployment of future molecular interventions targeting this interaction.

Novel Allosteric Effectors Targeting Human Transcription Factor TEAD.

The Hippo pathway is an evolutionary conserved signaling network involved in several cellular regulatory processes. Dephosphorylation and overexpression of Yes-associated proteins (YAPs) in the Hippo-off state are common in several types of solid tumors. YAP overexpression results in its nuclear translocation and interaction with transcriptional enhanced associate domain 1-4 (TEAD1-4) transcription factors. Covalent and non-covalent inhibitors have been developed to target several interaction sites between TEAD and YAP. The most targeted and effective site for these developed inhibitors is the palmitate-binding pocket in the TEAD1-4 proteins. Screening of a DNA-encoded library against the TEAD central pocket was performed experimentally to identify six new allosteric inhibitors. Inspired by the structure of the TED-347 inhibitor, chemical modification was performed on the original inhibitors by replacing secondary methyl amide with a chloromethyl ketone moiety. Various computational tools, including molecular dynamics, free energy perturbation, and Markov state model analysis, were employed to study the effect of ligand binding on the protein conformational space. Four of the six modified ligands were associated with enhanced allosteric communication between the TEAD4 and YAP1 domains indicated by the relative free energy perturbation to original molecules. Phe229, Thr332, Ile374, and Ile395 residues were revealed to be essential for the effective binding of the inhibitors.

Structure-based discovery of a novel small-molecule inhibitor of TEAD palmitoylation with anticancer activity.

The paralogous oncogenic transcriptional coactivators YAP and TAZ are the distal effectors of the Hippo signaling pathway, which plays a critical role in cell proliferation, survival and cell fate specification. They are frequently deregulated in most human cancers, where they contribute to multiple aspects of tumorigenesis including growth, metabolism, metastasis and chemo/immunotherapy resistance. Thus, they provide a critical point for therapeutic intervention. However, due to their intrinsically disordered structure, they are challenging to target directly. Since YAP/TAZ exerts oncogenic activity by associating with the TEAD1-4 transcription factors, to regulate target gene expression, YAP activity can be controlled indirectly by regulating TEAD1-4. Interestingly, TEADs undergo autopalmitoylation, which is essential for their stability and function, and small-molecule inhibitors that prevent this posttranslational modification can render them unstable. In this article we report discovery of a novel small molecule inhibitor of YAP activity. We combined structure-based virtual ligand screening with biochemical and cell biological studies and identified JM7, which inhibits YAP transcriptional reporter activity with an IC50 of 972 nMoles/Ltr. Further, it inhibits YAP target gene expression, without affecting YAP/TEAD localization. Mechanistically, JM7 inhibits TEAD palmitoylation and renders them unstable. Cellular thermal shift assay revealed that JM7 directly binds to TEAD1-4 in cells. Consistent with the inhibitory effect of JM7 on YAP activity, it significantly impairs proliferation, colony-formation and migration of mesothelioma (NCI-H226), breast (MDA-MB-231) and ovarian (OVCAR-8) cancer cells that exhibit increased YAP activity. Collectively, these results establish JM7 as a novel lead compound for development of more potent inhibitors of TEAD palmitoylation for treating cancer.

Emerging Mechanisms of Growth and Patterning Regulation by Dachsous and Fat Protocadherins.

Dachsous (Ds) and Fat are evolutionarily conserved cell adhesion molecules that play a critical role in development of multiple organ systems, where they coordinate tissue growth and morphogenesis. Much of our understanding of Ds-Fat signaling pathway comes from studies in Drosophila, where they initiate a signaling pathway that regulate growth by influencing Hippo signaling and morphogenesis by regulating Planar Cell Polarity (PCP). In this review, we discuss recent advances in our understanding of the mechanisms by which Ds-Fat signaling pathway regulates these critical developmental processes. Further, we discuss the progress in our understanding about how they function in mammals.

Assays Used for Discovering Small Molecule Inhibitors of YAP Activity in Cancers.

YAP/TAZ are transcriptional coactivators that function as the key downstream effectors of Hippo signaling. They are commonly misregulated in most human cancers, which exhibit a higher level of expression and nuclear localization of YAP/TAZ, and display addiction to YAP-dependent transcription. In the nucleus, these coactivators associate with TEA domain transcription factors (TEAD1-4) to regulate the expression of genes that promote cell proliferation and inhibit cell death. Together, this results in an excessive growth of the cancerous tissue. Further, YAP/TAZ play a critical role in tumor metastasis and chemotherapy resistance by promoting cancer stem cell fate. Furthermore, they affect tumor immunity by promoting the expression of PD-L1. Thus, YAP plays an important role in multiple aspects of cancer biology and thus, provides a critical target for cancer therapy. Here we discuss various assays that are used for conducting high-throughput screens of small molecule libraries for hit identification, and subsequent hit validation for successful discovery of potent inhibitors of YAP-transcriptional activity. Furthermore, we describe the advantages and limitations of these assays.

Early girl is a novel component of the Fat signaling pathway.

The Drosophila protocadherins Dachsous and Fat regulate growth and tissue polarity by modulating the levels, membrane localization and polarity of the atypical myosin Dachs. Localization to the apical junctional membrane is critical for Dachs function, and the adapter protein Vamana/Dlish and palmitoyl transferase Approximated are required for Dachs membrane localization. However, how Dachs levels are regulated is poorly understood. Here we identify the early girl gene as playing an essential role in Fat signaling by limiting the levels of Dachs protein. early girl mutants display overgrowth of the wings and reduced cross vein spacing, hallmark features of mutations affecting Fat signaling. Genetic experiments reveal that it functions in parallel with Fat to regulate Dachs. early girl encodes an E3 ubiquitin ligase, physically interacts with Dachs, and regulates its protein stability. Concomitant loss of early girl and approximated results in accumulation of Dachs and Vamana in cytoplasmic punctae, suggesting that it also regulates their trafficking to the apical membrane. Our findings establish a crucial role for early girl in Fat signaling, involving regulation of Dachs and Vamana, two key downstream effectors of this pathway.

The Hippo Signaling Network and Its Biological Functions.

Hippo signaling is an evolutionarily conserved network that has a central role in regulating cell proliferation and cell fate to control organ growth and regeneration. It promotes activation of the LATS kinases, which control gene expression by inhibiting the activity of the transcriptional coactivator proteins YAP and TAZ in mammals and Yorkie in Drosophila. Diverse upstream inputs, including both biochemical cues and biomechanical cues, regulate Hippo signaling and enable it to have a key role as a sensor of cells' physical environment and an integrator of growth control signals. Several components of this pathway localize to cell-cell junctions and contribute to regulation of Hippo signaling by cell polarity, cell contacts, and the cytoskeleton. Downregulation of Hippo signaling promotes uncontrolled cell proliferation, impairs differentiation, and is associated with cancer. We review the current understanding of Hippo signaling and highlight progress in the elucidation of its regulatory mechanisms and biological functions.

Audio-visual distraction as an adjunct to standby anaesthesia in persons with paraplegia: a case series of five operations.

INTRODUCTION: Persons with paraplegia present complex challenges to anaesthetists. Complications experienced by these patients can require major orthoplastic surgery such as excision of infected bone and soft tissue due to pressure sores and soft tissue reconstruction. Anaesthetic strategies deemed both safe and acceptable to this population are essential. CASE PRESENTATION: We report a case series of five procedures in four patients with complete chronic spinal cord injury (CSCI) who underwent operations in lateral position under standby anaesthesia and audio-visual distraction (AVD) with minimal sedation. Patients' experience was formally assessed as part of the ongoing AVD service evaluation in our institution. All stated that they were not concerned in the operating theatre and felt "comfortable" or "very comfortable" throughout. All patients rated the experience as "better" than their previous experience with general anaesthesia and felt "very satisfied" with their anaesthetic. Importantly, all patients would recommend sedation with AVD to other patients. DISCUSSION: To our knowledge, this is the first report of AVD application as an adjunct to standby anaesthesia during major surgery in persons with paraplegia. Patient feedback was extremely positive, therefore it is likely that by making standby anaesthesia more acceptable to patients the use of AVD could reduce anaesthetic risk in this complex patient group.

Vamana Couples Fat Signaling to the Hippo Pathway.

The protocadherins Dachsous and Fat initiate a signaling pathway that controls growth and planar cell polarity by regulating the membrane localization of the atypical myosin Dachs. How Dachs is regulated by Fat signaling has remained unclear. Here we identify the vamana gene as playing a crucial role in regulating membrane localization of Dachs and in linking Fat and Dachsous to Dachs regulation. Vamana, an SH3-domain-containing protein, physically associates with and co-localizes with Dachs and promotes its membrane localization. Vamana also associates with the Dachsous intracellular domain and with a region of the Fat intracellular domain that is essential for controlling Hippo signaling and levels of Dachs. Epistasis experiments, structure-function analysis, and physical interaction experiments argue that Fat negatively regulates Dachs in a Vamana-dependent process. Our findings establish Vamana as a crucial component of the Dachsous-Fat pathway that transmits Fat signaling by regulating Dachs.

Constitutive activation of the Nrf2/Keap1 pathway in insecticide-resistant strains of Drosophila.

Pesticide resistance poses a major challenge for the control of vector-borne human diseases and agricultural crop protection. Although a number of studies have defined how mutations in specific target proteins can lead to insecticide resistance, much less is known about the mechanisms by which constitutive overexpression of detoxifying enzymes contributes to metabolic pesticide resistance. Here we show that the Nrf2/Keap1 pathway is constitutively active in two laboratory-selected DDT-resistant strains of Drosophila, 91R and RDDTR, leading to the overexpression of multiple detoxifying genes. Disruption of the Drosophila Nrf2 ortholog, CncC, or overexpression of Keap1, is sufficient to block this transcriptional response. In addition, a CncC-responsive reporter is highly active in both DDT-resistant strains and this response is dependent on the presence of an intact CncC binding site in the promoter. Microarray analysis revealed that ∼20% of the genes differentially expressed in the 91R strain are known CncC target genes. Finally, we show that CncC is partially active in these strains, consistent with the fitness cost associated with constitutive activation of the pathway. This study demonstrates that the Nrf2/Keap1 pathway contributes to the widespread overexpression of detoxification genes in insecticide-resistant strains and raises the possibility that inhibitors of this pathway could provide effective synergists for insect population control.

Transcriptional regulation of xenobiotic detoxification in Drosophila.

Living organisms, from bacteria to humans, display a coordinated transcriptional response to xenobiotic exposure, inducing enzymes and transporters that facilitate detoxification. Several transcription factors have been identified in vertebrates that contribute to this regulatory response. In contrast, little is known about this pathway in insects. Here we show that the Drosophila Nrf2 (NF-E2-related factor 2) ortholog CncC (cap 'n' collar isoform-C) is a central regulator of xenobiotic detoxification responses. A binding site for CncC and its heterodimer partner Maf (muscle aponeurosis fibromatosis) is sufficient and necessary for robust transcriptional responses to three xenobiotic compounds: phenobarbital (PB), chlorpromazine, and caffeine. Genetic manipulations that alter the levels of CncC or its negative regulator, Keap1 (Kelch-like ECH-associated protein 1), lead to predictable changes in xenobiotic-inducible gene expression. Transcriptional profiling studies reveal that more than half of the genes regulated by PB are also controlled by CncC. Consistent with these effects on detoxification gene expression, activation of the CncC/Keap1 pathway in Drosophila is sufficient to confer resistance to the lethal effects of the pesticide malathion. These studies establish a molecular mechanism for the regulation of xenobiotic detoxification in Drosophila and have implications for controlling insect populations and the spread of insect-borne human diseases.

Effect of a single point mutation on the stability, residual structure and dynamics in the denatured state of GED: relevance to self-assembly.

The GTPase effector domain (GED) of dynamin forms large soluble oligomers in vitro, while its mutant--I697A--lacks this property at low concentrations. With a view to understand the intrinsic structural characteristics of the polypeptide chain, the global unfolding characteristics of GED wild type (WT) and I697A were compared using biophysical techniques. Quantitative analysis of the CD and fluorescence denaturation profiles revealed that unfolding occurred by a two-state process and the mutant was less stable than the WT. Even in the denatured state, the mutation caused chemical shift perturbations and significant differences were observed in the 15N transverse relaxation rates (R2), not only at the mutation site but all around. These results demonstrate that the hydrophobic change associated with the mutation perturbs the structural and motional preferences locally, which are then relayed via different folding pathways along the chain and the property of oligomerization in the native state is affected.

1H, 15N, 13C resonance assignment of folded and 8 M urea-denatured state of SUMO from Drosophila melanogaster.

SUMO, an important post-translational modifier of variety of substrate proteins, regulates different cellular functions. Here, we report the NMR resonance assignment of the folded and 8 M urea-denatured state of SUMO from Drosophila melanogaster (dsmt3).