Pannexin 1 binds ß-catenin to modulate melanoma cell growth and metabolism.

Melanoma is the most aggressive skin malignancy with increasing incidence worldwide. Pannexin1 (PANX1), a member of the pannexin family of channel-forming glycoproteins, regulates cellular processes in melanoma cells including proliferation, migration, and invasion/metastasis. However, the mechanisms responsible for coordinating and regulating PANX1 function remain unclear. Here, we demonstrated a direct interaction between the C-terminal region of PANX1 and the N-terminal portion of ß-catenin, a key transcription factor in the Wnt pathway. At the protein level, ß-catenin was significantly decreased when PANX1 was either knocked down or inhibited by two PANX1 blockers, Probenecid and Spironolactone. Immunofluorescence imaging showed a disrupted pattern of ß-catenin localization at the cell membrane in PANX1-deficient cells, and transcription of several Wnt target genes, including MITF, was suppressed. In addition, a mitochondrial stress test revealed that the metabolism of PANX1-deficient cells was impaired, indicating a role for PANX1 in the regulation of the melanoma cell metabolic profile. Taken together, our data show that PANX1 directly interacts with ß-catenin to modulate growth and metabolism in melanoma cells. These findings provide mechanistic insight into PANX1-mediated melanoma progression and may be applicable to other contexts where PANX1 and ß-catenin interact as a potential new component of the Wnt signaling pathway.

Macrophage calcium reporter mice reveal immune cell communication in vitro and in vivo.

Cell communication underlies emergent functions in diverse cell types and tissues. Recent evidence suggests that macrophages are organized in communicating networks, but new tools are needed to quantitatively characterize the resulting cellular conversations. Here, we infer cell communication from spatiotemporal correlations of intracellular calcium dynamics that are non-destructively imaged across cell populations expressing genetically encoded calcium indicators. We describe a hematopoietic calcium reporter mouse (Csf1rCreGCaMP5fl) and a computational analysis pipeline for inferring communication between reporter cells based on "excess synchrony." We observed signals suggestive of cell communication in macrophages treated with immune-stimulatory DNA in vitro and tumor-associated immune cells imaged in a dorsal window chamber model in vivo. Together, the methods described here expand the toolkit for discovery of cell communication events in macrophages and other immune cells.

The myeloid type I interferon response to myocardial infarction begins in bone marrow and is regulated by Nrf2-activated macrophages.

Sterile tissue injury is thought to locally activate innate immune responses via damage-associated molecular patterns (DAMPs). Whether innate immune pathways are remotely activated remains relatively unexplored. Here, by analyzing ~145,000 single-cell transcriptomes at steady state and after myocardial infarction (MI) in mice and humans, we show that the type I interferon (IFN) response, characterized by expression of IFN-stimulated genes (ISGs), begins far from the site of injury, in neutrophil and monocyte progenitors within the bone marrow. In the peripheral blood of patients, we observed defined subsets of ISG-expressing neutrophils and monocytes. In the bone marrow and blood of mice, ISG expression was detected in neutrophils and monocytes and their progenitors, intensified with maturation at steady-state and after MI, and was controlled by Tet2 and Irf3 transcriptional regulators. Within the infarcted heart, ISG-expressing cells were negatively regulated by Nrf2 activation in Ccr2- steady-state cardiac macrophages. Our results show that IFN signaling begins in the bone marrow, implicate multiple transcriptional regulators (Tet2, Irf3, and Nrf2) in governing ISG expression, and provide a clinical biomarker (ISG score) for studying IFN signaling in patients.

Mechanism of cognate sequence discrimination by the ETS-family transcription factor ETS-1.

Functional evidence increasingly implicates low-affinity DNA recognition by transcription factors as a general mechanism for the spatiotemporal control of developmental genes. Although the DNA sequence requirements for affinity are well-defined, the dynamic mechanisms that execute cognate recognition are much less resolved. To address this gap, here we examined ETS1, a paradigm developmental transcription factor, as a model for which cognate discrimination remains enigmatic. Using molecular dynamics simulations, we interrogated the DNA-binding domain of murine ETS1 alone and when bound to high-and low-affinity cognate sites or to nonspecific DNA. The results of our analyses revealed collective backbone and side-chain motions that distinguished cognate versus nonspecific as well as high- versus low-affinity cognate DNA binding. Combined with binding experiments with site-directed ETS1 mutants, the molecular dynamics data disclosed a triad of residues that respond specifically to low-affinity cognate DNA. We found that a DNA-contacting residue (Gln-336) specifically recognizes low-affinity DNA and triggers the loss of a distal salt bridge (Glu-343/Arg-378) via a large side-chain motion that compromises the hydrophobic packing of two core helices. As an intact Glu-343/Arg-378 bridge is the default state in unbound ETS1 and maintained in high-affinity and nonspecific complexes, the low-affinity complex represents a unique conformational adaptation to the suboptimization of developmental enhancers.

Inhibition of Pannexin 1 Reduces the Tumorigenic Properties of Human Melanoma Cells.

Pannexin 1 (PANX1) is a channel-forming glycoprotein expressed in many tissues including the skin. PANX1 channels allow the passage of ions and molecules up to 1 kDa, including ATP and other metabolites. In this study, we show that PANX1 is highly expressed in human melanoma tumors at all stages of disease progression, as well as in patient-derived cells and established melanoma cell lines. Reducing PANX1 protein levels using shRNA or inhibiting channel function with the channel blockers, carbenoxolone (CBX) and probenecid (PBN), significantly decreased cell growth and migration, and increased melanin production in A375-P and A375-MA2 cell lines. Further, treatment of A375-MA2 tumors in chicken embryo xenografts with CBX or PBN significantly reduced melanoma tumor weight and invasiveness. Blocking PANX1 channels with PBN reduced ATP release in A375-P cells, suggesting a potential role for PANX1 in purinergic signaling of melanoma cells. In addition, cell-surface biotinylation assays indicate that there is an intracellular pool of PANX1 in melanoma cells. PANX1 likely modulates signaling through the Wnt/ß-catenin pathway, because ß-catenin levels were significantly decreased upon PANX1 silencing. Collectively, our findings identify a role for PANX1 in controlling growth and tumorigenic properties of melanoma cells contributing to signaling pathways that modulate melanoma progression.

A One-Step Chemoenzymatic Labeling Strategy for Probing Sialylated Thomsen-Friedenreich Antigen.

Abnormal expression of sialylated Thomsen-Friedenreich antigen (Neu5Acα2-3Galß1-3GalNAcα-O-Ser/Thr, sialyl-T) has a strong relationship with various types of human cancers and many other diseases. However, the size and structural complexity, and relatively lower abundance of sialyl-T have posed a significant challenge to its detection. Therefore, details about the role of sialyl-T in a variety of physiological and pathological processes are still poorly understood. Here, a one-step chemoenzymatic labeling strategy to probe sialyl-T is described. This approach enables the sensitive, selective, and rapid detection of sialyl-T, and global profiling and identification of unknown sialyl-T-attached glycoproteins, which are potential therapeutic targets or biomarkers. The use of one-step labeling strategy not only has a higher sensitivity than a typical two-step reporter strategy but also avoids undergoing an additional chemical reaction step to introduce a reporter group after the labeling reaction, making it particularly useful for detecting low-abundance glycan epitopes on living cells.

Pharmacological inhibition of the transcription factor PU.1 in leukemia.

The transcription factor PU.1 is often impaired in patients with acute myeloid leukemia (AML). Here, we used AML cells that already had low PU.1 levels and further inhibited PU.1 using either RNA interference or, to our knowledge, first-in-class small-molecule inhibitors of PU.1 that we developed specifically to allosterically interfere with PU.1-chromatin binding through interaction with the DNA minor groove that flanks PU.1-binding motifs. These small molecules of the heterocyclic diamidine family disrupted the interaction of PU.1 with target gene promoters and led to downregulation of canonical PU.1 transcriptional targets. shRNA or small-molecule inhibition of PU.1 in AML cells from either PU.1lo mutant mice or human patients with AML-inhibited cell growth and clonogenicity and induced apoptosis. In murine and human AML (xeno)transplantation models, treatment with our PU.1 inhibitors decreased tumor burden and resulted in increased survival. Thus, our study provides proof of concept that PU.1 inhibition has potential as a therapeutic strategy for the treatment of AML and for the development of small-molecule inhibitors of PU.1.

Structure based design, synthesis and activity studies of small hybrid molecules as HDAC and G9a dual inhibitors.

Aberrant enzymatic activities or expression profiles of epigenetic regulations are therapeutic targets for cancers. Among these, histone 3 lysine 9 methylation (H3K9Me2) and global de-acetylation on histone proteins are associated with multiple cancer phenotypes including leukemia, prostatic carcinoma, hepatocellular carcinoma and pulmonary carcinoma. Here, we report the discovery of the first small molecule capable of acting as a dual inhibitor targeting both G9a and HDAC. Our structure based design, synthesis, and screening for the dual activity of the small molecules led to the discovery of compound 14 which displays promising inhibition of both G9a and HDAC in low micro-molar range in cell based assays.

Distinct Roles for Interfacial Hydration in Site-Specific DNA Recognition by ETS-Family Transcription Factors.

The ETS family of transcription factors is a functionally heterogeneous group of gene regulators that share a structurally conserved, eponymous DNA-binding domain. Unlike other ETS homologues, such as Ets-1, DNA recognition by PU.1 is highly sensitive to its osmotic environment due to excess interfacial hydration in the complex. To investigate interfacial hydration in the two homologues, we mutated a conserved tyrosine residue, which is exclusively engaged in coordinating a well-defined water contact between the protein and DNA among ETS proteins, to phenylalanine. The loss of this water-mediated contact blunted the osmotic sensitivity of PU.1/DNA binding, but did not alter binding under normo-osmotic conditions, suggesting that PU.1 has evolved to maximize osmotic sensitivity. The homologous mutation in Ets-1, which was minimally sensitive to osmotic stress due to a sparsely hydrated interface, reduced DNA-binding affinity at normal osmolality but the complex became stabilized by osmotic stress. Molecular dynamics simulations of wildtype and mutant PU.1 and Ets-1 in their free and DNA-bound states, which recapitulated experimental features of the proteins, showed that abrogation of this tyrosine-mediated water contact perturbed the Ets-1/DNA complex not through disruption of interfacial hydration, but by inhibiting local dynamics induced specifically in the bound state. Thus, a configurationally identical water-mediated contact plays mechanistically distinct roles in mediating DNA recognition by structurally homologous ETS transcription factors.

Discovery of novel small molecule inhibitors of lysine methyltransferase G9a and their mechanism in leukemia cell lines.

Lysine methyltransferase G9a regulates the transcription of multiple genes by primarily catalyzing mono- and di-methylation of histone H3 lysine 9, as well as several non-histone lysine sites. An attractive therapeutic target in treating leukemia, knockout studies of G9a in mice have found dramatically slowed proliferation and self-renewal of acute myeloid leukemia (AML) cells due to the attenuation of HoxA9-dependent transcription. In this study, a series of compounds were identified as potential inhibitors through structure-based virtual screening. Among these compounds, a new G9a inhibitor, DCG066, was confirmed by in vitro biochemical, and cell based enzyme assays. DCG066 has a novel molecular scaffold unlike other G9a inhibitors presently available. Similar to G9a's histone substrate, DCG066 can bind directly to G9a and inhibit methyltransferase activity in vitro. In addition to suppressing G9a methyltransferase activity and reducing histone H3 methylation levels, DCG066 displays low cytotoxicity in leukemia cell lines with high levels of G9a expression, including K562. This work presents DCG066 as an inhibitor of G9a with a novel structure, providing both a lead in G9a inhibitor design and a means for probing the functionality of G9a.

Discovery of novel aminoquinazolin-7-yl 6,7-dihydro-indol-4-ones as potent, selective inhibitors of heat shock protein 90.

A novel class of Hsp90 inhibitors, structurally distinct from previously reported scaffolds, was developed from rational design and optimization of a compound library screen hit. These aminoquinazoline derivatives, represented by compound 15 (SNX-6833) or 1-(2-amino-4-methylquinazolin-7-yl)-3,6,6-trimethyl-6,7-dihydro-1H-indol-4(5H)-one, selectively bind to Hsp90 and inhibit its cellular activities at concentrations as low as single digit nanomolar.

Application of chemoproteomics to drug discovery: identification of a clinical candidate targeting hsp90.

A chemoproteomics-based drug discovery strategy is presented that utilizes a highly parallel screening platform, encompassing more than 1000 targets, with a focused chemical library prior to target selection. This chemoproteomics-based process enables a data-driven selection of both the biological target and chemical hit after the screen is complete. The methodology has been exemplified for the purine binding proteome (proteins utilizing ATP, NAD, FAD). Screening of an 8000 member library yielded over 1500 unique protein-ligand interactions, which included novel hits for the oncology target Hsp90. The approach, which also provides broad target selectivity information, was used to drive the identification of a potent and orally active Hsp90 inhibitor, SNX-5422, which is currently in phase 1 clinical studies.

Discovery of novel 2-aminobenzamide inhibitors of heat shock protein 90 as potent, selective and orally active antitumor agents.

A novel class of heat shock protein 90 (Hsp90) inhibitors was developed from an unbiased screen to identify protein targets for a diverse compound library. These indol-4-one and indazol-4-one derived 2-aminobenzamides showed strong binding affinity to Hsp90, and optimized analogues exhibited nanomolar antiproliferative activity across multiple cancer cell lines. Heat shock protein 70 (Hsp70) induction and specific client protein degradation in cells on treatment with the inhibitors supported Hsp90 inhibition as the mechanism of action. Computational chemistry and X-ray crystallographic analysis of selected member compounds clearly defined the protein-inhibitor interaction and assisted the design of analogues. 4-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl]-2-[(trans-4-hydroxycyclohexyl)amino]benzamide (SNX-2112, 9) was identified as highly selective and potent (IC(50) Her2 = 11 nM, HT-29 = 3 nM); its prodrug amino-acetic acid 4-[2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-phenylamino]-cyclohexyl ester methanesulfonate (SNX-5422, 10) was orally bioavailable and efficacious in a broad range of xenograft tumor models (e.g. 67% growth delay in a HT-29 model) and is now in multiple phase I clinical trials.

Small molecule inhibitors of Hsp90 potently affect inflammatory disease pathways and exhibit activity in models of rheumatoid arthritis.

OBJECTIVE: To evaluate the ability of SNX-7081, a novel small molecule inhibitor of Hsp90, to block components of inflammation, including cytokine production, protein kinase activity, and angiogenic signaling. A close analog was evaluated in preclinical in vivo models of rheumatoid arthritis (RA). METHODS: SNX-7081 binding to Hsp90 was characterized in Jurkat cells and RA synovial fibroblasts (RASFs). Inhibition of NF-kappaB nuclear translocation was evaluated in cellular systems, using lipopolysaccharide (LPS), tumor necrosis factor alpha, or interleukin-1beta stimulation. Suppression of cytokine production in THP-1 cells, human umbilical vein endothelial cells, and RASFs was studied. Disruption of MAPK signaling cascades by SNX-7081 following growth factor stimulation was assessed. SNX-7081 was tested in 2 relevant angiogenesis assays: platelet-derived growth factor activation of fibroblasts and LPS-induced nitric oxide (NO) release in J774 macrophages. A close analog, SNX-4414, was evaluated in rat collagen-induced arthritis and adjuvant-induced arthritis, following oral treatment. RESULTS: SNX-7081 showed strong binding affinity to Hsp90 and expected induction of Hsp70. NF-kappaB nuclear translocation was blocked by SNX-7081 at nanomolar concentrations, and cytokine production was potently inhibited. Growth factor activation of ERK and JNK signaling was significantly reduced by SNX-7081. NO production was also sharply inhibited. In animal models, SNX-4414 fully inhibited paw swelling and improved body weight. Scores for inflammation, pannus formation, cartilage damage, and bone resorption returned to normal. CONCLUSION: The present results demonstrate that a small molecule Hsp90 inhibitor can impact inflammatory disease processes. The strong in vivo efficacy observed with SNX-4414 provides preclinical validation for consideration of Hsp90 inhibitors in the treatment of RA.

Discovery of benzamide tetrahydro-4H-carbazol-4-ones as novel small molecule inhibitors of Hsp90.

Hsp90 maintains the conformational stability of multiple proteins implicated in oncogenesis and has emerged as a target for chemotherapy. We report here the discovery of a novel small molecule scaffold that inhibits Hsp90. X-ray data show that the scaffold binds competitively at the ATP site on Hsp90. Cellular proliferation and client assays demonstrate that members of the series are able to inhibit Hsp90 at nanomolar concentrations.