The With-No-Lysine (K) (WNK) kinases are a family of serine/threonine kinases, firsted in a screen for novel mitogen-activated protein kinase (MAPK) kinases. Another unique feature of WNKs is their regulation by chloride, which directly binds to the kinase active site and inhibits autophosphorylation and kinase activation. The best-understood function of WNKs is their ability to phosphorylate SPAK and OSR1. Activated SPAK and OSR1 phosphorylate members of the SLC12 family of cation-chloride cotransporters. These include the three related sodium-coupled chloride cotransporters, NCC (sodium chloride cotransporter), and the sodium-potassium-2-chloride cotransporters NKCC1 and NKCC2, as well as the potassium-coupled chloride cotransporters, KCC1–4 (potassium chloride cotransporters).
Phosphorylation of NCC, NKCC1 and NKCC2 results in transporter activation, whereas phosphorylation of KCCs results in transporter inactivation. Regulation of these transporters by WNKs are important for cell volume control, transepithelial ion transport, and the regulation of intracellular chloride concentration. In neurons, for example, intracellular chloride concentration determines whether activation of ligand-gated chloride channels, such as the GABAA or glycine receptors, results in a hyperpolarizing or depolarizing effect.
1.Rodan AR,et al. Curr Top Dev Biol. 2017;123:1–47.
Leukotrienes (LTs) are a class of mediators derived from arachidonic acid by the initiating activity of 5-lipoxygenase and 5-lipoxygenase–activating protein (FLAP). They are involved in self-defense systems against foreign bodies or microorganisms, but overproduction causes a variety of immune and inflammatory diseases. Two G protein–coupled receptors (GPCRs) have been cloned as receptors for leukotriene B4 (LTB4). The first, BLT1, known as a high-affinity LTB4 receptor, is expressed in various subsets of leukocytes and is responsible for LTB4-dependent leukocyte migration. The second, BLT2, was originally reported as a low-affinity LTB4 receptor and is now considered as a receptor for various oxidized fatty acids, including 12-hydroxyheptadecatrienoic acid (12-HHT) and hydroxyeicosatetraenoic acids (HETEs). BLT2 is expressed in epidermal keratinocytes and epithelial cells of intestine, cornea, and lung and is responsible for wound healing and epidermal barrier function.
Human BLT1 consists of 352 amino acids and is mainly expressed in various subsets of leukocytes, including granulocytes, eosinophils, and effector-type CD4+ and CD8+ T cells, as well as certain subsets of dendritic cells and macrophages. BLT1 stimulation in leukocytes leads to degranulation through the production of phosphatidylinositol tris-phosphates (IP3) via activation of phosphatidylinositol-3-kinase (PI3 kinase). LTB4 also activates phagocytosis in macrophages through the activation of Gi, PI3 kinase, Rac, and Syk. Recently, the receptor for advanced glycation end products (RAGE) was identified as a BLT1-binding protein that regulates BLT1 signaling. RAGE functions as a molecular switch for BLT1, inhibits BLT1-dependent NF-κB activation, and stimulates BLT1-dependent chemotaxis. RAGE was also shown to bind to GPCRs other than BLT1 and is a new class of GPCR modulator and a new target of GPCR study. Five CysLT receptors have been identified: CysLT1, CysLT2, P2Y12, GPR99, and GPR17. CysLT1 is expressed in a variety of inflammatory cells, i.e., neutrophils, mast cells, and monocytes/macrophages, activation of CysLT1 by LTD4 results in the production of several second intracellular messengers through phospholipase Cβ.CysLT2 negatively regulates the development of Th2 pulmonary inflammation by inhibiting the CysLT1 functions on dendritic cells.
1.Yokomizo T,et al. J Clin Invest. 2018;128(7):2691–2701.
PD-1 or programmed death-1 was first identified and cloned in 1992 as a gene transcriptionally induced upon programmed cell death in T cells. It was subsequently found to be an expressed on activated T, B and myeloid cells, and its cytoplasmic region contains an immunoreceptor tyrosine-based inhibitory motif. PD-1 was a T cell molecule that limited T cell activation and proliferation and promoted tolerance. PD-1 is expressed at low levels by resting T cells and has been shown to be expressed by what some investigators have referred to as “exhausted” T cells in animal models of chronic infection.
In patients with multiple different types of epithelial, hematologic and other malignancies, high levels of PD-1 are detected in circulating and tumor-infiltrating lymphocytes including T cells that are tumor antigen specific, presumably due to chronic antigenic stimulation. These tumor-associated PD-1 expressing effector cells are dysfunctional, and their biological activity can be partially recovered using PD-1 or PD-L1 blocking antibodies. The ligands for the PD-1 receptor are PD-L1 and PD-L2, which are molecules found on antigen-presenting cells, and in the case of PD-L1, it is often expressed on epithelial and other types of tumor cells, resulting in an inhibitory interaction within the tumor microenvironment. The induction of PD-L1 expression by tumor cells may be an adaptive resistance mechanism for tumor cells in response to a developing antitumor immune response. In addition, oncogenic PTEN loss is associated with increased PD-L1 expression in glioma and triple-negative breast cancer cells.
PD-L1 expression can thus also be up-regulated by intrinsic oncogenic pathways as well as interferon-gamma secreted by infiltrating immune cells. PD-L1 and PD-1 are expressed by different cellular components in the tumor microenvironment, where their interaction can inhibit T cell immunity. The engagement of PD-1 by its principal ligand PD-L1 may result in apoptosis in T cells, anergy, “exhaustion”, and secretion of interleukin-10. PD-L1 expression may protect tumor cells expressing it from CD8+T cell-mediated lysis. In addition to PD-1, an interaction between PD-L1 and CD80 has been demonstrated in mouse models. PD-L1 acts as a receptor to “back” transmit signals into T cells and tumor cells to impact on their survival, whose mechanism is yet to be determined. Finally, PD-1 and PD-L1 can be expressed on T regulatory cells and may control their function, and innate immune effectors like NKT cells may also involve the PD-1/PD-L1 circuit.
1.Balar AV,et al. Cancer Immunol Immunother. 2017;66(5):551–564.
STING (Stimulator of Interferon Genes, also known as TMEM173, MITA, ERIS, and MPYS) is an adapter transmembrane protein that resides in the endoplasmic reticulum (ER). In eukaryotic cells, activation of STING occurs when double stranded DNA gains access to the cytosol. This pathway was originally uncovered in search for a mechanism by which DNA viruses could be sensed by the host immune system. STING pathway activation also can occur with certain bacterial and parasitic infections, and more recently has been described to occur under conditions when mammalian DNA itself can attain access to the cytosol. Cytosolic DNA is detected upon binding to the sensor cyclic-GMP-AMP synthase (cGAS, MB21D1), which catalyzes the synthesis of cyclic GMP-AMP (cGAMP) from guanosine triphosphate (GTP) and adenosine triphosphate (ATP). cGAMP functions as a second messenger that binds and activates STING. Upon binding of cGAMP, STING undergoes conformational changes that trigger its trafficking from the ER to the Golgi to perinuclear endosomes. Consequently, STING recruits tank-binding kinase 1 (TBK1) and is, in turn, phosphorylated by TBK1, which renders it accessible for the binding of the transcription factor interferon regulatory factor 3 (IRF3). TBK1 then phosphorylates IRF3 which translocates to the nucleus to drive transcription of IFN-β and other genes.
1.Leticia Corrales,et al. Clin Cancer Res. 2015 Nov 1; 21(21): 4774–4779.
The translocator protein (18 kDa) (TSPO) is a five transmembrane domain protein that is localized primarily in the outer mitochondrial membrane and is expressed predominantly in steroid-synthesizing tissues, including the brain. TSPO is involved in the translocation of cholesterol from the outer to the inner mitochondrial membrane, which is the rate-limiting step in the synthesis of steroids and neurosteroids. TSPO is currently under investigation as a biomarker of brain inflammation and reactive gliosis that are associated with various neuropathologies. Although TSPO is expressed in many organs, the highest levels are found in tissues containing steroidsynthesizing cells, such as adrenal, gonad and brain cells. As a major component of the outer mitochondrial membrane, TSPO mediates various mitochondrial functions, including cholesterol transport and steroid hormone synthesis, mitochondrial respiration, mitochondrial permeability transition (mPT) pore opening, apoptosis, and cell proliferation.
1.Rupprecht R, et al. Nat Rev Drug Discov. 2010;9(12):971–988.
Proteolysis targeting chimeras (PROTACs) are bivalent ligands in which a compound that binds to the protein target of interest is connected to a second molecule that binds an E3 ligase via a linker. Potentially, a mode of action whereby a target protein is degraded has several advantages over a more traditional therapeutic approach based on inhibition. First, a PROTAC compound only needs to bring the E3 protein close to the target of interest. The PROTAC ligand acts like a catalyst, and even sub-stoichiometric amounts of a PROTAC therapeutic can be expected to achieve (near) complete protein degradation. In the absence of small-molecule E3 binders, other peptidic PROTACs were developed that were sufficiently small to be cell permeable. E3 ligase ligands used for PROTACs including: thalidomide derivatives targeting Cereblon. Bestatin and compound 7 are ligands of cIAP, nutlin is a ligand of MDM2.
1.Scheepstra M,et al. Comput Struct Biotechnol J. 2019;17:160–176.
The ubiquitin--proteasome system (UPS) consists of a series of enzymatic reactions wherein ubiquitin, a 76-amino-acid polypeptide, is conjugated onto specific lysine residues of a variety of protein substrates via conjugation. The fate of ubiquitinated proteins is determined by: the number of ubiquitin residues (ub-chains), the position of conjugations, and the specific branching patterns of poly-ubiquitin chains. Protein ubiquitination (Ub) is a reversible mechanism (deubiquitination) regulated by DUBs, which has emerged as a cardinal regulatory target for posttranslational modifications which, like protein phosphorylation, adds to the complexity and sensitivity of the UPS. DUBs are a class of the human family of proteases that remove ubiquitin or ubiquitin-like proteins from a variety of protein substrates. DUBs selectively cleave the isopeptide bond present at the C-terminus of ubiquitin molecule. DUBs regulate diverse cellular processes and functions including processing or removal of polyubiquitin moieties or chains on proteins, thereby altering the targeted protein’s fate with regard to either degradation by proteasome and/or differential subcellular localization. In addition, DUBs regulate gene expression, apoptosis, cell cycle, DNA repair as well as cytokine signaling.
1.Farshi P,et al. Expert Opin Ther Pat. 2015;25(10):1191-1208.
Two transmembrane proteins are crucial for intracellular transmission of the Hh signal: the tumor suppressor membrane protein Patched (Ptch1) and the seven-transmembrane domain G-protein coupled receptor-like protein Smoothened (Smo). The activity of Smo affects the bifunctional glioma-associated oncogene transcription factors (Gli), which are transcriptional effectors of the Hh pathway. Gli proteins are members of the family of Kruppel-like factors with highly conserved Zn finger DNA-binding domains. In mammals, they are represented by three proteins: Gli1, Gli2 and Gli3. Gli1 acts principally as a transcriptional activator, whereas Gli2 and Gli3 display both activator and repressor functions. Gli proteins are extensively modified posttranslationally, and these modifications largely determine their trafficking in the cell and ultimate transcriptional output of the Hh pathway.
Among the posttranslational modifications of Gli1/2/3 are: phosphorylation, ubiquitination/proteasomal truncation, acetylation, sumoylation, methylation, and O-GlcNAcylation. In the absence of genetic or epigenetic alterations at the level of the canonical Hh pathway components, overactivation of Gli proteins may occur non-canonically via cross-talk with other signaling pathways. Among pro-tumorigenic pathways that regulate Gli proteins independently of the canonical Ptch1-Smo axis are: the mitogen-activated kinases (MAPK) cascade, the transforming growth factor beta (TGFβ) pathway, the epidermal growth factor receptor (EGFR) pathway, and the fibroblast growth factor receptor (FGFR) pathway.
1.Niewiadomski P, et al. Cells. 2019;8(2):147. Published 2019 Feb 11.
TNIK was originally identified as a new member of the Germinal Center Kinase (GCK) family. It is known that TNIK regulates the c‐Jun N‐terminal kinase (JNK) pathway through its C‐terminus and the nuclear factor‐κB (NF‐κB) signaling pathways through its N‐terminal kinase domain. In addition, TNIK has been shown to regulate the filamentous‐actin (F‐actin) cytoskeleton. TCF‐4, β‐catenin and TNIK proteins form a complex in colorectal cancer cells. TNIK phosphorylates the TCF‐4 protein at the conserved serine. This phosphorylation is essential for full activation of Wnt signaling. Knockdown of TNIK decreased the transcriptional activity of the TCF‐4 and β‐catenin complex and inhibited the growth of colorectal cancer cells and xenografts. This growth inhibition was abrogated by expression of the catalytic domain of TNIK.
The regulation of Wnt signaling by TNIK is conserved across species. Xenopus TNIK lacks the C‐terminal regulatory portion that is present in human TNIK, but the kinase domain is conserved. Xenopus TNIK is also essential for β‐catenin‐mediated determination of the dorsal axis. TNIK is an essential regulatory component of Wnt signaling, and colorectal cancer cells are highly dependent upon the expression and catalytic activity of TNIK for proliferation. Targeting of TNIK for pharmacological intervention was, thus, anticipated to inhibit Wnt signaling and suppress the growth of colorectal cancer cells.
1.Yamada T,et al. Cancer Sci. 2017;108(5):818–823.
AZ32 is an orally bioavailable and blood-brain barrier-penetratingATMinhibitor with anIC50of <6.2 nM for ATM enzyme, and anIC50of 0.31 μM for ATM in cell.
Astragaloside IV, an active component isolated from Astragalus membranaceus, suppresses the activation of ERK1/2 and JNK, and downregulates matrix metalloproteases (MMP)-2, (MMP)-9 in MDA-MB-231 breast cancer cells.
OTS514 is a highly effective TOPK(T-LAK cell-originated protein kinase) inhibitor (IC50: 2.6 nM).
SB 297006 is an antagonist of C-C chemokine receptor 3 (CCR3; IC50 = 39 nM), which normally is activated by eotaxin, eotaxin-3, MCP-3, MCP-4, RANTES, and MIP-1δ.
Vanillic acid is used as a condiment in food,which used in wine and vinegar. It is an intermediate in the production of vanillin from ferulic acid.
Calceolarioside B displays inhibition of aromatase. Calceolarioside B displays inhibition of human recombinant PKCalpha.