James C. Barrow

Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2 phosphorylation of Tti1/Tel2.

The apoptotic actions of p53 require its phosphorylation by a family of phosphoinositide-3-kinase-related-kinases (PIKKs), which include DNA-PKcs and ATM. These kinases are stabilized by the TTT (Tel2, Tti1, Tti2) cochaperone family, whose actions are mediated by CK2 phosphorylation. The inositol pyrophosphates, such as 5-diphosphoinositol pentakisphosphate (IP7), are generated by a family of inositol hexakisphosphate kinases (IP6Ks), of which IP6K2 has been implicated in p53-associated cell death. In the present study we report an apoptotic signaling cascade linking CK2, TTT, the PIKKs, and p53. We demonstrate that IP7, formed by IP6K2, binds CK2 to enhance its phosphorylation of the TTT complex, thereby stabilizing DNA-PKcs and ATM. This process stimulates p53 phosphorylation at serine 15 to activate the cell death program in human cancer cells and in murine B cells.

Inositol pyrophosphates promote tumor growth and metastasis by antagonizing liver kinase B1

Significance Inositol pyrophosphates are messenger molecules incorporating the energetic pyrophosphate bond. Although they have been implicated in diverse biologic processes, their physiologic functions remain enigmatic. We show that the catalytic activity of inositol hexakisphosphate kinase 2 (IP6K2), one of the principal enzymes generating the inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate), mediates cancer cell migration and tumor metastasis both in cell culture and intact mice. In this process, IP6K2 diminishes cell–cell adhesion, enabling cells to invade the intercellular matrix. Drugs that inhibit IP6K2 may be beneficial in cancer therapy. The inositol pyrophosphates, molecular messengers containing an energetic pyrophosphate bond, impact a wide range of biologic processes. They are generated primarily by a family of three inositol hexakisphosphate kinases (IP6Ks), the principal product of which is diphosphoinositol pentakisphosphate (IP7). We report that IP6K2, via IP7 synthesis, is a major mediator of cancer cell migration and tumor metastasis in cell culture and in intact mice. IP6K2 acts by enhancing cell-matrix adhesion and decreasing cell–cell adhesion. This action is mediated by IP7-elicited nuclear sequestration and inactivation of the tumor suppressor liver kinase B1 (LKB1). Accordingly, inhibitors of IP6K2 offer promise in cancer therapy.

KCNH2 -3.1 expression impairs cognition and alters neuronal function in a model of molecular pathology associated with schizophrenia

Overexpression in humans of KCNH2-3.1, which encodes a primate-specific and brain-selective isoform of the human ether-a-go-go-related potassium channel, is associated with impaired cognition, inefficient neural processing and schizophrenia. Here, we describe a new mouse model that incorporates the KCNH2-3.1 molecular phenotype. KCNH2-3.1 transgenic mice are viable and display normal sensorimotor behaviors. However, they show alterations in neuronal structure and microcircuit function in the hippocampus and prefrontal cortex, areas affected in schizophrenia. Specifically, in slice preparations from the CA1 region of the hippocampus, KCNH2-3.1 transgenic mice have fewer mature dendrites and impaired theta burst stimulation long-term potentiation. Abnormal neuronal firing patterns characteristic of the fast deactivation kinetics of the KCNH2-3.1 isoform were also observed in prefrontal cortex. Transgenic mice showed significant deficits in a hippocampal-dependent object location task and a prefrontal cortex-dependent T-maze working memory task. Interestingly, the hippocampal-dependent alterations were not present in juvenile transgenic mice, suggesting a developmental trajectory to the phenotype. Suppressing KCNH2-3.1 expression in adult mice rescues both the behavioral and physiological phenotypes. These data provide insight into the mechanism of association of KCNH2-3.1 with variation in human cognition and neuronal physiology and may explain its role in schizophrenia.

Development of an HTRF Assay for the Detection and Characterization of Inhibitors of Catechol-O-Methyltransferase

Catechol-O-methyltransferase (COMT) plays an important role in the deactivation of catecholamine neurotransmitters and hormones. Inhibitors of COMT, such as tolcapone and entacapone, are used clinically in the treatment of Parkinson’s disease. Discovery of novel inhibitors has been hampered by a lack of suitable assays for high-throughput screening (HTS). Although assays using esculetin have been developed, these are affected by fluorescence, a common property of catechol-type compounds. We have therefore evaluated a new homogenous time-resolved fluorescence (HTRF)–based assay from CisBio (Codolet, France), which measures the production of S-adenosyl-L-homocysteine (SAH). The assay has been run in both HTS and medium-throughput screening (MTS) modes. The assay was established using membranes expressing human membrane-bound COMT and was optimized for protein and time to give an acceptable signal window, good potency for tolcapone, and a high degree of translation between data in fluorescence ratio and data in terms of [SAH] produced. pIC50 values for the hits from the HTS mode were determined in the MTS mode. The assay also proved suitable for kinetic studies such as Km,app determination.

Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function

Significance The regulation of ubiquitylation is critical to maintain proteomic and cellular homeostasis. The cullin-RING E3 ubiquitin ligases (CRLs) mediate one-fifth of all ubiquitylation, but their regulation is largely unknown. Here, we describe how the generation of a small metabolite, inositol hexakisphosphate (IP6), locally switches two CRLs from their active to their inactive state by means of stabilizing their interaction with an inhibitor: the constitutive photomorphogenesis 9 signalosome. Furthermore, we demonstrate the physiologic consequences of IP6 depletion on CRL dysregulation, CRL substrate levels, and global cellular phenotypes. Targeting IP6 synthesis synergizes with the cytotoxic effect of CRL inhibition, which may have therapeutic relevance. The family of cullin-RING E3 Ligases (CRLs) and the constitutive photomorphogenesis 9 (COP9) signalosome (CSN) form dynamic complexes that mediate ubiquitylation of 20% of the proteome, yet regulation of their assembly/disassembly remains poorly understood. Inositol polyphosphates are highly conserved signaling molecules implicated in diverse cellular processes. We now report that inositol hexakisphosphate (IP6) is a major physiologic determinant of the CRL–CSN interface, which includes a hitherto unidentified electrostatic interaction between the N-terminal acidic tail of CSN subunit 2 (CSN2) and a conserved basic canyon on cullins. IP6, with an EC50 of 20 nM, acts as an intermolecular “glue,” increasing cullin–CSN2 binding affinity by 30-fold, thereby promoting assembly of the inactive CRL–CSN complexes. The IP6 synthase, Ins(1,3,4,5,6)P5 2-kinase (IPPK/IP5K) binds to cullins. Depleting IP5K increases the percentage of neddylated, active Cul1 and Cul4A, and decreases levels of the Cul1/4A substrates p27 and p21. Besides dysregulating CRL-mediated cell proliferation and UV-induced apoptosis, IP5K depletion potentiates by 28-fold the cytotoxic effect of the neddylation inhibitor MLN4924. Thus, IP5K and IP6 are evolutionarily conserved components of the CRL–CSN system and are potential targets for cancer therapy in conjunction with MLN4924.

Dissecting transcriptomic signatures of neuronal differentiation and maturation using iPSCs

Human induced pluripotent stem cells (hiPSCs) are a powerful model of neural differentiation and maturation. We present a hiPSC transcriptomics resource on corticogenesis from 5 iPSC donor and 13 subclonal lines across nine time points over 5 broad conditions: self-renewal, early neuronal differentiation, neural precursor cells (NPCs), assembled rosettes, and differentiated neuronal cells that were validated using electrophysiology. We identified widespread changes in the expression of individual transcript features and their splice variants, gene networks, and global patterns of transcription. We next demonstrated that co-culturing human NPCs with rodent astrocytes resulted in mutually synergistic maturation, and that cell type-specific expression data can be extracted using only sequencing read alignments without potentially disruptive cell sorting. We lastly developed and validated a computational tool to estimate the relative neuronal maturity of iPSC-derived neuronal cultures and human brain tissue, which were maturationally heterogeneous but contained subsets of cells most akin to adult human neurons.

Optimization of 8-Hydroxyquinolines as Inhibitors of Catechol O-Methyltransferase

A series of 8-hydroxy quinolines were identified as potent inhibitors of catechol O-methyltransferase (COMT) with selectivity for the membrane-bound form of the enzyme. Small substituents at the 7-position of the quinoline were found to increase metabolic stability without sacrificing potency. Compounds with good pharmacokinetics and brain penetration were identified and demonstrated in vivo modulation of dopamine metabolites in the brain. An X-ray cocrystal structure of compound 21 in the S-COMT active site shows chelation of the active site magnesium similar to catechol-based inhibitors. These compounds should prove useful for treatment of many neurological and psychiatric conditions associated with compromised cortical dopamine signaling.

Novel inhibitors of As(III) S-adenosylmethionine methyltransferase (AS3MT) identified by virtual screening

Due to increased interest in As(III) S-adenosylmethionine methyltransferase (AS3MT), a search for chemical probes that can help elucidate function was initiated. A homology model was built based on related enzymes, and virtual screening produced 426 potential hits. Evaluation of these compounds in a functional enzymatic assay revealed several modest inhibitors including an O-substituted 2-amino-3-cyano indole scaffold. Two iterations of near neighbor searches revealed compound 5 as a potent inhibitor of AS3MT with good selectivity over representative methyltransferases DOT1L and NSD2 as well as a representative set of diverse receptors. Compound 5 should prove to be a useful tool to investigate the role of AS3MT and a potential starting point for further optimization.