William D. Singer

Structure of the p115RhoGEF rgRGS domain–Gα13/i1 chimera complex suggests convergent evolution of a GTPase activator

p115RhoGEF, a guanine nucleotide exchange factor (GEF) for Rho GTPase, is also a GTPase-activating protein (GAP) for G12 and G13 heterotrimeric Gα subunits. The GAP function of p115RhoGEF resides within the N-terminal region of p115RhoGEF (the rgRGS domain), which includes a module that is structurally similar to RGS (regulators of G-protein signaling) domains. We present here the crystal structure of the rgRGS domain of p115RhoGEF in complex with a chimera of Gα13 and Gαi1. Two distinct surfaces of rgRGS interact with Gα. The N-terminal βN–αN hairpin of rgRGS, rather than its RGS module, forms intimate contacts with the catalytic site of Gα. The interface between the RGS module of rgRGS and Gα is similar to that of a Gα–effector complex, suggesting a role for the rgRGS domain in the stimulation of the GEF activity of p115RhoGEF by Gα13.

Regulation of Rho guanine nucleotide exchange factors by G proteins.

Monomeric Rho GTPases regulate cellular dynamics through remodeling of the cytoskeleton, modulation of immediate signaling pathways, and longer-term regulation of gene transcription. One family of guanine nucleotide exchange factors for Rho proteins (RhoGEFs) provides a direct pathway for regulation of RhoA by cell surface receptors coupled to heterotrimeric G proteins. Some of these RhoGEFs also contain RGS domains that can attenuate signaling by the G(12) and G(13) proteins. The regulation provided by these RhoGEFs is defined by their selective regulation by specific G proteins, phosphorylation by kinases, and potential localization with signaling partners. Evidence of their physiological importance is derived from gene knockouts in Drosophila and mice. Current understanding of the basic regulatory mechanisms of these RhoGEFs is discussed. An overview of identified interactions with other signaling proteins suggests the growing spectrum of their involvement in numerous signaling pathways.

The G protein G13 mediates inhibition of voltage-dependent calcium current by bradykinin

In neuroblastoma-glioma hybrid cells, bradykinin has dual modulatory effects on ion channels: it activates a K+ current as well as inhibits the voltage-dependent Ca2+ current (ICa,V). Both of these actions are mediated by pertussis toxin-insensitive G proteins. Antibodies raised against the homologous Gq and G11 proteins suppress only the activation of the K+ current; this suggested that at least two distinct G protein pathways transduce diverse effects of this transmitter. Here, we show that the inhibition of ICa,V by bradykinin is suppressed selectively by intracellular application of antibodies specific for G13. This novel G protein may play a general role in the inhibition of ICa,V by pathways resistant to pertussis toxin.

Recognition of the Activated States of Gα13 by the rgRGS Domain of PDZRhoGEF

G12 class heterotrimeric G proteins stimulate RhoA activation by RGS-RhoGEFs. However, p115RhoGEF is a GTPase Activating Protein (GAP) toward Galpha13, whereas PDZRhoGEF is not. We have characterized the interaction between the PDZRhoGEF rgRGS domain (PRG-rgRGS) and the alpha subunit of G13 and have determined crystal structures of their complexes in both the inactive state bound to GDP and the active states bound to GDP*AlF (transition state) and GTPgammaS (Michaelis complex). PRG-rgRGS interacts extensively with the helical domain and the effector-binding sites on Galpha13 through contacts that are largely conserved in all three nucleotide-bound states, although PRG-rgRGS has highest affinity to the Michaelis complex. An acidic motif in the N terminus of PRG-rgRGS occupies the GAP binding site of Galpha13 and is flexible in the GDP*AlF complex but well ordered in the GTPgammaS complex. Replacement of key residues in this motif with their counterparts in p115RhoGEF confers GAP activity.

Assays and characterization of mammalian phosphatidylinositol 4,5-bisphosphate-sensitive phospholipase D.

Phospholipase D (PLD) hydrolyzes phospholipids into phosphatidic acid (PA) and the base head groups. In mammalian cells, this activity plays an active role in signal transduction by acting as a mediator for a variety of extracellular stimuli. Two mammalian PLD genes have been identified and the encoded enzymes expressed and characterized. The activity of PLD1 is regulated by the ADPribosylation factor (ARF) and Rho families of monomeric GTPases, classic isoforms of protein kinase C (PKC), and the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ). PLD2, although highly sensitive to PIP 2 has shown only modest response to ARF and appears unresponsive to Rho and PKC, in vitro . Characterization of PLD activity and identification of various regulatory molecules were greatly facilitated by the establishment of an assay for in vitro studies. Alternative methods for the assay of PLD described in this chapter facilitate measurement of basal activity and regulation in the absence of PIP 2 .

Increased dietary cholesterol promotes enhanced mutagenesis in DNA polymerase kappa-deficient mice

DNA polymerase kappa (Polκ) bypasses planar polycyclic N2-guanine adducts in an error-free manner. Cholesterol derivatives may interact with DNA to form similarly bulky lesions. In accordance, these studies examined whether increased mutagenesis of DNA accompanies hypercholesterolemia in Polk-/- mice. These mice also carried apoE gene knockouts to ensure increased levels of plasma cholesterol following exposure to a high cholesterol diet. The mice carried a reporter transgene (the λ-phage cII gene) for subsequent quantitative analysis of mutagenesis in various tissues. We observed significantly increased mutation frequencies in several organs of apoE-/-Polk-/- mice following a high cholesterol diet, compared to those remaining on a standard diet. Regardless of dietary regime, the mutation frequency in many organs was significantly higher in apoE-/-Polk-/- than in apoE-/-Polk+/+ mice. As expected for polycyclic guanine adducts, the mutations mainly consisted of G:C transversions. The life expectancy of apoE-/-Polk-/- mice maintained on a high cholesterol diet was reduced compared to apoE-/-Polk+/+ mice. Overall, this study demonstrates a role for Polκ in bypass of cholesterol-induced guanine lesions.

Structural and Biochemical Analyses Reveal the Mechanism of Glutathione S-Transferase Pi 1 Inhibition by the Anti-cancer Compound Piperlongumine

Glutathione S-transferase pi 1 (GSTP1) is frequently overexpressed in cancerous tumors and is a putative target of the plant compound piperlongumine (PL), which contains two reactive olefins and inhibits proliferation in cancer cells but not normal cells. PL exposure of cancer cells results in increased reactive oxygen species and decreased GSH. These data in tandem with other information led to the conclusion that PL inhibits GSTP1, which forms covalent bonds between GSH and various electrophilic compounds, through covalent adduct formation at the C7-C8 olefin of PL, whereas the C2-C3 olefin of PL was postulated to react with GSH. However, direct evidence for this mechanism has been lacking. To investigate, we solved the X-ray crystal structure of GSTP1 bound to PL and GSH at 1.1 Å resolution to rationalize previously reported structure activity relationship studies. Surprisingly, the structure showed that a hydrolysis product of PL (hPL) was conjugated to glutathione at the C7-C8 olefin, and this complex was bound to the active site of GSTP1; no covalent bond formation between hPL and GSTP1 was observed. Mass spectrometry (MS) analysis of the reactions between PL and GSTP1 confirmed that PL does not label GSTP1. Moreover, MS data also indicated that nucleophilic attack on PL at the C2-C3 olefin led to PL hydrolysis. Although hPL inhibits GSTP1 enzymatic activity in vitro, treatment of cells susceptible to PL with hPL did not have significant anti-proliferative effects, suggesting that hPL is not membrane-permeable. Altogether, our data suggest a model wherein PL is a prodrug whose intracellular hydrolysis initiates the formation of the hPL-GSH conjugate, which blocks the active site of and inhibits GSTP1 and thereby cancer cell proliferation.

Studies of TAK1-centered polypharmacology with novel covalent TAK1 inhibitors

Targeted polypharmacology provides an efficient method of treating diseases such as cancer with complex, multigenic causes provided that compounds with advantageous activity profiles can be discovered. Novel covalent TAK1 inhibitors were validated in cellular contexts for their ability to inhibit the TAK1 kinase and for their polypharmacology. Several inhibitors phenocopied reported TAK1 inhibitor 5Z-7-oxozaenol with comparable efficacy and complementary kinase selectivity profiles. Compound 5 exhibited the greatest potency in RAS-mutated and wild-type RAS cell lines from various cancer types. A biotinylated derivative of 5, 27, was used to verify TAK1 binding in cells. The newly described inhibitors constitute useful tools for further development of multi-targeting TAK1-centered inhibitors for cancer and other diseases.

Abstract A178: Structure guided development of irreversible inhibitors for TAK1

Background: Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is serine/threonine kinase belonging to mitogen-activated protein kinase kinase family that can promote tumor cell survival by modulation of the tumor microenvironment, mediation of stress responses and suppression of pro-apoptotic signaling. Moreover, TAK1 has been identified as essential for the survival of certain KRAS-dependent cancer cells and has therefore been studied as a therapeutic target. Several TAK1 inhibitors including LYTAK1, NG25 and fungal isolate 5Z-7-oxozeanol (5Z-7) have been reported, but these compounds are relatively non-selective as illustrated by 5Z-7 which inhibits numerous other kinases including PKD2, IKKα, Mnk2, Flt3, Flt4, KDR, Trk, PDGFRα, MKK4, NLK, MEK at modest concentrations. Furthermore, 5Z-7 is a resorcyclic lactone with a complex cyclic structure, making it difficult to synthesize derivatives that might have with better selectivity and potency. In an effort to develop new TAK1 inhibitors with excellent selectivity and synthetic accessibility, we studied a series of pyrimidine-based covalent inhibitors which target CYS-174, a cysteine adjacent to the ATP binding site of TAK1. Methods: Structure-guided compound evolution was used to guide design of compounds. Candidate covalent compounds were evaluated for relative binding affinity to TAK1, and the ability to covalently label recombinant TAK1 protein. Co-crystal x-ray structures of selected compounds were additionally solved to guide iterations of compound design. Compounds were evaluated for anti-proliferative activity in TAK1-dependent cell lines from 3 distinct cancer types. Results: Co-crystal structures of TAK1 in complex with the pyrrolopyrimidine-based compound CPT1691 showed an unexpected binding mode leading to the hypothesis that substitution of the pyrrolopyrimidine for a simpler pyrimidine would provide synthetically simpler routes to compounds which covalently bind to Cys-174. This substitution was tolerated, yielding potent TAK1-binding compounds which were further evolved to optimize differential selectivity between TAK1 and other highly related kinases such as MEK1, ERK2 and FLT3. Additional co-crystal structures of these compounds were solved. These inhibitors showed anti-proliferative activity in various TAK1-dependent colon cancer cells (LoVo, SW620, SK-CO-1), pancreatic cancer cells (PANC-1, AsPc-1 and Colo357FG cell lines) and renal cancer cells. Conclusions: Covalent pyrimidine-based TAK1 probes provide an effective means of TAK1 inhibition that may have value as therapeutic agents and scientific tools. Citation Format: Deepak Gurbani, Li Tan, Scott Ficarro, John C. Hunter, William Singer, Faviola B. Vazquez, Ting Xie, Sang Min Lim, Jarrod Marto, Nathanael S. Gray, Kenneth D. Westover. Structure guided development of irreversible inhibitors for TAK1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A178.