G protein betagamma subunits as targets for small molecule therapeutic development

G proteins mediate the action of G protein coupled receptors (GPCRs), a major target of current pharmaceuticals and a major target of interest in future drug development. Most pharmaceutical interest has been in the development of selective GPCR agonists and antagonists that activate or inhibit specific GPCRs. Some recent thinking has focused on the idea that some pathologies are the result of the actions of an array of GPCRs suggesting that targeting single receptors may have limited efficacy. Thus, targeting pathways common to multiple GPCRs that control critical pathways involved in disease has potential therapeutic relevance. G protein betagamma subunits released from some GPCRs upon receptor activation regulate a variety of downstream pathways to control various aspects of mammalian physiology. There is evidence from cell- based and animal models that excess Gbetagamma signaling can be detrimental and blocking Gbetagamma signaling has salutary effects in a number of pathological models. Gbetagamma regulates downstream pathways through modulation of enzymes that produce cellular second messengers or through regulation of ion channels by direct protein-protein interactions. Thus, blocking Gbetagamma functions requires development of small molecule agents that disrupt Gbetagamma protein interactions with downstream partners. Here we discuss evidence that small molecule targeting Gbetagamma could be of therapeutic value. The concept of disruption of protein-protein interactions by targeting a "hot spot" on Gbetagamma is delineated and the biochemical and virtual screening strategies for identification of small molecules that selectively target Gbetagamma functions are outlined. Evaluation of the effectiveness of virtual screening indicates that computational screening enhanced identification of true Gbetagamma binding molecules. However, further refinement of the approach could significantly improve the yield of Gbetagamma binding molecules from this screen that could result in multiple candidate leads for future drug development.     

State-selective binding peptides for heterotrimeric G-protein subunits: novel tools for investigating G-protein signaling dynamics

Heterotrimeric G-proteins, comprising Galpha, Gbeta, and Ggamma subunits, are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive state in which GDP-bound Galpha is complexed with the Gbetagamma dimer, and an active state in which GTP-bound Galpha is freed of its Gbetagamma binding partner. Structural studies have illustrated the basis for the distinct conformations of these states which are regulated by alterations in three precise 'switch regions' of the Galpha subunit. Discrete differences in conformation between GDP- and GTP-bound Galpha underlie its nucleotide-dependent protein-protein interactions (e.g., with Gbetagamma/receptor and effectors, respectively) that are critical for maintaining their proper nucleotide cycling and signaling properties. Recently, several screening approaches have been used to identify peptide sequences capable of interacting with Galpha (and free Gbetagamma) in nucleotide-dependent fashions. These peptides have demonstrated applications in direct modulation of the nucleotide cycle, assessing the structural basis for aspects of Galpha and Gbetagamma signaling, and serving as biosensor tools in assays for Galpha activation including high throughput drug screening. In this review, we highlight some of the methods used for such discoveries and discuss the insights that can be gleaned from application of these identified peptides.     

Foldable subunits of helix protein

Detection of foldable subunits in proteins is an important approach to understand their evolutions and find building motifs for de novo protein design. Using united-residue model, we simulated the folding of a six-helix protein with a length of 120 amino acids (C-terminal domain of Ku86). The folding behaviors, structural topology and sequence repetition of this protein all suggest that it may have a two-fold quasi-repetition or symmetry in its sequence and structure. Therefore, we simulated the folding of its two halves (1–60 and 61–120 amino acids) and find that they can fold into native conformations independently. It is also found that their folding behaviors are very similar to other three-helix bundles. This suggests that this protein may be divided into two foldable halves.     

Switching of pseudorotaxanes and catenanes incorporating a tetrathiafulvalene unit by redox and chemical inputs

An acyclic polyether 1a, incorporating a central tetrathiafulvalene (TTF) electron donor unit and two 4-tert-butylphenoxy groups at its termini, has been synthesized. Two macrocyclic polyethers containing two different electron donors, namely a TTF unit with, in one case, a 1,4-dioxybenzene ring (2a), and, in the other case (2b), a 1,5-dioxynaphthalene ring system, have also been synthesized. These two macrocyclic polyethers have been mechanically interlocked in kinetically controlled template-directed syntheses with cyclobis(paraquat-p-phenylene) cyclophane (3(4+)) to afford the [2]catenanes 2a/3(4+) and 2b/3(4+), respectively. X-ray crystallography reveals that the [2]-catenane 2b/3(4+) has the TTF unit of 2b located inside the cavity of 3(4+). The spectroscopic (UV/vis and 1H NMR) and electrochemical properties of compounds 1a, 2a, 2b, 2a/3(4+), and 2b/3(4+) and of the [2]pseudorotaxane 1a.3(4+) were investigated. The absorption and emission properties of the mono- and dioxidized forms of the TTF unit in these various species have also been studied. The results obtained in acetonitrile solution can be summarized as follows. (a) While TTF2+ exhibits a strong fluorescence, no emission can be observed for the TTF2+ units contained in the polyethers and in their pseudorotaxanes and catenanes. (b) A donor-acceptor absorption band is observed upon two-electron oxidation of the TTF unit in the macrocyclic polyethers 2a and 2b. (c) The spontaneous self-assembly of 1a and 3(4+) to give the [2]pseudorotaxane 1a.3(4+) is strongly favored (Kass. = 5 x 10(5) L mol-1) but slow (at 296 K, k = 11.3 L mol-1 s-1 and delta G++ = 15.9 kcal mol-1) because of the steric hindrance associated with the bulky end groups of 1a. (d) In the pseudorotaxane 1a.3(4+), the reversible displacement of the cyclophane from the TTF unit in the threadlike substrate occurs on oxidation/reduction of its electroactive components. (e) Switching between the two translational isomers of the catenanes 2a/3(4+) and 2b/3(4+) occurs by cyclic oxidation and reduction of the TTF unit contained in 2a and in 2b, respectively. (f) Addition of o-chloroanil to the pseudorotaxane 1a.3(4+) and to the catenanes 2a/3(4+) and 2b/3(4+) causes the displacement of the TTF unit from the cavity of the cyclophane 3(4+) because of the formation of an adduct between the TTF unit and o-chloroanil.     

Stimulatory heterotrimeric G protein augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 human lung cancer cells

Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the α subunit of Gs (GαsQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GαsQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked GαsQL-stimulated Bak reporter luciferase activity. Expression of GαsQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Gαs activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Gαs augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.     

Established and emerging fluorescence-based assays for G-protein function: heterotrimeric G-protein alpha subunits and regulator of G-protein signaling (RGS) proteins

Heterotrimeric G-proteins are molecular switches that couple serpentine receptors to intracellular effector pathways and the regulation of cell physiology. Ligand-bound receptors cause G-protein alpha subunits to bind guanosine 5'-triphosphate (GTP) and activate effector pathways. Signal termination is facilitated by the intrinsic GTPase activity of G-protein alpha subunits. Regulators of G-protein signaling (RGS) proteins accelerate the GTPase activity of the G-protein alpha subunit, and thus negatively regulate G-protein-mediated signal transduction. In vitro biochemical assays of heterotrimeric G-proteins commonly include measurements of nucleotide binding, GTPase activity, and interaction with RGS proteins. However, the conventional assays for most of these processes involve radiolabeled guanine nucleotide analogues and scintillation counting. In this article, we focus on fluorescence-based methodologies to study heterotrimeric G-protein alpha subunit regulation in vitro. Furthermore, we consider the potential of such techniques in high-throughput screening and drug discovery.     

Magnetic nanoparticles--templated assembly of protein subunits: a new platform for carbohydrate-based MRI nanoprobes

A new approach for the preparation of carbohydrate-coated magnetic nanoparticles is reported. In a first step, we show that the pH-driven assembly-disassembly natural process that occurs in apoferritin protein is effective for the encapsulation of maghemite nanoparticles of different sizes: 4 and 6 nm. In a second step, we demonstrate that the presence of functional amine groups in the outer shell of apoferritin allows functionalization with two carbohydrates, N-acetyl-D-glucosamine and d-mannose. High-resolution electron microscopy (HREM), high angle annular dark field scanning electron microscopy (HAADF-STEM), electron energy loss spectroscopy (EELS), X-ray diffraction (XRD), and SQUID technique have been used to characterize the magnetic samples, termed herein Apomaghemites. The in vivo magnetic resonance imaging (MRI) studies showed the efficiency in contrasting images for these samples; that is, the r(2) NMR relaxivities are comparable with Endorem (a commercial superparamagnetic MRI contrast agent). The r(2) relaxivity values as well as the pre-contrast and post-contrast T(2)*-weighted images suggested that our systems could be used as perspective superparamagnetic contrast agents for magnetic resonance imaging (MRI). The carbohydrate-functionalized Apomaghemite nanoparticles retained their recognition abilities, as demonstrated by the strong affinity with their corresponding carbohydrate-binding lectins.     

Identification of a novel protein regulating microtubule stability through a chemical approach

To identify novel proteins regulating the microtubule cytoskeleton, we screened a library of purine derivatives using mitotic spindle assembly in Xenopus egg extracts as an assay. Out of a collection of 1561 compounds, we identified 15 that destabilized microtubules without targeting tubulin directly, resulting in small spindles. Affinity chromatography with one compound, named diminutol, revealed a potential target as NQO1, an NADP-dependent oxidoreductase. A role for NQO1 in influencing microtubule polymerization was confirmed through inhibition studies using known inhibitors and immunodepletion. Therefore, this chemical approach has identified a novel factor required for microtubule morphogenesis and cell division.     

Simplified capillary isoelectric focusing with chemical mobilization for intact protein analysis

We report a capillary isoelectric focusing system based on a sequential injection method for simplified chemical mobilization. This system was coupled to an ion trap mass spectrometer with an electrokinetically pumped nanoelectrospray interface. The nanoelectrospray emitter employed an acidic sheath electrolyte. To simplify focusing and mobilization, a plug of ammonium hydroxide was first injected into the capillary, followed by a section of mixed sample and ampholyte. During focusing, the NH₃H₂O section worked as catholyte. As focusing progressed, the NH₃H₂O section was titrated to lower pH by the acidic sheath electrolyte. Chemical mobilization started automatically once the ammonium hydroxide was consumed by the acidic sheath flow electrolyte, which then acted as the mobilization solution. In this report, the lengths of the NH₃H₂O section and sample were optimized. With a 1 m long capillary, a relative short plug of the NH₃H₂O section (3 cm) produced both fast migration and reasonable separation resolution. The simplified capillary isoelectric focusing mass spectrometry system produced base peak intensity relative standard deviation of 8.5% and migration time relative standard deviation ≤0.6% for myoglobin and cytochrome C in triplicate runs.     

Probing structure in invisible protein states with anisotropic NMR chemical shifts

A general method for obtaining quantitative structural information on invisible, excited protein states by solution-based NMR spectroscopy is presented. The approach exploits relaxation dispersion techniques in which changes in chemical shifts between ground and excited states are monitored in solutions with and without small amounts of residual molecular alignment. This allows the calculation of differences in chemical shifts induced by alignment that can be directly related to molecular structure, in cases where the orientation and magnitude of the chemical-shift tensor are well defined. An example using carbonyl chemical shifts as probes of a protein-ligand binding reaction is presented to illustrate and validate the method.     

Heat shock protein 27 interacts with vimentin and prevents insolubilization of vimentin subunits induced by cadmium

Vimentin is an intermediate filament that regulates cell attachment and subcellular organization. In this study, vimentin filaments were morphologically altered, and its soluble subunits were rapidly reduced via cadmium chloride treatment. Cadmium chloride stimulated three major mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and led apoptotic pathway via caspase-9 and caspase-3 activations. In order to determine whether MAPKs were involved in this cadmium-induced soluble vimentin disappearance, we applied MAPK-specific inhibitors (PD98059, SP600125, SB203580). These inhibitors did not abolish the cadmium-induced soluble vimentin disappearance. Caspase and proteosome degradation pathway were also not involved in soluble vimentin disappearance. When we observed vimentin levels in soluble and insoluble fractions, soluble vimentin subunits shifted to an insoluble fraction. As we discovered that heat-shock protein 27 (HSP27) was colocalized and physically associated with vimentin in unstressed cells, the roles of HSP27 with regard to vimentin were assessed. HSP27-overexpressing cells prevented morphological alterations of the vimentin filaments, as well as reductions of soluble vimentin, in the cadmium-treated cells. Moreover, HSP27 antisense oligonucleotide augmented these cadmium-induced changes in vimentin. These findings indicate that HSP27 prevents disruption of the vimentin intermediate filament networks and soluble vimentin disappearance, by virtue of its physical interaction with vimentin in cadmium-treated SK-N-SH cells.     

A chemical reporter for protein AMPylation

Protein AMPylation is an emerging post-translational modification, which plays key roles in bacterial pathogenesis and cell biology. Enzymes with AMPylation activity, referred to as AMPylators, have been identified in several bacterial pathogens and eukaryotes. To facilitate the study of this unique modification, we developed an alkynyl chemical reporter for detection and identification of protein AMPylation substrates. Covalent functionalization of AMPylation substrates with the alkynyl reporter in lieu of adenylyl 5'-monophosphate (AMP) allows their subsequent bioorthogonal ligation with azide-fluorescent dyes or affinity enrichment tags. We show that this chemical reporter is transferred by a range of AMPylators onto their cognate protein substrates and allows rapid detection and identification of AMPylated substrates.     

Favoring heterotrimeric boroxine formation using an internal Lewis base: a computational study

Heterotrimeric arylboroxines can be favorably formed by designing one of the arylboronic acid monomers to contain a pendant Lewis base. Using density functional theory (B3LYP//6-311+G*) calculations including Poisson-Boltzmann implicit solvent, we found that AB2 trimeric arylboroxines were thermodynamically favored over A2B, A3, or B3, where A and B are monomeric arylboronic acids with and without a pendant Lewis base, respectively. The most stable AB2 trimers were formed when the B monomer contained electron-withdrawing substituents, particularly halogens in the para-position or pi-acceptors in the meta-position. On the other hand, adding different para-substituents to the A monomer did not significantly change the energetics. Our calculations also suggest that ABC trimers with three different monomers will not be significantly favored over AB2 trimers when making small electronic perturbations, by changing the substituents on each monomer.     

A Photoresponsive Receptor with a 105 Magnitude of Reversible Anion-Binding Switching

In a leap toward anion separation that uses only energy input for binding and release cycles, we report herein a new class of photoswitchable anion receptors featuring a diiminoguanidinium functionality that displays a change of more than five orders of magnitude in switched-off binding strength towards sulfate, a representative oxyanion, upon photoirradiation with UV light. The (E,E)-2-pyridyl-diiminoguanidinium cation, synthesized as the triflate salt, binds sulfate with extraordinary strength in [D₆]DMSO owing to its bidentate guanidinium hydrogen bonding, which can chelate the O−S−O edge of sulfate. Upon photoisomerization to the Z,Z isomer, the anion-binding site is essentially shut off by intramolecular hydrogen bonds to the 2-pyridyl substituents, as shown by anion-binding titrations, theoretical calculations, and X-ray structural analysis. This approach will allow the development of advanced anion-separation cycles that use only energy input and generate no chemical waste, and thus address challenging chemical separation problems in a more sustainable way.     

Switching Kr/Xe selectivity with temperature in a metal-organic framework

Krypton (Kr) and xenon (Xe) adsorption on two partially fluorinated metal-organic frameworks (FMOFCu and FMOFZn) with different cavity size and topologies are reported. FMOFCu shows an inversion in sorption selectivity toward Kr at temperatures below 0 °C while FMOFZn does not. The 1D microtubes packed along the (101) direction connected through small bottleneck windows in FMOFCu appear to be the reason for this peculiar behavior. The FMOFCu shows an estimated Kr/Xe selectivity of 36 at 0.1 bar and 203 K.