A novel approach to local similarity of protein binding sites substantially improves computational drug design results

We present a novel notion of binding site local similarity based on the analysis of complete protein environments of ligand fragments. Comparison of a query protein binding site (target) against the 3D structure of another protein (analog) in complex with a ligand enables ligand fragments from the analog complex to be transferred to positions in the target site, so that the complete protein environments of the fragment and its image are similar. The revealed environments are similarity regions and the fragments transferred to the target site are considered as binding patterns. The set of such binding patterns derived from a database of analog complexes forms a cloud-like structure (fragment cloud), which is a powerful tool for computational drug design. It has been shown on independent test sets that the combined use of a traditional energy-based score together with the cloud-based score responsible for the quality of embedding of a ligand into the fragment cloud improves the self-docking and screening results dramatically. The usage of a fragment cloud as a source of positioned molecular fragments fitting the binding protein environment has been validated by reproduction of experimental ligand optimization results.     

A monoclonal antibody directed against the catalytic site of Bacillus anthracis adenylyl cyclase identifies a novel mammalian brain catalytic subunit.

A brain adenylyl cyclase was shown to contain an epitope closely related to that specified by a conserved sequence containing a nucleotide-binding consensus sequence GXXXXGKS and located in the catalytic sites of bacterial, calmodulin-dependent adenylyl cyclases [Goyard, S., Orlando, C., Sabatier, J.-M., Labruyere, E., d'Alayer, J., Fontan, G., van Rietschoten, J., Mock, M., Danchin, A., Ullmann, A., & Monneron, A. (1989) Biochemistry 28, 1964-1967]. A monoclonal antibody, mab 164, produced against a peptide corresponding to this conserved sequence specifically inhibited the Bordetella pertussis adenylyl cyclase. It also specifically inhibited rat and rabbit brain synaptosomal adenylyl cyclases. The extent of inhibition depended upon the type of enzyme purification, reaching 90% for the calmodulin-sensitive species of enzyme and 20-35% for the forskolin-agarose-retained species. The extent of inhibition in a given fraction also depended upon the effector present. mab 164 reacted on Western blots of forskolin-agarose-retained fractions with a 175-kDa component and did not recognize the Gs alpha stimulatory subunit. Consequently, the 175-kDa protein was considered as a good candidate for an adenylyl cyclase catalyst. The adenylyl cyclase activity contained in forskolin-agarose-retained fractions was further purified on calmodulin-Sepharose. On Western blots of such fractions, mab 164 reacted with a 140-kDa protein, a component that appeared to derive from the 175-kDa protein enriched in the previous step. The kcat of this 140-kDa presumptive adenylyl cyclase was estimated to be of the order of 600 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exploring subtype selectivity and metabolic stability of a novel series of ligands for the benzodiazepine binding site of the GABAA receptor

A novel series of agonists at the benzodiazepine binding site of the GABA_A receptor was prepared by functionalizing a known template. Adding substituents to the pyrazolone-oxygen of CGS-9896 led to a number of compounds with selectivities for either α2- or α1-containing GABA_A receptor subtypes offering an entry into indications such as anxiety and insomnia. In this communication, structure-activity relationship and efforts to increase in vitro stabilities are discussed.     

Ligand binding and structural properties of segments of GABAA receptor alpha 1 subunit overexpressed in Escherichia coli

The gamma-aminobutyric acid, type A (GABA(A)), receptor is the target for numerous therapeutic compounds. In the present study, the Gln(28)-Leu(296), Gln(28)-Arg(276), Gln(28)-Arg(248), and Gln(28)-Glu(165) (numbering of bovine precursor protein) segments of its alpha(1) subunit were overexpressed in Escherichia coli, along with Cys(166)-Leu(296) produced previously, for structural analysis by circular dichroism and ligand binding studies by fluorescence spectroscopy. Results showed that the protein segments were rich in beta-sheet structures. Binding of the fluorescent benzodiazepine Bodipy-FL Ro-1986 was evident from fluorescence resonance energy transfer and fluorescence anisotropy measurements. The binding affinity was in the micromolar range. The binding was attributable more to Cys(166)-Leu(296) than to Gln(28)-Glu(165) and was inhibited by known central benzodiazepine site ligands. Three point mutations, Y187A, T234A, and Y237A, were found to perturb protein secondary structures. Studies with the single Trp mutants W198Y and W273Y indicated that Trp(273) was closer to the binding site than Trp(198).     

A novel substance P binding site in rat brain regions modulates TRH receptor binding

Binding sites for thyrotropin-releasing hormone (TRH) were labelled with [³H](2-Me-His³)TRH ([³H]MeTRH) on membranes from rat brain regions at 0°C for 5 h. Amygdaloid membranes bound [³H]MeTRH with high-affinity (K _d=3.1±0.5 nM (n=4)). Five TRH analogs competed for this binding with the same rank order and with affinities that matched the pharmacological specificity of pituitary TRH receptors. Substance P (SP) and its C-terminal fragments reduced amygdaloid TRH receptor binding in a concentration dependent manner (IC₅₀ for SP=65 M). The rank order of potency of SP analogs at inhibiting TRH receptor binding was: SP>nonapeptide (3–11)>hexapeptide (6–11)>heptapeptide (5–11)>pentapeptide (7–11). However, other tachykinins were inactive in this system. SP was a potent inhibitor of [³H]MeTRH binding in hippocampus> spinal cord>retina>n. accumbens>hypothalamus>amygdaloid>olfactory bulb pituitary>pons/medulla in parallel assays. In amygdaloid membranes SP (50 M) reduced the apparent maximum receptor density by 39% (p<0.01) without altering the binding affinity, and 100 M SP induced a biphasic dissociation of [³H]MeTRH with kinetics faster than those induced by both TRH (10 M) and serotonin (100 M). In contrast, other neuropeptides such as neurotensin, proctolin, angiotensin II, bombesin and luteinizing hormone releasing hormone did not significantly inhibit [³H]MeTRH binding to amydaloid membranes. Thus, the SP site with low affinity in the rat brain is not like any of the previously described tachykinin/neurokinin binding sites but resembles the site found on neuroblastoma cells (108CC15) and on adrenal chromaffin cells that modulate cation permeability and nicotinic receptors respectively. The physiological role of these atypical SP sites in the rat brain remains to be determined.A preliminary account of these studies has been presented to the British Pharmacological Society (9).     

The GABAA receptor alpha 1 subunit Pro174-Asp191 segment is involved in GABA binding and channel gating

The GABA-binding site undergoes structural rearrangements during the transition from agonist binding to channel opening. To define possible roles of the GABA(A) receptor alpha(1) subunit Pro(174)-Asp(191) segment in these processes, we used the substituted cysteine accessibility method to characterize this region. Each residue was individually mutated to cysteine, expressed with wild-type beta(2) subunits in Xenopus laevis oocytes, and examined using two-electrode voltage clamp. Most mutations did not alter GABA EC(50) values. The D183C mutation produced a 7-fold reduction in GABA sensitivity. There were no significant changes in the K(I) values for the competitive antagonist, SR-95531. N-Biotinylaminoethyl methanethiosulfonate modified P174C-, R176C-, S177C-, V178C-, V180C-, A181C-, D183C-, R186C- and N188C-containing receptors. The pattern of accessibility suggests that this protein segment is aqueous-exposed and adopts a random coil conformation. Both GABA and SR-95531 slowed covalent modification of V178C, V180C, and D183C, indicating that these residues may line the GABA-binding site. Further, pentobarbital-induced channel activation accelerated modification of V180C and A181C and slowed the modification of R186C, suggesting that this region of the alpha(1) subunit may act as a dynamic element during channel-gating transitions.     

Tagetone modulates the coupling of flunitrazepam and GABA binding sites at GABAA receptor from chick brain membranes

The effects of tagetone on flunitrazepam (FNTZ) binding to synaptosomal membranes from chick brains in the presence and absence of allosteric modulations induced by gamma-aminobutyric acid (GABA) were investigated. Tagetone, at 50 mu g/ml (final concentration), decreased the binding affinity of [3H]FNTZ to synaptosomal membranes form chick brain (Kd=3.34 +/- 0.36 nM without tagetone and Kd,t=5.86 +/- 0.86 nM with tagetone; p&lt;0.05, two tailed Student's t-test) without affecting maximal binding (Bmax=488 +/- 24 fmoles/mg protein, and Bmax,t=500 +/- 25 fmoles/ mg protein in the absence and in the presence of tagetone respectively). The potency of GABA to stimulate [3H]FNTZ binding increased in the presence of tagetone (EC50 values were 2.78 and 1.12 mu M with and without tagetone respectively). GABA was able to decrease merocyanine Delta A570-610 values in a concentration dependent manner; half maximal effect was attained at a GABA concentration of 34 +/- 13 mu M. Tagetone, at a concentration of 50 mu g/ml and in the presence of GABA 30 mu M or 60 mu M, enhanced the ability of GABA alone on decreasing Delta A570-610. Tagetone alone did not change Delta A570-610 values. FNTZ, a well known GABA modulator, could also potentiate the effect of GABA. Theoretical calculations indicate that the effects on merocyanine Delta A value are mainly exerted at the membrane potential level (Delta Psim). The present results strongly suggest that tagetone affected the function of GABAA receptor in a complex way: on the one hand it impaired FNTZ binding; on the other hand tagetone improved both the coupling between FNTZ and GABA binding sites and it enhanced GABA-induced chloride permeability. Changes in the geometrical and electrostatic properties of the self-organized membrane structure may account for these effects of tagetone.     

A computational screen for site selective A-to-I editing detects novel sites in neuron specific Hu proteins

Background  

Several bioinformatic approaches have previously been used to find novel sites of ADAR mediated A-to-I RNA editing in human. These studies have discovered thousands of genes that are hyper-edited in their non-coding intronic regions, especially in alu retrotransposable elements, but very few substrates that are site-selectively edited in coding regions. Known RNA edited substrates suggest, however, that site selective A-to-I editing is particularly important for normal brain development in mammals.     

GABAA receptor kinetics in the cerebellar nuclei: evidence for detection of transmitter from distant release sites

Neurons of the cerebellar nuclei receive GABAergic input from Purkinje cells. Purkinje boutons have several closely spaced presynaptic densities without GABA transporters, raising the possibility that neurotransmitter released by one presynaptic site diffuses to multiple postsynaptic sites. To test whether such local spillover may contribute to transmission, we studied gating of GABA(A) receptors at 31-33 degrees C in cerebellar nuclear neurons acutely dissociated from mice. Currents were evoked by rapid application of long steps, brief pulses, and high-frequency trains of GABA to outside-out patches. Receptors desensitized and deactivated rapidly, and dose-response measurements estimated an EC(50) of approximately 30 microM. From these data, a kinetic scheme was developed that replicated the recorded currents. Next, we simulated diffusion of GABA in the synaptic cleft, constrained by previous electron microscopic data, and drove the kinetic GABA(A) receptor model with modeled concentration transients. Simulations predicted receptor occupancies of approximately 100% directly opposite the release site and approximately 50% at distant postsynaptic densities, such that receptors up to 700 nm from a release site opened on the timescale of the inhibitory postsynaptic currents before desensitizing. Further simulations of probabilistic release from multiple-site boutons suggested that local spillover-mediated transmission slows the onset and limits the extent of depression during high-frequency signaling.     

Tagetone modulates the coupling of flunitrazepam and GABA binding sites at GABAA receptor from chick brain membranes.

The effects of tagetone on flunitrazepam (FNTZ) binding to synaptosomal membranes from chick brains in the presence and absence of allosteric modulations induced by gamma-aminobutyric acid (GABA) were investigated. Tagetone, at 50 micrograms/ml (final concentration), decreased the binding affinity of [3H]FNTZ to synaptosomal membranes form chick brain (Kd = 3.34 +/- 0.36 nM without tagetone and Kd,t = 5.86 +/- 0.86 nM with tagetone; p < 0.05, two tailed Student's t-test) without affecting maximal binding (Bmax = 488 +/- 24 fmoles/mg protein, and Bmax,t = 500 +/- 25 fmoles/mg protein in the absence and in the presence of tagetone respectively). The potency of GABA to stimulate [3H]FNTZ binding increased in the presence of tagetone (EC50 values were 2.78 and 1.12 microM with and without tagetone respectively). GABA was able to decrease merocyanine delta A570-610 values in a concentration dependent manner; half maximal effect was attained at a GABA concentration of 34 +/- 13 microM. Tagetone, at a concentration of 50 micrograms/ml and in the presence of GABA 30 microM or 60 microM, enhanced the ability of GABA alone on decreasing delta A570-610. Tagetone alone did not change delta A570-610 values. FNTZ, a well known GABA modulator, could also potentiate the effect of GABA. Theoretical calculations indicate that the effects on merocyanine delta A570-610 value are mainly exerted at the membrane potential level (delta psi m). The present results strongly suggest that tagetone affected the function of GABAA receptor in a complex way: on the one hand it impaired FNTZ binding: on the other hand tagetone improved both the coupling between FNTZ and GABA binding sites and it enhanced GABA-induced chloride permeability. Changes in the geometrical and electrostatic properties of the self-organized membrane structure may account for these effects of tagetone.     

Identification of novel cis-regulatory regions from the Notch receptor genes lin-12 and glp-1 of Caenorhabditis elegans

Notch signaling regulates various cellular processes such as growth, proliferation and differentiation, and plays a key role in tissue patterning during animal development. In humans, defects in Notch signaling have been implicated in cancer, stroke, neurodegeneration, as well as learning and memory deficits. The genome of the nematode Caenorhabditis elegans encodes two members of the Notch transmembrane receptor family, LIN-12 and GLP-1, which have both unique and shared developmental functions. LIN-12 affects diverse cell fate specification events at certain embryonic and larval stages, including the ABplp lineage (a neuronal precursor), intestinal primordium, gonadal anchor cell and secondary vulval precursor cells. In addition to developmental functions, it also operates in the adult nervous system to control locomotion, memory and chemosensory response. Although lin-12 expression was subjected to intense analysis, it was almost not demonstrable in neurons; occasional lin-12 expression was detected only in the two RIG interneurons of young larvae. Here we identify two cis-regulatory regions from lin-12, both of them are specified by the presence of a conserved EXD/HOX composite binding site. One of these regions is located in the first intron and required for driving transgene expression in vulval precursor cell lineages and specific gonadal cells. The other region is located in the second intron and can confer neuronal expression for lin-12 throughout life. The latter regulatory element is highly conserved in the paralogous glp-1 genomic environment, suggesting redundant developmental and physiological roles for the two Notch paralogs in the C. elegans nervous system.     

Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs

Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2). This comparison was extended to alpha(6)R100Cbeta(2)gamma(2) receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of alpha(6)R100Cbeta(2)gamma(2) enables best interaction with NCS compound, followed by alpha(3)H126Cbeta(2)gamma(2), alpha(1)H101Cbeta(2)gamma(2) and alpha(2)H101Cbeta(2)gamma(2), while alpha(5)H105Cbeta(2)gamma(2) receptors show little interaction. Our results allow conclusions about the relative apposition of alpha(1)H101 and homologous positions in alpha(2), alpha(3), alpha(5) and alpha(6) with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABA(A) receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the alpha/beta subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the alpha/gamma interface.     

Different functional sites on rIFN-alpha 2 and their relation to the cellular receptor binding site

Functional domains on the recombinant interferon-alpha 2 (rIFN-alpha 2) molecule, which are involved in antiviral and NK enhancing activities, have been defined by immunochemical mapping with MAb, and their relationship with the IFN cellular receptor binding site has been studied. With 20 different anti-IFN-alpha 2 MAb selected by their binding to 125I-labeled IFN and by immunoprecipitation of the 20 Kd IFN molecule, we have defined three spatially separated epitopes (designated as sites A, B, and C) and two partially overlapping antigenic determinants on the IFN-alpha 2 molecule. Functional relation of IFN-alpha 2 A, B, and C epitopes have been determined by assaying the effect of various anti-IFN MAb on IFN-mediated biologic activities. MAb directed to sites A and B neutralized the antiviral activity of IFN. Furthermore, the MAb specific for site B displayed a neutralizing potency threefold higher than MAb directed to site A. Site B was also involved in the enhancing activity of IFN on NK-mediated cell cytotoxicity, whereas site A was not. MAb directed to site C partially affected the IFN-boosted NK activity but did not neutralize the IFN antiviral activity. Inhibition studies of 125I-IFN binding to human U-937 myelomonocytic cells by anti-IFN MAb demonstrated that MAb directed to site B blocked different IFN biologic functions by preventing its binding to the cellular receptor, whereas MAb directed to sites A and C caused no inhibition and partial inhibition of this binding, respectively.     

Methamphetamine induces long-term changes in GABAA receptor alpha2 subunit and GAD67 expression

The present study investigated whether GABA(A) receptor alpha2 subunit and GAD(67) are involved in chronic high dose methamphetamine (METH)-induced sensitization and neurotoxicity. The METH sensitization was established in rats by 7-day pump infusion plus daily injection (25mg/kg/day) and a subsequent 28-day withdrawal period. Behavioral sensitization was assessed by behavioral ratings after challenge with METH (0.5mg/kg). The neurotoxicity was evaluated by the expression of glial fibrillary acidic protein (GFAP). Western blot assay showed that METH sensitization decreases GABA(A) alpha2 subunit and GAD(67) protein levels in the nucleus accumbens (NAc) core and shell, and conversely, these proteins were increased in the caudate. An upregulation of GFAP expression was observed in the caudate, but not in the NAc core and shell. These data suggest that inhibition of GABA transmission in the NAc is related to METH behavioral sensitization, whereas activation of GABA transmission in the caudate is associated with METH-induced neurotoxicity.