Search

GSK878 is a newly described HIV-1 inhibitor that binds to the mature capsid (CA) hexamer in a pocket originally identified as the binding site of the well-studied CA inhibitor PF-74. Here, we show that GSK878 is highly potent, inhibiting an HIV-1 reporter virus in MT-2 cells with a mean 50% effective concentration (EC 50 ) of 39 pM and inhibiting a panel of 48 chimeric viruses containing diverse CA sequences with a mean EC 50 of 94 pM. CA mutations associated with reduced susceptibility to other inhibitors that bind to PF-74 binding site (L56I, M66I, Q67H, N74D, T107N, and Q67H/N74D) also reduced susceptibility to GSK878, with M66I, Q67H/N74D, and L56I having the greatest impact on antiviral activity. Amino acid substitutions in the CA cyclophilin A (CypA) binding loop (H87P and P90A), distal from the inhibitor binding site and associated with reduced CA-CypA binding, subtly, but reproducibly, also decreased GSK878 potency. Mechanism-of-action studies showed that GSK878 blocked both early (preintegration) and late (postintegration) steps in HIV-1 replication, with the early inhibition primarily determining the compound's antiviral activity. The early inhibition results from blocks to HIV-1 nuclear import and proviral integration and is associated with altered stability of the HIV-1 CA core., Competing Interests: The authors declare a conflict of interest. At the time of this study, all authors were employees of ViiV Healthcare and C.W., K.P., M.I.C., J.F.K., E.P.G., M.K., and R.A.F. are shareholders in GSK.

GSK3532795, formerly known as BMS-955176 (1), is a potent, orally active, second-generation HIV-1 maturation inhibitor (MI) that advanced through phase IIb clinical trials. The careful design, selection, and evaluation of substituents appended to the C-3 and C-17 positions of the natural product betulinic acid (3) was critical in attaining a molecule with the desired virological and pharmacokinetic profile. Herein, we highlight the key insights made in the discovery program and detail the evolution of the structure-activity relationships (SARs) that led to the design of the specific C-17 amine moiety in 1. These modifications ultimately enabled the discovery of 1 as a second-generation MI that combines broad coverage of polymorphic viruses (EC 50 <15 nM toward a panel of common polymorphisms representative of 96.5% HIV-1 subtype B virus) with a favorable pharmacokinetic profile in preclinical species.

Immune clearance of Hepatitis B virus (HBV) is characterized by broad and robust antiviral T cell responses, while virus-specific T cells in chronic hepatitis B (CHB) are rare and exhibit immune exhaustion that includes programmed-death-1 (PD-1) expression on virus-specific T cells. Thus, an immunotherapy able to expand and activate virus-specific T cells may have therapeutic benefit for CHB patients. Like HBV-infected patients, woodchucks infected with woodchuck hepatitis virus (WHV) can have increased hepatic expression of PD-1-ligand-1 (PD-L1), increased PD-1 on CD8+ T cells, and a limited number of virus-specific T cells with substantial individual variation in these parameters. We used woodchucks infected with WHV to assess the safety and efficacy of anti-PD-L1 monoclonal antibody therapy (αPD-L1) in a variety of WHV infection states. Experimentally-infected animals lacked PD-1 or PD-L1 upregulation compared to uninfected controls, and accordingly, αPD-L1 treatment in lab-infected animals had limited antiviral effects. In contrast, animals with naturally acquired WHV infections displayed elevated PD-1 and PD-L1. In these same animals, combination therapy with αPD-L1 and entecavir (ETV) improved control of viremia and antigenemia compared to ETV treatment alone, but with efficacy restricted to a minority of animals. Pre-treatment WHV surface antigen (sAg) level was identified as a statistically significant predictor of treatment response, while PD-1 expression on peripheral CD8+ T cells, T cell production of interferon gamma (IFN-γ) upon in vitro antigen stimulation (WHV ELISPOT), and circulating levels of liver enzymes were not. To further assess the safety of this strategy, αPD-L1 was tested in acute WHV infection to model the risk of liver damage when the extent of hepatic infection and antiviral immune responses were expected to be the greatest. No significant increase in serum markers of hepatic injury was observed over those in infected, untreated control animals. These data support a positive benefit/risk assessment for blockade of the PD-1:PD-L1 pathway in CHB patients and may help to identify patient groups most likely to benefit from treatment. Furthermore, the efficacy of αPD-L1 in only a minority of animals, as observed here, suggests that additional agents may be needed to achieve a more robust and consistent response leading to full sAg loss and durable responses through anti-sAg antibody seroconversion.

A series of potent and novel acylsulfonamide-bearing triazines were synthesized and the structure-activity relationships (SARs) as HCV entry inhibitors were evaluated. This acylsulfonamide series was derived from an early lead, 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoic acid wherein the carboxylic acid was replaced with an acylsulfonamide moiety. This structural modification provided a class of compounds which projected an additional vector off the terminus of the acylsulfonamide functionality as a means to drive activity. This effort led to the discovery of potent analogues within this series that demonstrated sub-nanomolar EC 50 values in the HCV pseudotype particle (HCVpp) assay., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is described. The objective of this work was the identification of a drug with antiviral properties and toxicology parameters similar to 2, but with a preclinical pharmacokinetic (PK) profile that was predictive of once-daily dosing. Critical to this discovery process was the employment of an ex vivo cardiovascular (CV) model which served to identify compounds that, like 2, were free of the CV liabilities that resulted in the discontinuation of BMS-605339 (1) from clinical trials. Structure-activity relationships (SARs) at each of the structural subsites in 2 were explored with substantial improvement in PK through modifications at the P1 site, while potency gains were found with small, but rationally designed structural changes to P4. Additional modifications at P3 were required to optimize the CV profile, and these combined SARs led to the discovery of BMS-890068 (29).

HIV-1 maturation inhibition (MI) has been clinically validated as an approach to the control of HIV-1 infection. However, identifying an MI with both broad polymorphic spectrum coverage and good oral exposure has been challenging. Herein, we describe the design, synthesis, and preclinical characterization of a potent, orally active, second generation HIV-1 MI, BMS-955176 (2), which is currently in Phase IIb clinical trials as part of a combination antiretroviral regimen.

The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.

The dose response curve is the gold standard for measuring the effect of a drug treatment, but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns, but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose, calculate classical pharmacological parameters such as the EC50, and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib, nilotinib, dasatinib and PD0325901) across a six-logarithm dose range, using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets, dose-dependent effects on cellular processes were identified using automated pathway analysis, and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover, these methods are automated, robust to non-optimized assays, and could be applied to other sources of quantitative data.

Regulators of G-protein signaling (RGS) proteins are a novel family of GTPase-activating proteins that interact with Galpha subunits of the Gi/o, Gz, Gq and G(12/13) subfamilies to dampen G-protein-coupled receptor (GPCR)-mediated signaling by accelerating intrinsic Galpha-GTPase activity. In the present study, we report on messenger ribonucleic acid (mRNA) localization in rat brain of six RGS genes by in situ hybridization. The distribution patterns of RGS2, RGS13, RGS14 and GAIP (Galpha interacting protein) overlapped in most brain regions examined. Highest regional expression was observed for RGS2 in the cerebral cortical layers, striatum, hippocampal formation, several thalamic and hypothalamic nuclei and hindbrain regions such as the pontine, interpeduncular and dorsal raphe nuclei. Levels of RGS14 mRNA closely paralleled those of RGS2 expression levels throughout most brain regions. RGS13 mRNA was enriched in the hippocampal formation, amygdala, mammillary nuclei as well as the pontine and interpeduncular nuclei. GAIP expression levels were highest in the hippocampal formation with moderate to low levels present in all other regions studied. Of the six RGS genes probed, RGS16 mRNA displayed a discrete localization predominantly in the thalamic midline/intralaminar and principal relay nuclei, and the hypothalamic suprachiasmatic nucleus. RGS1 mRNA signal was not detected in brain. In conclusion, the in situ hybridization studies for RGS2, RGS13, RGS14, RGS16 and GAIP mRNAs extend our knowledge of the distribution of RGS genes expressed in the rat central nervous system, and indicate overlapping RGS-enriched regions that may be indicative of functional diversification in GPCR signaling pathway modulation.

Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins that modulate neurotransmitter and G protein signaling. RGS7 and its binding partners Galpha and Gbeta5 are enriched in brain, but biochemical mechanisms governing RGS7/Galpha/Gbeta5 interactions and membrane association are poorly defined. We report that RGS7 exists as one cytosolic and three biochemically distinct membrane-bound fractions (salt-extractable, detergent-extractable, and detergent-insensitive) in brain. To define factors that determine RGS7 membrane attachment, we examined the biochemical properties of recombinant RGS7 and Gbeta5 synthesized in Spodoptera frugiperda insect cells. We have found that membrane-bound but not cytosolic RGS7 is covalently modified by the fatty acid palmitate. Gbeta5 is not palmitoylated. Both unmodified (cytosolic) and palmitoylated (membrane-derived) forms of RGS7, when complexed with Gbeta5, are equally effective stimulators of Galpha(o) GTPase activity, suggesting that palmitoylation does not prevent RGS7/Galpha(o) interactions. The isolated core RGS domain of RGS7 selectively binds activated Galpha(i/o) in brain extracts and is an effective stimulator of both Galpha(o) and Galpha(i1) GTPase activities in vitro. In contrast, the RGS7/Gbeta5 complex selectively interacts with Galpha(o) only, suggesting that features outside the RGS domain and/or Gbeta5 association dictate RGS7-Galpha interactions. These findings define previously unrecognized biochemical properties of RGS7, including the first demonstration that RGS7 is palmitoylated.

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) are transducers in many cellular transmembrane signaling systems where regulators of G-protein signaling (RGS) act as attenuators of the G-protein signal cascade by binding to the Galpha subunit of G-proteins (G(i)(alpha)(1)) and increasing the rate of GTP hydrolysis. The high-resolution solution structure of free RGS4 has been determined using two-dimensional and three-dimensional heteronuclear NMR spectroscopy. A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 2871 experimental NMR restraints. The atomic rms distribution about the mean coordinate positions for residues 5-134 for the 30 structures is 0.47 +/- 0.05 A for the backbone atoms, 0. 86 +/- 0.05 A for all atoms, and 0.56 +/- 0.04 A for all atoms excluding disordered side chains. The NMR solution structure of free RGS4 suggests a significant conformational change upon binding G(i)(alpha)(1) as evident by the backbone atomic rms difference of 1. 94 A between the free and bound forms of RGS4. The underlying cause of this structural change is a perturbation in the secondary structure elements in the vicinity of the G(i)(alpha)(1) binding site. A kink in the helix between residues K116-Y119 is more pronounced in the RGS4-G(i)(alpha)(1) X-ray structure relative to the free RGS4 NMR structure, resulting in a reorganization of the packing of the N-terminal and C-terminal helices. The presence of the helical disruption in the RGS4-G(i)(alpha)(1) X-ray structure allows for the formation of a hydrogen-bonding network within the binding pocket for G(i)(alpha)(1) on RGS4, where RGS4 residues D117, S118, and R121 interact with residue T182 from G(i)(alpha)(1). The binding pocket for G(i)(alpha)(1) on RGS4 is larger and more accessible in the free RGS4 NMR structure and does not present the preformed binding site observed in the RGS4-G(i)(alpha)(1) X-ray structure. This observation implies that the successful complex formation between RGS4 and G(i)(alpha)(1) is dependent on both the formation of the bound RGS4 conformation and the proper orientation of T182 from G(i)(alpha)(1). The observed changes for the free RGS4 NMR structure suggest a mechanism for its selectivity for the Galpha-GTP-Mg(2+) complex and a means to facilitate the GTPase cycle.

Pindolol-insensitive [3H]-5-hydroxytryptamine ([3H]-5-HT) binding to rat hypothalamic membranes was pharmacologically and functionally characterized to resolve whether this procedure selectively labels 5-HT7 receptors. Consistent with a previous report, 3 microM and not 100 nM pindolol was required to occupy fully 5-HT1A and 5-HT1B receptors. Remaining [3H]-5-HT binding was saturable (KD, 1.59+/-0.21 nM; Bmax, 53.8+/-3.1 fmol x mg protein(-1)). Displacement of [3H]-5-HT with metergoline and 5-CT revealed shallow Hill slopes (<0.5) but seven other compounds had slopes >0.8 and pKi values and the rank order of affinity were significantly correlated (r = 0.81 and 0.93, respectively) with published [3H]-5-HT binding to rat recombinant 5-HT7 receptors. In the presence of pindolol, 5-HT-enhanced accumulation of [32P]-cyclic AMP was unaffected by the 5-HT4 antagonist RS39604 (0.1 microM) or the 5-ht6 antagonist Ro 04-6790 (1 microM) but significantly attenuated by mesulergine (250 nM), ritanserin (450 nM) or methiothepin (200 nM) which have high affinity for the 5-HT7 receptor. Intracerebroventricular pretreatment with the serotonergic neurotoxin 5,7-dihydroxytryptamine, 5,7-DHT, elevated the [3H]-5-HT Bmax 2 fold, indicating that the hypothalamic 5-HT7 receptor is post-synaptic to 5-HT nerve terminals and regulated by synaptic 5-HT levels. These results suggest that, in the presence of 3 microM pindolol, [3H]-5-HT selectively labels hypothalamic binding sites consistent with functional 5-HT7 receptors.

Genzyme is developing therapies to replace the defective forms of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein in CF patients. The company is developing a gene therapy, as well as a recombinant production of CFTR for protein replacement therapy. Both approaches have been granted orphan drug status by the FDA [156348]. The results of several clinical trials were discussed at the first annual meeting of the American Society of Gene Therapy in May 1998. A single dose nasal administration was well tolerated by volunteers, but had disappointing efficacy. In a study completed at the Royal Brompton Hospital, London, a single dose aerosol application of GL-67:DOPE was administered to eight patients, while another eight received GL-67:DOPE plus pCF1-CFTR. In the second group, a moderate increase in the potential difference in the lung was observed, with a slight trend towards bacterial adherence normalization in the airway cells. Seven of the patients in the second group, and three patients who received lipid alone, developed, flu-like symptoms within 24 h. A trial at the University of Alabama, using the same formulation, showed that flu-like symptoms developed in six of eight patients by day two, and in all patients by day seven [290120]. In 1995, the company began a clinical safety trial involving delivery of a normal CF gene to the patient's lungs via an adenovirus vector. The administration involves the inhalation of an aerosol containing the vector or, separately, delivery to one lobe of the patient's lung via a bronchoscope [191678]. To evaluate additional delivery methods for the gene, Genzyme has an exclusive research agreement for the use of Vical's cytofectins as non-viral delivery vectors for CFTR. Also under investigation are delivery systems for the nasal epithelium using liposomes or lipid-DNA complexes. These protocols are being developed in collaboration with the National Heart & Lung Institute, London, and an undisclosed partner [162590], [177633]. Following in vitro screenings by the company, two T-shaped molecules were identified (GL-67 and GL-53), the gene transfer activities of which could be enhanced by dioleoyl-PE (DOPE). A recently-completed clinical trial in 16 CF patients demonstrated that the GL-67:DOPE:DMPE-PEG5000-pCF1-CFTR compound accumulated in the lung with minimal toxicity and resulted in a 25% correction of CF symptoms [268093]. Genzyme has also developed recombinant cell lines that synthesize CFTR and has used transgenic expression techniques to breed mice, rabbits and goats which secrete the protein in their milk. Protein replacement therapy is currently in preclinical investigation and research efforts have been reduced infavor of the gene therapeutic approach [177633].

Regulators of G protein signaling (RGS) proteins serve as potent GTPase-activating proteins for the heterotrimeric G proteins alphai/o and aq/11. This study describes the immunohistochemical distribution of RGS7 throughout the adult rat brain and its cellular colocalization with Galphaq/11, an important G protein-coupled receptor signal transducer for phospholipase Cbeta-mediated activity. In general, both RGS7 and Galphaq/11 displayed a heterogeneous and overlapping regional distribution. RGS7 immunoreactivity was observed in cortical layers I-VI, being most intense in the neuropil of layer I. In the hippocampal formation, RGS7 immunoreactivity was concentrated in the strata oriens, strata radiatum, mossy fibers, and polymorphic cells, with faint to nondetectable immunolabeling within the dentate gyrus granule cells and CA1-CA3 subfield pyramidal cells. Numerous diencephalic and brainstem nuclei also displayed dense RGS7 immunostaining. Dual immunofluorescence labeling studies with the two protein-specific antibodies indicated a cellular selectivity in the colocalization between RGS7 and Galphaq/11 within many discrete brain regions, such as the superficial cortical layer I, hilus area of the hippocampal formation, and cerebellar Golgi cells. To assess the ability of Galphaq/11-mediated signaling pathways to modulate dynamically RGS expression, primary cortical neuronal cultures were incubated with phorbol 12,13-dibutyrate, a selective protein kinase C activator. A time-dependent increase in levels of mRNA for RGS7, but not RGS4, was observed. Our results provide novel information on the region- and cell-specific pattern of distribution of RGS7 with the transmembrane signal transducer, Galphaq/11. We also describe a possible RGS7-selective neuronal feedback adaptation on Galphaq/11-mediated pathway function, which may play an important role in signaling specificity in the brain.

The effect of a 5-hydroxytryptamine7 (5-HT7) receptor-directed antisense oligonucleotide on rat behaviour and neuroendocrine function was investigated. Six days of intracerebroventricular 5-HT7 antisense oligonucleotide treatment significantly reduced [3H]5-HT binding to hypothalamic 5-HT7 receptors, whereas cortical 5-HT2C density remained unchanged. In rats on a food-restricted diet, both antisense and mismatch oligonucleotides reduced food intake and body weight compared with that in vehicle-treated controls by day 4 of administration. 5-HT7 antisense oligonucleotide administration did not affect exploratory or locomotor activity in photocell activity monitors on day 4 or elevated plus-maze behaviour on day 6 of intracerebroventricular treatment. 5-HT7 antisense oligonucleotide did not affect plasma corticosterone or prolactin levels or 5-HT turnover in either 5-HT cell body or terminal areas. These data demonstrate that intracerebroventricular 5-HT7 antisense oligonucleotide administration selectively reduced rat hypothalamic 5-HT7 receptor density without affecting any of the biochemical or behavioural measures. The results suggest that this antisense protocol could be a valuable tool to investigate central 5-HT7 receptor functions, and that this receptor is not critical for the control of neuroendocrine function or food intake.

NTera-2/D1 (NT2) is a human teratocarcinoma cell line which can be cultured as a dividing population of precursor cells that can be manipulated with retinoic acid (RA) to yield post-mitotic neurons. Precursor cells were transfected with the human beta2 adrenergic receptor, controlled by the neuronal cell specific rat synapsin-1 promoter. Transfected precursor cells did not display elevated expression of the beta2 adrenergic receptor. Upon differentiation to a neuronal phenotype with RA, betaSyn2 and betaSyn4 (beta2Syn-NT2; ATCC CRL-12356) displayed elevated beta2 adrenergic receptor levels, and an elevated coupling to cAMP production. It was also observed that the elevated expression of the beta2 adrenergic receptor in the neuronal NT2 cells resulted in an enhanced level of neuronal differentiation compared to the wild type cells. These results demonstrate that the neuronally restricted expression of the human beta2 adrenergic receptor in NT2 neurons results in increased receptor levels and receptor stimulated generation of cAMP, this may result in the observed improvement in neuronal differentiation., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Heterotrimeric G-proteins, comprising alpha, beta and gamma subunits, have been shown to play a central role in coupling multiple receptors to a variety of enzymes and ion channels. In vitro studies have demonstrated the existence of selective interactions between various alpha, beta and gamma subunits, as well as between specific heterotrimers and target receptor and effector proteins. However, little is known of the physiological relevance of such associations, and the determinants of specificity in G-protein signaling within the brain remain largely unidentified. To investigate the possibility that specific heterotrimeric interactions result from discrete localizations of the G-protein subunits within the brain, we have used the technique of in situ hybridization to map the distribution of G-protein beta and gamma subunits in the rat brain. Beta1, beta2, beta3 and beta5 subunits were found to be widely expressed throughout the rat brain, whilst beta4 and the G-protein gamma subunit messenger RNAs generally showed more discrete expression patterns. The expression patterns for these subunits suggest that individual beta and gamma subunits may be co-expressed in certain cell types and brain regions; a particularly intriguing and striking co-localization was observed in the case of beta4 and gamma2 subunit messenger RNAs in layer VI of the occipital cortex. The localizations of the G-protein beta and gamma subunits, and their potential coupling to various receptor/effector systems, are discussed.

Cellular Replacement by Immunoisolatory Biocapsule (CRIB) is a technology under development by Cyto-Therapeutics for the delivery of therapeutic substances to the central nervous system (CNS). Preclinical studies with implants are underway for the potential treatment of Alzheimer's disease, Huntington's chorea and other neurodegenerative diseases [180367]. Theses implants contain cells that have been genetically engineered to secrete human nerve growth factor. Development of the CRIB implant is in collaboration with Genentech. CRIB consists of product-secreting cells encapsulated in a semipermeable polymer membrane. The device is surgically implanted into the target area of the brain, thus bypassing the blood-brain barrier, whilst isolating the foreign cells from the patient's immune system [180367]. CRIB implants secreting nerve growth factor (NGF) were effective in preventing nerve degeneration in a monkey model of Alzheimer's disease. The devices contained baby hamster kidney fibroblasts (genetically modified to secrete human NGF) and were implanted into brain ventricles of elderly rhesus monkeys whose cholinergic neurons had been cut. The monkeys treated with non-NGF secreting implants showed a decrease in the number of cholinergic neurons, while those treated with NGF-secreting implants showed a smaller loss of neurons and sprouting of cholinergic nerve fibres [200261]. CytoTherapeutics is working towards clinical trials with these implants in patients with Alzheimer's disease [168581]. Trials of NGF-secreting implants have also been carried out in animal models of Huntington's disease. The company demonstrated that in rodents whose brains had been lesioned with quinolinic acid to approximate the neuronal death which occurs in the disease, NGF-releasing implants protected many striatal neurons that would normally have died. In addition, it was shown that behaviors normally lost as a result of striatal neuron damage could be significantly attenuated. Further investigations are scheduled for the potential use of CRIB/NGF technology in Huntington's disease [180367]. The CRIB technology was originally developed at Brown University (Rhode Island, USA) which owns several patents that cover the technology. CytoTherapeutics has entered into collaborative agreements with the university for the use of the technology [199903] as well as with NeuroSpheres, Canada, for the use of their neural stem cells [199898].

The RGS proteins are a recently discovered family of G protein regulators that have been shown to act as GTPase-activating proteins (GAPs) on the G(alpha i) and G(alpha q) subfamilies of the heterotrimeric G proteins. Here, we demonstrate that RGS7 is a potent GAP in vitro on G(alpha i1), and G(alpha o) heterotrimeric proteins and that RGS7 acts to down-regulate G(alpha q)-mediated calcium mobilization in a whole-cell assay system using a transient expression protocol. This RGS protein and RGS4 are reported to be expressed predominantly in brain, and in situ hybridization studies have revealed similarities in the regional distribution of RGS and G(alpha q) mRNA expression. Our findings provide further evidence to support a functional role for RGS4 and RGS7 in G(alpha q)-mediated signaling in the CNS.

Heterotrimeric G proteins integrate signals between receptors and effector proteins. We have cloned the human beta 5 subunit from a human brain cDNA library. The clone has a 1059 bp open reading frame and is highly homologous to the murine clone. In contrast to the brain specific mouse beta 5, northern analysis showed it to be expressed in multiple tissues.

Heterotrimeric G proteins function as molecular relays, shuttling between cell surface receptors and intracellular effectors that propagate a signal. G protein signaling is governed by the rates of GTP binding (catalyzed by the receptor) and GTP hydrolysis. RGS proteins (regulators of G protein signaling) were identified as potent negative regulators of G protein signaling pathways in simple eukaryotes and are now known to act as GTPase-activating proteins (GAPs) for G protein alpha-subunits in vitro. It is not known, however, if Galpha GAP activity is responsible for the regulatory action of RGS proteins in vivo. We describe here a Galpha mutant in yeast (gpa1(sst)) that phenotypically mimics the loss of its cognate RGS protein (SST2). The gpa1(sst) mutant is resistant to an activated allele of SST2 in vivo and is unresponsive to RGS GAP activity in vitro. The analogous mutation in a mammalian Gqalpha is also resistant to RGS action in transfected cells. These mutants demonstrate that RGS proteins act through Galpha and that RGS-GAP activity is responsible for their desensitizing activity in cells. The Galphasst mutant will be useful for uncoupling RGS-mediated regulation from other modes of signal regulation in whole cells and animals.

Described in this report is a systematic study which led to the identification of two new dopamine D2 partial agonists (5 and 17). Phenols 5 and 17 represent prototypes of two new classes of D2 partial agonists as well as templates for the future design of novel dopaminergic agents.

The rationale for matrix metalloproteinase (MMP) inhibition as a means to treat disease progression in breast cancer stems from the apparent involvement of MMPs in the hydrolysis of basement membranes during tumour cell invasion and subsequent metastasis. MMP-mediated matrix remodelling also appears to promote the growth of tumour cells, possibly by facilitating the proliferation and migration of endothelial cells and the neovascularization of tumour tissue. We found that transfection of the C127 breast cancer cell line by MMP-2 (gelatinase A), but not by MMP-1 or MMP-3 (collagenase and stromelysin respectively), gave rise to an invasive and metastatic phenotype. We were surprised to find that this phenotype depended not only on the catalytic properties of MMP-2 but also on properties associated with the MMP-2 non-catalytic C-terminal domain. Experiments with a synthetic gelatinase inhibitor revealed that a single dose could prevent the lungs of nude mice being colonized by the MMP-2 transfectants, and that the inhibitor had to be administered during or shortly after injection of the cells, indicating that an early event, such as the extravasation of the cells into the lung, is gelatinase-dependent in this system. In other studies employing long-term treatment with CT1746, an orally active gelatinase inhibitor, we have previously demonstrated a reduction in primary tumour growth rates, localized spread, and spontaneous metastasis, even when the treatment was commenced several days after tumour implantation. Furthermore, additive effects were recorded when gelatinase inhibitor therapy was combined with cytotoxic drug treatment. Since the gelatinase inhibitors can also inhibit bone resorption in vitro, these observations point to their potential for delaying disease recurrence and reducing rates of bone loss following conventional therapeutic strategies, in metastatic breast cancer.

A series of 2-(aminomethyl)chromans (2-AMCs) was synthesized and evaluated for their affinity and selectivity for both the high- and low-affinity agonist states (D2High and D2Low, respectively) of the dopamine (DA) D2 receptor. The 7-hydroxy-2-(aminomethyl)chroman moiety was observed to be the primary D2 agonist pharmacophore. The 2-methylchroman moiety was discovered to be an entirely novel scaffold which could be used to access the D2 agonist pharmacophore. Attaching various simple alkyl and arylalkyl side chains to the 7-hydroxy 2-AMC nucleus had significant effects on selectivity for the D2High receptor vs the 5HT1A and alpha 1 receptors. A novel DA partial agonist, (R)-(-)-2-(benzylamino)methyl)chroman-7-ol [R-(-)-35c], was identified as having the highest affinity and best selectivity for the D2High receptor vs the alpha 1 and 5HT1A receptors. Several regions of the 2-AMC nucleus were modified and recognized as potential sites to modulate the level of intrinsic activity. The global minimum conformer of the 7-hydroxy-2-AMC moiety was identified as fulfilling the McDermed model D2 agonist pharmacophoric criteria and was proposed as the D2 receptor-bound conformation. Structure-activity relationships gained from these studies have aided in the synthesis of D2 partial agonists of varying intrinsic activity levels. These agents should be of therapeutic value in treating disorders resulting from hypo- and hyperdopaminergic activity, without the side effects associated with complete D2 agonism or antagonism.

The coexpression of the rat Kv beta 1 subunit with the mouse Kv1.1 (mKv1.1) K+ channel in Chinese hamster ovary cells caused an increase in the rate of inactivation of whole-cell current. Current decayed in a bi-exponential fashion with a fast voltage-dependent and a slower voltage-independent component. The inactivating current component accounted for around 40% of the total outward current. In contrast to previous studies using K+ channel alpha subunits, peptides based on the N-terminal of the Kv beta 1 subunit were unable to mimic the action of the entire subunit. The findings indicate differences between the inactivation induced by the Kv beta 1 subunit and the N-type inactivation mechanism associated with certain rapidly-inactivating cloned K+ channel alpha subunits.

Human gelatinase A, a member of the matrix metalloproteinase family, is secreted from cells as the M(r) 72,000 latent precursor, progelatinase A. The autolytic removal of an N-terminal propeptide generates the M(r) 66,000 active form. Mutants of recombinant progelatinase A, altered such that the proposed active site glutamic acid residue (E375) was replaced by either an aspartic acid (proE375-->D), an alanine (proE375-->A) or a glutamine (proE375-->Q), were purified from medium conditioned by transfected NS0 mouse myeloma cells. Like wild-type progelatinase A, the mutant proenzymes were inactive and could bind tissue inhibitor of metalloproteinases (TIMP)-2 but not TIMP-1 to their C-terminal domains. Their rates of autolytic processing induced by the organomercurial (4-aminophenyl) mercuric acetate, however, were markedly slower and, of the three M(r) 66,000 forms so produced, only E375-->D displayed any proteolytic activity against either a synthetic substrate (kcat/Km = 10% that of the wild-type enzyme) or denatured type I collagen (specific activity = 0.9% that of the wild-type enzyme). ProE375-->A and proE375-->Q could be more rapidly processed to their M(r) 66,000 forms by incubation with a deletion mutant of gelatinase A that has full catalytic activity but lacks the C-terminal domain [delta (418-631) gelatinase A]. These two M(r) 66,000 forms displayed low activity on a gelatin zymogram (approximately 0.01% that of the wild-type enzyme) but, like E375-->D were able to bind TIMP-1 with an affinity equal to that of the activated wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

The properties of a deletion mutant delta V191-Q364 of gelatinase A, which represents the removal of the fibronectin-like type II repeats defined by exons 5-7, were compared with those of full-length gelatinase A. Both enzymes underwent self-activation over a similar time course in the presence of 4-aminophenylmercuric acetate. The fully active enzymes had similar kcat/Km values for the cleavage of an octapeptide substrate, but the deletion mutant had 50% of the activity of wild type gelatinase A against beta-casein and 10% of the activity against gelatin. The cleavage pattern for gelatin was similar for both enzymes but differed for type IV collagen. Comparison of the rates of association of the tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 and their N-terminal domains to both forms of gelatinase indicated that the fibronectin-like domain plays little role in TIMP binding. The deletion mutant failed to bind to collagen, while the wild type gelatinase bound tightly, indicating that the fibronectin-like domain is the sole site of collagen binding. Both gelatinases could be activated by concanavalin A-activated fibroblasts, suggesting that the fibronectin-like domain is not required for the membrane-mediated activation process.

The matrix metalloproteinases gelatinase A and stromelysin-1 have definable N-terminal (catalytic) and C-terminal domains. In order to analyze their interactions with the N- and C-terminal domains of the tissue inhibitors of metalloproteinases TIMP-1 and -2, mutants of both the enzymes and the inhibitors were prepared in which the C-terminal domains had been deleted. Since the Ki values for TIMP inhibition of the matrix metalloproteinases are in the picomolar range, it was not possible to measure these accurately within the sensitivity of available activity assays. Rate constants for the association of the wild-type proteins were therefore determined and systematically compared with those for the deletion mutants. It was found that TIMP-1 binds more rapidly than TIMP-2 to stromelysin-1 and that the C-terminal domain of the enzyme does not affect the rate of association of enzyme and inhibitor. This is in contrast to gelatinase A, where the C-terminal domain has been shown to play an important role in increasing the rate of complex formation with the TIMPs (Willenbrock et al., 1993). The TIMPs are also comprised of an N- and C-terminal domain. By deletion mutagenesis, we found that the C-terminal domain of both TIMPs contributed less to the rate of complex formation with stromelysin-1 than to that with gelatinase A. Hybrids of the N- and C-terminal domains of gelatinase A and stromelysin-1 were prepared and used to analyze further the differences in domain interactions with the TIMPs. They demonstrated that the interactions between the C-terminal domains of enzyme and inhibitor can occur irrespective of the nature of the N-terminal domain. We can conclude that the TIMPs have two major binding regions which associate in different ways with the domains of the enzymes gelatinase A and stromelysin-1. The N-terminal domains of the TIMPs bind to the enzyme catalytic domains to inhibit activity. The TIMP C-terminal domain acts to increase the association rate constant by binding to the N-terminal domain of stromelysin or the C-terminal domain of gelatinase A.

A nonglycosylated (N30QN78Q) form of the human tissue inhibitor of metalloproteinases, TIMP-1, has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small single crystals have been grown using sodium tartrate as a precipitant. The crystals are in space group P2(1), with cell dimensions a = 35.28, b = 53.95, c = 48.56, and beta = 96.0 degrees. There is a single molecule of TIMP-1 in the asymmetric unit. The crystals diffract to at least 2.3 A resolution. Complete data have been collected to 2.9 A and a search for heavy-metal derivatives is in progress.

The cloning and expression of the full-length tissue inhibitor of metalloproteinase 2 (TIMP-2), delta 187-194TIMP-2, and delta 128-194TIMP-2 and the purification of these inhibitors and a cleaved version of TIMP-2 lacking nine C-terminal amino acids (delta 186-194TIMP-2) are described. The mechanism of inhibition of gelatinase A by the TIMPs was investigated by comparing the kinetics of association of TIMP-1, TIMP-2, the C-terminal deletions, and the mutants of both TIMPs which consisted of the N-terminal domain only. The full-length TIMPs inhibited gelatinase A rapidly with association constants of 3.2 x 10(6) M-1 s-1 for TIMP-1 and 2.1 x 10(7) M-1 s-1 for TIMP-2 at I = 0.2. The C-terminal peptide of TIMP-2 is proposed to exist as an exposed "tail" responsible for binding to progelatinase A and for increasing the rate of inhibition of active gelatinase A through electrostatic interactions with the C-terminal domain of the enzyme. The C-terminal domains of both TIMP-1 and TIMP-2 participate in low-affinity interactions with the C-terminal domain of gelatinase A which increase the rate of association by a factor of about 100 in both cases.

The tissue inhibitor of metalloproteinases-1 (TIMP-1) was subjected to single-site mutations within the N-terminal three loops using an oligonucleotide-directed polymerase chain reaction method. All the histidines, and a number of other residues conserved between TIMP-1 and TIMP-2, were individually modified and the mutant TIMPs expressed in mammalian cells. Purified mutant TIMPs were shown to be correctly folded by measuring the effect of guanidine hydrochloride on intrinsic fluorescence. Kinetic analyses of mutants using a quenched fluorescent peptide substrate and the metalloproteinase PUMP indicated that mutation of His7 and Gln9 caused an increase in the apparent dissociation constant, largely due to an increase in the rate of dissociation of complexes. The data indicate that the anchored sequence between Cys 3 and Cys 13 is a key region for interaction of TIMP-1 with metalloproteinases.

Recombinant human interstitial collagenase, an N-terminal truncated form, delta 243-450 collagenase, recombinant human stromelysin-1, and an N-terminal truncated form, delta 248-460 stromelysin, have been stably expressed in myeloma cells and purified. The truncated enzymes were similar in properties to their wild-type counterparts with respect to activation requirements and the ability to degrade casein, gelatin, and a peptide substrate, but truncated collagenase failed to cleave native collagen. Removal of the C-terminal domain from collagenase also modified its interaction with tissue inhibitor of metalloproteinases-1. Hybrid enzymes consisting of N-terminal (1-242) collagenase.C-terminal (248-460) stromelysin and N-terminal (1-233) stromelysin.C-terminal (229-450) collagenase, representing an exchange of the complete catalytic and C-terminal domains of the two enzymes, were expressed in a transient system using Chinese hamster ovary cells and purified. Both proteins showed similar activity to their N-terminal parent and neither was able to degrade collagen. Analysis of the ability of the different forms of recombinant enzyme to bind to collagen by ELISA showed that both pro and active stromelysin and N-terminal collagenase.C-terminal stromelysin bound to collagen equally well. In contrast, only the active forms of collagenase and N-terminal stromelysin.C-terminal collagenase bound well to collagen, as compared with their pro forms.

Recombinant 72 kDa gelatinase A and a truncated form lacking the C-terminal domain were shown to be activated by organomercurials and to possess similar activities towards a number of substrates. The truncated proenzyme differed from the full-length gelatinase in that it could not be activated by a membrane activator and did not bind tissue inhibitor of metalloproteinase (TIMP)-2. Kinetic studies also showed that the inhibition of the activated truncated enzyme, by both TIMP-1 and TIMP-2, was considerably decreased compared with the full-length enzyme. We conclude that the C-terminal domain plays an important role in the regulation of gelatinase A by a potential physiological activator and inhibitors.

Recombinant tissue inhibitor of metalloproteinases (TIMP-1) and a truncated version containing only the three N-terminal loops, delta 127-184TIMP, have been expressed in myeloma cells and purified by affinity chromatography and gel filtration. delta 127-184TIMP was found to exist as two main glycosylation variants of molecular mass 24 kD and 19.5 kDa and an unglycosylated form of 13 kDa. All forms of the truncated inhibitor were able to inhibit and form complexes with active forms of the matrix metalloproteinases, indicating that the major structural features for specific interaction with these enzymes resides in these three loops. Stable binding of delta 127-184TIMP to pro 95-kDa gelatinase was not demonstrable under the conditions for binding of full-length TIMP-1.

The abilities of the matrix metalloproteinases 95 kDa and 72 kDa gelatinases (type IV collagenases), stromelysins-1 and -2 and punctuated metalloproteinase (PUMP) to degrade insoluble elastin, type IV collagen films and proteoglycan have been compared. The gelatinases and PUMP were markedly more active in the degradation of elastin than were the stromelysins. PUMP and the stromelysins were more potent proteoglycan-degrading enzymes. All of the enzymes studied degraded soluble native type IV collagen, but the gelatinases were more effective at higher temperatures. These quantitative data allow an analysis of the potential relative roles of these metalloproteinases in the breakdown of the key components of connective tissue matrices.

Vectors expressing adenovirus 5 E1A or a domain 2 mutant E1A were introduced into CHO-K1 cells in order to transactivate the hCMV-MIE promoter in transient and stable transfections. Expression from the hCMV promoter was efficiently activated by both wild-type and mutant E1A in contrast to other viral promoters such as the SV40 early promoter which are repressed by E1A. E1A genes expressed from a strong promoter were inhibitory to the growth of CHO cells. Nevertheless, by the use of a weaker promoter, it was possible to isolate stably transfected cell lines containing a level of E1A compatible with both continued cell growth and significant transactivation of the hCMV promoter. By this means we have generated cell lines secreting tissue inhibitor of metalloproteinases (TIMP) at levels approaching those previously attained using gene amplification. CHO cell lines constitutively expressing wild-type and mutant E1A genes have been derived which can serve as new host cell lines for transient expression and efficient stable expression without gene amplification.

We have used a glutamine synthetase (GS) gene as an amplifiable marker in Chinese hamster ovary (CHO) cells. GS was combined with an efficient transcription unit to produce tissue inhibitor of metalloproteinases (TIMP). Initial transfectant cell-lines selected using a GS gene secreted up to 9 micrograms TIMP/10(6) cells/24h. After one round of GS gene amplification expression levels of 110 micrograms TIMP/10(6) cells/24h were achieved. These GS gene amplified CHO cells, when adapted to grow in suspension, accumulated 180mg/l in shake flask culture. This system therefore provides a rapid method of achieving high level gene expression in mammalian cells.

The latent forms of stromelysin and collagenase from human gingival fibroblasts were purified to homogeneity. These latent proenzymes underwent serial small reductions in Mr upon activation by treatment with either 4-aminophenylmercuric acetate or trypsin. Similar shifts in Mr and activation kinetics were observed upon identical treatments of either recombinant prostromelysin or procollagenase. Prostromelysin showed a lag between activation and Mr fall, suggesting an initial activation by conformational change. Collagenase activity was enhanced up to 12-fold by either natural or recombinant stromelysin in the presence of trypsin or 4-aminophenylmercuric acetate. Stromelysin caused a further apparent decrease in the Mr of procollagenase. Since these important connective-tissue-degrading enzymes are usually co-ordinately produced by cells, a cascade mechanism is proposed in which collagenase is activated by stromelysin.

The promoters from Drosophila and human 70,000-dalton heat shock protein (hsp70) genes were linked to human tissue plasminogen activator (tPA) cDNA. Mouse C127 cells were transformed with bovine papilloma virus (BPV) vectors carrying the hybrid hsp70/tPA genes. Stable BPV-transformed cell lines were selected and analyzed for tPA expression before and after heat shock. In most cell lines, there was a low level of tPA production even in the absence of heat shock or other obvious stress. After heat shock (42 degrees C, 2 hr), there was up to a 40-fold increase in tPA production. Production of tPA protein occurred within the first 5 h after the heat shock and was due to a burst of hsp70/tPA transcription during the heat shock. The hsp70/tPA transcripts appeared to have a short half-life. Thus, stable mouse cell lines carrying hsp70/tPA hybrid genes can be induced by a short heat shock to transcribe high levels of hsp70/tPA mRNAs and, subsequently, to produce elevated levels of tPA protein.