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HIV-associated neurocognitive disorder (HAND) remains highly prevalent in HIV infected individuals and represents a special group of neuropathological disorders, which are associated with HIV-1 viral proteins, such as transactivator of transcription (Tat) protein. Cocaine abuse increases the incidence of HAND and exacerbates its severity by enhancing viral replication. Perturbation of dopaminergic transmission has been implicated as a risk factor of HAND. The presynaptic dopamine (DA) transporter (DAT) is essential for DA homeostasis and dopaminergic modulation of the brain function including cognition. Tat and cocaine synergistically elevate synaptic DA levels by acting directly on human DAT (hDAT), ultimately leading to dysregulation of DA transmission. Through integrated computational modeling and experimental validation, key residues have been identified in hDAT that play a critical role in Tat-induced inhibition of DAT and induce transporter conformational transitions. This review presents current information regarding neurological changes in DAT-mediated dopaminergic system associated with HIV infection, DAT-mediated adaptive responses to Tat as well as allosteric modulatory effects of novel compounds on hDAT. Understanding the molecular mechanisms by which Tat induces DAT-mediated dysregulation of DA system is of great clinical interest for identifying new targets for an early therapeutic intervention for HAND. [ABSTRACT FROM AUTHOR]

Summary: Kinase targets are being pursued in a variety of diseases beyond cancer, including immune and metabolic as well as viral, parasitic, fungal and bacterial. In particular, there is a relatively recent interest in kinase and ATP-binding targets in Mycobacterium tuberculosis in order to identify inhibitors and potential drugs for essential proteins that are not targeted by current drug regimens. Herein, we report the high throughput screening results for a targeted library of approximately 26,000 compounds that was designed based on current kinase inhibitor scaffolds and known kinase binding sites. The phenotypic data presented herein may form the basis for selecting scaffolds/compounds for further enzymatic screens against specific kinase or other ATP-binding targets in Mycobacterium tuberculosis based on the apparent activity against the whole bacteria in vitro. [ABSTRACT FROM AUTHOR]

Summary: There is an urgent need for the discovery and development of new antitubercular agents that target novel biochemical pathways and treat drug-resistant forms of the disease. One approach to addressing this need is through high-throughput screening of drug-like small molecule libraries against the whole bacterium in order to identify a variety of new, active scaffolds that will stimulate additional biological research and drug discovery. Through the Molecular Libraries Screening Center Network, the NIAID Tuberculosis Antimicrobial Acquisition and Coordinating Facility tested a 215,110-compound library against Mycobacterium tuberculosis strain H37Rv. A medicinal chemistry survey of the results from the screening campaign is reported herein. [Copyright &y& Elsevier]

Summary: There is an urgent need for the discovery and development of new antitubercular agents that target new biochemical pathways and treat drug resistant forms of the disease. One approach to addressing this need is through high-throughput screening of medicinally relevant libraries against the whole bacterium in order to discover a variety of new, active scaffolds that will stimulate new biological research and drug discovery. Through the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (www.taacf.org), a large, medicinally relevant chemical library was screened against M. tuberculosis strain H37Rv. The screening methods and a medicinal chemistry analysis of the results are reported herein. [Copyright &y& Elsevier]

A series of pyridomorphinans possessing an aryl (10a–s) or heteroaryl (11a–h) substituent at the 5′-position of the pyridine ring of 17-cyclopropylmethyl-4,5α-epoxypyrido[2′,3′:6,7]morphinan was synthesized and evaluated for binding and functional activity at the opioid δ, μ, and κ receptors. All of these pyridomorphinans bound with higher affinity at the δ site than at μ or κ sites. The binding data on isomeric compounds revealed that there exists greater bulk tolerance for substituents placed at the o-position of the phenyl ring than at m- or p-positions. Among the ligands examined, the 2-chlorophenyl (10l), 2-nitrophenyl (10n), 2-pyridyl (11a), and 4-quinolinyl (11g) compounds bound to the δ receptor with subnanomolar affinity. Compound 10c with the p-tolyl substituent displayed the highest μ/δ selectivity (ratio=42) whereas compound 10l with the 2-chlorophenyl substituent displayed the highest κ/δ selectivity (ratio=42) whereas compound 10l with the 2-chlorophenyl substituent displayed the highest κ/δ selectivity (ratio=23). At 10 μM concentration, the in vitro functional activity determined using [35S]GTP-γ-S binding assays showed that all of the compounds were antagonists devoid of any significant agonist activity at the δ, μ, and κ receptors. Antagonist potency determinations of three selected ligands revealed that the p-tolyl compound 10c is a potent δ selective antagonist. In the [35S]GTP-γ-S assays this compound had a functional antagonist Ki value of 0.2, 4.52, and 7.62 nM at the δ, μ, and κ receptors, respectively. In the smooth muscle assays 10c displayed δ antagonist potency with a Ke value of 0.88 nM. As an antagonist, it was 70-fold more potent at the δ receptors in the MVD than at the μ receptors in the GPI. The in vitro δ antagonist profile of this pyridomorphinan 10c resembles that of the widely used δ selective antagonist ligand naltrindole. [Copyright &y& Elsevier]

A series of naltrexone-derived pyridomorphinans possessing various substituents at the 5′-position on the pyridine ring were synthesized and evaluated for opioid receptor binding in rodent brain membranes and functional activity in smooth muscle preparations. While the introduction of aromatic 1-pyrrolyl group (6h) improved the δ affinity and δ antagonist potency of the parent compound (3), the introduction of guanidine group (6i) transformed it to a κ selective ligand in opioid receptor binding and [35S]GTP-γ-S functional assays. [Copyright &y& Elsevier]

In an attempt to identify novel ligands for the dopamine transporter, a series of 4-substituted-2-phenylquinazolines were synthesized and evaluated. Among the compounds studied, 4-[(diphenylmethyl)amino]-2-phenylquinazoline (4g) was identified as a novel partial inhibitor of [125I]RTI-55 binding to the dopamine transporter and a partial inhibitor of [3H]dopamine uptake. [Copyright &y& Elsevier]

Among primary brain cancers, gliomas are the most deadly and most refractory to current treatment modalities. Previous reports overwhelmingly support the role of the RNA-binding protein Hu antigen R (HuR) as a positive regulator of glioma disease progression. HuR expression is consistently elevated in tumor tissues, and a cytoplasmic localization appears essential for HuR-dependent oncogenic transformation. Here, we report HuRaggregation (multimerization) in glioma and the analysis of this tumor-specific HuR protein multimerization in clinical brain tumor samples. Using a split luciferase assay, a bioluminescence resonance energy transfer technique, and site-directed mutagenesis, we examined the domains involved in HuR multimerization. Results obtained with the combination of the split HuR luciferase assay with the bioluminescence resonance energy transfer technique suggested that multiple (at least three) HuR molecules come together during HuR multimerization in glioma cells. Using these data, we developed a model of HuR multimerization in glioma cells. We also demonstrate that exposing glioma cells to the HuR inhibitor tanshinone group compound 15,16-dihydrotanshinone-I or to the newly identified compound 5 disrupts HuR multimerization modules and reduces tumor cell survival and proliferation. In summary, our findings provide new insights into HuR multimerization in glioma and highlight possible pharmacological approaches for targeting HuR domains involved in cancer cell-specific multimerization. [ABSTRACT FROM AUTHOR]

Background: Because the role of dopamine (DA) D3 receptors has been investigated primarily in relation to cocaine-related behaviors little is known of the role of these receptors in heroin seeking.Purposes: To investigate the effect of the selective DA D3 receptor antagonist, SR 21502, on cue-induced reinstatement of heroin seeking and heroin conditioned place preference (CPP).Methods: In experiment 1, rats were trained to self-administer intravenous heroin for 15 days followed by extinction. Following extinction animals were treated with one of several SR 21502 doses (0, 7.5, 10 or 15mg/kg) and a cue-induced reinstatement test was conducted. In experiment 2, animals were conditioned to experience heroin in one compartment of a CPP apparatus and saline in the other. On the test day animals were treated with 0, 3.75, 7.5, 10 or 15mg/kg of SR 21502 and tested for their CPP.Results: The results from experiment 1 showed a significant dose-related reduction in cue-induced reinstatement of active lever pressing in the 7.5 and 10mg groups and an absence of the reinstatement effect in the 15mg group. In experiment 2, animals treated with vehicle or 3.75mg of SR 21502 showed significant heroin place preferences but those treated with the higher doses showed no CPP.Conclusions: Our findings suggest that DA D3 receptors play a significant role in heroin approach behaviors driven by conditioned stimuli. As such, we propose that SR 21502 holds potential as an effective pharmacotherapeutic agent for relapse prevention and should be studied further. [ABSTRACT FROM AUTHOR]

Cocaine abuse increases the incidence of HIV-1-associated neurocognitive disorders. We have demonstrated that HIV-1 transactivator of transcription (Tat) allosterically modulates dopamine (DA) reuptake through the human DA transporter (hDAT), potentially contributing to Tat-induced cognitive impairment and potentiation of cocaine conditioned place preference (CPP). This study determined the effects of a novel allosteric modulator of DAT, SRI-32743 , on the interactions of HIV-1 Tat, DA, cocaine, and [3H]WIN35,428 with hDAT in vitro. SRI-32743 (50 nM) attenuated Tat-induced inhibition of [3H]DA uptake and decreased the cocaine-mediated dissociation of [3H]WIN35,428 binding in CHO cells expressing hDAT, suggesting a SRI-32743 -mediated allosteric modulation of the Tat-DAT interaction. In further in vivo studies utilizing doxycycline-inducible Tat transgenic (iTat-tg) mice, 14 days of Tat expression significantly reduced the recognition index by 31.7% in the final phase of novel object recognition (NOR) and potentiated cocaine-CPP 2.7-fold compared to responses of vehicle-treated control iTat-tg mice. The Tat-induced NOR deficits and potentiation of cocaine-CPP were not observed in saline-treated iTat-tg or doxycycline-treated G-tg (Tat-null) mice. Systemic administration (i.p.) of SRI-32743 prior to behavioral testing ameliorated Tat-induced impairment of NOR (at a dose of 10 mg/kg) and the Tat-induced potentiation of cocaine-CPP (at doses of 1 or 10 mg/kg). These findings demonstrate that Tat and cocaine interactions with DAT may be regulated by compounds interacting at the DAT allosteric modulatory sites, suggesting a potential therapeutic intervention for HIV-infected patients with concurrent cocaine abuse. Reuptake of physiological dopamine (DA) is mediated through the DA-binding site on DA transporter (DAT), where cocaine is a competitive inhibitor with DA for the binding site, reducing reuptake (a). HIV-1 Tat acts as a negative allosteric modulator that keeps DAT in an outward-facing state, also leading to a reduction of DA transport (b). SRI-32743 interacts with the allosteric modulatory site on DAT, competing with Tat to bind, resulting in the attenuation of Tat-inhibited DA transport (c). [Display omitted] • SRI-32743 shares the binding residues inside the pocket of DAT with Tat binding. • SRI-32743 inhibits DAT-mediated DA uptake and binding in an allosteric modulatory manner. • Tat-inhibited DA uptake through DAT and its binding sites are reversed by SRI-32743. • SRI-32743 ameliorates Tat-induced cognitive deficits and potentiation of cocaine reward. [ABSTRACT FROM AUTHOR]

Pathogenic mutations in the LRRK2 gene can cause late-onset Parkinson disease. The most common mutation, G2019S, resides in the kinase domain and enhances activity. LRRK2 possesses the unique property of cis-autophosphorylation of its own GTPase domain. Because high-resolution structures of the human LRRK2 kinase domain are not available, we used novel high-throughput assays that measured both cis-autophos-phorylation and trans-peptide phosphorylation to probe the ATP-binding pocket. We disclose hundreds of commercially available activity-selective LRRK2 kinase inhibitors. Some compounds inhibit cis-autophosphorylation more strongly than trans-peptide phosphorylation, and other compounds inhibit G2019S-LRRK2 more strongly than WT-LRRK2. Through exploitation of structure-activity relationships revealed through high-throughput analyses, we identified a useful probe inhibitor, SRI-29132 (11). SRI-29132 is exquisitely selective for LRRK2 kinase activity and is effective in attenuating proinflammatory responses in macrophages and rescuing neurite retraction phenotypes in neurons. Furthermore, the compound demonstrates excellent potency, is highly blood-brain barrier-permeant, but suffers from rapid first-pass metabolism. Despite the observed selectivity of SRI-29132, docking models highlighted critical interactions with residues conserved in many protein kinases, implying a unique structural configuration for the LRRK2 ATP-binding pocket. Although the human LRRK2 kinase domain is unstable and insoluble, we demonstrate that the LRRK2 homolog from ameba can be mutated to approximate some aspects of the human LRRK2 ATP-binding pocket. Our results provide a rich resource for LRRK2 small molecule inhibitor development. More broadly, our results provide a precedent for the functional interrogation of ATP-binding pockets when traditional approaches to ascertain structure prove difficult. [ABSTRACT FROM AUTHOR]

Abstract: Introduction of nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzofuran inhibitor 2, resulted in the discovery of the more potent pyridofuran analogue 5. Subsequent introduction of small alkyl and alkoxy ligands into the pyridine ring resulted in further improvements in replicon potency. Replacement of the 4-chloro moiety on the pyrimidine core with a methyl group, and concomitant monoalkylation of the C-2 amino moiety resulted in the identification of several inhibitors with desirable characteristics. Inhibitor 41, from the monosubstituted pyridofuran and inhibitor 50 from the disubstituted series displayed excellent potency, selectivity (GAPDH/MTS CC50) and PK parameters in all species studied, while the selectivity in the thymidine incorporation assay (DNA·CC50) was low. [Copyright &y& Elsevier]

Abstract: Compound 1 was identified as a HCV replication inhibitor from screening/early SAR triage. Potency improvement was achieved via modulation of substituent on the 5-azo linkage. Due to potential toxicological concern, the 5-azo linkage was replaced with 5-alkenyl or 5-alkynyl moiety. Analogs containing the 5-alkynyl linkage were found to be potent inhibitors of HCV replication. Further evaluation identified compounds 53 and 63 with good overall profile, in terms of replicon potency, selectivity and in vivo characteristics. Initial target engagement studies suggest that these novel carbanucleoside-like derivatives may inhibit the HCV replication complex (replicase). [Copyright &y& Elsevier]

Background: Naltrexone, a compound with high affinity for the μ opioid receptor (MOP-R) reduces alcohol consumption. SoRI-9409 is a derivative of naltrexone that has highest affinity at δ opioid receptors (DOP-Rs). We have investigated the effects of SoRI-9409 on ethanol consumption to determine the consequences of altering the naltrexone compound to a form with increased efficacy at DOP-Rs. Methods: Effects of the opioid receptor antagonists, SoRI-9409 (0–30 mg/kg, IP), naltrexone (0–30 mg/kg, IP), or naltrindole (0–10 mg/kg, IP) on ethanol consumption was measured in high- and low-ethanol–consuming rats with two different drinking paradigms. SoRI-9409-, naltrexone-, and naltrindole-mediated inhibition of DOP-R–stimulated [35S]GTPγS binding was measured in brain membranes prepared from high-ethanol–consuming rats. The effects of SoRI-9409 on morphine-mediated analgesia, conditioned place preference, and anxiety were also examined. Results: In high- but not low-ethanol–consuming animals, SoRI-9409 is threefold more effective and selective at reducing ethanol consumption when compared with naltrexone or naltrindole for up to 24 hours. SoRI-9409 administered daily for 28 days continuously reduced ethanol consumption, and when the administration of SoRI-9409 was terminated, the amount of ethanol consumed remained lower compared with vehicle-treated animals. Furthermore, SoRI-9409 inhibits DOP-R–stimulated [35S]GTPγS binding in brain membranes of high-ethanol–consuming rats. Conclusions: SoRI-9409 causes selective and long-lasting reductions of ethanol consumption. This suggests that compounds that have high affinity for DOP-Rs such as SoRI-9409 might be promising candidates for development as a novel therapeutic for the treatment of alcoholism. [Copyright &y& Elsevier]