SRI-32743, a novel allosteric modulator, attenuates HIV-1 Tat protein-induced inhibition of the dopamine transporter and alleviates the potentiation of cocaine reward in HIV-1 Tat transgenic mice.

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.

Targeting the HuR Oncogenic Role with a New Class of Cytoplasmic Dimerization Inhibitors.

The development of novel therapeutics that exploit alterations in the activation state of key cellular signaling pathways due to mutations in upstream regulators has generated the field of personalized medicine. These first-generation efforts have focused on actionable mutations identified by deep sequencing of large numbers of tumor samples. We propose that a second-generation opportunity exists by exploiting key downstream "nodes of control" that contribute to oncogenesis and are inappropriately activated due to loss of upstream regulation and microenvironmental influences. The RNA-binding protein HuR represents such a node. Because HuR functionality in cancer cells is dependent on HuR dimerization and its nuclear/cytoplasmic shuttling, we developed a new class of molecules targeting HuR protein dimerization. A structure-activity relationship algorithm enabled development of inhibitors of HuR multimer formation that were soluble, had micromolar activity, and penetrated the blood-brain barrier. These inhibitors were evaluated for activity validation and specificity in a robust cell-based assay of HuR dimerization. SRI-42127, a molecule that met these criteria, inhibited HuR multimer formation across primary patient-derived glioblastoma xenolines (PDGx), leading to arrest of proliferation, induction of apoptosis, and inhibition of colony formation. SRI-42127 had favorable attributes with central nervous system penetration and inhibited tumor growth in mouse models. RNA and protein analysis of SRI-42127-treated PDGx xenolines across glioblastoma molecular subtypes confirmed attenuation of targets upregulated by HuR. These results highlight how focusing on key attributes of HuR that contribute to cancer progression, namely cytoplasmic localization and multimerization, has led to the development of a novel, highly effective inhibitor. SIGNIFICANCE: These findings utilize a cell-based mechanism of action assay with a structure-activity relationship compound development pathway to discover inhibitors that target HuR dimerization, a mechanism required for cancer promotion.

Synthesis and Structure-Activity Relationships of 5'-Aryl-14-alkoxypyridomorphinans: Identification of a µ Opioid Receptor Agonist/δ Opioid Receptor Antagonist Ligand with Systemic Antinociceptive Activity and Diminished Opioid Side Effects.

We previously identified a pyridomorphinan (6, SRI-22138) possessing a 4-chlorophenyl substituent at the 5'-position on the pyridine and a 3-phenylpropoxy at the 14-position of the morphinan as a mixed µ opioid receptor (MOR) agonist and δ/κ opioid receptor (DOR/KOR) antagonist with potent antinociceptive activity and diminished tolerance and dependence in rodents. Structural variations at the 5'- and 14-positions of this molecule gave insights into the structure-activity relationships for binding and functional activity. Subtle structural changes exerted significant influence, particularly on the ability of the compounds to function as agonists at the MOR. In vivo evaluation identified compound 20 (SRI-39067) as a MOR agonist/DOR antagonist that produced systemically active potent antinociceptive activity in tail-flick assay in mice, with diminished tolerance, dependence/withdrawal, reward liability, and respiratory depression versus morphine. These results support the hypothesis that mixed MOR agonist/DOR antagonist ligands may emerge as novel opioid analgesics with reduced side effects.

A Novel Mu-Delta Opioid Agonist Demonstrates Enhanced Efficacy With Reduced Tolerance and Dependence in Mouse Neuropathic Pain Models.

Numerous studies have demonstrated a physiological interaction between the mu opioid receptor (MOR) and delta opioid receptor (DOR) systems. A few studies have shown that dual MOR-DOR agonists could be beneficial, with reduced tolerance and addiction liability, but are nearly untested in chronic pain models, particularly neuropathic pain. In this study, we tested the MOR-DOR agonist SRI-22141 in mice in the clinically relevant models of HIV Neuropathy and Chemotherapy-Induced Peripheral Neuropathy (CIPN). SRI-22141 was more potent than morphine in the tail flick pain test and had equal or enhanced efficacy versus morphine in both neuropathic pain models, with significantly reduced tolerance. SRI-22141 also produced no jumping behavior during naloxone-precipitated withdrawal in CIPN or naïve mice, suggesting that SRI-22141 produces little to no dependence. SRI-22141 also reduced tumor necrosis factor-α and cyclooxygenase-2 in CIPN in the spinal cord, suggesting an anti-inflammatory mechanism of action. The DOR-selective antagonist naltrindole strongly reduced CIPN efficacy and anti-inflammatory activity in the spinal cord, without affecting tail flick antinociception, suggesting the importance of DOR activity in these models. Overall, these results provide compelling evidence that MOR-DOR agonists could have strong efficacy with reduced side effects and an anti-inflammatory mechanism in the treatment of neuropathic pain. PERSPECTIVE: This study demonstrates that a MOR-DOR dual agonist given chronically in chronic neuropathic pain models has enhanced efficacy with strongly reduced tolerance and dependence, with a further anti-inflammatory effect in the spinal cord. This suggests that MOR-DOR dual agonists could be effective treatments for neuropathic pain with reduced side effects.

Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D2 Dopamine Receptor.

The dopamine D2 receptor (D2R) is known to elicit effects through activating two major signaling pathways mediated by either G proteins (Gi/o) or ß-arrestins. However, the specific role of each pathway in physiological or therapeutic activities is not known with certainty. One approach to the dissection of these pathways is through the use of drugs that can selectively modulate one pathway vs. the other through a mechanism known as functional selectivity or biased signaling. Our laboratory has previously described a G protein signaling-biased agonist, MLS1547, for the D2R using a variety of in vitro functional assays. To further evaluate the biased signaling activity of this compound, we investigated its ability to promote D2R internalization, a process known to be mediated by ß-arrestin. Using multiple cellular systems and techniques, we found that MLS1547 promotes little D2R internalization, which is consistent with its inability to recruit ß-arrestin. Importantly, we validated these results in primary striatal neurons where the D2R is most highly expressed suggesting that MLS1547 will exhibit biased signaling activity in vivo. In an effort to optimize and further explore structure-activity relationships (SAR) for this scaffold, we conducted an iterative chemistry campaign to synthesize and characterize novel analogs of MLS1547. The resulting analysis confirmed previously described SAR requirements for G protein-biased agonist activity and, importantly, elucidated new structural features that are critical for agonist efficacy and signaling bias of the MLS1547 scaffold. One of the most important determinants for G protein-biased signaling is the interaction of a hydrophobic moiety of the compound with a defined pocket formed by residues within transmembrane five and extracellular loop two of the D2R. These results shed new light on the mechanism of biased signaling of the D2R and may lead to improved functionally-selective molecules.

Reformulating a Pharmacophore for 5-HT2A Serotonin Receptor Antagonists.

Several pharmacophore models have been proposed for 5-HT2A serotonin receptor antagonists. These typically consist of two aromatic/hydrophobic moieties separated by a given distance from each other, and from a basic amine. Although specified distances might vary, the models are relatively similar in their general construction. Because our preliminary data indicated that two aromatic (hydrophobic) moieties might not be required for such action, we deconstructed the serotonin-dopamine antipsychotic agent risperidone (1) into four smaller structural fragments that were thoroughly examined in 5-HT2A receptor binding and functional (i.e., two-electrode voltage clamp (TEVC) and intracellular calcium release) assays. It was apparent that truncated risperidone analogues behaved as antagonists. In particular, 6-fluoro-3-(1-methylpiperidin-4-yl)benzisoxazole (4) displayed high affinity for 5-HT2A receptors (Ki of ca. 12 nM) relative to risperidone (Ki of ca. 5 nM) and behaved as a potent 5-HT2A serotonin receptor antagonist. These results suggest that multiple aromatic (hydrophobic) moieties are not essential for high-affinity 5-HT2A receptor binding and antagonist activity and that current pharmacophore models for such agents are very much in need of revision.

Hexafluoro-2-propanol-Promoted Intermolecular Friedel-Crafts Acylation Reaction.

The intermolecular Friedel-Crafts acylation was carried out in hexafluoro-2-propanol to yield aryl and heteroaryl ketones at room temperature without any additional reagents.

Abuse-related neurochemical and behavioral effects of cathinone and 4-methylcathinone stereoisomers in rats.

Cathinone and many of its analogs produce behavioral effects by promoting transporter-mediated release of the monoamine neurotransmitters dopamine, norepinephrine and/or serotonin. Stereoselectivity is one determinant of neurochemical and behavioral effects of cathinone analogs. This study compared effectiveness of the S(-) and R(+) enantiomers of cathinone and 4-methylcathinone to produce in vitro monoamine release and in vivo abuse-related behavioral effects in rats. For neurochemical studies, drug effects were evaluated on monoamine release through dopamine, norepinephrine, and serotonin transporters (DAT, NET and SERT, respectively) in rat brain synaptosomes. For behavioral studies, drug effects were evaluated on responding for electrical brain stimulation in an intracranial self-stimulation (ICSS) procedure. The cathinone enantiomers differed in potency [S(-)>R(+)], but both enantiomers were >50-fold selective at promoting monoamine release through DAT vs. SERT, and both enantiomers produced ICSS facilitation. The 4-methylcathinone enantiomers also differed in potency [S(-)>R(+)]; however, in neurochemical studies, the decrease in potency from S(-) to R(+)4-methylcathinone was less for DAT than for SERT, and as a result, DAT vs. SERT selectivity was greater for R(+) than for S(-)4-methylcathinone (4.1- vs. 1.2-fold). Moreover, in behavioral studies, S(-)4-methylcathinone produced only ICSS depression, whereas R(+)4-methylcathinone produced ICSS facilitation. This study provides further evidence for stereoselectivity in neurochemical and behavioral actions of cathinone analogs. More importantly, stereoselective 4-methylcathinone effects on ICSS illustrate the potential for diametrically opposite effects of enantiomers in a preclinical behavioral assay of abuse potential.

Intramolecular Friedel-Crafts Acylation Reaction Promoted by 1,1,1,3,3,3-Hexafluoro-2-propanol.

Simple dissolution of an arylalkyl acid chloride in 1,1,1,3,3,3-hexafluoro-2-propanol promotes an intramolecular Friedel-Crafts acylation without additional catalysts or reagents. This reaction is operationally trivial in both execution and product isolation (only requiring concentration followed by purification) and accommodates a broad range of substrates. Preliminary studies that bear upon potential reaction mechanisms are reported.

A concomitant allylic azide rearrangement/intramolecular azide-alkyne cycloaddition sequence.

An intramolecular Huisgen cycloaddition of an interconverting set of isomeric allylic azides with alkynes affords substituted triazoles in high yield. The stereoisomeric vinyl-substituted triazoloxazines formed depend on the rate of cycloaddition of the different allylic azide precursors when the reaction is carried out under thermal conditions. In contrast, dimerized macrocyclic products were obtained when the reaction was done using copper(I)-catalyzed conditions, demonstrating the ability to control the reaction products through changing conditions.

Mephedrone and methylenedioxypyrovalerone (MDPV), major constituents of "bath salts," produce opposite effects at the human dopamine transporter.

RATIONALE: Psychoactive "bath salts" represent a relatively new drug of abuse combination that was placed in Schedule I in October 2011. Two common ingredients of bath salts include the cathinone analogs: mephedrone and methylenedioxypyrovalerone (MDPV). The mechanism of action of these synthetic cathinone analogs has not been well investigated. MATERIALS AND METHODS: Because cathinone and methcathinone are known to act as releasing agents at the human dopamine transporter (hDAT), mephedrone and MDPV were investigated at hDAT expressed in Xenopus oocytes. RESULTS: Whereas mephedrone was found to have the signature of a dopamine-releasing agent similar to methamphetamine or methcathinone, MDPV behaved as a cocaine-like reuptake inhibitor of dopamine. CONCLUSIONS: Mephedrone and MDPV produce opposite electrophysiological signatures through hDAT expressed in oocytes. Implications are that the combination (as found in bath salts) might produce effects similar to a combination of methamphetamine and cocaine.