Total Syntheses of Arylindolizidine Alkaloids (+)-Ipalbidine and (+)-Antofine

This paper presents the first application of two recently developed reactions to natural product synthesis. The first method involves a 6-endo-trig cyclization to prepare a versatile chiral enaminone building block. The second is a direct C–H arylation reaction. As a showcase for the utility of these methods, (+)-antofine and (+)-ipalbidine were synthesized in only 8 steps and 24–26% overall yields

Phenanthropiperidine Alkaloids: Methodology Development, Synthesis and Biological Evaluation

This work is directed towards the development of safe phenanthropiperidines for the treatment of cancer. It focuses on synthetic methodologies that facilitate their preparation and biological studies to better understand the neurological side-effects of the only alkaloid within this class to enter clinical trials: tylocrebrine. The preparation of cyclic enaminones in high enantiomeric purity is made possible through a one-flask, two-step protocol that uses mild Boc-deprotection conditions to suppress racemization. Elaboration of the enaminone scaffold was made possible with a direct palladium(II)-catalyzed arylation using aryltrifluoroborates as coupling partners. These methods give access to enantiomerically pure 3-arylpiperidines which were used as precursors of phenanthropiperidine alkaloids. A small phenanthropiperidine library was prepared and studied to elucidate the cause of tylocrebrine's neurological side-effects. Although the causes remain elusive, tylocrebrine and several of its analogs were found to bind to key biogenic amine receptors, disclosing another potential risk factor for their therapeutic use

Synthesis of 6- and 7-membered cyclic enaminones: Scope and mechanism

Six- and seven-membered cyclic enaminones can be prepared using common, environmentally benign reagents. Amino acids are used as synthetic precursors allowing diversification and the incorporation of chirality. The key reaction in this multi-step process involves deprotection of Boc-aminoynones and subsequent treatment with methanolic K2CO3 to induce cyclization. A β-amino elimination side reaction was identified in a few labile substrates that led to either loss of stereochemical purity or degradation. This process can be mitigated in specific cases using mild deprotection conditions. NMR and deuterium labeling experiments provided valuable insight into the workings and limitations of this reaction. Although disguised as a 6-endo-dig cyclization, the reagents employed in the transformation play a direct role in bond-making and bond-breaking, thus changing the mode of addition to a 6-endo-trig cyclization. This method can be used to construct an array of monocyclic and bicyclic scaffolds, many of which are found in well-known natural products (e.g. indolizidine, quinolizidine and Stemona alkaloids)

Inhibitory Control Deficits Associated with Upregulation of CB1R in the HIV-1 Tat Transgenic Mouse Model of Hand

In the era of combined antiretroviral therapy, HIV-1 infected individuals are living longer lives; however, longevity is met with an increasing number of HIV-1 associated neurocognitive disorders (HAND) diagnoses. The transactivator of transcription (Tat) is known to mediate the neurotoxic effects in HAND by acting directly on neurons and also indirectly via its actions on glia. The Go/No-Go (GNG) task was used to examine HAND in the Tat transgenic mouse model. The GNG task involves subjects discriminating between two stimuli sets in order to determine whether or not to inhibit a previously trained response. Data reveal inhibitory control deficits in female Tat(+) mice (p = .048) and an upregulation of cannabinoid type 1 receptors (CB1R) in the infralimbic (IL) cortex in the same female Tat(+) group (p < .05). A significant negative correlation was noted between inhibitory control and IL CB1R expression (r = -.543, p = .045), with CB1R expression predicting 30% of the variance of inhibitory control (R(2) = .295, p = .045). Furthermore, there was a significant increase in spontaneous excitatory postsynaptic current (sEPSC) frequencies in Tat(+) compared to Tat(-) mice (p = .008, across sexes). The increase in sEPSC frequency was significantly attenuated by bath application of PF3845, a fatty acid amide hydrolase (FAAH) enzyme inhibitor (p < .001). Overall, the GNG task is a viable measure to assess inhibitory control deficits in Tat transgenic mice and results suggest a potential therapeutic treatment for the observed deficits with drugs which modulate endocannabinoid enzyme activity. Graphical Abstract Results of the Go/No-Go operant conditioning task reveal inhibitory control deficits in female transgenic Tat(+) mice without significantly affecting males. The demonstrated inhibitory control deficits appear to be associated with an upregulation of cannabinoid type 1 receptors (CB1R) in the infralimbic (IL) cortex in the same female Tat(+) group

Robust anti-nociceptive effects of MAG lipase inhibition in a model of osteoarthritis pain

BACKGROUND AND PURPOSE: Chronic pain is often a symptom of knee osteoarthritis (OA) for which current analgesics are either inadequate, or are associated with serious side effects. The endocannabinoid system may offer alternative targets for pain-relief. We evaluated the effects of a potent and selective MAG lipase inhibitor (MJN110) on OA pain behaviour, spinal mechanisms of action and joint histopathology in the rat. Experimental approach: Intra-articular injection of monosodium iodoacetate (MIA) models OA pain and mimics clinical joint pathology. Effects of MJN110 on MIA-induced weight bearing asymmetry and lowered paw withdrawal thresholds (PWTs), changes in spinal gene expression and brain levels of relevant lipids were determined. Key results: Acute MJN110 (5 mg·kg−1) significantly reversed MIA induced weight bearing asymmetry (MIA /vehicle: 68 ± 6g; MIA /MJN110: 35 ± 4g, p<0.05) and lowered ipsilateral PWTs (MIA /vehicle: 7 ± 0.8g; MIA /MJN110: 11 ± 0.6g, p<0.05), via both CB1 and CB2 receptors. Repeated treatment with MJN110 (5 mg·kg−1) resulted in anti-nociceptive tolerance. A lower dose of MJN110 (1 mg·kg−1) acutely inhibited pain behaviour, which was maintained for one week of repeated administration, but had no effect on joint histology. MJN110 significantly inhibited expression of MPGES1 (p<0.05) in the ipsilateral dorsal horn of the spinal cord of MIA rats, compared to vehicle treated MIA rats. Both doses of MJN110 significantly elevated brain levels of the endocannabinoid 2-AG. Conclusions and Implications: Our data support the further investigation of the therapeutic potential of MAG lipase inhibitors for the treatment of OA pain

Robust anti-nociceptive effects of MAG lipase inhibition in a model of osteoarthritis pain

BACKGROUND AND PURPOSE: Chronic pain is often a symptom of knee osteoarthritis (OA) for which current analgesics are either inadequate, or are associated with serious side effects. The endocannabinoid system may offer alternative targets for pain-relief. We evaluated the effects of a potent and selective MAG lipase inhibitor (MJN110) on OA pain behaviour, spinal mechanisms of action and joint histopathology in the rat. Experimental approach: Intra-articular injection of monosodium iodoacetate (MIA) models OA pain and mimics clinical joint pathology. Effects of MJN110 on MIA-induced weight bearing asymmetry and lowered paw withdrawal thresholds (PWTs), changes in spinal gene expression and brain levels of relevant lipids were determined. Key results: Acute MJN110 (5 mg·kg−1) significantly reversed MIA induced weight bearing asymmetry (MIA /vehicle: 68 ± 6g; MIA /MJN110: 35 ± 4g, p<0.05) and lowered ipsilateral PWTs (MIA /vehicle: 7 ± 0.8g; MIA /MJN110: 11 ± 0.6g, p<0.05), via both CB1 and CB2 receptors. Repeated treatment with MJN110 (5 mg·kg−1) resulted in anti-nociceptive tolerance. A lower dose of MJN110 (1 mg·kg−1) acutely inhibited pain behaviour, which was maintained for one week of repeated administration, but had no effect on joint histology. MJN110 significantly inhibited expression of MPGES1 (p<0.05) in the ipsilateral dorsal horn of the spinal cord of MIA rats, compared to vehicle treated MIA rats. Both doses of MJN110 significantly elevated brain levels of the endocannabinoid 2-AG. Conclusions and Implications: Our data support the further investigation of the therapeutic potential of MAG lipase inhibitors for the treatment of OA pain

Neuroprotective effects of fatty acid amide hydrolase catabolic enzyme inhibition in a HIV-1 Tat model of neuroAIDS

The HIV-1 transactivator of transcription (Tat) is a neurotoxin involved in the pathogenesis of HIV-1 associated neurocognitive disorders (HAND). The neurotoxic effects of Tat are mediated directly via AMPA/NMDA receptor activity and indirectly through neuroinflammatory signaling in glia. Emerging strategies in the development of neuroprotective agents involve the modulation of the endocannabinoid system. A major endocannabinoid, anandamide (N-arachidonoylethanolamine, AEA), is metabolized by fatty acid amide hydrolase (FAAH). Here we demonstrate using a murine prefrontal cortex primary culture model that the inhibition of FAAH, using PF3845, attenuates Tat-mediated increases in intracellular calcium, neuronal death, and dendritic degeneration via cannabinoid receptors (CB1R and CB2R). Live cell imaging was used to assess Tat-mediated increases in [Ca(2+)]i, which was significantly reduced by PF3845. A time-lapse assay revealed that Tat potentiates cell death while PF3845 blocks this effect. Additionally PF3845 blocked the Tat-mediated increase in activated caspase-3 (apoptotic marker) positive neurons. Dendritic degeneration was characterized by analyzing stained dendritic processes using Imaris and Tat was found to significantly decrease the size of processes while PF3845 inhibited this effect. Incubation with CB1R and CB2R antagonists (SR141716A and AM630) revealed that PF3845-mediated calcium effects were dependent on CB1R, while reduced neuronal death and degeneration was CB2R-mediated. PF3845 application led to increased levels of AEA, suggesting the observed effects are likely a result of increased endocannabinoid signaling at CB1R/CB2R. Our findings suggest that modulation of the endogenous cannabinoid system through inhibition of FAAH may be beneficial in treatment of HAND

Inhibitory Neurotransmission Is Sex-Dependently Affected by Tat Expression in Transgenic Mice and Suppressed by the Fatty Acid Amide Hydrolase Enzyme Inhibitor PF3845 via Cannabinoid Type-1 Receptor Mechanisms

(1) Background. The endocannabinoid (eCB) system, which regulates physiological and cognitive processes, presents a promising therapeutic target for treating HIV-associated neurocognitive disorders (HAND). Here we examine whether upregulating eCB tone has potential protective effects against HIV-1 Tat (a key HIV transactivator of transcription) protein-induced alterations in synaptic activity. (2) Methods. Whole-cell patch-clamp recordings were performed to assess inhibitory GABAergic neurotransmission in prefrontal cortex slices of Tat transgenic male and female mice, in the presence and absence of the fatty acid amide hydrolase (FAAH) enzyme inhibitor PF3845. Western blot and mass spectrometry analyses assessed alterations of cannabinoid receptor and enzyme protein expression as well as endogenous ligands, respectively, to determine the impact of Tat exposure on the eCB system. (3) Results. GABAergic activity was significantly altered upon Tat exposure based on sex, whereas the effectiveness of PF3845 to suppress GABAergic activity in Tat transgenic mice was not altered by Tat or sex and involved CB1R-related mechanisms that depended on calcium signaling. Additionally, our data indicated sex-dependent changes for AEA and related non-eCB lipids based on Tat induction. (4) Conclusion. Results highlight sex- and/or Tat-dependent alterations of GABAergic activity and eCB signaling in the prefrontal cortex of Tat transgenic mice and further increase our understanding about the role of FAAH inhibition in neuroHIV

Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity

Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub)(1). Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates(2). By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate(3,4). Additionally, RING-between-RING (RBR) E3 ligases have a canonical RING domain that is linked to an ancillary domain. This ancillary domain contains a catalytic cysteine that enables a hybrid RING-HECT mechanism(5). Ubiquitination is typically considered a post-translational modification of lysine residues, as there are no known human E3 ligases with non-lysine activity. Here we perform activity-based protein profiling of HECT or RBR-like E3 ligases and identify the neuron-associated E3 ligase MYCBP2 (also known as PHR1) as the apparent single member of a class of RING-linked E3 ligase with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues that relay ubiquitin to its substrate via thioester intermediates. Crystallographic characterization of this class of E3 ligase, which we designate RING-Cys-relay (RCR), provides insights into its mechanism and threonine selectivity. These findings implicate non-lysine ubiquitination in cellular regulation of higher eukaryotes and suggest that E3 enzymes have an unappreciated mechanistic diversity

Monoacylglycerol Lipase Inhibitor MJN110 Reduces Neuronal Hyperexcitability, Restores Dendritic Arborization Complexity, and Regulates Reward-Related Behavior in Presence of HIV-1 Tat

While current therapeutic strategies for people living with human immunodeficiency virus type 1 (HIV-1) suppress virus replication peripherally, viral proteins such as transactivator of transcription (Tat) enter the central nervous system early upon infection and contribute to chronic inflammatory conditions even alongside antiretroviral treatment. As demand grows for supplemental strategies to combat virus-associated pathology presenting frequently as HIV-associated neurocognitive disorders (HAND), the present study aimed to characterize the potential utility of inhibiting monoacylglycerol lipase (MAGL) activity to increase inhibitory activity at cannabinoid receptor-type 1 receptors through upregulation of 2-arachidonoylglycerol (2-AG) and downregulation of its degradation into proinflammatory metabolite arachidonic acid (AA). The MAGL inhibitor MJN110 significantly reduced intracellular calcium and increased dendritic branching complexity in Tat-treated primary frontal cortex neuron cultures. Chronic MJN110 administration in vivo increased 2-AG levels in the prefrontal cortex (PFC) and striatum across Tat(+) and Tat(–) groups and restored PFC N-arachidonoylethanolamine (AEA) levels in Tat(+) subjects. While Tat expression significantly increased rate of reward-related behavioral task acquisition in a novel discriminative stimulus learning and cognitive flexibility assay, MJN110 altered reversal acquisition specifically in Tat(+) mice to rates indistinguishable from Tat(–) controls. Collectively, our results suggest a neuroprotective role of MAGL inhibition in reducing neuronal hyperexcitability, restoring dendritic arborization complexity, and mitigating neurocognitive alterations driven by viral proteins associated with latent HIV-1 infection