Many cases of accidental death associated with drug overdose are due to chronic opioid use, tolerance, and addiction. Analgesic tolerance is characterized by a decreased response to the analgesic effects of opioids, requiring increasingly higher doses to maintain the desired level of pain relief. Overactivation of GluN2B-containing N-methyl-d-Aspartate receptors is thought to play a key role in mechanisms underlying cellular adaptation that takes place in the development of analgesic tolerance. Herein, we describe a novel GluN2B-selective negative allosteric modulator, EU93-108, that shows high potency and brain penetrance. We describe the structural basis for binding at atomic resolution. This compound possesses intrinsic analgesic properties in the rodent tail immersion test. EU93-108 has an acute and significant anodyne effect, whereby morphine when combined with EU93-108 produces a higher tail flick latency compared to that of morphine alone. These data suggest that engagement of GluN2B as a target has utility in the treatment of pain, and EU93-108 could serve as an appropriate tool compound to interrogate this hypothesis. Future structure-activity relationship work around this scaffold could give rise to compounds that can be co-administered with opioids to diminish the onset of tolerance due to chronic opioid use, thereby modifying their utility.

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission. Most native NMDA receptors are tetrameric assemblies of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. Co-assembly of the glycine-binding GluN1 with glycine-binding GluN3 subunits (GluN3A-B) creates glycine activated receptors that possess strikingly different functional and pharmacological properties compared to GluN1/GluN2 NMDA receptors. The role of GluN1/GluN3 receptors in neuronal function remains unknown, in part due to lack of pharmacological tools with which to explore their physiological roles. We have identified the negative allosteric modulator EU1180-438, which is selective for GluN1/GluN3 receptors over GluN1/GluN2 NMDA receptors, AMPA, and kainate receptors. EU1180-438 is also inactive at GABA, glycine, and P2X receptors, but displays inhibition of some nicotinic acetylcholine receptors. Furthermore, we demonstrate that EU1180-438 produces robust inhibition of glycine-activated current responses mediated by native GluN1/GluN3A receptors in hippocampal CA1 pyramidal neurons. EU1180-438 is a non-competitive antagonist with activity that is independent of membrane potential (i.e. voltage-independent), glycine concentration, and extracellular pH. Non-stationary fluctuation analysis of neuronal current responses provided an estimated weighted mean unitary conductance of 6.1 pS for GluN1/GluN3A channels, and showed that EU1180-438 has no effect on conductance. Site-directed mutagenesis suggests that structural determinants of EU1180-438 activity reside near a short pre-M1 helix that lies parallel to the plane of the membrane below the agonist binding domain. These findings demonstrate that structural differences between GluN3 and other glutamate receptor subunits can be exploited to generate subunit-selective ligands with utility in exploring the roles GluN3 in neuronal function.

We have developed a specific technique for imaging cancer in vivo using Cy5.5-labeled factor VIIa (fVIIa), clotting-deficient FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-tissue factor (TF) antibody. FVIIa is the natural ligand for TF. We took advantage of the fact that vascular endothelial cells (VECs) in cancer, but not normal tissue, aberrantly express TF due to its induction by vascular endothelial growth factor (VEGF). Under physiological conditions, TF is expressed by stromal cells and outer blood vessel layers (smooth muscle and adventitia), but not by VECs. We hypothesized that labeled fVIIa or anti-TF antibodies could be used to image the tumor vasculature in vivo. To test this, Cy5.5-labeled fVIIa, FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-TF antibody were developed and administered to athymic nude mice carrying xenografts including glioma U87EGFRviii, pancreatic cancer ASPC-1 and Mia PaCa-2, and squamous cell carcinoma KB-V1. Cy5.5 labeled with these targeting proteins specifically localized to the tumor xenografts for at least 14 days but unconjugated Cy5.5 did not localize to any xenografts or organs. This method of imaging TF in the tumor VECs may be useful in detecting primary tumors and metastases as well as monitoring in vivo therapeutic responses.

Here we describe the synthesis and structure-activity relationship for a class of pyrazoline-containing dihydroquinolone negative allosteric modulators of the NMDA receptor that show strong subunit selectivity for GluN2C- and GluN2D-containing receptors over GluN2A- and GluN2B-containing receptors. Several members of this class inhibit NMDA receptor responses in the nanomolar range and are more than 50-fold selective over GluN1/GluN2A and GluN1/GluN2B NMDA receptors, as well as AMPA, kainate, GABA, glycine, nicotinic, serotonin, and purinergic receptors. Analysis of the purified enantiomers of one of the more potent and selective compounds shows that the S-enantiomer is both more potent and more selective than the R-enantiomer. The S-enantiomer had an IC 50 of 0.17-0.22 μM at GluN2D- and GluN2C-containing receptors, respectively, and showed over 70-fold selectivity over other NMDA receptor subunits. The subunit selectivity of this class of compounds should be useful in defining the role of GluN2C- and GluN2D-containing receptors in specific brain circuits in both physiological and pathophysiological conditions.

Nuclear factor kappa B (NF-κB) is a key signaling molecule in the elaboration of the inflammatory response. Data indicate that curcumin, a natural ingredient of the curry spice turmeric, acts as a NF-κB inhibitor and exhibits both anti-inflammatory and anti-cancer properties. Curcumin analogues with enhanced activity on the NF-κB and other inflammatory signaling pathways have been developed including the synthetic monoketone compound termed 3,5-Bis(2-fluorobenzylidene)-4-piperidone (EF24). 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31) is a structurally-related curcumin analogue whose potency for NF-κB inhibition has yet to be determined. To examine the activity of EF31 compared to EF24 and curcumin, mouse RAW264.7 macrophages were treated with EF31, EF24, curcumin (1–100µM) or vehicle (DMSO 1%) for 1 hour. NF-κB pathway activity was assessed following treatment with lipopolysaccharide (LPS) (1µg/mL). EF31 (IC50 ~5µM) exhibited significantly more potent inhibition of LPS-induced NF-κB DNA binding compared to both EF24 (IC50~35µM) and curcumin (IC50 >50µM). In addition, EF31 exhibited significantly greater inhibition of NF-κB nuclear translocation as well as the induction of downstream inflammatory mediators including pro-inflammatory cytokine mRNA and protein (tumor necrosis factor-α, interleukin-1β, and interleukin-6). Regarding the mechanism of these effects on NF-κB activity, EF31 (IC50~1.92µM) exhibited significantly greater inhibition of IκB kinase β compared to EF24 (IC50~131µM). Finally, EF31 demonstrated potent toxicity in NF-κB-dependent cancer cell lines while having minimal and reversible toxicity in RAW264.7 macrophages. These data indicate that EF31 is a more potent inhibitor of NF-κB activity than either EF24 or curcumin while exhibiting both anti-inflammatory and anticancer activities. Thus, EF31 represents a promising curcumin analogue for further therapeutic development.

NMDA receptors are tetrameric complexes of NR1 and NR2A-D subunits that mediate excitatory synaptic transmission and play a role in neurological disorders. We have identified a novel subunit-selective potentiator of NMDA receptors containing the NR2C or NR2D subunit, which could allow selective modification of circuit function in regions expressing NR2C/D subunits. The substituted tetrahydroisoquinoline CIQ enhances receptor responses two-fold with an EC50 of 3 μM by increasing channel opening frequency without altering mean open time or EC50 values for glutamate or glycine. The actions of CIQ depend on a single residue in the M1 region (NR2D Thr592) and the linker between the amino terminal domain and agonist binding domain. CIQ potentiates native NR2D-containing NMDA receptor currents from subthalamic neurons. Our identification of a subunit-selective NMDA receptor modulator reveals a new class of pharmacological tools with which to probe the role of NR2C- and NR2D-containing NMDA receptors in brain function and disease.

The synthesis and structure–activity relationship analysis of a novel class of amide-based biaryl NR2B-selective NMDA receptor antagonists are presented. Some of the studied compounds are potent, selective, non-competitive, and voltage-independent antagonists of NR2B-containing NMDA receptors. Like the founding member of this class of antagonists (ifenprodil), several interesting compounds of the series bind to the amino terminal domain of the NR2B subunit to inhibit function. Analogue potency is modu-lated by linker length, flexibility, and hydrogen bonding opportunities. However, unlike previously described classes of NR2B-selective NMDA antagonists that exhibit off-target activity at a variety of monoamine receptors, the compounds described herein show much diminished effects against the hERG channel and α1-adrenergic receptors. Selections of the compounds discussed have acceptable half-lives in vivo and are predicted to permeate the blood–brain barrier. These data together suggest that masking charged atoms on the linker region of NR2B-selective antagonists can decrease undesirable side effects while still maintaining on-target potency.

Nucleoside- and nucleotide-based therapeutics are indispensable treatment options for patients suffering from malignant and viral diseases. These agents are most commonly administered to patients as prodrugs to maximize bioavailability and efficacy. While the literature provides a practical prodrug playbook to facilitate the delivery of nucleoside and nucleotide therapeutics, small context-dependent amendments to these popular prodrug strategies can drive dramatic improvements in pharmacokinetic (PK) profiles. Herein we offer a brief overview of current prodrug strategies, as well as a case study involving the fine-tuning of lipid prodrugs of acyclic nucleoside phosphonate tenofovir (TFV), an approved nucleotide HIV reverse transcriptase inhibitor (NtRTI) and the cornerstone of combination antiretroviral therapy (cART). Installation of novel lipid terminal motifs significantly reduced fatty acid hepatic ω-oxidation while maintaining potent antiviral activity. This work contributes important insights to the expanding repertoire of lipid prodrug strategies in general, but particularly for the delivery and distribution of acyclic nucleoside phosphonates.

Increasing attention has been given to the anti-cancer effects of curcumin and the ability of this natural product to inhibit cancer cell proliferation. New curcumin analogs have been developed to optimize the in vitro and in vivo activity of the parent compound yet retain the same safety profile. EF24, a fluorinated synthetic analog, surpasses curcumin in its ability to inhibit cancer cell viability and down-regulate TNFα-induced NF-κB activation. Here we report a critical role of the p38-mediated signaling pathway in the determination of lung cancer cell’s sensitivity to EF24. We have found that EF24-induced decease of lung cancer cell viability was accompanied by upregulated mitogen-activated protein kinases (MAPK) as evidenced by increased phosphorylation of ERK1/2, JNK, and p38. Pharmacological investigation led to our suggestion that EF24 triggers a negative feedback loop through p38 activation. In support of this model, inhibition of p38, either by small molecule inhibitors or through an RNAi-mediated knockdown approach, enhanced the EF24 induced apoptotic death of A549 cells. Thus, inhibition of p38 may boost the EF24 anticancer effect. Indeed, a combination of EF24 and SB203580, a p38 inhibitor, synergistically inhibited clonogenic activity of A549 lung cancer cells and induced their apoptosis as reflected by poly(ADP-ribose) polymerase cleavage, the accumulation of the sub-G1 fraction of cells, and apoptotic cell staining. These studies offer a novel strategy that combines the curcumin analog EF24 with a p38 inhibitor for potentially enhanced therapy in the treatment of lung cancer.

A family of cyclic 1-deoxysphingolipid derivatives of structure 4 has been designed and synthesized, which may serve as tumorigenesis suppressors for various cancers. Compound 4 is a second-generation analogue developed from sphingosine (1), in which a hydroxyl substituent is moved from C1 to C5 and a methylene is added for conformational rigidity between the C2-nitrogen substituent and C4. The synthetic chemistry for pyrrolidine ring closure at C3-C4 features ring-closing metathesis followed by hydroboration-oxidation.