The same T98G cell that is shown in Movie S1 imaged 10 min after the addition of 0.5 µM ST-11. Speed = 10Ã-.

Figure S1: Representative histological features of orthotropic DBT tumor 3 weeks after implantation Figure S2: Semi-quantitative analysis of microglial invasion of DBT tumors Figure S3: ST-11 reduces proliferation and triggers apoptosis in DBT cells in vitro Figure S4: ST-11 leaves interphase MT tips intact and mediates reversible effects on MT assembly Figure S5: ST-11 activates apoptosis in cultured T98G and DBT cells Figure S6: Paclitaxel reduces the viability of MDA-MB-231 cells Figure S7: Solubility and stability of ST-11 Movies: MT dynamics in T98G cells Supplemental materials & methods containing detailed experimental procedures Table S1: Response of T98G cells to 10 AI analogues as measured with WST-1

T98G cell transfected with GFP-EB3 (green) to label growing MT tips and RFP-CenpB (red) to label centromeres. Speed = 10Ã-.

GPR124 is involved in embryonic development and remains expressed by select organs. The importance of GPR124 during development suggests that its aberrant expression might participate in tumor growth. Here we show that both increases and decreases in GPR124 expression in glioblastoma cells reduce cell proliferation by differentially altering the duration mitotic progression. Using mass spectrometry-based proteomics, we discovered that GPR124 interacts with ch-TOG, a known regulator of both microtubule (MT)-plus-end assembly and mitotic progression. Accordingly, changes in GPR124 expression and ch-TOG similarly affect MT assembly measured by real-time microscopy in cells. Our study describes a novel molecular interaction involving GPR124 and ch-TOG at the plasma membrane that controls glioblastoma cell proliferation by modifying MT assembly rates and controlling the progression of distinct phases of mitosis.

Abstract Glioblastoma multiforme is a devastating and intractable type of cancer. Current antineoplastic drugs do not improve the median survival of patients diagnosed with glioblastoma multiforme beyond 14 to 15 months, in part because the blood–brain barrier is generally impermeable to many therapeutic agents. Drugs that target microtubules (MT) have shown remarkable efficacy in a variety of cancers, yet their use as glioblastoma multiforme treatments has also been hindered by the scarcity of brain-penetrant MT-targeting compounds. We have discovered a new alkylindole compound, ST-11, that acts directly on MTs and rapidly attenuates their rate of assembly. Accordingly, ST-11 arrests glioblastoma multiforme cells in prometaphase and triggers apoptosis. In vivo analyses reveal that unlike current antitubulin agents, ST-11 readily crosses the blood–brain barrier. Further investigation in a syngeneic orthotopic mouse model of glioblastoma multiforme shows that ST-11 activates caspase-3 in tumors to reduce tumor volume without overt toxicity. Thus, ST-11 represents the first member of a new class of brain-penetrant antitubulin therapeutic agents. Mol Cancer Ther; 15(9); 2018–29. ©2016 AACR.

G protein-coupled receptors (GPCRs) are essential membrane proteins that facilitate cell-to-cell communication and co-ordinate physiological processes. At least 30 human GPCRs contain a Type I PSD-95/DLG/Zo-1 (PDZ) ligand in their distal C-terminal domain; this four amino acid motif of X-[S/T]-X-[phi] sequence facilitates interactions with PDZ domain-containing proteins. Because PDZ protein interactions have profound effects on GPCR ligand pharmacology, cellular localization, signal-transduction effector coupling and duration of activity, we analyzed the importance of Type I PDZ ligands for the function of 23 full-length and PDZ-ligand truncated (Delta PDZ) human GPCRs in cultured human cells. SNAP-epitope tag polyacrylamide gel electrophoresis revealed most Type I PDZ GPCRs exist as both monomers and multimers; removal of the PDZ ligand played minimal role in multimer formation. Additionally, SNAP-cell surface staining indicated removal of the PDZ ligand had minimal effects on plasma membrane localization for most GPCR5 examined. Label-free dynamic mass redistribution functional responses, however, revealed diverging effects of the PDZ ligand. While no clear trend was observed across all GPCRs tested or even within receptor families, a subset of GPCRs displayed diminished agonist efficacy in the absence of a PDZ ligand (i.e. HT2RB, ADRB1), whereas others demonstrated enhanced agonist efficacies (i.e. LPAR2, SSTR5). These results demonstrate the utility of label-free functional assays to tease apart the contributions of conserved protein interaction domains for GPCR signal-transduction coupling in cultured cells. (C) 2016 Elsevier Ltd. All rights reserved.

Ligands targeting G protein-coupled receptors (GPCR) expressed by microglia have been shown to regulate distinct components of their activation process, including cell proliferation, migration and differentiation into M1 or M2 phenotypes. Cannabinoids, including the active component of the Cannabis plant, tetrahydrocannabinol (THC), and the synthetic alkylindole (AI) compound, WIN55212-2 (WIN-2), activate two molecularly identified GPCRs: CB1 and CB2 . Previous studies reported that WIN-2 activates an additional unknown GPCR that is not activated by plant-derived cannabinoids, and evidence indicates that microglia express these receptors. Detailed studies on the role of AI-sensitive receptors in microglial cell activation were difficult as no selective pharmacological tools were available. Here, three newly-developed AI analogues allowed us to determine if microglia express AI-sensitive receptors and if so, study how they regulate the microglial cell activation process. We found that mouse microglia in primary culture express functional AI-sensitive receptors as measured by radioligand binding and changes in intracellular cAMP levels, and that these receptors control both basal and ATP-stimulated migration. AI analogues inhibit cell proliferation stimulated by macrophage-colony stimulating factor (M-CSF) without affecting basal cell proliferation. Remarkably, AI analogues do not control the expression of effector proteins characteristic of M1 or M2 phenotypes; yet activating microglia with M1 and M2 cytokines reduces the microglial response to AI analogues. Our results suggest that microglia express functional AI-sensitive receptors that control select components of their activation process. Agonists of these novel targets might represent a novel class of therapeutics to influence the microglial cell activation process.

Glioblastoma multiforme (GBM) is the most common and aggressive brain neoplasm. Treatment options for this cancer are limited, and the five-year survival rate is less than 5%. Cannabinoid compounds have the ability to cross the blood brain barrier (BBB) and selectively kill tumor cells while maintaining a favorable safety profile, making them attractive candidates as novel GBM therapeutics. For example, the cannabinoid agonists WIN55212-2 (WIN-2) and Δ9-tetrahydrocannabinol (THC) induce GBM cell death both in vitro and reduce tumor growth in vivo, while sparing healthy cells. While cannabinoid compounds typically act through two G protein-coupled receptors (GPCR), CB1, and CB2, evidence suggests that WIN-2, an alkylindole (AI) cannabinoid, kills tumor cells through a separate, unknown mechanism. To explore this unknown mechanism, our lab developed a novel series of AI compounds that exhibit reduced affinity to CB1 and CB2 receptors. The model AI compound, ST-11, was found to destabilize microtubules (MTs) and promote both cell cycle arrest in prometaphase and caspase-dependent apoptosis. Recent studies have shown that GBMs are particularly sensitive to mitotic disruption; however, the use of MT-targeting agents is limited by toxic side effects and a general inability to cross the BBB. ST-11 was found to exhibit a promising safety profile in mice and did not induce overt toxicity in any major organs up to its maximal administrable dose of 240 mg/kg. Additionally, ST-11 readily crossed the BBB within 60 min of intraperitoneal injection and dose-dependently induced caspase activation and reduced tumor size in an orthotopic syngeneic glioma mouse model. Our findings suggest that AI compounds such as ST-11 belong to a new class of brain-penetrant MT-targeting agents that preferentially kill tumor cells.

The serine hydrolase α/β-hydrolase domain 6 (ABHD6) hydrolyzes the most abundant endocannabinoid (eCB) in the brain, 2-arachidonoylglycerol (2-AG), and controls its availability at cannabinoid receptors. We show that ABHD6 inhibition decreases pentylenetetrazole (PTZ)-induced generalized tonic-clonic and myoclonic seizure incidence and severity. This effect is retained in Cnr1(-/-) or Cnr2(-/-) mice, but blocked by addition of a subconvulsive dose of picrotoxin, suggesting the involvement of GABAA receptors. ABHD6 inhibition also blocked spontaneous seizures in R6/2 mice, a genetic model of juvenile Huntington's disease known to exhibit dysregulated eCB signaling. ABHD6 blockade retained its antiepileptic activity over chronic dosing and was not associated with psychomotor or cognitive effects. While the etiology of seizures in R6/2 mice remains unsolved, involvement of the hippocampus is suggested by interictal epileptic discharges, increased expression of vGLUT1 but not vGAT, and reduced Neuropeptide Y (NPY) expression. We conclude that ABHD6 inhibition may represent a novel antiepileptic strategy.

GPR124 is an orphan receptor that belongs to the Adhesion GPCR family. Members of this family are characterized by long N‐terminal segments that contain many of the same functional domains found in cadherins, integrins, and tyrosine kinases, but are typically absent in GPCRs belonging to other families. While little is known about adhesion GPCRs, recent results show that GPR124 plays a role in endothelial cell migration and differentiation during angiogenesis. We found that GPR124 mRNA is highly expressed by various human astrocytoma cell lines. Due to the high expression of GPR124 in GBM cells and its known role in cellular migration and differentiation, we hypothesize that these orphan receptors control the migration of GBM cells through specific signal transduction pathways and thus represent promising new therapeutic targets to treat brain tumors.To test our hypothesis, we used a proteomics approach to determine the effector proteins that couple to this GPCR and measured the migration of human GBM cell lines expressing various levels of GPR124. Myc‐tagged GPR124 was heterologously expressed in the human GBM cell line U87MG and the proteins associated with these receptors were analyzed by Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) mass spectrometry. We found a concise list of effector proteins that are known to control cell migration. To determine if GPR124 levels affect cell migration, we used a modified Boyden chamber assay that was developed in our laboratory. We found that GPR124 over‐expression increases the migration of U87MG cells stimulated by lysophosphatidic acid (LPA). Together, our results suggest that GPR124 is expressed by astrocytomas and tightly regulates how these cells migrate toward select chemokines.

Cannabinoid receptor 1 (CB(1) receptor) controls several neuronal functions, including neurotransmitter release, synaptic plasticity, gene expression and neuronal viability. Downregulation of CB(1) expression in the basal ganglia of patients with Huntington's disease (HD) and animal models represents one of the earliest molecular events induced by mutant huntingtin (mHtt). This early disruption of neuronal CB(1) signaling is thought to contribute to HD symptoms and neurodegeneration. Here we determined whether CB(1) downregulation measured in patients with HD and mouse models was ubiquitous or restricted to specific striatal neuronal subpopulations. Using unbiased semi-quantitative immunohistochemistry, we confirmed previous studies showing that CB(1) expression is downregulated in medium spiny neurons of the indirect pathway, and found that CB(1) is also downregulated in neuropeptide Y (NPY)/neuronal nitric oxide synthase (nNOS)-expressing interneurons while remaining unchanged in parvalbumin- and calretinin-expressing interneurons. CB(1) downregulation in striatal NPY/nNOS-expressing interneurons occurs in R6/2 mice, Hdh(Q150/Q150) mice and the caudate nucleus of patients with HD. In R6/2 mice, CB(1) downregulation in NPY/nNOS-expressing interneurons correlates with diffuse expression of mHtt in the soma. This downregulation also occludes the ability of cannabinoid agonists to activate the pro-survival signaling molecule cAMP response element-binding protein in NPY/nNOS-expressing interneurons. Loss of CB(1) signaling in NPY/nNOS-expressing interneurons could contribute to the impairment of basal ganglia functions linked to HD.