The conformational signature of ß-arrestin2 predicts its trafficking and signalling functions.

Arrestins are cytosolic proteins that regulate G-protein-coupled receptor (GPCR) desensitization, internalization, trafficking and signalling. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets the proteins for internalization via clathrin-coated pits. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G-protein effectors into GPCR-based 'signalsomes'. Although the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify these divergent functions is unclear. Using a panel of intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters to monitor conformational changes in ß-arrestin2, here we show that GPCRs impose distinctive arrestin 'conformational signatures' that reflect the stability of the receptor-arrestin complex and role of ß-arrestin2 in activating or dampening downstream signalling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in ß-arrestin2 conformation. Our findings demonstrate that information about ligand-receptor conformation is encoded within the population average ß-arrestin2 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands and in identifying factors that dictate arrestin conformation and function.

Application of Pharmacokinetic Prediction Platforms in the Design of Optimized Anti-Cancer Drugs.

Cancer is the second most common cause of death in the United States, accounting for 602,350 deaths in 2020. Cancer-related death rates have declined by 27% over the past two decades, partially due to the identification of novel anti-cancer drugs. Despite improvements in cancer treatment, newly approved oncology drugs are associated with increased toxicity risk. These toxicities may be mitigated by pharmacokinetic optimization and reductions in off-target interactions. As such, there is a need for early-stage implementation of pharmacokinetic (PK) prediction tools. Several PK prediction platforms exist, including pkCSM, SuperCypsPred, Pred-hERG, Similarity Ensemble Approach (SEA), and SwissADME. These tools can be used in screening hits, allowing for the selection of compounds were reduced toxicity and/or risk of attrition. In this short commentary, we used PK prediction tools in the optimization of mitogen activated extracellular signal-related kinase kinase 1 (MEK1) inhibitors. In doing so, we identified MEK1 inhibitors with retained activity and optimized predictive PK properties, devoid of hERG inhibition. These data support the use of publicly available PK prediction platforms in early-stage drug discovery to design safer drugs.

Receptor-interacting Ser/Thr kinase 1 (RIPK1) and myosin IIA-dependent ceramidosomes form membrane pores that mediate blebbing and necroptosis.

Formation of membrane pores/channels regulates various cellular processes, such as necroptosis or stem cell niche signaling. However, the roles of membrane lipids in the formation of pores and their biological functions are largely unknown. Here, using the cellular stress model evoked by the sphingolipid analog drug FTY720, we show that formation of ceramide-enriched membrane pores, referred to here as ceramidosomes, is initiated by a receptor-interacting Ser/Thr kinase 1 (RIPK1)-ceramide complex transported to the plasma membrane by nonmuscle myosin IIA-dependent trafficking in human lung cancer cells. Molecular modeling/simulation coupled with site-directed mutagenesis revealed that Asp147 or Asn169 of RIPK1 are key for ceramide binding and that Arg258 or Leu293 residues are involved in the myosin IIA interaction, leading to ceramidosome formation and necroptosis. Moreover, generation of ceramidosomes independently of any external drug/stress stimuli was also detected in the plasma membrane of germ line stem cells in ovaries during the early stages of oogenesis in Drosophila melanogaster Inhibition of ceramidosome formation via myosin IIA silencing limited germ line stem cell signaling and abrogated oogenesis. In conclusion, our findings indicate that the RIPK1-ceramide complex forms large membrane pores we named ceramidosomes. They further suggest that, in addition to their roles in stress-mediated necroptosis, these ceramide-enriched pores also regulate membrane integrity and signaling and might also play a role in D. melanogaster ovary development.

The NMR-based characterization of the FTY720-SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET-PP2A interaction.

The su(var)3-9, enhancer of zeste, trithorax (SET)/inhibitor 2 of protein phosphatase 2A (PP2A) oncoprotein binds and inhibits PP2A, composed of various isoforms of scaffolding, regulatory, and catalytic subunits. Targeting SET with a sphingolipid analog drug fingolimod (FTY720) or ceramide leads to the reactivation of tumor suppressor PP2A. However, molecular details of the SET-FTY720 or SET-ceramide, and mechanism of FTY720-dependent PP2A activation, remain unknown. Here, we report the first in solution examination of the SET-FTY720 or SET-ceramide complexes by NMR spectroscopy. FTY720-ceramide binding resulted in chemical shifts of residues residing at the N terminus of SET, preventing its dimerization or oligomerization. This then released SET from PP2ACα, resulting in PP2A activation, while monomeric SET remained associated with the B56γ. Our data also suggest that the PP2A holoenzyme, composed of PP2A-Aß, PP2A-B56γ, and PP2ACα subunits, is selectively activated in response to the formation of the SET-FTY720 complex in A549 cells. Various PP2A-associated downstream effector proteins in the presence or absence of FTY720 were then identified by stable isotope labeling with amino cells in cell culture, including tumor suppressor nonmuscle myosin IIA. Attenuation of FTY720-SET association by point mutations of residues that are involved in FTY720 binding or dephosphorylation of SET at Serine 171, enhanced SET oligomerization and the formation of the SET-PP2A inhibitory complex, leading to resistance to FTY720-dependent PP2A activation.-De Palma, R. M., Parnham, S. R., Li, Y., Oaks, J. J., Peterson, Y. K., Szulc, Z. M., Roth, B. M., Xing, Y., Ogretmen, B. The NMR-based characterization of the FTY720-SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET-PP2A interaction.

The Diverse Roles of Arrestin Scaffolds in G Protein-Coupled Receptor Signaling.

The visual/ß-arrestins, a small family of proteins originally described for their role in the desensitization and intracellular trafficking of G protein-coupled receptors (GPCRs), have emerged as key regulators of multiple signaling pathways. Evolutionarily related to a larger group of regulatory scaffolds that share a common arrestin fold, the visual/ß-arrestins acquired the capacity to detect and bind activated GPCRs on the plasma membrane, which enables them to control GPCR desensitization, internalization, and intracellular trafficking. By acting as scaffolds that bind key pathway intermediates, visual/ß-arrestins both influence the tonic level of pathway activity in cells and, in some cases, serve as ligand-regulated scaffolds for GPCR-mediated signaling. Growing evidence supports the physiologic and pathophysiologic roles of arrestins and underscores their potential as therapeutic targets. Circumventing arrestin-dependent GPCR desensitization may alleviate the problem of tachyphylaxis to drugs that target GPCRs, and find application in the management of chronic pain, asthma, and psychiatric illness. As signaling scaffolds, arrestins are also central regulators of pathways controlling cell growth, migration, and survival, suggesting that manipulating their scaffolding functions may be beneficial in inflammatory diseases, fibrosis, and cancer. In this review we examine the structure-function relationships that enable arrestins to perform their diverse roles, addressing arrestin structure at the molecular level, the relationship between arrestin conformation and function, and sites of interaction between arrestins, GPCRs, and nonreceptor-binding partners. We conclude with a discussion of arrestins as therapeutic targets and the settings in which manipulating arrestin function might be of clinical benefit.

Development of a Novel Humanized Monoclonal Antibody to Secreted Frizzled-Related Protein-2 That Inhibits Triple-Negative Breast Cancer and Angiosarcoma Growth In Vivo.

BACKGROUND: We previously reported that secreted frizzled-related protein-2 (SFRP2) is expressed in a variety of tumors, including sarcoma and breast carcinoma, and stimulates angiogenesis and inhibits tumor apoptosis. Therefore, we hypothesized that a humanized SFRP2 monoclonal antibody (hSFRP2 mAb) would inhibit tumor growth. METHODS: The lead hSFRP2 antibody was tested against a cohort of 22 healthy donors using a time course T-cell assay to determine the relative risk of immunogenicity. To determine hSFRP2 mAb efficacy, nude mice were subcutaneously injected with SVR angiosarcoma cells and treated with hSFRP2 mAb 4 mg/kg intravenously every 3 days for 3 weeks. We then injected Hs578T triple-negative breast cells into the mammary fat pad of nude mice and treated for 40 days. Control mice received an immunoglobulin (Ig) G1 control. The SVR and Hs578T tumors were then stained using a TUNEL assay to detect apoptosis. RESULTS: Immunogenicity testing of hSFRP2 mAb did not induce proliferative responses using a simulation index (SI) ≥ 2.0 (p < 0.05) threshold in any of the healthy donors. SVR angiosarcoma tumor growth was inhibited in vivo, evidenced by significant tumor volume reduction in the hSFRP2 mAb-treated group, compared with controls (n = 10, p < 0.001). Likewise, Hs578T triple-negative breast tumors were smaller in the hSFRP2 mAb-treated group compared with controls (n = 10, p < 0.001). The hSFRP2 mAb treatment correlated with an increase in tumor cell apoptosis (n = 11, p < 0.05). Importantly, hSFRP2 mAb treatment was not associated with any weight loss or lethargy. CONCLUSION: We present a novel hSFRP2 mAb with therapeutic potential in breast cancer and sarcoma that has no effect on immunogenicity.

Frizzled-5: a high affinity receptor for secreted frizzled-related protein-2 activation of nuclear factor of activated T-cells c3 signaling to promote angiogenesis.

Secreted frizzled-related protein 2 (SFRP2) is a pro-angiogenic factor expressed in the vasculature of a wide variety of human tumors, and modulates angiogenesis via the calcineurin-dependent nuclear factor of activated T-cells cytoplasmic 3 (NFATc3) pathway in endothelial cells. However, until now, SFRP2 receptor for this pathway was unknown. In the present study, we first used amino acid alignments and molecular modeling to demonstrate that SFRP2 interaction with frizzled-5 (FZD5) is typical of Wnt/FZD family members. To confirm this interaction, we performed co-immunofluorescence, co-immunoprecipitation, and ELISA binding assays, which demonstrated SFRP2/FZD5 binding. Functional knock-down studies further revealed that FZD5 is necessary for SFRP2-induced tube formation and intracellular calcium flux in endothelial cells. Using protein analysis on endothelial cell nuclear extracts, we also discovered that FZD5 is required for SFRP2-induced activation of NFATc3. Our novel findings reveal that FZD5 is a receptor for SFRP2 and mediates SFRP2-induced angiogenesis via calcineurin/NFATc3 pathway in endothelial cells.

Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning.

Understanding the processes of mitochondrial dynamics (fission, fusion, biogenesis, and mitophagy) has been hampered by the lack of automated, deterministic methods to measure mitochondrial morphology from microscopic images. A method to quantify mitochondrial morphology and function is presented here using a commercially available automated high-content wide-field fluorescent microscopy platform and R programming-language-based semi-automated data analysis to achieve high throughput morphological categorization (puncta, rod, network, and large & round) and quantification of mitochondrial membrane potential. In conjunction with cellular respirometry to measure mitochondrial respiratory capacity, this method detected that increasing concentrations of toxicants known to directly or indirectly affect mitochondria (t-butyl hydroperoxide [TBHP], rotenone, antimycin A, oligomycin, ouabain, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP]), decreased mitochondrial networked areas in cultured 661w cells to 0.60-0.80 at concentrations that inhibited respiratory capacity to 0.20-0.70 (fold change compared to vehicle). Concomitantly, mitochondrial swelling was increased from 1.4- to 2.3-fold of vehicle as indicated by changes in large & round areas in response to TBHP, oligomycin, or ouabain. Finally, the automated identification of mitochondrial location enabled accurate quantification of mitochondrial membrane potential by measuring intramitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence intensity. Administration of FCCP depolarized and administration of oligomycin hyperpolarized mitochondria, as evidenced by changes in intramitochondrial TMRM fluorescence intensities to 0.33- or 5.25-fold of vehicle control values, respectively. In summary, this high-content imaging method accurately quantified mitochondrial morphology and membrane potential in hundreds of thousands of cells on a per-cell basis, with sufficient throughput for pharmacological or toxicological evaluation.

The arrestin-selective angiotensin AT1 receptor agonist [Sar1,Ile4,Ile8]-AngII negatively regulates bradykinin B2 receptor signaling via AT1-B2 receptor heterodimers.

The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar(1),Ile(4),Ile(8)]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar(1),Ile(4), Ile(8)]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar(1),Ile(4),Ile(8)]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar(1),Ile(4),Ile(8)]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar(1),Ile(4),Ile(8)]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar(1),Ile(4),Ile(8)]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic actions.

Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy.

Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C(18)-pyridinium ceramide treatment or endogenous C(18)-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C(18)-ceramide-induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 ß-lipidation, forming LC3B-II, and selective targeting of mitochondria by LC3B-II-containing autophagolysosomes (mitophagy) through direct interaction between ceramide and LC3B-II upon Drp1-dependent mitochondrial fission, leading to inhibition of mitochondrial function and oxygen consumption. Accordingly, expression of mutant LC3B with impaired ceramide binding, as predicted by molecular modeling, prevented CerS1-mediated mitochondrial targeting, recovering oxygen consumption. Moreover, knockdown of CerS1 abrogated sodium selenite-induced mitophagy, and stable LC3B knockdown protected against CerS1- and C(18)-ceramide-dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a new receptor function of ceramide for anchoring LC3B-II autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.

Development of inhibitors of heterotrimeric Gαi subunits.

Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gαi by competing for the GPCR and Gßγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α2-adrenoceptor (α2AR) mediated decreases in cAMP in HEK293 cells at 100nM. We then sought to discover selective small molecule inhibitors for Gαi. Molecular docking was used to identify potential molecules that bind to and stabilize the Gαi-GDP complex by directly interacting with both Gαi and GDP. Gαi-GTP and Gαq-GDP were used as a computational counter screen and Gαq-GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gαi. Several compounds showed Gαi specific inhibition and were able to block α2AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease.

First analytical confirmation of drug-induced crystal nephropathy in felines caused by GS-441524, the active metabolite of Remdesivir.

GS-441524 is an adenosine nucleoside antiviral demonstrating significant efficacy in the treatment of feline infectious peritonitis (FIP), an otherwise fatal illness, resulting from infection with feline coronavirus. However, following the emergence of COVID-19, veterinary development was halted, and Gilead pursued clinical development of a GS-441524 pro-drug, resulting in the approval of Remdesivir under an FDA emergency use authorization. Despite lack of regulatory approval, GS-441524 is available without a prescription through various unlicensed online distributors and is commonly purchased by pet owners for the treatment of FIP. Herein, we report data obtained from the analytical characterization of two feline renal calculi, demonstrating the propensity for GS-441524 to cause renal toxicity through drug-induced crystal nephropathy in vivo. As definitive diagnosis of drug-induced crystal nephropathy requires confirmation of the lithogenic material to accurately attribute a mechanism of toxicity, renal stone composition and crystalline matrix were characterized using ultra-performance liquid chromatography photodiode array detection (UPLC-PDA), ultra-performance liquid chromatography mass spectrometry (LCMS), nuclear magnetic resonance (NMR) spectroscopy, X-ray powder diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). This work serves to provide the first analytical confirmation of GS-441524-induced crystal nephropathy in an effort to support toxicologic identification of adverse renal effects caused by administration of GS-441524 or any pro-drug thereof.

Inhibition of complement C3 prevents osteoarthritis progression in guinea pigs by blocking STAT1 activation.

Osteoarthritis (OA) is one of the leading causes of disability, affecting over 500 million adults worldwide. Previous studies have found that various inflammatory factors can contribute to the pathogenesis of OA, including complement factors in the synovial fluid of OA patients. However, the pathogenesis of this disease is still not known, and the only therapy of severe OA is total joint replacements. Total joint replacements are invasive, expensive, and affect quality of life. Here we show that when human articular chondrocytes are stimulated with pro-inflammatory mediator interleukin-1ß (IL-1ß) there is an increase in inflammatory factors including complement component 3 (C3). We also found the transcription factor, signal transducer and activator of transcription 1 (STAT1), is responsible for increased C3 expression after IL-1ß stimulation in human articular chondrocytes. A specific STAT1 inhibitor, fludarabine, attenuates the hyper-expression of C3 and delays/prevents spontaneous OA in Dunkin-Hartley guinea pigs. Since fludarabine is already clinically used for chemotherapy, this study has great translational potential as a unique disease-modifying osteoarthritis drug (DMOAD) in treating primary OA.

Opportunistic pathogen Porphyromonas gingivalis targets the LC3B-ceramide complex and mediates lethal mitophagy resistance in oral tumors.

Mechanisms by which Porphyromonas gingivalis (P. gingivalis) infection enhances oral tumor growth or resistance to cell death remain elusive. Here, we determined that P. gingivalis infection mediates therapeutic resistance via inhibiting lethal mitophagy in cancer cells and tumors. Mechanistically, P. gingivalis targets the LC3B-ceramide complex by associating with LC3B via bacterial major fimbriae (FimA) protein, preventing ceramide-dependent mitophagy in response to various therapeutic agents. Moreover, ceramide-mediated mitophagy is induced by Annexin A2 (ANXA2)-ceramide association involving the E142 residue of ANXA2. Inhibition of ANXA2-ceramide-LC3B complex formation by wild-type P. gingivalis prevented ceramide-dependent mitophagy. Moreover, a FimA-deletion mutant P. gingivalis variant had no inhibitory effects on ceramide-dependent mitophagy. Further, 16S rRNA sequencing of oral tumors indicated that P. gingivalis infection altered the microbiome of the tumor macroenvironment in response to ceramide analog treatment in mice. Thus, these data provide a mechanism describing the pro-survival roles of P. gingivalis in oral tumors.

High-throughput respirometric assay identifies predictive toxicophore of mitochondrial injury.

Many environmental chemicals and drugs negatively affect human health through deleterious effects on mitochondrial function. Currently there is no chemical library of mitochondrial toxicants, and no reliable methods for predicting mitochondrial toxicity. We hypothesized that discrete toxicophores defined by distinct chemical entities can identify previously unidentified mitochondrial toxicants. We used a respirometric assay to screen 1760 compounds (5 µM) from the LOPAC and ChemBridge DIVERSet libraries. Thirty-one of the assayed compounds decreased uncoupled respiration, a stress test for mitochondrial dysfunction, prior to a decrease in cell viability and reduced the oxygen consumption rate in isolated mitochondria. The mitochondrial toxicants were grouped by chemical similarity and two clusters containing four compounds each were identified. Cheminformatic analysis of one of the clusters identified previously uncharacterized mitochondrial toxicants from the ChemBridge DIVERSet. This approach will enable the identification of mitochondrial toxicants and advance the prediction of mitochondrial toxicity for both drug discovery and risk assessment.

ß2-Adrenoceptor agonists in the regulation of mitochondrial biogenesis.

The stimulation of mitochondrial biogenesis (MB) via cell surface G-protein coupled receptors is a promising strategy for cell repair and regeneration. Here we report the specificity and chemical rationale of a panel of ß2-adrenoceptor agonists with regards to MB. Using primary cultures of renal cells, a diverse panel of ß2-adrenoceptor agonists elicited three distinct phenotypes: full MB, partial MB, and non-MB. Full MB compounds had efficacy in the low nanomolar range and represent two chemical scaffolds containing three distinct chemical clusters. Interestingly, the MB phenotype did not correlate with reported receptor affinity or chemical similarity. Chemical clusters were then subjected to pharmacophore modeling creating two models with unique and distinct features, consisting of five conserved amongst full MB compounds were identified. The two discrete pharmacophore models were coalesced into a consensus pharmacophore with four unique features elucidating the spatial and chemical characteristics required to stimulate MB.

Bax and Bcl-xL exert their regulation on different sites of the ceramide channel.

The present study demonstrates the important structural features of ceramide required for proper regulation, binding and identification by both pro-apoptotic and anti-apoptotic Bcl-2 family proteins. The C-4=C-5 trans-double bond has little influence on the ability of Bax and Bcl-xL to identify and bind to these channels. The stereochemistry of the headgroup and access to the amide group of ceramide is indispensible for Bax binding, indicating that Bax may interact with the polar portion of the ceramide channel facing the bulk phase. In contrast, Bcl-xL binding to ceramide channels is tolerant of stereochemical changes in the headgroup. The present study also revealed that Bcl-xL has an optimal interaction with long-chain ceramides that are elevated early in apoptosis, whereas short-chain ceramides are not well regulated. Inhibitors specific for the hydrophobic groove of Bcl-xL, including 2-methoxyantimycin A3, ABT-737 and ABT-263 provide insights into the region of Bcl-xL involved in binding to ceramide channels. Molecular docking simulations of the lowest-energy binding poses of ceramides and Bcl-xL inhibitors to Bcl-xL were consistent with the results of our functional studies and propose potential binding modes.

A high-content, live-cell, and real-time approach to the quantitation of ligand-induced ß-Arrestin2 and Class A/Class B GPCR mobilization.

We report the development of a method to analyze receptor and ß-arrestin2 mobilization between Class A and B GPCRs via time-resolved fluorescent microscopy coupled with semiautomated high-content multiparametric analysis. Using transiently expressed, tagged ß2-adrenergic receptor (ß2-AR) or parathyroid hormone receptor type 1 (PTH1R), we quantified trafficking of the receptors along with the mobilization and colocalization of coexpressed tagged ß-arrestin2. This classification system allows for exclusion of cells with nonoptimal characteristics and calculation of multiple morphological and spatial parameters including receptor endosome formation, ß-arrestin mobilization, colocalization, areas, and shape. Stimulated Class A and B receptors demonstrate dramatically different patterns with regard to ß-arrestin interactions. The method provides high kinetic resolution measurement of receptor translocation, which allows for the identification of the fleeting ß-arrestin interaction found with ß2-AR agonist stimulation, in contrast to stronger mobilization and receptor colocalization with agonist stimulation of the PTH1R. Though especially appropriate for receptor kinetic studies, this method is generalizable to any dual fluorescence probe system in which quantification of object formation and movement is desired. These methodologies allow for quantitative, unbiased measurement of microscopy data and are further enhanced by providing real-time kinetics.

Stimulation of natural killer cells with small molecule inhibitors of CD38 for the treatment of neuroblastoma.

High-risk neuroblastoma (NB) accounts for 15% of all pediatric cancer deaths. Refractory disease for high-risk NB patients is attributed to chemotherapy resistance and immunotherapy failure. The poor prognosis for high-risk NB patients demonstrates an unmet medical need for the development of new, more efficacious therapeutics. CD38 is an immunomodulating protein that is expressed constitutively on natural killer (NK) cells and other immune cells in the tumor microenvironment (TME). Furthermore, CD38 over expression is implicated in propagating an immunosuppressive milieu within the TME. Through virtual and physical screening, we have identified drug-like small molecule inhibitors of CD38 with low micromolar IC50 values. We have begun to explore structure activity relationships for CD38 inhibition through derivatization of our most effective hit molecule to develop a new compound with lead-like physicochemical properties and improved potency. We have demonstrated that our derivatized inhibitor, compound 2, elicits immunomodulatory effects in NK cells by increasing cell viability by 190 ± 36% in multiple donors and by significantly increasing interferon gamma. Additionally, we have illustrated that NK cells exhibited enhanced cytotoxicity toward NB cells (14% reduction of NB cells over 90 minutes) when given a combination treatment of our inhibitor and the immunocytokine ch14.18-IL2. Herein we describe the synthesis and biological evaluation of small molecule CD38 inhibitors and demonstrate their potential utility as a novel approach to NB immunotherapy. These compounds represent the first examples of small molecules that stimulate immune function for the treatment of cancer.

A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B.

Familial hypercholesterolemia (FH) patients suffer from excessively high levels of Low Density Lipoprotein Cholesterol (LDL-C), which can cause severe cardiovascular disease. Statins, bile acid sequestrants, PCSK9 inhibitors, and cholesterol absorption inhibitors are all inefficient at treating FH patients with homozygous LDLR gene mutations (hoFH). Drugs approved for hoFH treatment control lipoprotein production by regulating steady-state Apolipoprotein B (apoB) levels. Unfortunately, these drugs have side effects including accumulation of liver triglycerides, hepatic steatosis, and elevated liver enzyme levels. To identify safer compounds, we used an iPSC-derived hepatocyte platform to screen a structurally representative set of 10,000 small molecules from a proprietary library of 130,000 compounds. The screen revealed molecules that could reduce the secretion of apoB from cultured hepatocytes and from humanized livers in mice. These small molecules are highly effective, do not cause abnormal lipid accumulation, and share a chemical structure that is distinct from any known cholesterol lowering drug.