ß-arrestin-biased Allosteric Modulator of Neurotensin Receptor 1 Reduces Ethanol Drinking and Responses to Ethanol Administration in Rodents.

Alcohol use disorders (AUDs) impose an enormous societal and financial burden, and world-wide, alcohol misuse is the 7th leading cause of premature death1. Despite this, there are currently only 3 FDA approved pharmacological treatments for the treatment of AUDs in the United States. The neurotensin (Nts) system has long been implicated in modulating behaviors associated with alcohol misuse. Recently, a novel compound, SBI-553, that biases the action of Nts receptor 1 (NTSR1) activation, has shown promise in preclinical models of psychostimulant misuse. Here we investigate the efficacy of this compound to alter ethanol-mediated behaviors in a comprehensive battery of experiments assessing ethanol consumption, behavioral responses to ethanol, sensitivity to ethanol, and ethanol metabolism. Additionally, we investigated behavior in avoidance and cognitive assays to monitor potential side effects of SBI-553. We find that SBI-553 reduces binge-like ethanol consumption in mice without altering avoidance behavior or novel object recognition. We also observe sex-dependent differences in physiological responses to sequential ethanol injections in mice. In rats, we show that SBI-553 attenuates sensitivity to the interoceptive effects of ethanol (using a Pavlovian drug discrimination task). Our data suggest that targeting NTSR1 signaling may be promising to attenuate alcohol misuse, and adds to a body of literature that suggests NTSR1 may be a common downstream target involved in the psychoactive effects of multiple reinforcing substances.

Concentration and proteolysis of CX3CL1 may regulate the microglial response to CX3CL1.

Fractalkine (FKN) is a membrane-bound chemokine that can be cleaved by proteases such as ADAM 10, ADAM 17, and cathepsin S to generate soluble fragments. Studies using different forms of the soluble FKN yield conflicting results in vivo. These observations prompted us to investigate the function and pharmacology of two commonly used isoforms of FKN, a human full-length soluble FKN (sFKN), and a human chemokine domain only FKN (cdFKN). Both are prevalent in the literature and are often assumed to be functionally equivalent. We observed that recombinant sFKN and cdFKN exhibit similar potencies in a cell-based cAMP assay, but binding affinity for CX3CR1 was modestly different. There was a 10-fold difference in potency between sFKN and cdFKN when assessing their ability to stimulate ß-arrestin recruitment. Interestingly, high concentrations of FKN, regardless of cleavage variant, were ineffective at reducing pro-inflammatory microglial activation and may induce a pro-inflammatory response. This effect was observed in mouse and rat primary microglial cells as well as microglial cell lines. The inflammatory response was exacerbated in aged microglia, which is known to exhibit age-related inflammatory phenotypes. We observed the same effects in Cx3cr1-/- primary microglia and therefore speculate that an alternative FKN receptor may exist. Collectively, these data provide greater insights into the function and pharmacology of these common FKN reagents, which may clarify conflicting reports and urge greater caution in the selection of FKN peptides for use in in vitro and in vivo studies and the interpretation of results obtained using these differing peptides.

An Optimized Dihydrodibenzothiazepine Lead Compound (SBI-0797750) as a Potent and Selective Inhibitor of Plasmodium falciparum and P. vivax Glucose 6-Phosphate Dehydrogenase 6-Phosphogluconolactonase.

In Plasmodium, the first two and rate-limiting enzymes of the pentose phosphate pathway, glucose 6-phosphate dehydrogenase (G6PD) and the 6-phosphogluconolactonase, are bifunctionally fused to a unique enzyme named GluPho, differing structurally and mechanistically from the respective human orthologs. Consistent with the enzyme's essentiality for malaria parasite proliferation and propagation, human G6PD deficiency has immense impact on protection against severe malaria, making PfGluPho an attractive antimalarial drug target. Herein we report on the optimized lead compound N-(((2R,4S)-1-cyclobutyl-4-hydroxypyrrolidin-2-yl)methyl)-6-fluoro-4-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide (SBI-0797750), a potent and fully selective PfGluPho inhibitor with robust nanomolar activity against recombinant PfGluPho, PvG6PD, and P. falciparum blood-stage parasites. Mode-of-action studies have confirmed that SBI-0797750 disturbs the cytosolic glutathione-dependent redox potential, as well as the cytosolic and mitochondrial H2O2 homeostasis of P. falciparum blood stages, at low nanomolar concentrations. Moreover, SBI-0797750 does not harm red blood cell (RBC) integrity and phagocytosis and thus does not promote anemia. SBI-0797750 is therefore a very promising antimalarial lead compound.

Discovery of a first-in-class inhibitor of sulfide:quinone oxidoreductase that protects against adverse cardiac remodelling and heart failure.

AIMS: Hydrogen sulfide (H2S) is a potent signalling molecule that activates diverse cardioprotective pathways by post-translational modification (persulfidation) of cysteine residues in upstream protein targets. Heart failure patients with reduced ejection fraction (HFrEF) exhibit low levels of H2S. Sulfide:quinone oxidoreductase (SQOR) catalyses the first irreversible step in the metabolism of H2S and plays a key role in regulating H2S-mediated signalling. Here, the aim of this study was to discover a first-in-class inhibitor of human SQOR and evaluate its cardioprotective effect in an animal model of HFrEF. METHODS AND RESULTS: We identified a potent inhibitor of human SQOR (STI1, IC50 = 29 nM) by high-throughput screening of a small-molecule library, followed by focused medicinal chemistry optimization and structure-based design. STI1 is a competitive inhibitor that binds with high selectivity to the coenzyme Q-binding pocket in SQOR. STI1 exhibited very low cytotoxicity and attenuated the hypertrophic response of neonatal rat ventricular cardiomyocytes and H9c2 cells induced by neurohormonal stressors. A mouse HFrEF model was produced by transverse aortic constriction (TAC). Treatment of TAC mice with STI1 mitigated the development of cardiomegaly, pulmonary congestion, dilatation of the left ventricle, and cardiac fibrosis and decreased the pressure gradient across the aortic constriction. Moreover, STI1 dramatically improved survival, preserved cardiac function, and prevented the progression to HFrEF by impeding the transition from compensated to decompensated left ventricle hypertrophy. CONCLUSION: We demonstrate that the coenzyme Q-binding pocket in human SQOR is a druggable target and establish proof of concept for the potential of SQOR inhibitors to provide a novel therapeutic approach for the treatment of HFrEF.

Synthesis and evaluation of potent novel inhibitors of human sulfide:quinone oxidoreductase.

Here we report the first small-molecule inhibitors of human sulfide:quinone oxidoreductase (SQOR) that decrease the rate of breakdown of hydrogen sulfide (H2S), a potent cardioprotective signaling molecule. SQOR is a mitochondrial membrane-bound protein that catalyzes a two-electron oxidation of H2S to sulfane sulfur (S0), using glutathione (or sulfite) and coenzyme Q (CoQ) as S0 and electron acceptor, respectively. Inhibition of SQOR may constitute a new approach for the treatment of heart failure with reduced ejection fraction. Starting from top hits identified in a high-throughput screen, we conducted SAR development guided by docking of lead candidates into our crystal structure of SQOR. We identified potent SQOR inhibitors such as 19 which has an IC50 of 29 nM for SQOR inhibition and favorable pharmacokinetic and ADME properties required for in vivo efficacy testing.

Discovery of Orally Bioavailable Purine-Based Inhibitors of the Low-Molecular-Weight Protein Tyrosine Phosphatase.

Obesity-associated insulin resistance plays a central role in the pathogenesis of type 2 diabetes. A promising approach to decrease insulin resistance in obesity is to inhibit the protein tyrosine phosphatases that negatively regulate insulin receptor signaling. The low-molecular-weight protein tyrosine phosphatase (LMPTP) acts as a critical promoter of insulin resistance in obesity by inhibiting phosphorylation of the liver insulin receptor activation motif. Here, we report development of a novel purine-based chemical series of LMPTP inhibitors. These compounds inhibit LMPTP with an uncompetitive mechanism and are highly selective for LMPTP over other protein tyrosine phosphatases. We also report the generation of a highly orally bioavailable purine-based analogue that reverses obesity-induced diabetes in mice.

Optimization of a urea-containing series of nicotinamide phosphoribosyltransferase (NAMPT) activators.

NAD+ is a crucial cellular factor that plays multifaceted roles in wide ranging biological processes. Low levels of NAD+ have been linked to numerous diseases including metabolic disorders, cardiovascular disease, neurodegeneration, and muscle wasting disorders. A novel strategy to boost NAD+ is to activate nicotinamide phosphoribosyltransferase (NAMPT), the putative rate-limiting step in the NAD+ salvage pathway. We previously showed that NAMPT activators increase NAD+ levels in vitro and in vivo. Herein we describe the optimization of our NAMPT activator prototype (SBI-0797812) leading to the identification of 1-(4-((4-chlorophenyl)sulfonyl)phenyl)-3-(oxazol-5-ylmethyl)urea (34) that showed far more potent NAMPT activation and improved oral bioavailability.

Interesting findings, questionable interpretation: Comment on Thornewill et al. (2020).

A survey by Thornewill et al. (2020) produced potentially useful information about how national security roles of psychologists are viewed both within and beyond the discipline. However, a closer examination of the data, and of the authors' interpretations of that data, raises some important questions. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Targeting eIF4F translation initiation complex with SBI-756 sensitises B lymphoma cells to venetoclax.

BACKGROUND: The BCL2 inhibitor venetoclax has shown efficacy in several hematologic malignancies, with the greatest response rates in indolent blood cancers such as chronic lymphocytic leukaemia. There is a lower response rate to venetoclax monotherapy in diffuse large B-cell lymphoma (DLBCL). METHODS: We tested inhibitors of cap-dependent mRNA translation for the ability to sensitise DLBCL and mantle cell lymphoma (MCL) cells to apoptosis by venetoclax. We compared the mTOR kinase inhibitor (TOR-KI) MLN0128 with SBI-756, a compound targeting eukaryotic translation initiation factor 4G1 (eIF4G1), a scaffolding protein in the eIF4F complex. RESULTS: Treatment of DLBCL and MCL cells with SBI-756 synergised with venetoclax to induce apoptosis in vitro, and enhanced venetoclax efficacy in vivo. SBI-756 prevented eIF4E-eIF4G1 association and cap-dependent translation without affecting mTOR substrate phosphorylation. In TOR-KI-resistant DLBCL cells lacking eIF4E binding protein-1, SBI-756 still sensitised to venetoclax. SBI-756 selectively reduced translation of mRNAs encoding ribosomal proteins and translation factors, leading to a reduction in protein synthesis rates in sensitive cells. When normal lymphocytes were treated with SBI-756, only B cells had reduced viability, and this correlated with reduced protein synthesis. CONCLUSIONS: Our data highlight a novel combination for treatment of aggressive lymphomas, and establishes its efficacy and selectivity using preclinical models.

Discovery of ß-Arrestin Biased, Orally Bioavailable, and CNS Penetrant Neurotensin Receptor 1 (NTR1) Allosteric Modulators.

Neurotensin receptor 1 (NTR1) is a G protein coupled receptor that is widely expressed throughout the central nervous system where it acts as a neuromodulator. Neurotensin receptors have been implicated in a wide variety of CNS disorders, but despite extensive efforts to develop small molecule ligands there are few reports of such compounds. Herein we describe the optimization of a quinazoline based lead to give 18 (SBI-553), a potent and brain penetrant NTR1 allosteric modulator.

X-Ray Structure of Human Sulfide:Quinone Oxidoreductase: Insights into the Mechanism of Mitochondrial Hydrogen Sulfide Oxidation.

Hydrogen sulfide (H2S) is a gasotransmitter exhibiting pivotal functions in diverse biological processes, including activation of multiple cardioprotective pathways. Sulfide:quinone oxidoreductase (SQOR) is an integral membrane flavoprotein that catalyzes the first step in the mitochondrial metabolism of H2S. As such, it plays a critical role in controlling physiological levels of the gasotransmitter and has attracted keen interest as a potential drug target. We report the crystal structure of human SQOR, unraveling the molecular basis for the enzyme's ability to catalyze sulfane sulfur transfer reactions with structurally diverse acceptors. We demonstrate that human SQOR contains unique features: an electropositive surface depression implicated as a binding site for sulfane sulfur acceptors and postulated to funnel negatively charged substrates to a hydrophilic H2S-oxidizing active site, which is connected to a hydrophobic internal tunnel that binds coenzyme Q. These findings support a proposed model for catalysis and open the door for structure-based drug design.

Identification and characterization of small molecule inhibitors of the ubiquitin ligases Siah1/2 in melanoma and prostate cancer cells.

Inhibition of ubiquitin ligases with small molecule remains a very challenging task, given the lack of catalytic activity of the target and the requirement of disruption of its interactions with other proteins. Siah1/2, which are E3 ubiquitin ligases, are implicated in melanoma and prostate cancer and represent high-value drug targets. We utilized three independent screening approaches in our efforts to identify small-molecule Siah1/2 inhibitors: Affinity Selection-Mass Spectrometry, a protein thermal shift-based assay and an in silico based screen. Inhibitors were assessed for their effect on viability of melanoma and prostate cancer cultures, colony formation, prolyl-hydroxylase-HIF1α signaling, expression of selected Siah2-related transcripts, and Siah2 ubiquitin ligase activity. Several analogs were further characterized, demonstrating improved efficacy. Combination of the top hits identified in the different assays demonstrated an additive effect, pointing to complementing mechanisms that underlie each of these Siah1/2 inhibitors.

Discovery of 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent and orally bioavailable tissue-nonspecific alkaline phosphatase (TNAP) inhibitor.

Tissue-nonspecific alkaline phosphatase (TNAP) is an ectoenzyme crucial for bone matrix mineralization via its ability to hydrolyze extracellular inorganic pyrophosphate (ePPi), a potent mineralization inhibitor, to phosphate (Pi). By the controlled hydrolysis of ePPi, TNAP maintains the correct ratio of Pi to ePPi and therefore enables normal skeletal and dental calcification. In other areas of the body low ePPi levels lead to the development of pathological soft-tissue calcification, which can progress to a number of disorders. TNAP inhibitors have been shown to prevent these processes via an increase of ePPi. Herein we describe the use of a whole blood assay to optimize a previously described series of TNAP inhibitors resulting in 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent, selective and oral bioavailable compound that robustly inhibits TNAP in vivo.

Unified clinical science, or paradigm diversity? Comment on Melchert (2016).

Drawing on Kuhn's (1970) analysis, Melchert (2016) argued that current professional psychology exists in a preparadigmatic state and that a transition to a unified clinical science based on the paradigm of the behavioral and neurosciences is now possible. But Melchert's analysis makes questionable assumptions about reducibility and neglects several crucial aspects of Kuhn's analysis. A close examination of psychological work on problems such as violence against women indicates that different research paradigms and their associated exemplars identify strengths and weaknesses of specific treatment resources that cannot be entirely encompassed within a single paradigmatic perspective (Jackson, 2015b) and additionally suggests that psychological knowledge is governed by at least 3 overarching research paradigms, as well as a variety of subparadigms encompassing applied and mixed methods research and many current orientations to professional psychology (Jackson, 2015a). (PsycINFO Database Record

Use of Tissue Metabolite Analysis and Enzyme Kinetics To Discriminate between Alternate Pathways for Hydrogen Sulfide Metabolism.

Hydrogen sulfide (H2S) is an endogenously synthesized signaling molecule that is enzymatically metabolized in mitochondria. The metabolism of H2S maintains optimal concentrations of the gasotransmitter and produces sulfane sulfur (S0)-containing metabolites that may be functionally important in signaling. Sulfide:quinone oxidoreductase (SQOR) catalyzes the initial two-electron oxidation of H2S to S0 using coenzyme Q as the electron acceptor in a reaction that requires a third substrate to act as the acceptor of S0. We discovered that sulfite is a highly efficient acceptor and proposed that sulfite is the physiological acceptor in a reaction that produces thiosulfate, a known metabolic intermediate. This model has been challenged by others who assume that the intracellular concentration of sulfite is very low, a scenario postulated to favor reaction of SQOR with a considerably poorer acceptor, glutathione. In this study, we measured the intracellular concentration of sulfite and other metabolites in mammalian tissues. The values observed for sulfite in rat liver (9.2 µM) and heart (38 µM) are orders of magnitude higher than previously assumed. We discovered that the apparent kinetics of oxidation of H2S by SQOR with glutathione as the S0 acceptor reflect contributions from other SQOR-catalyzed reactions, including a novel glutathione:CoQ reductase reaction. We used observed metabolite levels and steady-state kinetic parameters to simulate rates of oxidation of H2S by SQOR at physiological concentrations of different S0 acceptors. The results show that the reaction with sulfite as the S0 acceptor is a major pathway in liver and heart and provide insight into the potential dynamics of H2S metabolism.

Cell-Based High-Throughput Luciferase Reporter Gene Assays for Identifying and Profiling Chemical Modulators of Endoplasmic Reticulum Signaling Protein, IRE1.

Endoplasmic reticulum (ER) stress activates three distinct signal transducers on the ER membrane. Inositol-requiring protein 1 (IRE1), the most conserved signal transducer, plays a key role in ER stress-mediated signaling. During ER stress, IRE1 initiates two discrete signaling cascades: the "adaptive" signaling cascade mediated by the XBP1 pathway and the "alarm" signaling cascade mediated by stress-activated protein kinase pathways. Fine-tuning of the balance between the adaptive and alarm signals contributes significantly to cellular fate under ER stress. Thus, we propose that the design of high-throughput screening (HTS) assays to selectively monitor IRE1 mediated-signaling would be desirable for drug discovery. To this end, we report the generation of stable human neural cell lines and development of cell-based HTS luciferase (Luc) reporter gene assays for the identification of pathway-specific chemical modulators of IRE1. We implemented a cell-based Luc assay using a chimeric CHOP-Gal4 transcription factor in 384-well format for monitoring IRE1 kinase-mediated p38MAPK activation and an unfolded response pathway element (URPE)-Luc cell-based assay in 1536-well format for monitoring IRE1's RNase-mediated activation of XBP1. Chemical library screening was successfully conducted with both the CHOP/Gal4-Luc cells and UPRE-Luc engineered cells. The studies demonstrate the feasibility of using these HTS assays for discovery of pathway-selective modulators of IRE1.

Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes.

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

Role of human sulfide: quinone oxidoreductase in H2S metabolism.

The first step in the mammalian metabolism of H2S is catalyzed by sulfide:quinone oxidoreductase (SQOR). Human SQOR is an integral membrane protein, which presumably interacts with the inner mitochondrial membrane in a monotopic fashion. The enzyme is a member of a family of flavoprotein disulfide oxidoreductases (e.g., glutathione reductase) that utilize a Cys-S-S-Cys disulfide bridge as an additional redox center. SQOR catalyzes a two-electron oxidation of H2S to sulfane sulfur using coenzyme Q as electron acceptor. The enzyme also requires a third substrate to act as the acceptor of the sulfane sulfur from a cysteine persulfide intermediate. Here, we describe a method for the bacterial expression of human SQOR as a catalytically active membrane-bound protein, procedures for solubilization and purification of the recombinant protein to >95% homogeneity, and spectrophotometric assays to monitor SQOR-mediated H2S oxidation in reactions with different sulfane sulfur acceptors.

Pathophysiological role of vascular smooth muscle alkaline phosphatase in medial artery calcification.

Medial vascular calcification (MVC) is a pathological phenomenon that causes vascular stiffening and can lead to heart failure; it is common to a variety of conditions, including aging, chronic kidney disease, diabetes, obesity, and a variety of rare genetic diseases. These conditions share the common feature of tissue-nonspecific alkaline phosphatase (TNAP) upregulation in the vasculature. To evaluate the role of TNAP in MVC, we developed a mouse model that overexpresses human TNAP in vascular smooth muscle cells in an X-linked manner. Hemizygous overexpressor male mice (Tagln-Cre(+/-) ; Hprt(ALPL) (/Y) or TNAP-OE) show extensive vascular calcification, high blood pressure, and cardiac hypertrophy, and have a median age of death of 44 days, whereas the cardiovascular phenotype is much less pronounced and life expectancy is longer in heterozygous (Tagln-Cre(+/-) ; Hprt(ALPL) (/-) ) female TNAP-OE mice. Gene expression analysis showed upregulation of osteoblast and chondrocyte markers and decreased expression of vascular smooth muscle markers in the aortas of TNAP-OE mice. Through medicinal chemistry efforts, we developed inhibitors of TNAP with drug-like pharmacokinetic characteristics. TNAP-OE mice were treated with the prototypical TNAP inhibitor SBI-425 or vehicle to evaluate the feasibility of TNAP inhibition in vivo. Treatment with this inhibitor significantly reduced aortic calcification and cardiac hypertrophy, and extended lifespan over vehicle-treated controls, in the absence of secondary effects on the skeleton. This study shows that TNAP in the vasculature contributes to the pathology of MVC and that it is a druggable target.

Biosynthesis of a central intermediate in hydrogen sulfide metabolism by a novel human sulfurtransferase and its yeast ortholog.

Human sulfide:quinone oxidoreductase (SQOR) catalyzes the conversion of H2S to thiosulfate, the first step in mammalian H2S metabolism. SQOR's inability to produce the glutathione persulfide (GSS(-)) substrate for sulfur dioxygenase (SDO) suggested that a thiosulfate:glutathione sulfurtransferase (TST) was required to provide the missing link between the SQOR and SDO reactions. Although TST could be purified from yeast, attempts to isolate the mammalian enzyme were not successful. We used bioinformatic approaches to identify genes likely to encode human TST (TSTD1) and its yeast ortholog (RDL1). Recombinant TSTD1 and RDL1 catalyze a predicted thiosulfate-dependent conversion of glutathione to GSS(-). Both enzymes contain a rhodanese homology domain and a single catalytically essential cysteine, which is converted to cysteine persulfide upon reaction with thiosulfate. GSS(-) is a potent inhibitor of TSTD1 and RDL1, as judged by initial rate accelerations and ≥25-fold lower Km values for glutathione observed in the presence of SDO. The combined action of GSS(-) and SDO is likely to regulate the biosynthesis of the reactive metabolite. SDO drives to completion p-toluenethiosulfonate:glutathione sulfurtransferase reactions catalyzed by TSTD1 and RDL1. The thermodynamic coupling of the irreversible SDO and reversible TST reactions provides a model for the physiologically relevant reaction with thiosulfate as the sulfane donor. The discovery of bacterial Rosetta Stone proteins that comprise fusions of SDO and TSTD1 provides phylogenetic evidence of the association of these enzymes. The presence of adjacent bacterial genes encoding SDO-TSTD1 fusion proteins and human-like SQORs suggests these prokaryotes and mammals exhibit strikingly similar pathways for H2S metabolism.