Ken A. Witt

Somatostatin receptor-4 (SST₄) is a therapeutic target for several conditions, including Alzheimer's disease, seizures, neuropsychiatric disorders, and pain. Our previous work on 1,2,4-triazole derivatives led to enhanced SST₄ binding affinity, selectivity, and functional activity. Herein we report the discovery of 3-thio-1,2,4-triazole series as selective and high affinity SST₄ agonists. Thirty-three compounds show 300-fold selectivity over other SST subtypes. SST₄ cAMP inhibition assay activity data aligned with the ligand binding affinity. Comparative docking results of the ligands under investigation with the cryo-EM and most recent model-built SST₄ structures suggest similar trends in binding. Amino acids responsible for high and moderate affinity were identified, whereas poorer ligand conformations and limited interactions were observed with the low-affinity compounds. In summary, this study presents a novel series of high affinity SST₄ agonists with corresponding in vitro activity, demonstrating viable therapeutic potential.

Over 4 decades of research support the link between Alzheimer disease (AD) and somatostatin [somatotropin-releasing inhibitory factor (SRIF)]. SRIF and SRIF-expressing neurons play an essential role in brain function, modulating hippocampal activity and memory formation. Loss of SRIF and SRIF-expressing neurons in the brain rests at the center of a series of interdependent pathological events driven by amyloid-β peptide (Aβ), culminating in cognitive decline and dementia. The connection between the SRIF and AD further extends to the neuropsychiatric symptoms, seizure activity, and inflammation, whereas preclinical AD investigations show SRIF or SRIF receptor agonist administration capable of enhancing cognition. SRIF receptor subtype-4 activation in particular presents unique attributes, with the potential to mitigate learning and memory decline, reduce comorbid symptoms, and enhance enzymatic degradation of Aβ in the brain. Here, we review the links between SRIF and AD along with the therapeutic implications. Significance Statement Somatostatin and somatostatin-expressing neurons in the brain are extensively involved in cognition. Loss of somatostatin and somatostatin-expressing neurons in Alzheimer disease rests at the center of a series of interdependent pathological events contributing to cognitive decline and dementia. Targeting somatostatin-mediated processes has significant therapeutic potential for the treatment of Alzheimer disease.

ID 52588 Poster Board 214 Alzheimer’s disease (AD) is the leading cause of dementia, affecting nearly 50 million people worldwide. Despite an increased understanding of AD pathogenesis, the search for viable disease-modifying therapeutics continues. Sigma-1 and sigma-2 receptors, which are found in regions of the brain highly affected by AD, present novel targets with disease-modifying potential. While little is known about the mechanism of action of the sigma receptors, they are proposed to be involved with the regulation of many AD hallmarks. Investigations of sigma-1 receptor and sigma-2 receptor modulators have independently shown to improve learning and memory behaviors, as well as reduce neuroinflammation and amyloid-beta brain levels in AD mouse models (Antonini et al., Journal of Alzheimer’s Disease, 24(3), 569–586, 2011; Izzo et al., PLoS ONE, 9(11), 2014) . Herein, we investigated a novel molecule (BBZI) with high receptor affinity for both sigma-1 (Ki = 0.8nM) and sigma-2 (Ki=2.5nM) receptors as to learning and memory behavioral responses with chronic dosing in the senescence-accelerated prone mouse (SAMP8) model of age-related cognitive decline and AD. Twelve-month old SAMP8 were treated with BBZI over 28-days, i.p. daily dosing (0.001, 0.01, 0.1, 1.0, and 10 mg/kg) and vehicle controls. Testing battery included investigation of locomotion and anxiety through the open field activity test (day 15) and the elevated plus test (days 16-17), Y-maze for short term spatial memory (day 22), novel object recognition to test recognition memory (days 23-24), and the T-maze foot-shock avoidance test for long term spatial memory (days 20 & 27). The results indicated there is no difference in distance traveled or level of anxiety like behavior across the dosing groups as indicated by the open field activity test and the elevated plus test, respectively. There was also no significant improvement in Y-maze tested spatial memory or recognition memory as indicated novel object recognition test results. However, the T-maze foot-shock avoidance test results indicated BBZI treatment of 1.0 mg/kg with i.p. injection displayed significant positive results with acquisition learning testing (9.25 +/- 0.91 trials to first avoidance, p < 0.05) and retention memory testing (6.42 +/- 0.84 trials to criterion, p < 0.001). Additionally, BBZI treatment of 0.1 mg/kg with i.p. injection yielded positive results with retention memory testing (8.46 +/- 0.86 trials to criterion, p < 0.05). Our study revealed that at the doses specified, BBZI promotes enhanced long term memory effectiveness with the T Maze foot-shock avoidance test in a mouse model of AD, while having no impact on general activity or anxiety. This corroborates the hypothesis of sigma receptors’ involvement in learning and memory behaviors and further supports the potential of Sigma receptor target therapeutics for AD treatment.

ID 53923 Poster Board 137 Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive impairments and brain amyloid-beta (Aβ) aggregation. Neprilysin (NEP) is a primary Aβ-degrading enzyme, activated by the neuropeptide somatostatin (ssT) through a receptor mediated action (Saito et al., 2005, Nat Med 11(4):434-439). Of the five ssT receptors, the subtype-4 (ssTR4) shows the greatest promise for AD treatment targeting. ssTR4 has optimal brain localization, being highly expressed in neurons of the neocortex, and hippocampus (Viollet et al., 2008, Mol Cell Endocrinol 286(1-2):75-87). We previously showed ssTR4 agonist actions increase spatial and recognition memory in Senescence Accelerated Mouse-Prone 8 (SAMP8) and APPswe mouse models of AD, with capacity to reduce Aβ-oligomer protein expression (Sandoval et al., 2012 Eur J Pharmacol 683(1-3):116-124; Sandoval et al., 2013, Brain Res 1520:145-56). Herein, we evaluated the novel ssTR4 agonist SM-I-26 on memory behavior and NEP activity in SAMP8 mice. SM-I-26 has high ssTR4 affinity (Ki=12 nM), selectivity (>400-fold selective over other ssTR subtypes), and activity (EC50=17 nM). Male 12-month-old SAMP8 mice were administered 0.01, 0.1, or 1.0 μg (i.c.v.) of SM-I-26 or vehicle for memory retention testing using the T-maze foot-shock avoidance test (memory retention 1-week post-injection, n = 7-8/group). NEP activity was tested at 1.0 μg (i.c.v.) against vehicle control in cortical and hippocampal tissue 24-h post injection (n = 7/group). SM-I-26 improved behavioral retention of the T-maze shock avoidance task in a dose-responsive manner (PPP < 0.05, independent t-test) compared to vehicle controls. A similar but non-significant trend between SM-I-26 and NEP activity was observed in hippocampal tissue. Data shows i.c.v. administration of the ssTR4 agonist SM-I-26 improves memory behavior in SAMP8 mice and increases NEP activity in primary tissues associated with AD impairments. The potential of a ssTR4 agonist therapeutic for AD treatment is supported by this work. This work has been supported by the National Institutes of Health, National Institute on Aging, R01AG047858.

Somatostatin receptor subtype 4 (SSTR4) is expressed in BV2 microglia, suggesting that SSTR4 agonists may impact microglia function. This study assessed the high-affinity SSTR4 agonist SM-I-26 (SMI) (0 nM, 10 nM, 1000 nM) against lipopolysaccharide (LPS)-induced inflammation (0, 10 or 100 ng/ml) over 6 or 24 h in BV2 microglia. Cell viability, nitrite output and mRNA expression changes of genes associated with our target (Sstr4), inflammation (Tnf-α, Il-6, Il-1β, inos), anti-inflammatory and anti-oxidant actions (Il-10, Catalase), and mediators of Aβ binding/phagocytosis (Msr1, Cd33, Trem1, Trem2) were measured. At 6 h SMI showed no effect across all conditions. At 24 h SMI (10 and 1000 nM) upregulated Sstr4 expression under inflammatory and non-inflammatory conditions. At 24 h SMI downregulated expression of the inflammatory cytokines Tnf-α (1000 nM within all LPS concentrations) and Il-6 (10 nM within 0 and 10 ng/ml LPS). At 24 h 10 nM SMI upregulated Il-10, while 1000 nM upregulated Catalase under inflammatory and non-inflammatory conditions. At 24 h Msr1 and Cd33 were upregulated by 1000 nM SMI under non-inflammatory conditions, while Trem1 was downregulated by 10 and 1000 nM SMI under mildly inflammatory and non-inflammatory conditions. These results show that SMI had concentration and time-dependent effects on mRNA expression of genes associated with different states of microglial activation. The SMI reduced Tnf-α and Il-6 inflammatory gene expression, and increased Il-10 anti-inflammatory gene expression, identifies anti-inflammatory actions of SSTR4 agonists extend to microglia.

Somatostatin receptor-4 (SST₄) is highly expressed in brain regions affiliated with learning and memory. SST₄ agonist treatment may act to mitigate Alzheimer's disease (AD) pathology. An integrated approach to SST₄ agonist lead optimization is presented herein. High affinity and selective agonists with biological efficacy were identified through iterative cycles of a structure-based design strategy encompassing computational methods, chemistry, and preclinical pharmacology. 1,2,4-Triazole derivatives of our previously reported hit (4) showed enhanced SST₄ binding affinity, activity, and selectivity. Thirty-five compounds showed low nanomolar range SST₄ binding affinity, 12 having a K_i < 1 nM. These compounds showed >500-fold affinity for SST₄ as compared to SST_2A. SST₄ activities were consistent with the respective SST₄ binding affinities (EC₅₀ < 10 nM for 34 compounds). Compound 208 (SST₄K_i = 0.7 nM; EC₅₀ = 2.5 nM; >600-fold selectivity over SST_2A) display a favorable physiochemical profile, and was advanced to learning and memory behavior evaluations in the senescence accelerated mouse-prone 8 model of AD-related cognitive decline. Chronic administration enhanced learning with i.p. dosing (1 mg kg⁻¹) compared to vehicle. Chronic administration enhanced memory with both i.p. (0.01, 0.1, 1 mg kg⁻¹) and oral (0.01, 10 mg kg⁻¹) dosing compared to vehicle. This study identified a novel series of SST₄ agonists with high affinity, selectivity, and biological activity that may be useful in the treatment of AD.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder resulting in memory and cognitive impairment. The use of somatostatin receptor subtype-4 (SSTR₄) agonists have been proposed for AD treatment. This study investigated the effects of selective SSTR₄ agonist NNC 26-9100 on mRNA expression of key genes associated with AD pathology (microglia mediators of Aβ phagocytosis, amyloid-beta (Aβ)-degrading enzymes, anti-oxidant enzymes and pro-inflammatory cytokines) in 3xTg-AD mice. Mice were administered NNC 26-9100 (0.2 µg, i.c.v.) or vehicle control, with cortical and subcortical brain tissue collected at 6 h and 24 h post-treatment. At 6 h, NNC 26-9100 treatment decreased cortical expression of cluster of differentiation-33 (Cd33) by 25%, while increasing cortical and subcortical macrophage scavenger receptor-1 (Msr1) by 1.8 and 2.0-fold, respectively. The Cd33 downregulation and Msr1 upregulation support a state of microglia associated Aβ phagocytosis. At 24 h, NNC 26-9100 treatment increased the cortical expression of Sstr4 (4.9-fold), Aβ-degrading enzymes neprilysin (9.3-fold) and insulin degrading enzyme (14.8-fold), and the antioxidant catalase (3.6-fold). Similar effects at 24 h were found in subcortical tissue with NNC 26-9100 treatment, but did not reach statistical significance. No changes in pro-inflammatory cytokine expression were found. These data demonstrated NNC 26-9100 facilitates transcriptional changes in brain tissue identified with Aβ phagocytosis and clearance, further supporting SSTR₄ as a treatment target for AD.

Sigma receptor 1 (Sig1R) and 2 (Sig2R) are expressed in central nervous system, in neurons and microglia. Modulation of these receptors has been identified for the treatment of inflammatory and neurodegenerative disorders, including Alzheimer's disease (AD). This research evaluated the impact of BBZI, a novel pan‐selective Sig1R‐Sig2R ligand in mouse models of Alzheimer's disease and microglia activation. BBZI has high affinity Sig1R‐Sig2R (Sig1R: Ki=0.8nM; Sig2R: Ki=2.5nM), with 100‐fold selectivity over a panel of 30 other receptors. Twelve‐month old Senescence Accelerated Mouse Prone‐8 (SAMP8) and 3xTg AD mice were treated with BBZI (i.c.v.) across a dosing range (0, 0.001, 0.01, 0.1, 1.0μg), and assessed for memory response via T‐maze. BBZI was also evaluated in BV2 microglia cells concurrent with inflammatory activation via lipopolysaccharide (LPS). After plating for 24hrs, cells were treated for 24hrs with BBZI (0, 10, 100, 1000nM) against LPS (0, 10, 100ng/mL). Alamar Blue (cellular proliferation) and nitrite (surrogate of nitric oxide) output in media. BBZI significantly enhanced memory in SAMP8 mice at both 0.001μg and 0.01μg doses (pSupport or Funding Information This work was supported by the School of Pharmacy, Southern Illinois University Edwardsville. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Diet-mediated alterations of critical brain nutrient transporters, major facilitator super family domain-containing 2a (Mfsd2a) and glucose transporter 1 (Glut1), have wide reaching implications in brain health and disease. The aim of the study was to examine the impact of long-term low- and high-fat diets with lard or fish oil on critical brain nutrient transporters, Mfsd2a and Glut1. Eight-week-old male C57BL/6 mice were fed 1 of the following 4 diets for 32 wk: 10% of kcal from lard, 10% of kcal from fish oil, 41% of kcal from lard, or 41% of kcal from fish oil. Body weight and blood chemistries delineated dietary effects. Cortical and subcortical Mfsd2a and Glut1 mRNA and protein expression were evaluated, with other supportive nutrient-sensitive targets also assessed for mRNA expression changes. Fish-oil diets increased cortical Mfsd2a mRNA expression compared with lard diets. Subcortical Mfsd2a mRNA expression decreased as the percentage of fat in the diet increased. There was an interaction between the type and percentage of fat with cortical and subcortical Mfsd2a and cortical Glut1 protein expression. In the lard diet groups, protein expression of cortical and subcortical Mfsd2a and cortical Glut1 significantly increased as fat percentage increased. As the fat percentage increased in the fish-oil diet groups, protein expression of cortical and subcortical Mfsd2a and cortical Glut1 did not change. When comparing the fish-oil groups with 10% lard, cortical Mfsd2a protein expression was significantly higher in the 10% and 41% fish-oil groups, whereas cortical Glut1 protein expression was significantly higher in only the 10% fish-oil group. A positive correlation between cortical peroxisome proliferator–activated receptor γ mRNA expression and Mfsd2a protein expression was shown. Corresponding to chronic dietary treatment, an interaction between the type of fat and the percentage of fat exists respective to changes in brain expression of the key nutrient transporters Mfsd2a and Glut1.