Interaction of T-cell-specific adapter protein with Src- and Tec-family kinases.

Adapter proteins are flexible and dynamic modulators of cellular signalling that are important for immune cell function. One of these, the T-cell-specific adapter protein (TSAd), interacts with the non-receptor tyrosine kinases Src and Lck of the Src family kinases (SFKs) and Itk of the Tec family kinases (TFKs). Three tyrosine residues in the TSAd C-terminus are phosphorylated by Lck and serve as docking sites for the Src homology 2 (SH2) domains of Src and Lck. The TSAd proline-rich region (PRR) binds to the Src homology 3 (SH3) domains found in Lck, Src and Itk. Despite known interactors, the role TSAd plays in cellular signalling remains largely unknown. TSAd's ability to bind both SFKs and TFKs may point to its function as a general scaffold for both kinase families. Using GST-pulldown as well as peptide array experiments, we found that both the SH2 and SH3 domains of the SFKs Fyn and Hck, as well as the TFKs Tec and Txk, interact with TSAd. This contrasts with Itk, which interacts with TSAd only through its SH3 domain. Although our analysis showed that TSAd is both co-expressed and may interact with Fyn, we were unable to co-precipitate Fyn with TSAd from Jurkat cells, as detected by Western blotting and affinity purification mass spectrometry. This may suggest that TSAd-Fyn interaction in intact cells may be limited by other factors, such as the subcellular localization of the two molecules or the co-expression of competing binding partners.

Crosstalk among DNA Damage, Mitochondrial Dysfunction, Impaired Mitophagy, Stem Cell Attrition, and Senescence in the Accelerated Ageing Disorder Werner Syndrome.

Werner syndrome (WS) is an accelerated ageing disease caused by multiple mutations in the gene encoding the Werner DNA helicase (WRN). The major clinical features of WS include wrinkles, grey hair, osteoporosis, and metabolic phenomena such as atherosclerosis, diabetes, and fatty liver, and resemble those seen in normal ageing, but occur earlier, in middle age. Defective DNA repair resulting from mutations in WRN explain the majority of the clinical features of WS, but the underlying mechanisms driving the larger metabolic dysfunction remain elusive. Recent studies in animal models of WS and in WS patient cells and blood samples suggest the involvement of impaired mitophagy, NAD+ depletion, and accumulation of damaged mitochondria in metabolic dysfunction. This mini-review summarizes recent progress in the understanding of the molecular mechanisms of metabolic dysfunction in WS, with the involvement of DNA damage, mitochondrial dysfunction, mitophagy reduction, stem cell impairment, and senescence. Future studies on NAD+ and mitophagy may shed light on potential therapeutic strategies for the WS patients.

The amide linker in nonpeptide neurotensin receptor ligands plays a key role in calcium signaling at the neurotensin receptor type 2.

Compounds acting via the GPCR neurotensin receptor type 2 (NTS2) display analgesia in relevant preclinical models. The amide bond in nonpeptide NTS1 antagonists plays a central role in receptor recognition and molecular conformation. Using NTS2 FLIPR and binding assays, we found that it is also a key molecular structure for binding and calcium mobilization at NTS2. We found that reversed amides display a shift from agonist to antagonist activity and provided examples of the first competitive nonpeptide antagonists observed in the NTS2 FLIPR assay. These compounds will be valuable tools for determining the role of calcium signaling in vitro to NTS2 mediated analgesia.

Identification of N-{[6-chloro-4-(2,6-dimethoxyphenyl)quinazolin-2-yl]carbonyl}-l-leucine (NTRC-808), a novel nonpeptide chemotype selective for the neurotensin receptor type 2.

Compounds acting via the GPCR neurotensin receptor type 2 (NTS2) display analgesic effects in relevant animal models. Using a pharmacophore model based on known NT receptor nonpeptide compounds, we screened commercial databases to identify compounds that might possess activity at NTS2 receptor sites. Modification of our screening hit to include structural features known to be recognized by NTS1 and NTS2, led to the identification of the novel NTS2 selective nonpeptide, N-{[6-chloro-4-(2,6-dimethoxyphenyl)quinazolin-2-yl]carbonyl}-l-leucine (9). This compound is a potent partial agonist in the FLIPR assay with a profile of activity similar to that of the reference NTS2 analgesic nonpeptide levocabastine (5).

The importance of the 6- and 7-positions of tetrahydroisoquinolines as selective antagonists for the orexin 1 receptor.

Selective antagonism of the orexin 1 (OX1) receptor has been proposed as a potential mechanism for treatment of drug addiction. We have previously reported studies on the structure-activity relationships of tetrahydroisoquinoline-based antagonists. In this report, we elucidated the respective role of the 6- and 7-substitutions by preparation of a series of either 6-substituted tetrahydroisoquinolines (with no 7-substituents) or vice versa. We found that 7-substituted tetrahydroisoquinolines showed potent antagonism of OX1, indicating that the 7-position is important for OX1 antagonism (10 c, Ke = 23.7 nM). While the 6-substituted analogs were generally inactive, several 6-amino compounds bearing ester groups showed reasonable potency (26 a, Ke = 427 nM). Further, we show evidence that suggests several compounds initially displaying insurmountable antagonism at the OX1 receptor are competitive antagonists with slow dissociation rates.

High production of IL-12 by human dendritic cells stimulated with combinations of pattern-recognition receptor agonists.

The cytokine IL-12p70 is crucial for T helper 1 (Th1) polarization and the generation of type 1 immunity required to fight cancer and pathogens. Therefore, strategies to optimize the production of IL-12p70 by human dendritic cells (DCs) may significantly improve the efficacy of vaccines and immunotherapies. However, the rules governing the production of IL-12p70 remain obscure. Here, we stimulated pattern recognition receptors (PRRs) representing five families of PRRs, to evaluate their ability to elicit high production of IL-12p70 by monocyte-derived DCs. We used ten well-characterized agonists and stimulated DCs in vitro with either single agonists or 27 different combinations. We found that poly(I:C), which engages the RNA-sensing PRRs TLR3 and MDA5, and LPS which stimulates TLR4, were the only agonists that could elicit notable IL-12p70 production when used as single ligands. We identified six different combinations of PRR agonists, all containing either the TLR3/MDA5 agonist poly(I:C) or the TLR7/8 agonist R848, that could synergize to elicit high production of IL-12p70 by human DCs. Five of the six combinations also triggered high production of the antiviral and antitumor cytokine IFNß. Overall, the tested PRR ligands could be divided into three groups depending on whether they triggered production of both IL-12p70 and IFNß, only one of the two, or neither. Thus, combinations of PRR agonists were found to increase the production of IL-12p70 by human DCs in a synergistic manner, and we identified six PRR agonist combinations that may represent strong adjuvant candidates, in particular for therapeutic cancer vaccines.

Diaryl urea analogues of SB-334867 as orexin-1 receptor antagonists.

As a part of our program to develop OX1-CB1 bivalent ligands, we required a better understanding of the basic structure-activity relationships (SARs) of orexin antagonists. A series of SB-334867 analogues were synthesized and evaluated in calcium mobilization assays. SAR results suggest that the 2-methylbenzoxazole moiety may be replaced with a disubstituted 4-aminophenyl group without loss of activity and an electron-deficient system is generally preferred at the 1,5-naphthyridine moiety for OX1 antagonist activity. In particular, substitution of larger potential linkers such as n-hexyl provided compound 33 with equivalent activity at the OX1 receptor compared to the lead compound SB-334867. These compounds should be of value in the development of ligands targeting the orexin-1 receptor and its potential heterodimers.

Synthesis and evaluation of 1,2,4-methyltriazines as mGluR5 antagonists.

In previous studies we showed that 3-(substituted phenylethynyl)-5-methyl[1,2,4]triazine analogues of MPEP were potent antagonists of glutamate-mediated mobilization of internal calcium in an mGluR5 in vitro efficacy assay. In the present study we report the synthesis and evaluation of six 3-(substituted biphenylethynyl)-5-methyl[1,2,4]triazines (5a-f), and five 3-(substituted phenoxyphenylethynyl)-5-methyltriazines (6a-e). Compound 2-(4-fluorophenyl-5-[2-(5-methyl[1,2,4]triazine-3-yl)ethynyl]benzonitrile (5f) with an IC(50) of 28.2 nM was the most potent analogue.

Trace amine-associated receptor 1 is a stereoselective binding site for compounds in the amphetamine class.

The demonstrated ability of amphetamine to functionally activate the rat trace amine associated receptor 1 (rTAAR1) and the subsequent reports of amphetamine activation of TAAR1 in rhesus monkey mouse, human, and human-rat chimeric TAAR1-expressing cell lines has led to speculation as to the role of this receptor in the central nervous system (CNS) responses associated with amphetamine and its analogs. The agonist potencies of ten pairs of enantiomeric amphetamines, including several with known CNS activity, at primate TAAR1 stably expressed in RD-HGA16 cells, robustly indicate the S-configuration to be associated with higher potency. Moreover, the rank order of potency to activate TAAR1 parallels the stimulant action reported by humans for the specific amphetamines. Taken together, these data suggest that TAAR1 is a stereoselective binding site for amphetamine and that activation of TAAR1 is involved in the modulation of the stimulant properties of amphetamine and its congeners. In addition, the observed parallel between hTAAR1 and rhTAAR1 responses supports the rhesus monkey as a highly translational model for developing novel TAAR1-directed compounds as therapeutics for amphetamine-related addictions.

Preparation of a Series of 5-Methyl-3-(substituted)-[1,2,4]triazines.

Procedures for the synthesis of thirty-six 5-methyl-3-(substituted)-[1,2,4]triazines have been described. These compounds were evaluated for antagonism at metabotropic glutamate receptor subtype 5. Two compounds, 5b and 3c, were determined to be low micromolar inhibitors of mGluR5.

Validation of a High-Throughput Calcium Mobilization Assay for the Human Trace Amine-Associated Receptor 1.

The human trace amine-associated receptor 1 (hTAAR1) is a G protein-coupled receptor (GPCR) that is widely expressed in monoaminergic nuclei in the central nervous system and has therapeutic potential for multiple diseases, including drug addiction and schizophrenia. Thus, identification of novel hTAAR1 ligands is critical to advancing our knowledge of hTAAR1 function and to the development of therapeutics for a wide range of diseases. Herein we describe the development of a robust, 3-addition high-throughput screening (HTS) calcium mobilization assay using stable CHO-Gαq16-hTAAR1 cells, which functionally couple hTAAR1 to the promiscuous Gαq16 protein and thus allow signal transduction to occur through mobilization of internal calcium. Our previously established 96-well hTAAR1 assay was first miniaturized to the 384-well format and optimized to provide an assay with a Z' factor of 0.84, which is indicative of a robust HTS assay. Using the 3-addition protocol, 22,000 compounds were screened and yielded a ~1% agonist hit rate and a ~0.2% antagonist hit rate. Of the antagonist hits, two confirmed hits are the most potent hTAAR1 antagonists identified to date (IC50 = 206 and 281 nM). While scientists have been studying hTAAR1 for years, the lack of suitable hTAAR1 antagonists has been a major roadblock for studying the basic pharmacology of hTAAR1. Thus, these new ligands will serve as valuable tools to study hTAAR1-mediated signaling mechanisms, therapeutic potential, and in vivo functions.

Targeting NAD+ in translational research to relieve diseases and conditions of metabolic stress and ageing.

Nicotinamide adenine dinucleotide (NAD+) plays a fundamental role in life and health through the regulation of energy biogenesis, redox homeostasis, cell metabolism, and the arbitration of cell survival via linkages to apoptosis and autophagic pathways. The importance of NAD+ in ageing and healthy longevity has been revealed from laboratory animal studies and early-stage clinical testing. While basic researchers and clinicians have investigated the molecular mechanisms and translation potential of NAD+, there are still major gaps in applying laboratory science to design the most effective trials. This mini-review was based on the programme and discussions of the 3rd NO-Age Symposium held at the Akershus University Hospital, Norway on the 28th October 2019. This symposium brought together leading basic researchers on NAD+ and clinicians who are leading or are going to perform NAD+ augmentation-related clinical studies. This meeting covered talks about NAD+ synthetic pathways, subcellular homeostasis of NAD+, the benefits of NAD+ augmentation from maternal milk to offspring, current clinical trials of the NAD+ precursor nicotinamide riboside (NR) on Ataxia-Telangiectasia (A-T), Parkinson's disease (PD), post-sepsis fatigue, as well as other potential NR-based clinical trials. Importantly, a consensus is emerging with respect to the design of clinical trials in order to measure meaningful parameters and ensure safety.

A research agenda for ageing in China in the 21st century (2nd edition): Focusing on basic and translational research, long-term care, policy and social networks.

One of the key issues facing public healthcare is the global trend of an increasingly ageing society which continues to present policy makers and caregivers with formidable healthcare and socio-economic challenges. Ageing is the primary contributor to a broad spectrum of chronic disorders all associated with a lower quality of life in the elderly. In 2019, the Chinese population constituted 18 % of the world population, with 164.5 million Chinese citizens aged 65 and above (65+), and 26 million aged 80 or above (80+). China has become an ageing society, and as it continues to age it will continue to exacerbate the burden borne by current family and public healthcare systems. Major healthcare challenges involved with caring for the elderly in China include the management of chronic non-communicable diseases (CNCDs), physical frailty, neurodegenerative diseases, cardiovascular diseases, with emerging challenges such as providing sufficient dental care, combating the rising prevalence of sexually transmitted diseases among nursing home communities, providing support for increased incidences of immune diseases, and the growing necessity to provide palliative care for the elderly. At the governmental level, it is necessary to make long-term strategic plans to respond to the pressures of an ageing society, especially to establish a nationwide, affordable, annual health check system to facilitate early diagnosis and provide access to affordable treatments. China has begun work on several activities to address these issues including the recent completion of the of the Ten-year Health-Care Reform project, the implementation of the Healthy China 2030 Action Plan, and the opening of the National Clinical Research Center for Geriatric Disorders. There are also societal challenges, namely the shift from an extended family system in which the younger provide home care for their elderly family members, to the current trend in which young people are increasingly migrating towards major cities for work, increasing reliance on nursing homes to compensate, especially following the outcomes of the 'one child policy' and the 'empty-nest elderly' phenomenon. At the individual level, it is important to provide avenues for people to seek and improve their own knowledge of health and disease, to encourage them to seek medical check-ups to prevent/manage illness, and to find ways to promote modifiable health-related behaviors (social activity, exercise, healthy diets, reasonable diet supplements) to enable healthier, happier, longer, and more productive lives in the elderly. Finally, at the technological or treatment level, there is a focus on modern technologies to counteract the negative effects of ageing. Researchers are striving to produce drugs that can mimic the effects of 'exercising more, eating less', while other anti-ageing molecules from molecular gerontologists could help to improve 'healthspan' in the elderly. Machine learning, 'Big Data', and other novel technologies can also be used to monitor disease patterns at the population level and may be used to inform policy design in the future. Collectively, synergies across disciplines on policies, geriatric care, drug development, personal awareness, the use of big data, machine learning and personalized medicine will transform China into a country that enables the most for its elderly, maximizing and celebrating their longevity in the coming decades. This is the 2nd edition of the review paper (Fang EF et al., Ageing Re. Rev. 2015).

The identification of nonpeptide neurotensin receptor partial agonists from the potent antagonist SR48692 using a calcium mobilization assay.

In a search for nonpeptide agonists for the neurotensin receptor (NTR1), we replaced the adamantyl amino acid moiety found in the antagonist SR48692 (1a) with leucine and related alpha-alkylamino acids found in peptide agonists. When tested in a calcium mobilization assay, we found that both d- and l-leucine confer partial agonist activity to the pyrazole scaffold with the l-enantiomer (3a) providing a significantly greater response. A brief SAR survey demonstrated that the observed agonist activity was resilient to changes made to the dimethoxyaryl ring in 3a. The resulting compounds were less potent relative to 3a but showed greater agonist responses. The partial agonist activity was extinguished when the chloroquinoline ring was replaced with naphthalene. Thus, while l-leucine appears to possess a powerful agonist directing affect for the NTR1 receptor, its presence alone in the molecular architecture is not sufficient to insure agonist behavior.

Amiodarone and its putative metabolites fail to activate wild type hTAAR1.

The ability of amiodarone and its putative metabolites to activate unmodified human trace amine associated receptor 1 (hTAAR1) stably expressed in CHO cell lines was evaluated. Receptor activation was monitored by measuring the accumulation of cAMP, the putative hTAAR1 native second messenger, or calcium mobilization in cells where the receptor was coupled to the promiscuous Gq, Galpha16. Despite literature reports of activation of rodent TAAR1 by these agents, no response was seen in either cell line although robust activation was obtained with the endogenous ligand beta-phenethylamine. These results indicate that TAAR1 activation by amiodarone and its analogs is species specific.

Identifying structural features on 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones critical for Neuropeptide S antagonist activity.

A series of 7-substituted 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones were synthesized and tested for Neuropeptide S (NPS) antagonist activity. A concise synthetic route was developed, which features a DMAP catalyzed carbamate formation. 4-Fluorobenzyl urea (1c) and benzyl urea (1d) were identified as the most potent antagonists among the compounds examined. Structure-activity relationships (SARs) demonstrate that a 7-position urea functionality is required for potent antagonist activity and alkylation of the urea nitrogen (1e) or replacement with carbon or oxygen (5a) results in reduced potency. In addition, compounds with alpha-methyl substitution (1b) or elongated alkyl chains (1h and 1j) had reduced potency, indicating a limited tolerance for 7-position substituents.

A new synthesis of the ORL-1 antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) and activity in a calcium mobilization assay.

A new chiral synthesis of the ORL-1 antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (2, J-113397) was developed. J-113397 has a K(e)=0.85nM in an ORL-1 calcium mobilization assay and is 89-, 887-, and 227-fold selective for the ORL-1 receptor relative to the mu, delta, and kappa opioid receptors.

Synthesis and pharmacological evaluation of phenylethynyl[1,2,4]methyltriazines as analogues of 3-methyl-6-(phenylethynyl)pyridine.

Procedures were developed for the synthesis of 3-methyl-5-phenylethynyl[1,2,4]triazine (4), 6-methyl-3-phenylethynyl[1,2,4]triazine (5), and 5-methyl-3-phenylethynyl[1,2,4]triazine (6a) as analogues of 2-methyl-6-(phenylethynyl)pyridine (2). The compounds were evaluated for antagonism of glutamate-mediated mobilization of internal calcium in an mGluR5 in vitro efficacy assay. The most potent of the three analogues was 6a. Twenty additional analogues of 6a were synthesized and evaluated for mGluR5 antagonist efficacy. The most potent compounds were 3-(3-methylphenylethynyl)-5-methyl[1,2,4]triazine (6b), 5-(3-chlorophenylethynyl)-5-methyl[1,2,4]triazine (6c), and 3-(3-bromophenylethynyl)-5-methyl[1,2,4]triazine (6d).

A rapid functional assay for the human trace amine-associated receptor 1 based on the mobilization of internal calcium.

The molecular targets for trace amines (TAs) such as p-tyramine and beta-phenylethylamine have been recently discovered and have been shown to comprise a family of G-protein-coupled receptors based on DNA sequence homologies. These have been termed trace amine-associated receptors (TAARs) because TAs do not activate all of the identified receptors. Because TA may be involved in modulating a variety of behaviors including mood, cognition, and addiction, it is of interest to discover novel ligands for TAARs to probe the role TAs play in brain function. Pharmacophore development for the G(s)-coupled human TAAR1 (hTAAR1) would be aided by a rapid functional assay amenable to screening libraries of compounds. Accordingly, the authors used RD-HGA16 CHO-1 cells from Molecular Devices, which stably express the promiscuous G(q), G(alpha16), to create a cell line stably expressing hTAAR1, thereby coupling receptor activation to the mobilization of internal calcium. They used this cell line to develop a homogenous fluorometric imaging plate reader-based assay using the Calcium 3 fluorescent dye. The EC50 and Emax data obtained for known TAs are in close agreement with previous work using transient hTAAR1 expression systems or a chimeric receptor. These data indicate that the hTAAR1 retains its reported pharmacological characteristics when coupled to G(alpha16).

Identification of 2-({[1-(4-Fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazol-3-yl]carbonyl}amino)tricyclo[3.3.1.13,7]decane-2-carboxylic Acid (NTRC-844) as a Selective Antagonist for the Rat Neurotensin Receptor Type 2.

Neurotensin receptor type 2 (NTS2) compounds display analgesic activity in animal pain models. We have identified the first high-affinity NTS2-selective antagonist (8) that is active in vivo. This study also revealed that the NTS2 FLIPR assay designation for a compound, agonist, partial agonist, and so forth, did not correlate with its in vivo activity as observed in the thermal tail-flick acute model of pain. This suggests that calcium mobilization is not the signaling pathway involved in NTS2-mediated analgesia as assessed by the thermal tail-flick model. Finally, we found a significant bias between rat and human for compound 9 in the NTS2 binding assay.