Methods for negating the impact of zinc contamination to allow characterization of positive allosteric modulators of glycine receptors.

Zinc is a ubiquitous contaminant in many buffers, purified products and common labware that has previously been suggested to impact on the results of functional GlyR studies and may inadvertently cause the effectiveness of some GlyR modulators to be over-estimated. This could greatly impact the assessment of potential drug-candidates and contribute to the reduced effectiveness of compounds that reach clinical stages. This is especially true for GlyR modulators being developed for pain therapeutics due to the changes in spinal zinc concentrations that have been observed during chronic pain conditions. In this study we use two-electrode voltage clamp electrophysiology to evaluate the metal chelators tricine and Ca-EDTA, and show that tricine produces inhibitory effects at GlyRα1 that are not mediated by zinc. We also utilized the zinc insensitive W170S mutation as a tool to validate metal chelators and confirm that zinc contamination has not impacted the examination of lipid modulators previously developed by our lab. This study helps to further develop methods to negate the impact of contaminating zinc in functional studies of GlyRs which should be incorporated into future studies that seek to characterize the activity of novel modulators at GlyRs.

Sterol derivative binding to the orthosteric site causes conformational changes in an invertebrate Cys-loop receptor.

Cys-loop receptors or pentameric ligand-gated ion channels are mediators of electrochemical signaling throughout the animal kingdom. Because of their critical function in neurotransmission and high potential as drug targets, Cys-loop receptors from humans and closely related organisms have been thoroughly investigated, whereas molecular mechanisms of neurotransmission in invertebrates are less understood. When compared with vertebrates, the invertebrate genomes underwent a drastic expansion in the number of the nACh-like genes associated with receptors of unknown function. Understanding this diversity contributes to better insight into the evolution and possible functional divergence of these receptors. In this work, we studied orphan receptor Alpo4 from an extreme thermophile worm Alvinella pompejana. Sequence analysis points towards its remote relation to characterized nACh receptors. We solved the cryo-EM structure of the lophotrochozoan nACh-like receptor in which a CHAPS molecule is tightly bound to the orthosteric site. We show that the binding of CHAPS leads to extending of the loop C at the orthosteric site and a quaternary twist between extracellular and transmembrane domains. Both the ligand binding site and the channel pore reveal unique features. These include a conserved Trp residue in loop B of the ligand binding site which is flipped into an apparent self-liganded state in the apo structure. The ion pore of Alpo4 is tightly constricted by a ring of methionines near the extracellular entryway of the channel pore. Our data provide a structural basis for a functional understanding of Alpo4 and hints towards new strategies for designing specific channel modulators.

Novel Phenylene Lipids That Are Positive Allosteric Modulators of Glycine Receptors and Inhibitors of Glycine Transporter 2.

Chronic pain is a complex condition that remains resistant to current therapeutics. We previously synthesized a series of N-acyl amino acids (NAAAs) that inhibit the glycine transporter, GlyT2, some of which are also positive allosteric modulators of glycine receptors (GlyRs). In this study, we have synthesized a library of NAAAs that contain a phenylene ring within the acyl tail with the objective of improving efficacy at both GlyT2 and GlyRs and also identifying compounds that are efficacious as dual-acting modulators to enhance glycine neurotransmission. The most efficacious positive allosteric modulator of GlyRs was 2-[8-(2-octylphenyl)octanoylamino]acetic acid (8-8 OPGly) which potentiates the EC5 for glycine activation of GlyRα1 by 1500% with an EC50 of 664 nM. Phenylene-containing NAAAs with a lysine headgroup were the most potent inhibitors of GlyT2 with (2S)-6-amino-2-[8-(3-octylphenyl)octanoylamino]hexanoic acid (8-8 MPLys) inhibiting GlyT2 with an IC50 of 32 nM. The optimal modulator across both proteins was (2S)-6-amino-2-[8-(2-octylphenyl)octanoylamino]hexanoic acid (8-8 OPLys), which inhibits GlyT2 with an IC50 of 192 nM and potentiates GlyRs by up to 335% at 1 µM. When tested in a dual GlyT2/GlyRα1 expression system, 8-8 OPLys caused the greatest reductions in the EC50 for glycine. This suggests that the synergistic effects of a dual-acting modulator cause greater enhancements in glycinergic activity compared to single-target modulators and may provide an alternate approach to the development of new non-opioid analgesics for the treatment of chronic pain.

Development and early qualitative evidence of two novel patient-reported outcome instruments to assess daily functioning in people with early-stage Parkinson's.

BACKGROUND: Previous research on concepts that are important to people living with early-stage Parkinson's indicated that 'functional' slowness, fine motor skills, and subtle gait abnormalities are cardinal concepts that are not comprehensively captured by existing patient-reported outcome (PRO) instruments that are used in clinical practice and research to assess symptoms and daily functioning within this patient population. We sought to develop novel PRO instruments to address this unmet need. METHODS: PRO instrument development was led by a multidisciplinary research group, including people living with Parkinson's (termed 'patient experts'), as well as patient engagement and involvement, regulatory science, clinical, and outcome measurement experts. A first set of PRO instruments, termed Early Parkinson's Function Slowness (42 items) and Early Parkinson's Mobility (26 items), were drafted to capture 'functional' slowness, fine motor skills, and subtle gait abnormalities. These PRO instruments were used in cognitive debriefing interviews with people living with early-stage Parkinson's (who were not involved with the multidisciplinary research group) to identify issues with relevance, clarity, ease of completion, conceptual overlap, or missing concepts. RESULTS: Sixty people living with early-stage Parkinson's were interviewed, which led to refining the items to 45 for the Early Parkinson's Functional Slowness and 23 for the Early Parkinson's Mobility PRO instruments. Refinement included rewording items to address clarity issues, merging or splitting items to address overlap issues, and adding new items to address missing concepts. The Early Parkinson's Function Slowness PRO instrument resulted in a multidimensional instrument covering upper limb, complex/whole body, general activity, and cognitive functional slowness. The Early Parkinson's Mobility PRO instrument resulted in comprehensive coverage of everyday mobility tasks, with a focus on gait concepts, plus complex/whole body, balance, and lower limb mobility. CONCLUSIONS: The Early Parkinson's Function Slowness and Early Parkinson's Mobility PRO instruments aim to address gaps in existing PRO instruments to measure meaningful symptoms and daily functioning in people living with early-stage Parkinson's. Utilizing a meticulous study design led by a multidisciplinary research group that included patient experts helped to ensure that the PRO instruments were patient-centric, content valid, and meaningful from a clinical and measurement perspective.

Patient Experience in Early-Stage Parkinson's Disease: Using a Mixed Methods Analysis to Identify Which Concepts Are Cardinal for Clinical Trial Outcome Assessment.

INTRODUCTION: Qualitative research on patient experiences in early-stage Parkinson's disease (PD) is limited. It is increasingly acknowledged that clinical outcome assessments used in trials do not fully capture the range of symptoms/impacts that are meaningful to people with early-stage PD. We aimed to conceptualize the patient experience in early-stage PD and identify, from the patient perspective, those cardinal symptoms/impacts which might be more useful to measure in clinical trials. METHODS: In a mixed-methods analysis, 50 people with early-stage PD and nine relatives were interviewed. Study design and results interpretation were led by a multidisciplinary group of patient, clinical, regulatory, and outcome measurements experts, and patient organization representatives. Identification of the cardinal concepts was informed by the relative frequency of reported concepts combined with insights from patient experts and movement disorder specialists. RESULTS: A conceptual model of the patient experience of early-stage PD was developed. Concept elicitation generated 145 unique concepts mapped across motor and non-motor symptoms, function, and impacts. Bradykinesia/slowness (notably in the form of "functional slowness"), tremor, rigidity/stiffness, mobility (particularly fine motor dexterity and subtle gait abnormalities), fatigue, depression, sleep/dreams, and pain were identified as cardinal in early-stage PD. "Functional slowness" (related to discrete tasks involving the upper limbs, complex mobility tasks, and general activities) was deemed to be more relevant than "difficulty" to patients with early-stage PD, who report being slower at completing tasks rather than encountering significant impairment with task completion. CONCLUSION: Patient experiences in early-stage PD are complex and wide-ranging, and the currently available patient-reported outcome (PRO) instruments do not evaluate many early-stage PD concepts such as functional slowness, fine motor skills, and subtle gait abnormalities. The development of a new PRO instrument, created in conjunction with people with PD, that fully assesses symptoms and the experience of living with early-stage PD, is required.

We conducted research to find out about the experiences and symptoms that have the greatest impact on everyday living for people with early-stage Parkinson's disease. This research also looked at which symptoms patients think are important to be tracked in clinical trials. The research team running this study included people living with Parkinson's disease (called "patient experts"). The team also included technical experts and representatives of patient organizations. To begin with, people living with early-stage Parkinson's disease and relatives were interviewed. The interviews collected their thoughts on the impact of early-stage Parkinson's disease on their daily lives. These insights revealed which experiences and symptoms were most important. The research team analyzed ideas and quotes from the interviews to create a picture of early-stage Parkinson's disease. The symptoms that mattered the most to people living with early-stage Parkinson's disease were tremor, rigidity/stiffness, fatigue, depression, sleep/dreams, and pain. Another important symptom was slowness of movement (which is called "bradykinesia/slowness"), and in particular "functional slowness," which included tasks involving the upper limbs, complicated movement tasks, and general activities. Effects on mobility were also important, particularly fine motor skills and subtle walking abnormalities. This research shows the wide-ranging effects that early-stage Parkinson's disease has on patients from their perspective. It also shows which effects are important to capture in trials of therapies aimed at this patient group.

Positive Allosteric Modulators of Glycine Receptors and Their Potential Use in Pain Therapies.

Glycine receptors are ligand-gated ion channels that mediate synaptic inhibition throughout the mammalian spinal cord, brainstem, and higher brain regions. They have recently emerged as promising targets for novel pain therapies due to their ability to produce antinociception by inhibiting nociceptive signals within the dorsal horn of the spinal cord. This has greatly enhanced the interest in developing positive allosteric modulators of glycine receptors. Several pharmaceutical companies and research facilities have attempted to identify new therapeutic leads by conducting large-scale screens of compound libraries, screening new derivatives from natural sources, or synthesizing novel compounds that mimic endogenous compounds with antinociceptive activity. Advances in structural techniques have also led to the publication of multiple high-resolution structures of the receptor, highlighting novel allosteric binding sites and providing additional information for previously identified binding sites. This has greatly enhanced our understanding of the functional properties of glycine receptors and expanded the structure activity relationships of novel pharmacophores. Despite this, glycine receptors are yet to be used as drug targets due to the difficulties in obtaining potent, selective modulators with favorable pharmacokinetic profiles that are devoid of side effects. This review presents a summary of the structural basis for how current compounds cause positive allosteric modulation of glycine receptors and discusses their therapeutic potential as analgesics. SIGNIFICANCE STATEMENT: Chronic pain is a major cause of disability, and in Western societies, this will only increase as the population ages. Despite the high level of prevalence and enormous socioeconomic burden incurred, treatment of chronic pain remains limited as it is often refractory to current analgesics, such as opioids. The National Institute for Drug Abuse has set finding effective, safe, nonaddictive strategies to manage chronic pain as their top priority. Positive allosteric modulators of glycine receptors may provide a therapeutic option.

A System for Assessing Dual Action Modulators of Glycine Transporters and Glycine Receptors.

Reduced inhibitory glycinergic neurotransmission is implicated in a number of neurological conditions such as neuropathic pain, schizophrenia, epilepsy and hyperekplexia. Restoring glycinergic signalling may be an effective method of treating these pathologies. Glycine transporters (GlyTs) control synaptic and extra-synaptic glycine concentrations and slowing the reuptake of glycine using specific GlyT inhibitors will increase glycine extracellular concentrations and increase glycine receptor (GlyR) activation. Glycinergic neurotransmission can also be improved through positive allosteric modulation (PAM) of GlyRs. Despite efforts to manipulate this synapse, no therapeutics currently target it. We propose that dual action modulators of both GlyTs and GlyRs may show greater therapeutic potential than those targeting individual proteins. To show this, we have characterized a co-expression system in Xenopus laevis oocytes consisting of GlyT1 or GlyT2 co-expressed with GlyRα1. We use two electrode voltage clamp recording techniques to measure the impact of GlyTs on GlyRs and the effects of modulators of these proteins. We show that increases in GlyT density in close proximity to GlyRs diminish receptor currents. Reductions in GlyR mediated currents are not observed when non-transportable GlyR agonists are applied or when Na+ is not available. GlyTs reduce glycine concentrations across different concentration ranges, corresponding with their ion-coupling stoichiometry, and full receptor currents can be restored when GlyTs are blocked with selective inhibitors. We show that partial inhibition of GlyT2 and modest GlyRα1 potentiation using a dual action compound, is as useful in restoring GlyR currents as a full and potent single target GlyT2 inhibitor or single target GlyRα1 PAM. The co-expression system developed in this study will provide a robust means for assessing the likely impact of GlyR PAMs and GlyT inhibitors on glycine neurotransmission.

Identification of N-acyl amino acids that are positive allosteric modulators of glycine receptors.

Glycine receptors (GlyRs) mediate inhibitory neurotransmission within the spinal cord and play a crucial role in nociceptive signalling. This makes them primary targets for the development of novel chronic pain therapies. Endogenous lipids have previously been shown to modulate glycine receptors and produce analgesia in pain models, however little is known about what chemical features mediate these effects. In this study, we characterised lipid modulation of GlyRs by screening a library of N-acyl amino acids across all receptor subtypes and determined chemical features crucial for their activity. Acyl-glycine's with a C18 carbon tail were found to produce the greatest potentiation, and require a cis double bond within the central region of the carbon tail (ω6 - ω9) to be active. At 1 µM, C18 ω6,9 glycine potentiated glycine induced currents in α3 and α3ß receptors by over 50%, and α1, α2, α1ß and α2ß receptors by over 100%. C18 ω9 glycine (N-oleoyl glycine) significantly enhance glycine induced peak currents and cause a dose-dependent shift in the glycine concentration response. In the presence of 3 µM C18 ω9 glycine, the EC5o of glycine at the α1 receptor was reduced from 17 µM to 10 µM. This study has identified several acyl-amino acids which are positive allosteric modulators of GlyRs and make promising lead compounds for the development of novel chronic pain therapies.

Renal Transplantation Is Associated with Increased Complications Following Spinal Fusion Operations: Analysis of a National Database.

BACKGROUND: Improved postoperative care for renal transplant recipients has advanced both duration and overall quality of life. However, degenerative spinal pathology is increasingly prevalent after transplant. Outcomes following spinal fusion among the renal transplant population in the United States are rarely addressed. METHODS: The Healthcare Cost and Utilization Project National Inpatient Sample database was employed. Cases in years 2008-2014 for patients ≥18 years old receiving spinal fusion, exploration/decompression, and/or spinal revision/re-fusion surgeries were included. Cases were divided into kidney transplant recipients (KTR) and non-kidney transplant recipients. Complications, demographics, and socioeconomic outcomes were compared between cohorts. RESULTS: Of 579,726 patients who met inclusion criteria, 685 (0.1%) were KTRs. The KTR population was older and included more men compared with the non-kidney transplant recipient population (60.1 years vs. 56.6 years, P < 0.001; 58% male vs. 45.5% male, P < 0.001). KTRs experienced higher total complication rates (29.8% vs. 18.9%, P < 0.001). Prevalence of acute posthemorrhagic anemia and need for transfusion was markedly higher for KTRs (15.8% vs. 9.1%, P < 0.001; 13.6% vs. 6.2%, P < 0.001). Multivariate analysis revealed longer length of stay (median 1.23 days, interquartile range 0.94-1.53, P < 0.001), lower routine discharge (odds ratio = 0.57, 95% confidence interval 0.48-0.69, P < 0.001), and higher discharge to alternative care facilities (odds ratio = 1.91, 95% confidence interval 1.57-2.33, P < 0.001) for KTRs. The inpatient course for KTRs undergoing spinal operations was significantly costlier ($87,445 vs. $71,589, P < 0.001). CONCLUSIONS: History of renal transplant was associated with increased inpatient medical and socioeconomic complications following spinal fusion. Physicians and patients must understand and respect the potentially increased perioperative challenges facing KTRs.

Age Effects in Sequence-Construction for a Continuous Cognitive Task: Similar Sequence-Trends but Fewer Switch-Points.

OBJECTIVES: Many real-life settings require decision makers to sort a predetermined set of outcomes or activities into a preferred sequence and people vary in whether they prefer to tackle the most challenging aspects first, leave them for the last, or intersperse them with less challenging outcomes. Prior research on age differences in sequence-preferences has focused on discrete and hypothetical events. The present study expands this work by examining sequence-preferences for a realistic, continuous, sustained, and cognitively challenging task. METHODS: Participants (N = 121, aged 21-86) were asked to complete 10 min of a difficult cognitive task (2-back), 10 min of an easy cognitive task (1-back), and 10 min of rest over the course of a 30-min interval. They could complete the tasks in any order and switch tasks as often as they wished and they were rewarded for correct performance. Additional measures included affective and physiological responses, task accuracy, time-perspective, and demographics. RESULTS: The majority of participants constructed sequences with decreasing task difficulty. Preferences for the general trend of the sequence were not significantly related to age, but the number of switches among the tasks decreased with age, and task-switching tended to incur greater accuracy decrements among older as compared to younger adults. DISCUSSION: We address potential methodological concerns, discuss theoretical implications, and consider potential real-life applications.