Structural requirements for binding to the δ opioid receptor: Alkyl replacements at the third residue of deltorphin I

The naturally occurring heptapeptide deltorphin I (Tyr- d -Ala-Phe-Asp-Val-Val-Gly-NH 2 ) exhibits extremely high affinity and selectivity for the δ opioid receptor. In an ongoing investigation of the features of this compound that confer these properties, seven new analogs of the peptide, in which phenylalanine at position three was replaced with amino acids containing alkyl side chains, were synthesized and tested for binding to μ, δ, and κ opioid receptors. These substitutions, including tert -leucine, tert -butylalanine, α-aminobutyric acid, norvaline, norleucine, β-cyclopentylalanine and octahydroindole-2-carboxylic acid, assessed the importance of aromaticity and lipophilicity/steric distribution of the side chain at this position in the binding interaction. Findings indicated that: (i) aromaticity at position three is not required for binding, and (ii) hydrophobic character, size, steric distribution and conformational flexibility influence affinity at the δ receptor. The data suggest that substitutions at the β-carbon of this residue disrupt the binding conformation of the peptide and possibly provide adverse steric effects.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43173/1/10989_2004_Article_BF00142239.pd

COVID-19 trajectories among 57 million adults in England: a cohort study using electronic health records

BACKGROUND: Updatable estimates of COVID-19 onset, progression, and trajectories underpin pandemic mitigation efforts. To identify and characterise disease trajectories, we aimed to define and validate ten COVID-19 phenotypes from nationwide linked electronic health records (EHR) using an extensible framework. METHODS: In this cohort study, we used eight linked National Health Service (NHS) datasets for people in England alive on Jan 23, 2020. Data on COVID-19 testing, vaccination, primary and secondary care records, and death registrations were collected until Nov 30, 2021. We defined ten COVID-19 phenotypes reflecting clinically relevant stages of disease severity and encompassing five categories: positive SARS-CoV-2 test, primary care diagnosis, hospital admission, ventilation modality (four phenotypes), and death (three phenotypes). We constructed patient trajectories illustrating transition frequency and duration between phenotypes. Analyses were stratified by pandemic waves and vaccination status. FINDINGS: Among 57 032 174 individuals included in the cohort, 13 990 423 COVID-19 events were identified in 7 244 925 individuals, equating to an infection rate of 12·7% during the study period. Of 7 244 925 individuals, 460 737 (6·4%) were admitted to hospital and 158 020 (2·2%) died. Of 460 737 individuals who were admitted to hospital, 48 847 (10·6%) were admitted to the intensive care unit (ICU), 69 090 (15·0%) received non-invasive ventilation, and 25 928 (5·6%) received invasive ventilation. Among 384 135 patients who were admitted to hospital but did not require ventilation, mortality was higher in wave 1 (23 485 [30·4%] of 77 202 patients) than wave 2 (44 220 [23·1%] of 191 528 patients), but remained unchanged for patients admitted to the ICU. Mortality was highest among patients who received ventilatory support outside of the ICU in wave 1 (2569 [50·7%] of 5063 patients). 15 486 (9·8%) of 158 020 COVID-19-related deaths occurred within 28 days of the first COVID-19 event without a COVID-19 diagnoses on the death certificate. 10 884 (6·9%) of 158 020 deaths were identified exclusively from mortality data with no previous COVID-19 phenotype recorded. We observed longer patient trajectories in wave 2 than wave 1. INTERPRETATION: Our analyses illustrate the wide spectrum of disease trajectories as shown by differences in incidence, survival, and clinical pathways. We have provided a modular analytical framework that can be used to monitor the impact of the pandemic and generate evidence of clinical and policy relevance using multiple EHR sources. FUNDING: British Heart Foundation Data Science Centre, led by Health Data Research UK

Structural requirements for substitutions in delta opioid receptor selective, cyclic tetrapeptide dermorphin analogs: Divergence from related pentapeptide enkephalin analogs.

The conformationally restricted, delta opioid receptor selective tetrapeptide H-Tyr-D-$\rm C\overline{ys-Phe-{\it D}-Pe}nOH,$ cyclized by disulfide formation between side chains of residues 2 and 4, was modified at each residue to allow comparison with the cyclic disulfide-containing pentapeptide analogs of (D-Pen\sp2, D-Pen\sp5) enkephalin (DPDPE). Analogs of the template tetrapeptide were designed to explore the biological consequences of substitution of amino acids with varying steric, electronic, lipophilic, and/or chiral properties at each residue. Effects on binding affinity and selectivity at mu and delta opioid receptors and on bioassay potency were evaluated. Analogs investigating the effect of the orientation and the nature of side chain aromatic functional moieties were prepared. Modifications which were expected to influence backbone and side chain conformation at each residue, as well as variation in the cyclic portion of the molecule, also were examined. Data indicated that structure-activity relationships developed for pentapeptide enkephalins often were inapplicable to the tetrapeptide series. In general, where direct comparisons were possible, structural requirements for the Phe\sp3 residue in this tetrapeptide series were less stringent than in the related pentapeptide series. These findings suggest possible differences in the manner in which the Phe\sp3 residue of dermorphin-like tetrapeptides and the Phe\sp4 residue of enkephalin-like pentapeptides interact at delta opioid receptors; however, other interpretations are not excluded. Distinctions between the series in elements of the binding interaction also were notable at other residues. These peptides lack the central glycine residue of enkephalin analogs, therefore resembling dermorphin, and can be expected to possess a higher degree of rigidity and structural definition, an effect to which results may be attributed. They thereby represent a more credible series for analysis of the structural and conformational features required for peptide ligand binding at the opioid receptor types. In addition, comparison of the effects of analogous modification between the cyclic tetrapeptides and the restricted pentapeptides allows examination of the proposal that different subsites of the delta receptor may be involved in the binding of the two series.Ph.D.Pharmacy sciencesPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128749/2/9135607.pd

Opioid receptor affinity and selectivity effects of second residue and carboxy terminal residue variation in a cyclic disulfide-containing opioid tetrapeptide

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74202/1/j.1399-3011.1991.tb00274.x.pd

IGFBP-3 blocks hyaluronan-CD44 signaling, leading to increased acetylcholinesterase levels in A549 cell media and apoptosis in a p53-dependent manner

Insulin-like growth factor binding protein-3 (IGFBP-3) belongs to a family of six IGF binding proteins. We previously found that IGFBP-3 exerts its cytotoxic effects on A549 (p53 wild-type) cell survival through a mechanism that depends on hyaluronan-CD44 interactions. To shed light on the mechanism employed, we used CD44-negative normal human lung cells (HFL1), A549, and H1299 (p53-null) lung cancer cells. A synthetic IGFBP-3 peptide (215-KKGFYKKKQCRPSKGRKR-232) but not the mutant (K228AR230A), was able to bind hyaluronan more efficiently than the analogous sequences from the other IGFBPs. In a manner comparable to that of the IGFBP-3 protein, the peptide blocked hyaluronan-CD44 signaling, and more effectively inhibited viability of A549 cells than viability of either H1299 or HFL1 cell lines. Treatment with the IGFBP-3 protein or its peptide resulted in increased acetylcholinesterase concentration and activity in the A549 cell media but not in the media of either HFL1 or H1299, an effect that correlated with increased apoptosis and decreased cell viability. These effects were diminished upon the same treatment of A549 cells transfected with either p53 siRNA or acetylcholinesterase siRNA. Taken together, our results show that IGFBP-3 or its peptide blocks hyaluronan-CD44 signaling via a mechanism that depends on both p53 and acetylcholinesterase

Biochemical determinants of the IGFBP‐3–hyaluronan interaction

IGFBP-3, the most abundant IGFBP and the main carrier of insulin-like growth factor I (IGF-I) in the circulation, can bind IGF-1 with high affinity, which attenuates IGF/IGF-IR interactions, thereby resulting in antiproliferative effects. The C-terminal domain of insulin-like growth factor-binding protein-3 (IGFBP-3) is known to contain an 18-basic amino acid motif capable of interacting with either humanin (HN) or hyaluronan (HA). We previously showed that the 18-amino acid IGFBP-3 peptide is capable of binding either HA or HN with comparable affinities to the full-length IGFBP-3 protein and that IGFBP-3 can compete with the HA receptor, CD44, for binding HA. Blocking the interaction between HA and CD44 reduced viability of A549 human lung cancer cells. In this study, we set out to better characterize IGFBP-3-HA interactions. We show that both stereochemistry and amino acid identity are important determinants of the interaction between the IGFBP-3 peptide and HA and for the peptide\u27s ability to exert its cytotoxic effects. Binding of IGFBP-3 to either HA or HN was unaffected by glycosylation or reduction of IGFBP-3, suggesting that the basic 18-amino acid residue sequence of IGFBP-3 remains accessible for interaction with either HN or HA upon glycosylation or reduction of the full-length protein. Removing N-linked oligosaccharides from CD44 increased its ability to compete with IGFBP-3 for binding HA, while reduction of CD44 rendered the protein relatively ineffective at blocking IGFBP-3-HA interactions. We conclude that both deglycosylation and disulfide bond formation are important for CD44 to compete with IGFBP-3 for binding HA

Interaction of amyloid beta with humanin and acetylcholinesterase is modulated by ATP

Humanin (HN) is known to bind amyloid beta (Aβ)-inducing cytoprotective effects, while binding of acetylcholinesterase (AChE) to Aβ increases its aggregation and cytotoxicity. Previously, we showed that binding of HN to Aβ blocks aggregation induced by AChE and that HN decreases but does not abolish Aβ-AChE interactions in A549 cell media. Here, we set out to shed light on factors that modulate the interactions of Aβ with HN and AChE. We found that binding of either HN or AChE to Aβ is not affected by heparan sulfate, while ATP, thought to reduce misfolding of Aβ, weakened interactions between AChE and Aβ but strengthened those between Aβ and HN. Using media from either A549 or H1299 lung cancer cells, we observed that more HN was bound to Aβ upon addition of ATP, while levels of AChE in a complex with Aβ were decreased by ATP addition to A549 cell media. Exogenous addition of ATP to either A549 or H1299 cell media increased interactions of endogenous HN with Aβ to a comparable extent despite differences in AChE expression in the two cell lines, and this was correlated with decreased binding of exogenously added HN to Aβ. Treatment with exogenous ATP had no effect on cell viability under all conditions examined. Exogenously added ATP did not affect viability of cells treated with AChE-immunodepleted media, and there was no apparent protection against the cytotoxicity resulting from immunodepletion of HN. Moreover, exogenously added ATP had no effect on the relative abundance of oligomer versus total Aβ in either cell line