Differences In Receptor Binding Affinity Research Articles

Nanomechanical collective vibration of SARS-CoV-2 spike proteins.

The development of effective therapeutics against COVID-19 requires a thorough understanding of the receptor recognition mechanism of the SARS-CoV-2 spike (S) protein. Here the multidomain collective dynamics on the trimer of the spike protein has been analyzed using normal mode analysis (NMA). A common nanomechanical profile was identified in the spike proteins of SARS-CoV-2 and its variants. The profile involves collective vibrations of the receptor-binding domain (RBD) and the N-terminal domain (NTD), which may mediate the physical interaction process. Quantitative analysis of the collective modes suggests a nanomechanical property involving large-scale conformational changes, which explains the difference in receptor binding affinity among different variants. These results support the use of intrinsic global dynamics as a valuable perspective for studying the allosteric and functional mechanisms of the S protein. This approach also provides a low-cost theoretical toolkit for screening potential pathogenic mutations and drug targets.

Prostaglandin E2 Exerts Biphasic Dose Response on the PreBötzinger Complex Respiratory-Related Rhythm.

Inflammation in infants can cause respiratory dysfunction and is potentially life-threatening. Prostaglandin E2 (PGE2) is released during inflammatory events and perturbs breathing behavior in vivo. Here we study the effects of PGE2 on inspiratory motor rhythm generated by the preBötzinger complex (preBötC). We measured the concentration dependence of PGE2 (1 nM-1 μM) on inspiratory-related motor output in rhythmic medullary slice preparations. Low concentrations (1–10 nM) of PGE2 increased the duration of the inspiratory burst period, while higher concentrations (1 μM) decreased the burst period duration. Using specific pharmacology for prostanoid receptors (EP1-4R, FPR, and DP2R), we determined that coactivation of both EP2R and EP3R is necessary for PGE2 to modulate the inspiratory burst period. Additionally, biased activation of EP3 receptors lengthened the duration of the inspiratory burst period, while biased activation of EP2 receptors shortened the burst period. To help delineate which cell populations are affected by exposure to PGE2, we analyzed single-cell RNA-Seq data derived from preBötC cells. Transcripts encoding for EP2R (Ptger2) were differentially expressed in a cluster of excitatory neurons putatively located in the preBötC. A separate cluster of mixed inhibitory neurons differentially expressed EP3R (Ptger3). Our data provide evidence that EP2 and EP3 receptors increase the duration of the inspiratory burst period at 1–10 nM PGE2 and decrease the burst period duration at 1 μM. Further, the biphasic dose response likely results from differences in receptor binding affinity among prostanoid receptors.

Vascular and molecular pharmacology of the metabolically stable CGRP analogue, SAX

The main purpose of this study was to compare in vitro pharmacological properties of human αCGRP (CGRP) and a recently discovered metabolically stable CGRP analogue, SAX, in isolated rat and human artery segments.In rat, CGRP and SAX induced similar vasodilatory responses in isolated mesenteric artery with the potency of SAX being lower than that of CGRP (vasodilatory pEC50 8.2 ± 0.12 and 9.0 ± 0.11, respectively). A corresponding difference in receptor binding affinity of SAX and CGRP was determined in rat cerebral membranes (pKi 8.3 ± 0.19 and 9.3 ± 0.14, respectively). CGRP and SAX-induced vasodilation was antagonised with similar potencies by the CGRP receptor antagonist BIBN4096BS supporting a uniform receptor population for the agonists.In human tissue, SAX and CGRP induced similar pharmacological responses with different potencies in subcutaneous artery (vasodilatory pEC50 8.8 ± 0.18 and 9.5 ± 0.13, respectively) and human recombinant receptors (cAMP signalling pEC50 9.1 ± 0.16 and 10.2 ± 0.19). Like in the rat mesenteric artery, both SAX and CGRP-responses were inhibited by the CGRP receptor antagonist BIBN4096BS with similar antagonistic potencies.In conclusion, all pharmacological characteristics of SAX and CGRP in human and rat sources points towards action via a uniform BIBN4096BS sensitive receptor population with the potency of SAX being 5–10 fold lower than that of CGRP.

A demonstration of analytical similarity comparing a proposed biosimilar pegfilgrastim and reference pegfilgrastim

BackgroundRecombinant human granulocyte-colony stimulating factor (G-CSF, filgrastim) is used primarily to reduce incidence and duration of severe neutropenia and its associated complications in cancer patients that have received a chemotherapy regimen. The pegylated form of filgrastim, “pegfilgrastim”, is a long-acting form that requires only a once-per-cycle administration for the management of chemotherapy-induced neutropenia. Apobiologix, a division of ApoPharma USA, Inc., and Intas Pharmaceuticals Limited have co-developed a proposed pegfilgrastim biosimilar to US-licensed pegfilgrastim. MethodsThe analytical similarity of Apobiologix pegfilgrastim and US-licensed pegfilgrastim with respect to their physicochemical profile was established using a wide range of rigorous orthogonal analytical techniques. Biological function was compared using receptor binding analyses, in vitro proliferation assays, and in vivo hematopoietic progenitor mobilization. ResultsApobiologix pegfilgrastim and the US-licensed pegfilgrastim reference product were found to be highly similar analytically with respect to molecular mass, primary, secondary and tertiary protein structures, purity, charge, and hydrophobicity. No differences in receptor binding affinity were observed, and all samples demonstrated similar in vitro and in vivo bioactivity. ConclusionThese studies provide robust evidence supporting the structural and functional similarity between Apobiologix pegfilgrastim and the US-licensed reference pegfilgrastim, and hence their biosimilarity.

Role of VGF-derived carboxy-terminal peptides in energy balance and reproduction: analysis of "humanized" knockin mice expressing full-length or truncated VGF.

Targeted deletion of VGF, a secreted neuronal and endocrine peptide precursor, produces lean, hypermetabolic, and infertile mice that are resistant to diet-, lesion-, and genetically-induced obesity and diabetes. Previous studies suggest that VGF controls energy expenditure (EE), fat storage, and lipolysis, whereas VGF C-terminal peptides also regulate reproductive behavior and glucose homeostasis. To assess the functional equivalence of human VGF(1-615) (hVGF) and mouse VGF(1-617) (mVGF), and to elucidate the function of the VGF C-terminal region in the regulation of energy balance and susceptibility to obesity, we generated humanized VGF knockin mouse models expressing full-length hVGF or a C-terminally deleted human VGF(1-524) (hSNP), encoded by a single nucleotide polymorphism (rs35400704). We show that homozygous male and female hVGF and hSNP mice are fertile. hVGF female mice had significantly increased body weight compared with wild-type mice, whereas hSNP mice have reduced adiposity, increased activity- and nonactivity-related EE, and improved glucose tolerance, indicating that VGF C-terminal peptides are not required for reproductive function, but 1 or more specific VGF C-terminal peptides are likely to be critical regulators of EE. Taken together, our results suggest that human and mouse VGF proteins are largely functionally conserved but that species-specific differences in VGF peptide function, perhaps a result of known differences in receptor binding affinity, likely alter the metabolic phenotype of hVGF compared with mVGF mice, and in hSNP mice in which several C-terminal VGF peptides are ablated, result in significantly increased activity- and nonactivity-related EE.

Differences in receptor binding affinity of several phytocannabinoids do not explain their effects on neural cell cultures

Phytocannabinoids are potential candidates for neurodegenerative disease treatment. Nonetheless, the exact mode of action of major phytocannabinoids has to be elucidated, but both, receptor and non-receptor mediated effects are discussed. Focusing on the often presumed structure–affinity-relationship, Ki values of phytocannabinoids cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), THC acid (THCA) and THC to human CB1 and CB2 receptors were detected by using competitive inhibition between radioligand [3H]CP-55,940 and the phytocannabinoids. The resulting Ki values to CB1 range from 23.5μM (THCA) to 14,711μM (CBDV), whereas Ki values to CB2 range from 8.5μM (THCA) to 574.2μM (CBDV). To study the relationship between binding affinity and effects on neurons, we investigated possible CB1 related cytotoxic properties in murine mesencephalic primary cell cultures and N18TG2 neuroblastoma cell line. Most of the phytocannabinoids did not affect the number of dopaminergic neurons in primary cultures, whereas propidium iodide and resazurin formation assays revealed cytotoxic properties of CBN, CBDV and CBG. However, THC showed positive effects on N18TG2 cell viability at a concentration of 10μM, whereas CBC and THCA also displayed slightly positive activities. These findings are neither linked to structural characteristics nor to the receptor binding affinity therewith pointing to another mechanism than a receptor mediated one.

From receptor pharmacology to improved outcomes: individualising the selection, dosing, and switching of antipsychotics

Determinants of antipsychotic selection and response include parameters of the primary disorder, psychiatric and physical comorbidities, past treatment effects, patient preferences, availability and acceptability of different treatments and formulations, as well as expected efficacy and safety. In the absence of consistent and sufficiently large efficacy differences among antipsychotics (except for clozapine in refractory patients), and in view of a greater focus on physical health, functional outcomes and quality of life, relatively more predictable differences in adverse events have become an important management consideration. Treatments are often selected because of a relatively lower propensity for extrapyramidal and sexual side effects, sedation, and, especially, the reduced development of cardiometabolic risk states and disorders. In this context, differences in receptor binding affinity, intrinsic activity and in the half life of antipsychotics are crucial to adopt adequate dosing and switching strategies. Dopamine rebound phenomena can occur when the level of dopamine blockade is not kept relatively constant during the switch process. This can happen when the new antipsychotic is underdosed or not fully absorbed, the switch is too abrupt from an antipsychotic with a short half life to one with a much longer half life, or when a dopamine antagonist is switched too fast to an antipsychotic with markedly less dopamine affinity or to a partial dopamine agonist. Intra-switch destabilization can also occur when changing too quickly from antipsychotics with strong histaminergic and/or cholinergic blockade to ones with lower affinity to these receptors. Overlapping “plateau” switch strategies and transient co-treatment with calming medications, such as benzodiazepines, have emerged as potential solutions to these rebound phenomena that can complicate the early switch period. To optimize outcomes, antipsychotic treatment should be pharmacologically informed and measurement based, combining both acute and long-term management goals and balancing efficacy and adverse effect considerations.

Pharmacokinetic/pharmacodynamic evaluation of urinary cortisol suppression after inhalation of fluticasone propionate and mometasone furoate

Fluticasone propionate (FP) and mometasone furoate (MF) are inhaled corticosteroids that possess a high ratio of topical to systemic activity. The systemic bioavailability of MF has been claimed to be minimal (1%). FP has been shown to exhibit the same degree of systemic effects, but its systemic availability is between 13 and 17%. We hypothesize that FP and MF have comparable systemic availabilities that can explain their potential to cause systemic effects. Steady-state FP and MF trough plasma samples were determined from a clinical study by Fardon et al. in patients with persistent asthma (forced expiratory volume in 1 s = 91%). The percent plasma protein binding of FP and MF was measured using ultracentrifugation. Free FP plasma concentrations were normalized for their differences in receptor binding affinity compared with MF and linked to overnight urinary cortisol/creatinine with an inhibitory E(max). A plot of steady-state FP and MF total trough plasma concentrations vs. dose showed that both drugs exhibit dose linearity. MF has comparable bioavailability to FP based on the steady-state concentrations observed for the different doses. The free plasma concentration producing 50% of urinary cortisol suppression (IC(50)) for MF was not statistically different from the free, normalized IC(50) for FP. FP and MF have similar pulmonary deposition and the same potential to cause systemic side-effects due to their similar IC(50) values. The observed urinary cortisol suppression of FP and MF is in agreement with their systemic availability, their differences in plasma protein binding and receptor binding affinity.

Selective Serotonin 5-HT 3 Receptor Antagonists for Postoperative Nausea and Vomiting

Selective serotonin 5-HT(3) receptor antagonists have proven safe and effective for the management of postoperative nausea and vomiting. Dolasetron, granisetron, ondansetron and tropisetron selectively and competitively bind to 5-HT(3) receptors, blocking serotonin binding at vagal afferents in the gut and in the regions of the CNS involved in emesis, including the chemoreceptor trigger zone and the nucleus tractus solitarii. Despite their shared mechanism of action, 5-HT(3) receptor antagonists have different chemical structures and exhibit differences in receptor binding affinity, dose response and duration of effect. Furthermore, although dolasetron, granisetron, ondansetron and tropisetron are all extensively metabolised by the cytochrome P450 (CYP) system, different components of this system predominate in the metabolism of each of these agents. Hence, although these agents are considered equally effective in the overall population, their pharmacokinetic and pharmacodynamic differences may explain the variability in individual responses to these drugs. This review discusses the pharmacological profiles of dolasetron, granisetron, ondansetron and tropisetron, and the clinical implications of differences in their profiles.

Establishing a therapeutic index for the inhaled corticosteroids: Part I: Pharmacokinetic/pharmacodynamic comparison of the inhaled corticosteroids

The inhaled corticosteroids contain physicochemical differences that alter both glucocorticoid receptor-binding characteristics and the pharmacokinetic variables of these drugs. Differences in receptor-binding affinity translate into differences in potency for different drugs. Differences in pharmacokinetics, however, determine the topical effect to systemic effect ratio, or the “pulmonary targeting” of the drug. Beneficial pharmacokinetic properties that may improve pulmonary targeting include low oral bioavailability, rapid systemic clearance, and slow absorption from the lung. Delivery devices can produce clinically significant differences in topical activity by altering the dose deposited in the lung and, for orally absorbed drugs, the amount deposited in the oropharynx and swallowed. Clinical trials have confirmed that differences in potency or drug delivery of 2-fold or more can be detected in patients with asthma. However, because of the relatively flat nature of the dose-response curve for morning peak expiratory flow and forced expiratory volume in 1 second, the trials must be adequately powered and well controlled. The use of bronchial provocation measures are problematic because of the prolonged lag time for response. Study design flaws can lead to misinterpretation of results. Clinical studies have indicated the following relative potency differences: fluticasone propionate > budesonide = beclomethasone dipropionate > triamcinolone acetonide = flunisolide. Current evidence suggests that potency differences can be overcome by giving larger doses of the less potent drug. However, because of these potency differences, studies of systemic effects should not be done in isolation of adequate topical activity studies to define the pulmonary targeting of the drugs. (J Allergy Clin Immunol 1998;102:S36-S51)

Differences in temperature-sensitive receptor binding of glucocorticoids in spontaneously hypertensive and normotensive Wistar-Kyoto rats

Glucocorticoid receptor binding was compared in liver cytosol preparations from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats using homologous displacement of [ 3H]dexamethasone. At 5°C, there was no difference in receptor binding affinity or concentration between strains for dexamethasone, corticosterone or aldosterone. At 37°C, affinity for dexamethasone was lower than at 5°C for both rat strains and decreased with time. However, at this higher temperature, binding affinity in the SHR preparation was consistently higher than in the WKY preparation. The WKY preparation had a higher receptor concentration. The rate of dissociation of the [ 3H]dexamethasone-receptor complex prepared at 5°C and then incubated at 37°C was rapid but not different between strains. A possible explanation of these results is that the relationship of the heat shock proteins to the receptor heterocomplex is different between strains. Evidence exists of a genetic difference in Hsp 70 between SHR and WKY rats, although its cosegregation with blood pressure has not been established.

Alteration of insulin-receptor kinase activity by high-fat feeding.

It has been demonstrated in in vivo and in vitro experiments that high-fat (HF) feeding causes insulin resistance. To elucidate the mechanism for this effect, we have measured the kinase activity of the insulin receptor purified from livers of HF-fed rats that showed impaired insulin action in isolated rat adipocytes. In adipocyte experiments, HF feeding led to a 65% decrease in the maximal response stimulated by insulin in a 2-deoxyglucose uptake study. Although insulin binding to adipocytes of HF-fed rats also decreased to 50% of control due to decreased binding affinity, the postbinding defect should be accounted for by decreased insulin action in view of the presence of spare receptor. In contrast to adipocytes, insulin binding to the lectin-purified insulin receptor from livers showed no difference in receptor-binding affinity between HF-fed and control rats. Insulin-stimulated phosphorylation of the beta-subunit of the insulin receptor was decreased to almost 50% throughout the entire dose-response curve. The study of glutamine-tyrosine (4:1) phosphorylation by the insulin-receptor kinase showed results similar to those of the autophosphorylation study. These results suggest that an HF diet causes insulin resistance by affecting insulin-receptor kinase, which plays an important role in transmembrane signaling between insulin binding and insulin action.

The molecular structure of 16α-iodo-17β-estradiol, a high affinity ligand for the estrogen receptor

16α-Iodoestradiol is an estrogenic steroid with high affinity for the estrogen receptor. When labelled with a gamma emitting isotope, such as 125I, the resulting radioactive steroid is an excellent ligand for the sensitive analysis of the estrogen receptor. The X-ray study of the crystal structure of 16α-iodoestradiol reveals that the bond distances and angles of the iodinated estrogen are similar to those of estradiol and estriol. The substitution of iodine for the 16α-hydroxyl group has little effect on geometric and electronic properties of the molecule and does not interfere with the hydrogen bonding ability of the 17β-hydroxyl group. The difference in receptor binding affinity between 16α-estradiol and estriol may be due to competition for intermolecular hydrogen bond formation between the hydroxyls at C(16) and C(17) on estriol. X-Ray data indicate that the 16-hydroxyl can form significantly shorter and presumably stronger hydrogen bonds.

Receptor binding and biological potency of several split forms (conversion intermediates) of human proinsulin. Studies in cultured IM-9 lymphocytes and in vivo and in vitro in rats.

The biological activities of several derivatives of human proinsulin (HPI) containing peptide bond cleavages or peptide deletions in the connecting peptide region were examined in vivo in rats and in several in vitro systems. The two derivatives which were tested in vivo, split (32-33)HPI and des-(64,65)HPI, both demonstrated greater potency in lowering blood glucose than did intact HPI. The receptor binding affinities of split (65-66)HPI, des-(57-65)HPI, des-(64,65)HPI, des-(33-56)HPI, des-(31,32)HPI, split (32-33)HPI, and split (56-57)HPI were examined in cultured IM-9 lymphocytes, freshly isolated rat adipocytes, and purified rat liver membranes and were compared to the binding of intact HPI and insulin. In these systems, HPI averaged approximately 1% of the activity of insulin. Modification of proinsulin in the connecting peptide region near the A-chain of insulin to form split (65-66)HPI, des-(57-65)HPI, des-(64,65)HPI, or des-(33-56)HPI resulted in an increase in affinity for receptor binding ranging from 11 to 27-fold over that of intact HPI. In contrast, modifications near the B-chain of insulin to form either des-(31,32)HPI or split (32-33)HPI resulted in roughly a 5-fold increase in affinity, whereas a cleavage within the connecting peptide to form split (56-57)HPI showed only a 2-fold increase in affinity as compared to intact HPI. The biological potencies of these materials were examined in isolated rat adipocytes. At high concentrations (10(-7) M), each derivative produced the same maximal response. At lower concentrations, differences in the relative potencies paralleled the differences in receptor binding affinity previously noted.