Shift In Binding Affinity Research Articles

Qualification of a Biolayer Interferometry Assay to Support AZD7442 Resistance Monitoring.

AZD7442, a combination of two long-acting monoclonal antibodies (tixagevimab [AZD8895] and cilgavimab [AZD1061]), has been authorized for the prevention and treatment of coronavirus disease 2019 (COVID-19). The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants requires methods capable of quickly characterizing resistance to AZD7442. To support AZD7442 resistance monitoring, a biolayer interferometry (BLI) assay was developed to screen the binding of tixagevimab and cilgavimab to SARS-CoV-2 spike proteins to reduce the number of viral variants for neutralization susceptibility verification. Six spike variants were chosen to assess the assay's performance: four with decreased affinity for tixagevimab (F486S:D614G and F486W:D614G proteins) or cilgavimab (S494L:D614G and K444R:D614G proteins) and two reference proteins (wild-type HexaPro and D614G protein). Equilibrium dissociation constant (KD) values from each spike protein were used to determine shifts in binding affinity. The assay's precision, range, linearity, and limits of quantitation were established. Qualification acceptance criteria determined whether the assay was fit for purpose. By bypassing protein purification, the BLI assay provided increased screening throughput. Although limited correlation between pseudotype neutralization and BLI data (50% inhibitory concentration versus KD) was observed for full immunoglobulins (IgGs), the correlations for antibody fragments (Fabs) were stronger and reflected a better comparison of antibody binding kinetics with neutralization potency. Therefore, despite strong assay performance characteristics, the use of full IgGs limited the screening utility of the assay; however, the Fab approach warrants further exploration as a rapid, high-throughput variant-screening method for future resistance-monitoring programs. IMPORTANCE SARS-CoV-2 variants harbor multiple substitutions in their spike trimers, potentially leading to breakthrough infections and clinical resistance to immune therapies. For this reason, a BLI assay was developed and qualified to evaluate the reliability of screening SARS-CoV-2 spike trimer variants against anti-SARS-CoV-2 monoclonal antibodies (MAbs) tixagevimab and cilgavimab, the components of AZD7442, prior to in vitro pseudovirus neutralization susceptibility verification testing. The assay bypasses protein purification with rapid assessment of the binding affinity of each MAb for each recombinant protein, potentially providing an efficient preliminary selection step, thus allowing a reduced testing burden in the more technically complex viral neutralization assays. Despite precise and specific measures, an avidity effect associated with MAb binding to the trimer confounded correlation with neutralization potency, negating the assay's utility as a surrogate for neutralizing antibody potency. Improved correlation with Fabs suggests that assay optimization could overcome any avidity limitation, warranting further exploration to support future resistance-monitoring programs.

Development of light-responsive protein binding in the monobody non-immunoglobulin scaffold

Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools. The ability to reversibly control their binding activity to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, the development of a light-controlled monobody (OptoMB) that works in vitro and in cells and whose affinity for its SH2-domain target exhibits a 330-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. By virtue of their ability to be designed to bind any protein of interest, OptoMBs have the potential to find new powerful applications as light-switchable binders of untagged proteins with the temporal and spatial precision afforded by light.

χ-Space Screening of Dermorphin-Based Tetrapeptides through Use of Constrained Arylazepinone and Quinolinone Scaffolds.

Herein, the synthesis of novel conformationally constrained amino acids, 4-amino-8-bromo-2-benzazepin-3-one (8-Br-Aba), 3-amino-3,4-dihydroquinolin-2-one, and regioisomeric 4-amino-naphthoazepinones (1- and 2-Ana), is described. Introduction of these constricted scaffolds into the N-terminal tetrapeptide of dermorphin (i.e., H-Tyr-d-Ala-Phe-Gly-NH2) induced significant shifts in binding affinity, selectivity, and in vitro activity at the μ- and δ-opioid receptors (MOP and DOP, respectively). A reported constrained μ-/δ-opioid lead tetrapeptide H-Dmt-d-Arg-Aba-Gly-NH2 was modified through application of various constrained building blocks to identify optimal spatial orientations in view of activity at the opioid receptors. Interestingly, when the aromatic moieties were turned toward the C-terminus of the peptide sequences, (partial) (ant)agonism at MOP and weak (ant)agonism at DOP were noticed, whereas the incorporation of the 1-Ana residue led toward balanced low nanomolar MOP/DOP binding and in vitro agonism.

Modulation of vitamin D receptor and estrogen receptor by 1,25(OH)2-vitamin D3 in T-47D human breast cancer cells

1,25(OH)2-Vitamin D3 inhibits breast cancer cell proliferation through interaction with the vitamin D receptor (VDR). Regulation of VDR is under the influence of several factors which include the functional ligand for this receptor (1,25(OH)2-vitamin D3) as well as heterologous steroid hormones. We evaluated the nature of homologous regulation in T-47D human breast cancer cells with a radiolabelled ligand binding assay and a ribonuclease protection assay for VDR. Significant VDR up-regulation, as measured by hormone binding assays, occurred with pre-incubations with 10(-9)M through 10(-6)M 1,25(OH)2-vitamin D3 (P < 0.05). A 7-fold VDR up-regulation with 10(-8)M 1,25(OH)2-vitamin D3 occurred at 4 h treatment and was not associated with an increase in VDR mRNA expression on ribonuclease protection assay. This supports the hypothesis that up-regulation of VDR is probably the result of ligand-induced stabilization of pre-existing receptor. All-trans-retinoic acid, the progesterone analog R-5020, and prednisone were found to induce heterologous up-regulation of the VDR. We then determined with ligand binding assays whether 1,25(OH)2-vitamin D3 could influence receptor levels for another hormone in a manner analogous to the heterologous regulation of VDR. Regulation of estrogen receptor (ER) by 1,25(OH)2-vitamin D3 was studied in T-47D and MDA-MB-231 breast cancer cells. Incubation of T-47D cells, which are ER (+), with 10(-8)M 1,25(OH)2-vitamin D3 did not result in up-regulation of ER. Yet estrogen binding was significantly up-regulated in a cell line that is ER(-), MDA-MB-231. The increased estrogen binding was associated with a shift in binding affinity and ribonuclease protection assay showed absence of ER mRNA in these cells, suggesting an up-regulation of estrogen binding proteins and not of the ER itself.

The resolution and characterization of putative ubiquitin carrier protein isozymes from rabbit reticulocytes.

The covalent ligation of the 8.6-kDa polypeptide ubiquitin to various cellular target proteins is believed to represent a fundamental regulatory process. In this mechanism, the ATP-coupled activation and subsequent ligation of ubiquitin are catalyzed by separate enzymes (E1 and E3, respectively) functionally linked by ubiquitin carrier protein (E2). Carrier protein has been proposed to constitute a family of isozymes having molecular masses of 14, 17, 20, 24, and 32 kDa whose role is to shuttle activated polypeptide in the form of a high-energy thiol ester intermediate to the carboxyl terminus of ubiquitin. Using a combination of covalent affinity and high performance liquid chromatographic methods, the pututive E2 isozymes have been purified to apparent homogeneity. The E2(14kDa) isozyme resolved into two forms differing in net charge at pH 7.5. All of the E2 isozymes contained only one thiol ester site except for E2(17kDa) and E2(20kDa) which were capable of forming two such adducts per subunit. Thiol ester formation was rapid for the E2 isozymes and required the presence of activating enzyme. In contrast, the reverse reaction of thiol ester transfer from E2 to E1 was kinetically significant for only E2(14kDa), E2(20kDa), and E2(24kDa). The stability of E2(17kDa) and E2(32kDa) to such trapping may reflect a marked shift in binding affinity to E1 upon thiol ester formation. In addition, differential rates for thiol ester formation to each subunit of dimeric E2(14kDa) was also noted. The E2(14kDa) isoforms were approximately 10-fold more active in E3-dependent ubiquitin-protein ligation than either E2(20kDa) or E2(32kDa). Neither E2(17kDa) nor E2(24kDa) supported this reaction. In addition, the thiol ester formed to E2(14kDa) was inherently more reactive since its second order rate constant for the E3-independent transfer of ubiquitin to the small molecular weight nucleophile dithiothreitol was an order of magnitude greater than found for the other isozymes. If these proteins constitute a family of isozymes, they exhibit considerable catalytic diversity.

Guanine nucleotide-induced shift in binding affinity for β-adrenergic agonists in rat reticulocyte and turkey erythrocyte membranes

Guanosine triphosphate (GTP) is important for the expression of catecholamine-dependent adenylate cyclase activity in a wide variety of membrane preparations, including those obtained from rat reticulocytes and turkey erythrocytes. Another generally recognized effect of GTP—to decrease the binding affinity of β-adrenergic agonists for β receptor sites—has not been demonstrable, until recently, in these two membrane preparations. The present study was designed to evaluate possible guanine nucleotide effects on agonist binding under clearly defined experimental conditions. In the absence of magnesium and EDTA, the approximate binding constant K D(app) , for isoproterenol did not change significantly in the presence of the GTP analogue, guanyl-5′-yl-imidodiphosphate [Gpp(NH)p], for either rat reticulocytes (14.0 ± 3.6 vs 19.5 ± 1.8 μM; P > 0.10) or turkey erythrocytes (2.66 ± 0.92 vs 2.33 ± 0.07 μM; P > 0.10). When magnesium (10 mM) and EDTA (1 mM) were both present, the K D(app) for isoproterenol improved 23-fold in the rat reticulocyte to 0.62 ± 0.1 μM (P < 0.001) and 9-fold in the turkey erythrocyte to 0.30 ± 0.09 μM (P < 0.001). The effect of magnesium to improve binding affinity required EDTA and was dose-dependent, with the half-maximal decrease in the K D(app) for isoproterenol occurring at a magnesium concentration of 0.1 mM. When binding experiments were conducted in the presence of magnesium and EDTA, Gpp(NH)p caused a marked decrease in agonist affinity in membranes of rat reticulocytes (0.62 ± 0.10 to 9.3 ± 3.3 μM; P < 0.001) and in turkey erythrocytes (0.30 ± 0.09 to 0.91 ± 0.16 μM; P < 0.001). These results demonstrate in turkey and rat erythroid cell membranes that optimal binding affinity for isoproterenol requires magnesium and EDTA and that, under these conditions, both membrane preparations are markedly influenced by guanine nucleotides.

The influence of antigen on a model for analysis of multiple coordinate events associated with the primary immune response

The influence of sheep red blood cells (SRBC), a T-dependent antigen, was analyzed in an experimental model of multiple coordinate functions of the immune response. The influence of antigen dose on the kinetics with which splenic B and T antigen-binding lymphocytes (ABL) and plaque-forming cells (PFC) developed, and the relative ratio of each of these cellular subsets were analyzed during the primary immune response to SRBC. As a function of increased antigen dose: 1) the total number of ABL increased, with relative increases on day 3 < day 5 > day 7; 2) the number of direct PFC increased, with relative antigen-dependent increases on day 3 > day 5 > day7; and 3) mean PFC lytic plaque volume progressively decreased. At low antigen doses, T ABL preferentially increased relative to B ABL, whereas at high antigen doses the reverse was observed. Thus, the increased capacity for a secondary response after a primary immunization at low antigen doses could be a consequence of the greater numbers of T ABL induced by small quantities of antigen. Both the rate of proliferation of ABL and their differentiation to PFC were influened positively by increasing doses of antigen; however, the ratio of PFC to ABL was higher at low antigen doses, suggesting that the relative rate of differentiation of ABL to PFC was greater than the rate of proliferation of ABL under conditions of limited antigen dose. Mean lytic plaque volume decreased slightly with time and markedly with increasing antigen dose. This could reflect shifts in binding affinity, mean antibody secreted per antibody-forming cell, or both. Whereas each of these parameters have been separately reported, the coordinate use of each provides a more critical analysis of each of the antigen-specific cell populations that participate in a response and their modification by immunoregulatory factors or events.

Regulation of beta-adrenergic receptors by guanyl-5'-yl imidodiphosphate and other purine nucleotides.

Guanyl-5'-yl imidodiphosphate (Gpp(NH)p), GTP, and other purine nucleotides selectively decrease the binding affinity of the beta-adrenergic receptors of frog erythrocyte membranes for beta-adrenergic agonists but not antagonists. Shifts in binding affinity were assessed by determining the ability of unlabeled ligands to compete with (-)-[3H]dihydroalprenolol for the membrane-bound receptors. The magnitude of the"right" shift in the binding displacement curve for any of 13 ligands tested was directly related to the intrinsic activity (maximal stimulatory capacity) of that agent for stimulation of the frog erythrocyte membrane adenylate cyclase. Thus, Gpp(NH)p-induced shifts in binding affinity were greatest for full agonists such as isoproterenol, intermediate for partial agonists such as soterenol, and no shifts were observed for antagonists such as propranolol. Shifts in binding affinity were observed only in preparations where agonist binding to the receptors leads to "coupling" of the receptors with adenylate cyclase. In solubilized preparations where the beta-adrenergic receptors and adenylate cyclase are functionally "uncoupled", Gpp(NH)p did not cause right shifts in agonist receptor binding displacement curves. In particulate preparations the Km of Gpp(NH)p for stimulation of adenylate cyclase was identical with that for its effect on beta-adrenergic agonist binding affinity, 1 to 2 muM. Moreover, the ability of several other nucleotides to cause shifts in receptor binding affinity directly paralleled their previously determined affinities for the nucleotide regulatory sites on adenylate cyclase. Gpp(NH)p also shifted agonist dose-response curves for stimulation of adenylate cyclase, but to the left. As with the effects on the receptor binding curves, the effects of Gpp(NH)p on the "apparent affinities" of agonists for enzyme stimulation were directly related to their intrinsic activities. Gpp(NH)p also markedly increased the intrinsic activity of partial agonists. These results appear to indicate that conformational alterations in adenylate cyclase caused by occupation of nucleotide regulatory sites by Gpp(NH)p are capable of inducing alterations in the beta-adrenergic receptors. These receptor alterations are induced only when the receptors are "coupled" to the enzyme by virtue of agonist binding. The nucleotide-altered conformation of the beta-adrenergic receptors is characterized by decreased binding affinity for agonist but increased functional efficacy in stimulating the enzyme.