An integrated system for optical biomolecular interaction analysis

Prolonging the circulatory half-life of low mass protein drugs can be achieved either by administration of a pro-drug or through co-injection with a carrier protein that will slowly release the active protein. The rate of release is concentration and affinity dependent. The optimal relationship between these two in prolonging the half-life of a pro-drug is the focus of this work. Interferon (IFN) ␤ is one of the most widely used protein drugs in the clinic. Here, we show that the circulatory half-life of IFN␤ can be significantly extended by coadministration with the extracellular domain of the IFN receptor ifnar2 (ifnar2-EC). To investigate the concentration/affinity relation, a range of tighter binding ifnar2-EC mutants was designed that bind IFN␤, but not IFN␣2, up to 50-fold tighter compared with the wild-type ifnar2-EC. This increased affinity is related to a slower dissociation rate, whereas the association of IFN␤ with ifnar2-EC is already near optimum. Using the wild-type and mutant receptors, we investigated their potential in occluding IFN␤ from circulation in a tissue culture assay, as well as in rats. To determine the potential of ifnar2-EC as a carrier protein, we co-administered a mixture of IFN␤ and ifnar2-EC to rats both intravenously and subcutaneously, and followed the blood plasma concentrations of IFN␤ over time. The tighter binding ifnar2-EC mutant had a clear advantage in prolonging the half-life of IFN␤ in circulation, even when lower protein concentrations were administered. A numerical simulation of the in vivo data demonstrates that the optimal binding affinity of a carrier protein is around the concentration needed to obtain optimal activity of the ligand.The occurrence of soluble proteins corresponding to the extracellular domains of transmembrane receptors and adhesion molecules in biological fluids are widely spread. These proteins retain their binding characteristics and typically function as natural antagonists, carrier molecules, or chaperones, protecting their ligands from proteolytic degradation; in some cases, they are biological agonists (1-5). The cellular release of these soluble receptors is tightly regulated. It is typically controlled by one of two mechanisms. First, differential mRNA splicing that leads to the production and secretion of only the extracellular domain (i.e. interleukin 4, epidermal growth factor, and ifnar2 (6 -8)). Second, specific proteolysis of the receptor from the cell surface (i.e. interleukin 1, interleukin 2, and tumor necrosis factor-␣) (7).If binding is tight and discharge is slow, binding proteins maintain the cytokines in circulation, but inactive, resulting in an antagonistic behavior. However, the line between antagonist and agonist is blurred, and the same soluble receptor can act as both, depending on the circumstances (7). The factors that most influence the role of soluble receptors as agonists or antagonists are the concentrations of the ligands, binding proteins, and unbound cytokines required for activity. The exact definition of qua...

Section: Methodsmentioning

confidence: 99%

Optimizing the Binding Affinity of a Carrier Protein

Peleg-Shulman

Roisman

Zupkovitz

et al. 2004

“…This method detects biomolecular interaction at interfaces as a change in the apparent optical thickness of a thin silica layer. The complete experimental setup for this methodology has been previously described in detail (20,21). Binding to the surface is monitored as a shift in the interference spectrum.…”

Section: Methodsmentioning

confidence: 99%

New Structural and Functional Aspects of the Type I Interferon-Receptor Interaction Revealed by Comprehensive Mutational Analysis of the Binding Interface

Piehler¹,

Roisman

Schreiber

2000

Self Cite

140

163

Type I interferons bind to two cell surface receptors, ifnar1 and ifnar2, as the first step in the activation of several signal transduction pathways that elicit an antiviral state and an anti-proliferative response. Here, we quantitatively mapped the complete binding region of ifnar2 on interferon (IFN)␣2 by 35 individual mutations to alanine and isosteric residues. Of the six "hot-spot" residues identified (Leu-30, Arg-33, Arg-144, Ala-145, Met-148, and Arg-149), four are located on the E-helix, which is located at the center of the binding site flanked by residues on the A-helix and the AB-loop. The contribution of residues of the D-helix, which have been previously implicated in binding, proved to be marginal for the interaction with the extracellular domain of ifnar2. Interestingly, the ifnar2 binding site overlaps the largest continuous hydrophobic patch on IFN␣2. Thus, hydrophobic interactions seem to play a significant role stabilizing this interaction, with the charged residues contributing toward the rapid association of the complex. Relating the anti-viral and anti-proliferative activity of the various interferon mutants with their affinity toward ifnar2 results in linear function over the whole range of affinities investigated, suggesting that ifnar2 binding is the rate-determining step in cellular activation. Dose-time analysis of the anti-viral response revealed that shortening the incubation time of low-level activation cannot be compensated by higher IFN doses. Considering the strict dependence of the cellular response on affinity, these results suggest that for maintaining transcription of IFN-responsive genes over a longer time period, low but continuous signaling through the IFN receptor is essential. Type I interferons (IFN)1 are a family of homologous cytokines, which potently elicit an anti-viral and anti-proliferative state in cells. All human type I IFNs (IFN␣, ␤, , and ) bind to a cell surface receptor consisting of two transmembrane proteins, ifnar1 (1) and ifnar2 (2), which associate upon binding. Binding of IFN to its receptor mediates activation of numerous genes (3) through different signal transduction pathways (reviewed in Ref. 4). Intriguingly, different expression profiles are induced by different IFNs, apparently through the same receptor, with particular differences between IFN␣ and IFN␤ (3, 5). However, the molecular mechanisms underlying the functional differences between IFNs are still unknown. Type I IFNs belong to the class of helical cytokines (6) and are built by five helices. The structure of several type I IFNs have been resolved, such as murine IFN␤ (7), human IFN␣2 (8, 9), human IFN␤ (10), and ovine IFN (11). Mutational studies have revealed functionally important residues on IFNs (reviewed in Ref. 12), which can be ascribed to distinct functional epitopes that interact with ifnar1 and ifnar2 (13, 14). The binding site for ifnar2 was mainly mapped on the AB-loop and D-helix and the binding site for ifnar1 on the C-helix. However, comparison with homologous cytokines...

“…Binding of small molecules, such as BMS-791325, to the enzyme causes a shift in the interference pattern of incidence light that can be measured in real time (43,44). Both association and dissociation of BMS-791325 to/from WT, L30S, and P495L NS5B enzymes could be measured using this method.…”

Section: Table 2 Effect Of Bms-791325-ns5b Polymerase Preincubation Tmentioning

confidence: 99%

Mechanism of Inhibition for BMS-791325, a Novel Non-nucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase

Rigat

Lü

Wang

et al. 2014

Background: BMS-791325, a non-nucleoside inhibitor of HCV NS5B, has robust clinical efficacy. Results: Biochemical and biophysical methods revealed a non-competitive time-dependent inhibition mechanism and permitted complete parameterization of inhibitor binding kinetics. Conclusion: Thumb and finger variants affect BMS-791325 association rates. Significance: The impact of NS5B variants on BMS-791325 binding provides insight into the basis of inhibitor resistance and the process of replication complex formation.