Second-harmonic generation-based methods to detect and characterize ligand-induced RNA conformational changes.

Second-harmonic generation (SHG) is a biophysical tool that senses ligand-induced conformational changes in biomolecules. The Biodesy Delta™ has been developed as a high-throughput screening platform to monitor conformational changes in proteins and oligonucleotides by SHG to support drug discovery efforts. This work will outline (1) an overview of this technology, (2) detailed protocols for optimizing screening-ready SHG assays on RNA targets, (3) practical considerations for developing robust and informative SHG measurements, and (4) a case study that demonstrates the application of these recommendations on an RNA target.

Detection of Ligand-Induced Conformational Changes in Oligonucleotides by Second-Harmonic Generation at a Supported Lipid Bilayer Interface.

There is a high demand for characterizing oligonucleotide structural changes associated with binding interactions as well as identifying novel binders that modulate their structure and function. In this study, second-harmonic generation (SHG) was used to study RNA and DNA oligonucleotide conformational changes associated with ligand binding. For this purpose, we developed an avidin-based biotin capture surface based on a supported lipid bilayer membrane.

Protein Conformational Changes Are Detected and Resolved Site Specifically by Second-Harmonic Generation.

We present here a straightforward, broadly applicable technique for real-time detection and measurement of protein conformational changes in solution. This method is based on tethering proteins labeled with a second-harmonic generation (SHG) active dye to supported lipid bilayers. We demonstrate our method by measuring the conformational changes that occur upon ligand binding with three well-characterized proteins labeled at lysine residues: calmodulin (CaM), maltose-binding protein (MBP), and dihydrofolate reductase (DHFR).

Peginesatide and erythropoietin stimulate similar erythropoietin receptor-mediated signal transduction and gene induction events.

Peginesatide is a synthetic, PEGylated, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. Peginesatide has a unique structure that consists of a synthetic peptide dimer (with no sequence similarity to erythropoietin) conjugated to a 40-kDa PEG moiety. Peginesatide is being developed for the treatment of anemia associated with chronic kidney disease in dialysis patients.

Methods for detection and confirmation of Hematide™/peginesatide in anti-doping samples.

Since the 1990's, cheating athletes have abused substances to increase their oxygen transport capabilities; among these substances, recombinant EPO is the most well known. Currently, other investigational pharmaceutical products are able to produce an effect similar to EPO but without having chemical structures related to EPO; these are the synthetic erythropoiesis stimulating agents (ESAs). Peginesatide (also known as Hematide™) is being developed by Affymax and Takeda and, if approved by regulatory authorities, could soon be released on the international market.

Hematide is immunologically distinct from erythropoietin and corrects anemia induced by antierythropoietin antibodies in a rat pure red cell aplasia model.

Objective: To evaluate the potential of Hematide, a PEGylated, synthetic peptide-based erythropoiesis-stimulating agent that is in clinical development for the treatment of anemia associated with chronic kidney disease and cancer, to correct antierythropoietin antibody-associated pure red cell aplasia (PRCA).

Materials And Methods: The binding of anti-Hematide antibodies (mouse, rabbit, and monkey) to recombinant human erythropoietin (rHuEPO) and of anti-rHuEPO antibodies (mouse, goat, rat, and human) to Hematide were evaluated. An anti-EPO antibody-mediated anemia rat model was developed by subcutaneously administering rHuEPO to rats three times weekly for 4 weeks.

Preclinical evaluation of Hematide, a novel erythropoiesis stimulating agent, for the treatment of anemia.

Objective: To evaluate the preclinical erythropoiesis stimulating properties of Hematide, a novel, PEGylated, synthetic peptide for the treatment of anemia associated with chronic kidney disease and cancer.

Methods: The in vitro activity of Hematide was assessed in competitive binding, proliferation, signal transduction, and apoptosis assays, and in erythroid colony-forming assays with CD34(+) cells purified from human bone marrow. Erythropoiesis and pharmacokinetics were evaluated in rat, monkey, and a rat chronic renal insufficiency (CRI) model following single administration.