Leif Nørskov-Lauritsen

Scaffold hopping and optimization towards libraries of glycogen synthase kinase-3 inhibitors

Using a virtual screening strategy based on a methodology derived from the CATS molecular descriptor, a novel compound class with inhibitory activity against the GSK-3 enzyme was identified through scaffold hopping. These compounds were readily synthesized, either by solid-phase or solution-phase chemistry. Compounds with inhibitory activity below 1 microM were identified.

Novel peptide ligand with high binding capacity for antibody purification

Small synthetic ligands for protein purification have become increasingly interesting with the growing need for cheap chromatographic materials for protein purification and especially for the purification of monoclonal antibodies (mAbs). Today, Protein A-based chromatographic resins are the most commonly used capture step in mAb down stream processing; however, the use of Protein A chromatography is less attractive due to toxic ligand leakage as well as high cost. Whether used as an alternative to the Protein A chromatographic media or as a subsequent polishing step, small synthetic peptide ligands have an advantage over biological ligands; they are cheaper to produce, ligand leakage by enzymatic degradation is either eliminated or significantly reduced, and they can in general better withstand cleaning in place (CIP) conditions such as 0.1M NaOH. Here, we present a novel synthetic peptide ligand for purification of human IgG. Immobilized on WorkBeads, an agarose-based base matrix from Bio-Works, the ligand has a dynamic binding capacity of up to 48 mg/mL and purifies IgG from harvest cell culture fluid with purities and recovery of >93%. The binding affinity is ∼10⁵ M⁻¹ and the interaction is favorable and entropy-driven with an enthalpy penalty. Our results show that the binding of the Fc fragment of IgG is mediated by hydrophobic interactions and that elution at low pH is most likely due to electrostatic repulsion. Furthermore, we have separated aggregated IgG from non-aggregated IgG, indicating that the ligand could be used both as a primary purification step of IgG as well as a subsequent polishing step.

Crystal Structure of a Prolactin Receptor Antagonist Bound to the Extracellular Domain of the Prolactin Receptor

The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5Å resolution by x-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to the PRLR extracellular domain was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry, and NMR spectroscopy. Compared with the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Comparison of the structure of the receptor-bound PRL variant with the structure reported for the unbound form of a similar analogue ( Jomain, J. B., Tallet, E., Broutin, I., Hoos, S., van Agthoven, J., Ducruix, A., Kelly, P. A., Kragelund, B. B., England, P., and Goffin, V. (2007) J. Biol. Chem. 282, 33118-33131 ) demonstrates that receptor-induced changes in the backbone of the four-helix bundle are subtle, whereas large scale rearrangements and structuring occur in the flexible N-terminal part of loop 1. Hydrogen exchange mass spectrometry data imply that the dynamics of the four-helix bundle in solution generally become stabilized upon receptor interaction at binding site 1.

Discovery of the improved antagonistic prolactin variants by library screening

Prolactin (PRL), a potent growth stimulator of the mammary epithelium, has been suggested to be a factor contributing to the development and progression of breast and prostate cancer. Several PRL receptor (PRLR) antagonists have been identified in the past decades, but their in vivo growth inhibitory potency was restricted by low receptor affinity, rendering them pharmacologically unattractive for clinical treatment. Thus, higher receptor affinity is essential for the development of improved PRLR antagonistic variants with improved in vivo potency. In this study, we generated Site 1 focused protein libraries of human G129R-PRL mutants and screened for those with increased affinity to the human PRLR. By combining the mutations with enhanced affinities for PRLR, we identified a novel G129R-PRL variant with mutations at Site 1 that render nearly 50-fold increase in the antagonistic potency in vitro.

Small‐Molecule Affinity Ligands for Protein Purification: Combined Computational Enrichment and Automated In‐line Screening of an Optically Encoded Library

As a result of their complexity, the purification of protein biopharmaceuticals poses challenges not encountered with traditional small-molecule drugs and requires lengthy series of chromatographic steps. Affinity chromatography (AC) is perhaps the most powerful chromatographic technique available, but is limited in scope because of the lack of suitable ligands for proteins for which no natural small-molecule binding partner or inhibitor exists. Current AC ligands used for targeting protein surfaces are primarily other proteins, such as a monoclonal antibody (mAb) or another binding protein. These ligands are expensive and amenable to rapid degradation under common column-cleaning conditions. Selective, chemically robust small-molecule ligands would greatly expand the scope and use of AC for the purification of biopharmaceuticals. However, small-molecule ligands targeting large protein surfaces are rare, despite significant recent achievements. The reason remains the inherent difficulty in designing small molecules that bind tightly and specifically to a large, featureless, amphiphilic, and flexible protein surface. In this study we have focused on identifying smallmolecule affinity ligands for human growth hormone (hGH) through the screening of a combinatorial library of compounds designed to mimic the interactions with its native receptor. Pioneering work has shown that the majority of the binding energy of the initial interaction between hGH and the extracellular part of its receptor (hGHbp) is conferred by a central functional epitope, or “hot spot”, dominated by two tryptophan residues (Trp104 and Trp169) in close proximity on the receptor (Figure 1). 5] This ensemble of residues essential for binding suggests that small-molecule binders may be designed to mimic the interactions of the biological protein partner.

Nonclinical pharmacokinetic and pharmacodynamic characterisation of somapacitan: A reversible non-covalent albumin-binding growth hormone

OBJECTIVE Somapacitan is an albumin-binding growth hormone derivative intended for once weekly administration, currently in clinical development for treatment of adult as well as juvenile GH deficiency. Nonclinical in vivo pharmacological characterisation of somapacitan was performed to support the clinical trials. Here we present the pharmacokinetic and pharmacodynamic effects of somapacitan in rats, minipigs, and cynomolgus monkeys. METHODS Pharmacokinetic studies investigating exposure, absorption, clearance, and bioavailability after single intravenous (i.v.) and subcutaneous (s.c.) administration were performed in all species. A dose-response study with five dose levels and a multiple dose pharmacodynamic study with four once weekly doses was performed in hypophysectomised rats to evaluate the effect of somapacitan on growth and IGF-I production. RESULTS Pharmacokinetic profiles indicated first order absorption from the subcutaneous tissue after s.c. injections for somapacitan in all three species. Apparent terminal half-lives were 5-6h in rats, 10-12h in minipigs, and 17-20h in monkeys. Somapacitan induced a dose-dependent growth in hypophysectomised rats (p<0.001) and an increase in plasma IGF-I levels in rats (p<0.01), minipigs (p<0.01), and cynomolgus monkeys (p<0.05) after single dose administration. Multiple once weekly dosing of somapacitan in hypophysectomised rats induced a step-wise increase in body weight with an initial linear phase the first 3-4days in each dosing interval (p<0.001). CONCLUSION The nonclinical pharmacokinetic and pharmacodynamic studies of somapacitan showed similar pharmacokinetic properties, with no absorption-limited elimination, increased clearance and increased and sustained levels of IGF-I in plasma for up to 10days after a single dose administration in all three species. Somapacitan induced a dose-dependent increase in body weight and IGF-I levels in hypophysectomised rats. Multiple dosing of somapacitan in hypophysectomised rats suggested a linear growth for the first 3-4days in each weekly dosing interval, whereas daily hGH dosing showed linear growth for approximately two weeks before reaching a plateau level.

Long-Acting Human Growth Hormone Analogue by Noncovalent Albumin Binding

The present work describes a series of human growth hormone (hGH) albumin binder conjugates with an extended in vivo half-life. A broad range of different conjugates were studied by varying the albumin binder structure and conjugation site. Conjugates were conveniently obtained by reductive alkylation or by alkylation to introduced cysteines using functionalized albumin-binding side chains. In vitro and in vivo profiling provided the basis for identification of position L101C in human growth hormone as the most optimal position for conjugation, where both a sufficient level of receptor binding and a suitably long half-life could yield a molecule with potential for a once-weekly dosing regimen.