Hendrik Koliwer-Brandl

From a library of MAG antagonists to nanomolar CD22 ligands.

Siglec‐2, also known as CD22, is involved in the regulation and survival of B‐cells and has been successfully targeted in cell depletion therapies with antibody‐based approaches. Sialic acid derivatives, already known to bind with high affinity to myelin‐associated glycoprotein (MAG, Siglec‐4), were screened for their binding affinity for CD22 by surface plasmon resonance. The best compound identified was further modified with various hydrophobic substituents at the 2‐, 5‐, and 9‐positions of the sialic acid scaffold, leading to nanomolar derivatives, of which ligand 17 b shows the most promising pharmacodynamic and pharmacokinetic profiles. Isothermal titration calorimetry measurements demonstrate that the binding is enthalpy driven. Interestingly, the thermodynamic fingerprints reveal an excellent correlation between gains in enthalpy and compensation by increased entropy costs. Moreover, 17 b exhibits a residence time in the range of a few seconds, clearly prolonged relative to residence times typically observed for carbohydrate–lectin interactions. Finally, initial tests regarding drug‐like properties of 17 b demonstrate the required high plasma protein binding yet a lack of oral availability, although its distribution coefficient (log D) is in the required range.

Low Molecular Weight Antagonists of the Myelin-Associated Glycoprotein: Synthesis, Docking, and Biological Evaluation

The injured adult mammalian central nervous system is an inhibitory environment for axon regeneration due to specific inhibitors, among them the myelin-associated glycoprotein (MAG), a member of the Siglec family (sialic-acid binding immunoglobulin-like lectin). In earlier studies, we identified the lead structure 5, which shows a 250-fold improved in vitro affinity for MAG compared to the tetrasaccharide binding epitope of GQ1balpha (1), the best physiological MAG ligand described so far. By modifying the 2- and 5-position, the affinity of 5 could be further improved to the nanomolar range (-->19a). Docking studies to a homology model of MAG allowed the rationalization of the experimental binding properties. Finally, pharmacokinetic parameters (stability in the cerebrospinal fluid, logD and permeation through the BBB) indicate the drug-like properties of the high-affinity antagonist 19a.

C-4 Modified Sialosides Enhance Binding to Siglec-2 (CD22): Towards Potent Siglec Inhibitors for Immunoglycotherapy†

The regulatory functions of Siglecs (sialic acid binding immunoglobulin-like lectins) in the immune system provide opportunities for innovative therapeutic strategies for a wide range of immunological disorders or cancer (immunoglycotherapy). Siglec-2 (CD22), as a consequence of its pivotal role in B cell activation, has become an attractive target for therapies of autoimmune diseases and B cell-derived nonHodgkin s lymphoma (NHL). NHL is among the ten most common cancers with over 20000 deaths in 2010 for the US alone. Siglec-2 binds with high preference to a(2,6)-linked sialic acids (Sia), such as Neu5Aca(2,6)lactosamine (Scheme 1). Neu5Aca2Me (1) interacts with Siglec-2 mainly through 1) the negative charge on its carboxylate group, 2) the C-5 N-acetamido substituent, and 3) the glycerol side chain. Furthermore, replacement of the C-9 hydroxy group by an amino group did not interfere with binding to Siglecs. Crystallographic studies on Siglec-1 (sialoadhesin, Sn) demonstrated that acylation of this amino group enhances the overall affinity of the ligand for Siglecs by two to three orders of magnitude. The first breakthrough in the development of potent Siglec-2 inhibitors was the design of 9biphenylcarboxamido Neu5Aca2Me (9-BPC-Neu5Aca2Me, 2) which has a more than two orders of magnitude higher affinity to Siglec-2 than 1, and 2 has demonstrated potential to modulate signal transduction in B cells. Furthermore, based on 2, compounds were developed, which kill B cell lymphoma cells. Structural studies and modifications of the C-5 N-acyl substituent and the C-2 aglycon moiety of Nacetylneuraminic acid (Neu5Ac) have led to further improvement in affinity. Herein we report, for the first time the design, synthesis, and evaluation of a novel class of disubstituted Neu5Ac derivatives that is modified at the C-4 and C-9 positions of 1. Our structure-based design approach resulted in a promising novel lead compound 9-biphenylcarboxamido-4-m-nitrophenylcarboxamido-4,9-dideoxy Neu5Aca2Me (9-BPC-4mNPC-Neu5Aca2Me, 6b) that has sub-micromolar affinity for Siglec-2 and may provide a pathway for immunoglycotherapy strategies. An evaluation of our homology model (see Supporting Information) for Siglec-2 and other Siglecs led us to hypothesize that substituents at C-4 may provide additional interactions. To address this hypothesis we posed the following questions: 1) Can C-4 substituents enhance the interaction with Siglecs? 2) Do they interact specifically with the protein? 3) Do C-4 and C-9 modifications act synergistically? 4) Do the C-4 modified Neu5Ac derivatives bind to the same binding site as other Sia, such as 1?

Kinetic and thermodynamic properties of MAG antagonists

Paraplegia is caused by injuries of the central nervous system (CNS) and especially young people suffer from these severe consequences as, for example, the loss of motor functions. The lack of repair of the injured nerve strands originates from the inhibitory environment for axon regeneration in the CNS. Specific inhibitory proteins block the regrowth of nerve roots. One of these neurite outgrowth inhibitors is the myelin-associated glycoprotein (MAG), which is a member of the Siglec family (sialic acid-binding immunoglobulin-like lectin). In previous studies, we identified potent small molecule MAG antagonists. In this communication, we report new neuraminic acid derivatives modified in the 4- and 5-position, and the influence of various structural modifications on their kinetic and thermodynamic binding properties.

Biochemical characterization of trans-sialidase TS1 variants from Trypanosoma congolense

BackgroundAnimal African trypanosomiasis, sleeping sickness in humans and Nagana in cattle, is a resurgent disease in Africa caused by Trypanosoma parasites. Trans-sialidases expressed by trypanosomes play an important role in the infection cycle of insects and mammals. Whereas trans-sialidases of other trypanosomes like the American T. cruzi are well investigated, relatively little research has been done on these enzymes of T. congolense.ResultsBased on a partial sequence and an open reading frame in the WTSI database, DNA sequences encoding for eleven T. congolense trans-sialidase 1 variants with 96.3% overall amino acid identity were amplified. Trans-sialidase 1 variants were expressed as recombinant proteins, isolated and assayed for trans-sialylation activity. The purified proteins produced α2,3-sialyllactose from lactose by desialylating fetuin, clearly demonstrating their trans-sialidase activity. Using an HPLC-based assay, substrate specificities and kinetic parameters of two variants were characterized in detail indicating differences in substrate specificities for lactose, fetuin and synthetic substrates. Both enzymes were able to sialylate asialofetuin to an extent, which was sufficient to reconstitute binding sites for Siglec-4. A mass spectrometric analysis of the sialylation pattern of glycopeptides from fetuin revealed clear but generally similar changes in the sialylation pattern of the N-glycans on fetuin catalyzed by the trans-sialidases investigated.ConclusionsThe identification and characterization of a trans-sialidase gene family of the African parasite T. congolense has opened new perspectives for investigating the biological role of these enzymes in Nagana and sleeping sickness. Based on this study it will be interesting to address the expression pattern of these genes and their activities in the different stages of the parasite in its infection cycle. Furthermore, these trans-sialidases have the biotechnological potential to be used for enzymatic modification of sialylated glycoconjugates.

Impact of Strong and Weak Lipid–Protein Interactions on the Structure of a Lipid Bilayer on a Gold Electrode Surface

A lipid bilayer deposited on an electrode surface can serve as a benchmark system to investigate lipid-protein interactions in the presence of physiological electric fields. Recoverin and myelin-associated glycoprotein (MAG) are used to study the impact of strong and weak protein-lipid interactions on the structure of model lipid bilayers, respectively. The structural changes in lipid bilayers are followed using electrochemical polarization modulation infrared reflection-absorption spectroscopy (PM IRRAS). Recoverin contains a myristoyl group that anchors in the hydrophobic part of a cell membrane. Insertion of the protein into the 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)-cholesterol lipid bilayer leads to an increase in the capacitance of the lipid film adsorbed on a gold electrode surface. The stability and kinetics of the electric-field-driven adsorption-desorption process are not affected by the interaction with protein. Upon interaction with recoverin, the hydrophobic hydrocarbon chains become less ordered. The polar head groups are separated from each other, which allows for recoverin association in the membrane. MAG is known to interact with glycolipids present on the surface of a cell membrane. Upon probing the interaction of the DMPC-cholesterol-glycolipid bilayer with MAG a slight decrease in the capacity of the adsorbed lipid film is observed. The stability of the lipid bilayer increases towards negative potentials. At the molecular scale this interaction results in minor changes in the structure of the lipid bilayer. MAG causes small ordering in the hydrocarbon chains region and an increase in the hydration of the polar head groups. Combining an electrochemical approach with a structure-sensitive technique, such as PM IRRAS, is a powerful tool to follow small but significant changes in the structure of a supramolecular assembly.

Design, synthesis, biological evaluation, and modeling of a non-carbohydrate antagonist of the myelin-associated glycoprotein

Broad modifications of various positions of the minimal natural epitope recognized by the myelin-associated glycoprotein (MAG), a blocker of regeneration of neurite injuries, produced sialosides with nanomolar affinities. However, important pharmacokinetic issues, for example, the metabolic stability of these sialosides, remain to be addressed. For this reason, the novel non-carbohydrate mimic 3 was designed and synthesized from (-)-quinic acid. For the design of 3, previously identified beneficial modifications of side chains of Neu5Ac were combined with the replacement of the ring oxygen by a methylene group and the substitution of the C(4)-OH by an acetamide. Although docking experiments to a homology model of MAG revealed that mimic 3 forms all but one of the essential hydrogen bonds identified for the earlier reported lead 2, its affinity was substantially reduced. Extensive molecular-dynamics simulation disclosed that the missing hydrogen bond of the former C(8)-OH leads to a change of the orientation of the side chain. As a consequence, an important hydrophobic contact is compromised leading to a loss of affinity.