Multivalent carbohydrate-based ligands
were synthesized and evaluated
as inhibitors of the adhesion protein HA of the influenza A virus
(IAV). HA relies on multivalency for strong viral adhesion. While
viral adhesion inhibition by large polymeric molecules has proven
viable, limited success was reached for smaller multivalent compounds.
By linking of sialylated LAcNAc units to di- and trivalent scaffolds,
inhibitors were obtained with an up to 428-fold enhanced inhibition
in various assays.
Divalent
ligands were prepared as inhibitors for the adhesion protein
of the problematic
Pseudomonas aeruginosa
pathogen.
Bridging two binding sites enables simultaneous binding of two galactose
moieties, which strongly enhances binding. An alternating motif of
glucose and triazole and aryl groups was shown to have the right mix
of rigidity, solubility, and ease of synthesis. Spacers were varied
with respect to the core unit as well as the aglycon portions in an
attempt to optimize dynamics and enhance interactions with the protein.
Affinities of the divalent ligands were measured by ITC, and
K
d
’s as low as 12 nM were determined,
notably for a compounds with either a rigid (phenyl) or flexible (butyl)
unit at the core. Introducing a phenyl aglycon moiety next to the
galactoside ligands on both termini did indeed lead to a higher enthalpy
of binding, which was more than compensated by entropic costs. The
results are discussed in terms of thermodynamics and theoretical calculations
of the expected and observed multivalency effects.
A potent divalent ligand of the Pseudomonas aeruginosa adhesion lectin LecA was elaborated into a tetravalent version. A polyethylene glycol (PEG) spacer was introduced to link two divalent galactosides. Each of the two divalent ligands contained a rigid spacer with a central phenyl group that is bridged by the PEG moiety. The resulting tetravalent ligand was found to bind LecA in the nanomolar range involving all of its sugar (sub)ligands. Analytical ultracentrifugation studies clearly showed that the tetravalent ligand was capable of aggregation the LecA tetramers in contrast to the divalent ligands. The aggregator behavior was found to be of importance in P. aeruginosa biofilm formation inhibition. Despite the weaker affinity it was a considerably better biofilm inhibitor with half inhibitory values around the 28 micromolar range.
Inducible Nitric Oxide Synthase (iNOS) has been involved in a variety of diseases, and thus it is interesting to discover and optimize new iNOS inhibitors. In previous studies, a series of benzimidazole-quinolinone derivatives with high inhibitory activity against human iNOS were discovered. In this work, three-dimensional quantitative structure-activity relationships (3D-QSAR), molecular docking and molecular dynamics (MD) simulation approaches were applied to investigate the functionalities of active molecular interaction between these active ligands and iNOS. A QSAR model with R2 of 0.9356, Q2 of 0.8373 and Pearson-R value of 0.9406 was constructed, which presents a good predictive ability in both internal and external validation. Furthermore, a combined analysis incorporating the obtained model and the MD results indicates: (1) compounds with the proper-size hydrophobic substituents at position 3 in ring-C (R3 substituent), hydrophilic substituents near the X6 of ring-D and hydrophilic or H-bond acceptor groups at position 2 in ring-B show enhanced biological activities; (2) Met368, Trp366, Gly365, Tyr367, Phe363, Pro344, Gln257, Val346, Asn364, Met349, Thr370, Glu371 and Tyr485 are key amino acids in the active pocket, and activities of iNOS inhibitors are consistent with their capability to alter the position of these important residues, especially Glu371 and Thr370. The results provide a set of useful guidelines for the rational design of novel iNOS inhibitors.
Pseudomonas aeruginosa is a widespread opportunistic pathogen that is capable of colonizing various human tissues and is resistant to many antibiotics. LecA is a galactose binding tetrameric lectin involved in adhesion, infection and biofilm formation. This study reports on the binding characteristics of mono- and divalent (chelating) ligands to LecA using different techniques. These techniques include Affinity Capillary Electrophoresis (ACE), Bio Layer Interferometry (BLI), Native Mass Spectrometry and a Thermal Shift Assay. Aspects of focus include: affinity, selectivity, binding kinetics and residence time. The affinity of a divalent ligand was determined to be in the low nanomolar range for all of the used techniques and with a ligand residence time of approximately 7 hours, while no strong binding was seen to related lectin tetramers. Each of the used techniques provides a unique and complementary insight into the chelation based binding mode of the divalent ligand to the LecA tetramer.
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