Design, Synthesis and Thermodynamic Studies of Galectin Ligands

Abstract

The signaling within and between cells in biology is governed by molecular recognition between natural or synthetic ligands and proteins. This thesis project aimed to investigate the thermodynamic properties of specific interaction between synthetic ligands and galectin proteins. The choice of galectins as model proteins resides in the availability of applicability in a full range of complementary experimental and theoretical methods, such as X-ray crystallography and neutron diffraction, NMR, ITC and computational simulations accompanied by design and synthesis of ligands and a rapid method to establish binding specificity and affinity (competitive fluorescent polarization assay). Furthermore, galectins are key biological mediators in numerous
biological activities playing important roles in inflammation, immunity and cancer progression, which also makes galectins interesting as a pharmaceutical target. The thermodynamic properties of binding are sensitive to minute changes in the ligand
scaffold, hence we identified small modifications in the ligand design and analyzed effects of these through a multidisciplinary approach. We analyzed the importance of hydrogen bond contribution by comparing a pair of diastereomers with galectin-3C
derived by the removal of non-interacting atoms from a known potent ligand. Solvation effect and conformational entropy favored the diastereomer S over R in complex with galectin-3, even though both ligands showed similar free energy of binding.
However, the affinity of the S diastereomer was higher than that of R when one of the galectin-3 arginine residues that hydrogen bound to the ligand hydroxyl group was mutated. A similar effect was observed for the closely related galectin-1: affinity for the S was higher than for R diastereomer. The same hydrogen bond between the ligand and galectin-3 was weaker when the diastereomeric hydroxylated ligands were oxidized to the corresponding ketone. Furthermore, we investigated if the ligand
binding to galectins can be achieved is only parts of the galactopyranose ring of known high-affinity ligand are kept and noninteracting atoms are removed. Indeed, replacing the galactopyranose ring with an acyclic threitol moiety with a-D-galactosemimicking stereoconfiguration and combining it with an affinity-enhancing trifluorophenyltriazole moiety reached a remarkably good affinity towards galectin-1 and 3 terminal domains. This evidenced that ligand binding to the galactoside-recognizing galectin protein family does not need the D-galactopyranose per se, only ideally positioned ligand key hydrogen-bonding groups.
Finally, we turned our attention from hydrogen bonds to the close related halogen bond. The change of single ligand halogen resulted in significant differences in the thermodynamic characteristics of galectin-3 binding. The halogen atom strongly affected the solvation and the chemical shifts of the residuals involved in the binding pocket. Furthermore, the enthalpy of binding correlated with the halogen σ-hole size and showed and increasing contribution to the free energy of binding going from fluorine to iodine. However, the steric restraints imposed by the larger iodine atom resulted in a decrease of entropy, thus counterbalancing the strong enthalpy contribution by iodine. Altogether, our in-depth thermodynamic studies on ligandgalectin molecular interactions have advanced our fundamental understanding of ligand and protein dynamics, effects of hydrogen bond geometries and of ligand-protein halogen bonds. We expect that this knowledge advancement will be of interest
and value for drug design not only against galectin proteins, but also for drug design in general.

Abstract (Swedish)

The word “medicine” has its origin from the Latin word medeor or medicor, which means to heal or to cure. Since ancient times, medicines helped to soothe the pain, which eventually resulted in general improvement of life. In a course of a lifetime, the use of medicines can vary according to the necessity. We can have medicines, or drugs, for most of the diseases, and for many others the available remedies are inappropriate, because of toxicity or loss of activity. An outstanding example is the galloping resistance developed by bacteria towards antibiotics.
The drug development is a very long process that might take longer than twenty years and that is constantly challenged by safety testing. The cost of this process is enormous, and it goes together with the high risk of failure. The number of possible drugs from the early stages can mount up to around 10.000 different molecules, which it will be reduced to a few, one, or even zero in the later stages. Would it be possible to reduce the initial arsenal of molecules by identifying the most efficient and safe ones already in the early stages?
The answer might be hidden in a better understanding of the basic mechanisms that govern the actions of a drug in our body. Like a puzzle of thousands of pieces: the drugs, called also ligands, can interact with many other molecules in the body, for example proteins, in many different ways. From pharmaceutical point of view, finding the best fit to the pieces of the puzzle drugs, or ligands, binding to a certain protein is quite a tangle.
As for every puzzle, the beginning might look scary and overwhelming, but with a lot of patience and taking one thing at the time (or as Swedes say “en sak i taget”), the pieces will come together and show the bigger picture. In my thesis project I studied the basic mechanism of how ligands bind to a family of proteins, called galectins, that are ubiquitously distributed in our body. The design of the molecules that interact with galectins developed from modifications of natural molecules based on the sugar molecule galactose. We analyzed the interactions between the ligands and galectin-3 and aimed at reaching a better description of the driving forces behind binding are composed of different energic (enthalpy) contributions and changes in order (entropy) of the system when a ligand binds to a galectin protein. These two factors, enthalpy and entropy, are both involved in the binding of a ligand, but very often they counterbalance each other. By using a combination of several experimental and theoretical techniques, we discovered that minute changes in the ligands can have a profound influence in the internal mobility of the protein and of the water molecules, which affect both the change in energy (enthalpy) and change of order (entropy)
when a ligand binds to a galectin. Hence, we reached and improved understanding of
enthalpy and entropy changes, as well as how they influence each other, upon ligand binding to a protein. Nevertheless, although we contributed to a deeper understanding of the mechanisms behind ligand-protein interactions, more research is needed in order to fully understand how this can be applied in drug discovery.

• author: Verteramo, Maria Luisa
• supervisor: Ulf Nilsson; Mikael Akke; Ulf Ellervik; Fredrik Zetterberg
• opponent: Professor Strømgaard, Kristian, University of Copenhagen, Denmark
• organization: Centre for Analysis and Synthesis
• alternative title: Design, syntes och termodynamiska studier om galectin ligander
• publishing date: 2019-05-10
• type: Thesis
• publication status: published
• subject: Organic Chemistry; Pharmaceutical Chemistry
• keywords: galectin, conformational entropy, solvation, thermodynamic, interaction, molecular recognition, thiodigalactoside, diastereomer, structure-based design
• pages: 164 pages
• defense location: Lecture Hall K:C, Kemicentrum, Naturvetarvägen 14, Lund University, Faculty of Engineering LTH
• defense date: 2019-06-05 13:00:00
• ISBN: 978-91-7422-665-2
• ISBN: 978-91-7422-664-5
• language: English
• LU publication?: yes
• id: b3fd657f-9cab-4c02-b2a5-fe6fc55d6b4f
• date added to LUP: 2019-05-02 14:07:58
• date last changed: 2023-09-07 09:15:58