Exploring Weak Ligand-Protein Interactions by Long-Lived NMR States: Improved Contrast in Fragment-Based Drug Screening

Ligands that have an affinity for protein targets can be screened very effectively by exploiting favorable properties of long-lived states (LLS) in NMR spectroscopy. In this work, we describe the use of LLS for competitive binding experiments to measure accurate dissociation constants of fragments that bind weakly to the ATP binding site of the N-terminal ATPase domain of heat shock protein 90 (Hsp90), a therapeutic target for cancer treatment. The LLS approach allows one to characterize ligands with an exceptionally wide range of affinities, since it can be used for ligand concentrations [L] that are several orders of magnitude smaller than the dissociation constants K-D. This property makes the LLS method particularly attractive for the initial steps of fragment-based drug screening, where small molecular fragments that bind weakly to a target protein must be identified, which is a difficult task for many other biophysical methods.

Exploring Ligand Affinities for Proteins by NMR of Long-Lived States

Roberto Buratto

The detection of molecules that can bind to active sites of protein targets and the measurement of their affinities is a promising application of NMR. Nowadays, the screening of drug candidates is routinely done by NMR in pharmaceutical industry. We have proposed to use the relaxation of Long-Lived States (LLS) for drug screening by NMR. Long-lived states are nuclear spin states whose decay time constant Tlls can be much longer than the longitudinal relaxation time T1. LLS can be used to screen and determine the dissociation constant KD of molecular fragments that bind weakly to protein targets. The use of LLS for fragment screening leads to a spectacular increase in contrast between free and bound ligands, and thus allows one to characterize binding of fragments with very weak affinities, with KD in the millimolar range, which is difficult to achieve by other methods such as ITC. By exploiting the LLS behavior of a spy molecule, we experimentally demonstrate that it is possible to measure dissociation constants KD as large as 12 mM, corresponding to very weak binding, where most other biophysical techniques fail, including other NMR methods based on the observation of ligands. Furthermore, we have combined LLS for screening for improved contrast with 1H dissolution-DNP to enhance the sensitivity. DNP-enhanced screening for measuring LLS signals of a weak ligand allows one to use very low concentrations of ligands and proteins. We observed dramatic differences between the spectra of the ligand in the presence or absence of a protein, or in the presence of the protein combined with a stronger ligand. Moreover, we have explored LLS involving pairs of 19F nuclei to study binding phenomena. Indeed, fluorine detection is quite interesting because it offers the possibility to perform screening experiments without any problems due to overlapping signals. In a customdesigned fluorinated ligand that binds trypsin, we have observed a promising ratio Tlls / T1 > 4. This fluorinated ligand has been used as spy molecule in competition experiments, which allowed us to rank the affinities and estimate dissociation constants of arbitrary ligands that do not contain any fluorine.

EPFL2015

Supramolecular chemistry of 12-Metallacrown-3 complexes

Zacharias Immanuel Grote

12-Metallacrown-3 complexes were formed by self-assembly of organometallic half-sandwich complexes [(π-ligand)MCl2]2 (M = Ru, Rh, Ir) and 3-hydroxy-2-pyridone ligands in the presence of Cs2CO3 as base. Upon mixing of different macrocycles, reversible metal fragment exchange occured, resulting in the generation of small dynamic combinatorial libraries. Investigation of their adaptive behaviour revealed a strong bias for the amplification of hetero-assemblies, and especially of the member whose composition reflects the overall composition of the library. As a consequence, it is not necessarily the thermodynamically most stable member (e.g. the assembly with the highest affinity to a given target) that is amplified the most. Dynamic exchange processes of this kind have also been used to synthesise mixed-metal macrocycles by controlled desymmetrisation of sterically hindered 12-metallacrown-3 complexes. Using amino-substituted 3-hydroxy-2-pyridone ligands, monomeric O,O'-chelate complexes were formed in aqueous solution. Self-assembly into trimeric metallamacrocycles was observed upon addition of base and in phosphate buffer solution, as evidenced by NMR spectroscopy and single crystal X-ray analyses. The macrocycles were able to act as highly selective receptors for lithium ions. The binding constants depend on the nature of the halfsandwich complex, the ligand, and the pH. With a commercially available [(cymene)RuCl2]2 complex and 4-(N-methylpiperazine)-3-hydroxy-2-pyridone, a receptor with a Li+ binding constant of Ka = (5.8±1.0) × 104 M-1 and a Li+ to Na+ selectivity of 10000:1 has been obtained. The fact that the assembly process of the receptor is pH dependant has been used to detect the presence of lithium ions by a simple pH measurement. Furthermore, it was possible to transduce the binding of Li+ into a change of color by means of a chemical reaction with FeCl3. This allowed the detection of Li+ in the pharmacologically relevant concentration range of 0.5–1.5 mM by the 'naked eye'. Modifications of the bridging ligand allowed the design of a potential fluorescent PET sensor for Li+ ions in water. 7Li / 6Li isotope separation was achieved by complexation of LiCl with 12-metallacrown-3 complexes having sterically very demanding π-ligands. An enrichment of 7Li of 5.4 % was observed. Using ligands in which two 3-hydroxy-2-pyridone units were connected by a linker, hexanuclear structures of up to 2.8 nm have been obtained. These assemblies can be regarded as expanded triple-stranded helicates.

EPFL2006

Study of ligand-receptor interactions by fluorescence correlation spectroscopy with different fluorophores: Evidence that the homopentameric 5-hydroxytryptamine type 3(As) receptor binds only one ligand

Kirstin Friedrich, Rudolf Hovius, Horst Vogel

The 5-hydroxytryptamine receptor of type 3 was investigated by fluorescence correlation spectroscopy (FCS). Binding constants of fluorescently labeled ligands, the stoichiometry, and the mass of the receptor are readily accessible by this technique, while the duration of measurement is on the order of seconds to minutes. The receptor antagonist 1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-aminopropyl)-4H-carbazol-4-one (GR-H) was labeled with the fluorophores rhodamine 6G, fluorescein, N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl], and the cyanine dye Cy5. These labels cover a large part of the visible electromagnetic spectrum. It is shown that the photophysical and chemical properties have a direct influence on the measurement quality (duration of measurement, signal-to-noise ratio) and the ligand-receptor interactions (dissociation constants), respectively. This makes it necessary to choose a suitable label or a combination of labels for receptor studies. The affinities of the fluorescently labeled ligands determined by FCS were virtually identical to the values obtained by radioligand binding experiments. Moreover, the dissociation constant of a nonfluorescent receptor ligand was determined successfully by an FCS competition assay. The experimental results showed that only one antagonist binds to the receptor, in agreement with measurements previously published [Tairi et al. (1998) Biochemistry 37, 15850-15864].