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

Long-Lived States of Magnetically Equivalent Spins Populated by Dissolution-DNP and Revealed by Enzymatic Reactions

Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) offers a way of enhancing NMR signals by up to five orders of magnitude in metabolites and other small molecules. Nevertheless, the lifetime of hyperpolarization is inexorably limited, as it decays toward thermal equilibrium with the nuclear spin-lattice relaxation time. This lifetime can be extended by storing the hyperpolarization in the form of long-lived states (LLS) that are immune to most dominant relaxation mechanisms. Levitt and co-workers have shown how LLS can be prepared for a pair of inequivalent spins by D-DNP. Here, we demonstrate that this approach can also be applied to magnetically equivalent pairs of spins such as the two protons of fumarate, which can have very long LLS lifetimes. As in the case of para-hydrogen, these hyperpolarized equivalent LLS (HELLS) are not magnetically active. However, a chemical reaction such as the enzymatic conversion of fumarate into malate can break the magnetic equivalence and reveal intense NMR signals

Challenges in preparing, preserving and detecting para-water in bulk: overcoming proton exchange and other hurdles

Para-water is an analogue of para-hydrogen, where the two proton spins are in a quantum state that is antisymmetric under permutation, also known as singlet state. The populations of the nuclear spin states in para-water are believed to have long lifetimes just like other Long-Lived States (LLSs). This hypothesis can be verified by measuring the relaxation of an excess or a deficiency of para-water, also known as a "Triplet-Singlet Imbalance'' (TSI), i.e., a difference between the average population of the three triplet states T (that are symmetric under permutation) and the population of the singlet state S. In analogy with our recent findings on ethanol and fumarate, we propose to adapt the procedure for Dissolution Dynamic Nuclear Polarization (D-DNP) to prepare such a TSI in frozen water at very low temperatures in the vicinity of 1.2 K. After rapid heating and dissolution using an aprotic solvent, the TSI should be largely preserved. To assess this hypothesis, we studied the lifetime of water as a molecular entity when diluted in various solvents. In neat liquid H2O, proton exchange rates have been characterized by spin-echo experiments on oxygen-17 in natural abundance, with and without proton decoupling. One-dimensional exchange spectroscopy (EXSY) has been used to study proton exchange rates in H2O, HDO and D2O mixtures diluted in various aprotic solvents. In the case of 50 mM H2O in dioxane-d(8), the proton exchange lifetime is about 20 s. After dissolving, one can observe this TSI by monitoring intensities in oxygen-17 spectra of H2O (if necessary using isotopically enriched samples) where the AX(2) system comprising a "spy'' oxygen A and two protons X-2 gives rise to binomial multiplets only if the TSI vanishes. Alternatively, fast chemical addition to a suitable substrate (such as an activated aldehyde or ketone) can provide AX2 systems where a carbon-13 acts as a spy nucleus. Proton signals that relax to equilibrium with two distinct time constants can be considered as a hallmark of a TSI. We optimized several experimental procedures designed to preserve and reveal dilute para-water in bulk

Drug Screening Boosted by Hyperpolarized Long-Lived States in NMR

Transverse and longitudinal relaxation times (T1ρ and T1) have been widely exploited in NMR to probe the binding of ligands and putative drugs to target proteins. We have shown recently that long-lived states (LLS) can be more sensitive to ligand binding. LLS can be excited if the ligand comprises at least two coupled spins. Herein we broaden the scope of ligand screening by LLS to arbitrary ligands by covalent attachment of a functional group, which comprises a pair of coupled protons that are isolated from neighboring magnetic nuclei. The resulting functionalized ligands have longitudinal relaxation times T1(1H) that are sufficiently long to allow the powerful combination of LLS with dissolution dynamic nuclear polarization (D-DNP). Hyperpolarized weak “spy ligands” can be displaced by high-affinity competitors. Hyperpolarized LLS allow one to decrease both protein and ligand concentrations to micromolar levels and to significantly increase sample throughput

Long-lived nuclear spin states of magnetically equivalent protons by means of dissolution dynamic nuclear polarization

Nuclear Magnetic Resonance (NMR) is one of the most versatile techniques since it enables the characterization of solid, liquid and gaseous systems in a plethora of in-vitro and in-vivo experiments. Despite its multidisciplinary scope, it still suffers from two drawbacks, namely the intrinsic low sensitivity and the fast return to equilibrium. Dissolution Dynamic Nuclear Polarization (D-DNP) is a powerful scheme to enhance the signals of nuclear spins by up to five orders of magnitude and it is at the forefront of current research in NMR. The main detriment is that the great efforts spent in producing the hard-earned hyperpolarized signals fade because of rapid longitudinal relaxation with a time constant T1. Long-Lived States (LLS) are states with lifetimes that may be much longer than T1. In a two-spin system, they can be defined as a T/S Imbalance (TSI) between the average population of the three triplet (T) states on the one hand and the population of the singlet (S) state on the other. So far, para-hydrogen is the only molecule routinely used as a source of long-lived hyperpolarization. In recent years, it has been shown that it is possible to create a TSI in magnetically in-equivalent spins, by exploiting DNP to lower the spin temperature well below the Zeeman splitting. The present thesis is devoted to combine D-DNP with LLS in the perspective of preparing enhanced and long-lasting NMR signals in systems comprising magnetically equivalent protons. We proved that that is possible to induce the TSI via D-DNP in partly deuterated ethanol and in fumaric acid. In ethanol the TSI has been revealed by 1H-13C cross-relaxation that leads to population differences across observable transitions. In fumarate, we resorted to a symmetry-breaking addition of D2O catalysed by fumarase which makes the TSI observable on the two magnetically in-equivalent protons of malate. In principle, our strategy should allow one to prepare either an excess or a deficiency of para-water, which we like to refer to as âforbidden fruitsâ of spectroscopy. Water is challenging since its TSI may suffer from relaxation due to fast proton exchange and to spin-rotation. We assessed conditions suitable to slow down proton exchange by dilution in aprotic solvents, yielding lifetimes of water as a molecular entity on a timescale compatible with D-DNP experiments. We designed an arrangement of coaxial tubes to measure T1 of water vapour at known pressures and temperatures. The experimental T1 are rather small (i.e., tens of ms) and in fair agreement with predictions if spin-rotation is the dominant relaxation mechanism. Empirical evidence of the survival of the hyperpolarized signal of water after dissolution in our lab suggests that the exchange of water molecules between the gaseous and liquid phases is either inefficient or slow in most of cases we studied. I report preliminary results of the application of our D-DNP scheme to induce a TSI in water. Considering the complexity of the project, further investigation is needed to unravel and optimize recent observations in view of identifying an unambiguous fingerprint of dilute para-water

Ligand-Protein Affinity Studies Using Long-Lived States of Fluorine-19 Nuclei

The lifetimes T-LLS of long-lived states or T-LLC of long-lived coherences can be used for the accurate determination of dissociation constants of weak protein ligand complexes. The remarkable contrast between signals derived from LLS or LLC in free and bound ligands can be exploited to search for weak binders with large dissociation constants K-D > 1 mM that are important for fragment-based drug discovery but may escape detection by other screening techniques. Alternatively, the high sensitivity of the proposed method can be exploited to work with large ligand-to-protein ratios, with an evident advantage of reduced consumption of precious proteins. The detection of F-19-F-19 long-lived states in suitably designed fluorinated spy molecules allows one to perform competition binding experiments with high sensitivity while avoiding signal overlap that tends to hamper the interpretation of proton spectra of mixtures

Kinetic isotope effects for fast deuterium and proton exchange rates

International audienceBy monitoring the effect of deuterium decoupling on the decay of transverse 15N magnetization in D–15N spin pairs during multiple-refocusing echo sequences, we have determined fast D–D exchange rates kD and compared them with fast H–H exchange rates kH in tryptophan to determine the kinetic isotope effect as a function of pH and temperature

Collisional cross-section of water molecules in vapour studied by means of 1 H relaxation in NMR

International audienceIn gas phase, collisions that affect the rotational angular momentum lead to the return of the magnetization to its equilibrium (relaxation) in Nuclear Magnetic Resonance (NMR). To the best of our knowledge, the longitudinal relaxation rates R 1 = 1/T 1 of protons in H 2 O and HDO have never been measured in gas phase. We report R 1 in gas phase in a field of 18.8 T, i.e., at a proton Larmor frequency ν 0 = 800 MHz, at temperatures between 353 and 373 K and pressures between 9 and 101 kPa. By assuming that spin rotation is the dominant relaxation mechanism, we estimated the effective cross-section σ J for the transfer of angular momentum due to H 2 O-H 2 O and HDO-D 2 O collisions. Our results allow one to test theoretical predictions of the intermolecular potential of water in gas phase