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

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

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

EVOLUTION OF THE PRODUCTION AND PRICE OF FOREST BIOMASS FOR ENERGY

This study aimed to analyze the evolution of the production and price of biomass from native and planted forests of the state of Paraná (Brazil), between 1998 and 2015, based on the behavior of the prices of the products, according to variations of their supply or demand. The annual rates for growth of the price and quantity produced were calculated and related to the displacements of the supply and demand curves of the products. The results indicated a decrease in the quantity and an increase in the biomass price for native forests, which caused a shift in the supply curve to the left. For the biomass of planted forests, the demand curve shifted to the right due to the demand increase of this product for energy production. The behavior of both curves indicated a substitution of the biomass from native forests to biomass from planted forests due to factors related to the increase of environmental protection regarding the native forests located in the state of Paran

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

First dunk tournament of the Brazilian women's basketball league: conclusions about the height of the hoop

La variabilidad y hermosura de las acciones técnicas individuales del baloncesto se derivan de las necesidades tácticas impuestas a los jugadores durante el juego y son una parte importante del espectáculo, que impacta positivamente a los espectadores y a la prensa. Entre las muchas maneras de fintas, tiros, pases y entradas, están los mates, como un momento de gran notoriedad en el juego en el baloncesto masculino. Con el objetivo de celebrar el primer “Torneo de mates LBF 2018”, el objetivo del estudio fue determinar la altura del aro necesario para que las jugadoras brasileñas pudieran concretizar los mates. Utilizamos la altura alcanzada en la prueba de salto vertical progresivo de 91 jugadoras de los nueve equipos de la temporada 2018. Encontramos que el 25% de los jugadores masculinos brasileños de élite son capaces de hacer mates en los juegos oficiales. Por lo tanto, el criterio para definir la altura del aro fue que el mismo porcentaje de jugadoras femeninas pudiera concluye mates, considerando la altura alcanzada en la prueba de salto y la necesidad de pasar la pelota sobre el aro. Así, llegamos a la altura del aro desde 2,75 m del suelo. Ocho jugadoras participaron en el evento, cuatro brasileñas y cuatro argentinas, cinco de ellas lograron actuar mates. Cabe señalar que la altura del aro no fue cambiada para los partidos de la temporada, con lo cual esta altura puede no implicar la aparición de mates en esta condición, ya que la dinámica del juego es diferente de la dinámica en el Torneo de mates.The variability and beauty of basketball's individual technical actions arise from the tactical needs imposed on players during the game, as an important part of the spectacle that positively impacts spectators and media. Among the many feints, throws, passes and layups, are the slam dunks, a notorious moment in the game, and also theme of specific events in men's basketball. In view of the realization of the 1st Slam Dunk Tournament during the 2018 LBF All Stars Games, the aim of the study was to determine the ideal hoop height for Brazilian female players to be able to slam dunk. We analyzed the height achieved in the progressive vertical jump test of 91 female players participating in the nine teams in the 2018 season. Findings suggest that 25% of the elite Brazilian male players slam dunk in official games. Thus, the criteria for defining the hoop height was that the same percentage of female players could slam dunk, considering the height reached on the test and the need to take the ball over the hoop. This resulted in 2.75 m as the height of the rim from the ground. Eight players participated in the event, four Brazilian and four Argentinian, and five of them managed to dunk. It should benoted that the height of the rim has not changed for the regular games of the season, and that this change may not imply the emergence of dunks in official matches, as the game dynamics are different from the dynamics in the Dunk Tournament and that the players need some time to adapt.A variabilidade e beleza das ações técnicas individuais do basquetebol são decorrentes das necessidades táticas impostas aos jogadores durante o jogo e fazem parte importante do espetáculo, impactando positivamente os espectadores e mídia. Entre várias formas de fintas, arremessos, passes e bandejas, destacam-se as enterradas, enquanto momento de grande notoriedade no jogo, bem como tema de eventos específicos dedicados a ela no basquetebol masculino. Visando a realização do 1º Torneio de Enterradas no Jogo das Estrelas LBF 2018, o objetivo do estudo foi qual altura do aro necessária para que as jogadoras brasileiras pudessem realizar enterradas. Foi utilizada a altura alcançada no teste de impulsão vertical em progressão de 91 jogadoras participantes das nove equipes na temporada 2018. Verificamos que 25 % dos jogadores masculinos brasileiros de elite enterram em jogos oficiais. Assim, o critério para definir a altura do aro foi que a mesma porcentagem de jogadoras femininas pudessem enterrar, considerando a altura alcançada no teste e a necessidade de passar a bola sobre o aro. Foi determinada a altura de 2.75 m do aro em relação ao solo. Oito jogadoras participaram do evento, sendo quatro brasileiras e quatro argentinas, sendo que cinco delas conseguiram realizar enterradas. Deve-se ressaltar que a altura do aro não foi alterada para os jogos da temporada, e considerar que essa mudança pode não implicar no surgimento de enterradas em partidas oficiais, dado que a dinâmica do jogo é diferente da dinâmica no Torneio de Enterradas e que as jogadoras necessitam de um tempo de adaptação.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES). Liga de Basquete Feminino – Brasil (LBF): convênio nº 36P-18381/2017peerReviewe

Method for the nmr based determination of the affinity of drugs for a target protein

The document relates to a method for the determination of the binding properties of at least one first chemical compound to at least one second chemical compound between a free essentially dissociated state and a bound essentially associated state by using nuclear magnetic resonance (NMR) measurements, wherein at least one of the first chemical compound or the second chemical compound comprises two coupled 1/2 spins in inequivalent molecular sites, wherein the relaxation of a long-lived state of the spin pair is measured as a function of the concentration of at least one of said first chemical compound for the determination of the equilibrium constant (KD) between the free state and the bound state

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

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

Plasmocitose Cutânea: Desafio Diagnóstico e Terapêutico num Doente com Apresentação Atípica

Cutaneous plasmacytosis is a rare dermatosis that is classically characterized by the presence of multiple papules and reddish- -brown nodules mainly in the trunk. It most commonly affects elderly Asians, with few reports in Caucasians and young patients. On histopathology there is a polyclonal proliferation of mature plasm cells. The objective of this report is to present a case of atypical manifestation of this rare disease, both due to clinical and epidemiological aspects.A plasmocitose cutânea é uma dermatose rara que em sua forma clássica se caracteriza pela presença de múltiplas pápulas e nódulos castanho-avermelhados principalmente no tronco. Acomete mais frequentemente asiáticos idosos, com poucos relatos em caucasianos e em pacientes jovens. Na histopatologia observa-se proliferação policlonal de plasmócitos maduros. O objetivo deste relato é apresentar um caso de manifestação atípica desta rara doença, tanto pelos aspectos clínicos quanto epidemiológicos