Rapid Protein-Ligand Affinity Determination by Photoinduced Hyperpolarized NMR.

The binding affinity determination of protein-ligand complexes is a cornerstone of drug design. State-of-the-art techniques are limited by lengthy and expensive processes. Building upon our recently introduced novel screening method utilizing photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR, we provide the methodological framework to determine binding affinities within 5-15 min using 0.1 mg of protein. The accuracy of our method is demonstrated for the affinity constants of peptides binding to a PDZ domain and fragment ligands binding to the protein PIN1. The method can also be extended to measure the affinity of nonphoto-CIDNP-polarizable ligands in competition binding experiments. Finally, we demonstrate a strong correlation between the ligand-reduced signals in photo-CIDNP-based NMR fragment screening and the well-established saturation transfer difference (STD) NMR. Thus, our methodology measures protein-ligand affinities in the micro- to millimolar range in only a few minutes and informs on the binding epitope in a single-scan experiment, opening new avenues for early stage drug discovery approaches.

A Benchmark Study of Protein-Fragment Complex Structure Calculations with NMR2.

Protein-fragment complex structures are particularly sought after in medicinal chemistry to rationally design lead molecules. These structures are usually derived using X-ray crystallography, but the failure rate is non-neglectable. NMR is a possible alternative for the calculation of weakly interacting complexes. Nevertheless, the time-consuming protein signal assignment step remains a barrier to its routine application. NMR Molecular Replacement (NMR2) is a versatile and rapid method that enables the elucidation of a protein-ligand complex structure. It has been successfully applied to peptides, drug-like molecules, and more recently to fragments. Due to the small size of the fragments, ca < 300 Da, solving the structures of the protein-fragment complexes is particularly challenging. Here, we present the expected performances of NMR2 when applied to protein-fragment complexes. The NMR2 approach has been benchmarked with the SERAPhic fragment library to identify the technical challenges in protein-fragment NMR structure calculation. A straightforward strategy is proposed to increase the method's success rate further. The presented work confirms that NMR2 is an alternative method to X-ray crystallography for solving protein-fragment complex structures.

Two-dimensional chromatography for enantiomeric analysis of mitotane and its metabolite o,p'-DDA in patients with adrenocortical carcinoma indicates enantioselective metabolism.

Mitotane is a chiral drug used to treat adrenocortical carcinoma, being metabolized to the o,p'-dichlorodiphenyl acetic acid (o,p'-DDA), also a chiral compound. Despite of its therapeutic significance, the overall ratios and enantiomers have not been known. In this study, we analyzed the enantiomers of mitotane and o,p'-DDA in the plasma of patients by a newly developed chiral-phase method employed in two-dimensional chromatography. Important differences were observed in the ratio of (S)/(R)-mitotane, which varied substantially from 1:1.2 to 1:10 whereas the (S)/(R)-o,p'-DDA ratio was relatively conserved, at approximately 2:1. These findings provide evidence for the enantioselective metabolism and provide a method for further analyses of mitotane and metabolites, which can explain the variation in the therapeutic response.

Fragment Screening and Fast Micromolar Detection on a Benchtop NMR Spectrometer Boosted by Photoinduced Hyperpolarization.

Fragment-based drug design is a well-established strategy for rational drug design, with nuclear magnetic resonance (NMR) on high-field spectrometers as the method of reference for screening and hit validation. However, high-field NMR spectrometers are not only expensive, but require specialized maintenance, dedicated space, and depend on liquid helium cooling which became critical over the recurring global helium shortages. We propose an alternative to high-field NMR screening by applying the recently developed approach of fragment screening by photoinduced hyperpolarized NMR on a cryogen-free 80 MHz benchtop NMR spectrometer yielding signal enhancements of up to three orders in magnitude. It is demonstrated that it is possible to discover new hits and kick-off drug design using a benchtop NMR spectrometer at low micromolar concentrations of both protein and ligand. The approach presented performs at higher speed than state-of-the-art high-field NMR approaches while exhibiting a limit of detection in the nanomolar range. Photoinduced hyperpolarization is known to be inexpensive and simple to be implemented, which aligns greatly with the philosophy of benchtop NMR spectrometers. These findings open the way for the use of benchtop NMR in near-physiological conditions for drug design and further life science applications.

Ultrafast Fragment Screening Using Photo-Hyperpolarized (CIDNP) NMR.

While nuclear magnetic resonance (NMR) is regarded as a reference in fragment-based drug design, its implementation in a high-throughput manner is limited by its lack of sensitivity resulting in long acquisition times and high micromolar sample concentrations. Several hyperpolarization approaches could, in principle, improve the sensitivity of NMR also in drug research. However, photochemically induced dynamic nuclear polarization (photo-CIDNP) is the only method that is directly applicable in aqueous solution and agile for scalable implementation using off-the-shelf hardware. With the use of photo-CIDNP, this work demonstrates the detection of weak binders in the millimolar affinity range using low micromolar concentrations down to 5 µM of ligand and 2 µM of target, thereby exploiting the photo-CIDNP-induced polarization twice: (i) increasing the signal-to-noise by one to two orders in magnitude and (ii) polarization-only of the free non-bound molecule allowing identification of binding by polarization quenching, yielding another factor of hundred in time when compared with standard techniques. The interaction detection was performed with single-scan NMR experiments of a duration of 2 to 5 s. Taking advantage of the readiness of photo-CIDNP setup implementation, an automated flow-through platform was designed to screen samples at a screening rate of 1500 samples per day. Furthermore, a 212 compounds photo-CIDNP fragment library is presented, opening an avenue toward a comprehensive fragment-based screening method.

Valor diagnóstico de los síntomas y signos clinicos en el Síndrome Tunelar Carpiano / Diagnosis value of clinics symptoms and signs in the Carpal Tunnel Syndrome

El Síndrome Tunelar Carpiano (S.T.C.) es la neuropatía por atrapamiento más frecuente del miembro superior. Existe en la literatura considerable discrepancia respecto a la sensibilidad y especificidad de síntomas y signos relacionados al S.T.C.. El objetivo de esté estudio fue determinar la validez diagnóstica de los síntomas y signos clínicos típicos de S.T.C. en pacientes con diagnósticos positivos por Estudio neurofisiológico (Gold Standard diagnóstico). Se estudiaron prospectivamente 73 manos con diagnóstico presuntivo de S.T.C.. Los mejores predictores de diagnóstico positivo de S.T.C. fueron: parestesias nocturnas que mejoran con el "shaking", falta de discriminación de dos puntos y atrofia tenar.

Carpal Tunner Syndrome (C.T.S.) is the most frequently recognized entrapment neuropathy on upper limbs. In the literature, there is a considerable disagreement about signs` and symptoms` sensibility and specificity related to C.T.S.. Our objective was to determine the diagnosis validity of typical signs and symptoms of C.T.S. in patients with positive neurophysiologic diagnosis (Gold Standard diagnosis). In a prospective way, we studied 73 hands with presumptive diagnosis of C.T.S.. The best predictors for a positive diagnosis were: numbness at night that reduce with shaking, impossibility to discriminate two points and thenar atrophy.