NMR Spectroscopy Topical Group Meetings

Abstract: Hepatitis B Virus (HBV) is a double stranded DNA virus and belongs to hepadnavirdae family. HBV infection causes a severe liver infectious disease and contributed about half of hepatocellular carcinoma (HCC). At least 257 million people are chronically infected with HBV, with an estimated 650,000 deaths per year from HBV associated HCC, mainly in Asia. We focused our attention on metabolic reprogramming induced by HBV infection. We found that HBV replication induces the promotions of central carbon metabolism, biosynthesis of nucleotides and total fatty acids. HBV infection induces up-regulation of the biosynthesis of hexosamine and phosphatidylcholine through activating glutamine-fructose-6-phosphate amidotransferase 1 (GFAT1), and choline kinase ? (CHKA), PCYT1A and LPP1. In addition, we use stable isotope-based metabolomics to demonstrate that glutamine consumption and its flux to TCA cycle, non-essential amino acids, and nucleotide synthesis are increased in HBV-infected cells. However, HBV-infected cells are insensitive to exogenous glutamine deprivation. We discovered for the first time that reprogramming ammonia-recycling metabolism towards glutamine synthesis is vital for the survival of HBV-infected cells under glutamine starvation status. Finally, HBV-induced HCC was metabolically different from non-viral induced HCC. Our findings provided further understanding of HBV infection.

Abstract: The detection and characterization of intermediates in catalytic reactions is crucial for the rational optimization of reaction conditions. In photocatalysis mechanistic studies mainly focused on the initial photoexcitation steps via time resolved UV spectroscopy supported by its extreme time resolution and sensitivity. In this talk I will present tech­niques and methods to extend the power of NMR spectroscopy in terms of structure elucidation, mechanistic studies and detection of intermediates, aggregation or intermolecular interactions to photocatalysis despite its low sensitivity and time resolution. First our LED based NMR illumination device [1] and the principles of NMR in photocatalysis will be introduced on a flavin catalyzed photooxidation, which allow for new insights into one- versus two-electron processes usually inacces­sible to UV/Vis [2]. Next the sequencing of intermediates and light on/off studies will be discussed on an Aza Henry reaction, which allow for the first time to differ between proton and H-atom transfer pathways [3]. The triple combination illumination/NMR/UV [2] will be presented together with the inclusion of radical species into NMR reaction profiles and the structure elucida­tion of thermally labile photoswitches [4]. As further techniques the NMR access to intermediates below the detection limit [5] and the sequencing of tiny intermediates [6] will be shown as well as the importance of aggregation and H-bond networks in photocatalysis [7].
[1] C. Feldmeier, H. Bartling, E. Riedle, R.M. Gschwind, J. Magn. Res., 2013, 232, 39.
[2] C. Feldmeier, H. Bartling, K. Magerl, R.M. Gschwind, Angew. Chem. Int. Ed., 2015, 54, 1347.
[3] H. Bartling, A. Eisenhofer, B. König, R.M. Gschwind, JACS 2016, 138, 11860-11871.
[4] A. Seegerer, P. Nitschke, R.M. Gschwind, Angew. Chem. Int. Ed. 2018, 57, 7493-7497.
[5] L. Nanjundappa, A. Seegerer, J. Hioe, R.M. Gschwind, JACS 2018, 140, 1855-1862.
[6]. S. Wang, L. Nanjundappa, J. Hioe, R. M. Gschwind, B. König, Chem. Sci., 2019, 10, 4580.
[7] N. Berg, D. Horinek, R.M. Gschwind, submitted.

Abstract: PSYCHE1-3 has rapidly become one of the standard methods for measuring “pure shift” spectra – broadband homodecoupled spectra, in which multiplet structure is suppressed and a single peak appears for each distinct chemical shift. Among the numerous competing methods used for pure shift NMR,4-6 the sensitivity, generality, and relative freedom from artefacts of PSYCHE have made it the method of choice in many cases.
No experimental technique is perfect: any NMR experiment, from pulse-acquire up to the most sophisticated solid-state experiments, requires compromises to be made between sensitivity, resolution, ease of use, quantitativity, spectral purity, etc. PSYCHE is successful because it offers a particularly favourable trade-off between the first three of these desiderata and the remaining two. In particular, it allows the user to choose the most appropriate compromise between sensitivity and spectral purity by varying the flip angle ? of the two “chirp” pulses used. The intensity of the pure shift signals is proportional to sin2?, while that of “recoupling” artefacts at ±1/2 Hz either side is proportional to sin4?. For a dilute sample where we are struggling for signal-to-noise ratio we can use a high ?, since the artefacts will be hidden by the noise, but for a high dynamic range sample we would use a low ?, to avoid recoupling artefacts being confused with the signals of dilute species.
To date, the need for this compromise has simply been accepted. But if it were possible to suppress the recoupling artefacts, we could have a PSYCHE experiment with simultaneously improved sensitivity, resolution, and spectral purity . . .
1. “Ultra-high resolution NMR spectroscopy”. Mohammadali Foroozandeh, Ralph W. Adams, Nicola Meharry, Damien Jeannerat, Mathias Nilsson and Gareth A. Morris, Angew. Chem., Int. Ed. 53, 6990-6992 (2014). DOI: 10.1002/anie.201404111.
2. “Ultrahigh-resolution Total Correlation NMR Spectroscopy”. Mohammadali Foroozandeh, Ralph W. Adams, Mathias Nilsson and Gareth A. Morris, J. am. Chem. Soc. 136, 11867-11869 (2014). DOI: 10.1021/ja507201t.
3. “PSYCHE Pure Shift NMR Spectroscopy”. Mohammadali Foroozandeh, Gareth A. Morris and Mathias Nilsson, Chem. Eur. J. 24, 13988 – 14000 (2018). DOI: 10.1002/chem.201800524.
4. “Pure Shift NMR Spectroscopy”, Ralph W. Adams, eMagRes 3, 1–15 (2014). DOI 10.1002/9780470034590.emrstm1362.
5. “Broadband 1H homodecoupled NMR experiments: Recent developments, methods and applications”, Laura Castañar and Teodor Parella, Magn. Reson. Chem. 53, 399-426 (2015). DOI: 10.1002/mrc.4266.
6. “Pure shift NMR”, Klaus Zangger, Prog. Nucl. Magn. Reson. Spectrosc. 86–87, 1-20 (2015). DOI: 10.1016/j.pnmrs.2015.02.002.

Abstract: Signal processing methods evolved significantly since invention of Fourier NMR in the beginning of 70’s. Broad use of high-resolution two-and higher dimensional experiments evoked development of non-uniform sampling (NUS) techniques. Great progress in computer hardware and new mathematical algorithms allowed complex processing schemes such as compressed sensing, tensor decomposition, neural networks, etc. The lecture will provide overview of the latest trends and ideas in this field as well as example of applications in bimolecular NMR.

Abstract: A variety of intrinsically disordered proteins (IDPs) or protein regions (IDRs) with important, yet unexplored, functional and regulatory roles is emerging. NMR provides a unique tool to investigate structurally heterogeneous, highly flexible proteins. The contribution of exclusively heteronuclear NMR experiments based on 13C direct detection to access atomic resolution structural and dynamic information on IDPs/IDRs will be discussed. Several examples of particular features or motives that often occur in IDPs/IDRs will be presented, revealing novel structural and dynamic modules not yet described in the PDB.

Abstract: The prediction of the quaternary structure of biomolecular macromolecules is of paramount importance for fundamental understanding of cellular processes and drug design. In the era of integrative structural biology, one way of increasing the accuracy of modelling methods used to predict the structure of biomolecular complexes is to include as much experimental or predictive information as possible in the process.
We have developed for this purpose a versatile information-driven docking approach HADDOCK (https://www.bonvinlab.org/software), accessible through a user-friendly web portal at https://wenmr.science.uu.nl. HADDOCK can integrate information derived from biochemical, biophysical or bioinformatics methods to enhance sampling, scoring, or both. The information that can be integrated is quite diverse: interface restraints from NMR, mutagenesis experiments, or bioinformatics predictions; shape data from small-angle X-ray scattering and cryo-electron microscopy experiments. In my talk, I will introduce HADDOCK and illustrate its capabilities with various examples including some recent work on the use of shape information to guide the docking of both macromolecular and small molecule complexes.

Abstract: The Regulator of Ty1 Transposition protein 106 (Rtt109) is a fungal histone acetyltransferase required for histone H3 K9, K27 and K56 acetylation. These acetylation sites have been linked to processing and folding of nascent H3 and play an integral role in replication- and repair-coupled nucleosome assembly. Rtt109 is unique in its activation, performed by two structurally unrelated histone chaperones, Asf1 and Vps75. These proteins stimulate Rtt109 activity via different mechanisms1. Rtt109 - Asf1 association has been proposed to be responsible for K56 acetylation, while the Rtt109-Vps75 interaction is required for K9 acetylation2,3.
In our work we find that Rtt109, Vps75 and Asf1 are capable of assembling as a previously uncharacterized complex onto the substrate H3-H4 dimer. Using an integrative structural biology approach based on a powerful combination of solution state NMR and small angle neutron scattering (SANS) we solve the structure of this complex and provide a mechanistic explanation for the enzyme activity4. We show that Vps75 promotes acetylation of residues in the H3 N-terminal tail by engaging it in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.
References: 
1. D’Arcy S., Luger K. Curr Opin Struct Biol, 21(6), 728–734 (2011).
2. Fillingham J., Recht J., Greenblatt JF. Mol Cell Biol. 28, 4342–4353 (2008).
3. Driscoll R., Hudson A., Jackson SP. Science, 315, 649–652 (2007).
4. Danilenko N., Lercher L., Gabel F., Kirkpatrick J., Carlomagno T. Nature Commun.10, Article number: 3435 (2019)

Abstract: Recently, there has been increasing interest in the mechanochemical synthesis (MS) of different forms of active pharmaceutical ingredients (APIs), including crystalline polymorphs, solvates, amorphous solids, and pharmaceutical cocrystals (PCCs). MS may enable the rational design of new solid forms with tuneable properties (e.g., solubility, stability, and bioavailability), and adheres the tenets of green chemistry, including the use of little or no solvent, low energy inputs, reduced waste products, and facile scalability.
Crucial to the successful design and synthesis of new solid forms of APIs and related PCCs is their structural characterization and prediction. An emerging discipline for this purpose is NMR crystallography, which combines solid-state NMR (SSNMR) spectroscopy, X-ray diffraction (XRD), and plane-wave DFT computational methods. We are currently investigating the use of quadrupolar nuclei (i.e., nuclear spins > 1/2, including 35Cl, 14N, and 17O) for structural prediction and refinement in the context of NMR crystallographic methods. The reason for this is twofold: (i) electric field gradient (EFG) tensors at quadrupolar nuclei give rise to unique powder patterns for even the most subtle structural changes and/or differences, allowing for unambiguous structure-property correlations, and (ii) EFG tensors are facile to compute using DFT in comparison to magnetic shielding tensors.
In this lecture, I will discuss: (1) MS of solid forms of HCl salts of APIs, PCCs, and nutraceuticals (including novel cocrystals of HCl salts of fluoxetine, promazine, promethazine, and chlorpromazine) prepared by ball milling and their characterization by multinuclear SSNMR; and (2) the use of new dispersion-corrected DFT-D2* methods that utilize 35Cl, 14N, and 17O electric field gradient (EFG) tensors obtained from multinuclear SSNMR experiments to evaluate the soundness of structural refinements, and the development of a new NMR crystallographic protocol for prediction of unknown structures of HCl API salts.

Abstract: Non-invasive imaging of metabolic pathways in neurological disease has been a long-standing goal to monitor disease progression or therapy efficacy. Positron emission tomography (PET) in combination with 2-18F-fluoro-2-deoxy-D-glucose (FDG) is currently the only clinically viable metabolic imaging method. However, the high uptake of FDG by normal brain drastically reduces the image contrast and the usefulness of FDG-PET in studying neurological disease. MR-based methods (1H, 13C, hyperpolarized 13C MRS) are promising but have failed to reach clinical significance due to technical complexity and lack of robustness and/or sensitivity. Deuterium metabolic imaging (DMI) is a novel MR-based method that uses the favorable MR characteristics of deuterium to map metabolism in vivo in 3D. The low intrinsic sensitivity of 2H NMR is offset by favorable T1 and T2 relaxation times, a large nuclear spin and a sparse NMR spectrum devoid of strong water and lipid signals. By combining 2H NMR with deuterated glucose administration (oral or intravenous), MR spectroscopic imaging and signal quantification through spectral fitting we were able to generate deuterium metabolic images of brain, brain tumor and liver metabolism in vivo. Our first-in-human DMI maps of glucose metabolism in healthy brain and in patients with high grade brain tumors illustrate that DMI has the potential to become a robust and widely applicable brain imaging method with strong clinical utility.

Abstract: Highly complex biological samples present challenging analysis problems for the field of metabolomics. Ideally, platforms that provide broad metabolome coverage allow the opportunity for deep insights into biological problems, while excellent quantitation ensures good data quality and allows an ability to compare across studies. However, these goals can be difficult to achieve on a routine basis because the highly complex sample matrix often precludes reliable measurements of many metabolites and complicates quantitation efforts. Due to its exquisite quantitative capabilities and reproducibility, NMR spectroscopy has much to offer the field of metabolomics, especially in the area of new methods development.
We can exploit NMR’s quantitative abilities using a simple protein precipitation procedure that allows the absolute quantitation of over 70 metabolites using a single standard compound. These metabolites, including some at even sub-micromolar concentrations, span a broad range of classes and pathways, including organic and amino acids, as well as energy metabolites and co-enzymes (NAD+, NADH, NADP+, NADPH), which we show can be measured simultaneously in a variety of tissue extracts and even whole human blood. This quantitative NMR approach is also useful for calibrating quantitative mass spectrometry measurements, without the use of internal or external metabolite standards and has led to some unusual findings about the stability of well-known metabolites such as glutamine and others.
Combining NMR’s quantitative capabilities with statistical methods has led to an improved method to aid identification of metabolites in complex samples. In an approach we call “eRANSY”, a new solvent extraction protocol and Ratio Analysis is used to simplify the NMR spectra dramatically to allow for improved unknown identification. Finally, we are using an old econometrics approach called seemingly unrelated regression (SUR) that allows us to model the effects of so-called confounding factors such as age, BMI, gender, and etc., which have a significant effect on the measured metabolite levels. We show that SUR can unravel these effects, and by building models using groups of biologically related metabolites we can achieve improved differentiation of patients in the context of colon cancer and polyp detection.

Abstract: In natural products-based drug discovery investigations, determining the chemical structure of the targeted materials represents one of the more time consuming challenges. This is especially true when the molecular architecture is novel and the available material is present in limited quantity. To help meet this challenge, we developed a novel NMR-based machine-learning tool ‘Small Molecule Accurate Recognition Technology’ (SMART 2.0) for the automatic annotation of new molecular structures. This is based on query of the 1H-13C HSQC spectrum for a new compound with SMART 2.1, a convolutional neural net trained tool that was taught on more than 25,000 experimental spectra and then additionally populated with calculated HSQCs from another 90,000 natural products. When combined with the NUS-ASAP 1H-13C HSQC pulse sequences, the SMART tool is exceptionally quick, in some cases reducing NMR-based structure determination time to minutes. Moreover, recent advances of the SMART have revealed its utility in the analysis of mixtures. In this presentation, the exceptional capacity of SMART will be demonstrated with the rapid annotation of new natural products from various collections of marine cyanobacteria. This study exemplifies the revolutionary potential of artificial intelligence assisted analytical approaches to overcome long standing challenges in natural products science.

Abstract: Small molecule inhibitors targeting the deubiquitinase Ubiquitin Specific Protease 7 (USP7) have potential as cancer therapeutics. Multiple clinical trials have shown that its down stream effector, murine double minute 2 (MDM2) is a viable drug target (1). Inhibition of USP7 leads to the same decrease in levels of MDM2 as MDM2 inhibition (2). Inhibition of either proteins ultimately targets the function of the tumor suppressor and cellular stress response monitor P53 (3). Furthermore, USP7 has been implicated in the regulation of several other cell cycle, tumor suppressor and oncogenic proteins (4).
Using ligand-based NMR in an activity agnostic fragment-based lead identification (FBLD) effort (5), we have identified small molecule binders to several novel binding sites in USP7. Guided by NMR, protein-ligand complex X-ray structures and biochemical assays, several fragment classes were structurally characterized and chemically modified to bind USP7 with increased affinity and activity. Binders to unique site in the ""palm"" of USP7, the Ubiquitin binding region, were shown to be active in a biochemical and cellular context.
The palm series are of mechanistic interest as they imply a very distinct mechanism of action independent of the catalytic triad by binding in a region that is directly involved in USP7-Ubiquitin interaction. The palm fragments bind USP7 in a site near Asp 305 and Glu 308, residues whose side chains are involved in polar interactions with the side chain of Lys 48 in Ubiquitin present in the Ubiquitin complex crystal structure 1NBF (6). These results suggest a possible mechanism for both steric interference of Ubiquitin binding but also the means by which USP7 recognizes K48 linkages.
I will show using biophysical and biochemical methods as well as cellular assays the relevance of the identified ligands and their binding site and support the postulated mechanism of action with information of their effects on the interaction of USP7 with mono- and di-Ubiquitin.
1 Ray-Coquard, I. et al. Effect of the MDM2 antagonist RG7112 on the P53 pathway in patients with MDM2-amplified, well-differentiated or dedifferentiated liposarcoma: an exploratory proof-of-mechanism study. The lancet oncology (2012). doi:10.1016/S1470-2045(12)70474-6.
2 Colland, F. et al. Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells. Molecular Cancer Therapeutics 8, 2286–2295 (2009).
3 Brown, C. J., Lain, S., Verma, C. S., Fersht, A. R. & Lane, D. P. Awakening guardian angels: drugging the p53 pathway. Nature Reviews Cancer 9, 862–873 (2009).
4 Nicholson, B. & Suresh Kumar, K. G. The multifaceted roles of USP7: new therapeutic opportunities. Cell Biochem Biophys 60, 61–68 (2011).
5 Methods Enzymol. Vol. 493 (2011)
6 Hu, M. et al. Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell 111, 1041–1054 (2002).