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The main protease M^pro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with M^pro. These investigations resulted in the four-armed compound 35b that binds directly to M^pro. 35b could be cocrystallized with M^pro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.

Apotosis is an essential process tightly regulated by the Bcl-2 protein family where proapoptotic Bax triggers cell death by perforating the mitochondrial outer membrane. Although intensively studied, the molecular mechanism by which these proteins create apoptotic pores remains elusive. Here, we show that Bax creates pores by extracting lipids from outer mitochondrial membrane mimics by formation of Bax/lipid clusters that are deposited on the membrane surface. Time-resolved neutron reflectometry and Fourier transform infrared spectroscopy revealed two kinetically distinct phases in the pore formation process, both of which were critically dependent on cardiolipin levels. The initially fast adsorption of Bax on the mitochondrial membrane surface is followed by a slower formation of pores and Bax-lipid clusters on the membrane surface. Our findings provide a robust molecular understanding of mitochondrial membrane perforation by cell-killing Bax protein and illuminate the initial phases of programmed cellular death. Bax initiates apoptosis by perforating mitochondrial membranes via formation of pores and extramembranous Bax-lipid complexes.

Programmed cell death (apoptosis) is essential in life. In its intrinsic apoptotic pathway opposing members of the B-cell lymphoma 2 (Bcl-2) protein family control the permeability of the mitochondrial outer membrane (MOM) and the release of apoptotic factors such as cytochrome c. Any misregulation of this process can cause disorders most prominently cancer, where often upregulation of cell protecting (anti-apoptotic) Bcl-2 members such as the Bcl-2 membrane protein itself plays a notorious role by blocking MOM perforation by - often drug induced - apoptotic proteins such as Bax which would cause cancer cell death normally. Here, we apply neutron reflectometry (NR) on supported lipid bilayers which mimic MOM environment and solid state/liquid state NMR spectroscopy to unravel the molecular basis driving opposing proteins to interact with each other at the MOM; a mechanism which is not really understood yet due to lack of high-resolution structural insight. Based on our central hypothesis that Bcl-2 drives its cell-protecting function at a membrane-embedded location as revealed by NR (1), we focus i) to determine the structure of human Bcl-2 protein in its membrane setting by combining solution and solid-state NMR; ii) use NR to study the kinetics and lipid/protein pore assemblied upon binding of Bax to mitochondrial membranes and its membrane destroying activities there; and iii) unravel the nature of direct interaction between Bcl-2 and Bax to neutralize each other. Knowledge generated here, will be indispensable in understanding the regulative function of the Bcl-2 family at mitochondrial membranes.

Background: Prostate cancer (PC) is a heterogenous multifocal disease ranging from indolent to lethal states. For improved treatment-stratification, reliable approaches are needed to faithfully differentiate between high- and low-risk tumors and to predict therapy response at diagnosis.

Methods: A metabolomic approach based on high resolution magic angle spinning nuclear magnetic resonance (HR MAS NMR) analysis was applied on intact biopsies samples (n = 111) obtained from patients (n = 31) treated by prostatectomy, and combined with advanced multi- and univariate statistical analysis methods to identify metabolomic profiles reflecting tumor differentiation (Gleason scores and the International Society of Urological Pathology (ISUP) grade) and subtypes based on tumor immunoreactivity for Ki67 (cell proliferation) and prostate specific antigen (PSA, marker for androgen receptor activity).

Results: Validated metabolic profiles were obtained that clearly distinguished cancer tissues from benign prostate tissues. Subsequently, metabolic signatures were identified that further divided cancer tissues into two clinically relevant groups, namely ISUP Grade 2 (n = 29) and ISUP Grade 3 (n = 17) tumors. Furthermore, metabolic profiles associated with different tumor subtypes were identified. Tumors with low Ki67 and high PSA (subtype A, n = 21) displayed metabolite patterns significantly different from tumors with high Ki67 and low PSA (subtype B, n = 28). In total, seven metabolites; choline, peak for combined phosphocholine/glycerophosphocholine metabolites (PC + GPC), glycine, creatine, combined signal of glutamate/glutamine (Glx), taurine and lactate, showed significant alterations between PC subtypes A and B.

Conclusions: The metabolic profiles of intact biopsies obtained by our non-invasive HR MAS NMR approach together with advanced chemometric tools reliably identified PC and specifically differentiated highly aggressive tumors from less aggressive ones. Thus, this approach has proven the potential of exploiting cancer-specific metabolites in clinical settings for obtaining personalized treatment strategies in PC.

The Hsp100 family member ClpB is a protein disaggregase which solubilizes and reactivates stress-induced protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. In the pathogenic bacterium Francisella tularensis, ClpB is involved in type VI secretion system (T6SS) disassembly through depolymerization of the IglA-IglB sheath. This leads to recycling and reassembly of T6SS components and this process is essential for the virulence of the bacterium. Here we report the backbone chemical shift assignments and 15N relaxation-based backbone dynamics of the N-terminal substrate-binding domain of ClpB (1-156).

Programmed mammalian cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of dysfunctional cells. In its intrinsic (mitochondrial) pathway, opposing members of the Bcl-2 (B cell lymphoma 2) protein family meet at the mitochondrial outer membrane (MOM) to control its integrity. Any imbalance can cause disorders, with upregulation of the cell-guarding antiapoptotic Bcl-2 protein itself being common in many, often incurable, cancers. Normally, the Bcl-2 protein itself is embedded in the MOM where it sequesters cell-killing apoptotic proteins such as Bax (Bcl-2-associated X protein) that would otherwise perforate the MOM and subsequently cause cell death. However, the molecular basis of Bcl-2’s ability to recognize those apoptotic proteins via their common BH3 death motifs remains elusive due to the lack of structural insight. By employing nuclear magnetic resonance on fully functional human Bcl-2 protein in membrane-mimicking micelles, we identified glycine residues across all functional domains of the Bcl-2 protein and could monitor their residue-specific individual response upon the presence of a Bax-derived 36aa long BH3 domain. The observed chemical shift perturbations allowed us to determine the response and individual affinity of each glycine residue and provide an overall picture of the individual roles by which Bcl-2’s functional domains engage in recognizing and inhibiting apoptotic proteins via their prominent BH3 motifs. This way, we provide a unique residue- and domain-specific insight into the molecular functioning of Bcl-2 at the membrane level, an insight also opening up for interfering with this cell-protecting mechanism in cancer therapy.

The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.

Evasion from programmed cell death (apoptosis) is the main hallmark of cancer and a major cause of resistance to therapy. Many tumors simply ensure survival by over-expressing the cell-protecting (anti-apoptotic) Bcl-2 membrane protein involved in apoptotic regulation. However, the molecular mechanism by which Bcl-2 protein in its mitochondrial outer membrane location protects cells remains elusive due to the absence of structural insight; and current strategies to therapeutically interfere with these Bcl-2 sensitive cancers are limited. Here, we present an NMR-based approach to enable structural insight into Bcl-2 function; an approach also ideal as a fragment-based drug discovery platform for further identification and development of promising molecular Bcl-2 inhibitors. By using solution NMR spectroscopy on fully functional intact human Bcl-2 protein in a membrane-mimicking micellar environment, and constructs with specific functions remaining, we present a strategy for structure determination and specific drug screening of functional subunits of the Bcl-2 protein as targets. Using 19F NMR and a specific fragment library (Bionet) with fluorinated compounds we can successfully identify various binders and validate our strategy in the hunt for novel Bcl-2 selective cancer drug strategies to treat currently incurable Bcl-2 sensitive tumors.

1. With accelerated land conversion and global heating at northern latitudes, it becomes crucial to understand, how life histories of animals in extreme environments adapt to these changes. Animals may either adapt by adjusting foraging behavior or through physiological responses, including adjusting their energy metabolism or both. Until now, it has been difficult to study such adaptations in free‐ranging animals due to methodological constraints that prevent extensive spatiotemporal coverage of ecological and physiological data.

2. Through a novel approach of combining DNA‐metabarcoding and nuclear magnetic resonance (NMR)‐based metabolomics, we aim to elucidate the links between diets and metabolism in Scandinavian moose Alces alces over three biogeographic zones using a unique dataset of 265 marked individuals.

3. Based on 17 diet items, we identified four different classes of diet types that match browse species availability in respective ecoregions in northern Sweden. Individuals in the boreal zone consumed predominantly pine and had the least diverse diets, while individuals with highest diet diversity occurred in the coastal areas. Males exhibited lower average diet diversity than females.

4. We identified several molecular markers indicating metabolic constraints linked to diet constraints in terms of food availability during winter. While animals consuming pine had higher lipid, phospocholine, and glycerophosphocholine concentrations in their serum than other diet types, birch‐ and willow/aspen‐rich diets exhibit elevated concentrations of several amino acids. The individuals with highest diet diversity had increased levels of ketone bodies, indicating extensive periods of starvation for these individuals.

5. Our results show how the adaptive capacity of moose at the eco‐physiological level varies over a large eco‐geographic scale and how it responds to land use pressures. In light of extensive ongoing climate and land use changes, these findings pave the way for future scenario building for animal adaptive capacity.

In this pilot study, we carried out metabolic profiling of patients with rheumatoid arthritis (RA) starting therapy with biological disease-modifying antirheumatic drugs (bDMARDs). The main aim of the study was to assess the occurring metabolic changes associated with therapy success and metabolic pathways involved. In particular, the potential of the metabolomics profiles was evaluated as therapeutically valuable prognostic indicators of the effectiveness of bDMARD treatment to identify responders versus non-responders prior to implementing treatment.

Plasma metabolomic profiles of twenty-five patients with RA prior bDMARD treatment and after three months of therapy were obtained by 1H NMR, liquid chromatography - mass spectrometry, and gas chromatography - mass spectrometry and evaluated by statistical and multivariate analyses.

In the group of responders, significant differences in their metabolic patterns were seen after three months of the bDMARD therapy compared with profiles prior to treatment. We identified 24 metabolites that differed significantly between these two-time points mainly belonging to amino acid metabolism, peptides, lipids, cofactors, and vitamins and xenobiotics. Eleven metabolites differentiated responders versus non-responders before treatment. Additionally, N-acetylglucosamine and N-acetylgalactosamine (GlycA) and N-acetylneuraminic acid (GlycB) persisted significant in comparison responders to non-responders after three months of therapy. Moreover, those two metabolites indicated prediction of response potential by results of receiver-operating characteristic (ROC) curve analysis.

The applied analysis provides novel insights into the metabolic pathways involved in RA patient’s response to bDMARD and therapy effectiveness. GlycA and GlycB are promising biomarkers to identify responding patients prior onset of bDMARD therapy.