| 1. |
- Altincekic, Nadide, et al.
(författare)
-
Targeting the Main Protease (Mpro, nsp5) by Growth of Fragment Scaffolds Exploiting Structure-Based Methodologies
- 2024
-
Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 19:2, s. 563-574
-
Tidskriftsartikel (refereegranskat)abstract
- The main protease Mpro, 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 Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro 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.
|
|
| 2. |
- Chachaj, Annika, et al.
(författare)
-
Metabolomics of Interstitial Fluid, Plasma and Urine in Patients with Arterial Hypertension: New Insights into the Underlying Mechanisms
- 2020
-
Ingår i: Diagnostics (Basel). - : MDPI. - 2075-4418. ; 10:11
-
Tidskriftsartikel (refereegranskat)abstract
- There is growing evidence that lymphatic system plays a pivotal role in the pathogenesis of hypertension. Here, for the first time, the metabolome of interstitial fluid is analyzed in patients with arterial hypertension. Due to ethical issues to obtain human interstitial fluid samples, this study included only oncological patients after axillary lymph node dissection (ALND). These patients were matched into hypertensive (n = 29) and normotensive (n = 35) groups with similar oncological status. Simultaneous evaluation of interstitial fluid, plasma, and urine was obtained by combining high-resolution proton nuclear magnetic resonance (1H NMR) spectroscopy with chemometric analysis. Orthogonal partial least squares discriminant analysis (OPLS-DA) provided a clear differentiation between the hypertension and normotensive group, with the discrimination visible in each biofluid. In interstitial fluid nine potential metabolomic biomarkers for hypertension could be identified (creatinine, proline, pyroglutamine, glycine, alanine, 1-methylhistidine, the lysyl group of albumin, threonine, lipids), seven distinct markers in plasma (creatinine, mannose, isobutyrate, glycine, alanine, lactate, acetate, ornithine), and seven respectively in urine (methylmalonate, citrulline, phenylacetylglycine, fumarate, citrate, 1-methylnicotinamide, trans-aconitate). Biomarkers in plasma and urine allowed for the identification of specific biochemical pathways involved in hypertension, as previously suggested. Analysis of the interstitial fluid metabolome provided additional biomarkers compared to plasma or urine. Those biomarkers reflected primarily alterations in the metabolism of lipids and amino acids, and indicated increased levels of oxidative stress/inflammation in patients with hypertension.
|
|
| 3. |
- Clifton, Luke A., et al.
(författare)
-
Creation of distinctive Bax-lipid complexes at mitochondrial membrane surfaces drives pore formation to initiate apoptosis
- 2023
-
Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 9:22
-
Tidskriftsartikel (refereegranskat)abstract
- 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 whichwere 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.
|
|
| 4. |
- Clifton, Luke A., et al.
(författare)
-
Insight into Bcl-2 proteins' functioning at mitochondrial membrane level
- 2023
-
Ingår i: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 122:3S1, s. 232a-232a
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 5. |
- Dudka, Ilona, et al.
(författare)
-
Comprehensive metabolomics analysis of prostate cancer tissue in relation to tumor aggressiveness and TMPRSS2-ERG fusion status
- 2020
-
Ingår i: BMC Cancer. - : BioMed Central. - 1471-2407 .- 1471-2407. ; 20:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Prostate cancer (PC) can display very heterogeneous phenotypes ranging from indolent asymptomatic to aggressive lethal forms. Understanding how these PC subtypes vary in their striving for energy and anabolic molecules is of fundamental importance for developing more effective therapies and diagnostics. Here, we carried out an extensive analysis of prostate tissue samples to reveal metabolic alterations during PC development and disease progression and furthermore between TMPRSS2-ERG rearrangement-positive and -negative PC subclasses.Methods: Comprehensive metabolomics analysis of prostate tissue samples was performed by non-destructive high-resolution magic angle spinning nuclear magnetic resonance (H-1 HR MAS NMR). Subsequently, samples underwent moderate extraction, leaving tissue morphology intact for histopathological characterization. Metabolites in tissue extracts were identified by H-1/P-31 NMR and liquid chromatography-mass spectrometry (LC-MS). These metabolomics profiles were analyzed by chemometric tools and the outcome was further validated using proteomic data from a separate sample cohort.Results: The obtained metabolite patterns significantly differed between PC and benign tissue and between samples with high and low Gleason score (GS). Five key metabolites (phosphocholine, glutamate, hypoxanthine, arginine and alpha-glucose) were identified, who were sufficient to differentiate between cancer and benign tissue and between high to low GS. In ERG-positive PC, the analysis revealed several acylcarnitines among the increased metabolites together with decreased levels of proteins involved in beta-oxidation; indicating decreased acyl-CoAs oxidation in ERG-positive tumors. The ERG-positive group also showed increased levels of metabolites and proteins involved in purine catabolism; a potential sign of increased DNA damage and oxidative stress.Conclusions: Our comprehensive metabolomic analysis strongly indicates that ERG-positive PC and ERG-negative PC should be considered as different subtypes of PC; a fact requiring different, sub-type specific treatment strategies for affected patients.
|
|
| 6. |
- Dudka, Ilona, et al.
(författare)
-
Metabolomic profiles of intact tissues reflect clinically relevant prostate cancer subtypes
- 2023
-
Ingår i: Journal of Translational Medicine. - : BioMed Central (BMC). - 1479-5876 .- 1479-5876. ; 21:1
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 7. |
- Dudka, Ilona, et al.
(författare)
-
Metabolomic profiling reveals plasma GlycA and GlycB as a potential biomarkers for treatment efficiency in rheumatoid arthritis
- 2021
-
Ingår i: Journal of Pharmaceutical and Biomedical Analysis. - : Elsevier. - 0731-7085 .- 1873-264X. ; 197
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 8. |
- Fohringer, Christian, et al.
(författare)
-
Integrating omics to characterize eco‐physiological adaptations : How moose diet and metabolism differ across biogeographic zones
- 2021
-
Ingår i: Ecology and Evolution. - : John Wiley & Sons. - 2045-7758. ; 11:7, s. 3159-3183
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 9. |
- Gröbner, Gerhard, et al.
(författare)
-
Solid-state NMR methods for studying membrane systems
- 2020
-
Ingår i: Solid-state NMR. - : Institute of Physics Publishing (IOPP). - 9780750325301 - 9780750325325 ; , s. 1.1-1.30
-
Bokkapitel (refereegranskat)abstract
- Human life would not exist without biological membranes, which are fascinating lamellar bilayers composed of lipids and proteins. These incredibly thin leaflets are spontaneously formed in aqueous environment by lipids, which are amphiphilic molecules occurring in huge variations with respect to their hydrophilic headgroups and their hydrophobic fatty acid chains. Eukaryotic cells can easily obtain over a thousand different lipid variations. These lipids provide membranes with their basic physicochemical features, essential to exert their biological function in a concerted action with their inherent proteins. To understand the unique features of membranes-from the individual lipid to the cooperative structural and dynamic behavior of the entire leaflet -NMR has provided a wealth of atomic-level insight. In contrast to many other structural biology approaches, NMR can use non-perturbing reporters (naturally or synthetically introduced) to monitor the behavior of the membrane under physio±logical conditions, including elevated temperature, and it can follow the structural and dynamic response of membranes and their constituents upon physiological activities. This way it can elucidate the essential lipid-lipid and lipid-protein interactions that underpin the membrane's function. NMR is ideal as it can exploit the naturally occurring nuclei (31P, 14N, 13C, 1H) within the lipids as well as synthetically incorporated deuterons as non-perturbing reporters of the structure and dynamics within the bilayer. In this manner a wide range of membrane functions have been elucidated, ranging from basic interactions with ions, small drug molecules, but also to peptides and proteins involved in numerous biological events. NMR has also contributed to the study of domain formation in membranes, where nanodomains and rafts have emerged as important factors in the regulation of several important biological processes. Some of the recent exciting developments are summarized in this chapter.
|
|
| 10. |
- Nadeem, Aftab, et al.
(författare)
-
Protein-lipid interaction at low pH induces oligomerization of the MakA cytotoxin from Vibrio cholerae
- 2022
-
Ingår i: eLIFE. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 11
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
