Mapping of Methyl Epitopes of a Peptide-Drug with Its Receptor by 2D STDD-Methyl TROSY NMR Spectroscopy.

The current trend in the biopharmaceutical market has boosted the development and production of biological drugs with high efficacy and fidelity for receptor binding. While high-resolution structural insights into binding epitopes of the receptor are indispensable for better therapeutic design, it is tedious and costly. In this work, we develop a protocol by integrating two well-known NMR-based solution-state methods. Saturation transfer double-difference with methyl-TROSY (STDD-Methyl TROSY NMR) was used to probe methyl binding epitopes of the ligand in a label-free environment. This study was carried out with Human insulin as a model peptide drug, with the insulin growth factor receptor (IGFR), which is an off-target receptor for insulin. Methyl epitopes identified from STDD-Methyl TROSY NMR spectroscopy were validated through the HADDOCK platform to generate a drug-receptor model. Since this method can be applied at natural abundance, it has the potential to screen a large set of peptide-drug interactions for optimum receptor binding. Thus, we propose STDD-Methyl TROSY NMR spectroscopy as a technique for rapid screening of biologics for the development of optimized biopharmaceutics.

Identifying type of sugar adulterants in honey: Combined application of NMR spectroscopy and supervised machine learning classification.

Nuclear magnetic resonance (NMR) is a powerful analytical tool which can be used for authenticating honey, at chemical constituent levels by enabling identification and quantification of the spectral patterns. However, it is still challenging, as it may be a person-centric analysis or a time-consuming process to analyze many honey samples in a limited time. Hence, automating the NMR spectral analysis of honey with the supervised machine learning models accelerates the analysis process and especially food chemistry researcher or food industry with non-NMR experts would benefit immensely from such advancements. Here, we have successfully demonstrated this technology by considering three major sugar adulterants, i.e., brown rice syrup, corn syrup, and jaggery syrup, in honey at varying concentrations. The necessary supervised machine learning classification analysis is performed by using logistic regression, deep learning-based neural network, and light gradient boosting machines schemes.

BF3·OEt2-Catalyzed Unexpected Stereoselective Formation of 2,4-trans-Diallyl-2-methyl-6-aryltetrahydro-2H-pyrans with Quaternary Stereocenters.

The present manuscript describes a convenient, mild, and highly stereoselective method for the allylation of δ-hydroxy-α,ß-unsaturated ketones having a benzylic hydroxyl group at the δ-position using allyltrimethylsilane mediated by BF3·OEt2, leading to 2,4-diallyl-2-methyl-6-aryltetrahydro-2H-pyran ring systems with quaternary carbon stereogenic centers. This represents the first example of a tandem isomerization followed by one C-O and two C-C bond-forming reactions in one pot. The isolation of TMS-protected lactol as an intermediate from the reaction strongly supports the proposed mechanistic pathway.

Human islet amyloid polypeptide (hIAPP) - a curse in type II diabetes mellitus: insights from structure and toxicity studies.

The human islet amyloid polypeptide (hIAPP) or amylin, a neuroendocrine peptide hormone, is known to misfold and form amyloidogenic aggregates that have been observed in the pancreas of 90% subjects with Type 2 Diabetes Mellitus (T2DM). Under normal physiological conditions, hIAPP is co-stored and co-secreted with insulin; however, under chronic hyperglycemic conditions associated with T2DM, the overexpression of hIAPP occurs that has been associated with the formation of amyloid deposits; as well as the death and dysfunction of pancreatic ß-islets in T2DM. Hitherto, various biophysical and structural studies have shown that during this process of aggregation, the peptide conformation changes from random structure to helix, then to ß-sheet, subsequently to cross ß-sheets, which finally form left-handed helical aggregates. The intermediates, formed during this process, have been shown to induce higher cytotoxicity in the ß-cells by inducing cell membrane disruption, endoplasmic reticulum stress, mitochondrial dysfunction, oxidative stress, islet inflammation, and DNA damage. As a result, several research groups have attempted to target both hIAPP aggregation phenomenon and the destabilization of preformed fibrils as a therapeutic intervention for T2DM management. In this review, we have summarized structural aspects of various forms of hIAPP viz. monomer, oligomers, proto-filaments, and fibrils of hIAPP. Subsequently, cellular toxicity caused by toxic conformations of hIAPP has been elaborated upon. Finally, the need for performing structural and toxicity studies in vivo to fill in the gap between the structural and cellular aspects has been discussed.

Natural compound safranal driven inhibition and dis-aggregation of α-synuclein fibrils.

Self-assembly of α-synuclein (α-Syn) is linked with a variety of neurodegenerative diseases collectively called as α-synucleiopathies. Therefore, discovering suitable inhibitors for this self-association process of α-Syn is a subject of intense research. In this background, we have demonstrated here that the natural compound, Safranal, delays/inhibits α-Syn fibrillation/aggregation, and we have also characterized its mode of action. The α-Syn fibrillation/aggregation kinetics studies in combination with TEM studies demonstrated that Safranal effectively inhibits α-Syn fibrillation/aggregation. NMR studies revealed that Safranal binds with α-Syn and stabilizes the monomeric protein. ANS fluorescence and CD measurements indicated that Safranal binds to the hydrophobic residues of the protein and causes delay in the formation of ß-sheet rich structures which are crucial for the fibrillation to occur. The results obtained from fluorescence quenching, NMR and ANS binding assays, when analysed taking into consideration the molecular structure of Safranal provide valuable insights into the mechanism of inhibition of α-Syn fibrillation/aggregation. We infer that inhibition of α-Syn fibrillation/aggregation is primarily driven by hydrophobic interactions between Safranal and the protein. Further, Safranal is also seen to dis-aggregates pre-formed α-Syn fibrils. These findings implicate that Safranal could become a potent therapeutic intervention in Parkinson's disease and other protein aggregation related disorders.

Triphala inhibits alpha-synuclein fibrillization and their interaction study by NMR provides insights into the self-association of the protein.

The process of assembly and accumulation of the intrinsically disordered protein (IDP), alpha-synuclein (αSyn) into amyloid fibrils is a pathogenic process leading to several neurodegenerative disorders such as Parkinson's disease, multiple system atrophy and others. Although several molecules are known to inhibit αSyn fibrillization, the mechanism of inhibition is just beginning to emerge. Here, we report the inhibition of fibrillization of αSyn by Triphala, a herbal preparation in the traditional Indian medical system of Ayurveda. Triphala was found to be a rich source of polyphenols which are known to act as amyloid inhibitors. ThT fluorescence and TEM studies showed that Triphala inhibited the fibrillization of αSyn. However, it was observed that Triphala does not disaggregate preformed αSyn fibrils. Further, native-PAGE showed that Triphala reduces the propensity of αSyn to oligomerize during the lag phase of fibrillization. Our NMR results showed that certain stretches of residues in the N-terminal and NAC regions of αSyn play an anchor role in the self-association process of the protein, thereby providing mechanistic insights into the early events during αSyn fibrillization.

NMR supersequences with real-time homonuclear broadband decoupling: Sequential acquisition of protein and small molecule spectra in a single experiment.

NOAH (NMR byOrderedAcquisition using 1H-detection) type of pure shift NMR pulse scheme has been designed for the efficient utilization of magnetization that presents in a spin-system under consideration. The proposed strategy, PROSMASH-HSQC2 (PROtein-HSQC and SMAll molecule-HSQC Signals with Homodecoupling) uses the real-time BIRD pure shift NMR strategy and two HSQC spectra (13C-HSQC for small molecules and 15N-HSQC for 15N-isotopic labelled proteins) can be recorded in a single NMR experiment. Thus, this method permits precise determination of drug-protein interactions at atomic levels by monitoring the chemical shift perturbations, and will have potential applications in drug discovery programs.

An efficient combination of BEST and NUS methods in multidimensional NMR spectroscopy for high throughput analysis of proteins.

Application of Non Uniform Sampling (NUS) along with Band-selective Excitation Short-Transient (BEST) NMR experiments has been demonstrated for obtaining the important residue-specific atomic level backbone chemical shift values in short durations of time. This application has been demonstrated with both well-folded (ubiquitin) and unfolded (α-synuclein) proteins alike. With this strategy, the experiments required for determining backbone chemical shifts can be performed very rapidly, i.e., in ∼2 hours of spectrometer time, and this data can be used to calculate the backbone folds of proteins using well established algorithms. This will be of great value for structural proteomic investigations on one hand, where the speed of structure determination is a limiting factor and for application in the study of slow kinetic processes involving proteins, such as fibrillization, on the other hand.

Fast and simultaneous determination of 1 H-1 H and 1 H-19 F scalar couplings in complex spin systems: Application of PSYCHE homonuclear broadband decoupling.

The present manuscript focuses on fast and simultaneous determination of 1 H-1 H and 1 H-19 F scalar couplings in fluorinated complex steroid molecules. Incorporation of broadband PSYCHE homonuclear decoupling in the indirect dimension of zero-quantum filtered diagonal experiments (F1-PSYCHE-DIAG) suppresses 1 H-1 H scalar couplings; however, it retains 1 H-19 F scalar couplings (along F1 dimension) for the 19 F coupled protons while preserving the pure-shift nature for 1 H resonances uncoupled to 19 F. In such cases, along the direct dimensions, 1 H-1 H scalar coupling multiplets deconvolute and they appear as duplicated multiplets for the 19 F coupled protons, which facilitates unambiguous discrimination of 19 F coupled 1 H chemical sites from the others. Further, as an added advantage, data acquisition has been accelerated by invoking the known ideas of spectral aliasing in the F1-PSYCHE-DIAG scheme and experiments demand only ~10 min of spectrometer times.

Multiple homonuclear band-selective decoupling NMR: Fast and unambiguous determination of diastereomeric excess.

Discrimination and quantification of chiral stereoisomers have been studied by different analytical methods, and NMR has emerged as a powerful one with the advancements in pure-shift NMR methods. In the present manuscript, an al-F1F2-MHOBS-DIAG NMR method for the quantification of diastereomeric excess ratio (dr) has been proposed and demonstrated, using hesperidin and naringin mixtures. This method enables simultaneous quantification of dr at multiple resonances, in a single experiment, and it takes only 10 min to record. The present method uses spectral aliasing and thus demands only very few indirect dwell increments. Further, the measured dr values are very reliable, because we consider several spins for the quantification.

Precise Determination of Enantiomeric Excess by a Sensitivity Enhanced Two-Dimensional Band-Selective Pure-Shift NMR.

Unambiguous identification and precise quantification of enantiomers in chiral mixtures is crucial for enantiomer specific synthesis as well as chemical analysis. The task is often challenging for mixtures with high enantiomeric excess and for complex molecules with strong (1)H-(1)H scalar (J) coupling network. The recent advancements in (1)H-(1)H decoupling strategies to suppress the J-interactions offered new possibilities for NMR based unambiguous discrimination and quantification enantiomers. Herein, we discuss a high resolution two-dimensional pure-shift zCOSY NMR method with homonuclear band-selective decoupling in both the F1 and F2 dimensions (F1F2-HOBS-zCOSY). This advanced method shows a sharp improvement in resolution over the other COSY methods and also eliminates the problems associated with the overlapping decoupling sidebands. The efficacy of this method has been exploited for precise quantification of enantiomeric excess (ee) ratio (R/S) up to 99:1 in the presence of very low concentrations of chiral lanthanide shift reagents (CLSR) or chiral solvating agents (CSA). The F1F2-HOBS-zCOSY is simple and can be easily implemented on any modern NMR spectrometers, as a routine analytical tool.

Chemical shift assignments of zinc finger domain of methionine aminopeptidase 1 (MetAP1) from Homo sapiens.

Methionine aminopeptidase Type I (MetAP1) cleaves the initiator methionine from about 70 % of all newly synthesized proteins in almost every living cell. Human MetAP1 is a two domain protein with a zinc finger on the N-terminus and a catalytic domain on the C-terminus. Here, we report the chemical shift assignments of the amino terminal zinc binding domain (ZBD) (1-83 residues) of the human MetAP1 derived by using advanced NMR spectroscopic methods. We were able to assign the chemical shifts of ZBD of MetAP1 nearly complete, which reveal two helical fragments involving residues P44-L49 (α1) and Q59-K82 (α2). The protein structure unfolds upon complex formation with the addition of 2 M excess EDTA, indicated by the appearance of amide resonances in the random coil chemical shift region of (15)NHSQC spectrum.