Biological Evaluations, NMR Analyses, Molecular Modeling Studies, and Overview of the Synthesis of the Marine Natural Product (-)-Mucosin.

Natural products obtained from marine organisms continue to be a rich source of novel structural architecture and of importance in drug discovery, medicine, and health. However, the success of such endeavors depends on the exact structural elucidation and access to sufficient material, often by stereoselective total synthesis, of the isolated natural product of interest. (-)-Mucosin (1), a fatty acid derivative, previously presumed to contain a rare cis-bicyclo[4.3.0]non-3-ene moiety, has since been shown to be the trans-congener. Analytically, the fused bicyclic ring system in (-)-1 constitutes a particular challenge in order to establish its relative and absolute stereochemistry. Herein, data from biological evaluations, NMR and molecular modeling studies of (-)-1 are presented. An overview of the synthetic strategies enabling the exact structural elucidation of (-)-mucosin (1) is also presented.

Evaluation of NMR predictors for accuracy and ability to reveal trends in 1 H NMR spectra of fatty acids.

Four different nuclear magnetic resonance (NMR) predictors have been evaluated for their ability to predict 600-MHz 1 H spectra of free fatty acids and fatty acid methyl esters of 20 common fatty acids. The predictors were evaluated on two main criteria: (1) their accuracy in direct prediction of the spectra (absolute accuracy) and (2) the ability to reveal trends or predict the change that occurs in the spectra as a result of a change in the fatty acid carbon chain, or by esterification of the free fatty acids to methyl esters (relative accuracy). The absolute accuracy in chemical shift prediction for fatty acids was good, compared with previous reports on a broader range of compounds. All four predictors had median prediction errors for chemical shifts of the signals in fatty acid methyl esters well below 0.1 ppm and as low as 0.015 ppm for one of the predictors. However, all predictors also had outliers with errors far above the upper interquartile range. In general, they also fail to reproduce trends of diagnostic value that were observed in the experimental data or properly predict the result of a minor change in molecular structure. All four predictors depend on experimental data from different origins. This may be a limiting factor for the relative accuracy of the predictors.

A Pharmacological Chaperone Therapy for Acute Intermittent Porphyria.

Mutations in hydroxymethylbilane synthase (HMBS) cause acute intermittent porphyria (AIP), an autosomal dominant disease where typically only one HMBS allele is mutated. In AIP, the accumulation of porphyrin precursors triggers life-threatening neurovisceral attacks and at long-term, entails an increased risk of hepatocellular carcinoma, kidney failure, and hypertension. Today, the only cure is liver transplantation, and a need for effective mechanism-based therapies, such as pharmacological chaperones, is prevailing. These are small molecules that specifically stabilize a target protein. They may be developed into an oral treatment, which could work curatively during acute attacks, but also prophylactically in asymptomatic HMBS mutant carriers. With the use of a 10,000 compound library, we identified four binders that further increased the initially very high thermal stability of wild-type HMBS and protected the enzyme from trypsin digestion. The best hit and a selected analog increased steady-state levels and total HMBS activity in human hepatoma cells overexpressing HMBS, and in an Hmbs-deficient mouse model with a low-expressed wild-type-like allele, compared to untreated controls. Moreover, the concentration of porphyrin precursors decreased in liver of mice treated with the best hit. Our findings demonstrate the great potential of these hits for the development of a pharmacological chaperone-based corrective treatment of AIP by enhancing wild-type HMBS function independently of the patients' specific mutation.

Pharmacological Chaperoning: A Potential Treatment for PMM2-CDG.

The congenital disorder of glycosylation (CDG) due to phosphomannomutase 2 deficiency (PMM2-CDG), the most common N-glycosylation disorder, is a multisystem disease for which no effective treatment is available. The recent functional characterization of disease-causing mutations described in patients with PMM2-CDG led to the idea of a therapeutic strategy involving pharmacological chaperones (PC) to rescue PMM2 loss-of-function mutations. The present work describes the high-throughput screening, by differential scanning fluorimetry, of 10,000 low-molecular-weight compounds from a commercial library, to search for possible PCs for the enzyme PMM2. This exercise identified eight compounds that increased the thermal stability of PMM2. Of these, four compounds functioned as potential PCs that significantly increased the stability of several destabilizing and oligomerization mutants and also increased PMM activity in a disease model of cells overexpressing PMM2 mutations. Structural analysis revealed one of these compounds to provide an excellent starting point for chemical optimization since it passed tests based on a number of pharmacochemical quality filters. The present results provide the first proof-of-concept of a possible treatment for PMM2-CDG and describe a promising chemical structure as a starting point for the development of new therapeutic agents for this severe orphan disease.

Discovery of compounds that protect tyrosine hydroxylase activity through different mechanisms.

Pharmacological chaperones are small compounds that correct the folding of mutant proteins, and represent a promising therapeutic strategy for misfolding diseases. We have performed a screening of 10,000 compounds searching for pharmacological chaperones of tyrosine hydroxylase (TH), the tetrahydrobiopterin (BH4)-dependent enzyme that catalyzes the rate-limiting step in the synthesis of catecholamines. A large number of compounds bound to human TH, isoform 1 (hTH1), but only twelve significantly protected wild-type (hTH1-wt) and mutant TH-R233H (hTH1-p.R202H), associated to the rare neurological disorder TH deficiency (THD), from time-dependent loss of activity. Three of them (named compounds 2, 4 and 5) were subjected to detailed characterization of their functional and molecular effects. Whereas compounds 2 and 4 had a characteristic pharmacological chaperone (stabilizing) effect, compound 5 protected the activity in a higher extent than expected from the low conformational stabilization exerted on hTH1. Compounds 4 and 5 were weak competitive inhibitors with respect to the cofactor BH4 and, as seen by electron paramagnetic resonance, they induced small changes to the first coordination sphere of the catalytic iron. Molecular docking also indicated active-site location with coordination to the iron through a pyrimidine nitrogen atom. Interestingly, compound 5 increased TH activity in cells transiently transfected with either hTH1-wt or the THD associated mutants p.L205P, p.R202H and p.Q381K without affecting the steady-state TH protein levels. This work revealed different mechanisms for the action of pharmacological chaperones and identifies a subtype of compounds that preserve TH activity by weak binding to the catalytic iron. This article is part of a Special Issue entitled: Cofactor-dependent proteins: Evolution, chemical diversity and bio-applications.

Early Events in the Amyloid Formation of the A546T Mutant of Transforming Growth Factor ß-Induced Protein in Corneal Dystrophies Compared to the Nonfibrillating R555W and R555Q Mutants.

The human transforming growth factor ß-induced protein (TGFBIp) is involved in several types of corneal dystrophies where protein aggregation and amyloid fibril formation severely impair vision. Most disease-causing mutations are located in the last of four homologous fasciclin-1 (FAS1) domains of the protein, and it has been shown that when isolated, the fourth FAS1 domain (FAS1-4) mimics the behavior of full-length TGFBIp. In this study, we use molecular dynamics simulations and principal component analysis to study the wild-type FAS1-4 domain along with three disease-causing mutations (R555W, R555Q, and A546T) to decipher any internal difference in dynamical properties of the domains that may explain their varied stabilities and aggregation properties. In addition, we use a protein-protein docking method in combination with chemical cross-linking experiments and mass spectrometry of the cross-linked species to obtain information about interaction faces between identical FAS1-4 domains. The results show that the pathogenic mutations A546T and R555W affect the packing in the hydrophobic core of FAS1-4 in different directions. We further show that the FAS1-4 monomers associate using their ß-rich regions, consistent with peptides observed to be part of the amyloid fibril core in lattice corneal dystrophy patients.

Pharmacological chaperones as a potential therapeutic option in methylmalonic aciduria cblB type.

Methylmalonic aciduria (MMA) cblB type is caused by mutations in the MMAB gene. This encodes the enzyme ATP:cob(I)alamin adenosyltransferase (ATR), which converts reduced cob(I)alamin to an active adenosylcobalamin cofactor. We recently reported the presence of destabilizing pathogenic mutations that retain some residual ATR activity. The aim of the present study was to seek pharmacological chaperones as a tailored therapy for stabilizing the ATR protein. High-throughput ligand screening of over 2000 compounds was performed; six were found to enhance the thermal stability of purified recombinant ATR. Further studies using a well-established bacterial system in which the recombinant ATR protein was expressed in the presence of these six compounds, showed them all to increase the stability of the wild-type ATR and the p.Ile96Thr mutant proteins. Compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) significantly increased this stability and did not act as an inhibitor of the purified protein. Importantly, compound V increased the activity of ATR in patient-derived fibroblasts harboring the destabilizing p.Ile96Thr mutation in a hemizygous state to within control range. When cobalamin was coadministrated with compound V, mutant ATR activity further improved. Oral administration of low doses of compound V to C57BL/6J mice for 12 days, led to increase in steady-state levels of ATR protein in liver and brain (disease-relevant organs). These results hold promise for the clinical use of pharmacological chaperones in MMA cblB type patients harboring chaperone-responsive mutations.

Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization.

Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/ßig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3' containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions.

Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp.

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.

A vitellogenin polyserine cleavage site: highly disordered conformation protected from proteolysis by phosphorylation.

Vitellogenin (Vg) is an egg-yolk precursor protein in most oviparous species. In honeybee (Apis mellifera), the protein (AmVg) also affects social behavior and life-span plasticity. Despite its manifold functions, the AmVg molecule remains poorly understood. The subject of our structure-oriented AmVg study is its polyserine tract - a little-investigated repetitive protein segment mostly found in insects. We previously reported that AmVg is tissue specifically cleaved in the vicinity of this tract. Here, we show that, despite its potential for an open, disordered structure, AmVg is unexpectedly resistant to trypsin/chymotrypsin digestion at the tract. Our findings suggest that multiple phosphorylation plays a role in this resilience. Sequence variation is highly pronounced at the polyserine region in insect Vgs. We demonstrate that sequence differences in this region can lead to structural variation, as NMR and circular dichroism (CD) evidence assign different conformational propensities to polyserine peptides from the honeybee and the jewel wasp Nasonia vitripennis; the former is extended and disordered and the latter more compact and helical. CD analysis of the polyserine region of bumblebee Bombus ignitus and wasp Pimpla nipponica supports a random coil structure in these species. The spectroscopic results strengthen our model of the AmVg polyserine tract as a flexible domain linker shielded by phosphorylation.

In†situ solid-state NMR spectroscopy of protein in heterogeneous membranes: the baseplate antenna complex of Chlorobaculum tepidum.

A clever combination: an in situ solid-state NMR analysis of CsmA proteins in the heterogeneous environment of the photoreceptor of Chlorobaculum tepidum is reported. Using different combinations of 2D and 3D solid-state NMR spectra, 90 % of the CsmA resonances are assigned and provide on the basis of chemical shift data information about the structure and conformation of CsmA in the CsmA-bacteriochlorophyll a complex.

HTSDSF Explorer, A Novel Tool to Analyze High-throughput DSF Screenings.

The identification of new drugs for novel therapeutic targets requires the screening of libraries containing tens of thousands of compounds. While experimental screenings are assisted by high-throughput technologies, in target-based biophysical assays, such as differential scanning fluorimetry (DSF), the analysis steps must be calculated manually, often combining several software packages. To simplify the determination of the melting temperature (Tm) of the target and the change induced by ligand binding (ΔTm), we developed the HTSDSF explorer, a versatile, all-in-one, user-friendly application suite. Implemented as a server-client application, in the primary screenings, HTSDSF explorer pre-analyzes and displays the Tm and ΔTm results interactively, thereby allowing the user to study hundreds of conditions and select the primary hits in minutes. This application also allows the determination of preliminary binding constants (KD) through a series of subsequent dose-response assays on the primary hits, thereby facilitating the ranking of validated hits and the advance of drug discovery efforts.

Binding Specificity of ASHH2 CW Domain Toward H3K4me1 Ligand Is Coupled to Its Structural Stability Through Its α1-Helix.

The CW domain binds to histone tail modifications found in different protein families involved in epigenetic regulation and chromatin remodeling. CW domains recognize the methylation state of the fourth lysine on histone 3 and could, therefore, be viewed as a reader of epigenetic information. The specificity toward different methylation states such as me1, me2, or me3 depends on the particular CW subtype. For example, the CW domain of ASHH2 methyltransferase binds preferentially to H3K4me1, and MORC3 binds to both H3K4me2 and me3 modifications, while ZCWPW1 is more specific to H3K4me3. The structural basis for these preferential bindings is not well understood, and recent research suggests that a more complete picture will emerge if dynamical and energetic assessments are included in the analysis of interactions. This study uses fold assessment by NMR in combination with mutagenesis, ITC affinity measurements, and thermal denaturation studies to investigate possible couplings between ASHH2 CW selectivity toward H3K4me1 and the stabilization of the domain and loops implicated in binding. The key elements of the binding site-the two tryptophans and the α1-helix form and maintain the binding pocket- were perturbed by mutagenesis and investigated. Results show that the α1-helix maintains the overall stability of the fold via the I915 and L919 residues and that the correct binding consolidates the loops designated as η1 and η3, as well as the C-terminal. This consolidation is incomplete for H3K4me3 binding to CW, which experiences a decrease in overall thermal stability on binding. Loop mutations not directly involved in the binding site, nonetheless, affect the equilibrium positions of the key residues.

Large-scale modulation of thermodynamic protein folding barriers linked to electrostatics.

Protein folding barriers, which range from zero to the tens of RT that result in classical two-state kinetics, are primarily determined by protein size and structural topology [Plaxco KW, Simons KT, Baker D (1998) J Mol Biol 277:985-994]. Here, we investigate the thermodynamic folding barriers of two relatively large proteins of the same size and topology: bovine alpha-lactalbumin (BLA) and hen-egg-white lysozyme (HEWL). From the analysis of differential scanning calorimetry experiments with the variable-barrier model [Muñoz V, Sanchez-Ruiz JM (2004) Proc Natl Acad Sci USA 101:17646-17651] we obtain a high barrier for HEWL and a marginal folding barrier for BLA. These results demonstrate a remarkable tuning range of at least 30 kJ/mol (i.e., five to six orders of magnitude in population) within a unique protein scaffold. Experimental and theoretical analyses on these proteins indicate that the surprisingly small thermodynamic folding barrier of BLA arises from the stabilization of partially unfolded conformations by electrostatic interactions. Interestingly, there is clear reciprocity between the barrier height and the biological function of the two proteins, suggesting that the marginal barrier of BLA is a product of natural selection. Electrostatic surface interactions thus emerge as a mechanism for the modulation of folding barriers in response to special functional requirements within a given structural fold.

A Workup Protocol Combined with Direct Application of Quantitative Nuclear Magnetic Resonance Spectroscopy of Aqueous Samples from Large-Scale Steam Explosion of Biomass.

Methods for thermochemical conversion of biomass into renewable energy and materials rapidly increase in range and outreach. A focus on the target product streams for valorization is natural, yet several pretreatment steps and conversion methods also result in an aqueous byproduct, which has been given less attention. This paper aims to fill this knowledge gap in the existing literature on identification and quantification of organic components in such aqueous phases by reporting a fast and direct workup protocol combined with application of quantitative analytical nuclear magnetic resonance (NMR) spectroscopy. Laboratory workup procedures combined with subsequent proton NMR spectroscopy with water signal suppression using presaturation pulses during relaxation delay, noesygppr1d, have been established, evaluated, and approved by testing on three different Bruker BioSpin NMR spectrometers; an 850 MHz AVANCE III HD with a 5 mm TCI CryoProbe, a 600 MHz AVANCE NEO with a QCI CryoProbe, and a 500 MHz AVANCE with a 5 mm BBO room-temperature probe additionally confirmed the quantification method to be applicable. The analytical procedure identified furfural, methanol, acetic acid, and formic acid as the dominating compounds in the analyzed aqueous samples, which were process effluents generated by the patented Arbacore pellet production process using steam explosion of wood shavings. A selected range of quantitative results in the aqueous phase from large-scale steam explosion is included in the study. The described procedure provides excellent quantitative reproducibility with experimental series standard deviations of <1% (mM), is nondestructive, and can be automated on demand.

Reduction in flavor-intense components in fish protein hydrolysates by membrane filtration.

Enzymatic protein hydrolysates based on side stream materials from the fish-filleting industry are increasingly explored as food ingredients. However, intense sensory properties, and high salt contents, are often a limiting factor. Most of the sensory attributes, such as fish flavor and salty taste, can be ascribed to low-molecular-weight, water-soluble components, whereas bitterness is associated with small hydrophobic peptides. In this study, protein hydrolysates based on head and backbone residuals from Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua) were produced using two different enzymes. The effects of micro- and nanofiltration on the chemical composition, protein recovery, and sensory properties of the final products were investigated. The choice of raw material and enzyme had negligible effects, whereas nanofiltration caused a considerable reduction in metabolites, ash, and the intensity of several sensory attributes. The intensity of bitterness increased after nanofiltration, indicating that small peptides associated with bitter taste were retained by the membrane. Total protein yield after microfiltration was 24%-29%, whereas 19%-24% were recovered in the nanofiltration retentate. PRACTICAL APPLICATION: Enzymatic protein hydrolysates can be included in food products to increase the protein content, and as a nutritional supplement and/or functional ingredient; however, unpalatable and intense flavors limit applications. This study investigated the use of membrane filtration to improve flavor quality and reduce salt content in fish protein hydrolysates. Although some protein loss is unavoidable in micro- and nanofiltration, this study demonstrates the production of fish protein hydrolysates with >90% protein and peptide content, which is suitable for inclusion in foods.

Inhibition of the Human Hsc70 System by Small Ligands as a Potential Anticancer Approach.

Heat shock protein (Hsp) synthesis is upregulated in a wide range of cancers to provide the appropriate environment for tumor progression. The Hsp110 and Hsp70 families have been associated to cancer cell survival and resistance to chemotherapy. In this study, we explore the strategy of drug repurposing to find new Hsp70 and Hsp110 inhibitors that display toxicity against melanoma cancer cells. We found that the hits discovered using Apg2, a human representative of the Hsp110 family, as the initial target bind also to structural regions present in members of the Hsp70 family, and therefore inhibit the remodeling activity of the Hsp70 system. One of these compounds, the spasmolytic agent pinaverium bromide used for functional gastrointestinal disorders, inhibits the intracellular chaperone activity of the Hsp70 system and elicits its cytotoxic activity specifically in two melanoma cell lines by activating apoptosis. Docking and molecular dynamics simulations indicate that this compound interacts with regions located in the nucleotide-binding domain and the linker of the chaperones, modulating their ATPase activity. Thus, repurposing of pinaverium bromide for cancer treatment appears as a promising novel therapeutic approach.

An Experimental Toolbox for Structure-Based Hit Discovery for P.†aeruginosa FabF, a Promising Target for Antibiotics.

FabF (3-oxoacyl-[acyl-carrier-protein] synthase 2), which catalyses the rate limiting condensation reaction in the fatty acid synthesis II pathway, is an attractive target for new antibiotics. Here, we focus on FabF from P. aeruginosa (PaFabF) as antibiotics against this pathogen are urgently needed. To facilitate exploration of this target we have set up an experimental toolbox consisting of binding assays using bio-layer interferometry (BLI) as well as saturation transfer difference (STD) and WaterLOGSY NMR in addition to robust conditions for structure determination. The suitability of the toolbox to support structure-based design of FabF inhibitors was demonstrated through the validation of hits obtained from virtual screening. Screening a library of almost 5 million compounds resulted in 6 compounds for which binding into the malonyl-binding site of FabF was shown. For one of the hits, the crystal structure in complex with PaFabF was determined. Based on the obtained binding mode, analogues were designed and synthesised, but affinity could not be improved. This work has laid the foundation for structure-based exploration of PaFabF.

Emulsion and Surface-Active Properties of Fish Solubles Based on Direct Extraction and after Hydrolysis of Atlantic Cod and Atlantic Salmon Backbones.

The focus on natural foods and "clean" labeled products is increasing and encourages development of new biobased ingredients. Fish solubles derived from downstream processing of side stream materials in the fish filleting industries have potential as emulsifiers based on their surface-active and emulsion stabilizing properties. The aim of this study was to evaluate and compare emulsion properties and critical micelle concentration (CMC) of direct protein extracts and protein hydrolysates based on fish backbones, and to identify associations between molecular weight distribution and process yield with the studied physicochemical properties. Protein extracts and enzymatic protein hydrolysates were produced based on two raw materials (cod and salmon backbones), two enzymes with different proteolytic specificity, and varying hydrolysis time. Emulsion activity index (EAI), emulsion stability index (ESI) and CMC were measured and compared with casein as a reference to protein-based emulsifiers. Protein hydrolysis was found to have negative impact on EAI and CMC, likely due to generation of small peptides disrupting the amphiphilic balance. The direct protein extracts had comparable EAI with casein, but the latter had superior ESI values. Protein hydrolysates with acceptable EAI could only be obtained at the expense of product yield. The study emphasizes the complexity of physicochemical properties of protein hydrolysates and discusses the challenges of achieving both good surface-active properties and high product yield.