Mononuclear phagocyte morphological response to chemoattractants is dependent on geranylgeranyl pyrophosphate.

Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a.NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.

Broadening the Utility of Farnesyltransferase-Catalyzed Protein Labeling Using Norbornene-Tetrazine Click Chemistry.

Bioorthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational modification, in which one or two 15- or 20-carbon isoprenoid chains are transferred onto cysteine residues near the C-terminus of a target protein. The three main enzymes─protein farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase I), and geranylgeranyl transferase II (GGTase II)─that catalyze this process have been shown to tolerate numerous structural modifications in the isoprenoid substrate. This feature has previously been exploited to transfer an array of farnesyl diphosphate analogues with a range of functionalities, including an alkyne-containing analogue for copper-catalyzed bioconjugation reactions. Reported here is the synthesis of an analogue of the isoprenoid substrate embedded with norbornene functionality (C10NorOPP) that can be used for an array of applications, ranging from metabolic labeling to selective protein modification. The probe was synthesized in seven steps with an overall yield of 7% and underwent an inverse electron demand Diels-Alder (IEDDA) reaction with tetrazine-containing tags, allowing for copper-free labeling of proteins. The use of C10NorOPP for the study of prenylation was explored in the metabolic labeling of prenylated proteins in HeLa, COS-7, and astrocyte cells. Furthermore, in HeLa cells, these modified prenylated proteins were identified and quantified using label-free quantification (LFQ) proteomics with 25 enriched prenylated proteins. Additionally, the unique chemistry of C10NorOPP was utilized for the construction of a multiprotein-polymer conjugate for the targeted labeling of cancer cells. That construct was prepared using a combination of norbornene-tetrazine conjugation and azide-alkyne cycloaddition, highlighting the utility of the additional degree of orthogonality for the facile assembly of new protein conjugates with novel structures and functions.

Improved synthesis and application of an alkyne-functionalized isoprenoid analogue to study the prenylomes of motor neurons, astrocytes and their stem cell progenitors.

Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs, and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.

The McKenna reaction - avoiding side reactions in phosphonate deprotection.

The McKenna reaction is a well-known and popular method for the efficient and mild synthesis of organophosphorus acids. Bromotrimethylsilane (BTMS) is the main reagent in this reaction, which transforms dialkyl phosphonate esters into bis(trimethylsilyl)esters, which are then easily converted into the target acids. However, the versatile character of the McKenna reaction is not always used to its full extent, due to formation of side products. Herein, demonstrated by using model examples we have not only analyzed the typical side processes accompanying the McKenna reaction, but also uncovered new ones. Further, we discovered that some commonly recommended precautions did not always circumvent the side reactions. The proposed results and recommendations may facilitate the synthesis of phosphonic acids.

A Benefit of Using the IDSCRF- over UFF-Radii Cavities and Why Joint Correlations of NMR Chemical Shifts Can Be Advantageous: Condensed Pyridines as an IEF-PCM/GIAO/DFT Case Study.

Herein, an advantage of the use of IDSCRF- over UFF-radii-based solute cavities in GIAO/DFT calculations is presented for the 13C and especially 15N NMR chemical shifts made for several bicyclic aromatic nitrogen heterocycles in CDCl3 solution treated within the classical IEF-PCM solvation scheme. Successful application of the IDSCRF-radii in the non 1:1 joint multinuclear 1H/13C and particularly 1H/13C/15N correlations of the measured δH,C(,N) values to those obtained theoretically is also documented for a series of test systems (-268 ≤ δN ≤ -72 ppm). The experimentally yet unknown δN's were found in this way for the title compounds via a trinuclear eq 1 determined for an optimally chosen value of the multiplication factor of initial raw δH data (mH = 10). Such a simultaneous analysis of the δH,C(,N) data is proposed as a novel method to study the solution structure of the other similar conformationally homogeneous (bio)organic compounds. The issue of small spurious imaginary vibrational frequencies computed for a few molecular systems using the Gaussian 09 default UFF-radii is briefly considered as well.

Improved synthesis and application of an alkyne-functionalized isoprenoid analogue to study the prenylomes of motor neurons, astrocytes and their stem cell progenitors.

Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.

New reactions of N-tert-butylimines; formation of N-heterocycles by methyl radical elimination on flash vacuum thermolysis of N-benzylidene- and N-(2-pyridylmethylidene)-tert-butylamines.

Thermal reactions of N-benzylidene- and N-(2-pyridylmethylidene)-tert-butylamines (5 and 13) under FVT conditions have been investigated. Unexpectedly, at 800 °C, compound 5 yields 1,2-dimethylindole and 3-methylisoquinoline. In the reaction of 13 at 800 °C, 3-methylimidazo[1,5-a]pyridine was obtained as the major product. Mechanisms of these reactions have been proposed on the basis of DFT calculations. Furthermore, UV-photoelectron spectroscopy combined with FVT has been applied for direct monitoring and characterization of the thermolysis products in situ.

Synthesis, Enzymatic Peptide Incorporation, and Applications of Diazirine-Containing Isoprenoid Diphosphate Analogues.

Prenylated proteins contain C15 or C20 isoprenoids linked to cysteine residues positioned near their C-termini. Here we describe the preparation of isoprenoid diphosphate analogues incorporating diazirine groups that can be used to probe interactions between prenylated proteins and other proteins that interact with them. Studies using synthetic peptides and whole proteins demonstrate that these diazirine analogues are efficient substrates for prenyltransferases. Photo-cross-linking experiments using peptides incorporating the diazirine-functionalized isoprenoids selectively cross-link to several different proteins. These new isoprenoid analogues should be broadly useful in the studies of protein prenylation.

Synthesis of the 6-Substituted Imidazo[1,2-a]Pyridine-3-yl-2- Phosphonopropionic Acids as Potential Inhibitors of Rab Geranylgeranyl Transferase.

Twelve phosphonopropionates derived from 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid (3-IPEHPC) were synthesized and evaluated for their activity as inhibitors of protein geranylgeranylation. The nature of the substituent in the C6 position of imidazo[1,2-a]pyridine ring was responsible for the compound's activity against Rab geranylgeranyl transferase (RGGT). The most active inhibitors disrupted Rab11A prenylation in the human cervical carcinoma HeLa cell line. The esterification of carboxylic acid in the phosphonopropionate moiety turned the inhibitor into an inactive analog.

Sensory aspects of acceptability of bitter-flavoured 7.5 mm film-coated tablets in adults, preschool and school children.

There is great interest in demonstrating acceptability of solid oral formulations in paediatric populations. This study investigated the acceptability of small, 7.5 mm, bitter-flavoured, coated tablets in healthy children and adults. A randomised, double-blind acceptability test was performed involving 101 children (4-12 years) and 52 adults (18-75 years). Acceptability was measured by participants as sensory assessment of taste, mouthfeel and hedonic perception, and by researcher observations of ability to swallow the tablet and negative facial expressions. Additionally, the taste-masking effect of film coatings was assessed based on the intensity of bitterness perception. At least one tablet was voluntarily swallowed by 35.7% of 4-6-year olds, 74% of 7-12-year olds and 98% of adults. The bitterness of the tablet did not affect participants' ability to swallow it. The sensory properties determined whether the tablet was acceptable. The following factors: low bitterness, high smoothness, high slipperiness and pleasant aftertaste had a positive impact on overall palatability in both populations. The paediatric scores during sensory evaluation of tablets differed from adults, showing lower acceptability. This study demonstrates the multifactorial nature of palatability of tablets and highlights that adults' palatability evaluation cannot be directly translated to a paediatric population.