IRAK4 is a central kinase in innate immunity, but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown, and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase. Phosphorylation of any two of the residues Thr-342, Thr-345, and Ser-346 is required for full activity, and the death domain regulates the activation of IRAK4. Using antibodies against activated IRAK4, we demonstrate that IRAK4 becomes phosphorylated in human cells following stimulation by IL-1R and Toll-like receptor agonists, which can be blocked pharmacologically by a dual inhibitor of IRAK4 and IRAK1. Interestingly, in dermal fibroblasts, although complete inhibition of IRAK4 kinase activity does not inhibit IL-1-induced IL-6 production, NF-κB, or MAPK activation, there is complete ablation of these processes in IRAK4-deficient cells. In contrast, the inhibition of IRAK kinase activity in primary human monocytes reduces R848-induced IL-6 production with minimal effect on NF-κB or MAPK activation. Taken together, these studies define the mechanism of IRAK4 activation and highlight the differential role of IRAK4 kinase activity in different human cell types as well as the distinct roles IRAK4 scaffolding and kinase functions play.

Journal of Mass SpectrometryVolume 45, Issue 7 p. 820-823 JMS Letter Aspartocin cyclic lipopeptide antibiotics: mass spectral structural confirmations and the diagnostic role played by the α,β-diaminobutyric acid residue Marshall M. Siegel, Corresponding Author Marshall M. Siegel siegelmarshall@hotmail.com Pfizer (formerly Wyeth Research), Chemical Technologies Section, 401 N. Middletown Road, Pearl River, NY 10965, United StatesPfizer (formerly Wyeth Research), Chemical Technologies Section, 401 N. Middletown Road, Building 222/Room 1043, Pearl River, NY 10965, United States.===Search for more papers by this authorFangming Kong, Fangming Kong Pfizer (formerly Wyeth Research), Chemical Technologies Section, 401 N. Middletown Road, Pearl River, NY 10965, United StatesSearch for more papers by this authorGuy T. Carter, Guy T. Carter Pfizer (formerly Wyeth Research), Chemical Technologies Section, 401 N. Middletown Road, Pearl River, NY 10965, United StatesSearch for more papers by this author Marshall M. Siegel, Corresponding Author Marshall M. Siegel siegelmarshall@hotmail.com Pfizer (formerly Wyeth Research), Chemical Technologies Section, 401 N. Middletown Road, Pearl River, NY 10965, United StatesPfizer (formerly Wyeth Research), Chemical Technologies Section, 401 N. Middletown Road, Building 222/Room 1043, Pearl River, NY 10965, United States.===Search for more papers by this authorFangming Kong, Fangming Kong Pfizer (formerly Wyeth Research), Chemical Technologies Section, 401 N. Middletown Road, Pearl River, NY 10965, United StatesSearch for more papers by this authorGuy T. Carter, Guy T. Carter Pfizer (formerly Wyeth Research), Chemical Technologies Section, 401 N. Middletown Road, Pearl River, NY 10965, United StatesSearch for more papers by this author First published: 24 May 2010 https://doi.org/10.1002/jms.1755Citations: 2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume45, Issue7July 2010Pages 820-823 RelatedInformation

Fluorescent bisretinoids, such as A2E and all-trans-retinal dimer, form as a by-product of vitamin A cycling in retina and accumulate in retinal pigment epithelial (RPE) cells as lipofuscin pigments. These pigments are implicated in pathological mechanisms involved in several vision-threatening diseases including age-related macular degeneration. Efforts to understand damaging events initiated by these bisretinoids have revealed that photoexcitation of A2E by wavelengths in the visible spectrum leads to singlet oxygen production and photooxidation of A2E. Here we have employed liquid chromatography coupled to electrospray ionization mass spectrometry together with tandem mass spectrometry (MS/MS), to demonstrate that A2E also undergoes photooxidation-induced degradation and we have elucidated the structures of some of the aldehyde-bearing cleavage products. Studies in which A2E was incubated with a singlet oxygen generator yielded results consistent with a mechanism involving bisretinoid photocleavage at sites of singlet molecular oxygen addition. We provide evidence that one of the products released by A2E photodegradation is methylglyoxal, a low molecular weight reactive dicarbonyl with the capacity to form advanced glycation end products. Methylglyoxal is already known to be generated by carbohydrate and lipid oxidation; this is the first report of its production via bisretinoid photocleavage. It is significant that AGE-modified proteins are detected in deposits (drusen) that accumulate below RPE cells in vivo; drusen have been linked to age-related macular degeneration pathogenesis. Whereas various processes play a role in drusen formation, these findings are indicative of a contribution from lipofuscin photooxidation in RPE.

Secondary drug screening methods are described for determining the relative degree of non-covalent binding between drug candidates and a protein of therapeutic interest by gel centrifugation chromatography using GPC spin columns for isolating the protein-drug complexes, under native conditions, and reversed-phase HPLC coupled with ESI-MS for highly resolved and sensitive detection of the drug in the complex, under denaturing conditions. The necessary control samples and limitations of this work are fully described. The GPC spin column HPLC ESI-MS methodology for screening of drugs non-covalently bound to proteins is illustrated for the non-covalent binding of geldanamycin with Hsp90cat protein.

Bruton's tyrosine kinase (Btk) plays a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. A number of cell signaling studies clearly show that Btk is activated by Lyn, a Src family kinase, through phosphorylation on activation loop tyrosine 551 (Y(551)). However, the detailed molecular mechanism regulating Btk activation remains unclear. In particular, we do not fully understand the correlation of kinase activity with Y(551) phosphorylation, and the role of the noncatalytic domains of Btk in the activation process. Insect cell expressed full-length Btk is enzymatically active, but a truncated version of Btk, composed of only the kinase catalytic domain, is largely inactive. Further characterization of both forms of Btk by mass spectrometry showed partial phosphorylation of Y(551) of the full-length enzyme and none of the truncated kinase domain. To determine whether the lack of activity of the kinase domain was due to the absence of Y(551) phosphorylation, we developed an in vitro method to generate Y(551) monophosphorylated Btk kinase domain fragment using the Src family kinase Lyn. Detailed kinetic analyses demonstrated that the in vitro phosphorylated Btk kinase domain has a similar activity as the full-length enzyme while the unphosphorylated kinase domain has a very low k(cat) and is largely inactive. A divalent magnesium metal dependence study established that Btk requires a second magnesium ion for activity. Furthermore, our analysis revealed significant differences in the second metal-binding site among the kinase domain and the full-length enzyme that likely account for the difference in their catalytic profile. Taken together, our study provides important mechanistic insights into Btk kinase activity and phosphorylation-mediated regulation.

Three lipocyclopeptide antibiotics, aspartocins A (1), B (2), and C (3), were obtained from the aspartocin complex by HPLC separation methodology. Their structures were elucidated using previously published chemical degradation results coupled with spectroscopic studies including ESI-MS, ESI-Nozzle Skimmer-MSMS and NMR. All three aspartocin compounds share the same cyclic decapeptide core of cyclo [Dab2 (Asp1-FA)-Pip3-MeAsp4-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11]. They differ only in the fatty acid side chain moiety (FA) corresponding to (Z)-13-methyltetradec-3-ene-carbonyl, (+,Z)-12-methyltetradec-3-ene-carbonyl and (Z)-12-methyltridec-3-ene-carbonyl for aspartocins A (1), B (2), and C (3), respectively. All of the sequence ions were observed by ESI-MSMS of the doubly charged parent ions. However, a number of the sequence ions observed were of low abundance. To fully sequence the lipocyclopeptide antibiotic structures, these low abundance sequence ions together with complementary sequence ions were confirmed by ESI-Nozzle-Skimmer-MSMS of the singly charged linear peptide parent fragment ions H-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11-Dab2(1+)-Asp1-FA. Cyclization of the aspartocins was demonstrated to occur via the beta-amino group of Dab2 from ions of moderate intensity in the ESI-MSMS spectra. As the fatty acid moieties do not undergo internal fragmentations under the experimental ESI mass spectral conditions used, the 14 Da mass difference between the fatty acid moieties of aspartocins A (1) and B (2) versus aspartocin C (3) was used as an internal mass tag to differentiate fragment ions containing fatty acid moieties and those not containing the fatty acid moieties. The most numerous and abundant fragment ions observed in the tandem mass spectra are due to the cleavage of the tertiary nitrogen amide of the pipecolic acid residue-3 (16 fragment ions) and the proline residue-11 (7 fragment ions). In addition, the neutral loss of ethanimine from alpha,beta-diaminobutyric acid residue 9 was observed for the parent molecular ion and for 7 fragment ions.

Chapter 2 Drug Screening Using Gel Permeation Chromatography Spin Columns Coupled with ESI-MS Marshall M. Siegel, Marshall M. Siegel Wyeth Research, 401 N. Middletown Rd., Bldg. 222/Room 1043, Pearl River, NY 10965, USASearch for more papers by this author Marshall M. Siegel, Marshall M. Siegel Wyeth Research, 401 N. Middletown Rd., Bldg. 222/Room 1043, Pearl River, NY 10965, USASearch for more papers by this author Book Editor(s):Prof. Dr. Klaus T. Wanner, Prof. Dr. Klaus T. Wanner Ludwig-Maximilians-University Munich, Department of Pharmacy, Center of Drug Research, Butenandtstr., 81377 Munich, GermanySearch for more papers by this authorDr. Georg Höfner, Dr. Georg Höfner Ludwig-Maximilians-University Munich, Department of Pharmacy, Center of Drug Research, Butenandtstr., 81377 Munich, GermanySearch for more papers by this author First published: 23 February 2007 https://doi.org/10.1002/9783527610907.ch2Citations: 4Book Series:Methods and Principles in Medicinal Chemistry Series Editor(s): Prof. Dr. Raimund Mannhold, Prof. Dr. Raimund Mannhold Molecular Drug Research Group, Heinrich-Heine-Universität, Universitätsstrasse 1, 40225 Düsseldorf, GermanySearch for more papers by this authorProf. Dr. Hugo Kubinyi, Prof. Dr. Hugo Kubinyi Donnersbergerstrasse 9, 67256 Weisenheim am Sand, GermanySearch for more papers by this authorProf. Dr. Gerd Folkers, Prof. Dr. Gerd Folkers Collegium Helveticum, STW/ETH Zurich, 8092 Zurich, SwitzerlandSearch for more papers by this author AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Summary This chapter contains sections titled: Introduction Preface Direct and Indirect ESI-MS Analysis of Non-covalent Drug–Protein Complexes Advantages of GPC Spin Columns Application of Equilibrium and Non-equilibrium Theory for the Analysis of GPC Spin Column Eluates Sample Prepared Under Equilibrium Conditions Prior to Spin Column Treatment Calculation for Predicting the Concentration of Sample Complex Eluted From the Spin Column Estimation of Relative Binding Affinities from GPC Spin-Column/ESI-MS Data Experimental Determination of the Kd Value from GPC Spin-Column/ESI-MS Data Experimental Spin Columns Spin Column Media: Advantages and Disadvantages, Volatile vs Non-volatile Buffers Preparing Non-covalent Complexes in Protein Buffer; Protein Concentration, Ligand Concentration, Incubation Time Sample Organization: Single Samples vs Mixtures, Mixture Set-up: Compatibility of Components, Plate Set-up Pooling Spin Column Eluates for Higher Throughput Manual vs Robotic Instrumentation for Sample Preparation and Acquiring Spin Column Eluates ESI Mass Spectrometer: ESI, APCI, Photodissociation, Positive/Negative Ionization ESI Multi-sprayer (MUX) Technology; Sample Throughput; Protein Consumption Reversed Phase (RP) HPLC ESI-MS Considerations Protein Removal for Optimum Sensitivity Data Reduction and Automated Interpretation of GPC Spin Column/ESI-MS Data Results Secondary Screens GPC Spin Column/ESI-MS Drug Screening Demonstration Papers Estrogen Receptor Target Non-covalent Binding of Drugs to RNA/DNA Targets Amgen Secondary Screens Novartis Secondary Screens Primary Screens RGS4 Protein Target Amgen Primary Screens Novartis Primary Screens Additional Spin Column Methods Competition Experiments of Inhibitor Mixture with Protein Target GPC Spin Column/ESI-MS Determination of Binding Sites Obtaining MS EC50s and Kds for Ligands Non-covalently Bound to Protein Active Sites Multiple Passes Through Spin Columns – Finding Strongest Binders Reverse Screening with GPC Spin Columns Conclusions GPC Spin Column/ESI-MS: Ease of Use, Mixture Analysis, High Speed, Reliability, Uncoupling of GPC from ESI-MS and HPLC ESI-MS Comparison of GPC Spin Column/HPLC ESI-MS with Tandem Chromatographic Method of GPC/HPLC ESI-MS Future Developments MS and HPLC Improvements Use of Automated Nanospray for Greater Sensitivity and Smaller Sample Size (Less Protein/Drug) Microfluidic Systems: Sensitivity, High Speed GPC Spin Column Eluates Analyzed by ESI/Ion Mobility/Mass Spectrometry GPC Spin Columns with Matrixless MALDI-MS and Gyros GPC Microfluidic ESI/MALDI-MS System Citing Literature Mass Spectrometry in Medicinal Chemistry: Applications in Drug Discovery RelatedInformation

Two new peptaibols, septocylindrin A (1) and septocylindrin B (2), related to the well-studied membrane-channel-forming peptaibol alamethicin, were obtained from a terrestrial isolate of the fungus Septocylindrium sp. Both 1 and 2 are linear 19-amino acid peptides with a modified phenylalanine C-terminus. Analysis of the HRMS data indicated that they differ only in the 18th residue, where 1 contains Glu and 2 contains Gln. The structures of these two peptaibols were determined by extensive NMR and HRMS analysis. The absolute configurations of amino acids present in 1 were determined using Marfey's methodology. Both compounds were isolated through bioassay-guided fractionation and exhibited significant antibacterial and antifungal activity.

The mammalian protein kinase Polo-Like Kinase-1 (Plk-1) is an important regulator of mitosis. It is involved in the signaling networks that regulate many cell cycle processes such as centrosome maturation and separation, mitotic entry and exit, spindle formation and cytokinesis. Over-expression of Plk-1 was observed in a number of different tumors such as head and neck squamous cell carcinomas, ovarian and breast carcinomas and melanomas1. Cancer patients with over-expressed Plk-1 were predicted to have poor clinical outcome and shorten lifespan compared to those with moderate Plk-1 expression2. Plk-1 knockdown studies using anti-Plk-1 antibodies3 or Plk-1 siRNA4 showed mitotic arrest and apoptosis. This validates Plk-1 as a good candidate for small molecule inhibitor therapy to treat a wide range of cancer diseases. Human Plk-1 consists of a N-terminal Ser/Thr Kinase Domain (KD) and a regulatory C-terminal Polo-Box Domain (PBD). The PBD functions both as a Plk-1 cellular localization domain as well as an inhibitory domain of the kinase. Upon binding to a phosphopeptide, such as Cdc25, PBD relinquishes the inhibitory effect thus allowing for phosphorylation and activation of the kinase domain. The active Plk-1 then carries out the signaling cascade through phosphorylation of various downstream molecules such as Cdc2-CyclinB complex and Anaphase Promoting Complex (APC)5. Presented here is the purification and characterization of Plk-1 Full Length (expressed in Sf9 and E. coli cells) as well as Plk-1 Kinase Domain (expressed in E. coli).

Natural products are a source of unique chemical entities with specific biological activities of great value to the pharmaceutical industry. However, the determination of unknown structures is usually time consuming and often becomes a bottleneck in the effort to develop natural products into effective drugs. The high-performance features of high magnetic field FTMS have greatly alleviated the structural elucidation bottleneck to meet increasingly shorter discovery timelines for drug candidates based on natural products. The high-performance features of high field FTMS include unsurpassed mass measurement accuracy for elemental formula determination, ultra-high mass resolution for component separation, the ability to perform multiple levels of tandem mass spectrometry for structural elucidation, and moderate sensitivity for limited supply of isolates. A number of applications utilizing these properties of FTMS have been reported recently for the structural elucidation of novel natural product structures originating from terrestrial and marine microorganisms. In this review, FTMS methods and their applications for the structural elucidation and characterization of natural products will be reviewed. Figure Molecular structure and positive ion mode nanoelectrospray FTICR mass spectrum of methylspirastrellolide A ( 3 ). The inset shows the isotopic distribution with high abundance of the A + 2 peak, but less than the abundance of the A + 1 peak. The resolved isotopic fine structure of the A + 2 peak reveals the presence of one chlorine atom based on accurate mass assignment and the measured abundance ratio between the resolved 37 Cl peak and the monoisotopic peak

PKC isozymes can be divided into three subfamilies based on structural and enzymatic differences: conventional, novel and atypical. PKCδ is a member of the subfamily of novel PKC's. In an effort to obtain X-ray crystal structure of this nPKC family target, we expressed and purified a recombinant PKCδ kinase domain construct in E. coli. Here we show that the recombinant kinase domain has activity, is monomeric by size-exclusion chromatography (SEC), and can auto-phosphorylate heterogeneously within the cell during expression. PKCδ kinase domain (317–676) was expressed in BL21 cells with a C-terminal 6-His tag, and purified +/− ATP to >95% following a four-step protocol. The purified product eluted as a single, monomeric peak during SEC. Multiple phosphorylations (avg 5–7) were observed by ESI-MS for both of the +/− ATP preparations; indicating that all phosporylation events most likely occur during expression. In addition, both +/− ATP preparations displayed substrate inhibition with the PKC-specific FARK inhibitor peptide, while NMR saturation transfer difference (STD) binding experiments showed that AMP-PNP binds and competes with bisindolylmaleimide for the same active site of PKCδ (317–676). Although, the construct proved difficult to crystallize, we have successfully produced a method for purification of a PKCδ kinase domain from E. coli suitable for assay development, and selectivity studies with other nPKC family members.

A series of 2-(quinazolin-4-ylamino)-[1,4] benzoquinone derivatives that function as potent covalent-binding, irreversible inhibitors of the kinase domain of vascular endothelial growth factor receptor-2 (VEGFR-2) has been prepared by ceric ammonium nitrate oxidation of substituted (2,5-dimethoxyphenyl)(6,7-disubstituted-quinazolin-4-yl)amines and by displacement of the chlorine atom of substituted 2-chloro-5-(6,7-disubstituted-quinazolin-4-ylamino)-[1,4]benzoquinones with various amines, anilines, phenols, and alcohols. Enzyme studies were conducted in the absence and presence of glutathione and plasma. Several of the compounds inhibit VEGF-stimulated autophosphorylation in intact cells. Kinetic experiments were performed to study the reactivity of selected inhibitors toward glutathione. Reactivities correlated with LUMO energies calculated as averages of those of individual conformers weighted by the Boltzmann distribution. These results and molecular modeling were used to rationalize the biological observations. The compounds behave as non-ATP-competitive inhibitors. Unequivocal evidence, from mass spectral studies, indicates that these inhibitors form a covalent interaction with Cys-1045. One member of this series displays antitumor activity in an in vivo model.

The reactions of 6-methylidene penems 4-7 with beta-lactamases (TEM-1, SHV-1, Amp-C) were characterized by electrospray ionization mass spectrometry (ESI-MS). The kinetics of the reactions were monitored, demonstrating that only one penem molecule reacts to form an acyl-enzyme complex. For penem 5, the ESI-MS/MS spectrum of the hydrolysis product produced in the reaction was identical to the spectrum generated from a synthesized dihydro[1,4]thiazepine 10, confirming the rearrangement of the penem ring system to a seven-membered dihydro[1,4]thiazepine structure. Gas-phase ESI-MS/MS fragmentation data were rationalized due to tautomerization between imine and enamine substructures. ESI-MS/MS analysis of the T-6 trypsin-digested fragments of TEM-1 and SHV-1 demonstrated that the penems were only attached to Ser-70 of these class A beta-lactamases and that the penem ring structures were rearranged to seven-membered dihydro[1,4]thiazepines.