Daan Noort

Publications of Daan Noort

• Interactions of organophosphates with keratins in the cornified epithelium of human skin.

  Authors: Daan R W Verstappen, Albert G Hulst, Alex Fidder, N P E Vermeulen, Daan Noort

  Chemico-biological interactions. 04/2012;

  Methods to unequivocally assess and quantify exposure to organophosphate anti-cholinesterase agents are highly valuable, either from a biomonitoring or a forensic perspective. Since for both OPMethods to unequivocally assess and quantify exposure to organophosphate anti-cholinesterase agents are highly valuable, either from a biomonitoring or a forensic perspective. Since for both OP pesticides and various nerve agents the skin is a predominant route of entry, we hypothesized that proteins in the skin might represent an ideal source of unequivocal and persistent biomarkers for exposure to these compounds. In this exploratory study we show that keratin proteins in human skin are relevant binding sites for organophosphates. The thick cornified epithelium of human plantar skin (callus) was exposed to a selection of relevant organophosphorus compounds and keratin proteins were subsequently extracted. After carboxymethylation of cysteine residues, enzymatic digestion of the keratins with pronase and trypsin was performed and the resulting amino acid and peptides were analyzed to assess whether covalent adducts had formed. LC-tandem MS analysis of the pronase digests demonstrated that tyrosine and to a lesser extent serine residues were selectively modified by organophosphate pesticides (both phosphorothioates and the corresponding oxon forms) under physiological conditions. In addition, modification of tyrosine with the nerve agent VX was unequivocally assessed. In order to elucidate specific binding sites, LC-tandem MS analysis of trypsin digests showed two separate tryptic keratin fragments, i.e. LASY*LDK and SLY*GLGGSK, with Y* the modified tyrosine residues, originating from keratin 1/6 and keratin 10, respectively. These preliminary findings, revealing novel binding targets for anti-cholinesterase organophosphates, will form a firm basis for the development of novel (non-invasive) methods for assessment of exposure to organophosphates. Whether this binding will also have biological implications remains an issue for further investigations.

• Toxic effects following phosgene exposure of human epithelial lung cells in vitro using a CULTEX® system.

  Authors: Dorien Wijte, Marcel J Alblas, Daan Noort, Jan P Langenberg, Herman P M van Helden

  Toxicology in vitro : an international journal published in association with BIBRA. 09/2011; 25(8):2080-7.

  The aim of the present study was to investigate toxic effects following phosgene exposure of human epithelial lung cells (A549) in vitro using a CULTEX® system. In particular, toxic effects regardingThe aim of the present study was to investigate toxic effects following phosgene exposure of human epithelial lung cells (A549) in vitro using a CULTEX® system. In particular, toxic effects regarding early biomarkers emerging during the latency period following exposure might be of great value for medical treatment. Cells cultured on semi-permeable membranes were directly exposed at the liquid-air interface to different concentrations of phosgene, or dry medical air. Cell membrane integrity (leakage of LDH), metabolic activity (reduction of Alamar Blue), oxidative damage (GSH, and HO-1, in cell lysates), and release of IL-8, were studied. For most of the above-mentioned biological end-point markers, significant changes could be assessed following a 20 min exposure to 1.0 ppm and 2.0 ppm phosgene. Moreover, except for IL-8, all biological marker profiles showed to be in line with results obtained by others in animal studies. The C×t value of 40 ppm min appeared to be constant. The overall results suggest that at 4 h post-exposure a maximal level of toxicity was achieved. Our results demonstrate the suitability of a CULTEX® system to detect toxic effects induced by phosgene on human epithelial lung cells, which may contribute to the discovery of early biomarkers for new medical countermeasures.

• Synthesis and evaluation of strand and turn modified ring-extended gramicidin S derivatives.

  Authors: Annemiek D Knijnenburg, Varsha V Kapoerchan, Gijsbert M Grotenbreg, Emile Spalburg, Albert J de Neeling, Roos H Mars-Groenendijk, Daan Noort, José M Otero, Antonio L Llamas-Saiz, Mark J van Raaij, Bep Ravensbergen, Peter H Nibbering, Gijs A van der Marel, Herman S Overkleeft, Mark Overhand

  Bioorganic & medicinal chemistry. 06/2011; 19(11):3402-9.

  In this paper, we describe the crystal structure of previously reported ring-extended gramicidin S (GS) derivative 2 (GS14K4), containing a d-amino acid residue in one of the β-strand regions. ThisIn this paper, we describe the crystal structure of previously reported ring-extended gramicidin S (GS) derivative 2 (GS14K4), containing a d-amino acid residue in one of the β-strand regions. This structure is in agreement with a previously reported modeling study of the same molecule. The polar side chain of the additional d-amino acid residue is positioned at the same face of the molecule as the hydrophobic side chains, and we believe that because of this compound 2 is considerably less hydrophobic than extended GS derivatives in which the strand regions are exclusively composed of l-amino acids. Using this backbone structure as our benchmark we prepared a small series of ring-extended GS analogues featuring sugar amino acid dipeptide isosteres of varied hydrophobicity at the turn region. We show that via this approach hydrophobicity of extended GS analogues can be tuned without affecting the secondary structure (as observed from NMR and CD spectra). Biological evaluation reveals that hydrophobicity correlates to cell toxicity, but still bacteriolysis is induced with GS analogues that are too hydrophilic to efficiently lyse human red blood cells.

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• Peripheral site ligand conjugation to a non-quaternary oxime enhances reactivation of nerve agent-inhibited human acetylcholinesterase.

  Authors: Martijn C de Koning, Marco van Grol, Daan Noort

  Toxicology letters. 04/2011; 206(1):54-9.

  Commonly employed pyridinium-oxime (charged) reactivators of nerve agent inhibited acetylcholinesterase (AChE) do not readily pass the blood brain barrier (BBB) because of the presence of charge(s).Commonly employed pyridinium-oxime (charged) reactivators of nerve agent inhibited acetylcholinesterase (AChE) do not readily pass the blood brain barrier (BBB) because of the presence of charge(s). Conversely, non-ionic oxime reactivators often suffer from a lack of reactivating potency due to a low affinity for the active site of AChE. It was therefore hypothesized that an extra contribution in affinity may be achieved by covalently connecting a peripheral site ligand (PSL) to a non-ionic reactivator, which may result in a higher reactivation potency of the total construct. This validity of this approach, which proved successful for charged pyridinium oximes in earlier work, is now further exemplified with the covalent linkage of a neutral PSL via a spacer to a non-ionic and otherwise almost non-reactivating α-ketoaldoxime. It is demonstrated that the linkage of the PSL resulted in a remarkable increase in reactivation potency of the hybrid compounds. Although the molecules reported here are still inefficient reactivators compared to the current pyridinium oximes, the presented approach holds promise for the future design and synthesis of non-ionic oxime reactivators with improved BBB penetration and may be suited as well for non-oxime reactivators thus further widening the scope in the ongoing search for broad-spectrum reactivators.

• Exploring the conformational and biological versatility of β-turn-modified gramicidin S by using sugar amino acid homologues that vary in ring size.

  Authors: Annemiek D Knijnenburg, Adriaan W Tuin, Emile Spalburg, Albert J de Neeling, Roos H Mars-Groenendijk, Daan Noort, José M Otero, Antonio L Llamas-Saiz, Mark J van Raaij, Gijs A van der Marel, Herman S Overkleeft, Mark Overhand

  Chemistry (Weinheim an der Bergstrasse, Germany). 03/2011; 17(14):3995-4004.

  Monobenzylated sugar amino acids (SAAs) that differ in ether ring size (containing an oxetane, furanoid, and pyranoid ring) were synthesized and incorporated in one of the β-turn regions of theMonobenzylated sugar amino acids (SAAs) that differ in ether ring size (containing an oxetane, furanoid, and pyranoid ring) were synthesized and incorporated in one of the β-turn regions of the cyclo-decapeptide gramicidin S (GS). CD, NMR spectroscopy, modeling, and X-ray diffraction reveal that the ring size of the incorporated SAA moieties determines the spatial positioning of their cis-oriented carboxyl and aminomethyl substituents, thereby subtly influencing the amide linkages with the adjacent amino acids in the sequence. Unlike GS itself, the conformational behavior of the SAA-containing peptides is solvent dependent. The derivative containing the pyranoid SAA is slightly less hydrophobic and displays a diminished haemolytic activity, but has similar antimicrobial properties as GS.

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• Evaluation of readily accessible azoles as mimics of the aromatic ring of D-phenylalanine in the turn region of gramicidin S.

  Authors: Matthijs van der Knaap, Lianne T Lageveen, Henk J Busscher, Roos Mars-Groenendijk, Daan Noort, José M Otero, Antonio L Llamas-Saiz, Mark J van Raaij, Gijsbert A van der Marel, Herman S Overkleeft, Mark Overhand

  ChemMedChem. 03/2011; 6(5):840-7.

  The influence of replacing the d-phenylalanine residue with substituted and unsubstituted azoles on the structure and biological activity of the antibiotic gramicidin S was investigated against aThe influence of replacing the d-phenylalanine residue with substituted and unsubstituted azoles on the structure and biological activity of the antibiotic gramicidin S was investigated against a representative panel of Gram-positive and Gram-negative bacteria strains. Substituted triazole derivatives, obtained using a convergent synthetic strategy, are as active as gramicidin S, provided that any substituent on the triazole moiety is not too large. The unsubstituted triazole derivative was biologically less active than the parent natural product, gramicidin S. In general for the triazole series, the hemolytic activity could be correlated with the antibacterial activity, that is, the higher the antibacterial activity, the higher the toxicity towards blood cells. Interestingly, its imidazole counterpart showed high antibacterial activity, combined with significantly diminished hemolytic activity.

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• Structural study of the complex stereoselectivity of human butyrylcholinesterase for the neurotoxic V-agents.

  Authors: Marielle Wandhammer, Eugénie Carletti, Marcel Van der Schans, Emilie Gillon, Yvain Nicolet, Patrick Masson, Maurice Goeldner, Daan Noort, Florian Nachon

  The Journal of biological chemistry. 03/2011; 286(19):16783-9.

  Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can beNerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous time-dependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs. The stereoselectivity of BChE is an important parameter for its efficiency at scavenging the most toxic OPs enantiomer for AChE. Crystals of BChE inhibited in solution or in cristallo with racemic V-agents (VX, Russian VX, and Chinese VX) systematically show the formation of the P(S) adduct. In this configuration, no catalysis of aging seems possible as confirmed by the three-dimensional structures of the three conjugates incubated over a period exceeding a week. Crystals of BChE soaked in optically pure VX(R)-(+) and VX(S)-(-) solutions lead to the formation of the P(S) and P(R) adduct, respectively. These structural data support an in-line phosphonylation mechanism. Additionally, they show that BChE reacts with VX(R)-(+) in the presence of racemic mixture of V-agents, at odds with earlier kinetic results showing a moderate higher inhibition rate for VX(S)-(-). These combined results suggest that the simultaneous presence of both enantiomers alters the enzyme stereoselectivity. In summary, the three-dimensional data show that BChE reacts preferentially with P(R) enantiomer of V-agents and does not age, in complete contrast to AChE, which is selectively inhibited by the P(S) enantiomer and ages.

• An adamantyl amino acid containing gramicidin S analogue with broad spectrum antibacterial activity and reduced hemolytic activity.

  Authors: Varsha V Kapoerchan, Annemiek D Knijnenburg, Miquel Niamat, Emile Spalburg, Albert J de Neeling, Peter H Nibbering, Roos H Mars-Groenendijk, Daan Noort, José M Otero, Antonio L Llamas-Saiz, Mark J van Raaij, Gijs A van der Marel, Herman S Overkleeft, Mark Overhand

  Chemistry (Weinheim an der Bergstrasse, Germany). 10/2010; 16(40):12174-81.

  The cyclic cationic antimicrobial peptide gramicidin S (GS) is an effective topical antibacterial agent that is toxic for human red blood cells (hemolysis). Herein, we present a series of amphiphilicThe cyclic cationic antimicrobial peptide gramicidin S (GS) is an effective topical antibacterial agent that is toxic for human red blood cells (hemolysis). Herein, we present a series of amphiphilic derivatives of GS with either two or four positive charges and characteristics ranging between very polar and very hydrophobic. Screening of this series of peptide derivatives identified a compound that combines effective antibacterial activity with virtually no toxicity within the same concentration range. This peptide acts against both Gram-negative and Gram-positive bacteria, including several MRSA strains, and represents an interesting lead for the development of a broadly applicable antibiotic.

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• Tuning hydrophobicity of highly cationic tetradecameric Gramicidin S analogues using adamantane amino acids.

  Authors: Annemiek D Knijnenburg, Varsha V Kapoerchan, Emile Spalburg, Albert J de Neeling, Roos H Mars-Groenendijk, Daan Noort, Gijs A van der Marel, Herman S Overkleeft, Mark Overhand

  Bioorganic & medicinal chemistry. 09/2010; 18(23):8403-9.

  Ring extended Gramicidin S analogues containing adamantane amino acids and six cationic residues were designed and evaluated. Systematic replacement of the hydrophobic residues with adamantane aminoRing extended Gramicidin S analogues containing adamantane amino acids and six cationic residues were designed and evaluated. Systematic replacement of the hydrophobic residues with adamantane amino acids resulted in a small set of compounds with varying amphipathic character. It was found that the amphipathicity of these compounds is correlated to their biological activity. Several bacterial strains including MRSA strains were shown to be killed by the novel peptides. The most potent antibacterial peptides are tetradecameric GS analogues containing six positives charges and two adamantane moieties.

• Immunomagnetic separation and quantification of butyrylcholinesterase nerve agent adducts in human serum.

  Authors: Jennifer L S Sporty, Sharon W Lemire, Edward M Jakubowski, Julie A Renner, Ronald A Evans, Robert F Williams, Jurgen G Schmidt, Marcel J van der Schans, Daan Noort, Rudolph C Johnson

  Analytical chemistry. 08/2010; 82(15):6593-600.

  A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. AntibutyrylcholinesteraseA novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 microL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilution-tandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agent-exposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure.

• Gramicidin S derivatives containing cis- and trans-morpholine amino acids (MAAs) as turn mimetics.

  Authors: Varsha V Kapoerchan, Emile Spalburg, Albert J de Neeling, Roos H Mars-Groenendijk, Daan Noort, José M Otero, Patricia Ferraces-Casais, Antonio L Llamas-Saiz, Mark J van Raaij, Joop van Doorn, Gijs A van der Marel, Herman S Overkleeft, Mark Overhand

  Chemistry (Weinheim an der Bergstrasse, Germany). 03/2010; 16(14):4259-65.

  The cyclic decapeptide gramicidin S (GS) was used as a model for the evaluation of four turn mimetics. For this purpose, one of the D-Phe-Pro two-residue turn motifs in the rigid cyclic beta-hairpinThe cyclic decapeptide gramicidin S (GS) was used as a model for the evaluation of four turn mimetics. For this purpose, one of the D-Phe-Pro two-residue turn motifs in the rigid cyclic beta-hairpin structure of GS was replaced with morpholine amino acids (MAA 2-5), differing in stereochemistry and length of the side-chain. The conformational properties of the thus obtained GS analogues (6-9) was assessed by using NMR spectroscopy and X-ray crystallography, and correlated with their biological properties (antimicrobial and hemolytic activity). We show that compound 8, containing the dipeptide isostere trans-MAA 4, has an apparent high structural resemblance with GS and that its antibacterial activity against a panel of Gram positive and -negative bacterial strains is better than the derivatives 6, 7 and 9.

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• Ring-Extended Derivatives of Gramicidin S with Furanoid Sugar Amino Acids in the Turn Region Have Enhanced Antimicrobial Activity.

  Authors: Annemiek D Knijnenburg, Emile Spalburg, Albert J de Neeling, Roos H Mars-Groenendijk, Daan Noort, Gijsbert M Grotenbreg, Gijs A van der Marel, Herman S Overkleeft, Mark Overhand

  ChemMedChem. 10/2009;

• Structural and biological evaluation of some loloatin C analogues.

  Authors: Adriaan W Tuin, Gijsbert M Grotenbreg, Emile Spalburg, Albert J de Neeling, Roos H Mars-Groenendijk, Gijsbert A van der Marel, Daan Noort, Herman S Overkleeft, Mark Overhand

  Bioorganic & medicinal chemistry. 08/2009;

  Loloatin C is a cyclic cationic antimicrobial peptide which is active against Gram positive as well as certain Gram negative bacteria. Unfortunately, it is equally potent against human erythrocytes.Loloatin C is a cyclic cationic antimicrobial peptide which is active against Gram positive as well as certain Gram negative bacteria. Unfortunately, it is equally potent against human erythrocytes. To probe the structure-activity relationship of this promising antibiotic peptide, amino acid substitution and/or incorporation of a constraint sugar amino acid dipeptide isoster has been applied. Six new derivatives have been synthesized using SPPS and their solution structure investigated using NMR studies. Finally, the antimicrobial and the hemolytic activities have been determined.

• Synthesis and biological evaluation of asymmetric gramicidin S analogues containing modified d-phenylalanine residues.

  Authors: Matthijs van der Knaap, Eefje Engels, Henk J Busscher, José M Otero, Antonio L Llamas-Saiz, Mark J van Raaij, Roos H Mars-Groenendijk, Daan Noort, Gijsbert A van der Marel, Herman S Overkleeft, Mark Overhand

  Bioorganic & medicinal chemistry. 08/2009;

  The synthesis of new analogues of the cationic antimicrobial peptide gramicidin S, having a modified d-phenylalanine residue, their antibacterial properties against several Gram positive and negativeThe synthesis of new analogues of the cationic antimicrobial peptide gramicidin S, having a modified d-phenylalanine residue, their antibacterial properties against several Gram positive and negative strains, as well as their hemolytic activity is reported.

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• Assessment of nerve agent exposure: existing and emerging methods.

  Authors: Jan P Langenberg, Marcel J van der Schans, Daan Noort

  Bioanalysis. 07/2009; 1(4):729-39.

  The perceived threat of the use of nerve agents by terrorists against civilian targets implies the need for methods for point-of-care (POC) diagnosis. This review presents an overview of methods thatThe perceived threat of the use of nerve agents by terrorists against civilian targets implies the need for methods for point-of-care (POC) diagnosis. This review presents an overview of methods that are currently available for the assessment of exposure to nerve agents. Since these methods are mostly MS based, they require complex and expensive equipment and well-trained personnel and, consequently, they are not very suitable for rapid POC diagnosis. However, new technologies are emerging that allow, among others, immunochemical detection of acetylcholinesterase inhibited by nerve agents. Also, lab-on-a-chip methodologies are under development. It is anticipated that MS methods will be suitable for POC diagnosis within a few years, due to the miniaturization of equipment and the emergence of methodologies that enable mass spectrometric analysis with little sample pretreatment and that are potentially fieldable, such as direct analysis in real time and desorption electrospray ionization MS.

• Activity-Based Protein Profiling Reveals Broad Reactivity of the Nerve Agent Sarin.

  Authors: Adriaan W Tuin, Marijke A E Mol, Roland M van den Berg, A Fidder, Gijs A van der Marel, Herman S Overkleeft, Daan Noort

  Chemical research in toxicology. 03/2009;

  Elucidation of noncholinesterase protein targets of organophosphates, and nerve agents in particular, may reveal additional mechanisms for their high toxicity as well as clues for novel therapeuticElucidation of noncholinesterase protein targets of organophosphates, and nerve agents in particular, may reveal additional mechanisms for their high toxicity as well as clues for novel therapeutic approaches toward intoxications with these agents. Within this framework, we here describe the synthesis of the activity-based probe 3, which contains a phosphonofluoridate moiety, a P-Me moiety, and a biotinylated O-alkyl group, and its use in activity-based protein profiling with two relevant biological samples, that is, rhesus monkey liver and cultured human A549 lung cells. In this way, we have unearthed eight serine hydrolases (fatty acid synthase, acylpeptide hydrolase, dipeptidyl peptidase 9, prolyl oligopeptidase, carboxylesterase, long-chain acyl coenzyme A thioesterase, PAF acetylhydrolase 1b, and esterase D/S-formyl glutathione hydrolase) as targets that are modified by the nerve agent sarin. It is also shown that the newly developed probe 3 might find its way into the development of alternative, less laborious purification protocols for human butyrylcholinesterase, a potent bioscavenger currently under clinical investigation as a prophylactic/therapeutic for nerve agent intoxications.

• Development of an automated on-line pepsin digestion-liquid chromatography-tandem mass spectrometry configuration for the rapid analysis of protein adducts of chemical warfare agents.

  Authors: Jeroen Carol-Visser, Marcel van der Schans, Alex Fidder, Albert G Hulst, Ben L M van Baar, Hubertus Irth, Daan Noort

  Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. 08/2008; 870(1):91-7.

  Rapid monitoring and retrospective verification are key issues in protection against and non-proliferation of chemical warfare agents (CWA). Such monitoring and verification are adequatelyRapid monitoring and retrospective verification are key issues in protection against and non-proliferation of chemical warfare agents (CWA). Such monitoring and verification are adequately accomplished by the analysis of persistent protein adducts of these agents. Liquid chromatography-mass spectrometry (LC-MS) is the tool of choice in the analysis of such protein adducts, but the overall experimental procedure is quite elaborate. Therefore, an automated on-line pepsin digestion-LC-MS configuration has been developed for the rapid determination of CWA protein adducts. The utility of this configuration is demonstrated by the analysis of specific adducts of sarin and sulfur mustard to human butyryl cholinesterase and human serum albumin, respectively.

• Liquid chromatography/tandem mass spectrometry detection of covalent binding of acetaminophen to human serum albumin.

  Authors: Micaela C Damsten, Jan N M Commandeur, Alex Fidder, Albert G Hulst, Daan Touw, Daan Noort, Nico P E Vermeulen

  Drug metabolism and disposition: the biological fate of chemicals. 09/2007; 35(8):1408-17.

  Covalent binding of reactive electrophilic intermediates to proteins is considered to play an important role in the processes leading to adverse drug reactions and idiosyncratic drug reactions.Covalent binding of reactive electrophilic intermediates to proteins is considered to play an important role in the processes leading to adverse drug reactions and idiosyncratic drug reactions. Consequently, both for the discovery and the development of new drugs, there is a great interest in sensitive methodologies that enable the detection of covalent binding of drugs and drug candidates in vivo. In this work, we present a strategy for the generation and analysis of drug adducts to human serum albumin. Our methodology is based on the isolation of albumin from blood, its digestion to peptides by pronase E, and the sensitive detection of adducts to the characteristic cysteine-proline-phenylalanine (CPF) tripeptide by liquid chromatography/tandem mass spectrometry. We chose acetaminophen (APAP) as a model compound because this drug is known to induce covalent binding to proteins when bioactivated by cytochromes P450 to its reactive N-acetyl-p-benzoquinoneimine metabolite. First, by microsomal incubations of APAP in presence of CPF and/or intact albumin, in vitro reference adducts were generated to determine the mass spectrometric characteristics of the expected CPF adducts and to confirm their formation on pronase E digestion of the alkylated protein. When applying this methodology to albumin isolated from blood of patients exposed to APAP, we were indeed able to detect the corresponding CPF adducts. Therefore, this strategy could be seen as a potential biomonitoring tool to detect in vivo reactive intermediates of drugs and drug candidates, e.g., in the preclinical and clinical development phase.

• Detection of sulfur mustard adducts in human callus by phage antibodies.

  Authors: Floris J Bikker, Roos H Mars-Groenendijk, Daan Noort, Alex Fidder, Govert P van der Schans

  Chemical biology & drug design. 06/2007; 69(5):314-20.

  As part of a research program to develop novel methods for diagnosis of sulfur mustard exposure in the human skin the suitability of phage display was explored. Phage display is a relative new methodAs part of a research program to develop novel methods for diagnosis of sulfur mustard exposure in the human skin the suitability of phage display was explored. Phage display is a relative new method that enables researchers to quickly evaluate a huge range of potentially useful antibodies, thereby bypassing the more costly and time-consuming hybridoma technique. The Tomlinson I and J phage libraries were used to select phage antibodies exhibiting affinity for sulfur mustard adducts on keratins, isolated from human callus. Two kinds of phage antibodies were obtained: antibodies recognizing keratin and antibodies recognizing keratin which was exposed to sulfur mustard. These phage antibodies retained activity after repeated culturing and culturing in larger volumes. For the first time antibody phage display was successfully applied for immunodiagnostics of a chemical warfare agent.

• Evaluation of the antibacterial spectrum of drosocin analogues.

  Authors: Floris J Bikker, Wendy E Kaman-van Zanten, Anne-Marij B C de Vries-van de Ruit, Ingrid Voskamp-Visser, Peter A V van Hooft, Roos H Mars-Groenendijk, Peter C de Visser, Daan Noort

  Chemical biology & drug design. 10/2006; 68(3):148-53.

  Drosocin is a 19-mer, cationic antimicrobial peptide from Drosophila melanogaster. The aim of the study was to examine the antibacterial spectrum of unglycosylated drosocin analogues. Furthermore,Drosocin is a 19-mer, cationic antimicrobial peptide from Drosophila melanogaster. The aim of the study was to examine the antibacterial spectrum of unglycosylated drosocin analogues. Furthermore, the amino acid sequence of DnaK, drosocin's intracellular target, from susceptible species was aligned and studied for sequence homology. From this a panel of 31 bacterial strains, including Salmonella strains with truncated lipopolysaccharide structures, was tested for susceptibility towards three drosocin analogues. Available bacterial DnaK amino acid sequences were retrieved from the ExPASy proteomics server of the Swiss Institute of Bioinformatics studied for sequence homology. Seventeen of the 31 strains tested were susceptible for the drosocin analogues. Minimal inhibitory concentration values against mainly Gram-negative bacteria ranged from 3.1 to 100 microm. With the exception of Micrococcus luteus and Xanthomonas campestris all drosocin analogue-susceptible strains were Enterobacteriaceae showing a high DnaK amino acid sequence homology.