Biomedical subjects
Publications and source records attributed to Rob M J Liskamp.
The synthesis of a novel potent cyclic peptide MC4-ligand by ring-closing metathesis (RCM) is described. Based on the Ac-Nle-Gly-Lys-D-Phe-Arg-Trp-Gly-NH2-MC4 ligand, Ac-Nle-Alg-Lys-D-Phe-Arg-Trp-Alg-NH2 was designed and synthesized followed by cyclization using RCM. Both compounds are high affinity and selective MC4-R-agonists. The cyclic RCM-peptide was more potent in a rat-grooming assay.
All possible peptoid-peptide hybrids of an MC4 receptor agonist were synthesized and investigated on cells expressing different melanocortin (MC) receptor subtypes and for rat grooming behavior. In general, receptor selectivity remained while affinity and potency were decreased. The length of the functional group of Trp was more important for MC3 and MC5 than for MC4 receptor binding. In general, the potency of the peptoid-peptide hybrids to increase rat excessive grooming behavior correlated well with MC4 receptor pharmacology.
[reaction: see text] A highly efficient coupling of protected beta-substituted aminoethane sulfonyl azides with thio acids is reported. In the case of peptide thio acids, this method encompasses a new chemoselective ligation method. Furthermore, the resulting alpha-amino acyl sulfonamides can be alkylated with suitable electrophiles to obtain densely functionalized sulfonamide scaffolds.
Thermodynamic and kinetic studies of biomolecular interactions give insight into specificity of molecular recognition processes and advance rational drug design. Binding of phosphotyrosine (pY)-containing peptides to Src- and Grb2-SH2 domains was investigated using a surface plasmon resonance (SPR)-based method. This SPR assay yielded thermodynamic binding constants in solution, and the kinetic information contained in the SPR signal allowed kinetic analysis, which demonstrated distinct ways for pY ligands to interact with the SH2 domains. The results for binding to Src SH2 were consistent with sequestration of water molecules in the interface of the pYEEI peptide/Src SH2 complex. The results for a pYVNV peptide binding to Grb2 SH2 suggested a conformational change for Grb2 SH2 upon binding, which is not observed for Src SH2. Binding of a cyclic construct, allowing the pYVNV sequence in the bound conformation, did not have the expected entropy advantage. The results suggest an alternative binding mode for this construct, with the hydrophobic ring-closing part interacting with the protein. In all cases, except for full-length Grb2 protein, the affinity for the immobilized peptide at the SPR sensor and in solution was identical. This study demonstrates that SPR thermodynamic and kinetic analysis is a useful strategic tool in drug design.
To gain insight into the parameters that determine the arrangement of proteins in membranes, (2)H NMR experiments were performed to analyze tilt and rotation angles of membrane-spanning alpha-helical model peptides upon incorporation in diacylphosphatidylcholine bilayers with varying thickness. The peptides consisted of the sequence acetyl-GW(2)(LA)(8)LW(2)A-NH(2) (WALP23) and analogues thereof, in which the interfacial Trp residues were replaced by Lys (KALP23) and/or the hydrophobic sequence was replaced by Leu (WLP23 and KLP23). The peptides were synthesized with a single deuterium-labeled alanine at four different positions along the hydrophobic segment. For all peptides a small but systematic increase in tilt angle was observed upon decreasing the bilayer thickness. However, significantly larger tilt angles were obtained for the Lys-flanked KALP23 than for the Trp-flanked WALP23, suggesting that interfacial anchoring interactions of Trp may inhibit tilting. Increasing the hydrophobicity resulted in an increase in tilt angle for the Trp-flanked analogue only. For all peptides the maximum tilt angle obtained was remarkably small (less than 12 degrees ), suggesting that further tilting is inhibited, most likely due to unfavorable packing of lipids around a tilted helix. The results furthermore showed that the direction of tilt is determined almost exclusively by the flanking residues: Trp- and Lys-flanked peptides were found to have very different rotation angles, which were influenced significantly neither by hydrophobicity of the peptides nor by the extent of hydrophobic mismatch. Finally, very small changes in the side chain angles of the deuterated alanine probes were observed in Trp-flanked peptides, suggesting that these peptides may decrease their hydrophobic length to help them to adapt to thin membranes.
The synthesis of potential beta-turn mimetics based on cyclic sulfonamide peptoid/peptoid hybrids is described. These are readily synthesized using a solid phase protocol followed by cyclization in solution, and their suitability to combinatorial approaches is illustrated by the synthesis of a small but diversely functionalized library.
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The Syk tandem Src homology 2 domain (Syk tSH2) constitutes a flexible protein module involved in the regulation of Syk kinase activity. The Syk tSH2 domain is assumed to function by adapting the distance between its two SH2 domains upon bivalent binding to diphosphotyrosine ligands. A thermodynamic and kinetic analysis of ligand binding was performed by using surface plasmon resonance (SPR). Furthermore, the effect of binding on the Syk tSH2 structural dynamics was probed by hydrogen/deuterium exchange and electrospray mass spectrometry (ESI-MS). Two ligands were studied: 1, a flexible peptide derived from the tSH2 recognition ITAM sequence at the gamma chain of the FcepsilonRI-receptor, and 2, a ligand in which the amino acids between the two SH2 binding motifs in ligand 1 have been replaced by a rigid linker of comparable length. Both ligands display comparable affinity for Syk tSH2 at 25 degrees C, yet a major difference in thermodynamics is observed. Upon binding of the rigid ligand, 2, the expected entropy advantage is not realized. On the contrary, 2 binds with a considerably higher entropy price of approximately 9 kcal mol-1, which is attributed to a further decrease in protein flexibility upon binding to this rigid ligand. The significant reduction in deuterium incorporation in the Syk tSH2 protein upon binding of either 1 or 2, as monitored by ESI-MS, indicates a major reduction in protein dynamics upon binding. The results are consistent with a two-step binding model: after an initial binding step, a rapid structural change of the protein occurs, followed by a second binding step. Such a bivalent binding model allows high affinity and fast dissociation kinetics, which are very important in transient signal-transduction processes.
Gaining insight into phosphoproteomes is of the utmost importance for understanding regulation processes such as signal transduction and cellular differentiation. While the identification of phosphotyrosine-containing amino acid sequences in peptides and proteins is now becoming possible, mainly because of the availability of high-affinity antibodies, no general and robust methodology allowing the selective enrichment and analysis of serine- and threonine-phosphorylated proteins and peptides is presently available. The method presented here involves chemical modification of phosphorylated serine or threonine residues and their subsequent derivatization with the aid of a multifunctional probe molecule. The designed probe contains four parts: a reactive group that is used to bind specifically to the modified phosphopeptide, an optional part in which heavy isotopes can be incorporated, an acid-labile linker, and an affinity tag for the selective enrichment of modified phosphopeptides from complex mixtures. The acid-cleavable linker allows full recovery from the affinity-purified material and removal of the affinity tag prior to MS analysis. The preparation of a representative probe molecule containing a biotin affinity tag and its applicability in phosphoproteome analysis is shown in a number of well-defined model systems of increasing degrees of complexity. Amounts of phosphopeptide as low as 1 nmol can be modified and enriched from a mixture of peptides. During the development of the beta-elimination/nucleophilic addition protocol, special attention was paid to the different experimental parameters that might affect the chemical-modification steps carried out on phosphorylated residues.
Structural flexibility plays a crucial role in protein function. To assess whether specific structural changes are associated with the binding of an immunoreceptor tyrosine-based activation motif (ITAM) to the tandem Src homology-2 domains (tSH2) of the spleen tyrosine kinase [EC 2.7.7.112] (Syk), we used an approach based on protein hydrogen/deuterium exchange in the presence and absence of the diphosphorylated ITAM peptide. The protein deuterium uptake by the intact Syk protein was monitored in time by electrospray mass spectrometry, which revealed a dramatic relative decrease in deuterium uptake when the protein was bound to the ITAM peptide, suggesting an overall change in protein dynamics. Subsequently, the deuterium incorporation of individual segments of the protein was investigated using proteolysis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass-analysis, which revealed that several regions of Syk tSH2 are significantly more protected from exchange in the presence of the ITAM peptide. Four protected regions encompass the phosphotyrosine and hydrophobic binding sites on the SH2 domains, whereas two other protected regions are located in the inter-SH2 linker motif and do not make any direct contacts with the peptide. Interestingly, our data suggest that binding of the ITAM peptide to Syk tSH2 induces distal structural effects on the protein that stabilize the inter-SH2 linker region, possibly by raising the degree of helical structure upon binding.
Therapeutic intervention in experimental autoimmune diseases by modulation of the T cell mediated autoimmune response has been accomplished in the past using altered peptide ligands (APLs). These peptides are usually created by applying alterations to the T cell epitope recognized by the autoaggressive T cells. In this study, we investigated whether it was possible to design APLs in a rational way, using knowledge of molecular interaction in the MHC-peptide-T cell receptor (TCR) complex, for the therapeutic intervention in experimental autoimmune encephalomyelitis (EAE). Additionally, the value of peptidomimetic modification and alterations based on posttranslational modifications for the design of APLs was examined. Based on a molecular model of the MHC-peptide complex, the T cell receptor contact residues were identified and selected alterations were applied. The designed APLs were tested for MHC binding capacity, T cell recognition, blocking of the autoreactive T cell response, immunogenicity, encephalitogenicity, and therapeutic activity. Based on the results of the in vitro assays, it was expected that some of our APLs would be able to modulate EAE. Nevertheless, none of these APLs displayed clear therapeutic activity in vivo. Thus, rational design of modified peptides for immunotherapy has to await further insights into the relationships between structure and peptide/peptidomimetic induced T cell activation, and until that, there is no possibility to take advantage of the tailor made origin of peptidomimetics.
The balance between specific and degenerate T cell recognition of MHC class II bound peptides is crucial for T cell repertoire selection, and holds important implications for protective immunity versus autoimmunity. To investigate the degree of degeneracy in T cell recognition, we applied selected modifications to T cell receptor (TCR) contact residue amino acids in the MHC class II bound epitope gpMBP72-85. By using glycosylated amino acids, as an example of a posttranslational modification, large alterations were applied. Small modifications were accomplished by exchanging an arginine residue for a citrulline or an ornithine residue. Finally, the unmodified TCR contact residue side chains were shifted one atom position to the left, using peptoid residues. Both these large and subtle changes in the wild type (WT) peptide caused lack of recognition by WT peptide specific monoclonal and polyclonal T cells. Furthermore, T cells specific for the modified peptides did not cross recognize the WT peptide. Using a set of additional compounds, we investigated the specificity of these T cell populations into detail. Our data reveal a strongly limited plasticity in T cell recognition, and a high specificity for TCR contact residue side chains.
A series of mono-, di-, and tetravalent galabiose (Galalpha1-4Gal) compounds were synthesized in good yields by coupling of a general carboxylic acid-bearing sugar building block to dendritic scaffolds based on the 3,5-di-(2-aminoethoxy)benzoic acid branching unit. Furthermore, a poly(amidoamine)- (PAMAM-) based dendritic galabioside was synthesized containing eight galabiose units. All galabiosides were tested in a hemagglutination assay and a surface plasmon resonance (SPR) competition assay in order to establish their potency in the binding to the bacterial Gram-positive pathogen Streptococcus suis. A monovalent galabioside containing a short spacer was used as a reference compound in all the assays. Variations in the scaffold as well as in the spacer arms were introduced to determine their influence on the inhibition. The best inhibitor of hemagglutination was an octavalent galabioside with a minimal inhibitory concentration (MIC) of 0.3 nM, to the best of our knowledge the first example of inhibition of bacterial binding by a soluble carbohydrate at a subnanomolar concentration.
An alkene-bridged mimic of the complex DE-bisthioether-ring system of the antibiotic nisin was prepared in one step from the linear precursor.
Staphylococcus aureus excretes a factor that specifically and simultaneously acts on the C5aR and the formylated peptide receptor (FPR). This chemotaxis inhibitory protein of S. aureus (CHIPS) blocks C5a- and fMLP-induced phagocyte activation and chemotaxis. Monoclonal anti-CHIPS Abs inhibit CHIPS activity against one receptor completely without affecting the other receptor, indicating that two distinct sites are responsible for both actions. A CHIPS-derived N-terminal 6 aa peptide is capable of mimicking the anti-FPR properties of CHIPS but has no effect on the C5aR. Synthetic peptides in which the first 6 aa are substituted individually for all other naturally occurring amino acids show that the first and third residue play an important role in blocking the FPR. Using an Escherichia coli expression system, we created mutant CHIPS proteins in which these amino acids are substituted. These mutant proteins have impaired or absent FPR- but still an intact C5aR-blocking activity, indicating that the loss of the FPR-blocking activity is not caused by any structural impairment. This identifies the first and third amino acid, both a phenylalanine, to be essential for CHIPS blocking the fMLP-induced activation of phagocytes. The unique properties of CHIPS to specifically inhibit the FPR with high affinity (kd=35.4 +/- 7.7 nM) could be an important new tool to further stimulate the fundamental research on the mechanisms underlying the FPR and its role in disease processes.
The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) is predominantly alpha-helical, and of the four distinctly different transmembrane M-segments, only the helicity of M1 is ambiguous. In this study, we have investigated the conformation of a membrane-embedded synthetic M1 segment by solid-state nuclear magnetic resonance (NMR) methods. A 35-residue peptide representing the extended alphaM1 domain 206-240 of the Torpedo californica nAChR was synthesized with specific 13C - and 15N-labelled amino acids, and was incorporated in different phosphatidylcholine model membranes. The chemical shift of the isotopic labels was resolved by magic angle spinning (MAS) NMR and could be related to the secondary structure of the alphaM1 analog at the labelled sites. Our results show that the membrane-embedded alphaM1 segment forms an unstable alpha-helix, particularly near residue Leu18 (alphaLeu223 in the entire nAChR). This non-helical tendency was most pronounced when the peptide was incorporated in fully hydrated phospholipid bilayers, with an estimated 40-50% of the peptides having an extended conformation at position Leu18. We propose that the conserved proline residue at position 16 in the alphaM1 analog imparts a conformational flexibility on the M1 segments that could enable membrane-mediated modulation of nAChR activity.
This report describes the synthesis of eight N-terminally modified astressin analogs and their biochemical evaluation as corticotropin releasing factor (CRF) antagonists. The lipidated astressin derivatives were tested on rat CRF receptor type 1 and 2alpha and were found to be active as CRF antagonists (rCRFR1: pA(2)=7.5-8.3; rCRFR2alpha: pA(2)=7.5-9.0) with nearly equal activities as compared to unmodified astressin (rCRFR1: pA(2)=8.3+/-0.09; rCRFR2alpha: pA(2)=8.7+/-0.08).