Modulation of acute inflammation by targeting glycosaminoglycan-cytokine interactions.

Glycosaminoglycans (GAGs) located on cellular membranes and the extracellular matrix (ECM) are able to interact with chemokines and pro-inflammatory cytokines, leading to local cytokine/chemokine accumulation. The tissue-bound cytokines/chemokines function in promoting leukocyte migration and activation, contributing to local inflammation. Hence, targeting of GAG-cytokine interactions may provide an avenue for the attenuation of inflammatory responses. A cationic peptide (MC2) derived from the heparin-binding sequence of mouse IFN-gamma was previously shown by our laboratory to delay allograft rejection in an animal model. In order to further investigate potential anti-inflammatory properties of the MC2 peptide, we have studied its activity in an acute peritoneal inflammation model. Groups of C57Bl/6 mice were injected intraperitoneally with either ConA or thioglycollate and treated with saline (control), the MC2 peptide or two control cationic peptides, poly-l-lysine (PLL) and poly-l-arginine (PLA). Treatment with the MC2 peptide, but not PLA or PLL, resulted in statistically significant reductions in total cell numbers, concentration of total proteins and concentrations of pro-inflammatory cytokines (TNFalpha, IL-6 or IL-1 beta) in peritoneal lavage fluids, without alterations to the qualitative cellular composition of the exudate. These results suggest that targeting GAG-cytokine interaction is a viable approach to reduce inflammation.

Solid-phase synthesis of disulfide heterodimers of peptides.

[structure: see text] The synthesis of the orthogonal disulfide template 1 and its use to synthesize unsymmetrical intermolecular disulfide bond peptides on a solid support are described. Application of template 1 to synthesize bioconjugates of cell permeable moieties based on the disulfide bond is demonstrated.

Potent inhibitors of LXXLL-based protein-protein interactions.

Protein-protein interactions between estrogen receptors, ERalpha and ERbeta, and their coactivators (CoAs) are an attractive target for drug intervention. This interaction is mediated by a small pentapeptide motif (LXXLL), termed the NR box. Based on this motif, a variety of cyclic and linear peptides were synthesized in order to gain a better understanding of the association of CoA proteins with the ER isoforms. Utilizing a time-resolved florescence-based coactivator interaction assay, we determined the abilities of these peptides to inhibit this interaction. Using molecular modeling and CD spectroscopy, we have examined the structural basis of their bioactivities with both hormone receptor isoforms. Either homocysteine or penicillamine was utilized as a substitute for cysteine in the disulfide-bridged peptides, while tertiary leucine and neopentyl glycine were used as the surrogates for the NR box leucines. The most potent disufide-bridged peptide (K(i)= 70 pM, with ERalpha) incorporates neopentyl glycine in the NR box, while the most active peptide in this series with ERbeta (K(i)=350 pM) incorporates tertiary leucine. Surprisingly, several linear peptides containing a single cysteine residue showed activities with low nanomolar K(i) values. Collectively, our results suggest a synthetic approach for designing potent and selective peptidomimetics for ERalpha and ERbeta interactions with CoA proteins effecting estrogen action.

Understanding base-assisted desulfurization using a variety of disulfide-bridged peptides.

A recently rediscovered reaction of base-assisted lanthionine formation has been applied to several systems of disulfide-bridged peptides. In addition to previously described nonapeptides consisting of i, i+3 cystine linkages, the reaction has now been extended to systems consisting of shorter (i, i+2) and longer (i, i+4) disulfide bridges. The desulfurization reaction is also compatible with disulfide bridges formed through homocysteines and penicillamines, yielding unusual amino acids such as cystathionine and beta,beta-dimethyl lanthionine (referred to as "penthionine") in a peptide chain, respectively. Systematic study of this transformation has provided several new insights into its mechanism. We have observed formation of dehydroalanine and dehydrovaline residues resulting from i, i+2-bridged cysteines and i, i+3-bridged cysteine/penicillamine peptides, respectively, thereby supporting a beta-elimination/Michael-addition mechanism for this transformation. Amino acid analysis and NMR data from total correlation spectroscopy (TOCSY) and (1)H-(13)C heteronuclear single quantum correlation (HSQC) experiments show three diastereomeric lanthionine-bridged peptides in the product mixture. But in the case of desulfurization of a cysteine/homocysteine containing disulfide-bridged peptide, Michael addition appears to be stereoselective, yielding a single stereoisomer of cystathionine within the peptide. According to molecular modeling and CD spectroscopy, constrained peptides such as those containing penicillamine are less likely to undergo facile desulfurization. Flexibility of the torsional angles (C(alpha)H-C(alpha)-C(beta)-S) corresponding to the cysteine residues and temperature appear to be contributing factors determining the extent of desulfurization.

Synthesis and biological activities of YkFA analogues: effects of position 4 substitutions and altered ring size on in vitro opioid activity.

Substitution in position 4 of the potent opioid peptide YkFA with aliphatic hydrophobic residues resulted in compounds that retained low nanomolar activities at both mu and delta opioid receptors, while ring contraction by incorporation of diaminobutyric acid in position 2 resulted in a more pronounced decrease in potency at both receptors for the psi[CH(2)NH] pseudopeptide as compared to the all amide parent.

Inhibitors tethered near the acetylcholinesterase active site serve as molecular rulers of the peripheral and acylation sites.

The acetylcholinesterase (AChE) active site consists of a narrow gorge with two separate ligand binding sites: an acylation site (or A-site) at the bottom of the gorge where substrate hydrolysis occurs and a peripheral site (or P-site) at the gorge mouth. AChE is inactivated by organophosphates as they pass through the P-site and phosphorylate the catalytic serine in the A-site. One strategy to protect against organophosphate inactivation is to design cyclic ligands that will bind specifically to the P-site and block the passage of organophosphates but not acetylcholine. To accelerate the process of identifying cyclic compounds with high affinity for the AChE P-site, we introduced a cysteine residue near the rim of the P-site by site-specific mutagenesis to generate recombinant human H287C AChE. Compounds were synthesized with a highly reactive methanethiosulfonyl substituent and linked to this cysteine through a disulfide bond. The advantages of this tethering were demonstrated with H287C AChE modified with six compounds, consisting of cationic trialkylammonium, acridinium, and tacrine ligands with tethers of varying length. Modification by ligands with short tethers had little effect on catalytic properties, but longer tethering resulted in shifts in substrate hydrolysis profiles and reduced affinity for acridinium affinity resin. Molecular modeling calculations indicated that cationic ligands with tethers of intermediate length bound to the P-site, whereas those with long tethers reached the A-site. These binding locations were confirmed experimentally by measuring competitive inhibition constants KI2 for propidium and tacrine, inhibitors specific for the P- and A-sites, respectively. Values of KI2 for propidium increased 30- to 100-fold when ligands had either intermediate or long tethers. In contrast, the value of KI2 for tacrine increased substantially only when ligands had long tethers. These relative changes in propidium and tacrine affinities thus provided a sensitive molecular ruler for assigning the binding locations of the tethered cations.

Helix-stabilized cyclic peptides as selective inhibitors of steroid receptor-coactivator interactions.

The interaction between nuclear receptors and coactivators provides an arena for testing whether protein-protein interactions may be inhibited by small molecule drug candidates. We provide evidence that a short cyclic peptide, containing a copy of the LXXLL nuclear receptor box pentapeptide, binds tightly and selectively to estrogen receptor alpha. Furthermore, as shown by x-ray analysis, the disulfide-bridged nonapeptide, nonhelical in aqueous solutions, is able to adopt a quasihelical conformer while binding to the groove created by ligand attachment to estrogen receptor alpha. An i, i+3 linked analog, H-Lys-cyclo(d-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH2 (peptidomimetic estrogen receptor modulator 1), binds with a Ki of 25 nM, significantly better than an i, i+4 bridged cyclic amide, as predicted by molecular modeling design criteria. The induction of helical character, effective binding, and receptor selectivity exhibited by this peptide analog provide strong support for this strategy. The stabilization of minimalist surface motifs may prove useful for the control of other macromolecular assemblies, especially when an amphiphilic helix is crucial for the strong binding interaction between two proteins.