Macrocyclic Oxindole Peptide Epoxyketones-A Comparative Study of Macrocyclic Inhibitors of the 20S Proteasome.

Peptide macrocycles have recently gained attention as protease inhibitors due to their metabolic stability and specificity. However, the development of peptide macrocycles with improved binding potency has so far been challenging. Here we present macrocyclic peptides derived from the clinically applied proteasome inhibitor carfilzomib with an oxindole group that mimics the natural product TMC-95A. Fluorescence kinetic activity assays reveal a high potency of the oxindole group (IC50 = 0.19 µM) compared with agents lacking this motif. X-ray structures of the ligands with the ß5-subunit of the yeast 20S proteasome illustrate that the installed macrocycle forces strong hydrogen bonding of the oxindole group with ß5-Gly23NH. Thus, the binding of our designed oxindole epoxyketones is entropically and enthalpically favored in contrast to more flexible proteasome inhibitors such as carfilzomib.

Synthesis and Evaluation of Macrocyclic Peptide Aldehydes as Potent and Selective Inhibitors of the 20S Proteasome.

This research explores the first design and synthesis of macrocyclic peptide aldehydes as potent inhibitors of the 20S proteasome. Two novel macrocyclic peptide aldehydes based on the ring-size of the macrocyclic natural product TMC-95 were prepared and evaluated as inhibitors of the 20S proteasome. Both compounds inhibited in the low nanomolar range and proved to be selective for the proteasome over other serine and cysteine proteases, particularly when compared to linear analogues with similar amino acid sequences. In HeLa cells, both macrocycles efficiently inhibited activation of nuclear factor-κB (NF-κB) transcription factor by blocking proteasomal degradation of the inhibitor protein IκBα after cytokine stimulation. Due to their covalent mechanism of binding these compounds represent a 1000-fold increase in inhibitory potency over previously reported noncovalently binding TMC-95 analogues. Molecular modeling of the macrocyclic peptides confirms the preference of the large S3 pocket for large, hydrophobic residues and the ability to exploit this to improve selectivity of proteasome inhibitors.

Visible-light photocatalyzed cross-linking of diacetylene ligands by quantum dots to improve their aqueous colloidal stability.

Ligand cross-linking is known to improve the colloidal stability of nanoparticles, particularly in aqueous solutions. However, most cross-linking is performed chemically, in which it is difficult to limit interparticle cross-linking, unless performed at low concentrations. Photochemical cross-linking is a promising approach but usually requires ultraviolet (UV) light to initiate. Using such high-energy photons can be harmful to systems in which the ligand-nanoparticle bond is fairly weak, as is the case for the commonly used semiconductor quantum dots (QDs). Here, we introduce a novel approach to cross-link thiolated ligands on QDs by utilizing the photocatalytic activity of QDs upon absorbing visible light. We show that using visible light leads to better ligand cross-linking by avoiding the problem of ligand dissociation that occurs upon UV light exposure. Once cross-linked, the ligands significantly enhance the colloidal stability of those same QDs that facilitated cross-linking.

Li+ transport in poly(ethylene oxide) based electrolytes: neutron scattering, dielectric spectroscopy, and molecular dynamics simulations.

The dynamics of Li(+) transport in polyethylene oxide (PEO) and lithium bis(trifluoromethanesulfonyl)imde mixtures are investigated by combining neutron spin-echo (NSE) and dielectric spectroscopy with molecular dynamics (MD) simulations. The results are summarized in a relaxation time map covering wide ranges of temperature and time. The temperature dependence of the dc conductivity and the dielectric α relaxation time is found to be identical, indicating a strong coupling between both. The relaxation times obtained from the NSE measurements at 0.05 Å(-1)

Optimization of peptidyl allyl sulfones as clan CA cysteine protease inhibitors.

This research investigates the synthesis and inhibitory potency of a series of novel dipeptidyl allyl sulfones as clan CA cysteine protease inhibitors. The structure of the inhibitors consists of a R(1)-Phe-R(2)-AS-Ph scaffold (AS = allyl sulfone). R(1) was varied with benzyloxycarbonyl, morpholinocarbonyl, or N-methylpiperazinocarbonyl substituents. R(2) was varied with either Phe of Hfe residues. Synthesis involved preparation of vinyl sulfone analogues followed by isomerization to allyl sulfones using n-butyl lithium and t-butyl hydroperoxide. Sterics, temperature and base strength were all factors that affected the formation and stereochemistry of the allyl sulfone moiety. The inhibitors were assayed with three clan CA cysteine proteases (cruzain, cathepsin B and calpain I) as well as one serine protease (trypsin). The most potent inhibitor, (E)-Mu-Phe-Hfe-AS-Ph, displayed at least 10-fold selectivity for cruzain over clan CA cysteine proteases cathepsin B and calpain I with a (kobs)/[I] of 6080 ± 1390 M(-1)s(-1).

Aza-peptidyl Michael acceptors. A new class of potent and selective inhibitors of asparaginyl endopeptidases (legumains) from evolutionarily diverse pathogens.

Aza-peptide Michael acceptors with the general structure of Cbz-Ala-Ala-AAsn- trans-CH=CHCOR are a new class of inhibitors specific for the asparaginyl endopeptidases (AE) (legumains). Structure-activity relationships (SARs) were characterized for a set of 31 aza-peptide Michael acceptors with AEs derived from three medically important parasites: the protist Trichomonas vaginalis, the hard tick Ixodes ricinus, and the flatworm Schistosoma mansoni. Despite arising from phylogenetically disparate organisms, all three AEs shared a remarkably similar SAR with lowest IC50 values extending into the picomolar range. The results suggest an evolutionary constraint on the topography of the prime side of the active site. SAR also revealed that esters in the P1' position are more potent than disubstituted amides and that monosubstituted amides and alkyl derivatives show little or no inhibition. The preferred P1' residues have aromatic substituents. Aza-asparaginyl Michael acceptors react with thiols, which provides insight into the mechanism of their inhibition of asparaginyl endopeptidases.

Natural and artificial cystine knots for assembly of homo- and heterotrimeric collagen models.

Native collagens are molecules that are difficult to handle because of their high tendency towards aggregation and denaturation. It was discovered early on that synthetic collagenous peptides are more amenable to conformational characterization and thus can serve as useful models for structural and functional studies. Single-stranded collagenous peptides of high propensity to self-associate into triple-helical trimers were used for this purpose as well as interchain-crosslinked homotrimers assembled on synthetic scaffolds. With the growing knowledge of the biosynthetic pathways of natural collagens and the importance of their interchain disulfide crosslinks, which stabilize the triple-helical structure, native as well as de novo designed cystine knots have gained increasing attention in the assembly of triple-stranded collagen peptides. In addition, natural sequences of collagens were incorporated in order to biophysically characterize their functional epitopes. This review is focused on the methods developed over the years, and future perspectives for the production of collagen-mimicking synthetic and recombinant triple-helical homo- and heterotrimers.

Development of an immunofiltration-based antigen-detection assay for rapid diagnosis of Ebola virus infection.

Ebola virus (EBOV) has caused outbreaks of severe viral hemorrhagic fever in regions of Central Africa where medical facilities are ill equipped and diagnostic capabilities are limited. To obtain a reliable test that can be implemented easily under these conditions, monoclonal antibodies to the EBOV matrix protein (VP40), which previously had been found to work in a conventional enzyme-linked immunosorbent assay, were used to develop an immunofiltration assay for the detection of EBOV antigen in chemically inactivated clinical specimens. The assay was evaluated by use of defined virus stocks and specimens from experimentally infected animals. Its field application was tested during an outbreak of Ebola hemorrhagic fever in 2003. Although the original goal was to develop an assay that would detect all EBOV species, only the Zaire and Sudan species were detected in practice. The assay represents a first-generation rapid field test for the detection of EBOV antigen that can be performed in 30 min without electrical power or expensive or sensitive equipment.

Macrocyclic statine-based inhibitors of BACE-1.

Minimal sequence requirements for binding of substrate-derived statine peptides to the aspartyl enzyme were established on the basis of the X-ray cocrystal structure of the hydroxyethylene-octapeptide OM00-3 in complexation with BACE-1. With this information to hand, macrocyclic compounds that conformationally restrict and preorganize the peptide backbone for an entropically favoured binding to the enzyme active site cleft were designed. By means of a side chain-to-side chain ring closure between two aspartyl residues in the P2 and P3' positions through phenylene-1,3-dimethanamine, a 23-membered ring structure was obtained; this structure retained an extended conformation of the peptide backbone, including the transition state analogue statine for tight interactions with the two aspartyl residues of the active centre. The conformational preorganization of the inhibitor molecule was verified by NMR structural analysis and was then confirmed by the crystal structure of the BACE-1/inhibitor complex. Detailed insights into the binding mode of this macrocyclic inhibitor explained its moderate binding affinity in cell-free assays (K(i)=2.5 microM) and yielded precious information for possible structural optimization in view of the lack of steric clashes of the macrocycle with the flap domain of the enzyme.

TMC-95-based inhibitor design provides evidence for the catalytic versatility of the proteasome.

TMC-95's natural cyclic tripeptide metabolites represent potent competitive proteasome inhibitors. The constrained conformation of TMC-95 proteasomal inhibitors provides the driving force for entropically high-affinity binding. Based on the crystal structure of the proteasome:TMC-95A complex, the synthetically challenging TMC-95 core structure was used for the design and synthesis of less demanding biphenyl-ether macrocycles, in which the biphenyl-ether moiety functions as an endocyclic clamp restricting its tripeptide backbone. These simplified analogs allowed us to identify high plasticity of the proteasomal tryptic-like specificity pocket. Biphenyl-ether compounds extended with an amide group were hydrolyzed by the proteasome, although the crystal structure of such proteasome:biphenyl-ether complexes revealed quenching of proteolysis at the acyl-enzyme intermediate. Our data reveal that biphenyl-ether derivatives bind noncovalently to the proteasomal tryptic-like active site in a reversible substrate-like manner without allosteric changes of active site residues.

Synthesis of single- and multiple-stranded cystine-rich peptides.

The large abundance of bioactive single- and multiple-stranded cystine-rich peptides in nature has fostered the development of orthogonal thiol-protection schemes and of efficient chemistries for regioselective disulfide formation in synthetic replica for decades. In parallel to these entirely synthetic strategies, an increased knowledge of oxidative refolding mechanisms of proteins has been accumulated, and the collective experience with air oxidation of cysteine-rich peptides into their native disulfide frameworks have largely confirmed Anfinsen's principle of the self-assembly of polypeptide chains. In fact, a continuously growing number of cysteine-rich bioactive peptides from the most diverse sources and with differing cysteine patterns were found to retain the critical sequence-encoded structural information for correct oxidative folding into the native structures as dominant isomers, although in the biosynthetic pathways the mature peptide forms are mostly generated by posttranslational processing of folded precursors. Such self-assembly processes can be optimized by opportune manipulation of the experimental conditions or by induction of productive intermediates. But there are also numerous cases where folding and disulfide formation are thermodynamically not coupled and where the application of a defined succession of regioselective cysteine pairings still represents the method of choice to install the desired native or non-native cystine frameworks. Among our contributions to the state of the art in the synthesis of cystine-rich peptides, we have mainly addressed the induction of correct oxidative refolding of single-stranded cysteine-rich peptides into their native structures by the use of selenocysteine and suitable strategies for disulfide-mediated assembly of monomers into defined oligomers as mimics of homo- and heterotrimeric collagens as a synthetic approach for the development of new biomaterials.

Peptidyl allyl sulfones: a new class of inhibitors for clan CA cysteine proteases.

A new series of peptidyl allyl sulfone inhibitors was discovered while trying to synthesize epoxy sulfone inhibitors from vinyl sulfones using basic oxidizing conditions. The various dipeptidyl allyl sulfones were evaluated with calpain I, papain, cathepsins B and L, cruzain and rhodesain and found to be potent inhibitors. In comparison to the previously developed class of vinyl sulfone inhibitors, the novel dipeptidyl allyl sulfones were more potent inhibitors than the corresponding dipeptidyl vinyl sulfones. It was observed that the stereochemistry of the vinyl sulfone precursor played a role in the potency of the dipeptidyl allyl sulfone inhibitor.

Design and evaluation of inhibitors for dipeptidyl peptidase I (Cathepsin C).

Dipeptidyl peptidase I (DPPI, cathepsin C) is a lysosomal cysteine protease that can activate zymogens of several different serine proteases by one step or sequential removal of dipeptides from the N-termini of the pro-protease protein substrates. To find DPPI inhibitors more suitable for cellular applications than diazomethyl ketones, we synthesized three types of inhibitors: dipeptide acyloxymethyl ketones, fluoromethyl ketones, and vinyl sulfones (VS). The acyloxymethyl ketones inhibited DPPI slowly and are moderate inhibitors of cellular DPPI. The fluoromethyl ketones were potent, but the inhibited DPPI regained activity quickly. The dipeptide vinyl sulfones were effective inhibitors for DPPI, but they also inhibited cathepsins B, H, and L weakly. The best inhibitor, Ala-Hph-VS-Ph, had a k2/K(I) of 2,000,000M(-1)s(-1). The vinyl sulfones also inhibited intracellular DPPI, and for this application the more stable inhibitors exhibit better potency. We conclude that vinyl sulfones are promising inhibitors to study the intracellular functions of DPPI.

Aza-peptide Michael acceptors: a new class of inhibitors specific for caspases and other clan CD cysteine proteases.

Aza-peptide Michael acceptors are a new class of irreversible inhibitors that are highly potent and specific for clan CD cysteine proteases. The aza-Asp derivatives were specific for caspases, while aza-Asn derivatives were effective legumain inhibitors. Aza-Lys and aza-Orn derivatives were potent inhibitors of gingipain K and clostripain. Aza-peptide Michael acceptors showed no cross reactivity toward papain, cathepsin B, and calpain.

Aza-peptide epoxides: potent and selective inhibitors of Schistosoma mansoni and pig kidney legumains (asparaginyl endopeptidases).

Aza-peptide epoxides are a new class of irreversible cysteine protease inhibitors. Derivatives containing a P1 aza-asparagine residue are specific for Schistosoma mansoni and pig kidney legumains, which are clan CD cysteine proteases. The inhibitors have second-order rate constants of up to 10(4) M(-1) s(-1) with pig kidney legumain and IC50 values as low as 45 nM with S. mansoni legumain. The most potent epoxides contain an ester moiety with S,S stereochemistry attached to the epoxide. Interestingly, amide and amino acid derivatives of the epoxysuccinate moiety were not inhibitors of legumain, while disubstituted amide derivatives are quite potent. The inhibitors have little or no inhibitory activity with other proteases such as caspases, chymotrypsin, papain, cathepsin B, granzyme B, and various aspartyl proteases.