RESUMO
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.
RESUMO
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).
Assuntos
Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Sulfonas/química , Calpaína/antagonistas & inibidores , Catepsina B/antagonistas & inibidores , Técnicas de Química Sintética/métodos , Cisteína Endopeptidases , Humanos , Cinética , Proteínas de Protozoários/antagonistas & inibidores , Estereoisomerismo , Relação Estrutura-Atividade , Temperatura , terc-Butil Hidroperóxido/químicaRESUMO
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.
Assuntos
Colágeno/química , Cistina/química , Modelos Moleculares , Sequência de Aminoácidos , Animais , Biopolímeros , Humanos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Compostos Aza/síntese química , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/síntese química , Ixodes/enzimologia , Oligopeptídeos/síntese química , Schistosoma mansoni/enzimologia , Trichomonas vaginalis/enzimologia , Animais , Compostos Aza/química , Biotina/química , Inibidores de Cisteína Proteinase/química , Ditiotreitol/química , Concentração Inibidora 50 , Oligopeptídeos/química , Relação Estrutura-Atividade , Compostos de Sulfidrila/químicaRESUMO
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.
RESUMO
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.
Assuntos
Compostos Aza/síntese química , Inibidores de Caspase , Inibidores de Cisteína Proteinase/síntese química , Peptídeos/síntese química , Adesinas Bacterianas , Compostos Aza/química , Caspases/química , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/química , Cisteína Endopeptidases Gingipaínas , Hemaglutininas/química , Cinética , Peptídeos/químicaRESUMO
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.
Assuntos
Acetileno/química , Coloides/química , Reagentes de Ligações Cruzadas/química , Pontos Quânticos/química , Compostos de Sulfidrila/química , Água/química , Catálise , Ligantes , Luz , Luminescência , SemicondutoresRESUMO
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.
Assuntos
Cisteína Endopeptidases/efeitos dos fármacos , Inibidores de Cisteína Proteinase/síntese química , Sulfonas/síntese química , Animais , Bovinos , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/síntese química , Dipeptídeos/farmacologia , Eritrócitos/enzimologia , Concentração Inibidora 50 , Cinética , Relação Estrutura-Atividade , Sulfonas/química , Sulfonas/farmacologiaRESUMO
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.
Assuntos
Catepsina C/antagonistas & inibidores , Cetonas/química , Sulfonas/química , Animais , Catepsina C/química , Linhagem Celular , Desenho de Fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Humanos , Cetonas/síntese química , Mastócitos/enzimologia , Ratos , Baço/enzimologia , Sulfonas/síntese químicaRESUMO
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.