RESUMEN
Lysocinâ E (1) is a structurally complex 37-membered depsipeptide comprising 12 amino-acid residues with an N-methylated amide and an ester linkage. Compound 1 binds to menaquinone (MK) in the bacterial membrane to exert its potent bactericidal activity. To decipher the biologically important functionalities within this unique antibiotic, we performed a comprehensive structure-activity relationship (SAR) study by systematically changing the side-chain structures of l-Thr-1, d-Arg-2, N-Me-d-Phe-5, d-Arg-7, l-Glu-8, and d-Trp-10. First, we achieved total synthesis of the 14 new side-chain analogues of 1 by employing a solid-phase strategy. We then evaluated the MK-dependent liposomal disruption and antimicrobial activity against Staphylococcus aureus by 1 and its analogues. Correlating data between the liposome and bacteria experiments revealed that membrane lysis was mainly responsible for the antibacterial functions. Altering the cationic guanidine moiety of d-Arg-2/7 to a neutral amide, and the C7-acyl group of l-Thr-1 to the C2 or C11 counterpart decreased the antimicrobial activities four- or eight-fold. More drastically, chemical mutation of d-Trp-10 to d-Ala-10 totally abolished the bioactivities. These important findings led us to propose the biological roles of the side-chain functionalities.
Asunto(s)
Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Cationes , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Molecular , Péptidos Cíclicos/síntesis química , Relación Estructura-ActividadRESUMEN
Lysocinâ E, a macrocyclic peptide, exhibits potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) through a novel mechanism. The first total synthesis of lysocinâ E was achieved by applying a full solid-phase strategy. The developed approach also provides rapid access to the enantiomeric, epimeric, and N-demethylated analogues of lysocinâ E. Significantly, the antibacterial activity of the unnatural enantiomer was comparable to that of the natural isomer, suggesting the absence of chiral recognition in its mode of action.
Asunto(s)
Antibacterianos/síntesis química , Péptidos Cíclicos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Catálisis , Complejos de Coordinación/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Paladio/química , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
To obtain therapeutically effective new antibiotics, we first searched for bacterial culture supernatants with antimicrobial activity in vitro and then performed a secondary screening using the silkworm infection model. Through further purification of the in vivo activity, we obtained a compound with a previously uncharacterized structure and named it 'lysocin E'. Lysocin E interacted with menaquinone in the bacterial membrane to achieve its potent bactericidal activity, a mode of action distinct from that of any other known antibiotic, indicating that lysocin E comprises a new class of antibiotic. This is to our knowledge the first report of a direct interaction between a small chemical compound and menaquinone that leads to bacterial killing. Furthermore, lysocin E decreased the mortality of infected mice. To our knowledge, lysocin E is the first compound identified and purified by quantitative measurement of therapeutic effects in an invertebrate infection model that exhibits robust in vivo effects in mammals.
Asunto(s)
Antibacterianos/farmacología , Membrana Celular/efectos de los fármacos , Descubrimiento de Drogas/métodos , Bacterias Grampositivas/efectos de los fármacos , Péptidos Cíclicos/farmacología , Vitamina K 2/antagonistas & inhibidores , Animales , Antibacterianos/aislamiento & purificación , Antibacterianos/uso terapéutico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriólisis/efectos de los fármacos , Bombyx/microbiología , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Bacterias Grampositivas/genética , Bacterias Grampositivas/metabolismo , Lysobacter/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Péptidos Cíclicos/aislamiento & purificación , Péptidos Cíclicos/uso terapéutico , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Vitamina K 2/metabolismoRESUMEN
Thioflavin-T is one of the most important amyloid specific dyes and has been used for more than 50 years; however, the molecular mechanism of staining is still not understood. Chemically synthesized short polyglutamine peptides (Q(n), n = 5-10) were subjected to the thioflavin-T (ThT) staining assay. It was found that the minimum Q(n) peptide that stained positive to ThT was Q(6). Two types of ThT-binding sites, a high-affinity site (k(d1) = 0.1-0.17 µM) and a low-affinity site (k(d2) = 5.7-7.4 µM), were observed in short polyQs (n = 6-9). (13)C{(2)H}REDOR NMR experiments were carried out to extract the local structure of ThT binding sites in Q(8) peptide aggregates by observing the intermolecular dipolar coupling between [3-Me-d(3)]ThT and natural abundance Q(8) or residue-specific [1,2-(13)C(2)] labeled Q(8)s. (13)C{(2)H}REDOR difference spectra of the [3-Me-d(3)]ThT/natural abundance Q(8) (1/9) complex indicated that all of the five carbons of the glutamine residue participated in the formation of ThT-binding sites. (13)C{(2)H}DQF-REDOR experiments of [3-Me-d(3)]ThT/residue-specific [1,2-(13)C(2)] labeled Q(8) (1/50) complexes demonstrated that the N-terminal glutamine residue had direct contact with the ThT molecule at the high-affinity ThT-binding sites.