RESUMEN
We compared the full-length capsid maturational protease (pPR, pUL80a) of human cytomegalovirus with its proteolytic domain (assemblin) for the ability to cleave two biological substrates, and we found that pPR is more efficient with both. Affinity-purified, refolded enzymes and substrates were combined under defined reaction conditions, and cleavage was monitored and quantified following staining of the resulting electrophoretically separated fragments. The enzymes were stabilized against self-cleavage by a single point mutation in each cleavage site (ICRMT-pPR and IC-assemblin). The substrates were pPR itself, inactivated by replacing its catalytic nucleophile (S132A-pPR), and the sequence-related assembly protein precursor (pAP, pUL80.5). Our results showed that (i) ICRMT-pPR is 5- to 10-fold more efficient than assemblin for all cleavages measured (i.e., the M site of pAP and the M, R, and I sites of S132A-pPR). (ii) Cleavage of substrate S132A-pPR proceeded M>R>I for both enzymes. (iii) Na(2)SO(4) reduced M- and R-site cleavage efficiency by ICRMT-pPR, in contrast to its enhancing effect for both enzymes on I site and small peptide cleavage. (iv) Disrupting oligomerization of either the pPR enzyme or substrate by mutating Leu382 in the amino-conserved domain reduced cleavage efficiency two- to fourfold. (v) Finally, ICRMT-pPR mutants that include the amino-conserved domain, but terminate with Pro481 or Tyr469, retain the enzymatic characteristics that distinguish pPR from assemblin. These findings show that the scaffolding portion of pPR increases its enzymatic activity on biologically relevant protein substrates and provide an additional link between the structure of this essential viral enzyme and its biological mechanism.
Asunto(s)
Citomegalovirus/enzimología , Endopeptidasas/metabolismo , Serina Endopeptidasas/metabolismo , Proteínas Virales/metabolismo , Dominio Catalítico , Cromatografía de Afinidad/métodos , Endopeptidasas/aislamiento & purificación , Humanos , Serina Endopeptidasas/aislamiento & purificación , Proteínas Virales/aislamiento & purificaciónRESUMEN
Combinatorial biosynthesis meets combinatorial pharmacology, cyanobacterial style: A new antimitotic natural product with features of both dolastatins 10 and 15 was isolated from the same Floridian Symploca sp. sample that produced the histone deacetylase inhibitor largazole. Both agents in combination are more effective in inhibiting cancer cell proliferation than either agent alone.
Asunto(s)
Antimitóticos/química , Protocolos de Quimioterapia Combinada Antineoplásica/química , Cianobacterias/química , Depsipéptidos/farmacología , Inhibidores de Histona Desacetilasas , Péptidos/química , Tiazoles/farmacología , Péptidos Catiónicos Antimicrobianos , Antimitóticos/farmacología , Antimitóticos/toxicidad , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ciclo Celular , Línea Celular Tumoral , Depsipéptidos/química , Histona Desacetilasas/metabolismo , Humanos , Péptidos/farmacología , Tiazoles/químicaRESUMEN
Investigation of an extract from the marine cyanobacterium Lyngbya semiplena, collected in Tumon Bay, Guam, led to the identification of three new cyclodepsipeptides, lyngbyastatins 8-10 (1-3). The structures of 1-3 were determined by NMR, MS, ESIMS fragmentation and chemical degradation. Compounds 1-3 are closely related to lyngbyastatins 4-7. Like the latter compounds, we found 1-3 to inhibit porcine pancreatic elastase, with IC(50) values of 123 nM, 210 nM and 120 nM, respectively.
Asunto(s)
Cianobacterias/química , Depsipéptidos/aislamiento & purificación , Elastasa Pancreática/antagonistas & inhibidores , Animales , Depsipéptidos/química , Relación Dosis-Respuesta a Droga , Guam , Espectroscopía de Resonancia Magnética , Espectrometría de Masa por Ionización de Electrospray , PorcinosRESUMEN
Full details of the concise and convergent synthesis (eight steps, 19% overall yield), its extension to the preparation of a series of key analogues, and the molecular target and pharmacophore of largazole are described. Central to the synthesis of largazole is a macrocyclization reaction for formation of the strained 16-membered depsipeptide core followed by an olefin cross-metathesis reaction for installation of the thioester. The biological evaluation of largazole and its key analogues, including an acetyl analogue, a thiol analogue, and a hydroxyl analogue, suggested that histone deacetylases (HDACs) are molecular targets of largazole and largazole is a class I HDAC inhibitor. In addition, structure-activity relationship (SAR) studies revealed that the thiol group is the pharmacophore of the natural product. Largazole's HDAC inhibitory activity correlates with its antiproliferative activity.
Asunto(s)
Depsipéptidos/síntesis química , Inhibidores Enzimáticos/síntesis química , Inhibidores de Histona Desacetilasas , Tiazoles/síntesis química , Antineoplásicos/síntesis química , Ciclización , Depsipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Relación Estructura-Actividad , Tiazoles/farmacologíaRESUMEN
Two cyclodepsipeptides named kempopeptins A (1) and B (2) were isolated from a collection of a Floridian marine cyanobacterium, Lyngbya sp., that had previously afforded the structurally related potent elastase inhibitors lyngbyastatin 7 and somamide B. The structures of 1 and 2 were elucidated mainly by 1D and 2D NMR spectroscopy, and the absolute configuration was established by chiral HPLC and Marfey's analysis of the degradation products. Kempopeptin A (1) exhibited an IC50 against elastase of 0.32 microM and against chymotrypsin of 2.6 microM, while kempopeptin B (2) inhibited trypsin with an IC50 of 8.4 microM.
Asunto(s)
Cianobacterias/química , Depsipéptidos/aislamiento & purificación , Depsipéptidos/farmacología , Inhibidores de Serina Proteinasa/aislamiento & purificación , Inhibidores de Serina Proteinasa/farmacología , Animales , Bovinos , Quimotripsina/antagonistas & inhibidores , Depsipéptidos/química , Florida , Concentración 50 Inhibidora , Biología Marina , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Páncreas/enzimología , Elastasa Pancreática/antagonistas & inhibidores , Inhibidores de Serina Proteinasa/química , PorcinosAsunto(s)
Proliferación Celular/efectos de los fármacos , Cianobacterias/química , Depsipéptidos/química , Depsipéptidos/farmacología , Tiazoles/química , Tiazoles/farmacología , Animales , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Depsipéptidos/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células Epiteliales/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/normas , Espectrometría de Masas/métodos , Ratones , Conformación Molecular , Células 3T3 NIH , Estándares de Referencia , Tiazoles/aislamiento & purificaciónRESUMEN
We discovered new structural diversity to a prevalent, yet medicinally underappreciated, cyanobacterial protease inhibitor scaffold and undertook comprehensive protease profiling to reveal potent and selective elastase inhibition. Structure-activity relationship (SAR) studies and X-ray cocrystal structure analysis allowed a detailed assessment of critical and tunable structural elements. To realize the therapeutic potential of these cyclodepsipeptides, we probed the cellular effects of a novel and representative family member, symplostatin 5 (1), which attenuated the downstream cellular effects of elastase in an epithelial lung airway model system, alleviating clinical hallmarks of chronic pulmonary diseases such as cell death, cell detachment, and inflammation. This compound attenuated the effects of elastase on receptor activation, proteolytic processing of the adhesion protein ICAM-1, NF-κB activation, and transcriptomic changes, including the expression of pro-inflammatory cytokines IL1A, IL1B, and IL8. Compound 1 exhibited activity comparable to the clinically approved elastase inhibitor sivelestat in short-term assays and demonstrated superior sustained activity in longer-term assays.
Asunto(s)
Antiinflamatorios/farmacología , Bronquios/efectos de los fármacos , Cianobacterias/química , Inhibidores Enzimáticos/farmacología , Elastasa Pancreática/antagonistas & inhibidores , Bronquios/citología , Línea Celular , Cristalografía por Rayos X , Inhibidores Enzimáticos/aislamiento & purificación , Células Epiteliales/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Three new analogues of dolastatin 13, termed lyngbyastatins 5-7 ( 1- 3), were isolated from two different collections of marine cyanobacteria, Lyngbya spp., from South Florida. Their planar structures were deduced by a combination of NMR techniques, and the absolute configurations were established by modified Marfey's analysis of the acid hydrolyzates. The related cyclodepsipeptide somamide B ( 4), previously reported from a Fijian cyanobacterium, has also been found in one of the extracts, and its absolute stereochemistry was unambiguously assigned for the first time. Compounds 1- 4 were found to selectively inhibit elastase over several other serine proteases, with IC50 values for porcine pancreatic elastase ranging from 3 to 10 nM.