RESUMEN
Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Células Epiteliales/parasitología , Bibliotecas de Moléculas Pequeñas/farmacología , Tioléster Hidrolasas/antagonistas & inhibidores , Toxoplasma/efectos de los fármacos , Toxoplasma/enzimología , Animales , Línea Celular , Cumarinas/química , Cumarinas/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismo , Toxoplasma/metabolismo , Toxoplasma/patogenicidadRESUMEN
The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical biology strategy that has uncovered the first essential protease for this organism. Identification and application of a unique CtHtrA inhibitor (JO146) to cultures of Chlamydia resulted in a complete loss of viable elementary body formation. JO146 treatment during the replicative phase of development resulted in a loss of Chlamydia cell morphology, diminishing inclusion size, and ultimate loss of inclusions from the host cells. This completely prevented the formation of viable Chlamydia elementary bodies. In addition to its effect on the human Chlamydia trachomatis strain, JO146 inhibited the viability of the mouse strain, Chlamydia muridarum, both in vitro and in vivo. Thus, we report a chemical biology approach to establish an essential role for Chlamydiaâ CtHtrA. The function of CtHtrA for Chlamydia appears to be essential for maintenance of cell morphology during replicative the phase and these findings provide proof of concept that proteases can be targeted for antimicrobial therapy for intracellular pathogens.
Asunto(s)
Antibacterianos/metabolismo , Chlamydia trachomatis/efectos de los fármacos , Chlamydia trachomatis/enzimología , Dipéptidos/metabolismo , Cuerpos de Inclusión/microbiología , Viabilidad Microbiana/efectos de los fármacos , Organofosfonatos/metabolismo , Serina Proteasas/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Línea Celular , Chlamydia trachomatis/genética , Genes Esenciales , Hepatocitos/microbiología , Humanos , MicroscopíaRESUMEN
MARTX toxins modulate the virulence of a number of Gram-negative Vibrio species. This family of toxins is defined by the presence of a cysteine protease domain (CPD), which proteolytically activates the Vibrio cholerae MARTX toxin. Although recent structural studies of the CPD have uncovered a new allosteric activation mechanism, the mechanism of CPD substrate recognition or toxin processing is unknown. Here we show that interdomain cleavage of MARTXVc enhances effector domain function. We also identify the first small-molecule inhibitors of this protease domain and present the 2.35-A structure of the CPD bound to one of these inhibitors. This structure, coupled with biochemical and mutational studies of the toxin, reveals the molecular basis of CPD substrate specificity and underscores the evolutionary relationship between the CPD and the clan CD caspase proteases. These studies are likely to prove valuable for devising new antitoxin strategies for a number of bacterial pathogens.
Asunto(s)
Toxina del Cólera/química , Toxina del Cólera/metabolismo , Proteasas de Cisteína/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Vibrio cholerae/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Western Blotting , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia , Espectroscopía Infrarroja por Transformada de Fourier , Especificidad por Sustrato , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/enzimologíaRESUMEN
Newly replicated Plasmodium falciparum parasites escape from host erythrocytes through a tightly regulated process that is mediated by multiple classes of proteolytic enzymes. However, the identification of specific proteases has been challenging. We describe here a forward chemical genetic screen using a highly focused library of more than 1,200 covalent serine and cysteine protease inhibitors to identify compounds that block host cell rupture by P. falciparum. Using hits from the library screen, we identified the subtilisin-family serine protease PfSU B1 and the cysteine protease dipeptidyl peptidase 3 (DPAP3) as primary regulators of this process. Inhibition of both DPAP3 and PfSUB1 caused a block in proteolytic processing of the serine repeat antigen (SERA) protein SERA5 that correlated with the observed block in rupture. Furthermore, DPAP3 inhibition reduced the levels of mature PfSUB1. These results suggest that two mechanistically distinct proteases function to regulate processing of downstream substrates required for efficient release of parasites from host red blood cells.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Eritrocitos/parasitología , Malaria Falciparum/parasitología , Plasmodium falciparum/enzimología , Serina Endopeptidasas/metabolismo , Animales , Antígenos de Protozoos/efectos de los fármacos , Antígenos de Protozoos/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Eritrocitos/metabolismo , Interacciones Huésped-Parásitos/efectos de los fármacos , Humanos , Isocumarinas/química , Isocumarinas/farmacología , Malaria Falciparum/metabolismo , Conformación Molecular , Pruebas de Sensibilidad Parasitaria , Péptidos/química , Péptidos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/fisiología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas , Estereoisomerismo , Subtilisinas/antagonistas & inhibidores , Subtilisinas/química , Subtilisinas/metabolismo , Sulfonas/química , Sulfonas/farmacologíaRESUMEN
Besides functioning as the plasma transporter of retinol and thyroxine, TTR (transthyretin) is a protease, cleaving apoA-I (apolipoprotein A-I) after a phenylalanine residue. In the present study, we further investigated TTR substrate specificity. By using both P-diverse libraries and a library of phosphonate inhibitors, a TTR preference for a lysine residue in P1 was determined, suggesting that TTR might have a dual specificity and that, in addition to apoA-I, other TTR substrates might exist. Previous studies revealed that TTR is involved in the homoeostasis of the nervous system, as it participates in neuropeptide maturation and enhances nerve regeneration. We investigated whether TTR proteolytic activity is involved in these functions. Both wild-type TTR and TTR(prot-) (proteolytically inactive TTR) had a similar effect in the expression of peptidylglycine alpha-amidating mono-oxygenase, the rate-limiting enzyme in neuropeptide amidation, excluding the involvement of TTR proteolytic activity in neuropeptide maturation. However, TTR was able to cleave amidated NPY (neuropeptide Y), probably contributing to the increased NPY levels reported in TTR-knockout mice. To assess the involvement of TTR proteolytic activity in axonal regeneration, neurite outgrowth of cells cultivated with wild-type TTR or TTR(prot-), was measured. Cells grown with TTR(prot-) displayed decreased neurite length, thereby suggesting that TTR proteolytic activity is important for its function as a regeneration enhancer. By showing that TTR is able to cleave NPY and that its proteolytic activity affects axonal growth, the present study shows that TTR has natural substrates in the nervous system, establishing further its relevance in neurobiology.
Asunto(s)
Sistema Nervioso/metabolismo , Prealbúmina/metabolismo , Animales , Apolipoproteína A-I/metabolismo , Línea Celular Tumoral , Humanos , Ratones , Ratones Noqueados , Estructura Molecular , Neuritas/metabolismo , Neuropéptido Y/metabolismo , Prealbúmina/genética , Especificidad por Sustrato , Tiroxina/metabolismoRESUMEN
The SARS coronavirus main peptidase (SARS-CoV M(pro)) plays an essential role in the life-cycle of the virus and is a primary target for the development of anti-SARS agents. Here, we report the crystal structure of M(pro) at a resolution of 1.82 Angstroms, in space group P2(1) at pH 6.0. In contrast to the previously reported structure of M(pro) in the same space group at the same pH, the active sites and the S1 specificity pockets of both protomers in the structure of M(pro) reported here are in the catalytically competent conformation, suggesting their conformational flexibility. We report two crystal structures of M(pro) having an additional Ala at the N terminus of each protomer (M(+A(-1))(pro)), both at a resolution of 2.00 Angstroms, in space group P4(3)2(1)2: one unbound and one bound by a substrate-like aza-peptide epoxide (APE). In the unbound form, the active sites and the S1 specificity pockets of both protomers of M(+A(-1))(pro) are observed in a collapsed (catalytically incompetent) conformation; whereas they are in an open (catalytically competent) conformation in the APE-bound form. The observed conformational flexibility of the active sites and the S1 specificity pockets suggests that these parts of M(pro) exist in dynamic equilibrium. The structural data further suggest that the binding of APE to M(pro) follows an induced-fit model. The substrate likely also binds in an induced-fit manner in a process that may help drive the catalytic cycle.
Asunto(s)
Compuestos Epoxi/metabolismo , Péptidos/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , Proteínas de la Matriz Viral/química , Sitios de Unión , Proteínas M de Coronavirus , Cristalografía por Rayos X , Compuestos Epoxi/química , Modelos Moleculares , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Protones , Electricidad Estática , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Compuestos Aza/síntesis química , Cisteína Endopeptidasas/química , Inhibidores de Cisteína Proteinasa/síntesis química , Ixodes/enzimología , Oligopéptidos/síntesis química , Schistosoma mansoni/enzimología , Trichomonas vaginalis/enzimología , Animales , Compuestos Aza/química , Biotina/química , Inhibidores de Cisteína Proteinasa/química , Ditiotreitol/química , Concentración 50 Inhibidora , Oligopéptidos/química , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/químicaRESUMEN
The main peptidase (M(pro)) from the coronavirus (CoV) causing severe acute respiratory syndrome (SARS) is one of the most attractive molecular targets for the development of anti-SARS agents. We report the irreversible inhibition of SARS-CoV M(pro) by an aza-peptide epoxide (APE; k(inact)/K(i) = 1900(+/-400) M(-1) s(-1)). The crystal structures of the M(pro):APE complex in the space groups C2 and P2(1)2(1)2(1) revealed the formation of a covalent bond between the catalytic Cys145 S(gamma) atom of the peptidase and the epoxide C3 atom of the inhibitor, substantiating the mode of action of this class of cysteine-peptidase inhibitors. The aza-peptide component of APE binds in the substrate-binding regions of M(pro) in a substrate-like manner, with excellent structural and chemical complementarity. In addition, the crystal structure of unbound M(pro) in the space group C2 revealed that the "N-fingers" (N-terminal residues 1 to 7) of both protomers of M(pro) are well defined and the substrate-binding regions of both protomers are in the catalytically competent conformation at the crystallization pH of 6.5, contrary to the previously determined crystal structures of unbound M(pro) in the space group P2(1).
Asunto(s)
Cisteína Endopeptidasas/química , Inhibidores de Proteasas/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Sitios de Unión , Catálisis , Proteasas 3C de Coronavirus , Cristalografía por Rayos X , Compuestos Epoxi/química , Concentración de Iones de Hidrógeno , Estructura Molecular , Péptidos/químicaRESUMEN
Aza-peptide Michael acceptors are a novel class of inhibitors that are potent and specific for caspases-2, -3, -6, -7, -8, -9, and -10. The second-order rate constants are in the order of 10(6) M(-1) s(-1). The aza-peptide Michael acceptor inhibitor 18t (Cbz-Asp-Glu-Val-AAsp-trans-CH=CH-CON(CH(2)-1-Naphth)(2) is the most potent compound and it inhibits caspase-3 with a k(2) value of 5620000 M(-1) s(-1). The inhibitor 18t is 13700, 190, 6.4, 594, 37500, and 173-fold more selective for caspase-3 over caspases-2, -6, -7, -8, -9, and -10, respectively. Aza-peptide Michael acceptors designed with caspase specific sequences are selective and do not show any cross reactivity with clan CA cysteine proteases such as papain, cathepsin B, and calpains. High-resolution crystal structures of caspase-3 and caspase-8 in complex with aza-peptide Michael acceptor inhibitors demonstrate the nucleophilic attack on C2 and provide insight into the selectivity and potency of the inhibitors with respect to the P1' moiety.
Asunto(s)
Compuestos Aza/síntesis química , Inhibidores de Caspasas , Oligopéptidos/síntesis química , Compuestos Aza/química , Caspasa 10 , Caspasa 2 , Caspasa 3 , Caspasa 6 , Caspasa 7 , Caspasa 9 , Caspasas/química , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Oligopéptidos/química , Relación Estructura-ActividadRESUMEN
Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb "Hydrolase important for pathogenesis 1" (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections.
Asunto(s)
Antituberculosos/química , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/química , Mycobacterium tuberculosis/enzimología , Serina Proteasas/metabolismo , Tuberculosis/microbiología , Animales , Antituberculosos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Estructura Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Serina Proteasas/química , Serina Proteasas/genética , Especificidad por SustratoRESUMEN
Aza-peptide epoxides, a novel class of irreversible protease inhibitors, are specific for the clan CD cysteine proteases. Aza-peptide epoxides with an aza-Asp residue at P1 are excellent irreversible inhibitors of caspases-1, -3, -6, and -8 with second-order inhibition rates up to 1 910 000 M(-1) s(-1). In general, the order of reactivity of aza-peptide epoxides is S,S > R,R > trans > cis. Interestingly, some of the R,R epoxides while being less potent are actually more selective than the S,S epoxides. Our aza-peptide epoxides designed for caspases are stable, potent, and specific inhibitors, as they show little to no inhibition of other proteases such as the aspartyl proteases porcine pepsin, human cathepsin D, plasmepsin 2 from P. falciparum, HIV-1 protease, and the secreted aspartic proteinase 2 (SAP-2) from Candida albicans; the serine proteases granzyme B and alpha-chymotrypsin; and the cysteine proteases cathepsin B and papain (clan CA), and legumain (clan CD).
Asunto(s)
Compuestos Aza/síntesis química , Inhibidores de Caspasas , Compuestos Epoxi/síntesis química , Oligopéptidos/síntesis química , Compuestos Aza/química , Caspasa 1/química , Caspasa 3 , Caspasa 6 , Caspasa 8 , Caspasas/química , Cristalografía por Rayos X , Diseño de Fármacos , Estabilidad de Medicamentos , Compuestos Epoxi/química , Humanos , Hidrólisis , Estructura Molecular , Oligopéptidos/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Aza-peptide epoxides, a new class of irreversible protease inhibitors, are specific for the clan CD cysteine proteases. The inhibitors have second-order rate constants up to 10(5) M(-1) s(-1), with the most potent epoxides having the S,S stereochemistry. The aza-Asn derivatives are effective legumain inhibitors, while the aza-Asp epoxides were specific for caspases. The inhibitors have little or no inhibition with other proteases such as chymotrypsin, papain, or cathepsin B.
Asunto(s)
Compuestos Aza/síntesis química , Inhibidores de Cisteína Proteinasa/síntesis química , Compuestos Epoxi/síntesis química , Oligopéptidos/síntesis química , Compuestos Aza/química , Inhibidores de Caspasas , Catepsina B/antagonistas & inhibidores , Cristalografía por Rayos X , Cisteína Endopeptidasas , Inhibidores de Cisteína Proteinasa/química , Compuestos Epoxi/química , Cinética , Oligopéptidos/química , Papaína/antagonistas & inhibidores , Proteínas de Plantas/antagonistas & inhibidores , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Compuestos Aza/síntesis química , Inhibidores de Caspasas , Inhibidores de Cisteína Proteinasa/síntesis química , Péptidos/síntesis química , Adhesinas Bacterianas , Compuestos Aza/química , Caspasas/química , Cisteína Endopeptidasas/química , Inhibidores de Cisteína Proteinasa/química , Cisteína-Endopeptidasas Gingipaínas , Hemaglutininas/química , Cinética , Péptidos/químicaRESUMEN
Small ubiquitin-related modifier (SUMO) is implicated in the regulation of numerous biological processes including transcription, protein localization, and cell cycle control. Protein modification by SUMO is found in Plasmodium falciparum; however, its role in the regulation of the parasite life cycle is poorly understood. Here we describe functional studies of a SUMO-specific protease (SENP) of P. falciparum, PfSENP1 (PFL1635w). Expression of the catalytic domain of PfSENP1 and biochemical profiling using a positional scanning substrate library demonstrated that this protease has unique cleavage sequence preference relative to the human SENPs. In addition, we describe a class of small molecule inhibitors of this protease. The most potent lead compound inhibited both recombinant PfSENP1 activity and P. falciparum replication in infected human blood. These studies provide valuable new tools for the study of SUMOylation in P. falciparum.
Asunto(s)
Hidrazinas/farmacología , Ácidos Ftálicos/farmacología , Plasmodium falciparum/enzimología , Inhibidores de Proteasas/farmacología , Proteínas Protozoarias/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Cisteína Endopeptidasas , Endopeptidasas/metabolismo , Humanos , Hidrazinas/química , Datos de Secuencia Molecular , Ácidos Ftálicos/química , Inhibidores de Proteasas/química , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
A series of peptidyl alpha-ketoamides with the general structure Cbz-L-Leu-D,L-AA-CONH-R were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, and cathepsin B. Nucleobases, methylpiperazine, and dimethylaminoalkyl groups were incorporated into the primed region of the inhibitors to generate compounds that potentially cross the blood-brain barrier. Two of these compounds (Cbz-Leu-D,L-Abu-CONH-(CH(2))(3)-adenin-9-yl and Cbz-Leu-D,L-Abu-CONH-(CH(2))(3)-(4-methylpiperazin-1-yl) have been shown to have useful concentrations in the brain in animals. The best inhibitor for calpain I was Cbz-Leu-D,L-Abu-CONH-(CH(2))(3)-2-methoxyadenin-9-yl (K(i) = 23 nM), and the best inhibitor for calpain II was Cbz-Leu-D,L-Phe-CONH-(CH(2))(3)-adenin-9-yl (K(i) = 68 nM). On the basis of the crystal structure obtained with heterocyclic peptidyl alpha-ketoamides, we have improved inhibitor potency by introducing a small hydrophobic group on the adenine ring. These inhibitors have good potential to be used in the treatment of neurodegenerative diseases.
Asunto(s)
Adenina/análogos & derivados , Calpaína/antagonistas & inhibidores , Dipéptidos/síntesis química , Piperazinas/síntesis química , Adenina/síntesis química , Adenina/química , Adenina/farmacocinética , Animales , Barrera Hematoencefálica/metabolismo , Calpaína/química , Catepsina B/antagonistas & inhibidores , Catepsina B/química , Dipéptidos/química , Dipéptidos/farmacocinética , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Piperazinas/química , Piperazinas/farmacocinética , Unión Proteica , Relación Estructura-Actividad , Porcinos , Distribución TisularRESUMEN
Aza-peptide Michael acceptors and epoxides with the general structure of YCO-Ala-Ala-AAsn-trans-CH horizontal lineCHCOR and YCO-Ala-Ala-AAsn-EP-COR, respectively, are shown to be potent inhibitors of asparaginyl endopeptidases (legumains) from the bloodfluke, Schistosoma mansoni (SmAE), and the hard tick, Ixodes ricinus (IrAE). Structure-activity relationships (SARs) were determined for a set of 41 aza-peptide Michael acceptors and eight aza-peptide epoxides. Both enzymes prefer disubstituted amides to monosubstituted amides in the P1' position, and potency increased as we increased the hydrophobicity of the inhibitor in this position. Extending the inhibitor to P5 resulted in increased potency, especially against IrAE, and both enzymes prefer small over large hydrophobic residues at P2. Aza-peptide Michael acceptor inhibitors are more potent than aza-peptide epoxide inhibitors, and for some of these compounds, second-order inhibiton rate constants are the fastest yet discovered. Given the central functions of these enzymes in both parasites, the data presented here may facilitate the eventual design of selective antiparasitic drugs.
Asunto(s)
Compuestos Aza/química , Cisteína Endopeptidasas/metabolismo , Compuestos Epoxi/química , Ixodes/enzimología , Oligopéptidos/química , Oligopéptidos/farmacología , Schistosoma mansoni/enzimología , Secuencia de Aminoácidos , Animales , Disponibilidad Biológica , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Cinética , Oligopéptidos/síntesis química , Oligopéptidos/farmacocinéticaRESUMEN
Calpains are intracellular cysteine proteases that catalyze the cleavage of target proteins in response to Ca(2+) signaling. When Ca(2+) homeostasis is disrupted, calpain overactivation causes unregulated proteolysis, which can contribute to diseases such as postischemic injury and cataract formation. Potent calpain inhibitors exist, but of these many cross-react with other cysteine proteases and will need modification to specifically target calpain. Here, we present crystal structures of rat calpain 1 protease core (muI-II) bound to two alpha-ketoamide-based calpain inhibitors containing adenyl and piperazyl primed-side extensions. An unexpected aromatic-stacking interaction is observed between the primed-side adenine moiety and the Trp298 side chain. This interaction increased the potency of the inhibitor toward muI-II and heterodimeric m-calpain. Moreover, stacking orients the adenine such that it can be used as a scaffold for designing novel primed-side address regions, which could be incorporated into future inhibitors to enhance their calpain specificity.
Asunto(s)
Calpaína/química , Carbamatos/antagonistas & inhibidores , Dipéptidos/antagonistas & inhibidores , Dipéptidos/química , Glicoproteínas/química , Aminoácidos Aromáticos/química , Animales , Calpaína/antagonistas & inhibidores , Cristalografía por Rayos X , Dipéptidos/farmacología , Conformación Proteica , Ratas , Relación Estructura-ActividadRESUMEN
MHP-133 is one of a novel series of compounds designed to target multiple brain substrates expected to have synergistic actions in the treatment of cognitive and neurodegenerative disorders such as Alzheimer's disease. The strategy was to develop compounds with multiple targets relevant for enhancing cognition and memory, but avoiding the serious side effects attributed to high potency cholinergic agonists. MHP-133 was shown to interact with subtypes of cholinergic, serotonergic, and imidazoline receptors and to weakly inhibit acetylcholinesterase activity. In vitro, the drug enhanced nerve growth factor (TrkA) receptor expression; it prevented excitotoxicity in a hippocampal slice preparation; and increased the secretion of soluble (non-toxic) amyloid precursor protein. MHP-133 also enhanced cognitive performance by rats and by non-human primate in tasks designed to assess working memory. The results of this study are consistent with the potential use of MHP-133 in the treatment of neurodegenerative disorders such as Alzheimer's disease.
Asunto(s)
Sistema Nervioso Central/efectos de los fármacos , Cognición/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Compuestos de Piridinio/farmacología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Sistema Nervioso Central/anatomía & histología , Sistema Nervioso Central/metabolismo , Antagonistas Colinérgicos/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Macaca mulatta , Masculino , Aprendizaje por Laberinto , Estructura Molecular , Enfermedades Neurodegenerativas/tratamiento farmacológico , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Células PC12 , Compuestos de Piridinio/química , Compuestos de Piridinio/metabolismo , Compuestos de Piridinio/uso terapéutico , Ratas , Ratas Sprague-Dawley , Receptor trkA/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Caspase-3 is a prototypic executioner caspase that plays a central role in apoptosis. Aza-peptide epoxides are a novel class of irreversible inhibitors that are highly specific for clan CD cysteine proteases. The five crystal structures of caspase-3-aza-peptide epoxide inhibitor complexes reported here reveal the structural basis for the mechanism of inhibition and the specificities at the S1' and the S4 subsites. Unlike the clan CA cysteine proteases, the catalytic histidine in caspase-3 plays a critical role during protonation and subsequent ring opening of the epoxide moiety and facilitates the nucleophilic attack by the active site cysteine. The nucleophilic attack takes place on the C3 carbon atom of the epoxide and results in an irreversible alkylation of the active site cysteine residue. A favorable network of hydrogen bonds involving the oxyanion hole, catalytic histidine, and the atoms in the prime site of the inhibitor enhance the binding affinity and specificity of the aza-peptide epoxide inhibitors toward caspase-3. The studies also reveal that subtle movements of the N-terminal loop of the beta-subunit occur when the P4 Asp is replaced by a P4 Ile, whereas the N-terminal loop and the safety catch Asp179 are completely disordered when the P4 Asp is replaced by P4 Cbz group.
Asunto(s)
Compuestos Aza/síntesis química , Compuestos Aza/metabolismo , Inhibidores de Caspasas , Caspasas/metabolismo , Compuestos Epoxi/síntesis química , Oligopéptidos/síntesis química , Sitios de Unión/efectos de los fármacos , Caspasa 3 , Caspasas/química , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Compuestos Epoxi/metabolismo , Humanos , Oligopéptidos/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Serine proteases are typically synthesized as proteolytically inactive zymogens that often become activated in a limited and highly localized manner. Consequently, determination of the spatial and temporal activation pattern of these molecules is of great importance to understanding the biological processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce a technique using fluorescent synthetic and protein-based inhibitors. With this approach we have detected a novel serine protease activity with a relative mobility of 37 kDa, localized to the surface of pole cells, the germ-line precursors, in embryos between nuclear cycles 11 and 14 in development. A second novel cell-specific protease activity was localized to the tissues of early gastrulating embryos. Microinjection of inhibitors into the perivitelline space of stage 2 embryos perturbed normal embryonic development. Fluorescein-conjugated chymotrypsin inhibitor and Bowman-Birk inhibitor labeled protease activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos.