RESUMEN
Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/metabolismo , Compuestos Azo , Fibroblastos/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Oxidorreductasas Intramoleculares , Factores Inhibidores de la Migración de Macrófagos , Azul de Tripano/química , Azul de Tripano/farmacología , Regulación Alostérica/efectos de los fármacos , Antígenos de Diferenciación de Linfocitos B/química , Compuestos Azo/química , Compuestos Azo/farmacología , Células Cultivadas , Fibroblastos/citología , Antígenos de Histocompatibilidad Clase II/química , Humanos , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/química , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Factores Inhibidores de la Migración de Macrófagos/química , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Cuaternaria de ProteínaRESUMEN
Oxazolidinones possessing a C-5 carboxamide functionality (reverse amides) represent a new series of compounds that block bacterial protein synthesis. These reverse amides also exhibited less potency against monoamine oxidase (MAO) enzymes and thus possess less potential for the side effects associated with MAO inhibition. The title compound (14) showed reduced in vivo myelotoxicity compared to linezolid in a 14-day safety study in rats, potent in vivo efficacy in murine systemic infection models, and excellent pharmacokinetic properties.
Asunto(s)
Antibacterianos/síntesis química , Óxidos S-Cíclicos/síntesis química , Oxazolidinonas/síntesis química , Acetamidas/farmacología , Administración Oral , Animales , Antibacterianos/farmacología , Antibacterianos/toxicidad , Disponibilidad Biológica , Óxidos S-Cíclicos/farmacología , Óxidos S-Cíclicos/toxicidad , Perros , Farmacorresistencia Bacteriana , Femenino , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Inyecciones Intravenosas , Linezolid , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Inhibidores de la Monoaminooxidasa/síntesis química , Inhibidores de la Monoaminooxidasa/farmacología , Inhibidores de la Monoaminooxidasa/toxicidad , Oxazolidinonas/farmacología , Oxazolidinonas/toxicidad , Ratas , Ratas Sprague-Dawley , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus , Infecciones Estreptocócicas/tratamiento farmacológico , Streptococcus pyogenes , Relación Estructura-ActividadRESUMEN
The genus Mycobacterium includes non-pathogenic species such as M. smegmatis, and pathogenic species such as M. tuberculosis, the causative agent of tuberculosis (TB). Treatment of TB requires a lengthy regimen of several antibiotics, whose effectiveness has been compromised by the emergence of resistant strains. New antibiotics that can shorten the treatment course and those that have not been compromised by bacterial resistance are needed. In this study, we report that thiadiazolidinones, a relatively little-studied heterocyclic class, inhibit the activity of mycobacterial alanine racemase, an essential enzyme that converts l-alanine to d-alanine for peptidoglycan synthesis. Twelve members of the thiadiazolidinone family were evaluated for inhibition of M. tuberculosis and M. smegmatis alanine racemase activity and bacterial growth. Thiadiazolidinones inhibited M. tuberculosis and M. smegmatis alanine racemases to different extents with 50% inhibitory concentrations (IC50) ranging from <0.03 to 28µM and 23 to >150µM, respectively. The compounds also inhibited the growth of these bacteria, including multidrug resistant strains of M. tuberculosis. The minimal inhibitory concentrations (MIC) for drug-susceptible M. tuberculosis and M. smegmatis ranged from 6.25µg/ml to 100µg/ml, and from 1.56 to 6.25µg/ml for drug-resistant M. tuberculosis. The in vitro activities of thiadiazolidinones suggest that this family of compounds might represent starting points for medicinal chemistry efforts aimed at developing novel antimycobacterial agents.
Asunto(s)
Alanina Racemasa/antagonistas & inhibidores , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Tiadiazoles/farmacología , Alanina Racemasa/química , Alanina Racemasa/metabolismo , Secuencia de Aminoácidos , Catálisis , Datos de Secuencia Molecular , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/enzimología , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen and a major cause of hospital-acquired infections. New antibacterial agents that have not been compromised by bacterial resistance are needed to treat MRSA-related infections. We chose the S. aureus cell wall synthesis enzyme, alanine racemase (Alr) as the target for a high-throughput screening effort to obtain novel enzyme inhibitors, which inhibit bacterial growth. Among the 'hits' identified was a thiadiazolidinone with chemical properties attractive for lead development. This study evaluated the mode of action, antimicrobial activities, and mammalian cell cytotoxicity of the thiadiazolidinone family in order to assess its potential for development as a therapeutic agent against MRSA. The thiadiazolidones inhibited Alr activity with 50% inhibitory concentrations (IC50) ranging from 0.36 to 6.4 µM, and they appear to inhibit the enzyme irreversibly. The series inhibited the growth of S. aureus, including MRSA strains, with minimal inhibitory concentrations (MICs) ranging from 6.25 to 100 µg/ml. The antimicrobial activity showed selectivity against Gram-positive bacteria and fungi, but not Gram-negative bacteria. The series inhibited human HeLa cell proliferation. Lead development centering on the thiadiazolidinone series would require additional medicinal chemistry efforts to enhance the antibacterial activity and minimize mammalian cell toxicity.
Asunto(s)
Alanina Racemasa/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/enzimología , Tiadiazoles/química , Tiadiazoles/farmacología , Alanina Racemasa/metabolismo , Antibacterianos/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Células HeLa , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Tiadiazoles/clasificaciónRESUMEN
BACKGROUND: In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target. METHODOLOGY/PRINCIPAL FINDINGS: To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the 'hits' for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells. CONCLUSIONS/SIGNIFICANCE: This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.
Asunto(s)
Alanina Racemasa/antagonistas & inhibidores , Antiinfecciosos/farmacología , Inhibidores Enzimáticos/clasificación , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Alanina-Deshidrogenasa/metabolismo , Alanina Racemasa/química , Alanina Racemasa/metabolismo , Alanina Racemasa/farmacología , Antiinfecciosos/análisis , Antiinfecciosos/química , Antiinfecciosos/clasificación , Muerte Celular/efectos de los fármacos , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/química , Células HeLa , Humanos , Concentración 50 Inhibidora , Cinética , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Especificidad por Sustrato/efectos de los fármacosRESUMEN
With nearly one-third of the global population infected by Mycobacterium tuberculosis, TB remains a major cause of death (1.7 million in 2006). TB is particularly severe in parts of Asia and Africa where it is often present in AIDS patients. Difficulties in treatment are exacerbated by the 6-9 month treatment times and numerous side effects. There is significant concern about the multi-drug-resistant (MDR) strains of TB (0.5 million MDR-TB cases worldwide in 2006). The rifamycins, long considered a mainstay of TB treatment, were a tremendous breakthrough when they were developed in the 1960's. While the rifamycins display many admirable qualities, they still have a number of shortfalls including: rapid selection of resistant mutants, hepatotoxicity, a flu-like syndrome (especially at higher doses), potent induction of cytochromes P450 (CYP) and inhibition of hepatic transporters. This review of the state-of-the-art regarding rifamycins suggests that it is quite possible to devise improved rifamycin analogs. Studies showing the potential of shortening the duration of treatment if higher doses could be tolerated, also suggest that more potent (or less toxic) rifamycin analogs might accomplish the same end. The improved activity against rifampin-resistant strains by some analogs promises that further work in this area, especially if the information from co-crystal structures with RNA polymerase is applied, should lead to even better analogs. The extensive drug-drug interactions seen with rifampin have already been somewhat ameliorated with rifabutin and rifalazil, and the use of a CYP-induction screening assay should serve to efficiently identify even better analogs. The toxicity due to the flu-like syndrome is an issue that needs effective resolution, particularly for analogs in the rifalazil class. It would be of interest to profile rifalazil and analogs in relation to rifampin, rifapentine, and rifabutin in a variety of screens, particularly those that might relate to hypersensitivity or immunomodulatory processes.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Rifamicinas/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Pulmonar/tratamiento farmacológico , África/epidemiología , Antituberculosos/uso terapéutico , Asia/epidemiología , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Humanos , Mycobacterium tuberculosis/inmunología , Rifamicinas/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Tuberculosis Resistente a Múltiples Medicamentos/inmunología , Tuberculosis Pulmonar/inmunologíaRESUMEN
Novel benzazepine oxazolidinone antibacterials were synthesized and evaluated against clinically relevant susceptible and resistant organisms. The effect of ring nitrogen position and N-substitution on antibacterial activity is examined.