RESUMEN
Apoptosis Signal-Regulating Kinase-1 (ASK1) is a known member of the Mitogen-Activated Protein Kinase Kinase Kinase (MAP3K) family and upon stimulation will activate the p38- and JNK-pathways leading to cardiac apoptosis, fibrosis, and hypertrophy. Using Structure-Based Drug Design (SBDD) in parallel with deconstruction of a published compound, a novel series of ASK1 inhibitors was optimized, which incorporated a saturated heterocycle proximal to the hinge-binding motif. This yielded a unique chemical series with excellent selectivity across the broader kinome, and desirable drug-like properties. The lead compound (10) is highly soluble and permeable, and exhibits a cellular EC50 = 24 nM and Kd < 1 nM. Of the 350 kinases tested, 10 has an IC50 ≤ 500 nM for only eight of them. This paper will describe the design hypotheses behind this series, key data points during the optimization phase, as well as a possible structural rationale for the kinome selectivity. Based on crystallographic data, the presence of an aliphatic cycle adjacent to the hinge-binder in the active site of the protein kinase showed up in <1% of the >5000 structures in the Protein Data Bank, potentially conferring the selectivity seen in this series.
Asunto(s)
MAP Quinasa Quinasa Quinasa 5/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Animales , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Imidazoles/química , Imidazoles/metabolismo , Imidazoles/uso terapéutico , Concentración 50 Inhibidora , MAP Quinasa Quinasa Quinasa 5/metabolismo , Ratones , Simulación de Dinámica Molecular , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Target-engagement pharmacodynamic (PD) biomarkers are valuable tools in the prioritization of drug candidates, especially for novel, first-in-class mechanisms whose robustness to alter disease outcome is unknown. Methionine aminopeptidase 2 (MetAP2) is a cytosolic metalloenzyme that cleaves the N-terminal methionine from nascent proteins. Inhibition of MetAP2 leads to weight loss in obese rodents, dogs and humans. However, there is a need to develop efficacious compounds that specifically inhibit MetAP2 with an improved safety profile. The objective of this study was to identify a PD biomarker for selecting potent, efficacious compounds and for predicting clinical efficacy that would result from inhibition of MetAP2. Here we report the use of NMet14-3-3γ for this purpose. Treatment of primary human cells with MetAP2 inhibitors resulted in an approx. 10-fold increase in NMet14-3-3γ levels. Furthermore, treatment of diet-induced obese mice with these compounds reduced body weight (approx. 20%) and increased NMet14-3-3γ (approx. 15-fold) in adipose tissues. The effects on target engagement and body weight increased over time and were dependent on dose and administration frequency of compound. The relationship between compound concentration in plasma, NMet14-3-3γ in tissue, and reduction of body weight in obese mice was used to generate a pharmacokinetic-pharmacodynamic-efficacy model for predicting efficacy of MetAP2 inhibitors in mice. We also developed a model for predicting weight loss in humans using a target engagement PD assay that measures inhibitor-bound MetAP2 in blood. In summary, MetAP2 target engagement biomarkers can be used to select efficacious compounds and predict weight loss in humans. SIGNIFICANCE STATEMENT: The application of target engagement pharmacodynamic biomarkers during drug development provides a means to determine the dose required to fully engage the intended target and an approach to connect the drug target to physiological effects. This work exemplifies the process of using target engagement biomarkers during preclinical research to select new drug candidates and predict clinical efficacy. We determine concentration of MetAP2 antiobesity compounds needed to produce pharmacological activity in primary human cells and in target tissues from an appropriate animal model and establish key relationships between pharmacokinetics, pharmacodynamics, and efficacy, including the duration of effects after drug administration. The biomarkers described here can aid decision-making in early clinical trials of MetAP2 inhibitors for the treatment of obesity.
Asunto(s)
Clorobencenos/farmacología , Cinamatos/farmacología , Ciclohexanos/farmacología , Compuestos Epoxi/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Metionil Aminopeptidasas/antagonistas & inhibidores , Metionil Aminopeptidasas/metabolismo , Sesquiterpenos/farmacología , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Animales , Biomarcadores/metabolismo , Clorobencenos/química , Cinamatos/química , Ciclohexanos/química , Relación Dosis-Respuesta a Droga , Compuestos Epoxi/química , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Valor Predictivo de las Pruebas , Sesquiterpenos/química , Resultado del TratamientoRESUMEN
The RAS/RAF/MEK/ERK signal transduction cascade plays an important role in the regulation of critical cellular processes such as cell proliferation, migration, and differentiation. The up-regulation of this pathway can negatively affect cell homeostasis and is responsible for the development of various forms of cancer and inflammation processes. Therefore, there is a strong interest in pursuing drug programs targeting some of the enzymes involved in this pathway. In addition to the determination of Ki, Kd, IC50, and/or EC50, a more thorough kinetic analysis can provide useful information for the selection of the best lead series during the early stage of the drug discovery process. This study describes a medium-throughput fluorescent probe displacement assay for the rapid determination of the k(off) constant, residence time, and kinetic efficiency for ERK (extracellular signal-regulated kinase) inhibitors. Using this method, we have identified several inhibitors that we have subjected to further kinetic analysis by comparing k(off) constants determined for these time-dependent inhibitors using either the active or inactive form of ERK2.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Colorantes Fluorescentes/química , Cinética , Proteína Quinasa 1 Activada por Mitógenos/químicaRESUMEN
Polymerase ß (POLB), with dual functionality as a lyase and polymerase, plays a critical role in the base excision repair (BER) pathway to maintain genomic stability. POLB knockout and rescue studies in BRCA1/2-mutant cancer cell lines revealed that inhibition of lyase and polymerase activity is required for the synthetic lethal interaction observed with PARP inhibitors, highlighting POLB as a valuable therapeutic target. Traditional biochemical assays to screen for enzyme inhibitors focus on a single substrate to product relationship and limit the comprehensive analysis of enzymes such as POLB that utilize multiple substrates or catalyze a multi-step reaction. This report describes the first high-throughput mass spectrometry-based screen to measure the two distinct biochemical activities of POLB in a single assay using a duplexed self-assembled monolayer desorption ionization (SAMDI) mass spectrometry methodology. A multiplexed assay for POLB dual enzymatic activities was developed optimizing for kinetically balanced conditions and a collection of 200,000 diverse small molecules was screened in the duplexed format. Small molecule modulators identified in the screen were confirmed in a traditional fluorescence-based polymerase strand-displacement assay and an orthogonal label-free binding assay using SAMDI affinity selection mass spectrometry (ASMS). This work demonstrates the flexibility of high-throughput mass spectrometry approaches in drug discovery and highlights a novel application of SAMDI technology that opens new avenues for multiplexed high-throughput screening.
RESUMEN
Selective inhibition of the RGD (Arg-Gly-Asp) integrin αvß1 has been recently identified as an attractive therapeutic approach for the treatment of liver fibrosis given its function, target expression, and safety profile. Our identification of a non-RGD small molecule lead followed by focused, systematic changes to the core structure utilizing a crystal structure, in silico modeling, and a tractable synthetic approach resulted in the identification of a potent small molecule exhibiting a remarkable affinity for αvß1 relative to several other integrin isoforms measured. Azabenzimidazolone 25 demonstrated antifibrotic efficacy in an in vivo rat liver fibrosis model and represents a tool compound capable of further exploring the biological consequences of selective αvß1 inhibition.
Asunto(s)
Diseño de Fármacos , Receptores de Vitronectina , Animales , Ratas , Humanos , Receptores de Vitronectina/antagonistas & inhibidores , Receptores de Vitronectina/metabolismo , Relación Estructura-Actividad , Cirrosis Hepática/tratamiento farmacológico , Modelos Moleculares , Descubrimiento de Drogas , Ratas Sprague-Dawley , Masculino , Cristalografía por Rayos X , Bencimidazoles/farmacología , Bencimidazoles/química , Bencimidazoles/síntesis químicaRESUMEN
N-(Pyridin-2-yl) arylsulfonamides 1 and 2 (PF-915275) were identified as potent inhibitors of 11ß-hydroxysteroid dehydrogenase type 1. A screen for bioactivation revealed that these compounds formed glutathione conjugates. This communication presents the results of a risk benefit analysis carried out to progress 2 (PF-915275) to a clinical study and the strategies used to eliminate reactive metabolites in this series of inhibitors. Based on the proposed mechanism of bioactivation and structure-activity relationships, design efforts led to N-(pyridin-2-yl) arylsulfonamides such as 18 and 20 that maintained potent 11ß-hydroxysteroid dehydrogenase type 1 activity, showed exquisite pharmacokinetic profiles, and were negative in the reactive metabolite assay.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Aminopiridinas/farmacocinética , Sulfonamidas/farmacocinética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Aminopiridinas/química , Aminopiridinas/farmacología , Glutatión/farmacocinética , Células HEK293 , Humanos , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.
Asunto(s)
Adenina/análogos & derivados , Pruebas de Enzimas/métodos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirazinas/farmacología , Transducción de Señal , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Adenina/química , Adenina/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , Histidina/genética , Histidina/metabolismo , Humanos , Cinética , Oligopéptidos/genética , Oligopéptidos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Pirazinas/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Phosphoinositide-dependent kinase-1 (PDK1) is a critical enzyme in the PI3K/AKT pathway and to the activation of AGC family protein kinases, including S6K, SGK, and PKC. Dysregulation of this pathway plays a key role in cancer cell growth, survival and tumor angiogenesis. As such, inhibitors of PDK1 offer the promise of a new therapeutic modality for cancer treatment. Fragment based drug screening has recently become a viable entry point for hit identification. In this work, NMR spectroscopy fragment screening of PDK1 afforded novel chemotypes as orthogonal starting points from HTS screening hits. Compounds identified as hits by NMR spectroscopy were tested in a biochemical assay, and fragments with activity in both assays were clustered. The Pfizer compound file was mined via substructure and 2D similarity search, and the chemotypes were prioritized by ligand efficiency (LE), SAR mining, chemical attractiveness, and chemical enablement of promising vectors. From this effort, an isoquinolone fragment hit, 5 (IC(50) 870 µM, LE = 0.39), was identified as a novel, ligand efficient inhibitor of PDK1 and a suitable scaffold for further optimization. Initially in the absence of crystallographic data, a fragment growing approach efficiently explored four vectors of the isoquinolone scaffold via parallel synthesis to afford a compound with crystallographic data, 16 (IC(50) 41.4 µM, LE = 0.33). Subsequent lead optimization efforts provided 24 (IC(50) 1.8 µM, LE = 0.42), with greater than fivefold selectivity against other key pathway kinases.
Asunto(s)
Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Ensayos Analíticos de Alto Rendimiento , Humanos , Enlace de Hidrógeno , Ligandos , Imagen por Resonancia Magnética , Fragmentos de Péptidos/química , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Piruvato Deshidrogenasa Quinasa Acetil-TransferidoraRESUMEN
Two different signaling pathways lead to the activation of the transcription factor NF-κB, initiating distinct biological responses: The canonical NF-κB pathway activation has been implicated in host immunity and inflammatory responses, whereas the noncanonical pathway activation has been involved in lymphoid organ development and B-cell maturation, as well as in the development of chronic inflammatory diseases and some hematologic cancers. The NF-κB-inducing kinase (NIK) is a cytoplasmic Ser/Thr kinase and is a key regulator of the noncanonical pathway. NIK activation results in the processing of the p100 subunit to p52, leading to the formation of the RelB/p52 complex and noncanonical pathway activation. Because of its role in the development of lymphoid malignancies, this kinase has always been considered as an attractive target for the treatment of certain types of cancers and immune diseases. We at Takeda have pursued a drug discovery program to identify small-molecule inhibitors against NIK. This report provides an overview of the data generated from our screening campaign using a small fragment library. Most importantly, we also provide a kinetic analysis of published compounds and chemical series developed at Takeda that are associated with a slow tight-binding mechanism and excellent cellular potency.
Asunto(s)
Descubrimiento de Drogas/métodos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Unión Proteica , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas , Quinasa de Factor Nuclear kappa BRESUMEN
The design and development of a series of highly selective pyrrolidine carboxamide 11beta-HSD1 inhibitors are described. These compounds including PF-877423 demonstrated potent in vitro activity against both human and mouse 11beta-HSD1 enzymes. In an in vivo assay, PF-877423 inhibited the conversion of cortisone to cortisol. Structure guided optimization effort yielded potent and stable 11beta-HSD1 selective inhibitor 42.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Adamantano/análogos & derivados , Amidas/química , Inhibidores Enzimáticos/química , Hipoglucemiantes/química , Pirrolidinas/química , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Adamantano/síntesis química , Adamantano/química , Adamantano/farmacología , Amidas/síntesis química , Amidas/farmacología , Animales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Cobayas , Humanos , Hipoglucemiantes/síntesis química , Hipoglucemiantes/farmacología , Ratones , Microsomas Hepáticos/metabolismo , Pirrolidinas/síntesis química , Pirrolidinas/farmacología , Relación Estructura-ActividadRESUMEN
Checkpoint kinase 1 (CHK1) is a key element in the DNA damage response pathway and plays a crucial role in the S-G(2)-phase checkpoint. Inhibiting CHK1 is a therapeutic strategy involving abrogation of the G2/M mitotic checkpoint defense of tumor cells toward lethal damage induced by DNA-directed chemotherapeutic agents. To date, most CHK1 inhibition approaches have involved targeting the ATP site of this kinase. In this study, we provide crystallographic and kinetic characterization of two small molecule inhibitors that bind to an allosteric site in the proximity of the CHK1 substrate site. Analysis of kinetic and biophysical data has led to the conclusion that these small molecule allosteric site inhibitors of CHK1 are reversible and are neither ATP- nor peptide substrate-competitive. K(i) values of 1.89 and 0.15 microM, respectively, have been determined for these compounds using a mixed inhibitor kinetic analysis. Cocrystal structures of the inhibitors bound to CHK1 reveal an allosteric site, unique to CHK1, located in the C-terminal domain and consisting of a shallow groove linked to a small hydrophobic pocket. The pocket displays induced fit characteristics in the presence of the two inhibitors. These findings establish the potential for the development of highly selective CHK1 inhibitors.
Asunto(s)
Proteínas Quinasas/metabolismo , Regulación Alostérica , Sitios de Unión , Dominio Catalítico , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Clonación Molecular , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Modelos Moleculares , Fragmentos de Péptidos/química , Conformación Proteica , Proteínas Quinasas/química , Proteínas Quinasas/genética , Resonancia por Plasmón de SuperficieRESUMEN
N-(Pyridin-2-yl) arylsulfonamides are identified as inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1), an enzyme that catalyzes the reduction of the glucocorticoid cortisone to cortisol. Dysregulation of glucocorticoids has been implicated in the pathogenesis of diabetes and the metabolic syndrome. In this Letter, we present the development of an initial lead to an efficient ligand with improved physiochemical properties using a deletion strategy. This strategy allowed for further optimization of potency leading to the discovery of the clinical candidate PF-915275.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Aminopiridinas/síntesis química , Inhibidores Enzimáticos/síntesis química , Sulfonamidas/síntesis química , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Aminopiridinas/química , Aminopiridinas/farmacocinética , Animales , Línea Celular , Simulación por Computador , Cricetinae , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Humanos , Ratas , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacocinéticaRESUMEN
Increased incidence of type 2 diabetes mellitus and obesity has elevated the medical need for new agents to treat these disease states. Resistance to the hormones insulin and leptin are hallmarks of both type 2 diabetes and obesity. Drugs that can ameliorate this resistance should be effective in treating type 2 diabetes and possibly obesity. Protein tyrosine phosphatase 1B (PTP1B) is thought to function as a negative regulator of insulin and leptin signal transduction. This article reviews PTP1B as a novel target for type 2 diabetes, and looks at the challenges in developing small-molecule inhibitors of this phosphatase.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Obesidad/tratamiento farmacológico , Proteínas Tirosina Fosfatasas , Diabetes Mellitus Tipo 2/metabolismo , Inhibidores Enzimáticos/química , Humanos , Biología Molecular , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/fisiología , Relación Estructura-ActividadRESUMEN
Prolyl hydroxylases (PHDs) down-regulate the level of hypoxia-inducible factors (HIFs) by hydroxylating key proline residues that trigger the degradation of the protein and affect the cell and its ability to respond to hypoxic stress. Several small molecule PHD inhibitors are now in various preclinical and clinical stages for the treatment of anemia. The present study provides a detail kinetic analysis for some of these inhibitors. The data generated in the present study suggest that these compounds are reversible and compete directly with the co-substrate, 2-oxoglutarate (2-OG) for binding at the enzyme active site. Most of these compounds are pan PHD inhibitors and exhibit a time-dependent inhibition (TDI) mechanism due to an extremely slow dissociation rate constant, koff, and a long residence time.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Prolina Dioxigenasas del Factor Inducible por Hipoxia/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Dominio Catalítico , Inhibidores Enzimáticos/química , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/química , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Ácidos Cetoglutáricos/metabolismo , Cinética , Unión Proteica , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
[This corrects the article DOI: 10.1021/acsmedchemlett.6b00481.].
RESUMEN
Apoptosis signal-regulating kinase 1 (ASK1/MAP3K) is a mitogen-activated protein kinase family member shown to contribute to acute ischemia/reperfusion injury. Using structure-based drug design, deconstruction, and reoptimization of a known ASK1 inhibitor, a lead compound was identified. This compound displayed robust MAP3K pathway inhibition and reduction of infarct size in an isolated perfused heart model of cardiac injury.
RESUMEN
Analogues substituted with various amines at the 6-position of the pyrazine ring on (4-amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)pyrazin-2-ylmethanone were discovered as potent and selective inhibitors of PDK1 with potential as anticancer agents. An early lead with 2-pyridine-3-ylethylamine as the pyrazine substituent showed moderate potency and selectivity. Structure-based drug design led to improved potency and selectivity against PI3Kα through a combination of cyclizing the ethylene spacer into a saturated, five-membered ring and substituting on the 4-position of the aryl ring with a fluorine. ADME properties were improved by lowering the lipophilicity with heteroatom replacements in the saturated, five-membered ring. The optimized analogues have a PDK1 Ki of 1 nM and >100-fold selectivity against PI3K/AKT-pathway kinases. The cellular potency of these analogues was assessed by the inhibition of AKT phosphorylation (T308) and by their antiproliferation activity against a number of tumor cell lines.
Asunto(s)
Antineoplásicos/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/síntesis química , Pirroles/síntesis química , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Etilaminas/síntesis química , Etilaminas/química , Etilaminas/farmacología , Humanos , Modelos Moleculares , Fosforilación , Conformación Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/química , Piridinas/farmacología , Pirroles/química , Pirroles/farmacología , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Assay conditions for the 11beta-hydroxysteroid dehydrogenase have been optimized by adding phospholipids in the media buffer to increase and stabilize the enzymatic activity. The presence of phospholipids greatly facilitates the study of the binding of cortisone and NADPH at the enzyme catalytic site. Kinetic analyses conducted with the human and rabbit enzyme isoforms suggest that both enzymes behave according to an ordered sequential bi-bi mechanism where the NADPH is the first to bind at the active site followed by cortisone. The equilibrium dissociation constant, K(i)a as well as the apparent Michaelis-Menten constants K(m)a, K(m)b, k(cat)a, and k(cat)b for NADPH and cortisone, have been determined to be 147.5 microM, 14.4 microM, 43.8 nM, 0.21 min(-1), and 0.27 min(-1), respectively, for the human enzyme and 41.1 microM, 3.1 microM, 161.7 nM, 0.49 min(-1), and 0.52min(-1), respectively, for the rabbit enzyme.