RESUMEN
Aberrant activation of matrix metalloproteinases (MMPs) is a common feature of pathological cascades observed in diverse disorders, such as cancer, fibrosis, immune dysregulation, and neurodegenerative diseases. MMP-9, in particular, is highly dynamically regulated in several pathological processes. Development of MMP inhibitors has therefore been an attractive strategy for therapeutic intervention. However, a long history of failed clinical trials has demonstrated that broad-spectrum MMP inhibitors have limited clinical utility, which has spurred the development of inhibitors selective for individual MMPs. Attaining selectivity has been technically challenging because of sequence and structural conservation across the various MMPs. Here, through a biochemical and structural screening paradigm, we have identified JNJ0966, a highly selective compound that inhibited activation of MMP-9 zymogen and subsequent generation of catalytically active enzyme. JNJ0966 had no effect on MMP-1, MMP-2, MMP-3, MMP-9, or MMP-14 catalytic activity and did not inhibit activation of the highly related MMP-2 zymogen. The molecular basis for this activity was characterized as an interaction of JNJ0966 with a structural pocket in proximity to the MMP-9 zymogen cleavage site near Arg-106, which is distinct from the catalytic domain. JNJ0966 was efficacious in reducing disease severity in a mouse experimental autoimmune encephalomyelitis model, demonstrating the viability of this therapeutic approach. This discovery reveals an unprecedented pharmacological approach to MMP inhibition, providing an opportunity to improve selectivity of future clinical drug candidates. Targeting zymogen activation in this manner may also allow for pharmaceutical exploration of other enzymes previously viewed as intractable drug targets.
Asunto(s)
Precursores Enzimáticos/antagonistas & inhibidores , Precursores Enzimáticos/química , Metaloproteinasa 9 de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz/química , Regulación Alostérica , Animales , Células COS , Dominio Catalítico , Chlorocebus aethiops , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Humanos , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Dominios ProteicosRESUMEN
A series of indazoles have been discovered as KHK inhibitors from a pyrazole hit identified through fragment-based drug discovery (FBDD). The optimization process guided by both X-ray crystallography and solution activity resulted in lead-like compounds with good pharmaceutical properties.
Asunto(s)
Descubrimiento de Drogas , Fructoquinasas/antagonistas & inhibidores , Indazoles/farmacología , Pirazoles/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Indazoles/síntesis química , Indazoles/química , Modelos Moleculares , Estructura Molecular , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Guided by X-ray crystallography of thrombin-inhibitor complexes and molecular modeling, alkylation of the N1 nitrogen of the imidazole P1 ligand of the pyridinoneacetamide thrombin inhibitor 1 with various acetamide moieties furnished inhibitors with significantly improved thrombin potency, trypsin selectivity, functional in vitro anticoagulant potency and in vivo antithrombotic efficacy. In the pyrazinoneacetamide series, oral bioavailability was also improved.
Asunto(s)
Anticoagulantes/farmacología , Antitrombinas/farmacología , Diseño de Fármacos , Imidazoles/síntesis química , Imidazoles/farmacología , Trombina/antagonistas & inhibidores , Administración Oral , Animales , Anticoagulantes/síntesis química , Anticoagulantes/química , Anticoagulantes/farmacocinética , Antitrombinas/síntesis química , Antitrombinas/química , Antitrombinas/farmacocinética , Disponibilidad Biológica , Cloruros , Cristalografía por Rayos X , Perros , Compuestos Férricos/farmacología , Imidazoles/química , Imidazoles/farmacocinética , Macaca mulatta , Modelos Moleculares , Estructura Molecular , Tiempo de Tromboplastina Parcial , Ratas , Relación Estructura-Actividad , Trombina/química , Trombina/metabolismo , Tripsina/metabolismoRESUMEN
We report here the first inhibitor-bound structure of a mitotic motor protein. The 1.9 A resolution structure of the motor domain of KSP, bound with the small molecule monastrol and Mg2+ x ADP, reveals that monastrol confers inhibition by "induced-fitting" onto the protein some 12 A away from the catalytic center of the enzyme, resulting in the creation of a previously non-existing binding pocket. The structure provides new insights into the biochemical and mechanical mechanisms of the mitotic motor domain. Inhibition of KSP provides a novel mechanism to arrest mitotic spindle formation, a target of several approved and investigative anti-cancer agents. The structural information gleaned from this novel pocket offers a new angle for the design of anti-mitotic agents.
Asunto(s)
Cinesinas/antagonistas & inhibidores , Cinesinas/química , Pirimidinas/farmacología , Tionas/farmacología , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Humanos , Magnesio/metabolismo , Microtúbulos/química , Mitosis , Modelos Moleculares , Proteínas Motoras Moleculares , Unión Proteica/genética , Conformación Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
As many processes in the preclinical drug discovery process become highly parallel, the need to also produce a large number of different proteins in parallel has become acute, such as for protein crystallization and activity screening. In turn, the requisite DNA constructions to produce these proteins must now be done at a rate that requires automated cloning procedures, each with an intrinsic low failure probability per sample. The high-throughput cloning solutions presented here achieve production of 192 different expression plasmids at a success rate of greater than 95% of the targeted open reading frames. Time for completion of the set by one person is reduced to approximately 11 working days, starting with polymerase chain reactions for a number of source clones and ending with purified expression plasmids. Achievement of this throughput utilizes the following: (1) the Beckman Coulter (Fullerton, CA) Biomek FX liquid handler for most manipulations, (2) Gateway cloning technology (Invitrogen Corp., Carlsbad, CA), and (3) computer programs designed for parallel processing of all sample information, including primer design and the resulting DNA and protein sequence assembly. Exemplary data are presented for discovery of a form of the Rho-kinase that crystallizes (ROCK2).
Asunto(s)
Clonación Molecular/métodos , Plásmidos/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Robótica , Programas Informáticos , Automatización , Cristalización , Evaluación Preclínica de Medicamentos/métodos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Mutagénesis , Plásmidos/biosíntesis , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Robótica/instrumentación , Factores de Tiempo , Quinasas Asociadas a rhoRESUMEN
The design, synthesis, and biological evaluation of a series of HIV-1 protease inhibitors [(-)-6, (-)-7, (-)-23, (+)-24] based upon the 3,5,5-trisubstituted pyrrolin-4-one scaffold is described. Use of a monopyrrolinone scaffold leads to inhibitors with improved cellular transport properties relative to the earlier inhibitors based on bispyrrolinones and their peptide counterparts. The most potent inhibitor (-)-7 displayed 13% oral bioavailability in dogs. X-ray structure analysis of the monopyrrolinone compounds cocrystallized with the wild-type HIV-1 protease provided valuable information on the interactions between the inhibitors and the HIV-1 enzyme. In each case, the inhibitors assumed similar orientations for the P2'-P1 substituents, along with an unexpected hydrogen bond of the pyrrolinone NH with Asp225. Interactions with the S2 pocket, however, were not optimal, as illustrated by the inclusion of a water molecule in two of the three inhibitor-enzyme complexes. Efforts to increase affinity by displacing the water molecule with second and third generation inhibitors did not prove successful. Lack of success with this venture is a testament to the difficulty of accurately predicting the many variables that influence and build binding affinity. Comparison of the inhibitor positions in three complexes with that of Indinavir revealed displacements of the protease backbones in the enzyme flap region, accompanied by variations in hydrogen bonding to accommodate the monopyrrolinone ring. The binding orientation of the pyrrolinone-based inhibitors may explain their sustained efficacy against mutant strains of the HIV-1 protease enzyme as compared to Indinavir.
Asunto(s)
Carbamatos/síntesis química , Inhibidores de la Proteasa del VIH/síntesis química , Proteasa del VIH/química , Pirroles/síntesis química , Animales , Disponibilidad Biológica , Carbamatos/química , Carbamatos/farmacocinética , Cristalografía por Rayos X , Perros , Diseño de Fármacos , Proteasa del VIH/genética , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacocinética , Modelos Moleculares , Mutación , Unión Proteica , Pirroles/química , Pirroles/farmacocinética , Pirroles/farmacologíaRESUMEN
In an effort to discover potent, clinically useful thrombin inhibitors, a rapid analogue synthetic approach was used to explore the P(1) region. Various benzylamines were coupled to a pyridine/pyrazinone P(2)-P(3) template. One compound with an o-thiadiazole benzylic substitution was found to have a thrombin K(i) of 0.84 nM. A study of ortho-substituted five-membered-ring heterocycles was undertaken and subsequently demonstrated that the o-triazole and tetrazole rings were optimal. Combination of these potent P(1) aryl heterocycles with a variety of P(2)-P(3) groups produced a compound with an extraordinary thrombin inhibitory activity of 1.4 pM. It is hoped that this potency enhancement in P(1) will allow for more diversification in the P(2)-P(3) region to ultimately address additional pharmacological concerns.
Asunto(s)
Compuestos Heterocíclicos/síntesis química , Trombina/antagonistas & inhibidores , Bencilaminas/síntesis química , Bencilaminas/química , Sitios de Unión , Compuestos Heterocíclicos/química , Modelos Moleculares , Pirazinas/síntesis química , Pirazinas/química , Piridinas/síntesis química , Piridinas/química , Relación Estructura-Actividad , Tetrazoles/síntesis química , Tetrazoles/química , Tiadiazoles/síntesis química , Tiadiazoles/química , Trombina/química , Triazoles/síntesis química , Triazoles/químicaRESUMEN
This report describes the development of a cell-based assay for high-throughput screening and detection of small-molecule inhibitors for hepatitis C virus (HCV) NS2/3 protease. The HCV NS2/3 protease is essential for the normal infectious cycle of HCV. Generation of a cell-based assay for this cis-acting viral protease involved reporter constructs in which the NS2/3 protease sequence was inserted between the ,B-lactamase (BLA) reporter and a ubiquitin-based destabilization domain. In stable cell lines, NS2/3 cis cleavage of the NS2/3-BLA fusion protein resulted in differential stability of the cleaved versus uncleaved BLA reporter, providing a robust readout for protease activity. BLA reporter activity was shown to be a function of NS2/3-specific protease activity, by using genetic mutants of the NS2/3 sequence. In addition, the cell-based assay was validated and screened in a 384-well format on a fully automated robotic platform where small-molecule inhibitors of NS2/3 protease activity were identified.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Hepacivirus/metabolismo , Inhibidores de Proteasas/farmacología , Automatización , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Genes Reporteros , Humanos , Células Jurkat , Modelos Genéticos , Mutación , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Endocannabinoids such as 2-arachidonylglycerol (2-AG) are ligands for cannabinoid receptors that contribute to the transmission and modulation of pain signals. The antinociceptive effect of exogenous 2-AG suggests that inhibition of monoglyceride lipase (MGLL), the enzyme responsible for degrading 2-AG and arresting signaling, may be a target for pain modulation. Here we describe the characterization of MGLL ligands following a high-throughput screening campaign. Ligands were discovered using ThermoFluor, a label-free affinity-based screening tool that measures ligand binding via modulation of protein thermal stability. A kinetic fluorescent assay using the substrate 4-methylcoumarin butyrate was used to counterscreen confirmed HTS positives. A comparison of results from binding and inhibition assays allowed elucidation of compound mechanism of action. We demonstrate the limit of each technology and the benefits of using orthogonal assay techniques in profiling compounds.
Asunto(s)
Dominio Catalítico/efectos de los fármacos , Pruebas de Enzimas/métodos , Inhibidores Enzimáticos/farmacología , Monoacilglicerol Lipasas/antagonistas & inhibidores , Ácidos Araquidónicos/química , Endocannabinoides , Inhibidores Enzimáticos/química , Glicéridos/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Hidrólisis , Concentración 50 Inhibidora , Cinética , Monoacilglicerol Lipasas/química , Monoacilglicerol Lipasas/metabolismo , Unión Proteica , Solubilidad , Especificidad por SustratoRESUMEN
For anyone who has participated in a screening exercise in a pharmaceutical or biotech setting with the aim to discover hits against protein targets, it is evident that a single screening exercise does not always offer hits of sufficient quality to be progressed into a lead compound. Often, more than one screen is needed. The premise in conducting a new screening exercise is to find "better" hits that are chemically and pharmacologically more attractive. As we move into challenging, new target classes, the need of new methods to find tractable hits is ever more urgent and the availability of differentiated backup compounds is crucial to sustain a clinical program. The obvious alternate routes to conduct the new screen include an improved compound library, a larger compound library, and different or more sensitive detection methods. As many of us have experienced firsthand, repeating a screen without drastically changing the chemical nature of the compound library or information content of the readout likely will offer only variations of the original hits. This chapter describes the strategies to adopt in fragment-based lead discovery to avoid rediscovering the known.
Asunto(s)
Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Bibliotecas de Moléculas Pequeñas , Preparaciones Farmacéuticas/metabolismo , Farmacocinética , Unión ProteicaRESUMEN
A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.
Asunto(s)
Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ácidos Araquidónicos/química , Ácidos Araquidónicos/metabolismo , Moduladores de Receptores de Cannabinoides/química , Moduladores de Receptores de Cannabinoides/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Endocannabinoides , Glicéridos/química , Glicéridos/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Monoacilglicerol Lipasas/genética , Monoacilglicerol Lipasas/metabolismo , Mutagénesis Sitio-Dirigida , Unión Proteica , Electricidad EstáticaRESUMEN
A fragment-based drug design paradigm has been successfully applied in the discovery of lead series of ketohexokinase inhibitors. The paradigm consists of three iterations of design, synthesis, and X-ray crystallographic screening to progress low molecular weight fragments to leadlike compounds. Applying electron density of fragments within the protein binding site as defined by X-ray crystallography, one can generate target specific leads without the use of affinity data. Our approach contrasts with most fragment-based drug design methodology where solution activity is a main design guide. Herein we describe the discovery of submicromolar ketohexokinase inhibitors with promising druglike properties.
Asunto(s)
Fructoquinasas/antagonistas & inhibidores , Indazoles/síntesis química , Modelos Moleculares , Piperidinas/síntesis química , Animales , Células CACO-2 , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Electrones , Humanos , Técnicas In Vitro , Indazoles/química , Indazoles/farmacocinética , Masculino , Microsomas Hepáticos/metabolismo , Estructura Molecular , Piperidinas/química , Piperidinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations associated with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor ( 11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compounds from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp). Utilizing knowledge garnered from previous KSP inhibitors, we found that beta-fluorination modulated the p K a of the piperidine nitrogen and reduced Pgp efflux, but the resulting compound ( 14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compound 30 that has an optimal in vitro and metabolic profile. Compound 30 (MK-0731) was recently studied in a phase I clinical trial in patients with taxane-refractory solid tumors.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Cinesinas/antagonistas & inhibidores , Neoplasias/enzimología , Piperidinas/farmacología , Pirroles/farmacología , Taxoides/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológico , Taxoides/uso terapéuticoRESUMEN
The development of 2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-ones as inhibitors of Chk1 kinase is described. Introduction of a fused ring at the C7/C8 positions of the pyrazoloquinolinone provided an increase in potency while guidance from overlapping inhibitor bound Chk1 X-ray crystal structures contributed to the discovery of a potent and solubilizing propyl amine moiety in compound 52 (Chk1 IC(50)=3.1 nM).
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/efectos de los fármacos , Quinolonas/farmacología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía por Rayos X , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Quinolonas/química , Relación Estructura-ActividadRESUMEN
Inspired by previous efforts in the pyrazolobenzoxazine class of KSP inhibitors, the design and synthesis of 1,4-diaryl-4,5-dihydropyrazole inhibitors of KSP are described. Crystallographic evidence of binding mode and in vivo potency data is also highlighted.
Asunto(s)
Diseño de Fármacos , Hidrógeno/química , Cinesinas/antagonistas & inhibidores , Cinesinas/metabolismo , Mitosis/efectos de los fármacos , Pirazoles/química , Pirazoles/farmacología , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Molecular , Pirazoles/síntesis química , Relación Estructura-ActividadRESUMEN
Observations from two structurally related series of KSP inhibitors led to the proposal and discovery of dihydropyrazolobenzoxazines that possess ideal properties for cancer drug development. The synthesis and characterization of this class of inhibitors along with relevant pharmacokinetic and in vivo data are presented. The synthesis is highlighted by a key [3+2] cycloaddition to form the pyrazolobenzoxazine core followed by diastereospecific installation of a quaternary center.
Asunto(s)
Benzoxazinas/química , Benzoxazinas/farmacología , Diseño de Fármacos , Cinesinas/antagonistas & inhibidores , Cinesinas/metabolismo , Mitosis/efectos de los fármacos , Pirazoles/química , Animales , Benzoxazinas/síntesis química , Benzoxazinas/farmacocinética , Línea Celular , Perros , Humanos , Hidrógeno/química , Estructura Molecular , Relación Estructura-ActividadRESUMEN
A series of monopyrrolinone-based HIV-1 protease inhibitors possessing rationally designed P2' side chains have been synthesized and evaluated for activity against wild-type HIV-1 protease. The most potent inhibitor displays subnanomolar potency in vitro for the wild-type HIV-1 protease. Additionally, the monopyrrolinone inhibitors retain potency in cellular assays against clinically significant mutant forms of the virus. X-ray structures of these inhibitors bound in the wild-type enzyme reveal important insights into the observed biological activity.
Asunto(s)
Inhibidores de la Proteasa del VIH , Proteasa del VIH/efectos de los fármacos , Pirrolidinonas , Animales , Bovinos , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Técnicas In Vitro , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conformación Molecular , Mutación , Pirrolidinonas/síntesis química , Pirrolidinonas/química , Pirrolidinonas/farmacología , Relación Estructura-ActividadRESUMEN
The evolution of 2,4-diaryl-2,5-dihydropyrroles as inhibitors of KSP is described. Introduction of basic amide and urea moieties to the dihydropyrrole nucleus enhanced potency and aqueous solubility, simultaneously, and provided compounds that caused mitotic arrest of A2780 human ovarian carcinoma cells with EC(50)s<10nM. Ancillary hERG activity was evaluated for this series of inhibitors.
Asunto(s)
Cinesinas/antagonistas & inhibidores , Pirroles/química , Pirroles/farmacología , Línea Celular Tumoral , Femenino , Humanos , Modelos Moleculares , Neoplasias Ováricas/patología , Pirroles/síntesis química , Huso Acromático/químicaRESUMEN
Molecular modeling in combination with X-ray crystallographic information was employed to identify a region of the kinesin spindle protein (KSP) binding site not fully utilized by our first generation inhibitors. We discovered that by appending a propylamine substituent at the C5 carbon of a dihydropyrazole core, we could effectively fill this unoccupied region of space and engage in a hydrogen-bonding interaction with the enzyme backbone. This change led to a second generation compound with increased potency, a 400-fold enhancement in aqueous solubility at pH 4, and improved dog pharmacokinetics relative to the first generation compound.