RESUMEN
We report herein the discovery and extensive characterization of ARD-1676, a highly potent and orally efficacious PROTAC degrader of the androgen receptor (AR). ARD-1676 was designed using a new class of AR ligands and a novel cereblon ligand. It has DC50 values of 0.1 and 1.1 nM in AR+ VCaP and LNCaP cell lines, respectively, and IC50 values of 11.5 and 2.8 nM in VCaP and LNCaP cell lines, respectively. ARD-1676 effectively induces degradation of a broad panel of clinically relevant AR mutants. ARD-1676 has an oral bioavailability of 67, 44, 31, and 99% in mice, rats, dogs, and monkeys, respectively. Oral administration of ARD-1676 effectively reduces the level of AR protein in the VCaP tumor tissue in mice and inhibits tumor growth in the VCaP mouse xenograft tumor model without any sign of toxicity. ARD-1676 is a highly promising development candidate for the treatment of AR+ human prostate cancer.
RESUMEN
We report the discovery of ARD-2051 as a potent and orally efficacious androgen receptor (AR) proteolysis-targeting chimera degrader. ARD-2051 achieves DC50 values of 0.6 nM and Dmax >90% in inducing AR protein degradation in both the LNCaP and VCaP prostate cancer cell lines, potently and effectively suppresses AR-regulated genes, and inhibits cancer cell growth. ARD-2051 achieves a good oral bioavailability and pharmacokinetic profile in mouse, rat, and dog. A single oral dose of ARD-2051 strongly reduces AR protein and suppresses AR-regulated gene expression in the VCaP xenograft tumor tissue in mice. Oral administration of ARD-2051 effectively inhibits VCaP tumor growth and causes no signs of toxicity in mice. ARD-2051 is a promising AR degrader for advanced preclinical development for the treatment of AR+ human cancers.
Asunto(s)
Neoplasias de la Próstata , Receptores Androgénicos , Masculino , Humanos , Ratones , Ratas , Animales , Perros , Receptores Androgénicos/metabolismo , Quimera Dirigida a la Proteólisis , Proteolisis , Línea Celular Tumoral , Neoplasias de la Próstata/patologíaRESUMEN
Signal transducer and activator of transcription 5 (STAT5) is an attractive therapeutic target, but successful targeting of STAT5 has proved to be difficult. Here we report the development of AK-2292 as a first, potent and selective small-molecule degrader of both STAT5A and STAT5B isoforms. AK-2292 induces degradation of STAT5A/B proteins with an outstanding selectivity over all other STAT proteins and more than 6,000 non-STAT proteins, leading to selective inhibition of STAT5 activity in cells. AK-2292 effectively induces STAT5 depletion in normal mouse tissues and human chronic myeloid leukemia (CML) xenograft tissues and achieves tumor regression in two CML xenograft mouse models at well-tolerated dose schedules. AK-2292 is not only a powerful research tool with which to investigate the biology of STAT5 and the therapeutic potential of selective STAT5 protein depletion and inhibition but also a promising lead compound toward ultimate development of a STAT5-targeted therapy.
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Neoplasias , Factor de Transcripción STAT5 , Humanos , Ratones , Animales , Factor de Transcripción STAT5/metabolismoRESUMEN
STAT5 is an attractive therapeutic target for human cancers. We report herein the discovery of a potent and selective STAT5 degrader with strong antitumor activity in vivo. We first obtained small-molecule ligands with sub-micromolar to low micromolar binding affinities to STAT5 and STAT6 SH2 domains and determined co-crystal structures of three such ligands in complex with STAT5A. We successfully transformed these ligands into potent and selective STAT5 degraders using the PROTAC technology with AK-2292 as the best compound. AK-2292 effectively induces degradation of STAT5A, STAT5B, and phosphorylated STAT5 proteins in a concentration- and time-dependent manner in acute myeloid leukemia (AML) cell lines and demonstrates excellent degradation selectivity for STAT5 over all other STAT members. It exerts potent and specific cell growth inhibitory activity in AML cell lines with high levels of phosphorylated STAT5. AK-2292 effectively reduces STAT5 protein in vivo and achieves strong antitumor activity in mice at well-tolerated dose schedules.
Asunto(s)
Leucemia Mieloide Aguda , Factor de Transcripción STAT5 , Humanos , Animales , Ratones , Factor de Transcripción STAT5/metabolismo , Ligandos , Leucemia Mieloide Aguda/tratamiento farmacológico , Dominios Homologos src , Línea CelularRESUMEN
We report herein the discovery of exceptionally potent and orally bioavailable PROTAC AR degraders with ARD-2585 being the most promising compound. ARD-2585 achieves DC50 values of ≤0.1 nM in the VCaP cell line with AR gene amplification and in the LNCaP cell line carrying an AR mutation. It potently inhibits cell growth with IC50 values of 1.5 and 16.2 nM in the VCaP and LNCaP cell lines, respectively, and achieves excellent pharmacokinetics and 51% of oral bioavailability in mice. It is more efficacious than enzalutamide in inhibition of VCaP tumor growth and does not cause any sign of toxicity in mice. ARD-2585 is a promising AR degrader for extensive investigations for the treatment of advanced prostate cancer.
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Antineoplásicos/uso terapéutico , Ftalimidas/uso terapéutico , Piperidonas/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Proteolisis/efectos de los fármacos , Receptores Androgénicos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antagonistas de Receptores Androgénicos/síntesis química , Antagonistas de Receptores Androgénicos/farmacocinética , Antagonistas de Receptores Androgénicos/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Diseño de Fármacos , Humanos , Masculino , Ratones SCID , Estructura Molecular , Ftalimidas/síntesis química , Ftalimidas/farmacocinética , Piperidonas/síntesis química , Piperidonas/farmacocinética , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeting the menin-MLL protein-protein interaction is being pursued as a new therapeutic strategy for the treatment of acute leukemia carrying MLL-rearrangements (MLLr leukemia). Herein, we report M-1121, a covalent and orally active inhibitor of the menin-MLL interaction capable of achieving complete and persistent tumor regression. M-1121 establishes covalent interactions with Cysteine 329 located in the MLL binding pocket of menin and potently inhibits growth of acute leukemia cell lines carrying MLL translocations with no activity in cell lines with wild-type MLL. Consistent with the mechanism of action, M-1121 drives dose-dependent down-regulation of HOXA9 and MEIS1 gene expression in the MLL-rearranged MV4;11 leukemia cell line. M-1121 is orally bioavailable and shows potent antitumor activity in vivo with tumor regressions observed at tolerated doses in the MV4;11 subcutaneous and disseminated models of MLL-rearranged leukemia. Together, our findings support development of an orally active covalent menin inhibitor as a new therapy for MLLr leukemia.
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Antineoplásicos/farmacología , Descubrimiento de Drogas , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Modelos Moleculares , Estructura Molecular , Proteínas Proto-Oncogénicas/metabolismo , Relación Estructura-ActividadRESUMEN
G protein-coupled receptor (GPCR) kinases (GRKs) regulate the desensitization and internalization of GPCRs. Two of these, GRK2 and GRK5, are upregulated in heart failure and are promising targets for heart failure treatment. Although there have been several reports of potent and selective inhibitors of GRK2 there are few for GRK5. Herein, we describe a ligand docking approach utilizing the crystal structures of the GRK2-Gßγ·GSK180736A and GRK5·CCG215022 complexes to search for amide substituents predicted to confer GRK2 and/or GRK5 potency and selectivity. From this campaign, we successfully generated two new potent GRK5 inhibitors, although neither exhibited selectivity over GRK2.
Asunto(s)
Amidas/farmacología , Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Quinasa 5 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/farmacología , Amidas/síntesis química , Amidas/química , Relación Dosis-Respuesta a Droga , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
The neurotropic protozoan Toxoplasma gondii is the second leading cause of death due to foodborne illness in the US, and has been designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no treatment options exist for the chronic dormant-phase Toxoplasma infection in the central nervous system (CNS). T. gondii cathepsin L (TgCPL) has recently been implicated as a novel viable target for the treatment of chronic toxoplasmosis. In this study, we report the first body of SAR work aimed at developing potent inhibitors of TgCPL with selectivity vs the human cathepsin L. Starting from a known inhibitor of human cathepsin L, and guided by structure-based design, we were able to modulate the selectivity for Toxoplasma vs human CPL by nearly 50-fold while modifying physiochemical properties to be more favorable for metabolic stability and CNS penetrance. The overall potency of our inhibitors towards TgCPL was improved from 2⯵M to as low as 110â¯nM and we successfully demonstrated that an optimized analog 18b is capable of crossing the BBB (0.5â¯brain/plasma). This work is an important first step toward development of a CNS-penetrant probe to validate TgCPL as a feasible target for the treatment of chronic toxoplasmosis.
Asunto(s)
Antiprotozoarios/química , Catepsina L/antagonistas & inhibidores , Sistema Nervioso Central/metabolismo , Dipéptidos/química , Inhibidores de Proteasas/química , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Antiprotozoarios/metabolismo , Antiprotozoarios/farmacología , Sitios de Unión , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Dominio Catalítico , Catepsina L/metabolismo , Dipéptidos/metabolismo , Dipéptidos/farmacología , Semivida , Humanos , Concentración 50 Inhibidora , Ratones , Microsomas Hepáticos/metabolismo , Simulación de Dinámica Molecular , Permeabilidad/efectos de los fármacos , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Proteínas Protozoarias/metabolismo , Relación Estructura-Actividad , Toxoplasma/efectos de los fármacos , Toxoplasma/enzimologíaRESUMEN
The Mycobacterium tuberculosis (Mtb) DosRST two-component regulatory system promotes the survival of Mtb during non-replicating persistence (NRP). NRP bacteria help drive the long course of tuberculosis therapy; therefore, chemical inhibition of DosRST may inhibit the ability of Mtb to establish persistence and thus shorten treatment. Using a DosRST-dependent fluorescent Mtb reporter strain, a whole-cell phenotypic high-throughput screen of a â¼540,000 compound small-molecule library was conducted. The screen discovered novel inhibitors of the DosRST regulon, including three compounds that were subject to follow-up studies: artemisinin, HC102A and HC103A. Under hypoxia, all three compounds inhibit Mtb-persistence-associated physiological processes, including triacylglycerol synthesis, survival and antibiotic tolerance. Artemisinin functions by disabling the heme-based DosS and DosT sensor kinases by oxidizing ferrous heme and generating heme-artemisinin adducts. In contrast, HC103A inhibits DosS and DosT autophosphorylation activity without targeting the sensor kinase heme.
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Artemisininas/farmacología , Histidina Quinasa/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Artemisininas/química , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Histidina Quinasa/metabolismo , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
G protein-coupled receptors (GPCRs) are central to many physiological processes. Regulation of this superfamily of receptors is controlled by GPCR kinases (GRKs), some of which have been implicated in heart failure. GSK180736A, developed as a Rho-associated coiled-coil kinase 1 (ROCK1) inhibitor, was identified as an inhibitor of GRK2 and co-crystallized in the active site. Guided by its binding pose overlaid with the binding pose of a known potent GRK2 inhibitor, Takeda103A, a library of hybrid inhibitors was developed. This campaign produced several compounds possessing high potency and selectivity for GRK2 over other GRK subfamilies, PKA, and ROCK1. The most selective compound, 12n (CCG-224406), had an IC50 for GRK2 of 130 nM, >700-fold selectivity over other GRK subfamilies, and no detectable inhibition of ROCK1. Four of the new inhibitors were crystallized with GRK2 to give molecular insights into the binding and kinase selectivity of this class of inhibitors.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Bovinos , Células Cultivadas , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Conformación Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Neurotropic alphaviruses, including western, eastern, and Venezuelan equine encephalitis viruses, cause serious and potentially fatal central nervous system infections in humans for which no currently approved therapies exist. We previously identified a series of thieno[3,2-b]pyrrole derivatives as novel inhibitors of neurotropic alphavirus replication, using a cell-based phenotypic assay (W. Peng et al., J. Infect. Dis. 199:950-957, 2009, doi:http://dx.doi.org/10.1086/597275), and subsequently developed second- and third-generation indole-2-carboxamide derivatives with improved potency, solubility, and metabolic stability (J. A. Sindac et al., J. Med. Chem. 55:3535-3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; J. A. Sindac et al., J. Med. Chem. 56:9222-9241, 2013, http://dx.doi.org/10.1021/jm401330r). In this report, we describe the antiviral activity of the most promising third-generation lead compound, CCG205432, and closely related analogs CCG206381 and CCG209023. These compounds have half-maximal inhibitory concentrations of â¼1 µM and selectivity indices of >100 in cell-based assays using western equine encephalitis virus replicons. Furthermore, CCG205432 retains similar potency against fully infectious virus in cultured human neuronal cells. These compounds show broad inhibitory activity against a range of RNA viruses in culture, including members of the Togaviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Although their exact molecular target remains unknown, mechanism-of-action studies reveal that these novel indole-based compounds target a host factor that modulates cap-dependent translation. Finally, we demonstrate that both CCG205432 and CCG209023 dampen clinical disease severity and enhance survival of mice given a lethal western equine encephalitis virus challenge. These studies demonstrate that indole-2-carboxamide compounds are viable candidates for continued preclinical development as inhibitors of neurotropic alphaviruses and, potentially, of other RNA viruses. IMPORTANCE There are currently no approved drugs to treat infections with alphaviruses. We previously identified a novel series of compounds with activity against these potentially devastating pathogens (J. A. Sindac et al., J. Med. Chem. 55:3535-3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; W. Peng et al., J. Infect. Dis. 199:950-957, 2009, doi:http://dx.doi.org/10.1086/597275; J. A. Sindac et al., J. Med. Chem. 56:9222-9241, 2013, http://dx.doi.org/10.1021/jm401330r). We have now produced third-generation compounds with enhanced potency, and this manuscript provides detailed information on the antiviral activity of these advanced-generation compounds, including activity in an animal model. The results of this study represent a notable achievement in the continued development of this novel class of antiviral inhibitors.
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Antivirales/farmacología , Virus de la Encefalitis Equina del Oeste/efectos de los fármacos , Encefalomielitis Equina/tratamiento farmacológico , Indoles/farmacología , Piridinas/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/síntesis química , Bunyaviridae/efectos de los fármacos , Bunyaviridae/crecimiento & desarrollo , Línea Celular , Virus de la Encefalitis Equina del Oeste/crecimiento & desarrollo , Virus de la Encefalitis Equina del Oeste/patogenicidad , Encefalomielitis Equina/mortalidad , Encefalomielitis Equina/virología , Femenino , Indoles/síntesis química , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/virología , Paramyxoviridae/efectos de los fármacos , Paramyxoviridae/crecimiento & desarrollo , Picornaviridae/efectos de los fármacos , Picornaviridae/crecimiento & desarrollo , Biosíntesis de Proteínas/efectos de los fármacos , Piridinas/síntesis química , Replicón/efectos de los fármacos , Relación Estructura-Actividad , Análisis de SupervivenciaRESUMEN
The urinary tract is one of the most common sites of infection in humans, and uropathogenic Escherichia coli (UPEC) is the main causative agent of urinary tract infections. Bacteria colonizing the urinary tract face extremely low iron availability. To counteract this, UPEC expresses a wide variety of iron acquisition systems. To exploit iron acquisition in UPEC as a global target for small-molecule inhibition, we developed and carried out a whole-cell growth-based high throughput screen of 149,243 compounds. Our primary assay was carried out under iron-limiting conditions. Hits in the primary screen were assayed using two counterscreens that ruled out iron chelators and compounds that inhibit growth by means other than inhibition of iron acquisition. We determined dose-response curves under two different iron conditions and purchased fresh compounds for selected hits. After retesting dose-response relationships, we identified 16 compounds that arrest growth of UPEC only under iron-limiting conditions. All compounds are bacteriostatic and do not inhibit proton motive force. A loss-of-target strategy was employed to identify the cellular target of these inhibitors. Two compounds lost inhibitory activity against a strain lacking TonB and were shown to inhibit irreversible adsorption of a TonB-dependent bacteriophage. Our results validate iron acquisition as a target for antibacterial strategies against UPEC and identify TonB as one of the cellular targets. IMPORTANCE Half of women will suffer at least one episode of urinary tract infection (UTI) during their lifetime. The current treatment for UTI involves antibiotic therapy. Resistance to currently used antibiotics has steadily increased over the last decade, generating a pressing need for the development of new therapeutic agents. Since iron is essential for colonization and scarce in the urinary tract, targeting iron acquisition would seem to be an attractive strategy. However, the multiplicity and redundancy of iron acquisition systems in uropathogenic Escherichia coli (UPEC) make it difficult to pinpoint a specific cellular target. Here, we identified 16 iron acquisition inhibitors through a whole-cell high-throughput screen, validating iron acquisition as a target for antibacterial strategies against UPEC. We also identified the cellular target of two of the inhibitors as the TonB system.
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Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Transporte Biológico/efectos de los fármacos , Proteínas de Escherichia coli/antagonistas & inhibidores , Hierro/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Escherichia coli Uropatógena/efectos de los fármacos , Colifagos/fisiología , Evaluación Preclínica de Medicamentos , Escherichia coli Uropatógena/crecimiento & desarrollo , Escherichia coli Uropatógena/metabolismo , Acoplamiento ViralRESUMEN
Rifampin, a semisynthetic rifamycin, is the cornerstone of current tuberculosis treatment. Among many semisynthetic rifamycins, benzoxazinorifamycins have great potential for TB treatment due to their superior affinity for wild-type and rifampin-resistant Mycobacterium tuberculosis RNA polymerases and their reduced hepatic Cyp450 induction activity. In this study, we have determined the crystal structures of the Escherichia coli RNA polymerase complexes with two benzoxazinorifamycins. The ansa-naphthalene moieties of the benzoxazinorifamycins bind in a deep pocket of the ß subunit, blocking the path of the RNA transcript. The C3'-tail of benzoxazinorifamycin fits a cavity between the ß subunit and σ factor. We propose that in addition to blocking RNA exit, the benzoxazinorifamycin C3'-tail changes the σ region 3.2 loop position, which influences the template DNA at the active site, thereby reducing the efficiency of transcription initiation. This study supports expansion of structure-activity relationships of benzoxazinorifamycins inhibition of RNA polymerase toward uncovering superior analogues with development potential.
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ARN Polimerasas Dirigidas por ADN/química , Proteínas de Escherichia coli/química , Rifamicinas/química , Antibióticos Antituberculosos , Cristalografía por Rayos X , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana , Proteínas de Escherichia coli/genética , Holoenzimas/química , Modelos Moleculares , Mutación , Conformación Proteica , Rifamicinas/síntesis química , Transcripción GenéticaRESUMEN
CCG-1423 (1) is a novel inhibitor of Rho/MKL1/SRF-mediated gene transcription that inhibits invasion of PC-3 prostate cancer cells in a Matrigel model of metastasis. We recently reported the design and synthesis of conformationally restricted analogs (e.g., 2) with improved selectivity for inhibiting invasion versus acute cytotoxicity. In this study we conducted a survey of aromatic substitution with the goal of improving physicochemical parameters (e.g., ClogP, MW) for future efficacy studies in vivo. Two new compounds were identified that attenuated cytotoxicity even further, and were fourfold more potent than 2 at inhibiting PC-3 cell migration in a scratch wound assay. One of these (8a, CCG-203971, IC50=4.2 µM) was well tolerated in mice for 5 days at 100mg/kg/day i.p., and was able to achieve plasma levels exceeding the migration IC50 for up to 3 h.
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Amidas/farmacología , Antineoplásicos/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Ácidos Nipecóticos/farmacología , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Neoplasias de la Próstata/tratamiento farmacológico , Factor de Respuesta Sérica/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Amidas/síntesis química , Amidas/química , Anilidas/síntesis química , Anilidas/química , Anilidas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Benzamidas/síntesis química , Benzamidas/química , Benzamidas/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Masculino , Estructura Molecular , Metástasis de la Neoplasia/tratamiento farmacológico , Ácidos Nipecóticos/síntesis química , Ácidos Nipecóticos/química , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismo , Relación Estructura-Actividad , Transactivadores , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
ΔFosB protein accumulates in the striatum in response to chronic administration of drugs of abuse, L-DOPA, or stress, triggering long lasting neural and behavioral changes that underlie aspects of drug addiction, abnormal involuntary movements (dyskinesia), and depression. ΔFosB binds AP-1 DNA consensus sequences found in promoters of many genes and can both repress or activate gene transcription. In the striatum, ΔFosB is thought to dimerize with JunD to form a functional transcription factor, though strikingly JunD does not accumulate in parallel. One explanation is that ΔFosB can recruit different partners, including itself, depending on the neuron type in which it is induced and the chronic stimulus, generating protein complexes with different effects on gene transcription. To develop chemical probes to study ΔFosB, a high-throughput screen was carried out to identify small molecules that modulate ΔFosB function. Two compounds with low micromolar activity, termed C2 and C6, disrupt the binding of ΔFosB to DNA via different mechanisms, and in in vitro assays stimulate ΔFosB-mediated transcription. In cocaine-treated mice, C2 significantly elevates mRNA levels of the AMPA glutamate receptor GluR2 subunit with specificity, a known target gene of ΔFosB that plays a role in drug addiction and endogenous resilience mechanisms. C2 and C6 show different activities against ΔFosB homodimers compared to ΔFosB/JunD heterodimers, suggesting that these compounds can be used as probes to study the contribution of different ΔFosB-containing complexes on the regulation of gene transcription in biological systems and to assess the utility of ΔFosB as a therapeutic target.
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Preparaciones Farmacéuticas/química , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/fisiología , Evaluación Preclínica de Medicamentos/métodos , Insectos , Ratones , Preparaciones Farmacéuticas/metabolismo , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/fisiología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/fisiologíaRESUMEN
High-throughput screening (HTS) has historically been used by the pharmaceutical industry to rapidly test hundreds of thousands of compounds to identify potential drug candidates. More recently, academic groups have used HTS to identify new chemical probes or small interfering RNA (siRNA) that can serve as experimental tools to examine the biology or physiology of novel proteins, processes, or interactions. HTS presents a significant challenge with the vast and complex nature of data generated. This report describes MScreen, a Web-based, open-source cheminformatics application for chemical library and siRNA plate management, primary HTS and dose-response data handling, structure search, and administrative functions. Each project in MScreen can be secured with passwords or shared in an open-information environment that enables collaborators to easily compare data from many screens, providing a useful means to identify compounds with desired selectivity. Unique features include compound, substance, mixture, and siRNA plate creation and formatting; automated dose-response fitting and quality control (QC); and user, target, and assay method administration. MScreen provides an effective means to facilitate HTS information handling and analysis in the academic setting so that users can efficiently view their screening data and evaluate results for follow-up.
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Bases de Datos de Compuestos Químicos , Ensayos Analíticos de Alto Rendimiento , Almacenamiento y Recuperación de la Información , ARN Interferente Pequeño , Bibliotecas de Moléculas Pequeñas/farmacología , InternetRESUMEN
By utilization of three-dimensional structure information of rifamycins bound to RNA polymerase (RNAP) and the human pregnane X receptor (hPXR), representative examples (2b-d) of a novel subclass of benzoxazinorifamycins have been synthesized. Relative to rifalazil (2a), these analogues generally display superior affinity toward wild-type and Rif-resistant mutants of the Mycobacterium tuberculosis RNAP but lowered antitubercular activity in cell culture under both aerobic and anaerobic conditions. Lowered affinity toward hPXR for some of the analogues is also observed, suggesting a potential for reduced Cyp450 induction activity. Mouse and human microsomal studies of analogue 2b show it to have excellent metabolic stability. Mouse pharmacokinetics in plasma and lung show accumulation of 2b but with a half-life suggesting nonoptimal pharmacokinetics. These studies demonstrate proof of principle for this subclass of rifamycins and support further expansion of structure-activity relationships (SARs) toward uncovering analogues with development potential.
Asunto(s)
Rifamicinas/síntesis química , Animales , Antibióticos Antituberculosos/síntesis química , Antibióticos Antituberculosos/farmacocinética , Benzoxazinas/síntesis química , Benzoxazinas/metabolismo , Benzoxazinas/farmacocinética , ARN Polimerasas Dirigidas por ADN/metabolismo , Diseño de Fármacos , Farmacorresistencia Bacteriana , Ratones , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Rifampin/farmacología , Rifamicinas/metabolismo , Rifamicinas/farmacocinética , Rifamicinas/farmacologíaRESUMEN
Synthesis inhibition is the basis for the treatment of type 1 Gaucher disease by the glucosylceramide synthase (GCS) inhibitor eliglustat tartrate. However, the extended use of eliglustat and related compounds for the treatment of glycosphingolipid storage diseases with CNS manifestations is limited by the lack of brain penetration of this drug. Property modeling around the D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol (PDMP) pharmacophore was employed in a search for compounds of comparable activity against the GCS but lacking P-glycoprotein (MDR1) recognition. Modifications of the carboxamide N-acyl group were made to lower total polar surface area and rotatable bond number. Compounds were screened for inhibition of GCS in crude enzyme and whole cell assays and for MDR1 substrate recognition. One analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (CCG-203586), was identified that inhibited GCS at low nanomolar concentrations with little to no apparent recognition by MDR1. Intraperitoneal administration of this compound to mice for 3 days resulted in a significant dose dependent decrease in brain glucosylceramide content, an effect not seen in mice dosed in parallel with eliglustat tartrate.
Asunto(s)
Encéfalo/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glucosilceramidas/metabolismo , Glucosiltransferasas/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Encéfalo/metabolismo , Células Cultivadas , Dioxanos/síntesis química , Dioxanos/farmacología , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Indanos/síntesis química , Indanos/farmacología , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos C57BL , Morfolinas/química , Vinblastina/farmacocinéticaRESUMEN
A series of rifamycin S and rifampin analogues incorporating substituted 8-amino, 8-thio, and 1,8-pyrazole substituents has been synthesized. The compounds were made by activation of the C-8 phenol as a sulfonate ester, followed by displacement with selected nitrogen and sulfur nucleophiles. The analogues were screened in assays to quantify their antitubercular activity under both aerobic and anaerobic conditions, and for inhibition of wild-type Mycobacterium tuberculosis (MTB) RNAP and rifamycin-resistant MTB RNAP (S450L) via an in vitro rolling circle transcription assay. Additionally, the MIC(90) values were determined for these analogues against Escherichia coli strains. Although none of the analogues displayed superior enzymatic or microbiological activity to their parent scaffolds, the results are consistent with the Rif C-8 hydroxyl acting as a hydrogen bond acceptor with S450 and that Rif resistance in the S450L mutant is due to loss of this hydrogen bond. Representative analogues were also evaluated in the human pregnane X receptor (PXR) activation assay.
Asunto(s)
Antituberculosos/química , Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/química , Rifampin/farmacología , Rifamicinas/química , Rifamicinas/farmacología , Antituberculosos/síntesis química , Escherichia coli/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Pirazoles/síntesis química , Pirazoles/química , Pirazoles/farmacología , Rifampin/síntesis química , Rifamicinas/síntesis química , Tuberculosis/tratamiento farmacológicoRESUMEN
Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An 66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.