RESUMEN
Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/antagonistas & inhibidores , Malaria/tratamiento farmacológico , Malaria/parasitología , Plasmodium/efectos de los fármacos , Plasmodium/enzimología , 1-Fosfatidilinositol 4-Quinasa/química , 1-Fosfatidilinositol 4-Quinasa/genética , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Citocinesis/efectos de los fármacos , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/genética , Ácidos Grasos/metabolismo , Femenino , Hepatocitos/parasitología , Humanos , Imidazoles/metabolismo , Imidazoles/farmacología , Estadios del Ciclo de Vida/efectos de los fármacos , Macaca mulatta , Masculino , Modelos Biológicos , Modelos Moleculares , Fosfatos de Fosfatidilinositol/metabolismo , Plasmodium/clasificación , Plasmodium/crecimiento & desarrollo , Pirazoles/metabolismo , Pirazoles/farmacología , Quinoxalinas/metabolismo , Quinoxalinas/farmacología , Reproducibilidad de los Resultados , Esquizontes/citología , Esquizontes/efectos de los fármacos , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
A novel family of 1H-imidazol-2-yl-pyrimidine-4,6-diamines has been identified with potent activity against the erythrocyte-stage of Plasmodium falciparum (Pf), the most common causative agent of malaria. A systematic SAR study resulted in the identification of compound 40 which exhibits good potency against both wild-type and drug resistant parasites and exhibits good in vivo pharmacokinetic properties.
Asunto(s)
Antimaláricos/química , Plasmodium falciparum/efectos de los fármacos , Pirimidinas/química , Animales , Antimaláricos/farmacocinética , Antimaláricos/farmacología , Descubrimiento de Drogas , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
Farnesoid X receptor (FXR) agonists are emerging as important potential therapeutics for the treatment of nonalcoholic steatohepatitis (NASH) patients, as they exert positive effects on multiple aspects of the disease. FXR agonists reduce lipid accumulation in the liver, hepatocellular inflammation, hepatic injury, and fibrosis. While there are currently no approved therapies for NASH, the bile acid-derived FXR agonist obeticholic acid (OCA; 6-ethyl chenodeoxycholic acid) has shown promise in clinical studies. Previously, we described the discovery of tropifexor (LJN452), the most potent non-bile acid FXR agonist currently in clinical investigation. Here, we report the discovery of a novel chemical series of non-bile acid FXR agonists based on a tricyclic dihydrochromenopyrazole core from which emerged nidufexor (LMB763), a compound with partial FXR agonistic activity in vitro and FXR-dependent gene modulation in vivo. Nidufexor has advanced to Phase 2 human clinical trials in patients with NASH and diabetic nephropathy.
Asunto(s)
Benzotiazoles/uso terapéutico , Ácido Quenodesoxicólico/análogos & derivados , Dieta Alta en Grasa/efectos adversos , Isoxazoles/uso terapéutico , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/agonistas , Animales , Benzotiazoles/química , Ácido Quenodesoxicólico/química , Ácido Quenodesoxicólico/uso terapéutico , Perros , Humanos , Isoxazoles/química , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/etiología , Estructura Terciaria de Proteína , Ratas , Resultado del TratamientoRESUMEN
Screening our in-house compound collection using a cell based Plasmodium falciparum proliferation assay we discovered a known pan-kinase inhibitor scaffold as a hit. Further optimization of this series led us to a novel benzamide scaffold which was devoid of human kinase activity while retaining its antiplasmodial activity. The evolution of this compound series leading to optimized candidates with good cellular potency against multiple strains as well as decent in vivo profile is described in this Letter.
Asunto(s)
Antimaláricos/química , Benzamidas/química , Inhibidores Enzimáticos/química , Fosfotransferasas/antagonistas & inhibidores , Plasmodium falciparum/enzimología , Animales , Antimaláricos/síntesis química , Antimaláricos/farmacología , Benzamidas/síntesis química , Benzamidas/farmacología , Evolución Molecular Dirigida , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Plasmodium falciparum/efectos de los fármacosRESUMEN
Farnesoid X receptor (FXR) agonism is emerging as an important potential therapeutic mechanism of action for multiple chronic liver diseases. The bile acid-derived FXR agonist obeticholic acid (OCA) has shown promise in a phase 2 study in patients with nonalcoholic steatohepatitis (NASH). Here, we report efficacy of the novel nonbile acid FXR agonist tropifexor (LJN452) in two distinct preclinical models of NASH. The efficacy of tropifexor at <1 mg/kg doses was superior to that of OCA at 25 mg/kg in the liver in both NASH models. In a chemical and dietary model of NASH (Stelic animal model [STAM]), tropifexor reversed established fibrosis and reduced the nonalcoholic fatty liver disease activity score and hepatic triglycerides. In an insulin-resistant obese NASH model (amylin liver NASH model [AMLN]), tropifexor markedly reduced steatohepatitis, fibrosis, and profibrogenic gene expression. Transcriptome analysis of livers from AMLN mice revealed 461 differentially expressed genes following tropifexor treatment that included a combination of signatures associated with reduction of oxidative stress, fibrogenesis, and inflammation. Conclusion: Based on preclinical validation in animal models, tropifexor is a promising investigational therapy that is currently under phase 2 development for NASH.
RESUMEN
Structure-based design was utilized to guide the early stage optimization of a substrate-like inhibitor to afford potent peptidomimetic inhibitors of the channel-activating protease prostasin. The first X-ray crystal structures of prostasin with small molecule inhibitors bound to the active site are also reported.
Asunto(s)
Serina Endopeptidasas/efectos de los fármacos , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacología , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Imitación Molecular , Estructura Molecular , Conformación Proteica , Inhibidores de Serina Proteinasa/química , Relación Estructura-ActividadRESUMEN
The farnesoid X receptor (FXR) is a nuclear receptor that acts as a master regulator of bile acid metabolism and signaling. Activation of FXR inhibits bile acid synthesis and increases bile acid conjugation, transport, and excretion, thereby protecting the liver from the harmful effects of bile accumulation, leading to considerable interest in FXR as a therapeutic target for the treatment of cholestasis and nonalcoholic steatohepatitis. We identified a novel series of highly potent non-bile acid FXR agonists that introduce a bicyclic nortropine-substituted benzothiazole carboxylic acid moiety onto a trisubstituted isoxazole scaffold. Herein, we report the discovery of 1 (tropifexor, LJN452), a novel and highly potent agonist of FXR. Potent in vivo activity was demonstrated in rodent PD models by measuring the induction of FXR target genes in various tissues. Tropifexor has advanced into phase 2 human clinical trials in patients with NASH and PBC.
Asunto(s)
Benzotiazoles/farmacología , Colestasis/tratamiento farmacológico , Isoxazoles/farmacología , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/agonistas , Administración Oral , Animales , Benzotiazoles/uso terapéutico , Disponibilidad Biológica , Perros , Evaluación Preclínica de Medicamentos/métodos , Factores de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Isoxazoles/uso terapéutico , Masculino , Microsomas Hepáticos/efectos de los fármacos , Piperidinas/química , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Relación Estructura-Actividad , Triglicéridos/sangreRESUMEN
Our current understanding of the role and regulation of protease activity in normal and pathogenic processes is limited by our ability to measure and deconvolute their enzymatic activity. To address this limitation, an approach was developed that utilizes rhodamine-based fluorogenic substrates encoded with PNA tags. The PNA tags address each of the substrates to a predefined location on an oligonucleotide microarray through hybridization, thus allowing the deconvolution of multiple signals from a solution. A library of 192 protease substrates was prepared by split and mix combinatorial synthesis. The methodology and validation of this approach for profiling proteolytic activity from single proteases and from those in crude cell lysates as well as clinical blood samples is described.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Ácidos Nucleicos de Péptidos/genética , Humanos , Hidrólisis , Células Jurkat , Péptido Hidrolasas/química , Biblioteca de Péptidos , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/metabolismo , Proteómica/métodos , Especificidad por Sustrato/genéticaRESUMEN
Piezo ion channels are activated by various types of mechanical stimuli and function as biological pressure sensors in both vertebrates and invertebrates. To date, mechanical stimuli are the only means to activate Piezo ion channels and whether other modes of activation exist is not known. In this study, we screened ~3.25 million compounds using a cell-based fluorescence assay and identified a synthetic small molecule we termed Yoda1 that acts as an agonist for both human and mouse Piezo1. Functional studies in cells revealed that Yoda1 affects the sensitivity and the inactivation kinetics of mechanically induced responses. Characterization of Yoda1 in artificial droplet lipid bilayers showed that Yoda1 activates purified Piezo1 channels in the absence of other cellular components. Our studies demonstrate that Piezo1 is amenable to chemical activation and raise the possibility that endogenous Piezo1 agonists might exist. Yoda1 will serve as a key tool compound to study Piezo1 regulation and function.
Asunto(s)
Canales Iónicos/agonistas , Canales Iónicos/metabolismo , Mecanotransducción Celular/fisiología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Humanos , RatonesRESUMEN
A high throughput screening campaign identified 5-(2-chlorophenyl)indazole compound 4 as an antagonist of the transient receptor potential A1 (TRPA1) ion channel with IC50 = 1.23 µM. Hit to lead medicinal chemistry optimization established the SAR around the indazole ring system, demonstrating that a trifluoromethyl group at the 2-position of the phenyl ring in combination with various substituents at the 6-position of the indazole ring greatly contributed to improvements in vitro activity. Further lead optimization resulted in the identification of compound 31, a potent and selective antagonist of TRPA1 in vitro (IC50 = 0.015 µM), which has moderate oral bioavailability in rodents and demonstrates robust activity in vivo in several rodent models of inflammatory pain.
Asunto(s)
Indazoles/química , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Administración Oral , Analgésicos/química , Analgésicos/farmacocinética , Analgésicos/farmacología , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacocinética , Antiinflamatorios/farmacología , Disponibilidad Biológica , Células CHO , Canales de Calcio , Cricetulus , Adyuvante de Freund , Humanos , Hiperalgesia/inducido químicamente , Hiperalgesia/tratamiento farmacológico , Indazoles/farmacocinética , Indazoles/farmacología , Masculino , Ratones Endogámicos C57BL , Planta de la Mostaza , Aceites de Plantas , Ratas Wistar , Especificidad de la Especie , Relación Estructura-Actividad , Canal Catiónico TRPA1 , Canales Catiónicos TRPC/antagonistas & inhibidoresRESUMEN
Imidazopyridine 1 was identified from a phenotypic screen against P. falciparum (Pf) blood stages and subsequently optimized for activity on liver-stage schizonts of the rodent parasite P. yoelii (Py) as well as hypnozoites of the simian parasite P. cynomolgi (Pc). We applied these various assays to the cell-based lead optimization of the imidazopyrazines, exemplified by 3 (KAI407), and show that optimized compounds within the series with improved pharmacokinetic properties achieve causal prophylactic activity in vivo and may have the potential to target the dormant stages of P. vivax malaria.
RESUMEN
On the basis of the initial success of optimization of a novel series of imidazolopiperazines, a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties. These changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies. The optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency, in vitro metabolic stability profile and, as a result, enhanced oral exposure in vivo in mice. The optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.
Asunto(s)
Antimaláricos/farmacología , Imidazoles/farmacología , Malaria Falciparum/tratamiento farmacológico , Piperazinas/farmacología , Plasmodium falciparum/efectos de los fármacos , Animales , Antimaláricos/síntesis química , Antimaláricos/química , Antimaláricos/farmacocinética , Disponibilidad Biológica , Células CACO-2 , Humanos , Imidazoles/síntesis química , Imidazoles/química , Imidazoles/farmacocinética , Malaria Falciparum/parasitología , Ratones , Ratones Endogámicos BALB C , Piperazinas/síntesis química , Piperazinas/química , Piperazinas/farmacocinética , Plasmodium falciparum/metabolismo , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimization of cellular potency against wild-type and drug resistant parasites and improvement of physiochemical and pharmacokinetic properties. The lead compounds in this series showed good potencies in vitro and decent oral exposure levels in vivo. In a Plasmodium berghei mouse infection model, one lead compound lowered the parasitemia level by 99.4% after administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.
Asunto(s)
Antimaláricos/síntesis química , Imidazoles/síntesis química , Piperazinas/síntesis química , Aminoácidos/síntesis química , Aminoácidos/química , Aminoácidos/farmacología , Compuestos de Anilina/síntesis química , Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Animales , Antimaláricos/química , Antimaláricos/farmacología , Derivados del Benceno/síntesis química , Derivados del Benceno/química , Derivados del Benceno/farmacología , Línea Celular , Resistencia a Medicamentos , Femenino , Humanos , Imidazoles/química , Imidazoles/farmacología , Concentración 50 Inhibidora , Malaria/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Piperazinas/química , Piperazinas/farmacología , Plasmodium berghei , Plasmodium falciparum/efectos de los fármacos , Ratas , Relación Estructura-ActividadRESUMEN
Prostasin or human channel-activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d-FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca(+2). Comparison of the structures with each other and with other members of the trypsin-like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca(+2) cations. This induced fit active site provides a new possible mode of regulation of trypsin-like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.
Asunto(s)
Dominio Catalítico , Cationes Bivalentes/metabolismo , Serina Endopeptidasas/química , Aprotinina/metabolismo , Calcio/metabolismo , Quimotripsina/metabolismo , Cristalografía por Rayos X , Ésteres , Gabexato/análogos & derivados , Gabexato/metabolismo , Guanidinas , Humanos , Inhibidores de Proteasas/metabolismo , Conformación Proteica , Alineación de Secuencia , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Especificidad por SustratoRESUMEN
Peptidic, non-covalent inhibitors of lysosomal cysteine protease cathepsin S (1 and 2) were investigated due to low oral bioavailability, leading to an improved series of peptidomimetic inhibitors. Utilizing phenyl succinamides as the P2 residue increased the oral exposure of this lead series of compounds, while retaining selective inhibition of the cathepsin S isoform. Concurrent investigation of the P1 and P2 subsites resulted in the discovery of several potent and selective inhibitors of cathepsin S with good pharmacokinetic properties due to the elimination of saturated aliphatic P2 residues.
Asunto(s)
Amidas/síntesis química , Catepsinas/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , Amidas/química , Amidas/farmacocinética , Amidas/farmacología , Animales , Diseño de Fármacos , Masculino , Estructura Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacocinética , Inhibidores de Proteasas/farmacología , Ratas , Ratas Wistar , Relación Estructura-Actividad , SuccinatosRESUMEN
We report a novel series of noncovalent inhibitors of cathepsin S. The synthesis of the peptidomimetic scaffold is described and structure-activity relationships of P3, P1, and P1' subunits are discussed. Lead optimization to a non-peptidic scaffold has resulted in a new class of potent, highly selective, and orally bioavailable cathepsin S inhibitors.
Asunto(s)
Carbamatos/síntesis química , Carbamatos/farmacología , Catepsinas/antagonistas & inhibidores , Oligopéptidos/síntesis química , Inhibidores de Proteasas/síntesis química , Administración Oral , Animales , Disponibilidad Biológica , Carbamatos/farmacocinética , Humanos , Masculino , Imitación Molecular , Oligopéptidos/farmacología , Inhibidores de Proteasas/farmacocinética , Inhibidores de Proteasas/farmacología , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
A systematic study of anilines led to the discovery of a metabolically robust fluoroindoline replacement for the alkoxy aniline toxicophore in 1. Investigations of the P1 pocket resulted in the discovery of a wide tolerance of functionality leading to the discovery of 11 as a potent and selective inhibitor of cathepsin S.
Asunto(s)
Amidas/farmacología , Catepsinas/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , Diseño de Fármacos , Amidas/síntesis química , Amidas/química , Aminación , Derivados del Benceno/síntesis química , Derivados del Benceno/química , Derivados del Benceno/farmacología , Sitios de Unión , Catepsina K , Catepsina L , Catepsinas/química , Catepsinas/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/química , Etanol/química , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
A series of N(alpha)-2-benzoxazolyl-alpha-amino acid-(arylaminoethyl)amides were identified as potent, selective, and noncovalent inhibitors of cathepsin S. Structure-activity relationships including strategies for modulating the selectivities among cathepsins S, K, and L, and in vivo pharmacokinetics are discussed. A X-ray structure of compound 3 bound to the active site of cathepsin S is also reported.
Asunto(s)
Amidas/farmacología , Catepsinas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos/farmacología , Amidas/química , Animales , Catepsinas/química , Catepsinas/genética , Catepsinas/fisiología , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Compuestos Heterocíclicos/química , Ratones , Ratones Noqueados , Modelos Moleculares , RatasRESUMEN
The synthesis and structure-activity relationship of a series of arylaminoethyl amide cathepsin S inhibitors are reported. Optimization of P3 and P2 groups to improve overall physicochemical properties resulted in significant improvements in oral bioavailability over early lead compounds. An X-ray structure of compound 37 bound to the active site of cathepsin S is also reported.
Asunto(s)
Amidas/síntesis química , Amidas/farmacología , Catepsinas/antagonistas & inhibidores , Administración Oral , Amidas/farmacocinética , Animales , Sitios de Unión , Disponibilidad Biológica , Cristalografía por Rayos X , Éteres Cíclicos/síntesis química , Éteres Cíclicos/farmacología , Humanos , Masculino , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacocinética , Inhibidores de Proteasas/farmacología , Ratas , Ratas Wistar , Relación Estructura-Actividad , ZincRESUMEN
Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1' and P3'. In contrast, the P2' and P4' substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.