RESUMEN
Polycomb Repressive Complex 2 (PRC2) plays an important role in transcriptional regulation during animal development and in cell differentiation, and alteration of PRC2 activity has been associated with cancer. On a molecular level, PRC2 catalyzes methylation of histone H3 lysine 27 (H3K27), resulting in mono-, di-, or trimethylated forms of H3K27, of which the trimethylated form H3K27me3 leads to transcriptional repression of polycomb target genes. Previously, we have shown that binding of the low-molecular-weight compound EED226 to the H3K27me3 binding pocket of the regulatory subunit EED can effectively inhibit PRC2 activity in cells and reduce tumor growth in mouse xenograft models. Here, we report the stepwise optimization of the tool compound EED226 toward the potent and selective EED inhibitor MAK683 (compound 22) and its subsequent preclinical characterization. Based on a balanced PK/PD profile, efficacy, and mitigated risk of forming reactive metabolites, MAK683 has been selected for clinical development.
Asunto(s)
Histonas , Neoplasias , Animales , Inhibidores Enzimáticos , Histonas/metabolismo , Humanos , Metilación , Ratones , Neoplasias/tratamiento farmacológico , Complejo Represivo Polycomb 2RESUMEN
SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein-ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivo antitumor activity. These studies culminated in the discovery of TNO155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias/enzimología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Antineoplásicos/uso terapéutico , Perros , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Macaca fascicularis , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Ratas , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Protein tyrosine phosphatase SHP2 is an oncoprotein associated with cancer as well as a potential immune modulator because of its role in the programmed cell death PD-L1/PD-1 pathway. In the preceding manuscript, we described the optimization of a fused, bicyclic screening hit for potency, selectivity, and physicochemical properties in order to further expand the chemical diversity of allosteric SHP2 inhibitors. In this manuscript, we describe the further expansion of our approach, morphing the fused, bicyclic system into a novel monocyclic pyrimidinone scaffold through our understanding of SAR and use of structure-based design. These studies led to the identification of SHP394 (1), an orally efficacious inhibitor of SHP2, with high lipophilic efficiency, improved potency, and enhanced pharmacokinetic properties. We also report other pyrimidinone analogues with favorable pharmacokinetic and potency profiles. Overall, this work improves upon our previously described allosteric inhibitors and exemplifies and extends the range of permissible chemical templates that inhibit SHP2 via the allosteric mechanism.
Asunto(s)
Aminopiridinas/uso terapéutico , Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Neoplasias/tratamiento farmacológico , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Pirimidinonas/uso terapéutico , Administración Oral , Regulación Alostérica , Sitio Alostérico , Aminopiridinas/síntesis química , Aminopiridinas/farmacocinética , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Estructura Molecular , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Pirimidinonas/síntesis química , Pirimidinonas/farmacocinética , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
SHP2 is a nonreceptor protein tyrosine phosphatase within the mitogen-activated protein kinase (MAPK) pathway controlling cell growth, differentiation, and oncogenic transformation. SHP2 also participates in the programed cell death pathway (PD-1/PD-L1) governing immune surveillance. Small-molecule inhibition of SHP2 has been widely investigated, including in our previous reports describing SHP099 (2), which binds to a tunnel-like allosteric binding site. To broaden our approach to allosteric inhibition of SHP2, we conducted additional hit finding, evaluation, and structure-based scaffold morphing. These studies, reported here in the first of two papers, led to the identification of multiple 5,6-fused bicyclic scaffolds that bind to the same allosteric tunnel as 2. We demonstrate the structural diversity permitted by the tunnel pharmacophore and culminated in the identification of pyrazolopyrimidinones (e.g., SHP389, 1) that modulate MAPK signaling in vivo. These studies also served as the basis for further scaffold morphing and optimization, detailed in the following manuscript.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos con 2 Anillos/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinonas/farmacología , Regulación Alostérica , Sitio Alostérico , Animales , Línea Celular Tumoral , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Compuestos Heterocíclicos con 2 Anillos/síntesis química , Compuestos Heterocíclicos con 2 Anillos/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Pirazoles/síntesis química , Pirazoles/metabolismo , Pirimidinonas/síntesis química , Pirimidinonas/metabolismo , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Mutant isocitrate dehydrogenase 1 (IDH1) is an attractive therapeutic target for the treatment of various cancers such as AML, glioma, and glioblastoma. We have evaluated 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors that bind to an allosteric, induced pocket of IDH1R132H. This Letter describes SAR exploration focused on improving both the in vitro and in vivo metabolic stability of the compounds, leading to the identification of 19 as a potent and selective mutant IDH1 inhibitor that has demonstrated brain penetration and excellent oral bioavailability in rodents. In a preclinical patient-derived IDH1 mutant xenograft tumor model study, 19 efficiently inhibited the production of the biomarker 2-HG.
RESUMEN
SHP2 is a cytoplasmic protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell proliferation, differentiation, and survival. Recently, we reported an allosteric mechanism of inhibition that stabilizes the auto-inhibited conformation of SHP2. SHP099 (1) was identified and characterized as a moderately potent, orally bioavailable, allosteric small molecule inhibitor, which binds to a tunnel-like pocket formed by the confluence of three domains of SHP2. In this report, we describe further screening strategies that enabled the identification of a second, distinct small molecule allosteric site. SHP244 (2) was identified as a weak inhibitor of SHP2 with modest thermal stabilization of the enzyme. X-ray crystallography revealed that 2 binds and stabilizes the inactive, closed conformation of SHP2, at a distinct, previously unexplored binding site-a cleft formed at the interface of the N-terminal SH2 and PTP domains. Derivatization of 2 using structure-based design resulted in an increase in SHP2 thermal stabilization, biochemical inhibition, and subsequent MAPK pathway modulation. Downregulation of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520 cancer cells. Remarkably, simultaneous occupation of both allosteric sites by 1 and 2 was possible, as characterized by cooperative biochemical inhibition experiments and X-ray crystallography. Combining an allosteric site 1 inhibitor with an allosteric site 2 inhibitor led to enhanced pharmacological pathway inhibition in cells. This work illustrates a rare example of dual allosteric targeted protein inhibition, demonstrates screening methodology and tactics to identify allosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies.
Asunto(s)
Regulación Alostérica , Sitio Alostérico , Piperidinas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Pirimidinas/farmacología , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias/tratamiento farmacológico , Conformación Proteica , Estabilidad ProteicaRESUMEN
CRA-026440 is a novel, broad-spectrum, hydroxamic acid-based inhibitor of histone deacetylase (HDAC) that shows antitumor and antiangiogenic activities in vitro and in vivo preclinically. CRA-026440 inhibited pure recombinant isozymes HDAC1, HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-026440 resulted in the accumulation of acetylated histone and acetylated tubulin, leading to an inhibition of tumor cell growth and the induction of apoptosis. CRA-026440 inhibited ex vivo angiogenesis in a dose-dependent manner. CRA-026440 parenterally given to mice harboring HCT116 or U937 human tumor xenografts resulted in a statistically significant reduction in tumor growth. CRA-026440, when used in combination with Avastin, achieved greater preclinical efficacy in HCT 116 colorectal tumor model. Inhibition of tumor growth was accompanied by an increase in the acetylation of alpha-tubulin in peripheral blood mononuclear cells and an alteration in the expression of many genes in the tumors, including several involved in angiogenesis, apoptosis, and cell growth. These results reveal CRA-026440 to be a novel HDAC inhibitor with potent antitumor activity.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Neoplasias/enzimología , Acetilación , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacocinética , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Apoptosis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Femenino , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Histonas/efectos de los fármacos , Humanos , Ácidos Hidroxámicos/química , Indoles/química , Ratones , Ratones Endogámicos BALB C , Neoplasias/irrigación sanguínea , Neoplasias/genética , Poli Adenosina Difosfato Ribosa/efectos adversos , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PRC2 is a multisubunit methyltransferase involved in epigenetic regulation of early embryonic development and cell growth. The catalytic subunit EZH2 methylates primarily lysine 27 of histone H3, leading to chromatin compaction and repression of tumor suppressor genes. Inhibiting this activity by small molecules targeting EZH2 was shown to result in antitumor efficacy. Here, we describe the optimization of a chemical series representing a new class of PRC2 inhibitors which acts allosterically via the trimethyllysine pocket of the noncatalytic EED subunit. Deconstruction of a larger and complex screening hit to a simple fragment-sized molecule followed by structure-guided regrowth and careful property modulation were employed to yield compounds which achieve submicromolar inhibition in functional assays and cellular activity. The resulting molecules can serve as a simplified entry point for lead optimization and can be utilized to study this new mechanism of PRC2 inhibition and the associated biology in detail.
Asunto(s)
Inhibidores Enzimáticos/química , Epigénesis Genética , Metiltransferasas/antagonistas & inhibidores , Complejo Represivo Polycomb 2/química , Regulación Alostérica , Células CACO-2 , Cromatografía Liquida , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Espectrometría de Masas , Estructura Molecular , Espectroscopía de Protones por Resonancia Magnética , Relación Estructura-ActividadRESUMEN
Inhibition of mutant IDH1 is being evaluated clinically as a promising treatment option for various cancers with hotspot mutation at Arg132. Having identified an allosteric, induced pocket of IDH1R132H, we have explored 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors for in vivo modulation of 2-HG production and potential brain penetration. We report here optimization efforts toward the identification of clinical candidate IDH305 (13), a potent and selective mutant IDH1 inhibitor that has demonstrated brain exposure in rodents. Preclinical characterization of this compound exhibited in vivo correlation of 2-HG reduction and efficacy in a patient-derived IDH1 mutant xenograft tumor model. IDH305 (13) has progressed into human clinical trials for the treatment of cancers with IDH1 mutation.
RESUMEN
An extensive structural manifold of short hydrogen bond-mediated, active site-directed, serine protease inhibition motifs is revealed in a set of over 300 crystal structures involving a large suite of small molecule inhibitors (2-(2-phenol)-indoles and 2-(2-phenol)-benzimidazoles) determined over a wide range of pH (3.5-11.4). The active site hydrogen-bonding mode was found to vary markedly with pH, with the steric and electronic properties of the inhibitor, and with the type of protease (trypsin, thrombin or urokinase type plasminogen activator (uPA)). The pH dependence of the active site hydrogen-bonding motif is often intricate, constituting a distinct fingerprint of each complex. Isosteric replacements or minor substitutions within the inhibitor that modulate the pK(a) of the phenol hydroxyl involved in short hydrogen bonding, or that affect steric interactions distal to the active site, can significantly shift the pH-dependent structural profile characteristic of the parent scaffold, or produce active site-binding motifs unique to the bound analog. Ionization equilibria at the active site associated with inhibitor binding are probed in a series of the protease-inhibitor complexes through analysis of the pH dependence of the structure and environment of the active site-binding groups involved in short hydrogen bond arrays. Structures determined at high pH (>11), suggest that the pK(a) of His57 is dramatically elevated, to a value as high as approximately 11 in certain complexes. K(i) values involving uPA and trypsin determined as a function of pH for a set of inhibitors show pronounced parabolic pH dependence, the pH for optimal inhibition governed by the pK(a) of the inhibitor phenol involved in short hydrogen bonds. Comparison of structures of trypsin, thrombin and uPA, each bound by the same inhibitor, highlights important structural variations in the S1 and active sites accessible for engineering notable selectivity into remarkably small molecules with low nanomolar K(i) values.
Asunto(s)
Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Trombina/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Animales , Bencimidazoles/química , Bencimidazoles/farmacología , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Conformación Proteica , Electricidad Estática , Relación Estructura-Actividad , Trombina/química , Tripsina/química , Inhibidores de Tripsina/química , Inhibidores de Tripsina/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/químicaRESUMEN
[reaction: see text] A new approach to the synthesis of the C ring subunit of known and potential bryostatin analogues is described. The convergent approach, illustrated above, requires fewer steps and offers greater flexibility in rapidly accessing diverse C ring analogues.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/síntesis química , Lactonas/química , Lactonas/síntesis química , Brioestatinas , Macrólidos , Estructura MolecularRESUMEN
We report the discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(4-methylphenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (2a) as an inducer of apoptosis using our proprietary cell- and caspase-based HTS assay. Through structure activity relationship (SAR) studies, 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(2-methoxy-4-(methylthio)phenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (5d) was identified as a potent apoptosis inducer with an EC(50) value of 0.08 microM in T47D cells, which was >15-fold more potent than screening hit 2a. Compound 5d also was found to be highly active in a growth inhibition assay with a GI(50) value of 0.05 microM in T47D cells and to function as an inhibitor of tubulin polymerization.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Caspasas/metabolismo , Química Farmacéutica/métodos , Neoplasias/tratamiento farmacológico , Tiazepinas/química , Línea Celular Tumoral , Proliferación Celular , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Modelos Químicos , Relación Estructura-Actividad , Tiazepinas/farmacología , Tubulina (Proteína)/químicaRESUMEN
Novel analogs of (-)-saframycin A are described. The analogs are shown to be potent inhibitors of the in vitro growth of several tumor cells in a broad panel and promising as leads for further optimization. The first in vivo studies in a solid tumor model (HCT-116) reveal potent antitumor activity with associated toxicity of daily administration.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Isoquinolinas/síntesis química , Isoquinolinas/química , Isoquinolinas/farmacocinética , Isoquinolinas/farmacología , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The rational design and synthesis of beta-amino-alpha-hydroxy amide derivatives as reversible inhibitors of methionine aminopeptidase-2 (MetAP2) with anti-proliferative activity against human umbilical vein endothelial cells (HUVECs) is described.
Asunto(s)
Amidas/síntesis química , Aminopeptidasas/antagonistas & inhibidores , Diseño de Fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Glicoproteínas/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , Amidas/farmacología , Aminopeptidasas/metabolismo , Animales , Bovinos , Glicoproteínas/metabolismo , Humanos , Metionil Aminopeptidasas , Inhibidores de Proteasas/farmacologíaRESUMEN
The structure-based design of potent and selective urokinase-type plasminogen activator (uPA) inhibitors with 4-aminoarylamidine or 4-aminoarylguanidine S1 binding groups, is described.
Asunto(s)
Benzamidinas/química , Benzamidinas/farmacología , Guanidina/análogos & derivados , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Aminas/química , Aminas/farmacología , Benzamidinas/síntesis química , Sitios de Unión , Cristalografía por Rayos X , Guanidina/síntesis química , Guanidina/farmacología , Modelos Moleculares , Unión Proteica , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Inhibidores de Serina Proteinasa/síntesis química , Relación Estructura-Actividad , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
The development of potent and selective urokinase-type plasminogen activator (uPA) inhibitors based on the lead molecule 2-(2-hydroxy-3-ethoxyphenyl)-1H-benzimidazole-5-carboxamidine (3a) is described.