RESUMEN
In an effort to identify potent and isoform selective inhibitors of PI3Kδ, GNE-293 (34) was identified. Inhibitor 2 was found to induce micronuclei formation in both the MNT and HCA in vitro assays. Compounds testing negative for genotoxicity were successfully identified through modifications of the 2-benzimidazole substituent and the methylene moiety to disrupt planarity. A variety of heteroatom linkers were explored to examine their effect on potency and isoform selectivity by restricting torsional angles to favor ligand interactions with PI3Kδ's Trp760. These modifications also resulted in an improved in vivo pharmacokinetic profile.
Asunto(s)
Óxidos S-Cíclicos/química , Óxidos S-Cíclicos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Purinas/química , Purinas/farmacología , Animales , Línea Celular , Perros , Humanos , Simulación del Acoplamiento Molecular , Pruebas de Mutagenicidad , Fosfatidilinositol 3-Quinasas/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/toxicidad , Ratas , Relación Estructura-ActividadRESUMEN
A potent inhibitor of PI3Kδ that is ≥ 200 fold selective for the remaining three Class I PI3K isoforms and additional kinases is described. The hypothesis for selectivity is illustrated through structure activity relationships and crystal structures of compounds bound to a K802T mutant of PI3Kγ. Pharmacokinetic data in rats and mice support the use of 3 as a useful tool compound to use for in vivo studies.
Asunto(s)
Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Triptófano/química , Animales , Sitios de Unión , Simulación por Computador , Femenino , Inyecciones Intravenosas , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Mutación , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Innate pattern recognition receptor agonists, including Toll-like receptors (TLRs), alter the tumor microenvironment and prime adaptive antitumor immunity. However, TLR agonists present toxicities associated with widespread immune activation after systemic administration. To design a TLR-based therapeutic suitable for systemic delivery and capable of safely eliciting tumor-targeted responses, we developed immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 dual agonist conjugated to tumor-targeting antibodies. Systemically administered human epidermal growth factor receptor 2 (HER2)-targeted ISACs were well tolerated and triggered a localized immune response in the tumor microenvironment that resulted in tumor clearance and immunological memory. Mechanistically, ISACs required tumor antigen recognition, Fcγ-receptor-dependent phagocytosis and TLR-mediated activation to drive tumor killing by myeloid cells and subsequent T-cell-mediated antitumor immunity. ISAC-mediated immunological memory was not limited to the HER2 ISAC target antigen since ISAC-treated mice were protected from rechallenge with the HER2- parental tumor. These results provide a strong rationale for the clinical development of ISACs.
Asunto(s)
Inmunoterapia , Neoplasias , Inmunidad Adaptativa , Animales , Antígenos de Neoplasias , Inmunoterapia/métodos , Ratones , Neoplasias/tratamiento farmacológico , Microambiente TumoralRESUMEN
Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.
Asunto(s)
Azetidinas/farmacología , Benzamidas/farmacología , Descubrimiento de Drogas , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Sulfonamidas/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Animales , Azetidinas/química , Azetidinas/farmacocinética , Benzamidas/química , Benzamidas/farmacocinética , Células Cultivadas , Células HEK293 , Humanos , Piperidinas/química , Piperidinas/farmacocinética , Piperidinas/farmacología , Ratas Sprague-Dawley , Sulfonamidas/química , Sulfonamidas/farmacocinética , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacocinéticaRESUMEN
Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 Å, revealing the binding site and mechanism of action for this class of antagonists.
Asunto(s)
Asma/tratamiento farmacológico , Furanos/uso terapéutico , Purinas/uso terapéutico , Canal Catiónico TRPA1/antagonistas & inhibidores , Animales , Asma/inducido químicamente , Asma/complicaciones , Células CHO , Cricetulus , Furanos/síntesis química , Furanos/metabolismo , Cobayas , Humanos , Inflamación/tratamiento farmacológico , Inflamación/etiología , Ligandos , Masculino , Estructura Molecular , Ovalbúmina , Oxadiazoles/síntesis química , Oxadiazoles/metabolismo , Oxadiazoles/uso terapéutico , Unión Proteica , Purinas/síntesis química , Purinas/metabolismo , Ratas Sprague-Dawley , Relación Estructura-Actividad , Canal Catiónico TRPA1/metabolismoRESUMEN
Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide Nav1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of Nav1.7. Compound 24 showed a robust PK/PD response in a Nav1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/química , Sulfonamidas/química , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Analgésicos/química , Analgésicos/metabolismo , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Sitios de Unión , Línea Celular , Supervivencia Celular/efectos de los fármacos , Dolor Crónico/tratamiento farmacológico , Dolor Crónico/patología , Perros , Semivida , Humanos , Ligandos , Masculino , Ratones , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Ratas , Relación Estructura-Actividad , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Bloqueadores del Canal de Sodio Activado por Voltaje/metabolismo , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/uso terapéuticoRESUMEN
Herein, we report the discovery and optimization of a series of orally bioavailable acyl sulfonamide NaV1.7 inhibitors that are selective for NaV1.7 over NaV1.5 and highly efficacious in in vivo models of pain and hNaV1.7 target engagement. An analysis of the physicochemical properties of literature NaV1.7 inhibitors suggested that acyl sulfonamides with high fsp3 could overcome some of the pharmacokinetic (PK) and efficacy challenges seen with existing series. Parallel library syntheses lead to the identification of analogue 7, which exhibited moderate potency against NaV1.7 and an acceptable PK profile in rodents, but relatively poor stability in human liver microsomes. Further, design strategy then focused on the optimization of potency against hNaV1.7 and improvement of human metabolic stability, utilizing induced fit docking in our previously disclosed X-ray cocrystal of the NaV1.7 voltage sensing domain. These investigations culminated in the discovery of tool compound 33, one of the most potent and efficacious NaV1.7 inhibitors reported to date.
Asunto(s)
Analgésicos/química , Canal de Sodio Activado por Voltaje NAV1.7/química , Sulfonamidas/química , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Analgésicos/metabolismo , Analgésicos/uso terapéutico , Animales , Sitios de Unión , Diseño de Fármacos , Semivida , Humanos , Masculino , Ratones , Ratones Transgénicos , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Dolor/inducido químicamente , Dolor/tratamiento farmacológico , Dolor/patología , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Sulfonamidas/metabolismo , Sulfonamidas/uso terapéutico , Bloqueadores del Canal de Sodio Activado por Voltaje/metabolismo , Bloqueadores del Canal de Sodio Activado por Voltaje/uso terapéuticoRESUMEN
Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel expressed in sensory neurons where it functions as an irritant sensor for a plethora of electrophilic compounds and is implicated in pain, itch, and respiratory disease. To study its function in various disease contexts, we sought to identify novel, potent, and selective small-molecule TRPA1 antagonists. Herein we describe the evolution of an N-isopropylglycine sulfonamide lead (1) to a novel and potent (4 R,5 S)-4-fluoro-5-methylproline sulfonamide series of inhibitors. Molecular modeling was utilized to derive low-energy three-dimensional conformations to guide ligand design. This effort led to compound 20, which possessed a balanced combination of potency and metabolic stability but poor solubility that ultimately limited in vivo exposure. To improve solubility and in vivo exposure, we developed methylene phosphate prodrug 22, which demonstrated superior oral exposure and robust in vivo target engagement in a rat model of AITC-induced pain.
Asunto(s)
Profármacos/farmacología , Prolina/análogos & derivados , Prolina/farmacología , Sulfonamidas/farmacología , Canal Catiónico TRPA1/antagonistas & inhibidores , Animales , Perros , Descubrimiento de Drogas , Estabilidad de Medicamentos , Humanos , Ligandos , Células de Riñón Canino Madin Darby , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Conformación Molecular , Profármacos/síntesis química , Profármacos/química , Profármacos/farmacocinética , Prolina/síntesis química , Prolina/farmacocinética , Ratas , Solubilidad , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/química , Sulfonamidas/farmacocinética , Canal Catiónico TRPA1/químicaRESUMEN
Selective block of NaV1.7 promises to produce non-narcotic analgesic activity without motor or cognitive impairment. Several NaV1.7-selective blockers have been reported, but efficacy in animal pain models required high multiples of the IC50 for channel block. Here, we report a target engagement assay using transgenic mice that has enabled the development of a second generation of selective Nav1.7 inhibitors that show robust analgesic activity in inflammatory and neuropathic pain models at low multiples of the IC50. Like earlier arylsulfonamides, these newer acylsulfonamides target a binding site on the surface of voltage sensor domain 4 to achieve high selectivity among sodium channel isoforms and steeply state-dependent block. The improved efficacy correlates with very slow dissociation from the target channel. Chronic dosing increases compound potency about 10-fold, possibly due to reversal of sensitization arising during chronic injury, and provides efficacy that persists long after the compound has cleared from plasma.
Asunto(s)
Analgésicos/uso terapéutico , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Neuralgia/tratamiento farmacológico , Bloqueadores de los Canales de Sodio/uso terapéutico , Sulfonamidas/uso terapéutico , Analgésicos/farmacocinética , Animales , Sitios de Unión , Células Cultivadas , Células HEK293 , Humanos , Concentración 50 Inhibidora , Ratones , Canal de Sodio Activado por Voltaje NAV1.7/química , Unión Proteica , Bloqueadores de los Canales de Sodio/farmacocinética , Sulfonamidas/farmacocinéticaRESUMEN
The sodium channel NaV1.7 has emerged as a promising target for the treatment of pain based on strong genetic validation of its role in nociception. In recent years, a number of aryl and acyl sulfonamides have been reported as potent inhibitors of NaV1.7, with high selectivity over the cardiac isoform NaV1.5. Herein, we report on the discovery of a novel series of N-([1,2,4]triazolo[4,3- a]pyridin-3-yl)methanesulfonamides as selective NaV1.7 inhibitors. Starting with the crystal structure of an acyl sulfonamide, we rationalized that cyclization to form a fused heterocycle would improve physicochemical properties, in particular lipophilicity. Our design strategy focused on optimization of potency for block of NaV1.7 and human metabolic stability. Lead compounds 10, 13 (GNE-131), and 25 showed excellent potency, good in vitro metabolic stability, and low in vivo clearance in mouse, rat, and dog. Compound 13 also displayed excellent efficacy in a transgenic mouse model of induced pain.
Asunto(s)
Diseño de Fármacos , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Dolor/tratamiento farmacológico , Sulfonamidas/química , Sulfonamidas/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Secuencia de Aminoácidos , Animales , Perros , Estabilidad de Medicamentos , Humanos , Cinética , Ratones , Conformación Molecular , Dolor/metabolismo , Ratas , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapéutico , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacocinética , Bloqueadores del Canal de Sodio Activado por Voltaje/uso terapéuticoRESUMEN
A novel selective benzoxazepin inhibitor of PI3Kδ has been discovered. Beginning from compound 3, an αPI3K inhibitor, we utilized structure-based drug design and computational analysis of dihedral torsion angles to optimize for PI3Kδ isoform potency and isoform selectivity. Further medicinal chemistry optimization of the series led to the identification of 24, a highly potent and selective inhibitor of PI3Kδ.
RESUMEN
Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been synthesized via intermediate 19 for use in antibody-drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (6), triazole (7), or piperazine (8) link to the PBD. In vitro IC50 values were 11-48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and 0.10-1.73 µg/mL (7 inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5-1 mg/kg, 1 mg/kg, and 3-6 mg/kg for 6, 8, and 7, respectively. Tumor stasis at 2 mg/kg was observed for anti-CD22 6 in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Benzodiazepinas/química , Benzodiazepinas/uso terapéutico , Inmunoconjugados/química , Inmunoconjugados/uso terapéutico , Neoplasias/tratamiento farmacológico , Pirroles/química , Pirroles/uso terapéutico , Animales , Antineoplásicos/farmacología , Benzodiazepinas/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dimerización , Femenino , Humanos , Inmunoconjugados/farmacología , Ratones , Modelos Moleculares , Pirroles/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidoresRESUMEN
We report on a novel series of aryl sulfonamides that act as nanomolar potent, isoform-selective inhibitors of the human sodium channel hNaV1.7. The optimization of these inhibitors is described. We aimed to improve potency against hNaV1.7 while minimizing off-target safety concerns and generated compound 3. This agent displayed significant analgesic effects in rodent models of acute and inflammatory pain and demonstrated that binding to the voltage sensor domain 4 site of NaV1.7 leads to an analgesic effect in vivo. Our findings corroborate the importance of hNaV1.7 as a drug target for the treatment of pain.
RESUMEN
Voltage-gated sodium (Nav) channels propagate action potentials in excitable cells. Accordingly, Nav channels are therapeutic targets for many cardiovascular and neurological disorders. Selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Targeting the human Nav1.7 channel involved in pain perception, we present a protein-engineering strategy that has allowed us to determine crystal structures of a novel receptor site in complex with isoform-selective antagonists. GX-936 and related inhibitors bind to the activated state of voltage-sensor domain IV (VSD4), where their anionic aryl sulfonamide warhead engages the fourth arginine gating charge on the S4 helix. By opposing VSD4 deactivation, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism, likely by stabilizing inactivated states of the channel. Residues from the S2 and S3 helices are key determinants of isoform selectivity, and bound phospholipids implicate the membrane as a modulator of channel function and pharmacology. Our results help to elucidate the molecular basis of voltage sensing and establish structural blueprints to design selective Nav channel antagonists.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/química , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Tiadiazoles/química , Tiadiazoles/farmacología , Secuencia de Aminoácidos , Membrana Celular/química , Cristalización/métodos , Cristalografía por Rayos X , Análisis Mutacional de ADN , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Canal de Sodio Activado por Voltaje NAV1.7/genética , Percepción del Dolor/efectos de los fármacos , Ingeniería de Proteínas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The discovery and optimization of a series of 6,7-dihydro-5H-cyclopenta[d]pyrimidine compounds that are ATP-competitive, selective inhibitors of protein kinase B/Akt is reported. The initial design and optimization was guided by the use of X-ray structures of inhibitors in complex with Akt1 and the closely related protein kinase A. The resulting compounds demonstrate potent inhibition of all three Akt isoforms in biochemical assays and poor inhibition of other members of the cAMP-dependent protein kinase/protein kinase G/protein kinase C extended family and block the phosphorylation of multiple downstream targets of Akt in human cancer cell lines. Biological studies with one such compound, 28 (GDC-0068), demonstrate good oral exposure resulting in dose-dependent pharmacodynamic effects on downstream biomarkers and a robust antitumor response in xenograft models in which the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway is activated. 28 is currently being evaluated in human clinical trials for the treatment of cancer.
Asunto(s)
Adenosina Trifosfato/metabolismo , Unión Competitiva , Descubrimiento de Drogas , Piperazinas/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirimidinas/metabolismo , Pirimidinas/farmacología , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Piperazinas/química , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/química , Pirimidinas/química , Especificidad por SustratoRESUMEN
PI3Kδ is a lipid kinase and a member of a larger family of enzymes, PI3K class IA(α, ß, δ) and IB (γ), which catalyze the phosphorylation of PIP2 to PIP3. PI3Kδ is mainly expressed in leukocytes, where it plays a critical, nonredundant role in B cell receptor mediated signaling and provides an attractive opportunity to treat diseases where B cell activity is essential, e.g., rheumatoid arthritis. We report the discovery of novel, potent, and selective PI3Kδ inhibitors and describe a structural hypothesis for isoform (α, ß, γ) selectivity gained from interactions in the affinity pocket. The critical component of our initial pharmacophore for isoform selectivity was strongly associated with CYP3A4 time-dependent inhibition (TDI). We describe a variety of strategies and methods for monitoring and attenuating TDI. Ultimately, a structure-based design approach was employed to identify a suitable structural replacement for further optimization.