RESUMEN
Recurrent somatic mutations in the BRG1/BRM-associated factor (BAF) chromatin remodeling complex subunit ARID1A occur frequently in advanced urothelial, endometrial, and ovarian clear cell carcinomas, creating an alternative chromatin state that may be exploited therapeutically. The histone methyltransferase EZH2 has been previously identified as targetable vulnerability in the context of ARID1A mutations. In this study, we describe the discovery of tulmimetostat, an orally available, clinical stage EZH2 inhibitor, and it elucidates the aspects of its application potential in ARID1A mutant tumors. Tulmimetostat administration achieved efficacy in multiple ARID1A mutant bladder, ovarian, and endometrial tumor models and improved cisplatin response in chemotherapy-resistant models. Consistent with its comprehensive and durable level of target coverage, tulmimetostat demonstrated greater efficacy than other PRC2-targeted inhibitors at comparable or lower exposures in a bladder cancer xenograft mouse model. Tulmimetostat mediated extensive changes in gene expression, in addition to a profound reduction in global H3K27me3 levels in tumors. Phase I clinical pharmacokinetic and pharmacodynamic data indicated that tulmimetostat exhibits durable exposure and profound target engagement. Importantly, a tulmimetostat controlled gene expression signature identified in whole blood from a cohort of 32 patients with cancer correlated with tulmimetostat exposure, representing a pharmacodynamic marker for the assessment of target coverage for PRC2-targeted agents in the clinic. Collectively, these data suggest that tulmimetostat has the potential to achieve clinical benefit in solid tumors as a monotherapy but also in combination with chemotherapeutic agents, and may be beneficial in various indications with recurrent ARID1A mutations. Significance: The EZH2 inhibitor tulmimetostat achieves comprehensive target inhibition in ARID1A mutant solid tumor models and cancer patients that can be assessed with a pharmacodynamic gene signature in peripheral blood.
Asunto(s)
Proteínas de Unión al ADN , Proteína Potenciadora del Homólogo Zeste 2 , Mutación , Factores de Transcripción , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Animales , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Ratones , Factores de Transcripción/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Femenino , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Línea Celular Tumoral , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismoRESUMEN
Potent and highly selective small-molecule inhibitors are needed to unravel the biological complexities of histone methyltransferases and to reveal their therapeutic potential. A prerequisite to developing these inhibitors is the identification of validated chemical matter for initiating a medicinal chemistry campaign. For the most part, finding these initial starting points occurs through screening of large, unbiased compound libraries. The size and nature of these libraries, coupled with the complexities of the bisubstrate utilizing histone methyltransferases, necessitates that the primary screen and subsequent hit triage be carefully considered.In this chapter, using EZH2 as a representative example, we describe a screening and hit triage campaign that identified validated chemical matter allowing initiation of medicinal chemistry studies. Moreover, we discuss a cell-based assay to support lead identification and optimization. The approach described here entailing a mixture of biochemical, biophysical and cell-based assays should be applicable to identifying validated starting points for other histone methyltransferases.
Asunto(s)
Inhibidores Enzimáticos , Metiltransferasas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Histona MetiltransferasasRESUMEN
The histone methyltransferase EZH2 has been the target of numerous small-molecule inhibitor discovery efforts over the last 10+ years. Emerging clinical data have provided early evidence for single agent activity with acceptable safety profiles for first-generation inhibitors. We have developed kinetic methodologies for studying EZH2-inhibitor-binding kinetics that have allowed us to identify a unique structural modification that results in significant increases in the drug-target residence times of all EZH2 inhibitor scaffolds we have studied. The unexpected residence time enhancement bestowed by this modification has enabled us to create a series of second-generation EZH2 inhibitors with sub-pM binding affinities. We provide both biophysical evidence validating this sub-pM potency and biological evidence demonstrating the utility and relevance of such high-affinity interactions with EZH2.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Descubrimiento de Drogas , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Células HeLa , Humanos , Ratones SCID , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Histone methyltransferase EZH2, which is the catalytic subunit of the PRC2 complex, catalyzes the methylation of histone H3K27-a transcriptionally repressive post-translational modification (PTM). EZH2 is commonly mutated in hematologic malignancies and frequently overexpressed in solid tumors, where its expression level often correlates with poor prognosis. First generation EZH2 inhibitors are beginning to show clinical benefit, and we believe that a second generation EZH2 inhibitor could further build upon this foundation to fully realize the therapeutic potential of EZH2 inhibition. During our medicinal chemistry campaign, we identified 4-thiomethyl pyridone as a key modification that led to significantly increased potency and prolonged residence time. Leveraging this finding, we optimized a series of EZH2 inhibitors, with enhanced antitumor activity and improved physiochemical properties, which have the potential to expand the clinical use of EZH2 inhibition.
RESUMEN
Leveraging the catalytic machinery of LSD1 (KDM1A), a series of covalent styrenylcyclopropane LSD1 inhibitors were identified. These inhibitors represent a new class of mechanism-based inhibitors that target and covalently label the FAD cofactor of LSD1. The series was rapidly progressed to potent biochemical and cellular LSD1 inhibitors with good physical properties. This effort resulted in the identification of 34, a highly potent (<4 nM biochemical, 2 nM cell, and 1 nM GI50), and selective LSD1 inhibitor. In-depth kinetic profiling of 34 confirmed its covalent mechanism of action, validated the styrenylcyclopropane as an FAD-directed warhead, and demonstrated that the potency of this inhibitor is driven by improved non-covalent binding (K I). 34 demonstrated robust cell-killing activity in a panel of AML cell lines and robust antitumor activity in a Kasumi-1 xenograft model of AML when dosed orally at 1.5 mg/kg once daily.
RESUMEN
The histone acetyltransferases, CREB binding protein (CBP) and EP300, are master transcriptional co-regulators that have been implicated in numerous diseases, such as cancer, inflammatory disorders, and neurodegeneration. A novel, highly potent, orally bioavailable EP300/CBP histone acetyltransferase (HAT) inhibitor, CPI-1612 or 17, was developed from the lead compound 3. Replacement of the indole scaffold of 3 with the aminopyridine scaffold of 17 led to improvements in potency, solubility, and bioavailability. These characteristics resulted in a 20-fold lower efficacious dose for 17 relative to lead 3 in a JEKO-1 tumor mouse xenograft study.
RESUMEN
The biological function of bromodomains, epigenetic readers of acetylated lysine residues, remains largely unknown. Herein we report our efforts to discover a potent and selective inhibitor of the bromodomain of cat eye syndrome chromosome region candidate 2 (CECR2). Screening of our internal medicinal chemistry collection led to the identification of a pyrrolopyridone chemical lead, and subsequent structure-based drug design led to a potent and selective CECR2 bromodomain inhibitor (GNE-886) suitable for use as an in vitro tool compound.
RESUMEN
Polycomb repressive complex 2 (PRC2) has been shown to play a major role in transcriptional silencing in part by installing methylation marks on lysine 27 of histone 3. Dysregulation of PRC2 function correlates with certain malignancies and poor prognosis. EZH2 is the catalytic engine of the PRC2 complex and thus represents a key candidate oncology target for pharmacological intervention. Here we report the optimization of our indole-based EZH2 inhibitor series that led to the identification of CPI-1205, a highly potent (biochemical IC50 = 0.002 µM, cellular EC50 = 0.032 µM) and selective inhibitor of EZH2. This compound demonstrates robust antitumor effects in a Karpas-422 xenograft model when dosed at 160 mg/kg BID and is currently in Phase I clinical trials. Additionally, we disclose the co-crystal structure of our inhibitor series bound to the human PRC2 complex.
Asunto(s)
Antineoplásicos/farmacología , Ensayos Clínicos Fase I como Asunto , Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Indoles/farmacología , Linfoma de Células B/tratamiento farmacológico , Piperidinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Indoles/síntesis química , Indoles/química , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Piperidinas/síntesis química , Piperidinas/química , Ratas , Relación Estructura-ActividadRESUMEN
Features from a high throughput screening (HTS) hit and a previously reported scaffold were combined to generate 1,7-naphthyridones as novel KDM5 enzyme inhibitors with nanomolar potencies. These molecules exhibited high selectivity over the related KDM4C and KDM2B isoforms. An X-ray co-crystal structure of a representative molecule bound to KDM5A showed that these inhibitors are competitive with the co-substrate (2-oxoglutarate or 2-OG).
Asunto(s)
Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Naftiridinas/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Proteína 2 de Unión a Retinoblastoma/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Perros , Diseño de Fármacos , Humanos , Células de Riñón Canino Madin Darby , Naftiridinas/química , Relación Estructura-ActividadRESUMEN
This communication describes the identification and optimization of a series of pan-KDM5 inhibitors derived from compound 1, a hit initially identified against KDM4C. Compound 1 was optimized to afford compound 20, a 10nM inhibitor of KDM5A. Compound 20 is highly selective for the KDM5 enzymes versus other histone lysine demethylases and demonstrates activity in a cellular assay measuring the increase in global histone 3 lysine 4 tri-methylation (H3K4me3). In addition compound 20 has good ADME properties, excellent mouse PK, and is a suitable starting point for further optimization.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteína 2 de Unión a Retinoblastoma/antagonistas & inhibidores , Animales , Sitios de Unión , Western Blotting , Línea Celular , Descubrimiento de Drogas , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Humanos , Concentración 50 Inhibidora , Ratones , Microsomas Hepáticos/enzimología , Modelos Moleculares , RatasRESUMEN
The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteína 2 de Unión a Retinoblastoma/antagonistas & inhibidores , Antineoplásicos/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Proteína 2 de Unión a Retinoblastoma/metabolismo , Relación Estructura-ActividadRESUMEN
CBP and EP300 are highly homologous, bromodomain-containing transcription coactivators involved in numerous cellular pathways relevant to oncology. As part of our effort to explore the potential therapeutic implications of selectively targeting bromodomains, we set out to identify a CBP/EP300 bromodomain inhibitor that was potent both in vitro and in cellular target engagement assays and was selective over the other members of the bromodomain family. Reported here is a series of cell-potent and selective probes of the CBP/EP300 bromodomains, derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepin-2-one.
RESUMEN
Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.
Asunto(s)
Proteína de Unión a CREB/antagonistas & inhibidores , Proteína p300 Asociada a E1A/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Acetilación/efectos de los fármacos , Proteína de Unión a CREB/química , Proteína de Unión a CREB/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Proteína p300 Asociada a E1A/química , Proteína p300 Asociada a E1A/metabolismo , Factores de Transcripción Forkhead/metabolismo , Histonas/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína/efectos de los fármacos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Transcriptoma/efectos de los fármacosRESUMEN
Inhibition of the bromodomains of the BET family, of which BRD4 is a member, has been shown to decrease myc and interleukin (IL) 6 in vivo, markers that are of therapeutic relevance to cancer and inflammatory disease, respectively. Herein we report substituted benzo[b]isoxazolo[4,5-d]azepines and benzotriazolo[4,3-d][1,4]diazepines as fragment-derived novel inhibitors of the bromodomain of BRD4. Compounds from these series were potent and selective in cells, and subsequent optimization of microsomal stability yielded representatives that demonstrated dose- and time-dependent reduction of plasma IL-6 in mice.
RESUMEN
In recent years, inhibition of the interaction between the bromodomain and extra-terminal domain (BET) family of chromatin adaptors and acetyl-lysine residues on chromatin has emerged as a promising approach to regulate the expression of important disease-relevant genes, including MYC, BCL-2, and NF-κB. Here we describe the identification and characterization of a potent and selective benzoisoxazoloazepine BET bromodomain inhibitor that attenuates BET-dependent gene expression in vivo, demonstrates antitumor efficacy in an MV-4-11 mouse xenograft model, and is currently undergoing human clinical trials for hematological malignancies (CPI-0610).
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Azepinas/química , Azepinas/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Azepinas/farmacocinética , Azepinas/farmacología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Perros , Genes myc/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Ratas , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The discovery and optimization of a series of small molecule EZH2 inhibitors is described. Starting from dimethylpyridone HTS hit (2), a series of indole-based EZH2 inhibitors were identified. Biochemical potency and microsomal stability were optimized during these studies and afforded compound 22. This compound demonstrates nanomolar levels of biochemical potency (IC50=0.002 µM), cellular potency (EC50=0.080 µM), and afforded tumor regression when dosed (200 mpk SC BID) in an EZH2 dependent tumor xenograft model.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Indoles/química , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Línea Celular Tumoral , Técnicas de Química Sintética , Diseño de Fármacos , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Medicamentos , Proteína Potenciadora del Homólogo Zeste 2 , Células HeLa/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Ratones , Terapia Molecular Dirigida/métodos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The histone lysine methyltransferase (MT) Enhancer of Zeste Homolog 2 (EZH2) is considered an oncogenic driver in a subset of germinal center B-cell-like diffuse large B cell lymphoma (GCB-DLBCL) and follicular lymphoma due to the presence of recurrent, monoallelic mutations in the EZH2 catalytic domain. These genomic data suggest that targeting the EZH2 MT activity is a valid therapeutic strategy for the treatment of lymphoma patients with EZH2 mutations. Here we report the identification of highly potent and selective EZH2 small molecule inhibitors, their validation by a cellular thermal shift assay, application across a large cell panel representing various non-Hodgkin's lymphoma (NHL) subtypes, and their efficacy in EZH2mutant-containing GCB-DLBCL xenograft models. Surprisingly, our EZH2 inhibitors selectively affect the turnover of trimethylated, but not monomethylated histone H3 lysine 27 at pharmacologically relevant doses. Importantly, we find that these inhibitors are broadly efficacious also in NHL models with wild-type EZH2.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/toxicidad , Histonas/metabolismo , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/toxicidad , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Histonas/química , Humanos , Cinética , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/patología , Linfoma no Hodgkin/metabolismo , Linfoma no Hodgkin/patología , Metilación , Ratones , Ratones Desnudos , Mutación , Péptidos/análisis , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Trasplante HeterólogoAsunto(s)
Descubrimiento de Drogas/métodos , Epigenómica/métodos , Animales , Diseño de Fármacos , HumanosRESUMEN
Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.
Asunto(s)
LDL-Colesterol/sangre , Fragmentos Fab de Inmunoglobulinas/farmacología , Receptores de LDL/química , Serina Endopeptidasas/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Fluoroinmunoensayo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Macaca mulatta , Masculino , Ratones , Ratones Transgénicos , Proproteína Convertasa 9 , Proproteína Convertasas , Receptores de LDL/metabolismo , Serina Endopeptidasas/químicaRESUMEN
PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.