ABSTRACT
Compounds that inhibit glutathione peroxidase 4 (GPX4) hold promise as cancer therapeutics in their ability to induce a form of nonapoptotic cell death called ferroptosis. Our research identified 24, a structural analog of the potent GPX4 inhibitor RSL3, that has much better plasma stability (t1/2 > 5 h in mouse plasma). The bioavailability of 24 provided efficacious plasma drug concentrations with IP dosing, thus enabling in vivo studies to assess tolerability and efficacy. An efficacy study in mouse using a GPX4-sensitive tumor model found that doses of 24 up to 50 mg/kg were tolerated for 20 days but had no effect on tumor growth, although partial target engagement was observed in tumor homogenate.
Subject(s)
Ferroptosis , Neoplasms , Mice , Animals , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Biological AvailabilityABSTRACT
BACKGROUND/AIM: The therapeutic potential of bromodomain and extra-terminal motif (BET) inhibitors in hematological cancers has been well established in preclinical and early-stage clinical trials, although as of yet, no BETtargeting agent has achieved approval. To add insight into potential response to mivebresib (ABBV-075), a broadspectrum BET inhibitor, co-clinical modeling of individual patient biopsies was conducted in the context of a Phase I trial in acute myeloid leukemia (AML). MATERIALS AND METHODS: Co-clinical modeling involves taking the patient's biopsy and implanting it in mice with limited passage so that it closely retains the original characteristics of the malignancy and allows comparisons of response between animal model and clinical data. Procedures were developed, initially with neonate NOD/Shi-scid-IL2rγnull (NOG) mice and then optimized with juvenile NOG-EXL as host mice, eventually resulting in a robust rate of engraftment (16 out of 26, 62%). RESULTS: Results from the co-clinical AML patient-derived xenograft (PDX) modeling (6 with >60% inhibition of bone marrow blasts) were consistent with the equivalent clinical data from patients receiving mivebresib in monotherapy, and in combination with venetoclax. The modeling system also demonstrated the activity of a novel BD2-selective BET inhibitor (ABBV-744) in the preclinical AML setting. Both agents were also highly effective in inhibiting blast counts in the spleen (10/10 and 5/6 models, respectively). CONCLUSION: These findings confirm the validity of the model system in the co-clinical setting, establish highly relevant in vivo models for the discovery of cancer therapy, and indicate the therapeutic value of BET inhibitors for AML and, potentially, myelofibrosis treatment.
Subject(s)
Leukemia, Myeloid, Acute , Pyridones , Animals , Cell Line, Tumor , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Pyridones/pharmacology , Pyridones/therapeutic use , SulfonamidesABSTRACT
The coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a recently emerged human coronavirus. COVID-19 vaccines have proven to be successful in protecting the vaccinated from infection, reducing the severity of disease, and deterring the transmission of infection. However, COVID-19 vaccination faces many challenges, such as the decline in vaccine-induced immunity over time, and the decrease in potency against some SARS-CoV-2 variants including the recently emerged Omicron variant, resulting in breakthrough infections. The challenges that COVID-19 vaccination is facing highlight the importance of the discovery of antivirals to serve as another means to tackle the pandemic. To date, neutralizing antibodies that block viral entry by targeting the viral spike protein make up the largest class of antivirals that has received US FDA emergency use authorization (EUA) for COVID-19 treatment. In addition to the spike protein, other key targets for the discovery of direct-acting antivirals include viral enzymes that are essential for SARS-CoV-2 replication, such as RNA-dependent RNA polymerase and proteases, as judged by US FDA approval for remdesivir, and EUA for Paxlovid (nirmatrelvir + ritonavir) for treating COVID-19 infections. This review presents an overview of the current status and future direction of antiviral drug discovery for treating SARS-CoV-2 infections, covering important antiviral targets such as the viral spike protein, non-structural protein (nsp) 3 papain-like protease, nsp5 main protease, and the nsp12/nsp7/nsp8 RNA-dependent RNA polymerase complex.
Subject(s)
Antiviral Agents , COVID-19 Drug Treatment , Drug Discovery , Antiviral Agents/pharmacology , COVID-19 Vaccines , Coronavirus 3C Proteases/antagonists & inhibitors , Humans , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Viral Proteins/metabolismABSTRACT
With the morbidity and mortality associated with the COVID-19 pandemic that we are witnessing this year, the risks posed by emerging viral diseases to global health are all too obvious. This pandemic highlights the importance of antiviral drug discovery, which targets emerging viral pathogens, as well as existing pathogenic viruses that undergo continuous evolution. Drug discovery and development is a long and resource intensive process; however, the use of biomarkers can accelerate clinical development of antivirals by providing information regarding diagnosis of specific viral infections, status of infection, potential safety parameters, and antiviral responses. In clinical practice, many of the biomarkers initially utilized to support clinical development are also used for patient care. While viral load is a standard and essential biomarker used to detect the desired viral suppression induced by an antiviral agent, it has become apparent that additional biomarkers, whether related to the virus, the host or as a consequence of the drug's mechanistic effects, are also important for monitoring clinical outcomes associated with an antiviral therapy. This review summarizes the biomarkers used in the clinical development (as well as in clinical practice, where appropriate) of antiviral therapies for hepatitis C virus, hepatitis B virus, human immunodeficiency virus, and severe acute respiratory syndrome coronavirus 2.
Subject(s)
Antiviral Agents/therapeutic use , Biomarkers/analysis , Virus Diseases/drug therapy , Animals , Antiviral Agents/pharmacology , COVID-19/virology , Clinical Trials as Topic , Humans , SARS-CoV-2/physiology , COVID-19 Drug TreatmentABSTRACT
Dual bromodomain BET inhibitors that bind with similar affinities to the first and second bromodomains across BRD2, BRD3, BRD4, and BRDT have displayed modest activity as monotherapy in clinical trials. Thrombocytopenia, closely followed by symptoms characteristic of gastrointestinal toxicity, have presented as dose-limiting adverse events that may have prevented escalation to higher dose levels required for more robust efficacy. ABBV-744 is a highly selective inhibitor for the second bromodomain of the four BET family proteins. In contrast to the broad antiproliferative activities observed with dual bromodomain BET inhibitors, ABBV-744 displayed significant antiproliferative activities largely although not exclusively in cancer cell lines derived from acute myeloid leukemia and androgen receptor positive prostate cancer. Studies in acute myeloid leukemia xenograft models demonstrated antitumor efficacy for ABBV-744 that was comparable with the pan-BET inhibitor ABBV-075 but with an improved therapeutic index. Enhanced antitumor efficacy was also observed with the combination of ABBV-744 and the BCL-2 inhibitor, venetoclax compared with monotherapies of either agent alone. These results collectively support the clinical evaluation of ABBV-744 in AML (Clinical Trials.gov identifier: NCT03360006).
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Pyridines/pharmacology , Pyrroles/pharmacology , Sulfonamides/pharmacology , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Cell Proliferation , Drug Therapy, Combination , Female , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
The BET family of proteins consists of BRD2, BRD3, BRD4, and BRDt. Each protein contains two distinct bromodomains (BD1 and BD2). BET family bromodomain inhibitors under clinical development for oncology bind to each of the eight bromodomains with similar affinities. We hypothesized that it may be possible to achieve an improved therapeutic index by selectively targeting subsets of the BET bromodomains. Both BD1 and BD2 are highly conserved across family members (>70% identity), whereas BD1 and BD2 from the same protein exhibit a larger degree of divergence (â¼40% identity), suggesting selectivity between BD1 and BD2 of all family members would be more straightforward to achieve. Exploiting the Asp144/His437 and Ile146/Val439 sequence differences (BRD4 BD1/BD2 numbering) allowed the identification of compound 27 demonstrating greater than 100-fold selectivity for BRD4 BD2 over BRD4 BD1. Further optimization to improve BD2 selectivity and oral bioavailability resulted in the clinical development compound 46 (ABBV-744).
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Drug Discovery/methods , Pyridines/chemistry , Pyridines/metabolism , Pyrroles/chemistry , Pyrroles/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Animals , Female , HeLa Cells , Humans , Mice , Mice, SCID , Protein Domains/drug effects , Protein Domains/physiology , Protein Structure, Secondary , Protein Structure, Tertiary , Pyridines/pharmacology , Pyrroles/pharmacology , Xenograft Model Antitumor Assays/methodsABSTRACT
Proteins of the bromodomain and extra-terminal (BET) domain family are epigenetic readers that bind acetylated histones through their bromodomains to regulate gene transcription. Dual-bromodomain BET inhibitors (DbBi) that bind with similar affinities to the first (BD1) and second (BD2) bromodomains of BRD2, BRD3, BRD4 and BRDt have displayed modest clinical activity in monotherapy cancer trials. A reduced number of thrombocytes in the blood (thrombocytopenia) as well as symptoms of gastrointestinal toxicity are dose-limiting adverse events for some types of DbBi1-5. Given that similar haematological and gastrointestinal defects were observed after genetic silencing of Brd4 in mice6, the platelet and gastrointestinal toxicities may represent on-target activities associated with BET inhibition. The two individual bromodomains in BET family proteins may have distinct functions7-9 and different cellular phenotypes after pharmacological inhibition of one or both bromodomains have been reported10,11, suggesting that selectively targeting one of the bromodomains may result in a different efficacy and tolerability profile compared with DbBi. Available compounds that are selective to individual domains lack sufficient potency and the pharmacokinetics properties that are required for in vivo efficacy and tolerability assessment10-13. Here we carried out a medicinal chemistry campaign that led to the discovery of ABBV-744, a highly potent and selective inhibitor of the BD2 domain of BET family proteins with drug-like properties. In contrast to the broad range of cell growth inhibition induced by DbBi, the antiproliferative activity of ABBV-744 was largely, but not exclusively, restricted to cell lines of acute myeloid leukaemia and prostate cancer that expressed the full-length androgen receptor (AR). ABBV-744 retained robust activity in prostate cancer xenografts, and showed fewer platelet and gastrointestinal toxicities than the DbBi ABBV-07514. Analyses of RNA expression and chromatin immunoprecipitation followed by sequencing revealed that ABBV-744 displaced BRD4 from AR-containing super-enhancers and inhibited AR-dependent transcription, with less impact on global transcription compared with ABBV-075. These results underscore the potential value of selectively targeting the BD2 domain of BET family proteins for cancer therapy.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/chemistry , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Protein Domains/drug effects , Pyridines/pharmacology , Pyrroles/pharmacology , Transcription Factors/antagonists & inhibitors , Transcription Factors/chemistry , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Mice , Pyridines/adverse effects , Pyridines/toxicity , Pyrroles/adverse effects , Pyrroles/toxicity , Rats , Receptors, Androgen/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Novel conformationally constrained BET bromodomain inhibitors have been developed. These inhibitors were optimized in two similar, yet distinct chemical series, the 6-methyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-ones (A) and the 1-methyl-1H-pyrrolo[2,3-c]pyridin-7(6H)-ones (B). Each series demonstrated excellent activity in binding and cellular assays, and lead compounds from each series demonstrated significant efficacy in in vivo tumor xenograft models.
Subject(s)
Nuclear Proteins/antagonists & inhibitors , Pyridones/chemistry , Transcription Factors/antagonists & inhibitors , Animals , Binding Sites , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Drug Evaluation, Preclinical , Half-Life , Humans , Mice , Microsomes/metabolism , Molecular Dynamics Simulation , Multiple Myeloma/drug therapy , Nuclear Proteins/metabolism , Protein Structure, Tertiary , Pyridones/pharmacokinetics , Pyridones/pharmacology , Pyridones/therapeutic use , Structure-Activity Relationship , Transcription Factors/metabolism , Transplantation, HeterologousABSTRACT
Curative interferon and ribavirin sparing treatments for hepatitis C virus (HCV)-infected patients require a combination of mechanistically orthogonal direct acting antivirals. A shared component of these treatments is usually an HCV NS5A inhibitor. First generation FDA approved treatments, including the component NS5A inhibitors, do not exhibit equivalent efficacy against HCV virus genotypes 1-6. In particular, these first generation NS5A inhibitors tend to select for viral drug resistance. Ombitasvir is a first generation HCV NS5A inhibitor included as a key component of Viekira Pak for the treatment of patients with HCV genotype 1 infection. Since the launch of next generation HCV treatments, functional cure for genotype 1-6 HCV infections has been achieved, as well as shortened treatment duration across a wider spectrum of genotypes. In this paper, we show how we have modified the anchor, linker, and end-cap architecture of our NS5A inhibitor design template to discover a next generation NS5A inhibitor pibrentasvir (ABT-530), which exhibits potent inhibition of the replication of wild-type genotype 1-6 HCV replicons, as well as improved activity against replicon variants demonstrating resistance against first generation NS5A inhibitors.
Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Drug Design , Hepacivirus/drug effects , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Animals , Antiviral Agents/pharmacokinetics , Benzimidazoles/pharmacokinetics , Genotype , Hepacivirus/genetics , Hepacivirus/physiology , Mice , Pyrrolidines/pharmacokinetics , Structure-Activity Relationship , Tissue Distribution , Virus Replication/drug effectsABSTRACT
ABT-072 is a non-nucleoside HCV NS5B polymerase inhibitor that was discovered as part of a program to identify new direct-acting antivirals (DAAs) for the treatment of HCV infection. This compound was identified during a medicinal chemistry effort to improve on an original lead, inhibitor 1, which we described in a previous publication. Replacement of the amide linkage in 1 with a trans-olefin resulted in improved compound permeability and solubility and provided much better pharmacokinetic properties in preclinical species. Replacement of the dihydrouracil in 1 with an N-linked uracil provided better potency in the genotype 1 replicon assay. Results from phase 1 clinical studies supported once-daily oral dosing with ABT-072 in HCV infected patients. A phase 2 clinical study that combined ABT-072 with the HCV protease inhibitor ABT-450 provided a sustained virologic response at 24 weeks after dosing (SVR24) in 10 of 11 patients who received treatment.
Subject(s)
Cytosine/analogs & derivatives , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Hepacivirus/enzymology , Stilbenes/chemistry , Sulfonamides/chemical synthesis , Sulfonamides/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Administration, Oral , Biological Availability , Chemistry Techniques, Synthetic , Cytosine/chemical synthesis , Cytosine/chemistry , Cytosine/pharmacokinetics , Cytosine/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Humans , Permeability , Stereoisomerism , Sulfonamides/chemistry , Sulfonamides/pharmacokinetics , Tissue Distribution , Viral Nonstructural Proteins/chemistryABSTRACT
The development of bromodomain and extraterminal domain (BET) bromodomain inhibitors and their examination in clinical studies, particularly in oncology settings, has garnered substantial recent interest. An effort to generate novel BET bromodomain inhibitors with excellent potency and drug metabolism and pharmacokinetics (DMPK) properties was initiated based upon elaboration of a simple pyridone core. Efforts to develop a bidentate interaction with a critical asparagine residue resulted in the incorporation of a pyrrolopyridone core, which improved potency by 9-19-fold. Additional structure-activity relationship (SAR) efforts aimed both at increasing potency and improving pharmacokinetic properties led to the discovery of the clinical candidate 63 (ABBV-075/mivebresib), which demonstrates excellent potency in biochemical and cellular assays, advantageous exposures and half-life both in animal models and in humans, and in vivo efficacy in mouse models of cancer progression and inflammation.
Subject(s)
Drug Discovery , Proteins/antagonists & inhibitors , Pyridones/pharmacology , Sulfonamides/pharmacology , Animals , Cell Line, Tumor , Chromatography, High Pressure Liquid , Fluorescence Resonance Energy Transfer , Half-Life , Humans , Mass Spectrometry , Mice , Proton Magnetic Resonance Spectroscopy , Pyridones/chemistry , Pyridones/pharmacokinetics , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacokineticsABSTRACT
Ten percent to 15% of all lung cancers are small-cell lung cancer (SCLC). SCLC usually grows and metastasizes before it is diagnosed and relapses rapidly upon treatment. Unfortunately, no new targeted agent has been approved in the past 30 years for patients with SCLC. The BET (bromodomain and extraterminal) proteins bind acetylated histones and recruit protein complexes to promote transcription initiation and elongation. BET proteins have been shown to regulate expression of key genes in oncogenesis, such as MYC, CCND2, and BCL2L1 Here, we demonstrate that approximately 50% of SCLC cell lines are exquisitely sensitive to growth inhibition by the BET inhibitor, ABBV-075. The majority of these SCLC cell lines underwent apoptosis in response to ABBV-075 treatment via induction of caspase-3/7 activity. ABBV-075 enhanced the expression of proapoptotic protein BIM and downregulated antiapoptotic proteins BCL2 and BCLxl to a lesser extent. Furthermore, BET inhibition increased BCL2-BIM complex, thus priming the cells for apoptosis. Indeed, strong synergy was observed both in vitro and in vivo when cotreating the cells with BET inhibitor and the BH3-mimetic, BCL2 inhibitor venetoclax (ABT-199). ABBV-075 interaction with venetoclax positively correlated with BCL2 expression. Taken together, our studies provide a rationale for treating SCLC with BET and BCL2 inhibitors in tumors with high BCL2 protein expression. Mol Cancer Ther; 16(8); 1511-20. ©2017 AACR.
Subject(s)
Apoptosis , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Lung Neoplasms/drug therapy , Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Pyridones/therapeutic use , Small Cell Lung Carcinoma/drug therapy , Small Cell Lung Carcinoma/pathology , Sulfonamides/therapeutic use , Animals , Apoptosis/drug effects , Bcl-2-Like Protein 11/metabolism , Biomarkers, Tumor/metabolism , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Female , Humans , Lung Neoplasms/pathology , Mice, SCID , Proto-Oncogene Proteins c-bcl-2/metabolism , Pyridones/pharmacology , Sulfonamides/pharmacology , Treatment Outcome , Xenograft Model Antitumor Assays , bcl-X Protein/metabolismABSTRACT
Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A two-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenylpyridazinone fragment with a weak binding affinity (1, Ki = 160 µM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.
Subject(s)
Macrocyclic Compounds/chemistry , Macrocyclic Compounds/pharmacology , Pyridones/chemistry , Pyridones/pharmacology , Animals , Crystallography, X-Ray , Drug Discovery , Macrocyclic Compounds/pharmacokinetics , Molecular Structure , Pyridones/pharmacokinetics , Rats , Structure-Activity RelationshipABSTRACT
ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered phase I clinical trials. Comprehensive preclinical characterization of ABBV-075 demonstrated broad activity across cell lines and tumor models, representing a variety of hematologic malignancies and solid tumor indications. In most cancer cell lines derived from solid tumors, ABBV-075 triggers prominent G1 cell-cycle arrest without extensive apoptosis. In this study, we show that ABBV-075 efficiently triggers apoptosis in acute myeloid leukemia (AML), non-Hodgkin lymphoma, and multiple myeloma cells. Apoptosis induced by ABBV-075 was mediated in part by modulation of the intrinsic apoptotic pathway, exhibiting synergy with the BCL-2 inhibitor venetoclax in preclinical models of AML. In germinal center diffuse large B-cell lymphoma, BCL-2 levels or venetoclax sensitivity predicted the apoptotic response to ABBV-075 treatment. In vivo combination studies uncovered surprising benefits of low doses of ABBV-075 coupled with bortezomib and azacitidine treatment, despite the lack of in vitro synergy between ABBV-075 and these agents. The in vitro/in vivo activities of ABBV-075 described here may serve as a useful reference to guide the development of ABBV-075 and other BET family inhibitors for cancer therapy. Cancer Res; 77(11); 2976-89. ©2017 AACR.
Subject(s)
Androgen Antagonists/therapeutic use , Pyridones/therapeutic use , Sulfonamides/therapeutic use , Androgen Antagonists/pharmacology , Apoptosis , Cell Line, Tumor , Drug Synergism , Humans , Pyridones/pharmacology , Sulfonamides/pharmacology , TransfectionABSTRACT
An NMR fragment screen for binders to the bromodomains of BRD4 identified 2-methyl-3-ketopyrroles 1 and 2. Elaboration of these fragments guided by structure-based design provided lead molecules with significant activity in a mouse tumor model. Further modifications to the methylpyrrole core provided compounds with improved properties and enhanced activity in a mouse model of multiple myeloma.
Subject(s)
Antineoplastic Agents/chemistry , Nuclear Proteins/antagonists & inhibitors , Pyrroles/chemistry , Transcription Factors/antagonists & inhibitors , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Drug Design , Half-Life , Humans , Mice , Molecular Dynamics Simulation , Multiple Myeloma/drug therapy , Nuclear Proteins/metabolism , Pyrroles/chemical synthesis , Pyrroles/pharmacokinetics , Pyrroles/therapeutic use , Structure-Activity Relationship , Transcription Factors/metabolism , Transplantation, HeterologousABSTRACT
Pibrentasvir (ABT-530) is a novel and pan-genotypic hepatitis C virus (HCV) NS5A inhibitor with 50% effective concentration (EC50) values ranging from 1.4 to 5.0 pM against HCV replicons containing NS5A from genotypes 1 to 6. Pibrentasvir demonstrated similar activity against a panel of chimeric replicons containing HCV NS5A of genotypes 1 to 6 from clinical samples. Resistance selection studies were conducted using HCV replicon cells with NS5A from genotype 1a, 1b, 2a, 2b, 3a, 4a, 5a, or 6a at a concentration of pibrentasvir that was 10- or 100-fold over its EC50 for the respective replicon. With pibrentasvir at 10-fold over the respective EC50, only a small number of colonies (0.00015 to 0.0065% of input cells) with resistance-associated amino acid substitutions were selected in replicons containing genotype 1a, 2a, or 3a NS5A, and no viable colonies were selected in replicons containing NS5A from other genotypes. With pibrentasvir at 100-fold over the respective EC50, very few colonies (0.0002% of input cells) were selected by pibrentasvir in genotype 1a replicon cells while no colonies were selected in other replicons. Pibrentasvir is active against common resistance-conferring substitutions in HCV genotypes 1 to 6 that were identified for other NS5A inhibitors, including those at key amino acid positions 28, 30, 31, or 93. The combination of pibrentasvir with HCV inhibitors of other classes produced synergistic inhibition of HCV replication. In summary, pibrentasvir is a next-generation HCV NS5A inhibitor with potent and pan-genotypic activity, and it maintains activity against common amino acid substitutions of HCV genotypes 1 to 6 that are known to confer resistance to currently approved NS5A inhibitors.
Subject(s)
Antiviral Agents/pharmacology , Benzimidazoles/adverse effects , Benzimidazoles/pharmacology , Hepacivirus/drug effects , Pyrrolidines/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Amino Acid Substitution , Cell Line, Tumor , Drug Resistance, Viral , Hep G2 Cells , Hepacivirus/classification , Hepacivirus/genetics , Hepatitis C/drug therapy , Humans , Pyrrolidines/adverse effects , Viral Nonstructural Proteins/geneticsABSTRACT
Competitive inhibitors of acetyl-lysine binding to the bromodomains of the BET (bromodomain and extra terminal) family are being developed for the treatment of solid and hematologic malignancies. The function of BET family member BRD4 at enhancers/superenhancers has been shown to sustain signal-dependent or pathogenic gene expression programs. Here, the hypothesis was tested that the transcription factor drivers of castration-resistant prostate cancer (CRPC) clinical progression, including the androgen receptor (AR), are critically dependent on BRD4 and thus represent a sensitive solid tumor indication for the BET inhibitor ABBV-075. DHT-stimulated transcription of AR target genes was inhibited by ABBV-075 without significant effect on AR protein expression. Furthermore, ABBV-075 disrupted DHT-stimulated recruitment of BET family member BRD4 to gene-regulatory regions cooccupied by AR, including the well-established PSA and TMPRSS2 enhancers. Persistent BET inhibition disrupted the composition and function of AR-occupied enhancers as measured by a reduction in AR and H3K27Ac ChIP signal and inhibition of enhancer RNA transcription. ABBV-075 displayed potent antiproliferative activity in multiple models of resistance to second-generation antiandrogens and inhibited the activity of the AR splice variant AR-V7 and ligand-binding domain gain-of-function mutations, F877L and L702H. ABBV-075 was also a potent inhibitor of MYC and the TMPRSS2-ETS fusion protein, important parallel transcription factor drivers of CRPC. IMPLICATIONS: The ability of BET family inhibitor ABBV-075 to inhibit transcription activation downstream of the initiating events of transcription factors like AR and TMPRSS2:ETS fusion proteins provides a promising therapeutic option for CRPC patients who have developed resistance to second-generation antiandrogens. Mol Cancer Res; 15(1); 35-44. ©2016 AACR.
Subject(s)
Prostatic Neoplasms, Castration-Resistant/pathology , Pyridones/pharmacology , Sulfonamides/pharmacology , Transcription Factors/antagonists & inhibitors , Androgen Antagonists/pharmacology , Animals , Antineoplastic Agents/pharmacology , Benzamides , Cell Line, Tumor , Cell Proliferation/drug effects , Chromatin/metabolism , Dihydrotestosterone/pharmacology , Drug Resistance, Neoplasm/drug effects , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Inhibitory Concentration 50 , Ligands , Male , Mice , Nitriles , Phenotype , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Prostatic Neoplasms, Castration-Resistant/genetics , Protein Domains , Receptors, Androgen/chemistry , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Signal Transduction/drug effects , Transcription Factors/metabolism , Transcription, Genetic/drug effectsABSTRACT
An increasing number of BET family protein inhibitors have recently entered clinical trials. It has been reported that attempts of monitoring target engagement of the BET bromodomain inhibitor OTX015 using literature-described putative pharmacodynamic markers, such as c-Myc, BRD2, etc., failed to detect pharmacodynamic marker responses in AML patients treated at active dose and those with clinical responses. Here, we report the identification and characterization of HEXIM1 and other genes as robust pharmacodynamic markers for BET inhibitors. Global gene expression profiling studies were carried out using cancer cells and surrogate tissues, such as whole blood and skin, to identify genes that are modulated by BET family proteins. Candidate markers were further characterized for concentration- and time-dependent responses to the BET inhibitor ABBV-075 in vitro and in vivo HEXIM1 was found to be the only gene that exhibited robust and consistent modulation by BET inhibitors across multiple cancer indications and surrogate tissues. Markers such as SERPINI1, ZCCHC24, and ZMYND8 were modulated by ABBV-075 and other BET inhibitors across cancer cell lines and xenograft tumors but not in blood and skin. Significant downregulation of c-Myc, a well-publicized target of BET inhibitors, was largely restricted to hematologic cancer cell lines. Incorporating well-characterized pharmacodynamic markers, such as HEXIM1 and other genes described here, can provide a better understanding of potential efficacy and toxicity associated with inhibiting BET family proteins and informs early clinical decisions on BET inhibitor development programs. Mol Cancer Ther; 16(2); 388-96. ©2016 AACR.
Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers , Neoplasms/genetics , Nuclear Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , Animals , Antineoplastic Agents/pharmacokinetics , Biopsy , Cell Line, Tumor , Cluster Analysis , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Organ Specificity/genetics , RNA-Binding Proteins/metabolism , Transcription Factors , Xenograft Model Antitumor AssaysABSTRACT
Research toward a next-generation HCV NS5A inhibitor has identified fluorobenzimidazole analogs that demonstrate potent, broad-genotype in vitro activity against HCV genotypes 1-6 replicons as well as HCV NS5A variants that are orders of magnitude less susceptible to inhibition by first-generation NS5A inhibitors in comparison to wild-type replicons. The fluorobenzimidazole inhibitors have improved pharmacokinetic properties in comparison to non-fluorinated benzimidazole analogs. Discovery of these inhibitors was facilitated by exploring SAR in a structurally simplified inhibitor series.
Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Hepacivirus/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/pharmacokinetics , Benzimidazoles/pharmacokinetics , Dogs , Genotype , Halogenation , Hepacivirus/genetics , Hepacivirus/metabolism , Hepatitis C/drug therapy , Humans , Mice , Rats , Replicon/drug effects , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
Dasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC50) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC50s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC50 resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections.