Discovery of KB-0742, a Potent, Selective, Orally Bioavailable Small Molecule Inhibitor of CDK9 for MYC-Dependent Cancers.

Transcriptional deregulation is a hallmark of many cancers and is exemplified by genomic amplifications of the MYC family of oncogenes, which occur in at least 20% of all solid tumors in adults. Targeting of transcriptional cofactors and the transcriptional cyclin-dependent kinase (CDK9) has emerged as a therapeutic strategy to interdict deregulated transcriptional activity including oncogenic MYC. Here, we report the structural optimization of a small molecule microarray hit, prioritizing maintenance of CDK9 selectivity while improving on-target potency and overall physicochemical and pharmacokinetic (PK) properties. This led to the discovery of the potent, selective, orally bioavailable CDK9 inhibitor 28 (KB-0742). Compound 28 exhibits in vivo antitumor activity in mouse xenograft models and a projected human PK profile anticipated to enable efficacious oral dosing. Notably, 28 is currently being investigated in a phase 1/2 dose escalation and expansion clinical trial in patients with relapsed or refractory solid tumors.

A comprehensive evaluation in clinic and physiologically-based pharmacokinetic modeling and simulation to confirm lack of cytochrome P450-mediated drug-drug interaction potential for pomotrelvir.

Pomotrelvir is a new chemical entity and potent direct-acting antiviral inhibitor of the main protease of coronaviruses. Here the cytochrome P450 (CYP)-mediated drug-drug interaction (DDI) potential of pomotrelvir was evaluated for major CYP isoforms, starting with in vitro assays followed by the basic static model assessment. The identified CYP3A4-mediated potential DDIs were evaluated clinically at a supratherapeutic dose of 1050 mg twice daily (b.i.d.) of pomotrelvir, including pomotrelvir coadministration with ritonavir (strong inhibitor of CYP3A4) or midazolam (sensitive substrate of CYP3A4). Furthermore, a physiologically-based pharmacokinetic (PBPK) model was developed within the Simcyp Population-based Simulator using in vitro and in vivo information and validated with available human pharmacokinetic (PK) data. The PBPK model was simulated to assess the DDI potential for CYP isoforms that pomotrelvir has shown a weak to moderate DDI in vitro and for CYP3A4 at the therapeutic dose of 700 mg b.i.d. To support the use of pomotrelvir in women of childbearing potential, the impact of pomotrelvir on the exposure of the representative oral hormonal contraceptive drugs ethinyl estradiol and levonorgestrel was assessed using the PBPK model. The overall assessment suggested weak inhibition of pomotrelvir on CYP3A4 and minimal impact of a strong CYP3A4 inducer or inhibitor on pomotrelvir PK. Therefore, pomotrelvir is not anticipated to have clinically meaningful DDIs at the clinical dose. These comprehensive in vitro, in clinic, and in silico efforts indicate that the DDI potential of pomotrelvir is minimal, so excluding patients on concomitant medicines in clinical studies would not be required.

Metabolic disposition of H3B-8800, an orally available small-molecule splicing modulator, in rats, monkeys, and humans.

H3B-8800, a novel orally available modulator of the SF3b complex, which potently and preferentially kills spliceosome-mutant tumor cells, is in clinical development for the treatment of advanced myeloid malignancies. We characterized the pharmacokinetics, metabolism and disposition of H3B-8800 in rats, monkeys and humans.In vitro, H3B-8800 is a substrate of CYP3A4/5, flavin-containing monooxygenases (FMOs) and P-glycoprotein (P-gp), and showed a favorable drug-drug interaction profile as a perpetrator.Following oral dosing of 14C-H3B-8800 in bile-duct cannulated SD rats, 54.7% of the dosed radioactivity was excreted in the bile, with less found in feces (36.8%). The low amount in urine (3.7%), suggests that renal elimination is a minor pathway of clearance for H3B-8800.In Long-Evans rats, radioactivity derived from 14C-H3B-8800 was rapidly absorbed, with the highest distribution in the ocular, metabolic/excretory, and gastrointestinal tract tissues. No radioactivity was detected in the central nervous system.Seven metabolites were observed in human plasma following 4 daily doses of 40 mg H3B-8800. H3B-68736 (N-desmethyl), H3B-77176 (N-oxide), and unchanged H3B-8800 were the prominent components in human plasma, at 27.3%, 18.1%, and 33.2%, respectively, of the total drug-related material in a pooled AUC0-24h sample. The same 7 metabolites were observed in monkey plasma.

Anti-tumor Activity of the Type I PRMT Inhibitor, GSK3368715, Synergizes with PRMT5 Inhibition through MTAP Loss.

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection.

Nonclinical pharmacokinetics and in vitro metabolism of H3B-6545, a novel selective ERα covalent antagonist (SERCA).

PURPOSE: H3B-6545, a novel selective estrogen receptor (ER)α covalent antagonist (SERCA) which inactivates both wild-type and mutant ERα, is in clinical development for the treatment of metastatic breast cancer. Preclinical studies were conducted to characterize the pharmacokinetics and metabolism of H3B-6545 in rat and monkeys. METHODS: The clearance and metabolic profiles of H3B-6545 were studied using rat, monkey and human hepatocytes, and reaction phenotyping was done using recombinant human cytochrome P450 enzymes. Blood stability, protein binding, and permeability were also determined in vitro. Pharmacokinetics of H3B-6545 was assessed after both intravenous and oral dosing. A nonclinical PBPK model was developed to assess in vitro-in vivo correlation of clearance. RESULTS: H3B-6545 had a terminal elimination half-life of 2.4 h in rats and 4.0 h in monkeys and showed low to moderate bioavailability, in line with the in vitro permeability assessment. Plasma protein binding was similar across species, at 99.5-99.8%. Nine metabolites of H3B-6545 were identified in hepatocyte incubations, none of which were unique to humans. Formation of glutathione-related conjugate of H3B-6545 was minimal in vitro. H3B-6545, a CYP3A substrate, is expected to be mostly cleared via hepatic phase 1 metabolism. Hepatocyte clearance values were used to adequately model the time-concentration profiles in rat and monkey. CONCLUSIONS: We report on the absorption and metabolic fate and disposition of H3B-6545 in rats and dogs and illustrate that in vitro-in vivo correlation of clearance is possible for targeted covalent inhibitors, provided reactivity is not a predominant mechanism of clearance.

Effect of a high-fat meal on the relative bioavailability of H3B-6527, a novel FGFR4 inhibitor, in healthy volunteers.

PURPOSE: This Phase I study estimated the effect of a high-fat meal on the pharmacokinetics (PK) of H3B-6527, a covalent inhibitor of the fibroblast growth factor receptor (FGFR) 4 in clinical development for hepatocellular carcinoma and intrahepatic cholangiocarcinoma. METHODS: In this randomized, single center, single-dose, open-label, 2-period crossover study 12 healthy male volunteers, aged 18-55 years old, received a single 200-mg dose of H3B-6527 (capsule) following an overnight fast or a high-fat breakfast. PK samples were collected serially up to 36 h postdose. H3B-6527 concentrations were measured using a validated high-performance liquid chromatography tandem mass spectrometry method. PK data were analyzed using a noncompartmental approach based on a mixed-effects model. The safety and tolerability of H3B-6527 were also assessed. RESULTS: H3B-6527 plasma exposure increased after a high-fat meal with fed/fasted ratios of the geometric means (90% confidence interval) of 174% (102-298%) for Cmax and 246% (146-415%) for AUC0-t. Food delayed and prolonged absorption of H3B-6527, with a fed/fasted ratio for tmax of 200% (137-263%). PK variability was lower under the fed condition, as illustrated by the CV% for Cmax and AUC0-t of 41.9-54.5% (fed) versus 64.3-70.4% (fasted). CONCLUSIONS: A single 200 mg dose of H3B-6527 was safe and generally well tolerated when administered to healthy adult males. A high-fat meal significantly increased exposure to H3B-6527, from 1.5- to 2.5-fold in the systemic circulation, compared to administration under fasted conditions. Food delayed and prolonged absorption of H3B-6527. In general, lower inter-subject variability was observed in the fed state in healthy volunteers. TRIAL REGISTRATION: ClinicalTrials.gov.: NCT03424577.

Discovery of Selective Estrogen Receptor Covalent Antagonists for the Treatment of ERαWT and ERαMUT Breast Cancer.

Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA). In vitro comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERαWT and ERαMUT cell lines. In vivo, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERαWT and ERαY537S breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERαWT and ERαMUT cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs.Significance: Nearly 30% of endocrine therapy-resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERαWT and ERαMUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors. Cancer Discov; 8(9); 1176-93. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.

Small molecule inhibitors and CRISPR/Cas9 mutagenesis demonstrate that SMYD2 and SMYD3 activity are dispensable for autonomous cancer cell proliferation.

A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes.

Identification of a CARM1 Inhibitor with Potent In Vitro and In Vivo Activity in Preclinical Models of Multiple Myeloma.

CARM1 is an arginine methyltransferase with diverse histone and non-histone substrates implicated in the regulation of cellular processes including transcriptional co-activation and RNA processing. CARM1 overexpression has been reported in multiple cancer types and has been shown to modulate oncogenic pathways in in vitro studies. Detailed understanding of the mechanism of action of CARM1 in oncogenesis has been limited by a lack of selective tool compounds, particularly for in vivo studies. We describe the identification and characterization of, to our knowledge, the first potent and selective inhibitor of CARM1 that exhibits anti-proliferative effects both in vitro and in vivo and, to our knowledge, the first demonstration of a role for CARM1 in multiple myeloma (MM). EZM2302 (GSK3359088) is an inhibitor of CARM1 enzymatic activity in biochemical assays (IC50 = 6 nM) with broad selectivity against other histone methyltransferases. Treatment of MM cell lines with EZM2302 leads to inhibition of PABP1 and SMB methylation and cell stasis with IC50 values in the nanomolar range. Oral dosing of EZM2302 demonstrates dose-dependent in vivo CARM1 inhibition and anti-tumor activity in an MM xenograft model. EZM2302 is a validated chemical probe suitable for further understanding the biological role CARM1 plays in cancer and other diseases.

H3B-6527 Is a Potent and Selective Inhibitor of FGFR4 in FGF19-Driven Hepatocellular Carcinoma.

Activation of the fibroblast growth factor receptor FGFR4 by FGF19 drives hepatocellular carcinoma (HCC), a disease with few, if any, effective treatment options. While a number of pan-FGFR inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by dose-limiting toxicities mediated by FGFR1-3 receptors. To evade the potential limitations of pan-FGFR inhibitors, we generated H3B-6527, a highly selective covalent FGFR4 inhibitor, through structure-guided drug design. Studies in a panel of 40 HCC cell lines and 30 HCC PDX models showed that FGF19 expression is a predictive biomarker for H3B-6527 response. Moreover, coadministration of the CDK4/6 inhibitor palbociclib in combination with H3B-6527 could effectively trigger tumor regression in a xenograft model of HCC. Overall, our results offer preclinical proof of concept for H3B-6527 as a candidate therapeutic agent for HCC cases that exhibit increased expression of FGF19. Cancer Res; 77(24); 6999-7013. ©2017 AACR.

Aligning Potency and Pharmacokinetic Properties for Pyridine-Based NCINIs.

Optimization of pyridine-based noncatalytic site integrase inhibitors (NCINIs) based on compound 2 has led to the discovery of molecules capable of inhibiting virus harboring N124 variants of HIV integrase (IN) while maintaining minimal contribution of enterohepatic recirculation to clearance in rat. Structure-activity relationships at the C6 position established chemical space where the extent of enterohepatic recirculation in the rat is minimized. Desymmetrization of the C4 substituent allowed for potency optimization against virus having the N124 variant of integrase. Combination of these lessons led to the discovery of compound 20, having balanced serum-shifted antiviral potency and minimized excretion in to the biliary tract in rat, potentially representing a clinically viable starting point for a new treatment option for individuals infected with HIV.

CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice.

CD95 ligand (CD95L) is expressed by immune cells and triggers apoptotic death. Metalloprotease-cleaved CD95L (cl-CD95L) is released into the bloodstream but does not trigger apoptotic signaling. Hence, the pathophysiological role of cl-CD95L remains unclear. We observed that skin-derived endothelial cells from systemic lupus erythematosus (SLE) patients expressed CD95L and that after cleavage, cl-CD95L promoted T helper 17 (Th17) lymphocyte transmigration across the endothelial barrier at the expense of T regulatory cells. T cell migration relied on a direct interaction between the CD95 domain called calcium-inducing domain (CID) and the Src homology 3 domain of phospholipase Cγ1. Th17 cells stimulated with cl-CD95L produced sphingosine-1-phosphate (S1P), which promoted endothelial transmigration by activating the S1P receptor 3. We generated a cell-penetrating CID peptide that prevented Th17 cell transmigration and alleviated clinical symptoms in lupus mice. Therefore, neutralizing the CD95 non-apoptotic signaling pathway could be an attractive therapeutic approach for SLE treatment.

Novel Oxindole Sulfonamides and Sulfamides: EPZ031686, the First Orally Bioavailable Small Molecule SMYD3 Inhibitor.

SMYD3 has been implicated in a range of cancers; however, until now no potent selective small molecule inhibitors have been available for target validation studies. A novel oxindole series of SMYD3 inhibitors was identified through screening of the Epizyme proprietary histone methyltransferase-biased library. Potency optimization afforded two tool compounds, sulfonamide EPZ031686 and sulfamide EPZ030456, with cellular potency at a level sufficient to probe the in vitro biology of SMYD3 inhibition. EPZ031686 shows good bioavailability following oral dosing in mice making it a suitable tool for potential in vivo target validation studies.

Structure and Property Guided Design in the Identification of PRMT5 Tool Compound EPZ015666.

The recent publication of a potent and selective inhibitor of protein methyltransferase 5 (PRMT5) provides the scientific community with in vivo-active tool compound EPZ015666 (GSK3235025) to probe the underlying pharmacology of this key enzyme. Herein, we report the design and optimization strategies employed on an initial hit compound with poor in vitro clearance to yield in vivo tool compound EPZ015666 and an additional potent in vitro tool molecule EPZ015866 (GSK3203591).

Physiologically Based Pharmacokinetic Modeling in Pediatric Oncology Drug Development.

Childhood cancer represents more than 100 rare and ultra-rare diseases, with an estimated 12,400 new cases diagnosed each year in the United States. As such, this much smaller patient population has led to pediatric oncology drug development lagging behind that for adult cancers. Developing drugs for pediatric malignancies also brings with it a number of unique trial design considerations, including flexible enrollment approaches, age-appropriate formulation, acceptable sampling schedules, and balancing the need for age-stratified dosing regimens, given the smaller patient populations. The regulatory landscape for pediatric pharmacotherapy has evolved with U.S. Food and Drug Administration (FDA) legislation such as the 2012 FDA Safety and Innovation Act. In parallel, regulatory authorities have recommended the application of physiologically based pharmacokinetic (PBPK) modeling, for example, in the recently issued FDA Strategic Plan for Accelerating the Development of Therapies for Pediatric Rare Diseases. PBPK modeling provides a quantitative and systems-based framework that allows the effects of intrinsic and extrinsic factors on drug exposure to be modeled in a mechanistic fashion. The application of PBPK modeling in drug development for pediatric cancers is relatively nascent, with several retrospective analyses of cytotoxic therapies, and latterly for targeted agents such as obatoclax and imatinib. More recently, we have employed PBPK modeling in a prospective manner to inform the first pediatric trials of pinometostat and tazemetostat in genetically defined populations (mixed lineage leukemia-rearranged and integrase interactor-1-deficient sarcomas, respectively). In this review, we evaluate the application of PBPK modeling in pediatric cancer drug development and discuss the important challenges that lie ahead in this field.

Species differences in metabolism of EPZ015666, an oxetane-containing protein arginine methyltransferase-5 (PRMT5) inhibitor.

1. Metabolite profiling and identification studies were conducted to understand the cross-species differences in the metabolic clearance of EPZ015666, a first-in-class protein arginine methyltransferase-5 (PRMT5) inhibitor, with anti-proliferative effects in preclinical models of Mantle Cell Lymphoma. EPZ015666 exhibited low clearance in human, mouse and rat liver microsomes, in part by introduction of a 3-substituted oxetane ring on the molecule. In contrast, a higher clearance was observed in dog liver microsomes (DLM) that translated to a higher in vivo clearance in dog compared with rodent. 2. Structure elucidation via high resolution, accurate mass LC-MS(n) revealed that the prominent metabolites of EPZ015666 were present in hepatocytes from all species, with the highest turnover rate in dogs. M1 and M2 resulted from oxidative oxetane ring scission, whereas M3 resulted from loss of the oxetane ring via an N-dealkylation reaction. 3. The formation of M1 and M2 in DLM was significantly abrogated in the presence of the specific CYP2D inhibitor, quinidine, and to a lesser extent by the CYP3A inhibitor, ketoconazole, corroborating data from human recombinant isozymes. 4. Our data indicate a marked species difference in the metabolism of the PRMT5 inhibitor EPZ015666, with oxetane ring scission the predominant metabolic pathway in dog mediated largely by CYP2D.

Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound.

A novel aryl pyrazole series of arginine methyltransferase inhibitors has been identified. Synthesis of analogues within this series yielded the first potent, selective, small molecule PRMT6 inhibitor tool compound, EPZ020411. PRMT6 overexpression has been reported in several cancer types suggesting that inhibition of PRMT6 activity may have therapeutic utility. Identification of EPZ020411 provides the field with the first small molecule tool compound for target validation studies. EPZ020411 shows good bioavailability following subcutaneous dosing in rats making it a suitable tool for in vivo studies.

EPZ011989, A Potent, Orally-Available EZH2 Inhibitor with Robust in Vivo Activity.

Inhibitors of the protein methyltransferase Enhancer of Zeste Homolog 2 (EZH2) may have significant therapeutic potential for the treatment of B cell lymphomas and other cancer indications. The ability of the scientific community to explore fully the spectrum of EZH2-associated pathobiology has been hampered by the lack of in vivo-active tool compounds for this enzyme. Here we report the discovery and characterization of EPZ011989, a potent, selective, orally bioavailable inhibitor of EZH2 with useful pharmacokinetic properties. EPZ011989 demonstrates significant tumor growth inhibition in a mouse xenograft model of human B cell lymphoma. Hence, this compound represents a powerful tool for the expanded exploration of EZH2 activity in biology.

A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models.

Protein arginine methyltransferase-5 (PRMT5) is reported to have a role in diverse cellular processes, including tumorigenesis, and its overexpression is observed in cell lines and primary patient samples derived from lymphomas, particularly mantle cell lymphoma (MCL). Here we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with antiproliferative effects in both in vitro and in vivo models of MCL. EPZ015666 (GSK3235025) is an orally available inhibitor of PRMT5 enzymatic activity in biochemical assays with a half-maximal inhibitory concentration (IC50) of 22 nM and broad selectivity against a panel of other histone methyltransferases. Treatment of MCL cell lines with EPZ015666 led to inhibition of SmD3 methylation and cell death, with IC50 values in the nanomolar range. Oral dosing with EPZ015666 demonstrated dose-dependent antitumor activity in multiple MCL xenograft models. EPZ015666 represents a validated chemical probe for further study of PRMT5 biology and arginine methylation in cancer and other diseases.

Structural and Kinetic Characterization of a Novel N-acetylated Aliphatic Amine Metabolite of the PRMT Inhibitor, EPZ011652.

Pharmacokinetic and metabolite identification studies were conducted to understand the clearance pathways of EPZ011652 [(2-aminoethyl)(methyl)({3-[4-(propan-2-yloxy)phenyl]-1H-pyrazol-4-yl}methyl)amine], a potent protein arginine N-methyltransferase inhibitor. Metabolic clearance was the major pathway of EPZ011652 elimination in rats with structural elucidation of metabolites via liquid chromatography - mass spectrometry (LC-MS(n)) accurate mass measurement revealing the formation of a novel aliphatic N-acetylated metabolite (M1) located on the terminal nitrogen of the ethylene-diamine side chain. EPZ015564, a synthetic standard of the N-acetyl product, was prepared and was also generated by human and rat, but not dog hepatocytes. In rat hepatocytes, on incubation with EPZ011652, the concentration of EPZ015564 initially increased before decreasing with incubation time, suggesting that the metabolite is itself a substrate for other metabolizing enzymes, in agreement with the identification of metabolites M2, M3, and M4 in rat bile, all N-acetylated metabolites, undergoing sequential phase I (demethylation, oxidation) or phase II (sulfation) reactions. Reaction phenotyping with recombinant human N-acetyltransferase (NAT) isoforms revealed that both NAT1 and NAT2 are capable of acetylating EPZ011652, although with different catalytic efficiencies. Kinetic profiles of EPZ015564 formation followed classic Michaelis-Menten behavior with apparent Km values of >1000 µM for NAT1 and 165 ± 14.1 µM for NAT2. The in vitro intrinsic clearance for EPZ011652 by NAT2 (110 µL/min/mg) was 500-fold greater than by NAT1. In summary, we report the unusual N-acetylation of an aliphatic amine and discuss the implications for drug discovery and clinical development.