Conformational-Design-Driven Discovery of EZM0414: A Selective, Potent SETD2 Inhibitor for Clinical Studies.

SETD2, a lysine N-methyltransferase, is a histone methyltransferase that plays an important role in various cellular processes and was identified as a target of interest in multiple myeloma that features a t(4,14) translocation. We recently reported the discovery of a novel small-molecule SETD2 inhibitor tool compound that is suitable for preclinical studies. Herein we describe the conformational-design-driven evolution of the advanced chemistry lead, which resulted in compounds appropriate for clinical evaluation. Further optimization of this chemical series led to the discovery of EZM0414, which is a potent, selective, and orally bioavailable inhibitor of SETD2 with good pharmacokinetic properties and robust pharmacodynamic activity in a mouse xenograft model.

Discovery of a First-in-Class Inhibitor of the Histone Methyltransferase SETD2 Suitable for Preclinical Studies.

SET domain-containing protein 2 (SETD2), a histone methyltransferase, has been identified as a target of interest in certain hematological malignancies, including multiple myeloma. This account details the discovery of EPZ-719, a novel and potent SETD2 inhibitor with a high selectivity over other histone methyltransferases. A screening campaign of the Epizyme proprietary histone methyltransferase-biased library identified potential leads based on a 2-amidoindole core. Structure-based drug design (SBDD) and drug metabolism/pharmacokinetics (DMPK) optimization resulted in EPZ-719, an attractive tool compound for the interrogation of SETD2 biology that enables in vivo target validation studies.

Discovery of the Oxadiazine FRM-024: A Potent CNS-Penetrant Gamma Secretase Modulator.

The recent approval of aducanumab for Alzheimer's disease has heightened the interest in therapies targeting the amyloid hypothesis. Our research has focused on identification of novel compounds to improve amyloid processing by modulating gamma secretase activity, thereby addressing a significant biological deficit known to plague the familial form of the disease. Herein, we describe the design, synthesis, and optimization of new gamma secretase modulators (GSMs) based on previously reported oxadiazine 1. Potency improvements with a focus on predicted and measured properties afforded high-quality compounds further differentiated via robust Aß42 reductions in both rodents and nonhuman primates. Extensive preclinical profiling, efficacy studies, and safety studies resulted in the nomination of FRM-024, (+)-cis-5-(4-chlorophenyl)-6-cyclopropyl-3-(6-methoxy-5-(4-methyl-1H-imidazole-1-yl)pyridin-2-yl)-5,6-dihydro-4H-1,2,4-oxadiazine, as a GSM preclinical candidate for familial Alzheimer's disease.

Discovery of MK-8719, a Potent O-GlcNAcase Inhibitor as a Potential Treatment for Tauopathies.

Inhibition of O-GlcNAcase (OGA) has emerged as a promising therapeutic approach to treat tau pathology in neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Beginning with carbohydrate-based lead molecules, we pursued an optimization strategy of reducing polar surface area to align the desired drug-like properties of potency, selectivity, high central nervous system (CNS) exposure, metabolic stability, favorable pharmacokinetics, and robust in vivo pharmacodynamic response. Herein, we describe the medicinal chemistry and pharmacological studies that led to the identification of (3aR,5S,6S,7R,7aR)-5-(difluoromethyl)-2-(ethylamino)-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]thiazole-6,7-diol 42 (MK-8719), a highly potent and selective OGA inhibitor with excellent CNS penetration that has been advanced to first-in-human phase I clinical trials.

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 second-generation P2X3 antagonists for treatment of pain.

A second-generation small molecule P2X3 receptor antagonist has been developed. The lead optimization strategy to address shortcomings of the first-generation preclinical lead compound is described herein. These studies were directed towards the identification and amelioration of preclinical hepatobiliary findings, reducing potential for drug-drug interactions, and decreasing the projected human dose of the first-generation lead.

Design, Synthesis, and Evaluation of a Novel Series of Oxadiazine Gamma Secretase Modulators for Familial Alzheimer's Disease.

Herein we describe the design, synthesis, and evaluation of a novel series of oxadiazine-based gamma secretase modulators obtained via isosteric amide replacement and critical consideration of conformational restriction. Oxadiazine lead 47 possesses good in vitro potency with excellent predicted CNS drug-like properties and desirable ADME/PK profile. This lead compound demonstrated robust Aß42 reductions and subsequent Aß37 increases in both rodent brain and CSF at 30 mg/kg dosed orally.

Characterization of FRM-36143 as a new γ-secretase modulator for the potential treatment of familial Alzheimer's disease.

BACKGROUND: Familial Alzheimer's disease (FAD) is caused by mutations in the amyloid precursor protein (APP) or presenilin (PS). Most PS mutations, which account for the majority of FAD cases, lead to an increased ratio of longer to shorter forms of the amyloid beta (Aß) peptide. The therapeutic rationale of γ-secretase modulators (GSMs) for Alzheimer's disease is based on this genetic evidence as well as on enzyme kinetics measurements showing changes in the processivity of the γ-secretase complex. This analysis suggests that GSMs could potentially offset some of the effects of PS mutations on APP processing, thereby addressing the root cause of early onset FAD. Unfortunately, the field has generated few, if any, molecules with good central nervous system (CNS) drug-like properties to enable proof-of-mechanism studies. METHOD: We characterized the novel GSM FRM-36143 using multiple cellular assays to determine its in vitro potency and off-target activity as well as its potential to reverse the effect of PS mutations. We also tested its efficacy in vivo in wild-type mice and rats. RESULTS: FRM-36143 has much improved CNS drug-like properties compared to published GSMs. It has an in vitro EC50 for Aß42 of 35 nM in H4 cells, can reduce Aß42 to 58 % of the baseline in rat cerebrospinal fluid, and also increases the non-amyloidogenic peptides Aß37 and Aß38. It does not inhibit Notch processing, nor does it inhibit 24-dehydrocholesterol reductase (DHCR24) activity. Most interestingly, it can reverse the effects of presenilin mutations on APP processing in vitro. CONCLUSIONS: FRM-36143 possesses all the characteristics of a GSM in terms of Aß modulation Because FRM-36143 was able to reverse the effect of PS mutations, we suggest that targeting patients with this genetic defect would be the best approach at testing the efficacy of a GSM in the clinic. While the amyloid hypothesis is still being tested with ß-site APP-cleaving enzyme inhibitors and monoclonal antibodies in sporadic AD, we believe it is not a hypothesis for FAD. Since GSMs can correct the molecular defect caused by PS mutations, they have the promise to provide benefits to the patients when treated early enough in the course of the disease.

Intracellular Retention of Three Quinuclidine Derivatives in Caco-2 Permeation Experiments: Mechanisms and Impact on Estimating Permeability and Active Efflux Ratio.

BACKGROUND: Three quinuclidine derivatives (FRM-1, FRM-2 and FRM-3) were subject to significant mass loss to cellular retention in Caco-2 permeation experiments. The apparent permeability coefficient (Papp) calculated with either 'sink' (Papp,sink) or 'non-sink' (Papp,nonsink) method was significantly biased. As a result, a simplified 3-compartmental distribution model was applied in this study to derive the 'intrinsic' Papp (Papp,int) and to understand the impact of cellular retention on estimating Papp and active efflux ratio (ER) values. METHODS: Time-courses of the amount of test compounds in the donor, receiver and cells were determined in the presence and absence of bafilomycin A1 (BFA, 100 nM) and / or cyclosporine A (CsA, 10 .M). A mathematical model was constructed to describe the mass transfer of test compounds among three compartments. The temporal profiles of directional Papp,sink, Papp,nonsink and the corresponding of ER values were compared with the counterpart parameters derived from data-fitting to the mathematical model. Simulations were performed for a better understanding of experimental observations. RESULTS: The mass recovery of test compounds deteriorated with incubation time and was direction dependent. Based on the directional Papp,sink values, the resulting ER is close to unity for FRM-1, and approximately 2 and 3.5 for FRM-2 and FRM-3. Treatment with BFA considerably enhanced mass recovery for FRM-1 and FRM-3 (by 5- and 2-fold) but elicited no impact on FRM-2, while ER values largely unchanged. Expectedly, Papp,nonsink was higher than Papp,sink, but the resulting ER was lower in most cases. In contrast, the model-derived Papp,int was much greater than the values of Papp,sink and Papp,nonsink. The model also quantitatively unveiled the respective contributions of lysosomal sequestration and nonspecific binding to the cellular retention of the compounds. CONCLUSION: Our work reveals the different mechanisms involved in cellular retention of these quinuclidine derivatives, and more importantly, demonstrates the value of kinetic analyses with mathematical modeling in minimizing the bias in Papp estimation when assumptions for conventional calculations are violated.

Optimization of Novel Aza-benzimidazolone mGluR2 PAMs with Respect to LLE and PK Properties and Mitigation of CYP TDI.

Investigation of a novel amino-aza-benzimidazolone structural class of positive allosteric modulators (PAMs) of metabotropic glutamate receptor 2 (mGluR2) identified [2.2.2]-bicyclic amine 12 as an intriguing lead structure due to its promising physicochemical properties and lipophilic ligand efficiency (LLE). Further optimization led to chiral amide 18, which exhibited strong in vitro activity and attractive pharmacokinetic (PK) properties. Hypothesis-driven target design identified compound 21 as a potent, highly selective, orally bioavailable mGluR2 PAM, which addressed a CYP time-dependent inhibition (TDI) liability of 18, while maintaining excellent drug-like properties with robust in vivo activity in a clinically validated model of antipsychotic potential.

Discovery of 5-aryl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-ones as positive allosteric modulators of metabotropic glutamate subtype-2 (mGlu2) receptors with efficacy in a preclinical model of psychosis.

Optimization of a benzimidazolone template for potency and physical properties revealed 5-aryl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-ones as a key template on which to develop a new series of mGlu2 positive allosteric modulators (PAMs). Systematic investigation of aryl-SAR led to the identification of compound 27 as a potent and highly selective mGlu2 PAM with sufficient pharmacokinetics to advance to preclinical models of psychosis. Gratifyingly, compound 27 showed full efficacy in the PCP- and MK-801-induced hyperlocomotion assay in rats at CSF concentrations consistent with mGlu2 PAM potency.

Concentration-response relationship of the α7 nicotinic acetylcholine receptor agonist FRM-17874 across multiple in vitro and in vivo assays.

Pharmacological activation of α7 nicotinic acetylcholine receptors (α7 nAChRs) may improve cognition in schizophrenia and Alzheimer's disease. The present studies describe an integrated pharmacological analysis of the effects of FRM-17874, an analogue of encenicline, on α7 nAChRs in vitro and in behavioral and neurophysiological assays relevant to cognitive function. FRM-17874 demonstrated high affinity binding to human α7 nAChRs, displacing [(3)H]-methyllacaconitine (Ki=4.3nM). In Xenopus laevis oocytes expressing human α7 nAChRs, FRM-17874 acted as an agonist, evoking inward currents with an EC50 of 0.42µM. Lower concentrations of FRM-17874 (0.01-3nM) elicited no detectable current, but primed receptors to respond to sub-maximal concentrations of acetylcholine. FRM-17874 improved novel object recognition in rats, and enhanced memory acquisition and reversal learning in the mouse water T-maze. Neurophysiological correlates of cognitive effects of drug treatment, such as synaptic transmission, long-term potentiation, and hippocampal theta oscillation were also evaluated. Modulation of synaptic transmission and plasticity was observed in rat hippocampal slices at concentrations of 3.2 and 5nM. FRM-17874 showed a dose-dependent facilitation of stimulation-induced hippocampal theta oscillation in mice and rats. The FRM-17874 unbound brain concentration-response relationship for increased theta oscillation power was similar in both species, exhibited a biphasic pattern peaking around 3nM, and overlapped with active doses and exposures observed in cognition assays. In summary, behavioral and neurophysiological assays indicate a bell-shaped effective concentration range and this report represents the first attempt to explain the concentration-response function of α7 nAChR-mediated pro-cognitive effects in terms of receptor pharmacology.

Drug-drug interactions related to altered absorption and plasma protein binding: theoretical and regulatory considerations, and an industry perspective.

Drug-drug interactions (DDIs) related to altered drug absorption and plasma protein binding have received much less attention from regulatory agencies relative to DDIs mediated via drug metabolizing enzymes and transporters. In this review, a number of theoretical bases and regulatory framework are presented for these DDI aspects. Also presented is an industry perspective on how to approach these issues in support of drug development. Overall, with the exception of highly permeable and highly soluble (BCS 1) drugs, DDIs related to drug-induced changes in gastrointestinal (GI) physiology can be substantial, thus warranting more attentions. For a better understanding of absorption-associated DDI potential in a clinical setting, mechanistic studies should be conducted based on holistic integration of the pharmaceutical profiles (e.g., pH-dependent solubility) and pharmacological properties (e.g., GI physiology and therapeutic margin) of drug candidates. Although majority of DDI events related to altered plasma protein binding are not expected to be of clinical significance, exceptions exist for a subset of compounds with certain pharmacokinetic and pharmacological properties. Knowledge of the identity of binding proteins and the binding extent in various clinical setting (including disease states) can be valuable in aiding clinical DDI data interpretations, and ensuring safe and effective use of new drugs.

Neuropharmacokinetics of two investigational compounds in rats: divergent temporal profiles in the brain and cerebrospinal fluid.

Two investigational compounds (FRM-1, (R)-7-fluoro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide and FRM-2, (R)-7-cyano-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide) resided in rat brain longer than in systemic circulation. In Caco-2 directional transport studies, they both showed good intrinsic passive permeability but differed significantly in efflux susceptibility (efflux ratio of <2 and ∼7, respectively), largely attributed to P-glycoprotein (P-gp). Capitalizing on these interesting properties, we investigated how cerebrospinal fluid (CSF) concentration (CCSF) would be shaped by unbound plasma concentration (Cu,p) and unbound brain concentration (Cu,b) in disequilibrium conditions and at steady state. Following subcutaneous administration, FRM-1CCSF largely followed Cu,p initially and leveled between Cu,p and Cu,b. However, it gradually approached Cu,b and became lower than, but parallel to Cu,b at the terminal phase. In contrast, FRM-2CCSF temporal profile mostly paralleled the Cu,p but was at a much lower level. Upon intravenous infusion to steady state, FRM-1CCSF and Cu,b were similar, accounting for 61% and 69% of the Cu,p, indicating a case of largely passive diffusion-governed brain penetration where CCSF served as a good surrogate for Cu,b. On the contrary, FRM-2CCSF and Cu,b were remarkably lower than Cu,p (17% and 8% of Cu,p, respectively), suggesting that FRM-2 brain penetration was severely impaired by P-gp-mediated efflux and CCSF underestimated this impact. A semi-physiologically based pharmacokinetic (PBPK) model was constructed that adequately described the temporal profiles of the compounds in the plasma, brain and CSF. Our work provided some insight into the relative importance of blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) in modulating CCSF.

Prednisone has no effect on the pharmacokinetics of CYP3A4 metabolized drugs - midazolam and odanacatib.

We evaluated the effect of prednisone on midazolam and odanacatib pharmacokinetics. In this open-label, 2-period crossover study in healthy male subjects, midazolam 2 mg was administered (Day -1) followed by odanacatib 50 mg (Day 1) during Part 1. In Period 2, prednisone 10 mg once daily (qd) was administered on Days 1-28; odanacatib was co-administered on Day 14 and midazolam 2 mg was co-administered on Days 1 and 28. Subjects were administered midazolam 2 mg on Days 42 and 56. Safety and tolerability were assessed throughout the study. A physiologically-based pharmacokinetic (PBPK) model was also built. There were 15 subjects enrolled; mean age was 31 years. The odanacatib AUC(0- ∞) GMR (90% CI) [odanacatib + prednisone (Day 14, Period 2)/odanacatib alone (Day 1, Period 1] was 1.06 (0.96, 1.17). AUC(0-∞) GMR (90%CI) [midazolam + prednisone (Day 28, Period 2)/midazolam alone (Day -1, Period 1] was 1.08 (0.93,1.26). There were no serious AEs or AEs leading to discontinuation. PBPK modeling showed that prednisone does not cause significant effects on the exposure of sensitive CYP3A4 substrates in vivo at therapeutic doses. Co-administration of prednisone 10 mg qd had no effect on pharmacokinetics of either odanacatib 10 mg or midazolam 2 mg.

Discovery of 2,5-diarylnicotinamides as selective orexin-2 receptor antagonists (2-SORAs).

The orexin (or hypocretin) system has been identified as a novel target for the treatment of insomnia due to the wealth of biological and genetic data discovered over the past decade. Recently, clinical proof-of-concept was achieved for the treatment of primary insomnia using dual (OX1R/OX2R) orexin receptor antagonists. However, elucidation of the pharmacology associated with selective orexin-2 receptor antagonists (2-SORAs) has been hampered by the lack of orally bioavailable, highly selective small molecule probes. Herein, the discovery and optimization of a novel series of 2,5-diarylnicotinamides as potent and orally bioavailable orexin-2 receptor selective antagonists is described. A compound from this series demonstrated potent sleep promotion when dosed orally to EEG telemetrized rats.

A dynamic view to the modulation of phosphorylation and O-GlcNAcylation by inhibition of O-GlcNAcase.

Protein phosphorylation and O-GlcNAcylation are reciprocally regulated. As hyperphosphorylation is implicated in tau pathology, approaches have been exploited to reduce the magnitude of tau phosphorylation by increasing the level of tau O-GlcNAcylation. With mathematic models constructed to describe different kinetic scenarios, we analyzed the temporal change of an O-GlcNAcylated protein in contrast to that of the phosphorylated form upon inhibition of O-GlcNAcase (OGA). The analyses indicate that when degradation of the modified protein is negligible relative to the naked one, the magnitude of O-GlcNAcylated protein increase is proportional to the level of inhibition, while the extent of phosphorylated protein decline varies due to other factors. Furthermore, the increase of O-GlcNAcylated protein parallels with the decrease of phosphorylated form upon acute or short-term inhibition of OGA, as observed in many in vitro and short term in vivo studies. However, phosphorylated protein is predicted to return to its initial level while O-GlcNAcylated protein to achieve a higher steady level under sustained inhibition. This simulated result is in line with a recent report on long-term inhibition of OGA in transgenic mice. Noticeably, inhibition withdrawal is anticipated to cause a transient rise of phosphorylated protein. If degradation of modified proteins proceeds in addition to the naked one, the characteristic temporal profiles of each form in response to OGA inhibition would depend on the relative importance of individual degradation pathways. The models described herein may serve as a useful investigational tool that will provide insight into pharmacological intervention for tauopathies in particular and for reciprocally modulated reactions in general.

In vitro assessment of drug-drug interaction potential of boceprevir associated with drug metabolizing enzymes and transporters.

The inhibitory effect of boceprevir (BOC), an inhibitor of hepatitis C virus nonstructural protein 3 protease was evaluated in vitro against a panel of drug-metabolizing enzymes and transporters. BOC, a known substrate for cytochrome P450 (P450) CYP3A and aldo-ketoreductases, was a reversible time-dependent inhibitor (k(inact) = 0.12 minute(-1), K(I) = 6.1 µM) of CYP3A4/5 but not an inhibitor of other major P450s, nor of UDP-glucuronosyltransferases 1A1 and 2B7. BOC showed weak to no inhibition of breast cancer resistance protein (BCRP), P-glycoprotein (Pgp), or multidrug resistance protein 2. It was a moderate inhibitor of organic anion transporting polypeptide (OATP) 1B1 and 1B3, with an IC(50) of 18 and 4.9 µM, respectively. In human hepatocytes, BOC inhibited CYP3A-mediated metabolism of midazolam, OATP1B-mediated hepatic uptake of pitavastatin, and both the uptake and metabolism of atorvastatin. The inhibitory potency of BOC was lower than known inhibitors of CYP3A (ketoconazole), OATP1B (rifampin), or both (telaprevir). BOC was a substrate for Pgp and BCRP but not for OATP1B1, OATP1B3, OATP2B1, organic cation transporter, or sodium/taurocholate cotransporting peptide. Overall, our data suggest that BOC has the potential to cause pharmacokinetic interactions via inhibition of CYP3A and CYP3A/OATP1B interplay, with the interaction magnitude lower than those observed with known potent inhibitors. Conversely, pharmacokinetic interactions of BOC, either as a perpetrator or victim, via other major P450s and transporters tested are less likely to be of clinical significance. The results from clinical drug-drug interaction studies conducted thus far are generally supportive of these conclusions.

ADME of biologics-what have we learned from small molecules?

Thorough characterization and in-depth understanding of absorption, distribution, metabolism, and elimination (ADME) properties of a drug candidate have been well recognized as an important element in small molecule (SM) drug discovery and development. This has been the area of focus for drug metabolism and pharmacokinetics (DMPK) scientists, whose role has been evolving over the past few decades from primarily being involved in the development space after a preclinical candidate was selected to extending their involvement into the discovery stage prior to candidate selection. This paradigm shift has ensured the entry into development of the best candidates with optimal ADME properties, and thus has greatly impacted SM drug development through significant reduction of the failure rate for pharmacokinetics related reasons. In contrast, the sciences of ADME and DMPK have not been fully integrated into the discovery and development processes for large molecule (LM) drugs. In this mini-review, we reflect on the journey of DMPK support of SM drug discovery and development and highlight the key enablers that have allowed DMPK scientists to make such impacts, with the aim to provide a perspective on relevant lessons learned from SM drugs that are applicable to DMPK support strategies for LMs.

Theoretical analysis of interplay of therapeutic protein drug and circulating soluble target: temporal profiles of 'free' and 'total' drug and target.

PURPOSE: To systemically investigate, for a therapeutic protein with a circulating soluble target, how the interplay of target dynamics and drug pharmacokinetics defines the 'total' and 'free' drug and target temporal profiles. METHOD: By extending the established rapid-binding target-mediated drug disposition (TMDD) pharmacokinetic model to circulating soluble targets, the temporal profiles of 'total' and 'free' drug and target were simulated with varying binding affinity (K(D)), target baseline (R(ss)), target turnover, and drug dose level. Two sets of published experimental data were compared with the simulated results. RESULTS: Binding to a circulating soluble target could lead to a divergence of the 'free' drug from the 'total' drug. Simulations show this divergent magnitude determined by K(D) and R(ss), with the temporal profile being defined by target turnover and drug dose level. As divergence proceeds, starting at the distribution phase, 'free' drug would decline faster but eventually parallel 'total' drug at the terminal phase, giving rise to a steeper distribution phase and comparable terminal half-life, relative to the 'total' form. The model also allows for estimation of the dynamic change of 'total' and 'free' target in response to the treatment of a therapeutic protein drug, facilitating dose level and regimen design to achieve desired 'free' target suppression. Model predictions compared favorably with two sets of published experimental data. CONCLUSIONS: Theoretical analyses identified key variables governing the different temporal profiles of 'total' and 'free' drug and target. The rapid-binding TMDD model reasonably captured the features of the interplay of drug pharmacokinetics and target dynamics for two reported cases.