GDF15 is a dynamic biomarker of the integrated stress response in the central nervous system.

AIM: Characterize Growth Differentiation Factor 15 (GDF15) as a secreted biomarker of the integrated stress response (ISR) within the central nervous system (CNS). METHODS: We determined GDF15 levels utilizing in vitro and in vivo neuronal systems wherein the ISR was activated. Primarily, we used the murine model of vanishing white matter disease (VWMD), a neurological disease driven by persistent ISR in the CNS, to establish a link between levels of GDF15 in the cerebrospinal fluid (CSF) and ISR gene expression signature in the CNS. GDF15 was also determined in the CSF of VWM patients. RESULTS: GDF15 expression was increased concomitant to ISR activation in stress-induced primary astrocytes as well as in retinal ganglion cells following optic nerve crush, while treatment with 2Bact, a specific eIF2B activator, suppressed both the ISR and GDF15. In the VWMD model, CSF GDF15 levels corresponded with the magnitude of the ISR and were reduced by 2BAct. In VWM patients, mean CSF GDF15 was elevated >20-fold as compared to healthy controls, whereas plasma GDF15 was undifferentiated. CONCLUSIONS: These data suggest that CSF GDF15 is a dynamic marker of ISR activation in the CNS and may serve as a pharmacodynamic biomarker for ISR-modulating therapies.

Discovery and SAR of 4-aminopyrrolidine-2-carboxylic acid correctors of CFTR for the treatment of cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease resulting from mutations on both copies of the CFTR gene. Phenylalanine deletion at position 508 of the CFTR protein (F508del-CFTR) is the most frequent mutation in CF patients. Currently, the most effective treatments of CF use a dual or triple combination of CFTR correctors and potentiators. In triple therapy, two correctors (C1 and C2) and a potentiator are employed. Herein, we describe the identification and exploration of the SAR of a series of 4-aminopyrrolidine-2-carboxylic acid C2 correctors of CFTR to be used in conjunction with our existing C1 corrector series for the treatment of CF.

Biological Characterization of F508delCFTR Protein Processing by the CFTR Corrector ABBV-2222/GLPG2222.

Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease in Caucasians caused by pathogenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (CFTR). Significant small molecule therapeutic advances over the past two decades have been made to target the defective CFTR protein and enhance its function. To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in CF, two biomolecular activities are required, namely, correctors to increase the amount of properly folded F508delCFTR levels at the cell surface and potentiators to allow the effective opening, i.e., function of the F508delCFTR channel. Combined, these activities enhance chloride ion transport yielding improved hydration of the lung surface and subsequent restoration of mucociliary clearance. To enhance clinical benefits to CF patients, a complementary triple combination therapy consisting of two corrector molecules, type 1 (C1) and type 2, with additive mechanisms along with a potentiator are being investigated in the clinic for maximum restoration of mutated CFTR function. We report the identification and in vitro biologic characterization of ABBV-2222/GLPG2222 (4-[(2R,4R)-4-({[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-yl]benzoic acid),-a novel, potent, and orally bioavailable C1 corrector developed by AbbVie-Galapagos and currently in clinical trials-which exhibits substantial improvements over the existing C1 correctors. This includes improvements in potency and drug-drug interaction (DDI) compared with 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (VX-809, Lumacaftor) and improvements in potency and efficacy compared with 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)indol-5-yl]cyclopropane-1-carboxamide (VX-661, Tezacaftor). ABBV-2222/GLPG2222 exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR with an EC50 value <10 nM. SIGNIFICANCE STATEMENT: To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in cystic fibrosis, AbbVie-Galapagos has developed ABBV-2222/GLPG2222, a novel, potent, and orally bioavailable C1 corrector of this protein. ABBV-2222/GLPG2222, which is currently in clinical trials, exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR and substantial improvements over the existing C1 correctors.

Discovery of ABBV/GLPG-3221, a Potent Corrector of CFTR for the Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is a genetic disorder that affects multiple tissues and organs. CF is caused by mutations in the CFTR gene, resulting in insufficient or impaired cystic fibrosis transmembrane conductance regulator (CFTR) protein. The deletion of phenylalanine at position 508 of the protein (F508del-CFTR) is the most common mutation observed in CF patients. The most effective treatments of these patients employ two CFTR modulator classes, correctors and potentiators. CFTR correctors increase protein levels at the cell surface; CFTR potentiators enable the functional opening of CFTR channels at the cell surface. Triple-combination therapies utilize two distinct corrector molecules (C1 and C2) to further improve the overall efficacy. We identified the need to develop a C2 corrector series that had the potential to be used in conjunction with our existing C1 corrector series and provide robust clinical efficacy for CF patients. The identification of a pyrrolidine series of CFTR C2 correctors and the structure-activity relationship of this series is described. This work resulted in the discovery and selection of (2S,3R,4S,5S)-3-(tert-butyl)-4-((2-methoxy-5-(trifluoromethyl)pyridin-3-yl)methoxy)-1-((S)-tetrahydro-2H-pyran-2-carbonyl)-5-(o-tolyl)pyrrolidine-2-carboxylic acid (ABBV/GLPG-3221), which was advanced to clinical trials.

eIF2B activator prevents neurological defects caused by a chronic integrated stress response.

The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts.

Identification and Characterization of Novel CFTR Potentiators.

There is still a high unmet need for the treatment of most patients with cystic fibrosis (CF). The identification and development of new Cystic Fibrosis Transmembrane conductance Regulator (CFTR) modulators is necessary to achieve higher clinical benefit in patients. In this report we describe the characterization of novel potentiators. From a small screening campaign on F508del CFTR, hits were developed leading to the identification of pre-clinical candidates GLPG1837 and GLPG2451, each derived from a distinct chemical series. Both drug candidates enhance WT CFTR activity as well as low temperature or corrector rescued F508del CFTR, and are able to improve channel activity on a series of Class III, IV CFTR mutants. The observed activities in YFP halide assays translated well to primary cells derived from CF lungs when measured using Trans-epithelial clamp circuit (TECC). Both potentiators improve F508del CFTR channel opening in a similar manner, increasing the open time and reducing the closed time of the channel. When evaluating the potentiators in a chronic setting on corrected F508del CFTR, no reduction of channel activity in presence of potentiator was observed. The current work identifies and characterizes novel CFTR potentiators GLPG1837 and GLPG2451, which may offer new therapeutic options for CF patients.

Discovery of 4-[(2R,4R)-4-({[1-(2,2-Difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-yl]benzoic Acid (ABBV/GLPG-2222), a Potent Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Corrector for the Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is a multiorgan disease of the lungs, sinuses, pancreas, and gastrointestinal tract that is caused by a dysfunction or deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an epithelial anion channel that regulates salt and water balance in the tissues in which it is expressed. To effectively treat the most prevalent patient population (F508del mutation), two biomolecular modulators are required: correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective opening of the CFTR channel. Despite approved potentiator and potentiator/corrector combination therapies, there remains a high need to develop more potent and efficacious correctors. Herein, we disclose the discovery of a highly potent series of CFTR correctors and the structure-activity relationship (SAR) studies that guided the discovery of ABBV/GLPG-2222 (22), which is currently in clinical trials in patients harboring the F508del CFTR mutation on at least one allele.

Discovery of a potent and selective ROMK inhibitor with improved pharmacokinetic properties based on an octahydropyrazino[2,1-c][1,4]oxazine scaffold.

Following the discovery of small molecule acyl piperazine ROMK inhibitors, the acyl octahydropyrazino[2,1-c][1,4]oxazine series was identified. This series displays improved ROMK/hERG selectivity, and as a consequence, the resulting ROMK inhibitors do not evoke QTc prolongation in an in vivo cardiovascular dog model. Further efforts in this series led to the discovery of analogs with improved pharmacokinetic profiles. This new series also retained comparable ROMK potency compared to earlier leads.

Discovery of MK-7145, an Oral Small Molecule ROMK Inhibitor for the Treatment of Hypertension and Heart Failure.

ROMK, the renal outer medullary potassium channel, is involved in potassium recycling at the thick ascending loop of Henle and potassium secretion at the cortical collecting duct in the kidney nephron. Because of this dual site of action, selective inhibitors of ROMK are expected to represent a new class of diuretics/natriuretics with superior efficacy and reduced urinary loss of potassium compared to standard-of-care loop and thiazide diuretics. Following our earlier work, this communication will detail subsequent medicinal chemistry endeavors to further improve lead selectivity against the hERG channel and preclinical pharmacokinetic properties. Pharmacological assessment of highlighted inhibitors will be described, including pharmacodynamic studies in both an acute rat diuresis/natriuresis model and a subchronic blood pressure model in spontaneous hypertensive rats. These proof-of-biology studies established for the first time that the human and rodent genetics accurately predict the in vivo pharmacology of ROMK inhibitors and supported identification of the first small molecule ROMK inhibitor clinical candidate, MK-7145.

Differentiation of ROMK potency from hERG potency in the phenacetyl piperazine series through heterocycle incorporation.

Following the discovery of small molecule acyl piperazine ROMK inhibitors and their initial preclinical validation as a novel diuretic agent, our group set out to discover new ROMK inhibitors with reduced risk for QT effects, suitable for further pharmacological experiments in additional species. Several strategies for decreasing hERG affinity while maintaining ROMK inhibition were investigated and are described herein. The most promising candidate, derived from the newly discovered 4-N-heteroaryl acetyl series, improved functional hERG/ROMK ratio by >10× over the previous lead. In vivo evaluation demonstrated comparable diuretic effects in rat with no detectable QT effects at the doses evaluated in an in vivo dog model.

Discovery of a Potent and Selective ROMK Inhibitor with Pharmacokinetic Properties Suitable for Preclinical Evaluation.

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.

Characterization of the triazine, T4, a representative from a novel series of CaV2 inhibitors with strong state-dependence, poor use-dependence, and distinctively fast kinetics.

There is strong pharmacological, biological, and genetic evidence supporting the role of N-type calcium channels (CaV2.2) in nociception. There is also human validation data from ziconotide, the CaV2.2-selective peptidyl inhibitor used clinically to treat refractory pain. Unfortunately, ziconotide utility is limited by its narrow therapeutic window and required intrathecal route of administration. A major focus has been placed on identifying state-dependent CaV2.2 inhibitors to improve safety margins. Much less attention, however, has been given to characterizing the kinetics of CaV2.2 inhibitors as a means to further differentiate compounds and maximize therapeutic potential. Here we provide a detailed characterization of the CaV2.2 inhibitor T4 in terms of its state-dependence, use-dependence, kinetics, and mechanism of inhibition. Compound T4 displayed a >20-fold difference in potency when measured under inactivating conditions (IC50=1.1 µM) as compared to closed-state conditions (IC50=25 µM). At 3 µM, T4 produced a 15-fold hyperpolarizing shift in the inactivation curve for CaV2.2 while having no effect on channel activation. To assess the kinetic properties of T4 in a more physiological manner, its inhibition kinetics were assessed at 32°C using 2 mM Ca(2+) as the charge carrier. Surprisingly, the repriming rate for CaV2.2 channels at hyperpolarized potentials was similar in both the presence and absence of T4. This was in contrast to other compounds which markedly delayed repriming. Furthermore, T4 inhibited CaV2.2 channels more potently when channel inactivation was driven through a tonic sub-threshold depolarization rather than through a use-dependent protocol, despite similar levels of inactivation.

Mechanistic insights into the analgesic efficacy of A-1264087, a novel neuronal Ca(2+) channel blocker that reduces nociception in rat preclinical pain models.

UNLABELLED: Voltage-gated Ca(2+) channels play an important role in nociceptive transmission. There is significant evidence supporting a role for N-, T- and P/Q-type Ca(2+) channels in chronic pain. Here, we report that A-1264087, a structurally novel state-dependent blocker, inhibits each of these human Ca(2+) channels with similar potency (IC50 = 1-2 µM). A-1264087 was also shown to inhibit the release of the pronociceptive calcitonin gene-related peptide from rat dorsal root ganglion neurons. Oral administration of A-1264087 produces robust antinociceptive efficacy in monoiodoacetate-induced osteoarthritic, complete Freund adjuvant-induced inflammatory, and chronic constrictive injury of sciatic nerve-induced, neuropathic pain models with ED50 values of 3.0, 5.7, and 7.8 mg/kg (95% confidence interval = 2.2-3.5, 3.7-10, and 5.5-12.8 mg/kg), respectively. Further analysis revealed that A-1264087 also suppressed nociceptive-induced p38 and extracellular signal-regulated kinase 1/2 phosphorylation, which are biochemical markers of engagement of pain circuitry in chronic pain states. Additionally, A-1264087 inhibited both spontaneous and evoked neuronal activity in the spinal cord dorsal horn in complete Freund adjuvant-inflamed rats, providing a neurophysiological basis for the observed antihyperalgesia. A-1264087 produced no alteration of body temperature or motor coordination and no learning impairment at therapeutic plasma concentrations. PERSPECTIVE: The present results demonstrate that the neuronal Ca(2+) channel blocker A-1264087 exhibits broad-spectrum efficacy through engagement of nociceptive signaling pathways in preclinical pain models in the absence of effects on psychomotor and cognitive function.

Discovery of a novel sub-class of ROMK channel inhibitors typified by 5-(2-(4-(2-(4-(1H-Tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one.

A sub-class of distinct small molecule ROMK inhibitors were developed from the original lead 1. Medicinal chemistry endeavors led to novel ROMK inhibitors with good ROMK functional potency and improved hERG selectivity. Two of the described ROMK inhibitors were characterized for the first in vivo proof-of-concept biology studies, and results from an acute rat diuresis model confirmed the hypothesis that ROMK inhibitors represent new mechanism diuretic and natriuretic agents.

Synthesis and SAR of 4-aminocyclopentapyrrolidines as orally active N-type calcium channel inhibitors for inflammatory and neuropathic pain.

A novel series of N-type calcium channel inhibitors have been discovered. Optimization of potency and HT-ADME properties provides 4-aminocyclopentapyrrolidines with analgesic efficacy after oral dosing.

Establishment of a secondary screening assay for P/Q-type calcium channel blockers.

Development of calcium channel blockers is attractive, but has in the past been hampered by lack of high throughput electrophysiological technology. This limitation has been overcome by the implementation of automated patch clamp systems that allow identification of state-dependent compounds, which preferentially target pathologically overactive channels. We recently presented a fluorescence-based high-throughput screen for P/Q-type calcium channels followed by automated electrophysiology. Here, we provide a detailed description of the development of the secondary screen, and show the full analysis of the inactivation kinetics of the recombinant P/Q channel that served as a basis for the automated patch clamp protocol. Increasing the length of pre-depolarization shifted the inactivation to more hyperpolarized potentials. No steady-state inactivation was reached up to pre-depolarization durations of 3 min, while stability of the recordings progressively declined. As a compromise, a 3s pre-depolarization protocol was proposed for functional screening. In order to validate the electrophysiological screening, we compared kinetics and pharmacology of recombinant P/Q-type channels between automated and manual patch clamp measurements. Channel activation was similar under both conditions. By contrast, inactivation occurred at more hyperpolarized potentials in the automated system. Therefore, P/Q-type calcium channel inactivation is sensitive to the applied technological platform and needs to be adjusted when performing automated patch clamp recordings. Our results indicate that a thorough analysis of the inactivation kinetics is mandatory, when establishing an electrophysiological screening protocol for calcium channel blockers. As some data obtained by automated recordings may not be identical to manual patch clamp analysis, we recommend a proper initial validation of the screening assay and--if necessary--a posthoc adjustment of automated patch clamp values. The protocol presented here supports hit-to-lead and lead optimization efforts during the development of novel P/Q-type calcium channel blockers, and may be valuable for the generation of assays in other ion channel programs.

Improved Cav2.2 Channel Inhibitors through a gem-Dimethylsulfone Bioisostere Replacement of a Labile Sulfonamide.

We report the investigation of sulfonamide-derived Cav2.2 inhibitors to address drug-metabolism liabilities with this lead class of analgesics. Modification of the benzamide substituent provided improvements in both potency and selectivity. However, we discovered that formation of the persistent 3-(trifluoromethyl)benzenesulfonamide metabolite was an endemic problem in the sulfonamide series and that the replacement of the center aminopiperidine scaffold failed to prevent this metabolic pathway. This issue was eventually addressed by application of a bioisostere strategy. The new gem-dimethyl sulfone series retained Cav2.2 potency without the liability of the circulating sulfonamide metabolite.

Aminopiperidine sulfonamide Cav2.2 channel inhibitors for the treatment of chronic pain.

The voltage-gated calcium channel Ca(v)2.2 (N-type calcium channel) is a critical regulator of synaptic transmission and has emerged as an attractive target for the treatment of chronic pain. We report here the discovery of sulfonamide-derived, state-dependent inhibitors of Ca(v)2.2. In particular, 19 is an inhibitor of Ca(v)2.2 that is selective over cardiac ion channels, with a good preclinical PK and biodistribution profile. This compound exhibits dose-dependent efficacy in preclinical models of inflammatory hyperalgesia and neuropathic allodynia and is devoid of ancillary cardiovascular or CNS pharmacology at the doses tested. Importantly, 19 exhibited no efficacy in Ca(v)2.2 gene-deleted mice. The discovery of metabolite 26 confounds further development of members of this aminopiperidine sulfonamide series. This discovery also suggests specific structural liabilities of this class of compounds that must be addressed.

An automated electrophysiological assay for differentiating Ca(v)2.2 inhibitors based on state dependence and kinetics.

Ca(V)2.2 (N-type) calcium channels are key regulators of neurotransmission. Evidence from knockout animals and localization studies suggest that Ca(V)2.2 channels play a critical role in nociceptive transmission. Additionally, ziconotide, a selective peptide inhibitor of Ca(V)2.2 channels, is clinically used to treat refractory pain. However, the use of ziconotide is limited by its low therapeutic index, which is believed, at least in part, to be a consequence of ziconotide inhibiting Ca(V)2.2 channels regardless of the channel state. Subsequent efforts have focused on the discovery of state-dependent inhibitors that preferentially bind to the inactivated state of Ca(V)2.2 channels in order to achieve an improved safety profile relative to ziconotide. Much less attention has been paid to understanding the binding kinetics of these state-dependent inhibitors. Here, we describe a novel electrophysiology-based assay on an automated patch platform designed to differentiate Ca(V)2.2 inhibitors based on their combined state dependence and kinetics. More specifically, this assay assesses inactivated state block, closed state block, and monitors the kinetics of recovery from block when channels move between states. Additionally, a use-dependent assay is described that uses a train of depolarizing pulses to drive channels to a similar level of inactivation for comparison. This use-dependent protocol also provides information on the kinetics of block development. Data are provided to show how these assays can be utilized to screen for kinetic diversity within and across chemical classes.

Development and validation of a fluorescence-based HTS assay for the identification of P/Q-type calcium channel blockers.

Dysfunction of P/Q-type calcium channels is thought to underlie a variety of neurological diseases. There is evidence that migraine, Alzheimer's disease, and epilepsy involve a gain-of-function of the channel, leading to abnormal presynaptic vesicle release. P/Q-channel blockers may normalize current flow and consequently lead to an alleviation of disease symptoms. Although the medical need is high, there are no such compounds on the market. Here we describe a high throughput screen (HTS) for P/Q-type calcium channel blockers and the confirmation of hits by automated electrophysiology. We generated a HEK293 cell line stably expressing the α1A subunit of the P/Q-type calcium channel under control of a tetracycline (Tet) promoter. The accessory ß1.1 and α2δ1 subunits were co-expressed constitutively. The cell line was pharmacologically characterized by ion channel specific modulators, and revealed functional P/Q-type calcium currents. Using a fluorescence imaging plate reader (FLIPR), an assay for P/Q-type calcium channels was established based on a calcium sensitive dye. HTS of a 150,000 compound-containing sub-library led to the identification of 3262 hits that inhibited the fluorescence signal with potencies below 10 µM. Hit-to-lead (HTL) efforts identified 12,400 analogues. Compounds were clustered into 37 series, and 8 series of interest were prioritized. An electrophysiological secondary screen, providing a more direct measure of channel function, was implemented into the HTL process. 27 selected exemplars of different chemotypes were validated by automated whole-cell patch clamp analysis at inactivated channel state. The discovery of P/Q-channel blockers may foster the development of new therapeutics for a variety of neurological diseases.