Inhibition of salt-inducible kinases (SIKs) SIK1, SIK2, and SIK3 represents a new potential therapeutic approach for autoimmune and inflammatory disease treatment via modulation of pro-inflammatory and immunoregulatory pathways, particularly inhibition of SIK2 and SIK3. After discovering a new chemotype for SIK inhibition, further optimization of potency, selectivity, ADMET and PK properties resulted in a 1,6-naphtyridine containing molecule GLPG4876 (7). However, 7 was clastogenic when examined in vivo in rat micronucleus assays, preventing further development. Overlay of 7 with GLPG3970 (6) within the SIK3 protein structure inspired the design of pyridine derivatives, leading to the identification of GLPG4970 (8). Compound 8 was negative in genotoxicity screening assays and demonstrated potent SIK2/SIK3 inhibition, for which isoform selectivity was determined in a cellular context. Compound 8 displayed improved potency compared with previously reported SIK inhibitors in biochemical and phenotypic cellular assays, and showed dose-dependent activity in disease relevant mouse pharmacological models of colitis.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. This epithelial anion channel regulates the active transport of chloride and bicarbonate ions across membranes. Mutations result in reduced surface expression of CFTR channels with impaired functionality. Correctors are small molecules that support the trafficking of CFTR to increase its membrane expression. Such correctors can have different mechanisms of action. Combinations may result in a further improved therapeutic benefit. We describe the identification and optimization of a new pyrazolol3,4-bl pyridine-6-carboxylic acid series with high potency and efficacy in rescuing CFTR from the cell surface. Investigations showed that carboxylic acid group replacement with acylsulfonamides and acylsulfonylureas improved ADMET and PK properties, leading to the discovery of the structurally novel co-corrector GLPG2737. The addition of GLPG2737 to the combination of the potentiator GLPG1837 and C1 corrector 4 led to an 8-fold increase in the F508del CFTR activity.

Cystic fibrosis (CF) is a life-threatening recessive genetic disease caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR). With the discovery of Ivacaftor and Lumacaftor, it has been shown that administration of one or more small molecules can partially restore the CFTR function. Correctors are small molecules that enhance the amount of CFTR on the cell surface, while potentiators improve the gating function of the CFTR channel. Herein, we describe the discovery and optimization of a novel potentiator series. Scaffold hopping, focusing on retaining the different intramolecular contacts, was crucial in the whole discovery process to identify a novel series devoid of genotoxic liabilities. From this series, the clinical candidate GLPG2451 was selected based on its pharmacokinetic properties, allowing QD dosing and based on its low CYP induction potential.

The deletion of phenylalanine at position 508 (F508del) in cystic fibrosis transmembrane conductance regulator (CFTR) causes a severe defect in folding and trafficking of the chloride channel resulting in its absence at the plasma membrane of epithelial cells leading to cystic fibrosis. Progress in the understanding of the disease increased over the past decades and led to the awareness that combinations of mechanistically different CFTR modulators are required to obtain meaningful clinical benefit. Today, there remains an unmet need for identification and development of more effective CFTR modulator combinations to improve existing therapies for patients carrying the F508del mutation. Here, we describe the identification of a novel F508del corrector using functional assays. We provide experimental evidence that the clinical candidate GLPG/ABBV-2737 represents a novel class of corrector exerting activity both on its own and in combination with VX809 or GLPG/ABBV-2222.

BackgroundN1303K, one of the common, severe disease-causing mutations in the CFTR gene, causes both defective biogenesis and gating abnormalities of the CFTR protein. The goals of the present study are to quantitatively assess the gating defects associated with the N1303K mutation and its pharmacological response to CFTR modulators including potentiators VX-770 and GLPG1837 and correctors VX-809, and VX-661.MethodsGating behavior and pharmacological responses to CFTR potentiators were assessed using patch-clamp technique in the excised, inside-out mode. We also examined the effects of GLPG1837, VX-770, VX-809 and VX-661 on N1303K-CFTR surface expression using Western blot analysis.ResultsLike wild-type (WT) CFTR, N1303K-CFTR channels were activated by protein kinase A-dependent phosphorylation, but the open probability (Po) of phosphorylated N1303K-CFTR was extremely low (~0.03 vs ~0.45 in WT channels). N1303K mutants showed abnormal responses to ATP analogs or mutations that disrupt ATP hydrolysis and/or dimerization of CFTR's two nucleotide-binding domains (NBDs). However, the Po of N1303K-CFTR was dramatically increased by GLPG1837 (~17-fold) and VX-770 (~8-fold). VX-809 or VX-661 enhanced N1303K-CFTR maturation by 2–3 fold, and co-treatment with GLPG1837 or VX-770 did not show any negative drug-drug interaction.ConclusionN1303K has a severe gating defect, reduced ATP-dependence and aberrant response to ATP analogs. These results suggest a defective function of the NBDs in N1303K-CFTR. An improvement of channel function by GLPG1837 or VX-770 and an increase of Band C protein by VX-809 or VX-661 support a therapeutic strategy of combining CFTR potentiator and corrector for patients carrying the N1303K mutation.

Cystic fibrosis (CF) is caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR). With the discovery of Ivacaftor and Orkambi, it has been shown that CFTR function can be partially restored by administering one or more small molecules. These molecules aim at either enhancing the amount of CFTR on the cell surface (correctors) or at improving the gating function of the CFTR channel (potentiators). Here we describe the discovery of a novel potentiator GLPG1837, which shows enhanced efficacy on CFTR mutants harboring class III mutations compared to Ivacaftor, the first marketed potentiator. The optimization of potency, efficacy, and pharmacokinetic profile will be described.

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 preclinical 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.

Acrolein is a toxic and highly reactive unsaturated aldehyde, often found in cigarette smoke and vehicle exhaust gases. Likewise, acrolein derived from cyclophosphamide-treated patients constitutes the major culprit of bladder irritation during chemotherapy in cancer patient. Although, initially, its toxicity and inflammatory properties have been related to the activation of the transient receptor potential A1 (TRPA1) in nociceptive neurons, recent evidences suggests that other receptor may also play a role in acrolein-induced toxicity. Here we show that, acrolein induces chemical irritation and nocifensive response in the absence of TRPA1. Ratiometric calcium measurements and patch-clamp demonstrate that acrolein activates TRPV1 but, unlike TRPA1 that desensitizes immediately after activation, acrolein-induced activation of TRPV1 is prolonged in time. Furthermore, we identify the N-terminal amino acid residue C157 as key for acrolein-induced TRPV1 activation. Taken together, our results reveal a mechanism underlying the major role of TRPV1 as mediator for the acrolein-induced toxicity, unveiling TRPV1 as a potential therapeutic target in a wide spectrum of noxious conditions, from exposure to smoke to cancer treatment.

Various TRP channels act as polymodal sensors of thermal and chemical stimuli, but the mechanisms whereby chemical ligands impact on TRP channel gating are poorly understood. Here we show that AITC (allyl isothiocyanate; mustard oil) and menthol represent two distinct types of ligands at the mammalian cold sensor TRPM8. Kinetic analysis of channel gating revealed that AITC acts by destabilizing the closed channel, whereas menthol stabilizes the open channel, relative to the transition state. Based on these differences, we classify agonists as either type I (menthol-like) or type II (AITC-like), and provide a kinetic model that faithfully reproduces their differential effects. We further demonstrate that type I and type II agonists have a distinct impact on TRPM8 currents and TRPM8-mediated calcium signals in excitable cells. These findings provide a theoretical framework for understanding the differential actions of TRP channel ligands, with important ramifications for TRP channel structure-function analysis and pharmacology.

Allyl isothiocyanate (AITC; mustard oil) confers the typical pungency to condiments such as wasabi and mustard, and has been widely used as a pharmacological tool to study pain and neurogenic inflammation. The somatosensory effects of AITC have been largely attributed to direct activation of TRPA1 and TRPV1, two excitatory Transient Receptor Potential (TRP) ion channels expressed in nociceptor neurons. Nevertheless, a subset of sensory neurons responds to AITC in a TRPA1- and TRPV1-independent manner. We show that AITC activates TRPA1/TRPV1-deficient sensory neurons through activation of TRPM8, a TRP channel involved in cool/cold sensation. Whole-cell and inside-out patch-clamp recordings revealed that AITC rapidly and reversibly activates heterologously expressed TRPM8 in a membrane-delimited manner. Moreover, we found that, at higher doses, AITC induced channel activation is followed by channel block, a process that is more pronounced in the human TRPM8 than in the mouse orthologue. Analysis of chimeric channels combining human and mouse TRPM8 orthologues cytosolic revealed domains that determine this AITC-induced channel blockade. TRPM8 may contribute to the complex psychophysical effects of AITC.

Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a Ca(2+)-activated nonselective cation channel involved in the transduction of sweet, bitter, and umami tastes. We previously showed that TRPM5 is a locus for the modulation of taste perception by temperature changes, and by quinine and quinidine, 2 bitter compounds that suppress gustatory responses. Here, we determined whether other bitter compounds known to modulate taste perception also affect TRPM5. We found that nicotine inhibits TRPM5 currents with an effective inhibitory concentration of ~1.3mM at -50 mV. This effect may contribute to the inhibitory effect of nicotine on gustatory responses in therapeutic and experimental settings, where nicotine is often employed at millimolar concentrations. In addition, it implies the existence of a TRPM5-independent pathway for the detection of nicotine bitterness. Nicotine seems to act from the extracellular side of the channel, reducing the maximal whole-cell conductance and inducing an acceleration of channel closure that leads to a negative shift of the activation curve. TRPM5 currents were unaffected by nicotine's metabolite cotinine, the intensive sweetener saccharin or by the bitter xanthines caffeine, theobromine, and theophylline. We also tested the effects of bitter compounds on another essential element of the sweet taste transduction pathway, the type 3 IP3 receptor (IP3R3). We found that IP3R3-mediated Ca(2+) flux is slightly enhanced by nicotine, not affected by saccharin, modestly inhibited by caffeine, theobromine, and theophylline, and strongly inhibited by quinine. Our results demonstrate that bitter compounds have differential effects on key elements of the sweet taste transduction pathway, suggesting for heterogeneous mechanisms of bitter-sweet taste interactions.

1704-Pos Board B434 Two Distinct Modes of Action of TRPM8 Agonists Thomas Voets, Annelies Janssens. KU Leuven, Leuven, Belgium. TRPM8, a transient receptor potential (TRP) cation channel expressed in sensory neurons, functions both as a cold sensor and as an ionotropic receptor for various natural and synthetic ligands, including menthol, eucalyptol, icilin and mustard oil (AITC). The mechanisms whereby TRPM8 agonists act on the channel are incompletely understood. Here we analyzed in detail the changes in kinetics of TRPM8 gating induced by different ligands, and identified two clearly distinct modes of agonist action. The majority of agonists (type I), including menthol, cause a prominent slowing of the channel relaxation kinetics in response to voltage steps, whereas AITC (type II) causes a clear acceleration. These results can be reproduced using a Monod-Wyman-Changeux model, where each subunit can bind a single ligand. In this model, type I agonists cause an stabilization of the open state, while type II ligands destabilize the closed state. These results indicate that agonists can exert energetically distinct effects on the TRPM8 channel protein, and suggest that ‘‘equipotent’’ concentrations of type I and type II agonists may differentially affect electrical activity in sensory neurons.

Allyl isothiocyanate (AITC, or oil) is a powerful plant-derived irritant that functions as a defensive trait against herbivores and confers pungency to mustard and wasabi. AITC is extensively used experimentally to induce acute pain and neurogenic inflammation, which are largely mediated by the activation of nociceptive cation channels TRPA1 and TRPV1. We have recently shown that AITC activates TRPV1 through a mechanism that is similar to that underlying the activation induced by capsaicin. In the present study, we tested whether AITC sensitizes TRPV1 for activation by extracellular acidosis and heat. Patch-clamp experiments in TRPV1-expressing HEK293T cells revealed that AITC enhances the responses to low pH and heat. These results were confirmed with intracellular calcium imaging experiments in the same cells and in dorsal root ganglion (DRG) neurons isolated from Trpa1 knockout (KO) mice. The responses to low pH and heat in the presence of AITC were strongly reduced by the TRPV1 inhibitor capsazepine and nearly absent in DRG neurons isolated from Trpa1/Trpv1 KO mice. The mechanism of cross sensitization between AITC, low pH and heat seem to occur via the induction of additive shifts of the voltage dependence of channel activation. These findings indicate that TRPV1 is a locus for cross sensitization between AITC, acidosis and heat in nociceptive neurons and help understanding the molecular bases underlying the role of this channel as mediator of the algesic properties of AITC.

The powerful plant-derived irritant allyl isothiocyanate (AITC, aka mustard oil) induces hyperalgesia to heat in rodents and humans through mechanisms that are not yet fully understood. It is generally believed that AITC activates the broadly tuned chemosensory cation channel transient receptor potential cation channel subfamily A member 1 (TRPA1), triggering an inflammatory response that sensitizes the heat sensor transient receptor potential cation channel subfamily V member 1 (TRPV1). In the view of recent data demonstrating that AITC can directly activate TRPV1, we here explored the possibility that this compound sensitizes TRPV1 to heat stimulation in a TRPA1-independent manner. Patch-clamp recordings and intracellular Ca2+ imaging experiments in HEK293T cells over-expressing mouse TRPV1 revealed that the increase in channel activation induced by heating is larger in the presence of AITC than in control conditions. The analysis of the effects of AITC and heat on the current–voltage relationship of TRPV1 indicates that the mechanism of sensitization is based on additive shifts of the voltage dependence of activation towards negative voltages. Finally, intracellular Ca2+ imaging experiments in mouse sensory neurons isolated from Trpa1 KO mice yielded that AITC enhances the response to heat, specifically in the subpopulation expressing TRPV1. Furthermore, this effect was strongly reduced by the TRPV1 inhibitor capsazepine and virtually absent in neurons isolated from double Trpa1/Trpv1 KO mice. Taken together, these findings demonstrate that TRPV1 is a locus for cross sensitization between AITC and heat in sensory neurons and may help explaining, at least in part, the role of this channel in AITC-induced hyperalgesia to heat.