DLK (Ki = 0.5 nM); p-JNK (IC50 = 30 nM); DRG (IC50 = 107 nM); MKK4 (IC50 ＞5000 nM); MKK7 (IC50 ＞5000 nM); JNK1 (IC50 = 129 nM); JNK2 (IC50 = 514 nM); JNK3 (IC50 = 364 nM); MLK1 (IC50 = 67.8 nM); MLK2 (IC50 = 767 nM); MLK3 (IC50 = 602 nM)

GNE-3511 has a IC50 of 30 nM for p-JNK and 107 nM for DRG, respectively, which indicates inhibitory activity[1].
GNE-3511 has kinase selectivity for MKK4, MKK7, JNK1, JNK2, JNK3, MLK1, MLK2 and MLK3 with IC50 values of ＞5000 nM, ＞5000 nM, 129 nM, 514 nM, 364 nM, 67.8 nM, 767 nM and 602 nM, respectively[1].
GNE-3511 shows concentration-dependent inhibition of neuronal aging in vitro[1].

GNE-3511 (oral gavage; 75 mg/kg; single) inhibits DLK in mice to reduce CYP-induced nociceptive behavior[2].
GNE-3511 (oral gavage; 75 mg/kg; single) reduces the edema and bleeding that CYP causes in the mouse bladder[2].
GNE-3511 (iv.; 1 mg/kg or po.; 5 mg/kg) has moderate plasma clearances in vivo, moderate volumes of distribution, brief half-lives, and moderate brain penetration[2].

p-JNK Cell Assay[1]
7500 HEK293 cells stably transfected with Dox-inducible human DLK in 40 μL of DMEM with 10% serum were seeded into each well of 384-well poly-d-lysine coated plates. The plates were incubated at 37 °C for 20–24 h prior to the addition of 5 μL of 60 μM doxycycline in DMEM. After incubation with doxycycline at 37 °C for approximately 20 h, 5 μL of DLK inhibitors in DMEM was added, and cells were incubated at 37 °C for an additional 5.5 h. The cells were then washed with PBS, permeabilized with 0.1% Triton X-100, blocked for 1 h with SuperBlock before the overnight incubation with the primary antibodies at 4 °C. The secondary antibodies were incubated for 2 h, washed with PBS, and then stained with Hoechst 33342 dye. The cell plates were imaged on Opera Imaging Platform.

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In Vitro Axon Degeneration Cell Assay[1]
Assay was conducted as previous described (14, 64) with the following modifications. Dorsal root ganglion (DRG) neurons were freshly dissected from E14.5 rat embryos. The resulting cell suspension was filtered through a 50 μm sieve to remove remaining tissue pieces, centrifuged 5 min at 1000 rpm, and resuspended in DRG culture medium (DMEM/F12 containing 1× N3 supplement, 0.18% glucose, 25 ng/mL NGF). Neurons were then plated on a 384-well dish at a density of 1200–2000 cells per well on top of the astrocyte monolayer. To inhibit cell proliferation, medium was supplied with 200 μM uridine and 100 μM 5-fluorodeoxyuridine the next day. DRGs were cultured for 4 days prior to the assay.

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In Vitro Transporter Assays[1]
Madin–Darby kidney cells (MDCK) stably transfected with human MDR1 (Pgp) were obtained from the National Institutes of Health. Cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% FBS, 80 ng/mL colchicine, and 5 μg/mL Plasmocin. Cells were harvested with trypsin and seeded on Millipore Millicell 24-well plates at initial concentrations of 2.0 × 105 cells/mL and allowed to grow for 5 days. Cell monolayers were equilibrated in transport buffer (Hank’s balanced salt solution with 10 mM Hepes, pH 7.4) for 60 min at 37 °C with 5% CO2 and 95% relative humidity prior to the experiment. Dose solutions were prepared in transport buffer and consisted of test compounds (5 μM) and the monolayer integrity marker lucifer yellow (100 μM). The dose solutions were added to the donor chambers, and transport buffer was added to all receiver chambers. The transport was examined in the apical to basolateral (A–B) and basolateral to apical (B–A) directions. The receiver chambers were sampled (50 μL) at 60, 120, and 180 min and were replenished with fresh transport buffer after the 60 and 120 min samplings. Lucifer yellow permeability was used as a marker of monolayer integrity during the experiment. Compound concentrations in the donor and receiving compartments were determined by LC–MS/MS analysis. The apparent permeability (Papp) in the apical to basal A–B and basal to apical B–A directions was calculated as follows: Papp = (dQ/dt)[1/(AC0)], where dQ/dt = rate of compound appearance in the receiver compartment, A = surface area of the inset, and C0 = initial substrate concentration at T = 0. The efflux ratio (ER) was calculated as Papp(B–A)/Papp(A–B).

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GNE-3511 is a novel, highly potent and selective zipper kinase (e.g. DLK, MAP3K12) inhibitor with IC50 of 0.107 uM for DLK. Recently, it was discovered that Dual Leucine Zipper Kinase (DLK, MAP3K12) is an important regulator of neuronal degeneration in various contexts. The phosphorylation of c-Jun was completely suppressed by GNE-3511 at high doses, while at low doses, the proportion of p-c-Jun positive cells was reduced to a moderate level.

In each well of 384-well poly-d-lysine coated plates, hek293 cells were seeded with 7500 Dox-inducible human DLK-transfected cells in 40 μL of DMEM with 10% serum. Before adding 5 μL of DMEM containing 60 μM doxycycline, the plates were incubated at 37 °C for 20–24 hours. 5 μL of DLK inhibitors in DMEM were added after the cells had been incubated with doxycycline at 37 °C for roughly 20 h. The cells were then incubated for an additional 5.5 h. at this temperature. Following permeabilization with 0.1% Triton X-100 and blocking for 1 hour with SuperBlock, the cells were then washed with PBS before being incubated with the primary antibodies at 4 °C for the duration of the next day. The secondary antibodies were incubated for 2 hours, followed by a PBS wash and Hoechst 33342 dye staining. Opera Imaging Platform was used to image the cell plates.

Cystitis mouse model[1]
75 mg/kg
oral gavage;75 mg/kg; single

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All experiments with mice were performed under animal protocols approved by the Animal Care and Use Committee at Genentech and adhere to ACS Ethical Guidelines for animal studies. For all in vivo studies, C57Bl/6 mice were dosed with GNE-3511orally as an MCT suspension. Optic nerve crush studies were conducted as described, except p-c-Jun was measured at 6 h by MSD ELISA. For MPTP studies, animals were administered four ip doses of 20 mg/kg MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), with each dose separated by 4 h. Twenty-five hours after the first MPTP dose, animals were sacrificed and processed essentially as described. For each animal, the p-c-Jun positive cell number/mm2 in five sections was averaged to generate a value for that animal.[1]

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To determine the effect of DLK inhibition in mice with cystitis, a single dose of DLK inhibitor GNE-3511 was administered at a dose of 75 mg/kg with 10 mg/kg of 7.5 mg/mL GNE-3511 solution by oral gavage at 2 h prior to CYP injection. Mice were housed in an environmentally controlled facility with a 12 h/12 h light/dark cycle and free access to water and food. [2]

[1]. Discovery of dual leucine zipper kinase (DLK, MAP3K12) inhibitors with activity in neurodegeneration models. J Med Chem. 2015 Jan 8;58(1):401-18.

[2]. Neuronal Dual Leucine Zipper Kinase Mediates Inflammatory and Nociceptive Responses in Cyclophosphamide-Induced Cystitis. J Innate Immun. 2021;13(5):259-268.

Interstitial cystitis is associated with neurogenic inflammation and neuropathic bladder pain. Dual leucine zipper kinase (DLK) expressed in sensory neurons is implicated in neuropathic pain. We hypothesized that neuronal DLK is involved in the regulation of inflammation and nociceptive behavior in cystitis. Mice deficient in DLK in sensory neurons (cKO) were generated by crossing DLK floxed mice with mice expressing Cre recombinase under Advillin promoter. Cystitis was induced by cyclophosphamide (CYP) administration in mice. Nociceptive behavior, bladder inflammation, and pathology were assessed following cystitis induction in control and cKO mice. The role of DLK in CYP-induced cystitis was further determined by pharmacological inhibition of DLK with GNE-3511. Deletion of neuronal DLK attenuated CYP-induced pain-like nociceptive behavior and suppressed histamine release from mast cells, neuronal activation in the spinal cord, and bladder pathology. Mice deficient in neuronal DLK also showed reduced inflammation induced by CYP and reduced c-Jun activation in the dorsal root ganglia (DRG). Pharmacological inhibition of DLK with GNE-3511 recapitulated the effects of neuronal DLK depletion in CYP treatment mice. Our study suggests that DLK is a potential target for the treatment of neuropathic pain and bladder pathology associated with cystitis.[2]

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Dual leucine zipper kinase (DLK, MAP3K12) was recently identified as an essential regulator of neuronal degeneration in multiple contexts. Here we describe the generation of potent and selective DLK inhibitors starting from a high-throughput screening hit. Using proposed hinge-binding interactions to infer a binding mode and specific design parameters to optimize for CNS druglike molecules, we came to focus on the di(pyridin-2-yl)amines because of their combination of desirable potency and good brain penetration following oral dosing. Our lead inhibitor GNE-3511 (26) displayed concentration-dependent protection of neurons from degeneration in vitro and demonstrated dose-dependent activity in two different animal models of disease. These results suggest that specific pharmacological inhibition of DLK may have therapeutic potential in multiple indications.[1]

C23H26F2N6O

440.49

440.213

C, 62.71; H, 5.95; F, 8.63; N, 19.08; O, 3.63

1496581-76-0

72547959

Light yellow to yellow solid powder

1.4±0.1 g/cm3

579.0±50.0 °C at 760 mmHg

304.0±30.1 °C

0.0±1.6 mmHg at 25°C

1.630

2.01

1

9

5

32

689

0

FC1(C([H])([H])C([H])([H])N(C1([H])[H])C1=C([H])C(=C([H])C(N([H])C2C([H])=C(C#N)C([H])=C([H])N=2)=N1)C1([H])C([H])([H])C([H])([H])N(C([H])([H])C1([H])[H])C1([H])C([H])([H])OC1([H])[H])F

RHFIAUKMKYHHFA-UHFFFAOYSA-N

InChI=1S/C23H26F2N6O/c24-23(25)4-8-31(15-23)22-11-18(17-2-6-30(7-3-17)19-13-32-14-19)10-21(29-22)28-20-9-16(12-26)1-5-27-20/h1,5,9-11,17,19H,2-4,6-8,13-15H2,(H,27,28,29)

2-[[6-(3,3-difluoropyrrolidin-1-yl)-4-[1-(oxetan-3-yl)piperidin-4-yl]pyridin-2-yl]amino]pyridine-4-carbonitrile

GNE 3511; GNE3511; 2-((6-(3,3-difluoropyrrolidin-1-yl)-4-(1-(oxetan-3-yl)piperidin-4-yl)pyridin-2-yl)amino)isonicotinonitrile; GNE 3511; 2-[[6-[3,3-Bis(Fluoranyl)pyrrolidin-1-Yl]-4-[1-(Oxetan-3-Yl)piperidin-4-Yl]pyridin-2-Yl]amino]pyridine-4-Carbonitrile; CHEMBL3393333; GNE3511; compound 26 [PMID: 25341110]; GNE-3511

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.72 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)