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 targets dual leucine zipper kinase (DLK, MAP3K12) with an IC50 of 1.9 nM (recombinant human DLK) and a Ki value of 0.6 nM [1]
GNE-3511 shows high selectivity for DLK over other MAP kinases (e.g., LZK, MLK1-4, JNK1-3) with IC50 > 1000 nM [1]

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].

---

In recombinant human DLK kinase assay, GNE-3511 (0.1-100 nM) dose-dependently inhibited DLK activity, with an IC50 of 1.9 nM; it did not significantly inhibit 45 other kinases at concentrations up to 1 μM [1]
- In primary rat cortical neurons, GNE-3511 (1-100 nM) protected against axonal damage induced by nerve growth factor (NGF) withdrawal; 10 nM increased intact axon length by 62% compared to vehicle (p < 0.01) [1]
- GNE-3511 (0.3-10 μM) dose-dependently reduced phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun (DLK downstream substrates) in NGF-deprived neurons, with 10 μM inhibiting p-JNK by 75% (western blot analysis) [1]
- In mouse bladder epithelial cells (MBECs) stimulated with lipopolysaccharide (LPS), GNE-3511 (1-10 μM) decreased mRNA expression of pro-inflammatory cytokines TNF-α (48% reduction at 10 μM) and IL-6 (52% reduction at 10 μM) (p < 0.05) [2]

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].

---

In a mouse model of axonal injury (optic nerve crush), intraperitoneal administration of GNE-3511 (30 mg/kg twice daily for 7 days) preserved retinal ganglion cell (RGC) survival; RGC density was 58% higher than vehicle control (p < 0.01) [1]
- In cyclophosphamide-induced cystitis (CIC) mice, oral administration of GNE-3511 (10, 30 mg/kg once daily for 7 days) reduced bladder inflammation; 30 mg/kg decreased bladder wet weight by 32% and TNF-α protein levels by 45% (p < 0.05) [2]
- GNE-3511 (30 mg/kg, p.o.) ameliorated nociceptive responses in CIC mice, reducing voiding frequency by 38% and pelvic hypersensitivity to von Frey stimulation by 42% (p < 0.01) [2]
- In the optic nerve crush model, GNE-3511 (30 mg/kg, i.p.) maintained axonal integrity, with a 47% increase in intact axons at the optic nerve midpoint compared to vehicle [1]

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.

---

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.

---

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).

---

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.

---

Recombinant DLK kinase activity assay: Purified recombinant human DLK was incubated with GNE-3511 (0.01 nM to 1 μM) and a fluorescently labeled peptide substrate in kinase assay buffer at 30°C for 60 minutes; kinase activity was measured by detecting fluorescence polarization of phosphorylated substrate; IC50 values were calculated from dose-response curves [1]
- Kinase selectivity assay: GNE-3511 (1 μM) was screened against a panel of 46 human kinases (including MAP kinases, JNKs, and MLKs); kinase activity was assessed using the same fluorescent peptide assay protocol, and selectivity was determined by comparing inhibition rates relative to DLK [1]

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.

---

Primary cortical neuron axonal protection assay: Cortices from embryonic day 18 rat embryos were dissected, dissociated, and plated on poly-L-lysine-coated coverslips; neurons were cultured in NGF-containing medium for 7 days, then NGF was withdrawn and GNE-3511 (1-100 nM) was added; after 48 hours, neurons were fixed, immunostained for β-tubulin III (axon marker), and intact axon length was quantified by image analysis [1]
- Western blot for DLK downstream signaling: NGF-deprived cortical neurons were treated with GNE-3511 (0.3-10 μM) for 24 hours; cells were lysed, proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-JNK, JNK, p-c-Jun, c-Jun, and GAPDH (loading control); band intensities were quantified by densitometry [1]
- MBEC inflammatory response assay: Mouse bladder epithelial cells were seeded in 24-well plates and cultured to confluence; cells were pretreated with GNE-3511 (1-10 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 6 hours; total RNA was extracted, reverse-transcribed to cDNA, and TNF-α/IL-6 mRNA levels were measured by qPCR (normalized to GAPDH) [2]

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

---

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]

---

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]

---

Mouse optic nerve crush model: 8-week-old C57BL/6 male mice were anesthetized, and the right optic nerve was crushed with forceps for 10 seconds; mice were randomly divided into 2 groups (n=10 per group): vehicle control and GNE-3511 30 mg/kg [1]
- GNE-3511 was formulated in 10% DMSO, 40% polyethylene glycol 400, and 50% saline; mice were administered the drug via intraperitoneal injection twice daily for 7 consecutive days, starting 1 hour post-injury [1]
- RGC survival assessment: At 7 days post-injury, mice were euthanized, retinas were isolated, and RGCs were labeled with a fluorescent tracer; RGC density was counted in retinal flat mounts under a fluorescence microscope [1]
- Cyclophosphamide-induced cystitis model: 8-week-old female C57BL/6 mice were intraperitoneally injected with cyclophosphamide (150 mg/kg) to induce cystitis; 24 hours later, mice were randomly divided into 3 groups (n=8 per group): vehicle control, GNE-3511 10 mg/kg, 30 mg/kg [2]
- GNE-3511 was formulated in 0.5% methylcellulose and 0.1% Tween 80 in water; mice were administered the drug via oral gavage once daily for 7 days [2]
- Cystitis assessment: At study end, mice were euthanized, bladders were excised and weighed; bladder tissue was homogenized for TNF-α protein quantification by ELISA, and paraffin sections were stained with HE for histological analysis [2]
- Nociception testing: Voiding frequency was recorded for 2 hours before euthanasia; pelvic hypersensitivity was assessed using von Frey filaments (0.02-4 g) to measure withdrawal threshold [2]

In rats, the bioavailability of GNE-3511 at an oral dose of 10 mg/kg was 42%[1]
- The terminal elimination half-life (t1/2) of GNE-3511 in rats was 3.2 hours and in mice it was 4.5 hours[1]
- After oral administration of 30 mg/kg to mice, the peak plasma concentration (Cmax) was 2.8 μg/mL and the time to peak concentration (Tmax) was 1 hour[2]
- GNE-3511 has good tissue penetration, with a brain-to-plasma concentration ratio of 0.7 in mice and a bladder-to-plasma concentration ratio of 1.2 in CIC mice[1][2]
- The plasma protein binding rate of GNE-3511 in human plasma was 92% (equilibrium dialysis method)[1]

In repeated-dose toxicity studies in rats (up to 100 mg/kg/day, intraperitoneal injection) and mice (up to 150 mg/kg/day, oral administration) over a period of 2 weeks, GNE-3511 did not cause significant changes in body weight, food intake, or clinical chemical parameters (ALT, AST, creatinine, BUN)[1][2]. - No histopathological abnormalities were observed in the major organs (brain, liver, kidney, heart, bladder) of animals receiving therapeutic doses[1][2]. - At concentrations up to 100 μM, GNE-3511 did not induce cytotoxicity in primary cortical neurons or MBECs after 72 hours of incubation[1][2]. - No significant drug interactions with CYP3A4, CYP2D6, or CYP2C9 were found in human liver microsomes.[1]

[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. Dileucine zipper kinase (DLK), expressed in sensory neurons, is involved in neuropathic pain. We hypothesized that neuronal DLK is involved in the regulation of inflammatory and nociceptive behaviors in cystitis. Sensory neuron DLK-deficient mice (cKO) were constructed by crossing DLK conditional knockout mice with mice expressing Cre recombinase under the control of the Advilin promoter. Cystitis was induced in these mice using cyclophosphamide (CYP). Nociceptive behaviors, bladder inflammation, and pathological changes following cystitis induction were assessed in control and cKO mice. Pharmacological inhibition of DLK using GNE-3511 further determined the role of DLK in CYP-induced cystitis. Neuronal DLK deficiency attenuated CYP-induced pain-like nociceptive behaviors and inhibited mast cell histamine release, spinal cord neuron activation, and bladder pathological changes. Neuronal DLK-deficient mice also exhibited reduced CYP-induced inflammatory responses and decreased c-Jun activation in the dorsal root ganglion (DRG). Pharmacological inhibition of DLK using GNE-3511 reproduced the effects of neuronal DLK deficiency in CYP-treated mice. Our study suggests that DLK is a potential target for treating neuropathic pain and cystitis-related bladder pathological changes. [2]

---

Dielusine zipper kinase (DLK, MAP3K12) has recently been identified as an important regulator of neuronal degeneration in various conditions. This paper describes the process of constructing highly effective and selective DLK inhibitors from lead compounds obtained from high-throughput screening. By using speculative hinge-binding interactions to infer binding modes and specific design parameters to optimize central nervous system drug molecules, we ultimately focused on di(pyridin-2-yl)amine compounds, which have both desirable pharmacological effects and good oral brain penetration. Our lead inhibitor GNE-3511 (26) exhibited concentration-dependent neurodegenerative protective effects in vitro and showed dose-dependent activity in two different animal disease models. These results suggest that specific pharmacological inhibition of DLK may have therapeutic potential in a variety of indications. [1]

---

GNE-3511 is a potent, selective, and orally bioavailable DLK inhibitor that has been developed for the treatment of neurodegenerative and inflammatory diseases. [1][2]
- Its mechanism of action involves inhibiting DLK-mediated activation of the JNK/c-Jun signaling pathway, thereby preventing axonal degeneration and reducing the production of pro-inflammatory cytokines. [1][2]
- GNE-3511 has shown neuroprotective activity in axonal injury models, supporting its potential application in diseases involving neuronal loss, such as Alzheimer's disease and glaucoma. [1]
- In cyclophosphamide-induced cystitis, GNE-3511 alleviates bladder inflammation and nociception by inhibiting DLK-dependent inflammatory signaling, suggesting its potential therapeutic value. Inflammatory Bladder Diseases [2]

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.

---

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