Hematopoietic cell kinase (HCK)

BT424 had more selective inhibition of HCK at EC50 of 12 uM as compared to other SFKs (Fig. 7B, C, D). Researchers performed cytotoxicity studies of BT424 and did not reveal any cytotoxicity at 50 uM for RAW264.7 macrophages and podocytes (Fig. 7E, F). Researchers performed stability test for BT424 by diluting 100 mM stock in DMSO to 25 uM with water and keep it at room temperature for 24 h, HPLC assay didn’t find degradation (less than 3%). These data indicate that researchers developed HCK more selected inhibitor BT424 and could be the target-to-hit compound.[1]

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HCK inhibitor BT424 decreased macrophage M1 polarization, proliferation and migration [1]
Firstly, researchers tested BT424 effects on autophagy in BMDMs. BT424 is insoluble in water, researchers made the stock in DMSO with 100 mM and diluted to 25 uM in medium to treat cells. Researchers found that BT424 treatment could increase autophagy activity with higher LC3II/LC3I ratio and lower P62 levels (Fig. 8A). Inhibition of HCK activation with BT424 decreased macrophage M1 pro-inflammatory polarization and increased M2 polarization in BMDM as shown by western blot of M1 and M2 markers of iNOS and CD206 (Fig. 8B). Next, researchers measured the effects of BT424 on macrophage proliferation. Researchers first performed MTT assay in Raw264.7 (Fig. 8C) cells and found that BT424 significantly decreased proliferation of these cells. Researchers also used propidium iodide (PI) in Raw264.7 to measure cell cycle and found BT424 treatment increased G0/G1 phases and decreased G2/M phases (Fig. 8D). Click-iT™ EdU cell proliferation assay (Fig. 8E) and Ki67 IF staining (Fig. 8F) were performed in Raw264.7. EdU and Ki67 positive cells were decreased in BT424 treatment, compared to control cells. Researchers performed scratch assay in Raw264.7 cells and found that BT424 significantly decreased migration of these cells (Fig. 8G). These data demonstrated that by inhibiting HCK activity BT424 could inhibit macrophage M1 pro-inflammatory polarization, proliferation and migration.[1]

HCK specific inhibitor BT424 ameliorate inflammation and kidney fibrosis in UUO model [1]
Researchers first tested the toxicity of the HCK inhibitor by daily gavage in WT mice with BT424 at 25 mg/kg for 1 month. They did not observe any obvious toxicity based on the body weight changes, behavior, and physical activity of the mice. In addition, Researchers performed Routine Chemistry Panel test from IDEXX BioAnalytics (Westbrook, ME 04092) with the serum from vehicle- and BT424-treated mice and did not see noticeable changes in liver enzymes, renal function, lipid profile, and glucose levels (Supplementary Table S1). These data indicate that treatment of BT424 does not induce significant toxicity in these mice. Researchers then tested the effect of BT424 in UUO-induced fibrosis. The mice were treated with BT424 at 25 mg/kg or vehicle (n = 5) by daily gavage starting one day prior to the surgery to 7 days post-surgery. In H&E staining, we found that BT424 treatment reduced morphologic tubular injury in the UUO kidney vs vehicle-treated mice (Fig. 9A). Masson and Col1A1-IF staining confirmed that BT424 treatment reduced renal fibrosis in the UUO kidneys (Fig. 9A, B). Col1a1, Fibronectin and FSP-1 mRNA expression were significantly decreased in the UUO kidney of BT424-treated mice (Fig. 9C). F4/80-positive macrophage population was significantly decreased in BT424 treated mice with UUO (Fig. 9D). Western blot demonstrated that iNOS decreased due to the reduction of total macrophage number and reducing M1 polarization (Fig. 9E). CD206 reduced in BT424 treated kidneys due to the reduction of total macrophage number but increased after macrophage number adjustment as BT424 regulating macrophage suggesting skewing from M1 toward M2 polarization (Fig. 9E). Autophagy activity was increased in macrophages from the UUO kidneys of BT424 treated mice as reflected by co-staining LC3 and F4/80 and quantification of LC3 in macrophages (Fig. 9F). These data confirmed that BT424 attenuates fibrosis by reduced macrophage number and reduce macrophage inflammatory M1 polarization through autophagy in UUO model.

Autophagy measurement [1]
Autophagy LC3 HiBiT reporter was transfected to HEK293 cells with PloyJet following the manufacture’s protocol. Then LC3 reporter was detected with Nano-Glo HiBiT fluorescent reagent. The fluorescent signal was adjusted by load protein amount. For Raw264.7 and BMDM, autophagy inducer PP242 at 1 uM, autophagosome degradation inhibitor Bafilomycin A1 (BafA1) at 100 nM, and autophagy inhibiter 3-Methyladenine (3MA) at 5 mM treated for 24 h for LC3 and P62 measurement with WB. HCK inhibitors BT424 and dasatinib were added to pretreat cells 2 h before autophagy stimulation.

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EGFR and integrin signaling pathway studies [1]
BMDMs from WT and HCK KO mice were starving overnight with 1% FBS DMEM with L929 on day 5. Then EGF at 100 ng/ml was used to treat cells for 15 and 30 min before harvesting on ice with cold PBS wash. The cell lysate was then used for western blot assay to measure EGFR signaling-related proteins. For the integrin signaling, BMDMs were plated to collagen pre-coated 10 cm dishes for 1 h following the paper16. F-actin and pY20 with SYK were measured by IF stain and western blot.

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Chemokine array [1]
The chemical array was performed with cultured medium from BMDM from WT and HCK KO mice using the Proteome Profiler Mouse Chemokine Array Kit following the manufacturer’s instructions. BMDMs at 7-day were polarized to M1 with 100 ng/ml LPS with 50 ng/ml IFNγ for 24 h, WT BMDM were pre-treated with HCK inhibitor dasatinib for 2 h before M1 polarization.

MTT and propidium iodide flow for cell proliferation [1]
BMDMs cultured for 4 days were used for MTT and PI flow assay. Propidium Iodide staining solution and MTT were used and following the manufactory’s protocols. Attune Flow Cytometer and FlowJo v10.8.0 were used for flow data analysis. BT424 treated Raw 264.7 for 24 h before the cell proliferation assay.

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2D and 3D migration assay [1]
Scratch Assays. RAW 264.7 cells and BMDMs were plated on 6 well plates one day prior to the assay day and starved overnight. Starving medium with 0.5% FBS was used to reduce cell proliferation. Scratches were made by using 200 ul pipet tips. Starving medium with 0.5 FBS was replaced for full culture medium to reduce the cell proliferation. Images were taken at 0 h, 1 h, 3 h, 6 h, 12 h and 24 h after the scratch. The distance of unhealed area was measured with ImageJ package “Wound_healing_size_tool”.

Animal studies [1]
HCK exon3 loxp flanked KO mouse were developed at EuMMCR in Germany (HCK ES Cell Clone: HEPD0510). These mice were crossed with tissue specific Cre mice (CMV-cre B6.C-Tg (CMV-cre)1Cgn/J006054; LysM-cre B6.129P2-Lyz2tm1(cre)Ifo/J) to generate HCK KO specifically in tubular cells and macrophages. C57BL/6 J WT mice also from Jackson Laboratory. All mice were maintained in our animal facility at Mount Sinai under controlled environmental conditions: 12/12 light/dark cycle, ambient temperature 20–25 °C.
UUO model in WT, HCK KO, and HCK inhibitor mice was performed following our previous paper14. BT424 was made in 250 mg/ml in DMSO as stock, then was diluted to 2.5 mg/ml with PBS before gavage mice for final concentration of 25 mg/kg body weight. Unilateral IRI with contralateral nephrectomy (uIRIx) model was performed following the papers69,70, briefly, artery and vein of right kidney was tied, and the right kidney was removed. Then the left kidney’s artery and vein were clamped for 25 min and released. At the end of mice models, the mice were IP injected with 100 mg/kg ketamine and 10 mg/kg xylazine and then perfused with cold PBS for tissue collection.

[1]. HCK induces macrophage activation to promote renal inflammation and fibrosis via suppression of autophagy. Nat Commun. 2023 Jul 18;14(1):4297.

Renal inflammation and fibrosis are common pathways leading to the progression of chronic kidney disease (CKD). We previously found that hematopoietic cell kinase (HCK) is upregulated in human chronic allogeneic transplantation injury, promoting renal fibrosis; however, its cellular origin and molecular mechanisms remain unclear. In this study, using immunostaining and single-cell sequencing data, we found that HCK is highly enriched in pro-inflammatory macrophages of diseased kidneys. HCK knockout (KO) or HCK inhibitors reduced macrophage M1-like pro-inflammatory polarization, proliferation, and migration in RAW264.7 cells and bone marrow-derived macrophages (BMDM). We found that HCK interacts with two autophagy-related proteins, ATG2A and CBL, thereby inhibiting macrophage autophagy flux. In vivo, both systemic and myeloid-specific HCK knockout reduced renal inflammation and fibrosis, specifically manifested as reduced macrophage numbers, pro-inflammatory polarization, and decreased migration to unilateral ureterally obstructed (UUO) kidneys and unilateral ischemia-reperfusion injury (IRI) models. In addition, we developed a selective boron-containing HCK inhibitor that reduced the pro-inflammatory activity, proliferation and migration of macrophages in vitro and alleviated renal fibrosis in UUO mice. This study elucidated the downstream mechanism by which HCK regulates macrophage activation and polarization in CKD through autophagy and pointed out that selective HCK inhibitors are promising new therapies for the treatment of renal fibrosis. [1] In summary, we revealed that SFK HCK participates in the progression of renal interstitial fibrosis/tubular atrophy (IF/TA) in CKD and chronic kidney disease (CAI) by regulating autophagy within macrophages, altering their polarization, proliferation and migration to damaged kidneys. We also developed a non-toxic, specific HCK inhibitor that targets the lead compound BT424 and demonstrated that it can modulate macrophage function to alleviate progressive renal fibrosis. [1] Our study showed that among the SFK family members, HCK expression is highly regulated in macrophages of diseased kidneys. On the other hand, FYN is mainly expressed in podocytes and regulates podocyte function by phosphorylating renin. We have previously demonstrated that dasatinib can induce proteinuria in mice with lupus nephritis by inhibiting FYN-induced podocyte injury. Therefore, developing more selective HCK inhibitors that do not affect FYN activity is crucial for us. This paper describes a relatively selective HCK inhibitor, BT424, which does not affect podocyte injury, and we therefore believe that BT424 is a better HCK-targeting drug for treating patients with kidney disease with anti-inflammatory and anti-fibrotic effects.
Our study has some limitations. We confirmed that HCK is the major SFK in macrophages and is highly regulated in kidney disease, but we cannot rule out the role of other SFK members in kidney disease. We also recognize that macrophages have multiple functions in kidney disease. Recently, studies have reported the transdifferentiation of macrophages into myofibroblasts, and we will examine whether this process is also regulated by HCK in future studies. In addition, in HCK knockout mice with unilateral ureteral obstruction (UUO), the total number of F4/80 positive cells, including macrophages (infiltrating and resident) and some dendritic cells, was significantly reduced. Future research will focus on the interaction between macrophages, renal tubular cells and fibroblasts in the context of HCK knockout. LysM-cre is not macrophage-specific because the LysM promoter is also expressed in neutrophils. Previous studies have shown that HCK regulates the activation and migration of neutrophils. Therefore, further research is needed to distinguish the role of HCK in macrophages and neutrophils during renal inflammation and fibrosis. We treated mice with the HCK inhibitor BT424 before injury, and the results showed that the inhibitor could prevent renal injury or fibrosis in these mouse models. Further research is needed to determine whether treatment of mice after the onset of the disease can reverse renal injury and fibrosis. In summary, we have demonstrated that HCK plays a key role in regulating macrophage function in the context of renal inflammation and fibrosis. By constructing systemic and cell-specific HCK knockout models and developing selective boron-containing HCK inhibitors, we have demonstrated the potential value of this pathway in the treatment of progressive kidney disease. [1]

C22H15BCL2N2O2

421.08

420.0603

2755180-37-9

168510578

White to off-white solid powder

1

3

3

29

649

0

B1(N=C(NO1)C2=CC3=C(C(=CC(=C3)Cl)Cl)OC2C4=CC=CC=C4)C5=CC=CC=C5

SYIZHZBXDOQNIR-UHFFFAOYSA-N

InChI=1S/C22H15BCl2N2O2/c24-17-11-15-12-18(22-26-23(29-27-22)16-9-5-2-6-10-16)20(14-7-3-1-4-8-14)28-21(15)19(25)13-17/h1-13,20H,(H,26,27)

4-(6,8-dichloro-2-phenyl-2H-chromen-3-yl)-2-phenyl-5H-1,3,5,2-oxadiazaborole

BT424; BT-424; 2755180-37-9;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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