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. 2024 Jul 15;7(10):e202402625.
doi: 10.26508/lsa.202402625. Print 2024 Oct.

c-KIT inhibitors reduce pathology and improve behavior in the Tg(SwDI) model of Alzheimer's disease

Max Stevenson  1 , Michaeline L Hebron  1 , Xiaoguang Liu  1 , Kaluvu Balaraman  2 , Christian Wolf  2 , Charbel Moussa  3
Affiliations

c-KIT inhibitors reduce pathology and improve behavior in the Tg(SwDI) model of Alzheimer's disease

Max Stevenson et al. Life Sci Alliance. .

Abstract

Treatments for Alzheimer's disease have primarily focused on removing brain amyloid plaques to improve cognitive outcomes in patients. We developed small compounds, known as BK40143 and BK40197, and we hypothesize that these drugs alleviate microglial-mediated neuroinflammation and induce autophagic clearance of neurotoxic proteins to improve behavior in models of neurodegeneration. Specificity binding assays of BK40143 and BK40197 showed primary binding to c-KIT/Platelet Derived Growth Factor Receptors (PDGFR)α/β, whereas BK40197 also differentially binds to FYVE finger-containing phosphoinositide kinase (PIKFYVE). Both compounds penetrate the CNS, and treatment with these drugs inhibited the maturation of peripheral mast cells in transgenic mice, correlating with cognitive improvements on measures of memory and anxiety. In the brain, microglial activation was profoundly attenuated and amyloid-beta and tau were reduced via autophagy. Multi-kinase inhibition, including c-KIT, exerts multifunctional effects to reduce neurodegenerative pathology via autophagy and microglial activity and may represent a potential therapeutic option for neurodegeneration.

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Conflict of interest statement

C Moussa, K Balaraman, and C Wolf, are inventors on several US and international Georgetown University patents to use BK40143 and BK40197 and other tyrosine kinase inhibitors as a treatment for neurodegenerative diseases. Georgetown University spun out the technology (April 2020) to a start‐up company, KeifeRx, in which it holds equity and for which C Moussa is a co-founder, shareholder, and consultant. The remaining authors do not have any potential conflicts of interest to declare.

Figures

None
Graphical abstract
Figure 1.
Figure 1.. BK40143 and BK40197, two novel c-KIT inhibitors.
(A, B, C, D) Chemical structures of (A) BK40143 and (B) BK40197, two novel inhibitors of c-KIT, with corresponding competitive binding assay values for select kinases ((C) BK40143, (D) BK40197). Source data are available for this figure.
Figure 2.
Figure 2.. In vitro target engagement analysis of BK40143 and BK40197.
(A, B, C, D) Flow cytometric analysis of SH-SY5Y cells transduced with α-synuclein revealed increased production of autophagic vacuoles in cells treated with (A, B) BK40143 (P = 0.001 two-tailed t test) and (C, D) BK40197 (P = 0.004, two-tailed t test). Source data are available for this figure.
Figure 3.
Figure 3.. BK40143 and BK40197 inhibit mast cell proliferation in vivo.
(A, B, C, D, E) Flow cytometric analysis using antibodies conjugated against c-KIT (CD117) and FcεR1 revealed that TgAPP mice injected with DMSO displayed (A) decreased ratios of immature versus mature mast cells compared with mice treated with (B, D) 5 mg/kg BK40143 (P = 0.03, two-tailed t test) and (C, E) 45 mg/kg BK40197 (P = 0.07, two-tailed t test). (F, G, H) Western blot revealed decreased levels of phospho-c-KIT in the brains of TgAPP mice treated with (G) 5 mg/kg BK40143 (P = 0.03, two-tailed t test) and (H) 45 mg/kg BK40197 (P = 0.19, two-tailed t test). Source data are available for this figure.
Figure 4.
Figure 4.. Pharmacokinetic properties of BK40197.
(A, B) Pharmacokinetic analysis revealed (A, B) brain penetrance of BK40197 at multiple doses. (C, D) Administration of (C) BK40143 and (D) BK40197 in WT mice revealed the maximum tolerable dose for each drug in vivo. Source data are available for this figure.
Figure 5.
Figure 5.. BK40143 and BK40197 improve behavioral outcomes in animal models of proteinopathies.
(A, B) Male (blue) and female (red) 12-mo-old TgAPP mice tested via novel object recognition displayed significantly improved outcomes when treated with (A) 5 mg/kg BK40143 (P = 0.0003) or (B) 45 mg/kg BK40197 (P = 0.006) compared with DMSO-treated controls. (C, D, E, F) These mice also displayed improved recall during (C) Morris water maze training trials (P = 0.04), whereas mice treated with 5 mg/kg BK40143 showed similar improvements on (D, E, F) Morris water maze endpoint measures (time in correct quadrant: P = 0.02, quadrant entries: P = 0.03. platform latency: P = 0.05). (G, H, I, J) TgAPP mice treated with 45 mg/kg BK40197 (G) spend more time in the open arm of the elevated plus maze compared to DMSO-treated controls (P = 0.02). 12-mo-old A53T mice treated with 5 mg/kg BK40143 display improved outcomes on (H) novel object recognition (P = 0.03), while A53T mice treated with (I) 5 mg/kg BK40143 (P = 0.04) or (J) 45 mg/kg BK40197 (P = 0.05) showed significant improvements in nesting behavior. Source data are available for this figure.
Figure 6.
Figure 6.. BK40143 and BK40197 reduce amyloid-beta concentrations and induce autophagy in vivo.
(A, B, C, D, E, F, G) TgAPP mice injected with DMSO displayed significantly greater levels of amyloid-beta (A) 3, 3′ diaminobenzidine staining 20x, (B) AlexaFluor 488 40x compared with mice treated with (C, D) 5 mg/kg BK40143 or (E, F) 45 mg/kg BK40197, quantified in (G) (143: P = 0.001, 197: P ≤ 0.0001). (H, I) ELISA for (H) amyloid-beta in TgAPP mice (1.25 mg/kg: P = 0.0001, 2.5 mg/kg: P = 0.05) and (I) pTau in Tg4510 mice (1.25 mg/kg: P = 0.05, 2.5 mg/kg: P = 0.04) treated with BK40143 revealed decreases in protein levels compared with DMSO-treated controls. (J, K) ELISA for (J) amyloid-beta in TgAPP mice (P = 0.003) and (K) pTau in Tg4510 mice (5.0 mg/kg: P = 0.03) treated with BK40197 revealed decreases in protein levels compared with DMSO-treated controls. (L, M, N, O) Western blot revealed increased beclin-1 expression in TgAPP mice treated with (L, N) 5 mg/kg BK40143 (P = 0.004) and (M, O) 45 mg/kg BK40197 (P = 0.05).
Figure 7.
Figure 7.. BK40143 and BK40197 alleviate microglial inflammatory morphology.
(A, B, C, E, F, G, I, J, K) Immunohistochemical staining of IBA1+ microglia revealed increased staining intensity in TgAPP mice treated with DMSO ((A) 20x, (B) 40x, (C) 63x z-stack) compared with mice treated with (E, F, G) 5 mg/kg BK40143 and (I, J, K) 45 mg/kg BK40197. (D, H, L, M, N) IMARIS 3D reconstruction and analysis revealed microglia from (D) DMSO-treated mice displayed significantly greater surface area than microglia from mice treated with (H, M) 5 mg/kg BK40143 (P = 0.01) and (L, N) 45 mg/kg BK40197 (P = 0.004).
Figure S1.
Figure S1.. BK40143 and BK40197 reduce microglial PAR2-tryptase in TgAPP mice.
(A, B, C, D) Proximity ligation assay showing direct PAR2-tryptase interactions in (A, D) DMSO-treated mice compared with (B, D) BK40143-treated and (C, D) BK40197-treated mice compared with. One-tailed unpaired t test.
Figure S2.
Figure S2.. c-KIT is expressed on microglia in Tg APP mice.
(A, B, C, D) Immunohistochemical staining for (A) IBA1 and (B) c-KIT revealed (C, D) expression of c-KIT on microglia in TgAPP mice.
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