VX-765 (Belnacasan)

Biological Activity

VX-765 is a novel and irreversible IL-converting enzyme/caspase-1 inhibitor with an IC50 of 0.8 nM. VRT-043198, a potent and selective inhibitor of ICE/caspase-1 sub-family caspases is an active metabolite VX-765. VRT-043198 exhibits 100-10,000-fold selectivity against other caspase-3 and -6-9. VX-765 was efficiently converted to VRT-043198 when administered orally to mice and inhibited LPS-induced cytokine secretion. VX-765 has been shown to inhibit acute partial seizures in preclinical models and has shown activity in preclinical models of chronic partial epilepsy that do not respond to currently available medicines for epilepsy. In addition, VX-765 reduced disease severity and the expression of inflammatory mediators in models of rheumatoid arthritis and skin inflammation.

Product Citations

• 

  EMBO Mol Med. 2020 Jan 9;12(1):e9386.

  Glutathione peroxidase 8 negatively regulates caspase-4/11 to protect against colitis.
  VX-765 (Belnacasan) purchased from AbMole

Customer Product Validations & Biological Datas

	Source	EMBO Mol Med (2020 Jan). Figure 6. VX-765 (AbMole BioScience, Houston, TX, USA)
	Method	intraperitoneal injections
	Cell Lines	male GPx8-/- mice
	Concentrations	20 mg/kg
	Incubation Time	14 days
	Results	Consistently, VX-765 treatment was also associated with lower mortality and less disease severity in mice with DSS-induced colitis.

	Source	Brain Behav Immun (2016). Figure 7. VX-765
	Method	i.v.
	Cell Lines	C57BL/6 mice
	Concentrations	25 mg/kg/day and 50 mg/kg/day
	Incubation Time	four weeks
	Results	As shown in Fig. 7A–B, with administration of VX-765 (25 mg/kg/day and 50 mg/kg/day), the mice showed decreased levels of mature IL-1b and IL-18 as compared to those with vehicle treatment (P < 0.01).

	Source	Brain Behav Immun (2016). Figure 6. VX-765
	Method	i.v.
	Cell Lines	C57BL/6 mice
	Concentrations	50 mg/kg/day
	Incubation Time	four weeks
	Results	Administration of VX-765 ameliorated depression- and anxiety-like behavior in OVX mice. VX-765 (50 mg/kg/day) significantly increased percentage of sucrose consumption in OVX mice as compared to vehicletreated OVX group.

Protocol

Cell Experiment
Cell lines	human PBMCs
Preparation method	A total of 2×105 cells/well (100 μL cell suspension) is distributed in triplicate in flat-bottom 96-well plates. Either 50 μL of Belnacasan (40 μM in RPMI 1640 complete medium containing 0.2% DMSO) or vehicle control is added to appropriate wells. Following a 30-min incubation at 37°C, 50 μL of LPS diluted in RPMI 1640 complete medium is added at final concentrations varying from 0.001 to 10 ng/mL. Cells are returned to a 37°C incubator. At 4 h after LPS addition, 75 μL of supernatant is removed from wells, cleared by centrifugation for 5 min at 1500 rpm, and stored at 4°C until assayed. Cells are returned to a 37°C incubator until 24 h after LPS addition, at which time 100 μL of supernatant is removed, cleared by centrifugation, and stored at 4°C. Supernatants are tested using ELISA kits for IL-1β, IL-6, IL-18, and IL-1α.
Concentrations	40 μM
Incubation time	4 h, 24 h

Animal Experiment
Animal models	Collagen-Induced Arthritis Model in Mouse
Formulation	25% Cremophor EL
Dosages	10-100 mg/kg twice daily
Administration	oral gavage

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

Chemical Information

References

Resibufogenin suppresses growth and metastasis through inducing caspase-1-dependent pyroptosis via ROS-mediated NF-κB suppression in non-small cell lung cancer
Hui Yin, et al. Anat Rec (Hoboken). 2021 Feb;304(2):302-312. PMID: 32396707.

Polyphyllin VI Induces Caspase-1-Mediated Pyroptosis via the Induction of ROS/NF-κB/NLRP3/GSDMD Signal Axis in Non-Small Cell Lung Cancer
Jin-Feng Teng, et al. Cancers (Basel). 2020 Jan 13;12(1):193. PMID: 31941010.

(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765), an orally available selective interleukin (IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting the release of IL-1beta and IL-18.
Wannamaker W, et al. J Pharmacol Exp Ther. 2007 May;321(2):509-16. PMID: 17289835.