F3II (IC50 = 4 µM); MDA-MB-231 (IC50 = 21 µM); Rac1; Apoptosis
RAC1 (Ras-related C3 botulinum toxin substrate 1) GTPase, specifically its interaction with Guanine Exchange Factors (GEFs) such as TIAM1, Vav1, Vav2, Vav3, and DBL.
The binding is dependent on the Tryptophan 56 (W56) residue of RAC1.

1A-116 (48 hours) has a concentration-dependent effect on the proliferation of MDA-MB-231 and F3II cells, with IC50 values of 4 μM and 21 μM, respectively [1]. 1A-116 (1, 10 μM; 12 h) dramatically increases Rac1 in F3II cells and, in a concentration-dependent way, decreases intracellular Rac1-GTP levels [1]. 1A-116 bursts the Rac1-P-Rex1 response at 50 and 100 μM over a 12-hour period [1]. LN229 cell proliferation is inhibited in a circadian manner by 1A-116 (20 μM; 5-hour interval over 25 h) [2]. At 10 HPS, 1A-116 (10 μM; 16 h) dramatically decreased cell migration, demonstrating temporal regulation. Hours post synchronization (HPS): Following serum shock, the elapsed time is measured in hours [2]. 1A-116 (20, 50 μM; 6 h) senses sterilization of cells in a manner that is dependent on the circadian rhythm [2]. At the GEF-Rac1 level, 1A-116 (100 nM) shows inhibitory Rac1 activity and decreases the thickness of the flipped layer mediated by Vav2 and Rac1, but not PAK1 [3]. 1][2]

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In LN229 human glioblastoma cells, 1A-116 (20 µM) inhibited proliferation in a circadian-dependent manner, with maximum inhibition at 10 hours post-synchronization (HPS) and minimum at 23 HPS. The IC50 for proliferation inhibition was 10.93 ± 0.9 µM at 10 HPS and 30.85 ± 1.78 µM at 23 HPS. These circadian effects were lost in BMAL1-deficient LN229E1 cells. [2]
In LN229 cells, 1A-116 (20 µM for 6 hours) induced early apoptosis (Annexin V staining) more effectively when applied at 10 HPS compared to 23 HPS. At a higher concentration (50 µM), the time-dependent difference was not statistically significant. [2]
1A-116 (10 µM) significantly inhibited LN229 cell migration into a cell-free area only when applied at 10 HPS, not at 23 HPS. This concentration showed no effects on cell viability. [2]
In COS-1 cells, 1A-116 (50 µM for 24h) inhibited SRE-activation mediated by constitutively active Rac1 (Q61L) by approximately 40%. It also inhibited SRE-activation by active versions of GEFs: Vav1 Δ1-189 (40-50%), Vav2 Δ1-186 (40-50%), Vav3 DH-PH-ZF (40-50%), oncogenic Dbl (60%), and Tiam1 C1199 (75%). [3]
1A-116 (50 µM) inhibited the activity of the oncogenic Rac1 P29S mutant in an SRE-luciferase reporter assay. [3]
In a pull-down assay using COS-1 cells expressing Rac1 Q61L, treatment with 1A-116 (50 µM for 24h) decreased the levels of active Rac1-GTP. [3]
In COS-1 cells expressing Rac1 Q61L-EGFP, 1A-116 (50 µM for 16h) abolished the formation of peripheral ruffles and lamellipodia induced by Rac1. [3]
In 3D organotypic cultures of human keratinocytes (Ker-CT), 1A-116 (100 nM) reversed the hyperplastic and disorganized epithelium caused by the stable overexpression of active Vav2 or Rac1 F28L, but did not affect the phenotype driven by active PAK1 (Y423). [3]

1A-116 (intravenous; 3 mg/kg; once day for 21 days) showed strong anti-metastatic action without causing appreciable damage, with a 60% decrease in the total number of metastatic lung colonies in vivo. [1] 1A -116 (20 mg/kg; intraperitoneal; once daily; for ZT12, 73 days; for ZT3, 68 days) In tumor-bearing mice, treatment with ZT12 increased longevity in comparison to ZT3. Zeitgeber Time 12 (ZT12) denotes the time at which the lights go out (7 p.m. tonight), while ZT0 denotes the time at which they come on (7 a.m. tonight). [2]. Good wall availability is shown in 1A-116 [3].

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In a nude mouse model bearing intracranial LN229 human glioblastoma xenografts, daily intraperitoneal injection of 1A-116 (20 mg/kg) at ZT12 (lights off) significantly increased median survival (73 days) compared to treatment at ZT3 (lights on, 68 days) or vehicle groups (ZT12: 64.5 days, ZT3: 63.5 days). [2]

In silico docking experiments were performed using AutoDock Vina. The binding pocket was centered on the C-alpha of the W56 residue of Rac1 with a grid size of 14 Å. 1A-116 was docked against 50 conformers of Rac1 from the PDB, showing a mean binding affinity of -6.02 ± 0.315 kcal/mol. [3]
Docking to the maximum RMSD pair of Rac1 conformers (1E96A and 2YINC) showed that 1A-116 forms a hydrogen bond with W56 via its guanidine group. The binding affinity was -5.867 ± 0.1803 kcal/mol for 1E96A and -6.122 ± 0.2539 kcal/mol for 2YINC. [3]
Docking experiments with Rac1 W56F and Cdc42 F56W mutants showed reduced binding affinity for Rac1 W56F (-5.59 ± 0.0139 kcal/mol vs. -6.08 ± 0.226 kcal/mol for wild-type) and increased affinity for Cdc42 F56W (-6.09 ± 0.00994 kcal/mol vs. -5.69 ± 0.0170 kcal/mol for wild-type Cdc42), confirming the critical role of W56. The predicted binding affinity for the Rac1 P29S mutant was -6.18 ± 0.0402 kcal/mol. [3]

Cell Proliferation Assay[1][2]
Cell Types: MDA-MB-231, F3II, LN229 Cell
Tested Concentrations: 20 µM
Incubation Duration: 48 hrs (hours); 5 hrs (hours) apart within 25 hrs (hours).
Experimental Results: Inhibition of cell proliferation in a concentration-dependent and circadian manner.

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Cell viability assay [3]
Cell Types: Ker-CT Human Keratinocytes with Oncogenic Vav2/Rac1 F28L/PAK1 Tyrosine 423
Tested Concentrations: 100 nM
Incubation Duration:
Experimental Results: Inhibition of Rac1 activity at the GEF-Rac1 level.

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Cell migration assay[2]
Cell Types: LN229 Cell
Tested Concentrations: 10 µM
Incubation Duration: 16 hrs (hours)
Experimental Results: Cell migration was diminished at 10 HPS, showing time dependence.

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Apoptosis analysis[2]
Cell Types: LN229 Cell
Tested Concentrations: 20, 50 µM
Incubation Duration: 6 hrs (hours)
Experimental Results: Apoptosis is induced in a circadian rhythm-dependent manner.

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Western Blot Analysis[1]
Cell Types: F3II Cell
Tested Concentrations: 1, 10 µM
Incubation Duration: 12 hrs (hours)
Experimental Results: Blocks Rac1-P-Rex1 interaction. Reduces intracellular Rac1-GTP levels in a concentration-dependent manner.

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For proliferation assays, LN229 or LN229E1 cells (3x10^3 cells/well in 96-well plates) were synchronized by a 2-hour serum shock (50% FBS). At different hours post-synchronization (HPS), cells were treated with 1A-116 (0-100 µM) or vehicle for 72h. Cell growth was measured by 0.1% crystal violet uptake (absorbance at 570 nm) or Calcein AM staining (fluorescence at 520 nm). Inhibition of proliferation (Ip) was calculated as Ip = 1 - (drug-treated uptake / vehicle-treated uptake). [2]
For viability assays, synchronized LN229 cells (2.5x10^3 cells/well in 96-well plates) were treated with 1A-116 (5 or 10 µM) or vehicle at 10 or 23 HPS. Cell viability was measured 16h later by counting Calcein AM-stained fluorescent cells. [2]
For migration assays, LN229 cells (2.5x10^5 cells/well in 12-well plates) were synchronized and plated with a silicon stopper to create a cell-free area. The stopper was removed at treatment time, and cells were treated with 1A-116 (10 µM) or vehicle at 10 or 23 HPS. After 16h, cells were stained with Calcein AM, and the area covered by migrated cells was measured using ImageJ software. [2]
For apoptosis assays, synchronized LN229 cells (3x10^3 cells/well in 96-well plates) were treated with 1A-116 (20 or 50 µM) or vehicle for 6h at 10 or 23 HPS. Early apoptosis was measured using fluorescent-labeled Annexin V. [2]
For quantitative real-time PCR (qPCR), synchronized LN229 cells (8x10^5 cells/well in 6-well plates) were harvested at 4h intervals over 24h. RNA was extracted, and cDNA was synthesized. qPCR was performed using SYBR Green to measure mRNA levels of Bmal1, Per1, Rac1, Tiam1, normalized to Actin. [2]
For In-Cell Western assays, synchronized LN229 cells (3x10^3 cells/well in 96-well plates) were fixed at 3h intervals over 24h and stained with monoclonal antibodies against TIAM1, BMAL1, or PER1. [2]
For SRE-luciferase assays, COS-1 cells (4x10^5 cells/well in 6-well plates) were co-transfected with plasmids encoding active GEFs or GTPases, along with SRE-Luc and Renilla reporter plasmids. Cells were then treated with 1A-116 (50 µM) or vehicle for 24h, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System. [3]
For Rac1 pull-down assays, COS-1 cells expressing Rac1 Q61L were treated with 1A-116 (50 µM) or vehicle for 24h. Lysates were incubated with GST-PAK1 coupled to Glutathione Sepharose beads to pull down active Rac1-GTP. Bound Rac1 was detected by western blot with an anti-Rac1 antibody. [3]
For 3D organotypic cultures, Ker-CT cells (2x10^5) were seeded on polycarbonate inserts and cultured for 2 days, then switched to 3D-Barrier medium and air-lift was performed. On day 6 post air-lift, cultures were treated with 1A-116 (100 nM). After 11 days, cultures were fixed, paraffin-embedded, sectioned, and stained with hematoxylin and eosin. [3]
For immunofluorescence, COS-1 cells expressing Rac1 Q61L-EGFP were seeded on polylysine-coated coverslips and treated with 1A-116 (50 µM) or vehicle for 24h. Cells were fixed, stained with Alexa Fluor 635-phalloidin (for F-actin) and DAPI (for nuclei), and analyzed by confocal microscopy. [3]

Animal/Disease Models: Female BALB/c inbred mice (8 to 10 weeks old; average 20 g) [1]
Doses: 3 mg/kg
Route of Administration: intravenous (iv) (iv)injection; one time/day for 21 days.
Experimental Results: demonstrated high antimetastatic activity.

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Animal/Disease Models: Male NIH Swiss FoxN1(Δ/Δ) nude mice (2 months old; GBM model) [2].
Doses: 20 mg/kg
Route of Administration: intraperitoneal (ip) injection (ZT3, ZT12); one time/day, ZT12 for 73 days, ZT3 for 68 days.
Experimental Results: ZT12-treated tumor-bearing mice survived longer compared with ZT3 treatment.

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For the glioblastoma xenograft model, 2-month-old male athymic nude mice (NIH Swiss foxN1Δ/Δ) were housed under a 12h light:12h dark cycle (lights on at ZT0, local time 7 a.m.; lights off at ZT12, local time 7 p.m.). Animals were kept for one week for acclimation. Approximately 2x10^5 viable LN229 cells were implanted intracranially into the right striatum using a stereotaxic device (coordinates from Bregma: ML: -2, AP: 0, DV: -3.4). Surgeries were performed between ZT3 and ZT11. Mice were allowed to recover for at least 7 days, receiving subcutaneous ampicillin (6 mg/kg) and ibuprofen (0.05 mg/mL) in drinking water. For chronopharmacological treatment, mice received a daily intraperitoneal injection of 1A-116 (20 mg/kg in 200 µL vehicle, which was H₂O) or vehicle at either ZT3 or ZT12. Survival time was measured twice a week, and the presence of the tumor was confirmed by necropsy and histological analysis with cresyl violet staining. [2]

[1]. Preclinical development of novel Rac1-GEF signaling inhibitors using a rational design approach in highly aggressive breast cancer cell lines. Anticancer Agents Med Chem. 2014;14(6):840-51.

[2]. Timing of Novel Drug 1A-116 to Circadian Rhythms Improves Therapeutic Effects against Glioblastoma. Pharmaceutics. 2021 Jul 16;13(7):1091.

[3]. Computational and in vitro Pharmacodynamics Characterization of 1A-116 Rac1 Inhibitor: Relevance of Trp56 in Its Biological Activity. Front Cell Dev Biol. 2020 Apr 15;8:240.

1A-116 is a small molecule Rac1 inhibitor developed by rational design using a docking-based virtual screening approach. It is designed to block the protein-protein interaction (PPI) between Rac1 and its GEFs by targeting the Trp56 residue on Rac1. [3]
1A-116 meets Lipinski's "Rule of Five" and Veber's rules for drug-likeness (e.g., MW 307.32 g/mol, predicted logP 4.67, 2 H-bond donors, 3 H-bond acceptors, 3 rotatable bonds, polar surface area 50.41 Ų), suggesting good oral bioavailability potential. [3]
The compound has shown pro-apoptotic and anti-invasive activity in malignant glioma cells and anti-proliferative effects in various cancer cell lines. Its efficacy is modulated by the circadian clock through the rhythmic expression of its target, TIAM1. [2][3]
1A-116 can inhibit the activity of the oncogenic Rac1 P29S mutant, which is a driver mutation found in sun-exposed melanoma. [3]

C16H16F3N3

307.31355381012

307.129

C, 62.53; H, 5.25; F, 18.55; N, 13.67

1430208-73-3

1430208-73-3

71543346

White to off-white solid powder

4

2

4

3

22

385

0

DVIJFJSZZNOTLP-UHFFFAOYSA-N

InChI=1S/C16H16F3N3/c1-10-7-11(2)9-12(8-10)21-15(20)22-14-6-4-3-5-13(14)16(17,18)19/h3-9H,1-2H3,(H3,20,21,22)

1-(3,5-dimethylphenyl)-2-[2-(trifluoromethyl)phenyl]guanidine

1A 116; 1A116; 1A-116

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)

DMSO: 61~100 mg/mL (198.5~325.4 mM)
Ethanol: ~61 mg/mL (~198.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.14 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 25.0 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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Solubility in Formulation 2: 2.5 mg/mL (8.14 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.14 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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