human LIMK1 (IC50 = 38 nM)
The target of R-10015 is LIM domain kinase (LIMK), specifically human LIMK1. It inhibits LIMK1 enzyme activity with an IC₅₀ value of 38 ± 5 nM [1]

R-10015 (100 μM; 0-4 hours) directly prevents cofilin phosphorylation in CEM-SS T cells by inhibiting LIM kinase[1]. R-10015 prevents nuclear migration, virion release, and HIV-1 DNA synthesis[1]. R-10015 inhibits a number of viruses, such as herpes simplex virus 1 (HSV-1), Venezuelan equine encephalitis virus (VEEV), Rift Valley fever virus (RVFV), and Zaire ebolavirus (EBOV)[1].

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1. Inhibition of HIV-1 infection:
- In Rev-CEM-GFP-Luc cells (HIV Rev-dependent indicator T cells), pretreatment with R-10015 for 1 h followed by HIV-1(NL4-3) infection for 3 h inhibited HIV-dependent luciferase expression, with dose-dependent activity. It minimally inhibited VSV G-pseudotyped HIV infection, indicating specificity for native HIV entry-related processes [1]
- In resting CD4⁺ T cells and CD45RO⁺ memory CD4 T cells, R-10015 (100 μM) pretreatment for 1 h inhibited latent infection by X4 HIV-1(NL4-3) and R5 HIV-1(AD8), respectively. After 5 days of culture and activation with CD3/CD28 beads, viral p24 release was significantly reduced without affecting T cell activation (assessed by CD25/CD69 expression) [1]
- In primary PBMCs, R-10015 (100 μM) pretreatment for 1 h inhibited infection by HIV-1 primary isolates (92/BR/018 and 93UG070), with reduced p24 levels in supernatants after 3 days of culture [1]
2. Inhibition of other viruses:
- EBOV (Zaire): HFF-1 cells treated with R-10015 (50 μM) for 2 h followed by EBOV infection (MOI = 2.5) for 48 h showed reduced EBOV GP protein expression (assessed by confocal imaging with Alexa 488-labeled antibody) [1]
- RVFV: Vero cells treated with R-10015 followed by RVFV-Luc(MP12) infection (MOI = 0.1) showed inhibited viral replication, quantified by luciferase assay [1]
- VEEV: Vero cells treated with R-10015 followed by infection with VEEV-Luc(TC-83), VEEV(TC-83), or VEEV(TrD) (MOI = 0.1) showed reduced viral load, measured by luciferase assay or plaque assay at 24 h [1]
- HSV-1: Vero cells treated with R-10015 (100 μM) for 2 h followed by HSV-1 infection showed reduced viral plaque formation after culture without the drug [1]
3. Effects on LIMK/cofilin pathway and actin dynamics:
- In CEM-SS T cells, R-10015 (100 μM) treatment blocked cofilin serine 3 phosphorylation (assessed by Western blotting and intracellular staining with anti-p-cofilin antibody plus flow cytometry) without affecting LIMK or PAK2 phosphorylation [1]
- In Jurkat T cells, R-10015 (100 μM) inhibited chemotaxis in response to SDF-1 (40 ng/ml) in a transwell assay, with reduced cell migration to the lower chamber after 2 h of incubation at 37°C [1]
- In resting CD4 T cells, R-10015 (100 μM) inhibited SDF-1-induced actin polymerization (assessed by FITC-phalloidin staining, flow cytometry, and confocal fluorescence microscopy) [1]
4. Mechanism-related antiviral effects:
- R-10015 (100 μM) did not affect surface CD4/CXCR4 expression or viral entry (assessed by BlaM-Vpr-tagged HIV-1 infection) in CEM-SS T cells [1]
- It inhibited HIV-1 DNA synthesis and 2-LTR circle formation (assessed by real-time PCR) in CEM-SS T cells treated for 1 h before single-cycle HIV-1(Env) infection [1]
- It inhibited virion release: in HIV-1(Env)-infected CEM-SS T cells (treated after 12 h of infection), chronically infected ACH2 cells (treated with various doses), and HEK293 cells transfected with pHIV-1(NL4-3) (treated for 48 h), R-10015 reduced p24 levels in supernatants (assessed by ELISA) [1]

R-10015 (10 mg/kg; i.p.) shows no signs of toxicity. The outcome raises the prospect of using LIMK inhibitors for a brief period of time to prevent viral infections[1].

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R-10015 was administered by intraperitoneal injection at 10 mg/kg daily for 7 days. Mice were weighed daily and observed for stress signs. [1]

A ten-dose inhibition assay was performed to evaluate the activity of R-10015 against purified recombinant LIMK1 enzyme. The compound was tested at multiple concentrations to generate an inhibition curve, and the IC₅₀ value was calculated as 38 ± 5 nM. Molecular docking simulation confirmed that R-10015 binds to the ATP-binding pocket of LIMK1 (PDB accession no. 3S95, chain A), acting as a typical type I ATP-competitive kinase inhibitor [1]

Cell Line: CEM-SS T cells
Concentration: 100 μM
Incubation Time: 0 hour,0.5 hour,1 hour,2 hours,4 hours
Result: Inhibited cofilin phosphorylation directly through blocking LIM kinase in CEM-SS T cells.

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1. HIV infection inhibition assay in Rev-CEM-GFP-Luc cells:
- Seed Rev-CEM-GFP-Luc cells and pretreat with different doses of R-10015 or DMSO for 1 h.
- Infect cells with HIV-1(NL4-3) or VSV G-pseudotyped HIV for 3 h, then wash to remove virus and drug.
- Incubate for 48 h, then quantify HIV-dependent luciferase expression (for efficacy) and assess cell viability by PI staining (for cytotoxicity) [1]
2. Western blot and flow cytometry for cofilin phosphorylation:
- Treat CEM-SS T cells with R-10015 (100 μM) for a time course, then lyse cells to extract proteins.
- Perform Western blotting to detect phosphorylation of cofilin, LIMK, and PAK2 (GAPDH as loading control).
- For flow cytometry: fix and permeabilize treated cells, stain with anti-p-cofilin antibody, and analyze fluorescence intensity (background staining without the antibody as control) [1]
3. Chemotaxis and actin polymerization assays:
- Chemotaxis assay: Treat Jurkat T cells with R-10015 (100 μM), add to the upper chamber of a 24-well transwell plate, and fill the lower chamber with SDF-1 (40 ng/ml). Incubate at 37°C for 2 h, then count cells migrated to the lower chamber [1]
- Actin polymerization assay: Treat resting CD4 T cells with R-10015 (100 μM), expose to SDF-1, stain with FITC-phalloidin, and quantify F-actin intensity by flow cytometry and confocal fluorescence microscopy [1]
4. Viral replication and virion release assays:
- EBOV inhibition: Treat HFF-1 cells with R-10015 (50 μM) for 2 h, infect with EBOV (MOI = 2.5) for 48 h, fix cells, stain with anti-EBOV GP antibody (Alexa 488-labeled) and Hoechst, then image by confocal microscopy [1]
- RVFV/VEEV/HSV-1 inhibition: Treat Vero cells with R-10015, infect with respective viruses (MOI = 0.1 for RVFV/VEEV, no MOI specified for HSV-1), and quantify viral load by luciferase assay (RVFV/VEEV-Luc), plaque assay (VEEV/HSV-1) [1]
- Virion release: Treat HIV-1-infected CEM-SS T cells (12 h post-infection), chronically infected ACH2 cells (various doses), or HEK293 cells transfected with pHIV-1(NL4-3) (48 h treatment) with R-10015, then measure p24 in supernatants by ELISA [1]

6-8 weeks female C3H/HeN mice[1]
10 mg/kg
Intraperitoneal injection

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A toxicity observation study was conducted in C3H/HeN mice. R-10015 was administered by intraperitoneal injection at a dose of 10 mg/kg once daily for 7 days. Control groups included mice treated with DMSO or PBS. Mice were monitored daily for body weight changes and signs of stress (e.g., abnormal behavior, lethargy) [1]

1. In vitro toxicity: - R-10015 did not show significant cytotoxicity at concentrations up to 100 μM in Rev-CEM-GFP-Luc, CEM-SS T, Vero, and HFF-1 cells (assessed by PI staining, flow cytometry, and multiple cytotoxicity assays based on luciferase; 1% saponin was used as a positive control for cytotoxicity) [1] 2. In vivo toxicity: - No significant toxicity was observed in C3H/HeN mice treated with R-10015 (10 mg/kg, intraperitoneal injection, 7 days). Body weight remained stable, and no signs of stress were detected during the observation period [1]

[1]. Discovery of Novel Small-Molecule Inhibitors of LIM Domain Kinase for Inhibiting HIV-1. J Virol. 2017 Jun 9;91(13). pii: e02418-16.

1. Mechanism of Action: R-10015 is a type I ATP-competitive kinase inhibitor that binds to the ATP-binding pocket of LIMK1. By inhibiting LIMK activity, it blocks the phosphorylation of cofilin serine 3, thereby disrupting the dynamic changes of the actin cytoskeleton. This interferes with the processes by which the virus enters the cell, migrates intracellularly, and releases viral particles—key steps that depend on actin dynamics and are crucial for HIV-1 and other viruses [1]. 2. Broad-spectrum antiviral potential: R-10015 inhibits a variety of enveloped viruses, including HIV-1 (X4/R5 strains and primary isolates), Zaire Ebola virus (EBOV), Rift Valley fever virus (RVFV), Venezuelan equine encephalitis virus (VEEV), and herpes simplex virus type 1 (HSV-1), supporting its potential as a broad-spectrum antiviral drug [1]. 3. Therapeutic significance: The LIMK/cofilin pathway is a host-dependent factor in HIV-1 infection (the virus hijacks this pathway for intracellular migration). R-10015 targets this host pathway, which reduces the risk of viral resistance compared to directly targeting viral proteins [1].

C20H19CLN6O2

410.856862306595

410.125

C, 58.47; H, 4.66; Cl, 8.63; N, 20.46; O, 7.79

2097938-51-5

2097938-51-5

129896912

Off-white to pink solid powder

3.4

2

6

4

29

604

0

MGRJCGXCUUCOQG-UHFFFAOYSA-N

InChI=1S/C20H19ClN6O2/c1-29-20(28)12-2-3-14-15(8-12)26-17(25-14)11-4-6-27(7-5-11)19-16-13(21)9-22-18(16)23-10-24-19/h2-3,8-11H,4-7H2,1H3,(H,25,26)(H,22,23,24)

methyl 2-[1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-yl]-3H-benzimidazole-5-carboxylate

R10015; R-10015; R 10015

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: 62.5~82 mg/mL (199.6~152.1 mM)
Water ˂1 mg/mL
Ethanol: ~10 mg/mL (24.3 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (12.17 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 50.0 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: ≥ 5 mg/mL (12.17 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 50.0 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: ≥ 5 mg/mL (12.17 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 50.0 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.)