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Purity: ≥98%
LMI-070 (Branaplam; NVS-SM1) is a potent, orally bioactive, small-molecule enhancer of SMN2 (survival of motor neuron-2) splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. The transient double-strand RNA structure created by the U1 small nuclear ribonucleic protein (snRNP) complex and SMN2 pre-mRNA is stabilized as the molecular mechanism of action. Separate from constitutive recognition, the binding affinity of U1 snRNP to the 5' splice site is enhanced in a sequence-specific manner. This novel mechanism highlights the viability of sequence-selective splice modulation mediated by small molecules and the possibility of applying this tactic to other splicing disorders.
| Targets |
SMN ( IC50 = 20 nM ); hERG ( EC50 = 6.3 μM )
Branaplam (LMI-070; NVS-SM1) treatment alters the expression of 175 genes in human fibroblasts[1]. |
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| ln Vitro |
Branaplam (LMI-070; NVS-SM1) treatment alters the expression of 175 genes in human fibroblasts[1].
In a high-throughput screen using an NSC34 motor neuron cell line expressing an SMN2 mini-gene reporter, hits were selected based on complementary changes in forward and reverse reporter activity. Compounds scoring positively and showing dose responsiveness were confirmed by qPCR for increased SMN2 exon 7 inclusion and by ELISA for increased SMN protein levels in SMNΔ7 mouse myoblasts. Compound optimization led to the identification of NVS-SM1 (1) and NVS-SM2 (2) as active SMN2 splicing modulators, and NVS-SM3 (3) and NVS-SM4 (4) as inactive analogs. SMN ELISA assays in mouse myoblasts and SMA patient fibroblasts guided the optimization. In patient-derived fibroblasts, treatment with 100 nM NVS-SM1 for 24 hours significantly increased the ratio of exon 7-included to exon 7-excluded SMN2 transcripts compared to DMSO control, as measured by RT-qPCR. The inactive analog NVS-SM3 (5 µM) did not show this effect. In SMNΔ7 mouse myoblasts, NVS-SM1 and NVS-SM2 treatment increased SMN protein levels in a dose-dependent manner as measured by ELISA. In hiPSC-derived neurons from SMA patients, NVS-SM1 treatment showed robust activity in elevating SMN2 full-length transcript levels. In peripheral blood mononuclear cells (PBMCs) from SMA type III patients, NVS-SM1 treatment induced the desired transcript response, suggesting its potential as a peripheral pharmacodynamic marker. Global RNAseq analysis in human fibroblasts treated with 100 nM NVS-SM1 for 24 hours showed changes in splicing for a discrete set of 39 events (affecting 35 genes), while changes in overall gene expression were limited (175 genes altered by NVS-SM1 vs. 23 by the inactive NVS-SM3). This indicates selective splicing modulation.[1] |
| ln Vivo |
Branaplam (LMI-070; NVS-SM1) hydrochloride (3, 10, 30 mg/kg; oral) causes dose-dependent increases in SMN2-FL transcript and SMN protein in brain and spinal cord[1]. Branaplam hydrochloride (1 mg/kg IV; 3 mg/kg PO) has an AUC of 3.03 μM•h and a CL of 25 mL/min/kg[2]. In C/+ mice, a single oral dose of 30 mg/kg of Branaplam hydrochloride causes a notable and long-lasting increase in SMN protein in the brain for up to 160 hours[1]. Branaplam hydrochloride (oral; 0.03, 0.1, 0.3, 1, 3 mg/kg) increases body weight and prolongs life in SMNΔ7 mice[1].
In the C/+ SMA mouse model, oral administration of NVS-SM1 or NVS-SM2 produced dose-dependent elevations of SMN2 full-length transcript and SMN protein levels in the brain and spinal cord. In the C/+ model, a single oral dose of NVS-SM1 (30 mg/kg) resulted in a significant and durable elevation of SMN protein in the brain for up to 160 hours. In the severe SMNΔ7 mouse model, oral administration of NVS-SM1 led to a dose-dependent increase in SMN protein in the brain, improved body weight, and extended lifespan. Specifically, 50% of animals in the 1 mg/kg group and 62% in the 3 mg/kg group showed increased survival. A study with two additional cohorts of SMNΔ7 mice treated with 1 mg/kg/day NVS-SM1 showed that beneficial effects on body weight and survival were sustained even after drug withdrawal from day 36 to 49, indicating durable efficacy from early postnatal treatment.[1] |
| Enzyme Assay |
Binding of compounds to the U1 snRNP-RNA complex was assessed using size-exclusion chromatography coupled with mass spectrometry (SEC-MS). Enriched U1 snRNP from HeLa nuclear extracts was incubated with biotinylated SMN2 exon 7 5'ss RNA and compounds. NVS-SM2 bound specifically to the U1 snRNP-RNA complex but not to U1 snRNP or RNA alone. The inactive analog NVS-SM4 did not bind any component.[1]
Surface plasmon resonance (SPR) was used to analyze the kinetics of U1 snRNP binding to immobilized 5'ss RNA. U1 snRNP was diluted in SPR buffer containing either DMSO or compound and injected over a streptavidin chip with captured RNA. Co-injection experiments allowed testing of NVS-SM2 in both association and dissociation phases. NVS-SM2 (1 µM) markedly slowed the dissociation of U1 snRNP from the wild-type SMN2 5'ss RNA (approximately fourfold) but had no observable effect on binding to mutant sequences (-1A→C or -2G→C). The inactive NVS-SM4 had no effect.[1] |
| Cell Assay |
For the high-throughput screen, NSC34 cells were transfected with SMN2 mini-gene reporter constructs (full-length and Δ7 reporters). Cells were plated in 96-well plates and treated with compounds 24 hours post-transfection. After 24 hours of treatment, cells were lysed, and cDNA was synthesized. Splicing outcomes (exon inclusion/exclusion) were quantified using PCR followed by fragment analysis on a LabChip system. Percentage exon inclusion was calculated.[1]
For validation of splicing modulation in minigene systems, NSC34 cells were transfected with various SMN2-BRCA1 chimeric constructs or mutant minigenes. Cells were treated with compounds (NVS-SM2) for 24 hours, followed by cell lysis, cDNA synthesis, and RT-PCR analysis. PCR products were separated and quantified using a LabChip system to determine the percentage of exon inclusion.[1] For measuring endogenous SMN2 splicing and protein, SMA patient fibroblasts or mouse myoblasts were seeded in microtiter plates. Cells were treated with compounds (e.g., 100 nM NVS-SM1 or 5 µM NVS-SM3) for 24 hours. Total RNA was isolated, reverse transcribed, and analyzed by TaqMan qPCR using probe/primer sets specific for SMN2 exon 7-included and exon 7-excluded transcripts, normalized to a housekeeping gene (GusB). For protein analysis, cells were lysed, and SMN protein levels were quantified using a commercial SMN ELISA kit according to the manufacturer's instructions.[1] |
| Animal Protocol |
C/+ SMA mouse model
3, 10, 30 mg/kg Oral For pharmacokinetic and pharmacodynamic studies in the C/+ SMA mouse model, female C/+ mice (8-10 weeks old) were used. NVS-SM1 or NVS-SM2 was administered orally at specified doses. Animals were euthanized at indicated time points post-dose, and tissues (brain, spinal cord) were collected for analysis of SMN2 transcript ratio (by RT-qPCR) and SMN protein levels (by ELISA). Plasma was also collected for exposure analysis.[1] For efficacy studies in the severe SMNΔ7 mouse model, pups were treated orally with varying doses of NVS-SM1 (e.g., 1 mg/kg or 3 mg/kg body weight) or vehicle. Body weight was monitored regularly. Survival was tracked, and Kaplan-Meier survival curves were generated. In some studies, dosing was continued daily until a specified endpoint (e.g., day 49) or withdrawn after a period to assess sustained effects.[1] For the single-dose duration of response study in C/+ mice, animals received a single oral dose of NVS-SM1 (30 mg/kg) and were euthanized at various time points up to 160 hours post-dose for brain SMN protein analysis by ELISA.[1] |
| ADME/Pharmacokinetics |
NVS-SM1 and NVS-SM2 exhibited favorable pharmacokinetic characteristics in rodents. Both compounds showed high plasma exposure and good oral bioavailability.
Notably, both compounds were well distributed to the brain, which is the primary target tissue for SMA treatment. [1] |
| Toxicity/Toxicokinetics |
(The provided text does not contain specific toxicity, toxicokinetics, or safety data for NVS-SM1 or NVS-SM2, such as LD50, organ toxicity, drug interactions, or plasma protein binding.) [1]
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| References |
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| Additional Infomation |
Branaplam is being investigated in the clinical trial NCT02268552 (the LMI070 (Branaplam) open-label study for spinal muscular atrophy type 1 (SMA)). Drug Indication Treatment of Huntington's Disease Spinal muscular atrophy (SMA) is caused by a deficiency of the survival motor neuron (SMN) protein, and the deletion of SMN1 is the cause of SMN protein deficiency. SMN2 is a nearly identical copy of the gene that undergoes aberrant splicing, primarily resulting in the deletion of exon 7, producing a truncated, unstable protein. The small molecule splicing regulators described in this article, including NVS-SM1, enhance the splicing of exon 7 of SMN2, thereby increasing the production of functional full-length SMN protein.
Its mechanism of action involves a transient interaction between a stable U1 snRNP and the 5' splicing site of exon 7 of SMN2 (particularly the rare “nGA” motif). This enhances the binding affinity of the U1 snRNP in a sequence-selective manner, promoting proper splicing. In a severe SMA mouse model, NVS-SM1 demonstrated sustained in vivo efficacy following early postnatal oral administration, supporting the potential for therapeutic intervention before widespread neuronal loss. This work demonstrates the feasibility of using small molecules to achieve sequence-selective splicing regulation, with potential advantages in bioavailability and delivery compared to oligonucleotide-based approaches. [1] |
| Molecular Formula |
C22H27N5O2
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| Molecular Weight |
393.49
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| Exact Mass |
393.216
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| Elemental Analysis |
C, 67.15; H, 6.92; N, 17.80; O, 8.13
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| CAS # |
1562338-42-4
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| Related CAS # |
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| PubChem CID |
135565042
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| Appearance |
White to yellow solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
660.0±55.0 °C at 760 mmHg
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| Flash Point |
353.0±31.5 °C
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| Vapour Pressure |
0.0±2.1 mmHg at 25°C
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| Index of Refraction |
1.576
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| LogP |
2.86
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
29
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| Complexity |
541
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O(C1C([H])=C([H])C(C2C([H])=C([H])C(C3C([H])=NN([H])C=3[H])=C([H])C=2O[H])=NN=1)C1([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])N([H])C(C([H])([H])[H])(C([H])([H])[H])C1([H])[H]
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| InChi Key |
STWTUEAWRAIWJG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H27N5O2/c1-21(2)10-16(11-22(3,4)27-21)29-20-8-7-18(25-26-20)17-6-5-14(9-19(17)28)15-12-23-24-13-15/h5-9,12-13,16,27-28H,10-11H2,1-4H3,(H,23,24)
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| Chemical Name |
5-(1H-pyrazol-4-yl)-2-[6-(2,2,6,6-tetramethylpiperidin-4-yl)oxypyridazin-3-yl]phenol
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1 mg/mL (2.54 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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. Solubility in Formulation 2: ≥ 0.71 mg/mL (1.80 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 7.1 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. View More
Solubility in Formulation 3: ≥ 0.71 mg/mL (1.80 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: ≥ 0.57 mg/mL (1.45 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 5: 1% DMSO 99% Saline |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5414 mL | 12.7068 mL | 25.4136 mL | |
| 5 mM | 0.5083 mL | 2.5414 mL | 5.0827 mL | |
| 10 mM | 0.2541 mL | 1.2707 mL | 2.5414 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT05111249 | Completed | Drug: Branaplam Drug: Placebo |
Early Manifest Huntington Disease |
Novartis Pharmaceuticals | December 8, 2021 | Phase 2 |
| NCT02268552 | Completed | Drug: branaplam | Spinal Muscular Atrophy | Novartis Pharmaceuticals | April 2, 2015 | Phase 1 Phase 2 |
| NCT05330286 | Terminated | Drug: LMI070 | Healthy Volunteers | Novartis Pharmaceuticals | April 13, 2022 | Phase 1 |
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