TGF-β; Alk4/5
BIO-013077-01 primarily targets the transforming growth factor-β (TGF-β) receptors, specifically the activin receptor-like kinase 5 (ALK5, also known as TGF-β receptor type I) and ALK4. TGF-β transduces signals through a complex of two distinct serine/threonine kinase receptors termed type I and type II. By acting as an antagonist of these receptors, the compound inhibits the canonical TGF-β signaling cascade. This blockade prevents the phosphorylation of the type I receptor by the constitutively active type II receptor, thereby inhibiting the subsequent phosphorylation and activation of downstream Smad proteins. Through this mechanism, BIO-013077-01 effectively modulates the expression of genes regulated by the TGF-β superfamily, impacting key cellular processes like proliferation, differentiation, and apoptosis.

Members of the TGF-β superfamily govern a number of biological processes, including adhesion, migration, apoptosis, differentiation, and proliferation of cells. There are three main TGF-β isoforms that are expressed in mammals: TGF-β1, TGF-β2, and TGF-β3. Type I and type II serine/threonine kinase receptors, which are similar but have different structures and functions, form a complex that allows TGF-β to transduce signals. Numerous human disorders, including as cancer, pancreatitis, and hematological malignancies, have also been linked to dysregulation of TGF-β signaling [1].
In vitro, BIO-013077-01 functions as a potent antagonist of the TGF-β type I receptors (ALK4/5), effectively inhibiting TGF-β-mediated signal transduction. By blocking this pathway, it modulates the expression of genes involved in a wide range of cellular functions, including cell proliferation, differentiation, adhesion, migration, and apoptosis. In cell-based assays, the compound is used to study the role of TGF-β signaling in various biological processes and disease models. Research indicates that members of the TGF-β superfamily govern numerous biological processes, and dysregulation of this signaling pathway is associated with various human diseases including cancer, pancreatic diseases, and hematological malignancies. BIO-013077-01 is used as a tool to dissect these pathways and evaluate the therapeutic potential of TGF-β inhibition.

Specific in vivo activity data for BIO-013077-01 are not detailed in the available literature. However, as a potent TGF-β inhibitor, its mechanism of action suggests potential in vivo applications for studying diseases associated with TGF-β dysregulation. TGF-β signaling is known to play a crucial role in a variety of pathological conditions, including cancer, fibrosis, and inflammatory diseases. The compound could be investigated in animal models to assess its efficacy in modulating these disease processes. For in vivo studies, it can be formulated, for example, by dissolving in DMSO, PEG300, Tween 80, and saline. Comprehensive pharmacokinetic and pharmacodynamic studies would be required to fully characterize its in vivo profile and therapeutic potential.

The transforming growth factor-β (TGF-β) superfamily members, which include TGF-βs, activins, bone morphogenetic proteins (BMPs), growth and differentiation factors, and Mullerian inhibiting substance are structurally related secreted cytokines found in species ranging from worms and insects to mammals. A wide range of cellular functions such as cell proliferation, differentiation, adhesion, migration, and apoptosis are regulated by TGF-β superfamily members. The TGF-βs include the three major TGF-β isoforms, TGF-β1, TGF-β2, and TGF-β3 which are expressed in mammals. Among them, TGF-β1 is the prototypic member of this family of cytokines and is the major isoform. TGF-β1 transduces signals through a complex of two related but structurally and functionally distinct serine/threonine kinase receptors, termed type 1 and type II. The signaling cascade is promoted by the binding of ligand to the constitutively active type II receptors on the cell surface, which further recruits the type I receptors, also called as activin receptor-like kinase 5 (ALK5), into the complex. Subsequently, ALK5 is phosphorylated in the juxtamembrane GS domain by the type II receptors thereby creating a binding site for Smad proteins and stimulating its kinase activity. The activated ALK5 phosphorylates Smad2 and Smad3 proteins thereby causing their dissociation from the receptor and heteromeric complex formation with Smad4. These Smad complexes translocate to the nucleus, assemble with specific DNA-binding co-factors and co-modulators to finally activate several hundred genes involved in cell differentiation, proliferation, apoptosis, migration, and extracellular matrix production. TGF-β1 plays a critical role in the initiation and progression of fibrosis in various organ systems such as kidney, heart, lung, and liver. Deregulation of TGF-β signaling has been also implicated in various human diseases including cancer, pancreatic diseases, and hematological malignancies [1].
In vitro enzyme assays for BIO-013077-01 involve measuring its ability to inhibit the activity of TGF-β type I receptors (ALK5 and ALK4). These assays typically use recombinant kinase domains of the receptors and evaluate phosphorylation of specific substrates in the presence of varying concentrations of the compound. The assay protocols are based on the known mechanism of TGF-β signal transduction, where the binding of a TGF-β ligand to the type II receptor recruits and phosphorylates the type I receptor (ALK5), which then propagates the signal downstream. By acting as an antagonist of ALK5 and ALK4, BIO-013077-01's inhibitory activity is quantified, providing an IC50 value that reflects its potency in blocking this critical enzymatic step in the TGF-β pathway.

Luciferase reporter assay [1]
Biological activity of the test compounds was determined by measuring their ability to inhibit TGF-β-induced p3TP-luciferase reporter activity in HaCaT stable cells transfected with p3TP-Luc. HaCaT cells were seeded at concentrations of 3 × 104 in 96-well plates. The next day, when they reach approximately 90% confluency, various concentrations of ALK5 inhibitors and 2 ng/mL of TGF-β were added to the cells. After 24 h, cell lysates were harvested using Luciferase assay kit according to the manufacturer’s instruction, and luminescence was measured by a luminometer Micro Lumat Plus.
In vitro cell-based assays for BIO-013077-01 evaluate its effects on TGF-β-mediated cellular functions, such as proliferation, differentiation, adhesion, migration, and apoptosis. Researchers treat various cell lines with the compound and assess changes in these processes. A wide range of cellular functions are regulated by TGF-β superfamily members. The compound's ability to inhibit TGF-β signaling is typically confirmed by measuring the expression of downstream target genes or the phosphorylation of Smad proteins, which are key mediators of the TGF-β pathway. These cell-based assays are crucial for understanding the functional consequences of ALK4/5 inhibition and for validating the compound's mechanism of action in a cellular context.

Information regarding specific in vivo animal studies for BIO-013077-01 is limited. However, given its mechanism as a TGF-β inhibitor, it has the potential to be studied in animal models of diseases where TGF-β signaling is dysregulated, such as cancer, fibrosis, and inflammatory conditions. In such studies, the compound would be administered to disease model animals via various routes, such as intraperitoneal injection, and its effects on disease progression, biomarkers, and survival would be assessed. The compound is a solid powder, and for in vivo administration, it can be formulated in vehicles like 0.5% CMC Na for oral administration or DMSO/PEG300/Tween 80/Saline for injection.

Detailed pharmacokinetic (PK) data for BIO-013077-01 are not available in the provided references. As a research-grade small molecule, its ADME (Absorption, Distribution, Metabolism, Excretion) properties would need to be characterized for any in vivo applications. The compound has a molecular weight of 287.32 g/mol and a molecular formula of C17H13N5, which suggests it could have favorable properties for cell permeability. For research use, it is typically dissolved in DMSO for in vitro studies. A comprehensive PK study would involve administering the compound to animal models and measuring its concentration in plasma and tissues over time to determine its half-life, clearance, and bioavailability.

The toxicity profile of BIO-013077-01 is not extensively detailed in the provided literature. As a potent inhibitor of the TGF-β pathway, its safety would need to be carefully evaluated in preclinical studies. The compound is intended for research use only and is not for human or veterinary use. Standard safety precautions should be followed when handling this chemical in a laboratory setting, as it is a research-grade compound. Any comprehensive toxicological assessment would require dedicated studies to evaluate potential off-target effects and to establish a safe dosage range for in vivo applications.

[1]. Synthesis and biological evaluation of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)pyrazoles as transforming growth factor-β type 1 receptor kinase inhibitors. Eur J Med Chem. 2011 Sep;46(9):3917-25.

series of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)pyrazole compounds, 14a–d, 15a–d, 17a, 17b, 18a–d, 19a, and 19b, were synthesized and their inhibitory activity against ALK5 was evaluated in enzyme activity assays and cell-based luciferase reporter gene assays. 2-[3-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-pyrazol-1-yl]-N-phenylethylthioamide (18a) showed an IC50 value of 0.013 μM for inhibiting ALK5 phosphorylation and achieved an 80% inhibition rate at 0.1 μM in a luciferase reporter gene assay performed on HaCaT cells permanently transfected with the p3TP-luc reporter gene construct. [1]
BIO-013077-01 is a pyrazole-based small molecule identified as a potent inhibitor of the TGF-β signaling pathway. Its mechanism of action involves antagonizing the type I TGF-β receptors ALK5 and ALK4, which are key serine/threonine kinases in the TGF-β signal transduction cascade. By inhibiting this pathway, the compound affects a wide range of cellular functions, including proliferation, differentiation, adhesion, migration, and apoptosis. The TGF-β superfamily is involved in numerous physiological and pathological processes, making inhibitors like BIO-013077-01 valuable tools for research in areas such as oncology and fibrosis. It is exclusively a research chemical and has not been approved for clinical trials or therapeutic use.

C17H13N5

287.33

287.117

C, 71.06; H, 4.56; N, 24.37

746667-48-1

746667-48-1

10469294

Light yellow to brown solid powder

3.39

1

4

2

22

376

0

CC1=NC(=CC=C1)C2=C(C=NN2)C3=CC4=NC=CN=C4C=C3

VXJLYXCHOKEODY-UHFFFAOYSA-N

InChI=1S/C17H13N5/c1-11-3-2-4-15(21-11)17-13(10-20-22-17)12-5-6-14-16(9-12)19-8-7-18-14/h2-10H,1H3,(H,20,22)

6-[3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]quinoxaline

BIO013077-01; BIO-013077; BIO 013077; BIO-013077-01; BIO 013077-01; 6-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)quinoxaline; BIO-013077-01; 6-[3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]quinoxaline; 6-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]quinoxaline; Quinoxaline, 6-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-; SCHEMBL4008166; DTXSID00440482; BIO013077

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 : ~100 mg/mL (~348.04 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.70 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 (8.70 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 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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 (Please use freshly prepared in vivo formulations for optimal results.)