srk1 Antibody

What is ASK1 and what role does it play in cellular signaling?

ASK1 (Apoptosis Signal-regulating Kinase 1), also known as MAP3K5, is a serine/threonine kinase that functions as an essential component of the MAP kinase signal transduction pathway. It mediates cellular responses to environmental changes and plays crucial roles in determining cell fate through differentiation and survival pathways. ASK1 is particularly important in the apoptosis signal transduction pathway through mitochondria-dependent caspase activation. It's also required for the innate immune response, providing host defense against various pathogens .

What is SRK-181 and how does it differ from other TGFβ inhibitors?

SRK-181 is a fully human, selective, IgG4 monoclonal antibody that specifically targets latent TGFβ1. Unlike non-selective TGFβ inhibitors that target multiple TGFβ isoforms (which has led to dose-limiting cardiotoxicities in clinical settings), SRK-181 selectively binds to latent TGFβ1 and inhibits its activation. This selectivity potentially offers a better safety profile while still targeting the TGFβ1 isoform that drives tumor immune escape mechanisms .

What is the underlying mechanism of action for ASK1 in stress response pathways?

ASK1 operates as a stress-responsive kinase that gets activated in response to various stressors including oxidative stress, endoplasmic reticulum stress, and receptor-mediated inflammatory signals such as tumor necrosis factor (TNF) or lipopolysaccharide (LPS). Upon activation, ASK1 functions as an upstream activator of the MKK/JNK signal transduction cascade and the p38 MAPK signal transduction cascade through phosphorylation and activation of several MAP kinase kinases including MAP2K4/SEK1, MAP2K3/MKK3, MAP2K6/MKK6, and MAP2K7/MKK7. These MAP2Ks subsequently activate p38 MAPKs and c-jun N-terminal kinases (JNKs), which control the transcription factors activator protein-1 (AP-1) .

How can I effectively use ASK1 antibodies in my experimental workflow?

For ASK1 detection and analysis, researchers can employ various antibody-based techniques. The anti-ASK1 antibody [EP553Y] (ab45178) has been validated for multiple applications including Western blotting (WB; 1:500-1:1000 dilution), immunohistochemistry on paraffin sections (IHC-P), immunocytochemistry/immunofluorescence (ICC/IF), and flow cytometry (intracellular) . For phosphorylation-specific detection, the Anti-ASK1 (phospho Ser83) antibody can detect endogenous levels of ASK1 only when phosphorylated at Ser83, making it valuable for studying ASK1 regulation .

What experimental approaches are recommended for evaluating SRK-181 efficacy in cancer models?

Based on current research, SRK-181 efficacy can be evaluated through several methodological approaches:

• Combination therapy assessment: SRK-181 is primarily being investigated in combination with anti-PD-(L)1 therapies to overcome resistance to immune checkpoint inhibition. Experimental designs should include appropriate control groups (monotherapy, combination therapy, and vehicle controls) .

• Tumor microenvironment analysis: Since TGFβ1 drives tumor immune escape by promoting an immunosuppressive microenvironment, researchers should assess changes in the tumor immune landscape through biomarker analysis .

• Species-appropriate models: Researchers should note that SRK-181-mIgG1 (containing murine IgG constant domains to minimize immunogenicity) has been used successfully in mouse tumor models that recapitulate key features of clinical anti-PD-(L)1 resistance .

What is the significance of phosphorylation at Ser83 in ASK1 function?

Phosphorylation at Ser83 represents an important regulatory mechanism for ASK1 function. Specific antibodies against phospho-Ser83 ASK1 enable researchers to detect endogenous levels of ASK1 only when phosphorylated at this site . This phosphorylation event is particularly relevant for studying how ASK1 activity is modulated under different cellular conditions and stress responses. Understanding this post-translational modification provides insights into the molecular mechanisms regulating ASK1-mediated signaling cascades in both normal and pathological contexts.

What are the recommended storage and handling conditions for ASK1 antibodies?

For optimal performance of ASK1 antibodies, proper storage and handling are essential. The Anti-ASK1 (phospho Ser83) antibody should be shipped at 4°C and upon delivery, aliquoted and stored at -20°C. It's important to avoid freeze/thaw cycles to maintain antibody integrity. The formulation typically includes Phosphate Buffered Saline (without Mg²⁺ and Ca²⁺), pH 7.4, with 150mM NaCl, 0.02% Sodium Azide, and 50% Glycerol .

What quality control measures should be implemented when using SRK-181 in research?

When working with SRK-181 in research settings, several quality control measures are recommended:

• Validation of selectivity: Confirm SRK-181's selective binding to latent TGFβ1 versus other TGFβ isoforms.

• Functional activity assessment: Verify that SRK-181 inhibits latent TGFβ1 activation in your experimental system.

• Cross-species reactivity: SRK-181 has demonstrated binding to human and mouse latent TGFβ1. For studies in other species, proper validation of cross-reactivity is essential .

• Appropriate controls: Include isotype controls and positive/negative controls to validate experimental outcomes.

How do I select the appropriate secondary antibodies for ASK1 detection systems?

The selection of appropriate secondary antibodies is crucial for reliable ASK1 detection. For rabbit polyclonal anti-ASK1 antibodies, suitable secondary antibodies include Goat Anti-Rabbit IgG H&L Antibody conjugated with various detection systems such as alkaline phosphatase (AP), biotin, FITC, or HRP . The choice depends on your detection method (e.g., Western blot, immunohistochemistry, flow cytometry). Consider factors such as sensitivity requirements, signal amplification needs, and multiplexing capabilities of your experimental system when selecting secondary antibodies.

How does SRK-181 overcome resistance to immune checkpoint inhibitors at the molecular level?

SRK-181 addresses a key mechanism of resistance to immune checkpoint inhibition by targeting the TGFβ1 pathway. TGFβ1 specifically drives tumor immune escape by:

• Promoting an immunosuppressive pro-tumor microenvironment

• Reducing antigen presentation

• Impairing T-cell infiltration and tumor-killing activity

By selectively binding to latent TGFβ1 and preventing its activation, SRK-181 can potentially reverse these immunosuppressive effects. Preclinical studies with SRK-181-mIgG1 have demonstrated its ability to overcome primary resistance to anti-PD-1 therapy in mouse tumor models that recapitulate key features of clinical anti-PD-(L)1 resistance .

What is the relationship between ASK1 activation and apoptotic pathways in disease models?

ASK1 plays a crucial role in the apoptosis signal transduction pathway through mitochondria-dependent caspase activation. Mechanistically, ASK1 gets activated in response to various stress stimuli such as oxidative stress and endoplasmic reticulum stress, which significantly impact the regulation of programmed cell death . This activation leads to downstream signaling through the MKK/JNK and p38 MAPK pathways, ultimately affecting the transcription factor AP-1 and other regulators of apoptosis. In disease models, dysregulation of this pathway can contribute to pathological conditions characterized by either excessive cell death or inappropriate cell survival.

What is the comparative safety profile of SRK-181 against non-selective TGFβ inhibitors?

SRK-181's selective targeting of latent TGFβ1 provides important safety advantages over non-selective TGFβ inhibitors. Comprehensive preclinical assessments have shown that:

• In vitro studies demonstrated that SRK-181 has no effect on human platelet function and does not induce cytokine release in human peripheral blood.

• Four-week toxicology studies revealed that weekly intravenous administration of SRK-181 was well tolerated in both rats and monkeys, with no treatment-related adverse findings.

• The no-observed-adverse-effect levels were 200 mg/kg in rats and 300 mg/kg in monkeys (the highest doses tested), providing a nonclinical safety factor of up to 813-fold (based on Cmax) above the phase 1 starting dose of 80 mg every 3 weeks.

These data demonstrate that SRK-181 does not produce the nonclinical toxicities associated with non-selective TGFβ inhibition, which have included dose-limiting cardiotoxicities in clinical settings .

What is the current clinical development status of SRK-181?

SRK-181 is currently being evaluated in the DRAGON (NCT04291079) open-label, phase 1 clinical trial. The study has two parts: Part A (dose escalation) evaluated SRK-181 alone (Part A1) and SRK-181 plus anti-PD-(L)1 therapy (Part A2), while Part B (expansion phase) is administering SRK-181 (1500mg q3w) in combination with pembrolizumab in anti-PD-1 resistant patients with various cancer types including clear cell renal cell carcinoma (ccRCC), non-small cell lung cancer, melanoma, urothelial carcinoma, and head and neck cancer .

As of May 26, 2023, 20 anti-PD-1-refractory metastatic ccRCC patients had been enrolled. The most common treatment-related adverse events (>10%) of any grade were pruritus (15%), rash maculo-papular (15%), and rash (10%). No grade 4 or 5 treatment-related adverse events were observed. Among 16 response-evaluable patients, four had confirmed partial responses according to RECIST1.1 criteria .

How can ASK1 antibodies be used to identify potential therapeutic targets in stress-related pathologies?

ASK1 antibodies serve as valuable tools for identifying potential therapeutic targets in stress-related pathologies through several research approaches:

• Pathway mapping: Using ASK1 antibodies, researchers can map the activation state of ASK1-dependent signaling pathways in different disease contexts by detecting total and phosphorylated ASK1 levels .

• Protein interaction studies: Antibodies can help identify novel protein interactions with ASK1 in stress conditions, potentially revealing new regulatory mechanisms or therapeutic targets.

• Biomarker development: The phosphorylation status of ASK1 at sites like Ser83 can potentially serve as biomarkers for stress-activated states in various tissues .

• Target validation: ASK1 antibodies can be used in combination with inhibitors to validate the role of ASK1 in disease models and confirm the specificity of therapeutic interventions.

What are the key pharmacokinetic parameters to consider when designing experiments with SRK-181?

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When designing experiments with SRK-181, researchers should consider these pharmacokinetic parameters along with the sustained serum exposure observed with weekly intravenous administration. This information can guide appropriate dosing strategies for preclinical studies and help predict clinical translation potential .

How should I interpret variations in ASK1 phosphorylation patterns across different experimental conditions?

Variations in ASK1 phosphorylation patterns across different experimental conditions require careful interpretation:

• Site-specific effects: Different phosphorylation sites on ASK1 (such as Ser83) may have distinct functional consequences. Some sites may activate ASK1, while others may inhibit its function .

• Temporal dynamics: Consider the time course of phosphorylation events, as rapid and transient phosphorylation might indicate acute stress responses, while sustained phosphorylation may suggest chronic stress conditions.

• Context dependency: ASK1 phosphorylation patterns may vary depending on cell type, tissue context, and the nature of the stress stimulus (oxidative stress, inflammatory signals, etc.) .

• Pathway cross-talk: Interpret ASK1 phosphorylation in the context of other signaling pathways, as cross-talk between different stress-response mechanisms can influence phosphorylation patterns.

What biomarker strategies can be employed to monitor SRK-181 efficacy in experimental models?

Based on SRK-181's mechanism of action and ongoing clinical studies, several biomarker strategies can be employed to monitor its efficacy:

• TGFβ pathway activation markers: Measure changes in downstream signaling molecules in the TGFβ pathway before and after SRK-181 treatment.

• Immune cell infiltration and phenotyping: Assess changes in tumor-infiltrating lymphocytes and their functional status, as SRK-181 is expected to enhance T-cell infiltration and function by blocking TGFβ1-mediated immunosuppression .

• Tumor microenvironment analysis: Examine changes in the immunosuppressive characteristics of the tumor microenvironment, including regulatory T-cell populations and immunosuppressive cytokines.

• Treatment response biomarkers: In the DRAGON clinical trial, 4 out of 16 evaluable patients showed partial responses to SRK-181 combined with pembrolizumab, suggesting that identifying predictive biomarkers of response would be valuable for patient stratification .