What is Ac-RGK-AMC C26H38N8O6 (660846-99-1) and what are its primary applications in scientific research?

Ac-RGK-AMC C26H38N8O6, with the CAS number 660846-99-1, is a synthetic peptide used primarily in biochemical research and pharmaceutical development. This compound functions as a fluorogenic substrate for a variety of proteases, particularly those involved in the regulation and breakdown of intracellular proteins. In scientific laboratories, it is a valuable tool for studying enzyme activity, providing insights into the kinetics and specificity of proteases. By attaching a fluorogenic moiety, such as the amino-methylcoumarin (AMC), to the peptide chain, researchers can monitor enzymatic activity through fluorescence detection. This method allows for highly sensitive and real-time analysis of enzyme interactions, facilitating the characterization of biochemical pathways and molecular mechanisms underpinning various physiological and pathological processes.

The versatility of Ac-RGK-AMC C26H38N8O6 is further exemplified by its use in high-throughput screening for drug discovery. The ability to quantitatively measure protease activity using this substrate enables researchers to identify potential drug candidates that inhibit or modulate specific enzyme targets. Such applications are critical in the development of therapeutic agents for diseases where protease dysregulation is implicated, such as cancer, neurodegenerative conditions, and infectious diseases. In addition to drug discovery, this compound is also employed in investigating cellular processes like apoptosis, protein degradation, and signal transduction, providing valuable insights into cellular homeostasis and growth regulation. Its role in elucidating the function and behavior of biological macromolecules continues to advance our understanding of human health and disease, underscoring its importance in both basic and applied research settings.

How does Ac-RGK-AMC C26H38N8O6 contribute to enzyme assay development and what are its advantages?

Ac-RGK-AMC C26H38N8O6 is instrumental in the development of enzyme assays due to its properties as a fluorogenic substrate, which allows for meticulous evaluation of enzyme activity under various conditions. The incorporation of the amino-methylcoumarin (AMC) fluorophore facilitates the detection and quantification of enzymatic reactions through fluorescence emission, which can be measured with high sensitivity and specificity. This characteristic is particularly advantageous in enzyme kinetics studies, where understanding the rate and efficiency of substrate turnover is essential. By providing a real-time measure of enzymatic activity, Ac-RGK-AMC C26H38N8O6 enables researchers to dissect complex enzyme mechanisms, analyze substrate specificity, and determine catalytic efficiency under different experimental conditions.

One of the significant advantages of using Ac-RGK-AMC C26H38N8O6 in enzyme assays is its ability to generate quantitative data that can be easily correlated with enzyme concentration and activity levels. This quantitative capability is crucial in settings such as pharmaceutical development, where efficacy and potency of enzyme inhibitors or modulators need to be assessed accurately. Additionally, the high sensitivity of fluorogenic assays compared to colorimetric or radiometric methods allows for the detection of low enzyme concentrations, broadening the application range in both basic research and clinical diagnostics.

Moreover, Ac-RGK-AMC C26H38N8O6 is compatible with automation and high-throughput screening technologies, making it suitable for drug discovery programs where numerous compounds need to be screened rapidly for enzyme-inhibitory activities. The fluorescence-based detection is non-destructive, which means assays can be continuously monitored over time without terminating the reaction, providing dynamic insights into enzyme behavior. This feature enhances the reliability of data by reducing variability and increasing reproducibility across experiments. In summary, Ac-RGK-AMC C26H38N8O6 offers numerous technical and practical advantages in enzyme assay development, making it a preferred choice in biochemical research and drug discovery.

What are the structural features of Ac-RGK-AMC C26H38N8O6 that make it suitable for its role as a fluorogenic substrate?

The structural features of Ac-RGK-AMC C26H38N8O6 that contribute to its efficacy as a fluorogenic substrate lie primarily in its carefully designed peptide sequence and the attached fluorophore, which together facilitate specific interactions with target enzymes while enabling sensitive detection. The peptide chain, comprised of arginine (R), glycine (G), and lysine (K), provides specificity towards a range of enzymes, particularly serine and cysteine proteases that recognize and cleave these amino acid motifs. This sequence ensures that the substrate is selective for its intended targets, effectively functioning as a molecular key that fits into specific enzyme active sites.

Crucial to its function is the amino-methylcoumarin (AMC) moiety, which acts as a fluorogenic group. In its intact state, the AMC group remains non-fluorescent, thereby maintaining a low background signal. Upon enzymatic cleavage of the peptide bond adjacent to the AMC, the fluorophore is released and subsequently emits fluorescence when excited at a specific wavelength. This change in fluorescence serves as a direct indicator of substrate cleavage and hence enzyme activity. The ability to monitor reactions in real-time with high specificity makes this compound particularly valuable in studying enzyme kinetics and dynamics.

Furthermore, the acetyl group (Ac) at the N-terminus of the peptide chain serves a protective role, enhancing the stability of the molecule and preventing non-specific degradation by other proteolytic enzymes present in biological samples. This increases the shelf-life and usability of the substrate in various experimental conditions. The overall chemical structure of Ac-RGK-AMC C26H38N8O6, with its balance of stability, specificity, and detectability, makes it an ideal tool in enzymatic studies, enabling researchers to gain precise insights into the functions and regulatory mechanisms of enzymes in complex biochemical and physiological environments.

How is the use of Ac-RGK-AMC C26H38N8O6 impactful in the field of drug discovery and development?

Ac-RGK-AMC C26H38N8O6 plays a significant role in the field of drug discovery and development by providing a robust platform for screening potential therapeutic compounds that target proteases. Proteases are essential enzymes that participate in numerous biological processes, and their dysregulation is implicated in various diseases, including cancer, cardiovascular disorders, and infectious diseases. By serving as a fluorogenic substrate, Ac-RGK-AMC C26H38N8O6 enables the development of assays that quantitatively measure enzyme activity, offering a direct method to evaluate the efficacy of compounds designed to modulate enzyme function.

In the drug discovery pipeline, high-throughput screening (HTS) is a critical initial step that involves testing a large library of compounds to identify candidates that exhibit desired biological activity. Ac-RGK-AMC C26H38N8O6 is well-suited for HTS due to its compatibility with automated fluorescence detection systems, allowing for rapid and efficient screening processes. The ability to detect even minimal changes in enzyme activity through its sensitive fluorescence readout helps in identifying potent inhibitors or activators of target proteases, which are then subjected to further validation and optimization.

Beyond the initial screening phases, the use of Ac-RGK-AMC C26H38N8O6 extends to lead optimization and mechanism of action studies. By providing detailed kinetic data, researchers can understand how modifications to compound structure affect interaction with the enzyme, guiding medicinal chemistry efforts to enhance potency, selectivity, and pharmacokinetic properties. Additionally, the substrate’s ability to facilitate real-time monitoring of enzymatic reactions is invaluable in elucidating the pathways and biological contexts in which these targets operate, thus improving the understanding of disease mechanisms and aiding in the prediction of therapeutic efficacy and safety.

Overall, Ac-RGK-AMC C26H38N8O6 significantly advances drug discovery and development by offering a reliable and versatile tool for the investigation of protease targets, ultimately contributing to the creation of novel therapeutics that can alleviate human disease burden through controlled modulation of enzyme activity.

What factors should researchers consider when using Ac-RGK-AMC C26H38N8O6 in experimental assays?

When integrating Ac-RGK-AMC C26H38N8O6 into experimental assays, researchers should consider several critical factors to ensure accurate and reproducible results. One of the foremost considerations is the selection of appropriate assay conditions, including buffer composition, pH, and ionic strength. These parameters must be optimized to closely mimic the physiological environment of the target enzyme, as deviations can significantly impact enzyme activity and substrate interaction. Buffer components should also be chosen to minimize the risk of quenching the fluorescent signal or interfering with the enzymatic reaction.

Another important aspect is the determination of optimal substrate concentration. Ensuring a sufficient concentration of Ac-RGK-AMC C26H38N8O6 is crucial for accurately capturing enzyme kinetics. The substrate concentration should be close to or exceed the enzyme's Km value—the substrate concentration at which the reaction velocity is half of Vmax—to yield meaningful kinetic data. This not only aids in characterizing the catalytic efficiency of the enzyme but also assists in evaluating inhibitor potency during drug screening. However, care must be taken to avoid substrate inhibition, which can occur if concentrations are excessively high.

Sensitivity and detection limits of the fluorescence measuring equipment should also be assessed. Ensuring that instrumentation is well-calibrated and capable of detecting real-time fluorescence changes is essential for capturing dynamic enzymatic activity. Using a microplate reader or fluorescence spectrophotometer with appropriate excitation and emission filters is recommended to maximize signal-to-noise ratios and ensure reliable data collection.

Additionally, researchers should be mindful of potential non-specific interactions, which could lead to false positive or negative results. This requires careful experimental design, including the use of appropriate controls such as substrate and enzyme blanks, to account for background fluorescence and baseline enzyme activity. Implementing replicate assays is also advisable to enhance data reliability and statistical significance.

Lastly, storage and handling conditions of Ac-RGK-AMC C26H38N8O6 are pivotal for maintaining its stability and activity. The substrate should be protected from light and stored as per manufacturer instructions, typically at low temperatures, to prevent degradation of the fluorogenic moiety and peptide chain. By thoroughly considering these factors, researchers can harness the full potential of Ac-RGK-AMC C26H38N8O6 in elucidating enzyme function and dynamics in various biological and pharmaceutical research settings.