Determination and Measurement of Inhibitor Activity

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Accurate determination of inhibitor activity is a critical step in high-throughput screening workflows and downstream inhibitor development. At Creative Enzymes, we provide precise, reproducible, and comprehensive measurements of inhibitory effects across a wide variety of enzyme classes. By employing gold-standard methodologies, advanced instrumentation, and carefully validated assay designs, our services deliver quantitative data on potency, specificity, and mechanism of inhibition. These results guide clients in identifying the most promising candidates for further optimization and application.

Inhibitor Activity Determination: The Step After HTS

While high-throughput assays are effective in identifying potential hits, only detailed activity measurements can distinguish between weak, non-specific binders and genuine lead candidates. Determination of inhibitor activity involves not only quantifying inhibitory strength but also understanding how inhibitors interact with enzymes under physiologically or industrially relevant conditions.

Determination and measurement of enzyme inhibitor activity Figure 1: Inhibition of enzyme activity—% Activity vs log [inhibitor].

Traditional evaluation approaches, if poorly designed, may produce misleading results due to variability in assay conditions, poor sensitivity, or lack of reproducibility. To overcome these challenges, a specialized platform is needed to provide accurate kinetics, mechanistic information, and comparative analyses.

Creative Enzymes integrates enzymology expertise, state-of-the-art analytical tools, and rigorous quality control to provide trusted measurements that support both research and applied development pipelines.

Our Reliable Service Offerings

Our Determination and Measurement of Inhibitor Activity service is designed to deliver detailed, quantitative, and reproducible assessments of inhibitor performance.

Services We Offer

Service	Description
Potency Assessment	Determination of IC₅₀, EC₅₀, and K_i values using standard dose–response curves. Application of nonlinear regression models for accurate parameter estimation.
Kinetic Characterization	Michaelis–Menten and Lineweaver–Burk analyses to determine kinetic constants (K_M, V_max, k_cat). Differentiation of competitive, non-competitive, uncompetitive, and mixed inhibition mechanisms.
Selectivity and Specificity Testing	Cross-screening against related enzyme families to detect off-target effects. Multi-enzyme profiling for clients seeking highly selective inhibitors.
Stability and Robustness Studies	Evaluation of inhibitor activity across a range of temperatures, pH conditions, and cofactors. Testing in complex biological matrices (e.g., cell lysates, serum) for translational relevance.
Advanced Analytical Methods	Spectrophotometric, fluorometric, and luminescence-based readouts. Label-free technologies such as surface plasmon resonance (SPR) and biolayer interferometry (BLI). Mass spectrometry-based activity assays for high sensitivity and precision.

Deliverables

Raw and processed activity data with full traceability.
IC₅₀, K_i, K_M, and V_max values supported by clear dose–response curves.
Comparative inhibitor rankings and selectivity profiles.
Mechanism-of-action insights (competitive vs. allosteric inhibition).
A comprehensive final report with detailed assay conditions, reproducibility metrics, and recommendations for subsequent studies.

Contact Our Team

Our Advantages

Comprehensive Methodologies

A full spectrum of analytical techniques ensures flexibility and accuracy.

High Sensitivity and Precision

Advanced instrumentation minimizes variability and enhances reproducibility.

Mechanistic Insights

Beyond potency values, we deliver detailed information on inhibition mechanisms.

Customizable Testing Conditions

Assays tailored to specific enzymes, industries, or physiological environments.

Rigorous Quality Control

Replicates, controls, and orthogonal methods ensure data reliability.

Seamless Integration

Smooth connection with upstream HTS services and downstream SAR analysis.

Case Studies and Real-World Applications

Case 1: Measuring Inhibitory Activity of Pancreatic Lipase Inhibitors

This study investigated pancreatic lipase (PL) inhibition by compounds isolated from Andrographis paniculata root to explore anti-obesity potential. Using HPLC micro-fractionation and in vitro assays, several phytochemicals showed significant inhibitory activity, with IC₅₀ values ranging from 4.46–21.76 μg/mL. Among them, 7-O-methyl-dihydro wogonin (MDHW) was most potent (IC₅₀ = 14.86 μM), also demonstrating synergy with orlistat. Kinetic studies revealed competitive inhibition (K_i = 7.4 μM), with hydrophobic binding confirmed by spectroscopy and molecular docking. Structural analyses further highlighted stable MDHW-PL interactions, establishing its high inhibitory potency and potential as a pharmaceutical or nutraceutical anti-obesity agent.

Isolation and mechanistic study of a potent lipase inhibitor from Andrographis paniculata roots Figure 2. (A) Inhibition of PL activity by MDHW and orlistat at pH 8.0, 298 K, c(PL) = 5 µM, c(4-MUO) = 0.25 mM. (B) Reversibility of MDHW inhibition at varying concentrations. (C) Lineweaver–Burk plots with inset slope vs. [MDHW]. (D) Dose–effect curves of MDHW and orlistat on PL. (E) Fa–Cl plots for MDHW–orlistat combination. (F) Structure of 7-O-methyl-dihydrowogonin (MDHW). (Gaur et al., 2023)

Case 2: Measuring Inhibitory Activity of Selamectin Against DprE1 in Mycobacteria

Selamectin, an avermectin with anti-mycobacterial activity, was investigated for its inhibitory effect on Mycobacterium tuberculosis. Mutant strains with altered DprE1, an enzyme in arabinogalactan synthesis, showed increased susceptibility, suggesting a potential target. Biochemical assays confirmed selamectin binding to DprE1, while docking studies identified a loop region near Leu275 as the interaction site. Sequence alignment linked residue variations with MIC values, partially validated in engineered M. smegmatis mutants. However, metabolic labeling did not confirm typical DprE1 inhibition profiles. These findings indicate selamectin interacts with DprE1 in vitro but may act via multiple targets in vivo.

Selamectin identified as a novel dpre1 enzyme inhibitor in Mycobacterium tuberculosis Figure 3. Dose response inhibition of DprE1 by selamectin. IC₅₀ determination of selamectin against wild type (■), L282F (●), and L282V (▲) M. smegmatis DprE1. (Ezquerra-Aznárez et al., 2022)

FAQs About Inhibitor Activity Measurement

Q: What types of parameters can you measure?

A: We provide IC₅₀, EC₅₀, and K_i values, as well as kinetic constants such as K_M, V_max, and k_cat. Additionally, we assess inhibitor stability, selectivity, and mechanism of action under various conditions.
Q: Which assay formats are available?

A: We offer spectrophotometric, fluorometric, luminescent, and label-free methods (SPR, BLI), as well as mass spectrometry-based assays for high sensitivity. Assay formats are chosen based on enzyme class, inhibitor properties, and client goals.
Q: How do you ensure accuracy and reproducibility?

A: All assays are validated with appropriate controls, replicates, and reference standards. We calculate Z′ factors and other statistical quality metrics to confirm reliability. Orthogonal assays are also applied when necessary.
Q: Can testing conditions be customized?

A: Yes. We adapt assay conditions to client needs, including physiological (pH, cofactors, temperature) or industrial environments (organic solvents, high heat). This ensures data relevance to real-world applications.
Q: How are false positives or artifacts addressed?

A: False positives are minimized through replicate testing, inclusion of appropriate blanks and controls, and confirmation using orthogonal assay formats. Our data analysis pipelines are designed to filter out low-confidence results.
Q: What deliverables will I receive?

A: Clients receive complete raw data, processed datasets, dose–response curves, calculated parameters (IC₅₀, K_i, etc.), and a detailed final report. Recommendations for next steps, including SAR analysis or mechanistic studies, are also provided.

References:

Ezquerra-Aznárez JM, Degiacomi G, Gašparovič H, et al. The veterinary anti-parasitic selamectin is a novel inhibitor of the Mycobacterium tuberculosis Dpre1 enzyme. IJMS. 2022;23(2):771. doi:10.3390/ijms23020771
Gaur P, Khan F, Shanker K. Potential lipase inhibitor from underutilized part of Andrographis paniculata: Targeted isolation and mechanism of inhibition. Industrial Crops and Products. 2023;197:116623. doi:10.1016/j.indcrop.2023.116623

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