Confirm true inhibitors, reduce false positives, and move from screening hits to lead-ready data with orthogonal biochemical assays designed for drug discovery workflows.
Running a screen is only the beginning. The real challenge starts after the primary readout, when teams need to determine which compounds are real, which are artifacts, and which deserve more time and resources.
In enzyme screening workflows, biochemical assays are frequently used after screening to support hit confirmation, orthogonal validation, triaging, mechanism of action studies, and hit expansion. Biochemical activity assays are used repeatedly after the primary screen to help prioritize compounds and reduce risk.
Orthogonal assays are a standard and valuable part of post-screening workflows. Confirm screening hits with an orthogonal assay, ideally using a different detection mechanism when practical. This helps teams validate potency with higher confidence before committing additional chemistry, biology, and budget.
Most groups do not need to replace their screening method to benefit from orthogonal confirmation. In many workflows, the screening assay and the orthogonal assay play different roles:
That makes orthogonal validation one of the fastest ways to improve decision quality after screening.
Get BellBrook’s practical guide to how biochemical assays can be used after screening for hit confirmation, orthogonal validation, triaging, mechanism studies, hit expansion, and residence time analysis.
BellBrook biochemical assays are designed to help teams move quickly from screening hits to better decisions. In post-screen workflows, the ideal assay should be sensitive enough to detect low product formation, simple to run, and suitable for quantitative measurements such as IC50 determination. Sensitivity, simplicity, and minimal assay complexity matter because each additional reagent or signal-development step increases the potential for interference and error.
Direct biochemical detection can help reduce the risk of interference from complex coupled detection schemes. Direct detection methods are generally preferable to more complex coupled-enzyme cascades when possible.
Orthogonal assays should support quantitative follow-up, including dose-response testing and potency ranking. BellBrook assays are well suited for IC50 follow-up and broader hit characterization workflows.
BellBrook assays can support validation across multiple enzyme classes and post-screen applications, helping teams move from initial hits to confirmation, triage, and deeper characterization.
A strong orthogonal assay is more than a confirmation tool. It can support several critical steps after screening.
Use an orthogonal biochemical assay to reconfirm activity in dose-response and improve confidence in apparent hits before moving further into chemistry or biology.
Identify non-stoichiometric inhibitors and other artifacts, including detergent shift, enzyme titration, and hill slope analysis. These are important tools for reducing false positives early.
Post-screen biochemical assays can be used to evaluate competitive behavior, compare potency under different substrate conditions, and prioritize compounds with more confidence.
Jump-dilution biochemical assays are an alternative way to estimate inhibitor off-rates and residence time, providing another layer of post-screen differentiation.
As more teams use virtual screening and AI-guided prioritization to narrow compound sets before testing, the need for reliable biochemical validation becomes even more important.
This video is explicitly focused on how biochemical assays are used after either biochemical HTS or virtual screening, with applications in hit confirmation, triaging, hit expansion, mechanism studies, and residence time analysis.
If your team is moving from in silico prioritization to experimental validation, an orthogonal biochemical assay can help you:
Virtual screening is a powerful approach in small-molecule drug discovery. But identifying a hit is just the beginning. To move beyond predictions, you need biochemical validation. In this video, we walk through the critical steps for confirming virtual screening hits.
For teams that want to move quickly from hit validation into a more complete biochemical workflow, Enzolution can provide a faster path forward by pairing assay-ready detection with relevant enzyme systems.
This can be especially helpful when you need to validate hits rapidly, compare compounds across a defined target workflow, or generate more consistent data without building every assay component from scratch.
Use orthogonal confirmation to get clarity quickly, then move into a more complete workflow with Enzolution when needed.
Enzolution assay systems include validated enzyme, substrates, assay buffer, plates, and protocols required to run an enzyme activity assay in an orthogonal assay format. We’ve done the work so you don’t have to – saving you months of validation work!
Enzolution assay systems are the solution, saving you time and resources when launching your inhibitor screening campaign. Learn how Enzolution works in the short video.
Tell us what you are screening and we’ll recommend a practical orthogonal assay strategy for your workflow.
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We understand that confidentiality in this business is critical. Our goal is to accelerate your discovery by providing the best assay available, coupled with expert support. We can fast track an NDA if desired.
BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400
info@bellbrooklabs.com