Table 1. Common types of assay interference.

Assay interference described in detail in this Review are underlined. Missing fields in the Table indicate that a substantial amount of work remains to be done to characterize compounds used in HTS and determine the impact of various types of assay interference. Also not included in this Table, due to lack of available information, are the effects of metal ion interference on assays [4,5] and assay interference by compounds that affect cell membrane permeabilization [47].

Assay Interference	Effect on Assay:	Characteristics of note	Prevention	Prevalence in Library	Percent Enrichment of Actives	Ref
Aggregation	-Biochemical: Non-specific enzyme inhibition; protein sequestration -Cell-based: Protein sequestration	-Concentration-dependent -IC50 is sensitive to [E] -Efficacy is sensitive to detergent -Time-dependent inhibition -Inhibition curves have steep Hill slopes -Reversible by diluting compound	-Include 0.01–0.1% Triton X-100 in assay buffer	- 1.7–1.9%	-As high as 90–95% of actives in some biochemical assays	[11, 12, 30, 31, 33, 35, 48]
Compound Fluorescence	-Biochemical: Increase in light detected may affect apparent potency; Bleed-through fluorescence in adjacent wells -Cell-based: May affect apparent potency; General increase in light detected; Increase in light detected due to association of compound with cell/cellular structures	-Reproducible -Concentration-dependent	-Use orange/red-shifted fluorophores -Include a pre-read after compound addition but prior to fluorophore addition -Include time delay after excitation of fluorophore (time-resolved) -Use a ratiometric fluorescence output -Use previous profiling efforts to identify fluorescent compounds	-EX340nm/EM450nm ~2–5% -EX480nm/EM540nm ~0.01–0.2% -EX525nm/EM598nm ~0.004–0.01% -EX547nm/EM618nm ~0.001-.01% - EX570nm/EM670m ~0%	-Up to 50% of actives in certain assays involving blue-shifted spectral range	[9,11,14,18–20, 49–52]
Firefly Luciferase Inhibitors	-Biochemical: Inhibition -Cell-based: Inhibition or Activation	-In purified (biochemical) firefly luciferase assay with KM substrate, concentration-dependent inhibition of luciferase	-Test actives against purified firefly luciferase using KM levels of substrate -Use orthogonal assay with an alternate reporter to confirm activity -Use previous profiling efforts to identify FLuc inhibitors	-At least 3%	-As high as 60% of actives in some cell-based assays	[6,23]
Redox Cycling Compounds	-Biochemical: Inhibition -Cell-based: Inhibition or Activation	-Concentration-dependent -Potency depends on concentration of RCC and reducing reagent -Time dependent enzyme inactivation -pH sensitivity -Effect generally depends on strength of reducing reagent -If H2O2 is generated, elimination of activity upon catalase addition	-Replace DTT and TCEP in buffers with weaker reducing agents such as cysteine and glutathione -Use [DTT] ≥ 10mM -Use previous profiling efforts to identify RCCs	-Compounds that generate H2O2 at appreciable levels: ~0.03%	-Enrichment for a given assay found to be as high as 85%	[37 – 40, 42]
Oxidizers	-Biochemical: Inhibition -Cell-based: Inhibition or Activation	-Concentration-dependent -Potency lessened in the presence of reducing reagent -pH sensitivity -Time dependent enzyme inactivation -Reversible	-Measure caspase activity +/− DTT in presence of compound -Measure reactivity against glutathione		~20–50% enrichment for some assays	[45, 46, 53]
Covalent reactivity	-Biochemical and Cell-based: Inhibition or activation	-Generally, irreversible modification of enzymes/proteins	-Identify compounds with reactive functionalities	-Less than 0.65% (0.65% of compounds had reactive functional groups and inhibited the enzyme target-cruzain)		[4,10,11,13,54]
Cytotoxicity	-Biochemical: N/A -Cell-based: Apparent inhibition due to cell death	-Occurs more commonly at higher compound concentrations -May occur more commonly in assays with longer compound incubation times	-Test compounds for cytotoxicity using commercially available assays, dye uptake, or colorimetric assays -Use previous profiling efforts to identify potential candidates (PubChem AID 463)			[55 – 59]
Inner filter effect (absorption of light)	-Biochemical & Cell-based: Decrease in light detected may affect apparent potency	-Components of assay system are colored	-Check for colored compounds -Conduct excitation/emission at longer wavelengths			[9,19]
Light scattering	-Biochemical & Cell-Based: Decrease in light detected; Increase in fluorescent ratio for ratiometric assays	-Generally occurs at lower wavelengths of light (blue spectral region) and at higher concentrations of compound	-Conduct excitation/emission at longer wavelengths			[19]
Quenching	-Biochemical & Cell-Based: Decrease in light detected may affect apparent potency	-Reduction in the excited state of the fluorophore results in decreased light emission				[9,19,60]
Surfactant-like compounds	-Biochemical: Promiscuous activation of enzymes -Cell-based: Cell lysis and cytotoxic at high concentrations	-Concentration-dependent -Non-specific activation of enzymes	-Include 0.1% CHAPS or Triton X-100 in assay buffer			[11, 61, 62]