Assay interferences

In general, the attempt to retest every compound that passes a statistically defined threshold of activity for each assay and to implement a concomitant assay interference test has been rewarded by recovery of a full spectrum of biological activities and diverse chemotypes in the confirmed hit set. In many cases, the compounds that the medicinal chemists ultimately judge to be the best starting points for lead development exhibited only modest activity (e.g., IC50 values of 0.5-5 pM) in HTS. 

Comley, J., Assay interference a limiting factor in HTS , Drug Discov. World, 4,91, 2003. 

LVE region, see Linear viscoelastic region Lyase, pectic characterized, 342 PGase assay, interference in,... 

Scalbert et al. (1989) described several methods to determine the concentration of hydrolysable and condensed tannins (proanthocyanidins see Chapter 1, section 3.13.1) in wood extracts. Tannins are complex and heterogeneous In addition to the distinction between the flavonoid-based condensed tannins and the gallic acid-based hydrolysable tannins, each group can display a large degree of chemical variability that can affect the efficacy of the different assays. Interference of chemically related nontannin compounds, such as flavonoids, can in certain cases bias the results. 

Colorometric Studies. None of the solution components of the o-dianisidine activity assay interfere with the detection of chromophore formation at 500 nm. The UV/visible spectrum of PEUU (on Mylar backing) is approximately flat in the vicinity of 500 nm, indicating that PEUU should not interfere with the assay. Inclusion of PEUU in the reference cuvette further reduced the possibility of interference from PEUU. 

Propranolol 40-160 mg t.i.d. 320 mg/day <5% 100% 100% 100% In ESRD bioavailability of propranolol may increase metabolites may cause increased bilirubin by assay interference hvooaivcemiamav occur NC NC Dose for GFR10-50 ml/min... 

This number represents theoretical number samples that should be available for population PK analysis. Actual number may be lower due to various practical reasons (not recorded, assay interference, etc.). 

BaeM IB, Holloway GA (2010) New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem 53 2719-2740... 

Precision, accuracy, and specificity have meaning only for the concentration tested. Therefore, it is imperative to test the range of concentrations of drug and potentially interfering substances that will be encountered during sample analysis. For example, a cross-reactivity of 1% with cortisone concentrations of 1-50 ng/mL is acceptable for a prednisone RIA because after a standard 20-mg dose of prednisone, the prednisone concentrations in plasma will exceed or equal the cortisone concentrations. In contrast, similar cross-reactivity with cholesterol at these concentrations would render the assay useless without prior separation of prednisone and cholesterol, because cholesterol is present in concentrations of 150-250 mg/mL in plasma (—1000 X prednisone). It is apparent that crossreactivity per se is not the problem. Cross-reactivity in combination with the anticipated concentrations of the cross-reacting substance determines the resultant assay interference. 

Many types of reactive molecules are well known to medicinal chemists acyl halides, aldehydes, aliphatic esters, aliphatic ketones, alkyl halides, anhydrides, alpha-halocarbonyl compounds, aziridines, 1,2-dicarbonyl compounds, epoxides, halopyrimidines, heteroatom-heteroatom single bonds, imines, Michael acceptors and (l-heterosubstituted carbonyl compounds, perhalo ketones, phosphonate esters, thioesters, sulfonate esters, and sulfonyl halides, to name a few [14]. This is not to say that these functionalities are not useful - some even appear in approved drugs -but all of these can react covalently with proteins, and thus should be regarded with suspicion. However, molecules can react covalently with proteins even if they do not contain functionalities that raise alarm. Jonathan Baell has referred to these as pan assay interference compounds, or PAINS, and has published a list of moieties to watch out for, as well as strategies to detect them [15, 16]. 

When a compound interferes with an assay such that it leads to an apparently positive result, this is a screening artifact. There are various possible mechanisms of assay interference, and it is therefore important to understand the screening assay(s) and... 

Fibrin clots in plasma from overfilling interference of tube additive in test method hemolysis fiom underfilfing Assay interference hemolysate contains biomarker or releases intracellular proteases that degrade the biomarker Assay interference... 

Assay interference, altered biological response of biomarker... 

Contamination Exogenous feeal, food, bacteria, environmental, for example, chemical environmental cleaning agents (e.g., Clidox) Blood (cystocentesis) and lower urinary tract cells (catheterization) introduced by collection method Intrinsic sample differences crystals, casts, epithelial, and blood cells physicochemical properties (pH, albumin, hemoglobin) Collection procedure Addition or dilution of biomarkers biomarker stability assay interference... 

Sample processing Whole urine or centrifuged (supernatant) Potential for cellular contamination in whole urine (altering biomarker level assay interference)... 

The choice of urine collection containers is based on the sample collection volume needed (spot or time collection) and need for sterility and preservative. Sterile containers are preferable to prevent microbial growth and contamination. Preservatives may be a source of assay interference but necessary for some biomarkers. A standardized set of instructions for clinical volunteers describing how urine should be collected may be useful to include in each sample collection kit. 

An exception occurs for the compounds with extremely long residence time (slow koff). Such compoimds behave like or are covalent binders to the target [32]. In the case of reactive compounds, this type of behavior can lead to pan-assay interference (PAINS [33]) since such compounds frequently appear as actives against a broad range of targets. Such compounds are not always detected by a... 

All of this data supported a conclusion that we were looking at physicochemical compound property behavior or an unspecific assay interference property rather than specific interactions with a given receptor. It was surprising that all three assay technologies against various GPCRs showed a clear response to the compounds with exception of the antagonist screen. The conclusion was that in our hands the pepducin approach would not provide a clear rational approach to identify true hits. 

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