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Chemoinformatic methods for predicting interference in drug of abuse/toxicology immunoassays.

ABSTRACT: BACKGROUND:Immunoassays used for routine drug of abuse (DOA) and toxicology screening may be limited by cross-reacting compounds able to bind to the antibodies in a manner similar to the target molecule(s). To date, there has been little systematic investigation using computational tools to predict cross-reactive compounds. METHODS:Commonly used molecular similarity methods enabled calculation of structural similarity for a wide range of compounds (prescription and over-the-counter medications, illicit drugs, and clinically significant metabolites) to the target molecules of DOA/toxicology screening assays. We used various molecular descriptors (MDL public keys, functional class fingerprints, and pharmacophore fingerprints) and the Tanimoto similarity coefficient. These data were then compared with cross-reactivity data in the package inserts of immunoassays marketed for in vitro diagnostic use. Previously untested compounds that were predicted to have a high probability of cross-reactivity were tested. RESULTS:Molecular similarity calculated using MDL public keys and the Tanimoto similarity coefficient showed a strong and statistically significant separation between cross-reactive and non-cross-reactive compounds. This result was validated experimentally by discovery of additional cross-reactive compounds based on computational predictions. CONCLUSIONS:The computational methods employed are amenable toward rapid screening of databases of drugs, metabolites, and endogenous molecules and may be useful for identifying cross-reactive molecules that would be otherwise unsuspected. These methods may also have value in focusing cross-reactivity testing on compounds with high similarity to the target molecule(s) and limiting testing of compounds with low similarity and very low probability of cross-reacting with the assay.

SUBMITTER: Krasowski MD

PROVIDER: S-EPMC2855823 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Chemoinformatic methods for predicting interference in drug of abuse/toxicology immunoassays.

Krasowski Matthew D MD Siam Mohamed G MG Iyer Manisha M Pizon Anthony F AF Giannoutsos Spiros S Ekins Sean S

Clinical chemistry 20090402 6

<h4>Background</h4>Immunoassays used for routine drug of abuse (DOA) and toxicology screening may be limited by cross-reacting compounds able to bind to the antibodies in a manner similar to the target molecule(s). To date, there has been little systematic investigation using computational tools to predict cross-reactive compounds.<h4>Methods</h4>Commonly used molecular similarity methods enabled calculation of structural similarity for a wide range of compounds (prescription and over-the-counte ...[more]

PMID: 19342505

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Project description:BackgroundLaboratory tests for routine drug of abuse and toxicology (DOA/Tox) screening, often used in emergency medicine, generally utilize antibody-based tests (immunoassays) to detect classes of drugs such as amphetamines, barbiturates, benzodiazepines, opiates, and tricyclic antidepressants, or individual drugs such as cocaine, methadone, and phencyclidine. A key factor in assay sensitivity and specificity is the drugs or drug metabolites that were used as antigenic targets to generate the assay antibodies. All DOA/Tox screening immunoassays can be limited by false positives caused by cross-reactivity from structurally related compounds. For immunoassays targeted at a particular class of drugs, there can also be false negatives if there is failure to detect some drugs or their metabolites within that class.MethodsMolecular similarity analysis, a computational method commonly used in drug discovery, was used to calculate structural similarity of a wide range of clinically relevant compounds (prescription and over-the-counter medications, illicit drugs, and clinically significant metabolites) to the target ('antigenic') molecules of DOA/Tox screening tests. These results were compared with cross-reactivity data in the package inserts of immunoassays marketed for clinical testing. The causes for false positives for phencyclidine and tricyclic antidepressant screening immunoassays were investigated at the authors' medical center using gas chromatography/mass spectrometry as a confirmatory method.ResultsThe results illustrate three major challenges for routine DOA/Tox screening immunoassays used in emergency medicine. First, for some classes of drugs, the structural diversity of common drugs within each class has been increasing, thereby making it difficult for a single assay to detect all compounds without compromising specificity. Second, for some screening assays, common 'out-of-class' drugs may be structurally similar to the target compound so that they account for a high frequency of false positives. Illustrating this point, at the authors' medical center, the majority of positive screening results for phencyclidine and tricyclic antidepressants assays were explained by out-of-class drugs. Third, different manufacturers have adopted varying approaches to marketed immunoassays, leading to substantial inter-assay variability.ConclusionThe expanding structural diversity of drugs presents a difficult challenge for routine DOA/Tox screening that limit the clinical utility of these tests in the emergency medicine setting.

Project description:Background:Vilazodone is an FDA approved medication used to treat major depressive disorder. The authors describe two cases of accidental vilazodone exposure in toddlers who presented with symptoms similar to amphetamine exposure and also with unexplained positive amphetamine urine immunoassay drug screens. Given a lack of published data on cross-reactivity of vilazodone and its metabolites with drug of abuse screening tests, the authors investigated drug of abuse immunoassay cross-reactivity of vilazodone and metabolites using computational and empirical approaches. Methods:To ascertain the likelihood that vilazodone would cross-react with drug of abuse screening immunoassays, the authors assessed the two-dimensional (2D) similarity of the vilazodone parent molecule and known metabolites to an array of antigenic targets for urine immunoassay drug screens. To facilitate studies of the commercially unavailable M17 metabolite, it was prepared synthetically through a novel scheme. Urine and serum were spiked with vilazodone and M17 into urine (200-100,000 ng/mL) and serum (20-2000 ng/mL) samples and tested for cross-reactivity. Results:Computational analysis using 2D similarity showed that vilazodone and metabolites have generally low similarity to antigenic targets of common drug of abuse screening immunoassays, predicting weak or no cross-reactivity. The M17 metabolite had 2D similarity to amphetamines and tricyclic antidepressants in a range similar to some other compounds exhibiting weak cross-reactivity on these immunoassays. Cross-reactivity testing was therefore performed on two different urine amphetamines immunoassays and a serum tricyclic antidepressant immunoassay. However, actual testing of cross reactivity for vilazodone and the M17 metabolite did not detect cross-reactivity for any urine amphetamines screen at concentrations up to 100,000 ng/mL and for a serum tricyclic antidepressants assays at concentrations up to 2000 ng/mL. Conclusion:While the vilazodone metabolite M17 has weak 2D structural similarity to amphetamines and tricyclic antidepressants, the current study did not demonstrate any experimental cross-reactivity with two different urine amphetamines immunoassays and a serum tricyclic antidepressant immunoassay. Vilazodone ingestions in young children present a diagnostic challenge in their similarity to amphetamine ingestions and the lack of routine laboratory tests for vilazodone. Further work is needed to understand the metabolic profile for vilazodone in children versus adults.

Project description:The homeostasis between mature neurotrophin NGF and its precursor proNGF is thought to be crucial in physiology and in pathological states. Therefore, the measurement of the relative amounts of NGF and proNGF could serve as a footprint for the identification of disease states, for diagnostic purposes. Since NGF is part of proNGF, their selective identification with anti-NGF antibodies is not straightforward. Currently, many immunoassays for NGF measurement are available, while the proNGF assays are few and not validated by published information. The question arises, as to whether the commercially available assays are able to distinguish between the two forms. Also, since in biological samples the two forms coexist, are the measurements of one species affected by the presence of the other? We describe experiments addressing these questions. For the first time, NGF and proNGF were measured together and tested in different immunoassays. Unexpectedly, NGF and proNGF were found to reciprocally interfere with the experimental outcome. The interference also calls into question the widely used NGF ELISA methods, applied to biological samples where NGF and proNGF coexist. Therefore, an immunoassay, able to distinguish between the two forms is needed. We propose possible ways forward, toward the development of a selective assay. In particular, the use of the well validated anti-NGF ?D11 antibody in an alphaLISA assay with optimized incubation times would be a solution to avoid the interference in the measurement of a mixed sample containing NGF and proNGF. Furthermore, we explored the possibility of measuring proNGF in a biological sample. But the available commercial kit for the detection of proNGF does not allow the measurement of proNGF in mouse brain tissues. Therefore, we validated an SPR approach for the measurement of proNGF in a biological sample. Our experiments help in understanding the technical limits in the measurement of the NGF/proNGF ratio in biological samples, and propose concrete solutions toward the solution of this problem.

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Chemoinformatic methods for predicting interference in drug of abuse/toxicology immunoassays.

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Chemoinformatic methods for predicting interference in drug of abuse/toxicology immunoassays.

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