Development and evaluation of

in silico toxicity screening panelsWill Krawszik1, Maja Aleksic2, Paul Russell2, Jonathan G.L. Mullins3

1 – Moleculomics In Silico Discovery Inc (Canada), 500 Boulevard Cartier Ouest, Bureau 115, Laval, Quebec, H7V 5B7, Canada2 – Safety and Environmental Assurance Centre, Unilever, Colworth Science Park, Sharnbrook, Bedfordshire, MK44 1LQ, UK3 – Moleculomics Ltd (UK), Institute of Life Sciences, Swansea University Medical School, Singleton, Swansea, SA2 8PP, UK

Contact email: will@moleculomics.com Contact telephone: 514-701-2771

Background - Modelling receptor interactions is of significant interest to the scientific

community, with many computational tools available. However, current tools are designed for

the prediction of on-target effects and are widely used in the pharmaceutical industry, where

compounds are routinely screened for binding affinity to only a single receptor of interest.

In summary, the project developed a workflow capable of reliable prediction, at

>90% accuracy, of protein-ligand hits when compared with recorded in vitro

interactions, detailed in DrugBank and ToxCast. This is the first successful

implementation of an in silico panel for pharmacological profiling and is a highly

promising development.

Targets to provide an early assessment of the potential hazard of a compound or chemical series, as recommended by Bowes et al (2012), Nature reviews: Drug Discovery, 11: 909-922:

G protein-coupled receptorsAdenosine receptor A2A (ADORA2A)α1A-adrenergic receptor (ADRA1A)α2A-adrenergic receptor (ADRA2A)β1-adrenergic receptor (ADRB1)β2-adrenergic receptor (ADRB2)‡Cannabinoid receptor CB1 (CNR1)Cannabinoid receptor CB2 (CNR2)Cholecystokinin A receptor (CCKAR)Dopamine receptor D1 (DRD1)‡Dopamine receptor D2 (DRD2)‡Endothelin receptor A (EDNRA)Histamine H1 receptor (HRH1)‡Histamine H2 receptor (HRH2)δ-type opioid receptor (OPRD1)κ-type opioid receptor (OPRK1)‡μ-type opioid receptor (OPRM1)‡Muscarinic acetylcholine receptor M1 (CHRM1)Muscarinic acetylcholine receptor M2 (CHRM2)‡Muscarinic acetylcholine receptor M3 (CHRM3)5-HT1A (HTR1A) 5-HT1B (HTR1B) 5-HT2A (HTR2A)‡ 5-HT2B (HTR2B) High/Vasopressin V1A receptor (AVPR1A)

Ion channelsAcetylcholine receptor subunit α1 or α4 (CHRNA1 or CHRNA4)‡Voltage-gated calcium channel subunit α Cav1.2 (CACNA1C)‡GABAA receptor α1(GABRA1)‡Potassium voltage-gated channel subfamily H member 2; hERG (KCNH2)Potassium voltage gated channel KQT-like member 1 (KCNQ1) and minimal potassium channel MinK (KCNE1)NMDA receptor subunit NR1 (GRIN1)‡5-HT3 (HTR3A)‡ Voltage-gated sodium channel subunit α

EnzymesAcetylcholinesterase (ACHE)Cyclooxygenase 1;COX1 (PTGS1)Cyclooxygenase 2; COX2 (PTGS2)‡Monoamine oxidase A (MAOA)‡Phosphodiesterase 3A (PDE3A)Phosphodiesterase 4D (PDE4D)‡Lymphocyte-specific protein tyrosine kinase (LCK)

TransportersDopamine transporter (SLC6A3)Noradrenaline transporter (SLC6A2)‡Serotonin transporter (SLC6A4)‡

Nuclear receptorsAndrogen receptor (AR)Glucocorticoid receptor (NR3C1)

Conclusions - The project benefited from an extensive validation exercise,

involving large databases of in vitro results. This enabled analysis of the

accuracy of prediction with reference to in vitro results, for which the

prediction of hits was pleasingly high, although there remains work to

improve upon the delineation of misses. In addition, comparisons with the in

vitro data for the blind test compounds were promising. The resulting

technology system provides extremely valuable molecular knowledge which

provides the basis to a novel screening tool as it enables a paradigm shift

from reliance on observing effects at a system level, including a reduced

reliance upon animal testing, to predicting effects based on understanding at

the molecular level, whilst also reducing drug development costs through the

ability to screen for toxic or adverse reactions earlier within the drug

development cycle. Such molecular knowledge is a valuable commodity to a

range of industries including; agrochemical, biotech, synthetic biology and

medical/health research.

ToxCast compounds FDA Approved Drug Bank “experimental”

19 “test” compounds vs. Bowes et al “Panel 44 set”

Pathway Analysis

Approach - An off-target screening approach was adopted, screening compounds of interest against the structures of 44 receptors known to be associated with toxic or adverse reactions. This work involved extensive testing, comparison and cross-referencing of at least 3 independent docking methods to in vitro results. A fundamental feature of this work was establishing “hit thresholds” for both known toxins and FDA approved compounds to provide a useful reference for compounds of unknown efficacy or toxicity.

Stage 1: Structural modelling and validation

Stage 2: In silico screening of “blind test” compounds

Stage 3: Continued validation / refinement of the approach

Normalisation techniques – A number of techniques were applied to normalise both the in vitro control data and the results of a given docking in the context of other dockings as follows;Normalisation of the in vitro data – It was observed that the control data obtained from the FDA Approved and ToxCast compounds naturally featured more hits than misses. In vitro control data was normalised to ensure the resulting prediction was not biased towards the prediction of a hit over a miss or vice versa.Normalisation of the ligand results - Results were normalised due to the observation of the tendency of the in vitro studies to identify a higher level of larger molecule "hits" and smaller molecule "misses". The developed algorithm normalises the predicted energy of interaction independent of the number of atoms.Normalisation of the docking results – This was undertaken to account for scoring functions that take into account how tightly a drug binds relative to other ligands that are predicted to bind at the same site and; and for the purpose of comparison of the respective binding affinities at different sites within the same protein.

Heavy atoms in the receptor protein, indicated in Silver

Dockings extracted from training data sets, indicated by blue circles

Dockings of “low interactivity” and “interactivity” ligands from, indicated in yellow and finally;

Dockings of “high interactivity” ligands, indicated by red ‘x’s.

Cholecystokinin A receptor (CCKAR)

5-HT3 (HTR3A)

Cannabinoid receptor CB2 (CNR2)

Predicted interaction scores below are in the range [0, 1]. Results have been normalised such that >0.5 is the criterion for a predicted hit with a true positive rate of 90% and a true negative rate of 30-40% (estimated). To aid analysis of these results a colour coding system has been applied whereby blue denotes a score <0.5, and therefore a miss. Hits are indicated by a score >0.5 shaded by an increase in intensity of red towards a score of 1.0 (full confidence hit). This colour coding enables the user to readily identify trends within the results.

Figures below indicating high throughput docking locations of validation data sets and specific docking orientations of “blind test” compounds.