Privileged Structures and Polypharmacology within and between Protein Families

Polypharmacology is often a key contributor to the efficacy of a drug, but is also a potential risk. We investigated two hits discovered via a cell-based phenotypic screen, the CDK9 inhibitor CCT250006 (1) and the pirin ligand CCT245232 (2), to establish methodology to elucidate their secondary protein targets. Using computational pocket-based analysis, we discovered intrafamily polypharmacology for our kinase inhibitor, despite little overall sequence identity. The interfamily polypharmacology of 2 with B-Raf was used to discover a novel pirin ligand from a very small but privileged compound library despite no apparent ligand or binding site similarity. Our data demonstrates that in areas of drug discovery where intrafamily polypharmacology is often an issue, ligand dissimilarity cannot necessarily be used to assume different off-target profiles and that understanding interfamily polypharmacology will be important in the future to reduce the risk of idiopathic toxicity and in the design of screening libraries.


Pocket Analysis
The SiteHopper PatchScore represents a summation of four Color and Shape Tanimoto coefficients weighted 3:1 in favor of Color similarity. Five pharmacophoric pseudocenter types make up the color forcefield: hydrogen bond donor, hydrogen bond acceptor, anion, cation and hydrophobe. Binding sites were identified with fpocket (version 2.0) which was implemented for ligand-independent cavity detection using default settings with two parameter alterations; the −r flag was set to 3.0 (default 4.5) and the −n flag was set to 3 (default 2). For each protein structure, chains were treated independently. For each chain, the cavity with the greatest fpocket Score was considered as the binding sitethis was confirmed visually for binding sites pertinent to this analysis.

B-Raf Docking Study
An enzyme-inhibitor cocrystal structure of human B-Raf kinase with a bisamide chemotype (PDB 4G9C) [1] was prepared for modeling using Protein Preparation Wizard in Maestro. [2] To propose predicted binding modes of ligands, Glide (Grid-based Ligand Docking with Energetics) [3] was used for the docking experiments. The receptor grid was defined by a grid box of 30 × 30 × 30 Å 3 with a default inner box (10 × 10 × 10 Å 3 ) centered on the cocrystallized ligand in PDB 4G9C.
The dual pirin/B-Raf ligand compound 2 was prepared using LigPrep, [4] applying the OPLS_2005 force field with possible tautomeric and ionization states within pH range 5.0−9.0 generated. Using Glide Extra Precision (XP) settings, flexible docking of the ligand was conducted without any constraints. The docked pose with the lowest RMSD to the bisamide-containing aromatic ring of the crystallized ligand in PDB 4G9C was selected as the predicted binding pose.

Chemistry Experimental
All final compounds were screened through our in-house computational PAINS filter and gave no structural alerts as potential assay interference compounds. Unless otherwise stated, reactions were conducted in oven dried glassware under an atmosphere of nitrogen or argon using anhydrous solvents. All commercially obtained reagents and solvents were used as received. Thin layer chromatography (TLC) was performed on pre-coated aluminum sheets of silica (60 F254 nm, Merck) and visualized using short-wave UV light. Flash column chromatography was carried out on Merck silica gel 60 (particle size 40-65 μm). Column chromatography was also performed on Biotage SP1 or Isolera 4 purification systems using Biotage Flash silica cartridges (SNAP KP-Sil). Ion exchange chromatography was performed using acidic Biotage Isolute Flash SCX-2 columns.
Determination of optimal probe concentration: 5 μL of pirin (400nM in assay buffer) or 5 μL assay buffer and increasing concentrations of probe (5 μL, dilution series from 0.2 up to 1000nM) were added. From the plotted data, a probe concentration of 2 nM was selected and gave an assay window of 4.0 with a Z' of 0.74.

Fluorescent probe K D determination: 5 μL of probe molecule (4 nM in assay buffer) to increasing concentrations of the pirin protein (5 μL, dilution series from 0.2 to 200 nM). Fluorescence polarization values for tracer control wells
(2 nM probe in assay buffer only) were subtracted from each data point prior to data analysis. The K D determination was analyzed using non-linear regression analysis (one site-specific binding model, GraphPad Prism 6) and gave a K D of 12 nM.
Compound IC 50 determination: Compounds (0.2 μL at 50 x screening concentration in DMSO) were dispensed using an ECHO 550 Liquid Handler (Labcyte Inc.). To the corresponding wells, 5 μL of probe molecule (4 nM in assay buffer) and 5 μL of pirin protein (60 nM) were added. Tracer controls (2 nM probe molecule only) and bound tracer controls (2 nM probe in the presence of appropriate protein concentration) were included on each assay plate.
IC 50 determination was performed using non-linear least squares curve fitting (GraphPad Prism 6, log(inhibitor) vs.
The equation states that the IC 50 for a ligand that is competitive for binding with the assay probe is related to the binding affinity of the ligand (K i ), the bound fraction of the probe (f 0 ), the binding affinity of the probe (K d ) and the concentration of the probe (L 0 ). For competition experiments, it is recommended that a protein concentration giving a bound fraction between 0.5 and 0.8 be selected. A bound fraction below 0.7 will often result in an assay that is not statistically robust due to the decreased size of the binding window, however as the bound fraction approaches 1 the relationship between K i and IC 50 deviates from linear and the resolvable range of the assay decreases. For these reasons, a bound fraction of 0.72 was used for all assays.       For the screening protocol and assay conditions see: https://www.thermofisher.com/uk/en/home/life-science/drugdiscovery/target-and-lead-identification-and-validation/kinasebiology/kinase-activity-assays/z-lyte.html
Assay step-by-step process: 1. Upon receipt of your small molecule, staff at the ICKP will dilute each to the appropriate concentration (if required) 2. This compound is added to a 'mother plate' consisting of customer samples, controls and blanks  These serve as the source for 'daughter plates' which are stored at -20* until assay initiation  Note: All compounds are screened in duplicate 3. Next there will be 3 additions to the assay:  1. Bar codes assigned to each file ensure that data corresponding to the correct compound is being analysed 2. After completion of each assay, ICKP staff ensure that the run has passed standard quality control measures by examining reference compounds on the QC plate 3. Upon determination that the run has met QC standards, a Z-Prime (Z') value is calculated utilizing data from the controls/blanks on each individual plate  This QC measure is in place to ensure that each individual plate in the run has passed QC 4. Finally, a mean percentage activity is calculated for for each customer.
 A standard deviation for all the duplicates is also calculated