FRAGMENT SCREENING IN LEAD DISCOVERY BY WEAK AFFINITY CHROMATOGRAPHY (WAC ) SARomics Biostructures AB & Red Glead Discovery AB Medicon Village, Lund, Sweden
Fragment-based lead discovery The basic idea: Screen few fragments (100 1,000) - Less complex molecules higher probability to bind - Less resource intensive than HTS - Better sampling of chemical space Find weak but efficient i (high h binding energy per atom) binders Develop into more potent compounds using structural information HTS hit Fragment hits Boyd, Turnbull & Walse (2012) Fragment library design considerations, WIREs Comput. Mol. Sci., 2, 868-885.
Conventional HTS vs. fragment screening in drug discovery Figure adapted from Duong-Thi, 2013, Thesis.
Structure-based and fragment-based discovery of vemurafenib Vemurafenib (PLX4032) The usage of FBLD combined with SBDD was a critical success factor in the development of vemurafenib
Fragment-based lead discovery Fragment screening Medicinal chemistry Synthesis (small molecules & peptides) Analytical chemistry (NMR & MS/MS) In vitro biology In vitro ADME/physchem HTS/FBLG expertise Screening technologies In silico screening Thermoshift assay (DSF) NMR WAC Biochemical screening (HCS) X-ray crystallography MST Compounds Proprietary library Specifically ordered sets Client libraries Structure-based drug design X-ray crystallography Computational chemistry NMR spectroscopy Biophysics Protein chemistry
Primary screening techniques NMR screening Thermal shift assay / DSF Weak Affinity Chromatography Sample changer screens 96 samples overnight Cryoprobe equipped 800 MHz NMR instrument Ligand-observed 1 H NMR spectra NMR analysis/titration of selected hits Initial screening 0.1 mg g/ /ml 0.2 mg/ml ΔTm at 62.5 μm ΔTm at 62.5 μm RG200001 5.95 7.3 RG200001 6.3 6.4 RG200002 9.6 10.4 RG200002 10.2 10.7 RG200003-1.3-0.48 RG200003-0.9-0.76 Dose response curves Target protein immobilized on HPLC column MS-detection Physiological buffer as mobile phase Retention time of fragments related to selective interactions ti with targett Simultaneous identification of weak binders (mm) in complex mixtures
Secondary and follow-up techniques X-ray crystallography MST, microscale thermophoresis HCS high concentration biochemical screening High-throughput low volume crystallization Soaking pools of fragments Co-crystallization ti of selected fragments High-throughput data collection (autom. synchrotron beamlines) Structure determination and refinement through automated data pipelines pp EC50=44 µm Versatile plate readers FRET, TRF, AlphaLisa, fluorescence polarization etc Crystallization robotics MAX IV Measures the motion of molecules along microscopic i temperature t gradients K d, dissociation constant: nm to mm range Rapid assay optimization Fast measurement: K d in 10 min Low sample consumption: minimal concentration (nm) and small volume (<4 µl) 96-1536 well plate Development and optimization Buffer composition, Additions, Linearity, Kinetic parameters Screening Robustness (Z -factor), reagent stability, reproducibility (day-to-day variation), DMSO sensitivity, automation
Weak affinity chromatography (WAC ) Principles i of WAC Data analysis Affinity constant (KD) is calculated from the retention time difference ( t ret ) for the compounds on target and blank columns. Column with immobilized protein Fragment binding WAC attractive as primary fragment screening assay Column without protein (blank column) Column cross section Invented by Prof. Sten Ohlson (Linnaeus University, it Sweden) Commercial rights by Transientic Interactions AB (TI) SARomics and RGD in unique collaboration with TI offering exclusive service platform Fragment mix Affinity LC-MS column Schematic representation of screening using WAC 0.5 minutes retention time difference is significant Extracted ion chromatograms K D = B tot /( t ret flowrate)
Some technical details WAC Typical setup: Target protein coupled via lysine residues Mixes of 10-40 cmpds (1 µm, n=2) ~1-4 mg protein on LC column (e.g. 2.1 x 50 mm) Each LC run of 30-60 minutes Affinity range: 100 µm 4 mm (can be adjusted)
WAC data on bromodomain (BRD4) Application within Nile project
BRIEF SUMMARY WAC-screening of 155 fragments Fragment lib. MW: 123-308, average MW = 239 10-fragment-mixtures 20 mm sodium acetate buffer Robustness Unchanged performance of BRD4-column over time (>1 months at least) Re-test with every two 10-mixes pooled into one 20-mix provided very similar results RG210073 (IC50 = 40 µm in biochemical assay) included in all mixes as control; ( µ y) ; Retention time = 138 ± 5 minutes (%CV = 3.9%) on BRD4-column (n=18)
Actives Retention time difference was used to identify actives: Δt ret = t BRD4-col t control_column A fragment with Δt ret > 2 minutes considered to active (provided that also (Δt ret /t BRD4-col ) > 0.1) Data suggest that t WAC has a lower degree of false positives than other techniques such as DSF and high h concentration biochemical screening No screened 155 No hits (Δt ret > 2 min) 23 No strong hits (Δt ret > 5 min) 13 Hit rate 15% WAC only technique revealing RG200067 reported by Pfizer as BRD4 binding fragment Ref: Fish et al., J Med Chem (2012), 55(22), 9831 H N RG200067 O Br N
Comparison with other techniques BRD4 screening hits HSP90 screening hits Using a WAC -cutoff of t ret > 5 min, all fragments verified by other Screening of 111 fragments by WAC, techniques except: NMR, SPR, FP and Tm shift. Selected RG200054 and RG200067 (reference cmpd Pfizer-hit ) fragments also by ITC and X-ray RG210010 and RG200039 not tested in other assays crystallography. Fragments listed with decreasing affinities as measured by WAC. (Meiby et al. 2013, Anal. Chem., Compound ID Δt ret (min) WAC Biochem DSF NMR X-ray 85, 6756) RG210073 138.1 A (IC50 ~50 µm) A NT YES RG210080 87.2 A (56%@100µM) A NT YES RG210070 84.1 Tendency of activity A NT YES RG210074 71.4 A (54%@100µM) A NT YES RG210069 52.1 Tendency of activity A NT YES RG210081 35.1 A (45% inhib@100µm) A NT YES RG210079 28.3 A (31% inhib@100µm) A NT YES RG200067 18.8 NA NA NA 4HBV RG210019 10.7 NA A NA NT RG100104 7.6 NT NA A NT RG200054 7.3 NA NA NT NT RG100056 6.8 NT NT A NT RG210010 5.9 Not tested in any other assay RG200039 5.0 Not tested in any other assay A = active; NA = not active; NT = not tested
Hit rate BRD PPI 70% 60% 50% 40% 30% 20% 10% 0% 61% 12% < 0.25 min 0.25 to 0.5 min 9% 6% 7% 5% 0.5 to 1 min 1 to 2 min 2-5 min > 5min 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 86% 8% < 0.25 min 0.25 to 0.5 min 3% 2% 1% 0% 0.5 to 1 min 1 to 2 min 2-5 min > 5min Screens performed in duplicate
WAC for complex mixtures Reaction mixtures Extracts from natural sources, i.e. natural products Analysis of Protein Target Interactions of Synthetic y g y Mixtures by Affinity-LC/MS. Singh et al. SLAS Discov. 2017 Apr;22(4):440-446
Beneficial WAC characteristics Throughput mixes, detection of multiple binders Robustness low variation between replicates, long column life time Cost efficiency low consumption of protein and fragments, standard equipment Quality automatic quality control, not sensitive for impurities Ranking immediate ranking