Slide 1: Donated Chemical Probe Dual BCAT1/2 Probe BAY-069
Donated Chemical Probe Dual BCAT1/2 chemical probe Probe BAY-069 /////////// June, 2th 2020 Presenters: BCAT team
Slide 2: Scientific rationale
introduction Dual BCAT1/2 Probe BAY-069 Scientific rationale Broad indication space • BCAAs are essential amino acids that animals cannot synthesize de novo • Highly tumor specific: • BCAT1 is c-Myc driven • De novo expression in tumors (besides brain*, testis and uterus) • (*) Overexpressed in IDH1
Slide 3: Literature known compounds
introduction Dual BCAT1/2 Probe BAY-069 literature known compounds CAS: 1800024-45-6 Pfizer ERG240 Ergon compound 61 (GSK) BACT1 / BCAT2 92 nM / 26 nM 0.8 µM / 4.3 µM 50 µM / 50 µM BCAT1 IC50cell-MDA MB231 452 nM >30 µM > 30 µM BCAT1 IC50cell-U87MG 4.9 µM - - Caco2 (Papp (A-B)/ 116 / 131 / 1.1 207 /
Slide 4: Cellular mechanistic assay – BCAA measurement
Cellular activity Dual BCAT1/2 Probe BAY-069 Cellular mechanistic assay – BCAA measurement BCAT1 and BCAT2 protein expression (Western Blot) Enzymatic detection of BCAA in cell culture Medium without FCS MDA-MB-231 Increase of BCAAs indicates inhibition of U-87 MG BCAT 20 h sampling, Intra-
Slide 5: Cellular mechanistic assay – BCAA measurement
Cellular activity Dual BCAT1/2 Probe BAY-069 Cellular mechanistic assay – BCAA measurement Papp A-B < 25 >= 25 > 100 BAY-069 BAY-069 leads to increased BCAA levels in U-87-MG (high BCAT1 expressing) cells These results confirm on-target cellular activity Correlation between biochemical BCAT1 I
Slide 6: Cellular functional assay - 2D cell proliferation
Cellular activity Dual BCAT1/2 Probe BAY-069 Cellular functional assay - 2D cell proliferation experiments* MDA-MB-231 U-87-MG SEM CAL 51 HC33 NCI-H2110 IC50 IC90 IC50 IC90 IC50 IC90 IC50 IC90 IC50 IC90 IC50 IC90 >50 µM >50 µM >50 µM >50 µM >50 µM >50 µM 47.7 µM 50 µM 50 µM 50 µM 50 µM 50 µM BAY-069
Slide 7: Biochemical assay – effect of protein binding
Biochemical activity Dual BCAT1/2 Probe BAY-069 Biochemical assay – effect of protein binding BAY-069 BAY-069 Strong loss of potency in the presence of serum albumin for BAY-069 Might explain lack of effect in in-vitro assays with FCS, e.g. proliferation assays 7 /// Donated Chemical Probe BAY-0
Slide 8: Probe profile: Technical profile
Probe profile Dual BCAT1/2 Probe BAY-069 Technical profile POTENCY (IC50 [nM]) Properties & Physchem O BACT1 IC50 27 nM LogD @ pH 7.5 / pka 2.4 / 5.7 Cl O BACT2 IC50 130 nM fu [%] Williams_E / brain / Mouse 0.81 / 0.47 / 0.14 N F LLE / BEI 4.8 / 19.4 Sw powder @ pH 6.5 [mg/L] 140 F N O F H Mechan. I
Slide 9: Probe profile: Atropisomerism
Probe profile Dual BCAT1/2 Probe BAY-069 Atropisomerism Annulated phenyl induces axial chirality H-bond to Gln244 in X-ray allows unequivocal assignment of stereochemistry in active isomer Atropisomers has been separated via column chromatography Atropisomers show different biochemical profiles: BAY
Slide 10: Low dose in vivo rat PK and high dose mouse PK
Probe profile Dual BCAT1/2 Probe BAY-069 Low dose In vivo rat PK and High dose In vivo mouse PK BAY Number BAY-069 BAY-069 BAY-069 Dose 25 mg/kg 50 mg/kg 100 mg/kg Vehicle: PEG400/EtOH/water (50/10/40) AUC0-tlast 16 h*mg/L 53 h*mg/L 270 h*mg/L AUC0-tlast,norm 0,63 h*kg/L 1,1 h*kg/L 2,7 h*kg/L AUC0-t
Slide 11: Target engagement: X-ray structure of BAY-069
Target engagement O Dual BCAT1/2 Probe BAY-069 Cl O N F F X-ray structure of BAY-069 N O H F BAY-069 PLP ~ 75° Lys222 Resolution 1.85 Å, Rwork/Rcryst = 0.184 / 0.227 BAY-069 bound to BCAT1, chain A BAY-069 soaked into BCAT1 crystals grown with co-factor PLP (covalently linked to Lys222) & substrate
Slide 12: Selectivity
Selectivity O Cl Dual BCAT1/2 Probe BAY-069 O N F F N O Cavbase with close congener BAY-278 F H BAY-069 BAY-278 Searching across the X-ray characterized proteome (pocketome) with closely related variant BAY-278 Using in-house co-crystal structure with BAY-278 (identical binding mode) pick all pseudo
Slide 13: Selectivity profile: broader panels
Selectivity Dual BCAT1/2 Probe BAY-069 Selectivity Profile in more detail: broader panels Other Transaminases In-house protease panel (# 30 proteases) Safety screen (Eurofins, # 77 targets) Aspartate transaminase: All IC50 > 10 µM; one hit at 6 µM Clean (no relevant activity >50%) GOT1/2: IC50 > 50
Slide 14: In vitro technical profile of Negative Control BAY-771
Dual BCAT1/2 Probe BAY-069 In vitro technical profile of Negative Control BAY-771 POTENCY (IC50 [nM]) Properties & Physchem O BACT1 IC50 6.5 µM LogD @ pH 7.5 2.2 O BACT2 IC50 10.8 µM fu [%] Williams_E / rat / Mouse - N F Mechan. IC50 MDA-MB-231 n.d. Sw @ pH 6.5 [mg/L] >412 F N O H 6.2 µM MW / TPSA [
Slide 15: Summary / Conclusion: Probe criteria
Dual BCAT1/2 Probe BAY-069 Summary / Conclusion Probe criteria Inhibitor potency: goal is < 100 nM (IC50, Kd) meets criteria for BACT1; slightly above for BCAT2 Selectivity within target family: goal is > 30-fold meets criteria; selective to aspartate transaminase Selectivity outside target family:
Slide 16: Project Team / Acknowledgement
Dual BCAT1/2 Probe BAY-069 Project Team / Acknowledgement Martje Tönies Matthew Habgood Léa Bouché Irene Helbig Stefan Kaulfuss Bernhard Radlwimmer Katja Zimmermann Hartmut Rehwinkel Wolfgang Schwede Duy Nguyen Luisella Toschi Heike Petrul Roman Hillig Clara Lemos Judith Guenther Roland Neuhaus Chri
Slide 17: Thank You
Thank You ///////////
Slide 18: Synthesis of BAY-069
Dual BCAT1/2 Probe BAY-069 Synthesis of BAY-069 NBS Fe / AcOH BAY-5000 (racemate) 18 /// Donated Chemical Probe BAY-069 /// 2020
Slide 19: Synthesis of negative Control BAY-771
Dual BCAT1/2 Probe BAY-069 Synthesis of negative Control BAY-771 BAY-771 (racemate) 19 /// Donated Chemical Probe BAY-069 /// 2020
Slide 20: Selectivity: Cavbase search for co/anti-targets
Selectivity Dual BCAT1/2 Probe BAY-069 Selectivity Profile in more detail: use of Cavbase search for possible co/anti-targets for BAY-278 Results: All hits identified have very low scores for pocket match to query (see backup for more detail) No hints for any cross reactivity of probe BAY-069 (bas
Slide 21: Back-up: Cavbase search top scorer
Back-up Dual BCAT1/2 Probe BAY-069 Selectivity Profile in more detail: use of Cavbase search for possible co/anti-targets for BAY-078 Top Scorer: BCAT1 (X647) ↔ PDB1GYP Glycosomal Glyceraldehyde-3-Phosphate Dehydrogenase from LEISHMANIA MEXICANA Results: Visual inspection confirms irrelevance to mat
Slide 22: Back-up: Cavbase search top human scorer
Back-up Dual BCAT1/2 Probe BAY-069 Selectivity Profile in more detail: use of Cavbase search for possible co/anti-targets for BAY-078 Top human Scorer: BCAT1 (X647) ↔ PDB2JKV Human Phosphogluconate Dehydrogenase Results: Visual inspection confirms irrelevance to match 22 /// Donated Chemical Probe B
Slide 23: Back-up: safety screen (Eurofins, 77 targets)
Back-up Dual BCAT1/2 Probe BAY-069 Selectivity Profile in more detail: safety screen (Eurofins, # 77 targets) 23 /// Donated Chemical Probe BAY-069 /// 2020
Slide 24: Back-up: safety screen (Eurofins, 77 targets)
Back-up Dual BCAT1/2 Probe BAY-069 Selectivity Profile in more detail: safety screen (Eurofins, # 77 targets) 24 /// Donated Chemical Probe BAY-069 /// 2020
Slide 25: Back-up: in-house protease panel
Back-up Dual BCAT1/2 Probe BAY-069 Selectivity Profile in more detail: in-house protease panel Protease # Metallo protease 8 Serine protease 15 Cysteine protease 4 Aminopeptidase 3 25 /// Donated Chemical Probe BAY-069 /// 2020
Slide 26: Probe profile: Solubility
Probe profile Dual BCAT1/2 Probe BAY-069 Solubility Properties & Physchem O Sw @ pH 6.5 [mg/L] <0.10 Cl O Sw @ pH 6.5 PBS [mg/L] 93 N F Sw @ pH 4 citrate buffer [mg/L] 4.4 F N O F H Sw in PEG400/EtOH/H2O 60/10/30 [mg/L] >830 BAY-069 (en1) Sw in PEG400/H2O 60/40 [mg/L] >790 26 /// Donated Chemical Pr
Slide 27: Compound 61 (GSK) as additional BCAT1/2 probe
Probe profile Compound 61 (GSK) as additional BCAT1/2 probe Technical profile POTENCY (IC50 [nM]) Properties & Physchem BACT1 IC50 92 nM LogD @ pH 7.5 / pka 1.93 / 6.3 BACT2 IC50 26 nM fu [%] Williams_E 6.2 / 6.2 / 0.47 Mechan. IC50 MDA-MB-231 564 nM Sw powder @ pH 6.5 [mg/L] 151 Mechan. IC50 U-87-M
Slide 28: Compound 61 (GSK): Literature
Probe profile Compound 61 (GSK) as additional BCAT1/2 probe Literature Enzyme inhibition (BCAT2) activity comparable to in-house data (50 nM vs 26 nM) Cellular data slightly better (160 nM vs 452 nM) but measured in different cell type (primary adipocyte) 28 /// Donated Chemical Probe BAY-069 //
Slide 29: Compound 61 (GSK): safety screen
Back-up Compound 61 (GSK) as additional BCAT1/2 probe Selectivity Profile in more detail: safety screen (Eurofins, # 77 targets) 29 /// Donated Chemical Probe BAY-069 /// 2020
Slide 30: Compound 61 (GSK): safety screen
Back-up Compound 61 (GSK) as additional BCAT1/2 probe Selectivity Profile in more detail: safety screen (Eurofins, # 77 targets) 30 /// Donated Chemical Probe BAY-069 /// 2020