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Serine racemase: biochemistry,regulation and inhibition - Stefano Bruno

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Watching at the "D" side: D-amino acids and their significance in neurobiology
June 05 -June 09, 2016 – Lake Como School of Advanced Studies

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Serine racemase: biochemistry,regulation and inhibition - Stefano Bruno

  1. 1. D-serine Serine racemase Glutamic acid
  2. 2. Wolosker et al, 2008
  3. 3. PLP-enzymes
  4. 4. Percudani and Peracchi, EMBO Reports, 2003
  5. 5. Amino acid sequence Secondary structure elements Three dimensional structure FIVE FOLD TYPES
  6. 6.    
  7. 7.
  8. 8. N H C O P O O O O CH3 HC   H2 N (CH2)4 Enz H + R H C COO NH2 N H C O P O O O O CH3 HC   H2 N H C H + R COO EnzLysNH2 External aldimine Internal aldimine
  9. 9.
  10. 10. Phillips 2015
  11. 11. • •
  12. 12. Serineracemase + NH3 
  13. 13. Racemization -elimination derivatization HPLC
  14. 14. Closest mammalian paralogs Identity Serine Dehydratase 28% Cystathionine Beta-synthase 27%
  15. 15. Ligand Saccharomy ces pombe Rattus norvegicus Homo sapiens Zea mays Enterococcus faecalis
  16. 16. Smith et al, 2010 SMALL DOMAIN (a. a. 78-155) LARGE DOMAIN (a.a. 1-68, 157-340) 20°
  17. 17. +malonate
  18. 18. ATPbinding
  19. 19. REAZIONE KM (mM) kcat (min-1) kcat /KM (min-1·mM-1) -ATP +ATP -ATP +ATP -ATP +ATP L-Ser β-elimination 76  10 12  1 37  4 147  3 0.49 12.25 D-Ser β-elimination 56  12 144  27 0.9  0.1 6.6  0.6 0.016 0.046 L-Ser racemization 48  16 40  15 2.9  0.3 10  1 0.06 0.25 β-elimination activation by ATP: L-serine 25-fold D-serine 2.9-fold L-serine β-elimination/racemization: -ATP: 8.2 +ATP:50
  20. 20. L-serine beta elimination D-serine beta elimination L-serine racemization
  21. 21. - GLY + GLY Marchetti et al 2013 50 fold effect
  22. 22. Marchetti et al., 2013 15 fold effect +ATP -ATP Normalizedfluorescence [Glycine] (mM)
  23. 23. - ATP + ATP + malonate - malonate
  24. 24. 𝑦 = 𝑦0 + 𝑎 ∗ 𝐿 𝑀𝑊𝐶 𝑐𝛼 1 + 𝑐𝛼 + 𝛼(1 + 𝛼) (1 + 𝛼)2+𝐿 𝑀𝑊𝐶(1 + 𝑐𝛼)2 KD T = 1.8 ± 0.5 mM Intracellular [ATP] ~ 1-6 mM High affinity state Low affinity state KD R = 0.0115 mM MWC model
  25. 25. Ionbinding
  26. 26. S. pombe E. coli A. thaliana R. norvegicus M. musculus H. sapiens Conserved
  27. 27. Mg2+ EC50 = 17.3 ± 0.5 μM Hill coefficient =2.0 ± 0.1 Ca2+ EC50 194 ± 6 μM Hill coefficient of 1.9 ± 0.1 +Mg2+ +Ca2+ +Mg2+ +Ca2+
  28. 28. [Ca2+]free [Mg2+]free [ATP] Cytosol At rest ̴100 nM (Berridge et al. 2000) 400-600 M (Jahnen- Dechent and Ketteler 2012) ̴1-5 mM (Genc et al. 2011; Gribble et al. 2000) Activated 500-1000 nM (Berridge et al. 2000) Microdoma ins ̴100 M (Berridge et al. 2006; Rizzuto et al. 2006)
  29. 29. Mg2++ no ATPNo Mg2++ no ATP + Mg2++ + ATP
  30. 30. Cl- F- Br- I-
  31. 31. Marchetti et al., 2014 + EDTA
  32. 32. His tag hSR Tireodoxine Tev site Construct with cleavable His-tag His tag hSR Thrombin site
  33. 33. Inhibitors NMDARs Hyperstimulation • Alzheimer disease • Parkinson disease • ALS Hypostimulation • Schizophrenia
  34. 34. Strisovsky et al (2005)
  35. 35. Strisovsky et al (2005) Vorlova et al (2015) 2,2,-dichloromalonate
  36. 36. Hoffman et al (2015)
  37. 37. Virtual screening Ligands at the ATP binding site Malonate analogs
  38. 38. Structure-based virtual Screening • Fluorescence Rational Drug Design • Activity assays Excitation at 412 nm 𝑣0 = [𝑆] ∗ 𝑉𝑚𝑎𝑥 𝑆 + 𝐾 𝑀(1 + 𝐾𝑖 [𝐼] ) -elimination assays
  39. 39. Cyclopropane derivatives Substitutions on Cα Malonic acid Beato et al 2016
  40. 40. OH OH O O HN 11 B OH OH O O OH 2 B OH OH O O 1 B O O OH OH 3 A O O OH OH Maleic acid COMPOSTO Ki (μM) KD fluorescence (μM) Malonate 66 15.0±0.6 Maleate 600 352±14 3 A 1300 1000±100 8 A 2700 >5000 9 A 3500 >5000 5 A 3900 2500±500 1 A 3800 >5000 12 A >5000 >5000 11 B 70 32.9±2.7 2 B 90 12.0±0.6 1 B 620 432±18 20 B 3800 >3000 8 B >5000 ≥2500 19 B >5000 >2500 12 B >5000 >5000 Beato et al 2016
  41. 41. selection 11.000 compounds Docking 69 compounds Rescoring, post- filtering 26 compounds 8 potential inhibitors Database SPECS >300.000 compounds 3L6B (H. sapiens, closed conformation) 3HMK (R. norvegicus, open conformation) Template: Dellafiora et al 2015 4 moderate inhibitors
  42. 42. COMPO UND SPECS Structure Conformation Inhibition % 1 AG- 205/07908015 Open 70 2 AG- 670/36765032 Open 56 3 AI- 942/42301799 closed 59 4 AQ- 390/42133048 Open 70 5 AE- 641/00361044 Open - 6 AG- 690/36720008 Open - 7 AK- 968/41924790 Open - 8 AI- 240/31702043 Open -
  43. 43. Cpd 1 Cpd 2 Cpd 4 Cpd 5 Cpd 7 Cpd 8 Malonate
  44. 44. NADH ADP PPi NMN-red MNA-red
  45. 45. NADH MNA NMN
  46. 46. 𝑣 = 𝑉𝑚𝑎𝑥 𝑆 𝐾 𝑀 + 𝛽𝑉𝑚𝑎𝑥 𝑆 [𝐼] 𝛼𝐾 𝑀 𝐾𝐼 1 + [𝑆] 𝐾 𝑀 + [𝐼] 𝐾𝐼 + 𝑆 [𝐼] 𝛼𝐾 𝑀 𝐾𝐼 hyperbolic mixed-type inhibition KI=20 μM

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