Domainselective ACE inhibitors caught in the act

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Domain-selective ACE inhibitors caught in the act

• Jean Watermeyer

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Hypertension

• It is estimated that 1 in 4 adult South Africans
  suffers from hypertension a leading cause of
  cardiac disease
• Hypertension and cardiac disease are major causes
  of premature death worldwide
• 1950s Angiotensin discovered
• peptide in bloodstream of hypertensive patients
  that induced increase in blood pressure in test
  animals
• Two forms inactive AngI (10aa) and active AngII
  (8aa)
• AngI converted to AngII by angiotensin-converting
  enzyme (ACE)
• Today, ACE inhibitors are widely used to treat
  cardiac disease

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Blood pressure regulation RAS and KKS

• F.A. Sayed-Tabatabaei, et al Circ. Res.
  2006981123-1133

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ACE introduction

• zinc metalloprotease
• C-terminal dipeptidase
• expressed in many mammalian tissues, especially
  kidneys and lung endothelia
• Membrane bound shed into bloodstream
• Glycoprotein
• Two homologous domains (55)
• 2 active sites
• different specificities
• different Cl-dependence
• Testis isoform ? C domain

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ACE inhibitors
Figure from Acharya et al, 2003
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Novel domain-selective inhibitors
Nchinda et al, 2006
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Crystallisable ACE construct
Gordon et al, 2003

• Structure essentially identical to minimally
  glycosylated wild-type tACE (lacking res 1-36)

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tACE-G13 glycosylation mutant
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Aims of my work

• Obtain co-crystals of tACE-G13 with novel
  domain-specific inhibitors
• Collect good diffraction data
• Solve the phasing using molecular replacement
• Build structure models of tACE-G13 bound to
  inhibitors

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Co-crystallisation

• 4?l hanging drop at 16C
• protein stock purified tACE-G13 in 5mM HEPES
• inhibitor in excess (1.5 1000-fold)
• 500m?l Als oil over reservoir
• Streak-seeding

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Data collection
BM14 ESRF Grenoble, France
Data processing HKL2000 Refinement CNS Model
building O
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inhibitor N-ACE-84 density in the active site
2.1A resolution
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Precautions against model bias

• Use composite omit maps generated with simulated
  annealing rather than simple 2Fobs-Fcalc maps
• Refine selected areas while fixing others
• Reset and refine B-factors cautiously
• Use same Rfree reflection set that was used to
  solve the MR model.

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Domain-selectivity in the S2 pocket
N-ACE-84 C-selective Ki N 196 ?M Ki C 0.8 ?M
4.23A
4.59A
N-ACE-86 not selective Ki N 23.7 ?M Ki C
84.3 ?M
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Reaction mechanism
proton shuttle
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(No Transcript)
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Conclusions

• Gem-diol tetrahedral reaction intermediate
  visualised
• Binding of P2 backbone to beta 4 suggests
  strand-strand interaction with longer peptide
• Domain-selectivity is due to the P2 Trp residue
• bulkier, charged or polar residues hinder binding
  of N-84 to the N-domain
• smaller or more hydrophobic residues in the
  C-domain mean enhanced binding of N-84
• N-86 is non-selective because its smaller P2
  group doesnt fill the S2 pocket
• There is more space in the S2 pocket (GOL
  molecule) so inhibitors could be enlarged for
  greater selectivity

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Ongoing work

• Crystal structures of another two domain-specific
  inhibitors derived from lisinopril
• Epitope mapping of an antibody to the N-domain of
  ACE that has an inhibitory effect at high
  concentrations
• Other crystallisation trials ACE2, sACE

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ACE functions

• Classical ACE causes vasoconstriction
• ACE inhibitors are widely used to treat cardiac
  disease
• Some more recent discoveries
• role in cell growth and differentiation (Ac-SDKP
  substrate)
• ACE degrades amyloid beta peptide in vitro
• Cleavage of luteinising hormone-releasing hormone
• signalling by ACE
• inhibitor-induced phosphorylation of cytoplasmic
  tail by CK2 and JNK accumulation in nucleus
• Possible cleavage release of GPI-anchored
  proteins

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ACE Structure