H,KATPase and Antacid Drugs

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H,K-ATPase and Antacid Drugs
Richard J. Law Biomedical Division
Biopostdoc Symposium July 26th, 2006
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Acknowledgements

• Structures Mechanisms Group
• Felice Lightstone
• Ed Lau
• Brian Bennion

• UCLA collaborators The pump people
• Keith Munson
• Rachel Garcia
• George Sachs

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Gastric H,K-ATPase its what makes your stomach
acidic!

• Associated Conditions
• Heart burn
• Gastroesophageal Reflux Disease (GERD)
• Dyspepsia
• Gastric/duodenal Ulcers
• Zollinger-Ellison Syndrome
• Stomach/Esophageal cancer

Gastric lumen
H,K-ATPase
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H,K-ATPase as a Drug Target
(Most profitable drug target of all time!)
Two classes of drugs (theoretically) 1.
Proton-Pump-Inhibitors (PPIs)
The Purple Pill
Prevacid lansoprazole (TAP) Protonix
pantoprazole (Altana) Aciphex rabeprazole
(Eisai)
(omeprazole)
Proctor Gamble
2. H,K channel-Inhibitors
The Healing Purple Pill
?
(s-omeprazole)
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What we need to investigate?

• Mechanism of proton pumping inc. ATP cleavage
  conformational change
• Mechanism of K ion conduction
• Mechanism of PPI drugs
• Design of new K channel blocker drugs

N
2K
ATP
A
106x H
P
CYTOPLASM
X
X
GASTIC LUMEN
10x K
2H
KC inhibitors would block this pathway
PPIs inhibit this pathway
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Pumps are like double motorized barn doors, not
like leaf blowers!
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Pumps are like double motorized barn doors, not
like leaf blowers!
Exit Channel
Occlusion Site
Entry Channel
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Pump Cycle Structures
H (cytoplasm) ATP
Structures of H,K-ATPase are homology models
based on sr-Ca2-ATPase
K (cytoplasm)
ADP
E1
E2-Kocc
E1-P-H
P
E2-P
H (lumen)
K (lumen)
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H,K-ATPase System Set-up
For K ion conduction (and new inhibitor binding)
we need to study the E2-P state.

• Protein was placed in a POPC bilayer.
• Hydrated with TIP3P waters and ions added to
  neutralise charge and provide a 150nM solution.
• 2 hydronium ions were included in the pore.
• Several simulations were run with different
  starting positions for K ions in the pore.

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The E2 K entry channel

• The E2-P conformation presents an open channel
  on the lumen side. It can be solvated with water
  and ions.
• There is no channel wide enough to accommodate
  water or ions above a certain point.
• This is the entry channel for K ions.
• What happens to an ion placed in the entry
  channel in simulation? Will it find an occlusion
  site for us??

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The Entry channel K to the Occlusion Site

• Several simulations were run placing 1 and 2
  ions in the entry channel

• Simulations showed fast movement of ions to the
  end the channel to a location at the broken M4
  helix

• Backbone carbonyls coordinate the ions. This is
  the occlusion site.

• The waters do not appear to be very stable in
  the hydrophobic entry channel

Munson, K., Law, RJ., Sachs, G. (2006) Analysis
of the Gastric H,K ATPase E2P Conformation for
Ion pathways and Inhibitor Binding Sites
(Submitted to Biochemistry).
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Pump Cycle
H (cytoplasm) ATP
K (cytoplasm)
ADP
E1
E2-Kocc
E1-P-H
P
E2-P
H (lumen)
K (lumen)
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Conformational change (targeted MD) and double
gating.
Conformational change from E2-P to E1 state with
targeted MD

• Does this simulation help to define the double
  gate we need for pump function? Where are the
  gates?

• Do the ions leave the occlusion site and go into
  an exit channel??

• Does a solvated exit channel form?

• What happens to the occlusion site during the
  conformational change?

• Conformational change from E2-P to E1 state
  barely affects the entry channel at all. So how
  is the entry channel closed?

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Conformational change and ion exit (not!)
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Conformational change and ion exit (not!)
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Conformational change and gating.
Mutation of K743 to A or S give 0 and 4 of wt
K pump activity.
Exit Channel
L743 is the second gate
Occlusion Site
Entry Channel
The simulation shows that the entry gate is the
fracturing of the water in the entry channel.
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PPIs Prodrugs Mechanism

• Drugs like omeprazole and pantoprazole are
  actually pro-drugs.
• That is, they undergo significant chemical
  re-arrangement before they are active.

1.
Chemical Conversion (Acidic canalicular space of
the parietal cell or elsewhere)
Pantoprazole (Protonix)
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PPIs Prodrugs Mechanism

• Can we improve the mechanism of chemical
  conversion to the active form of the PPI drugs?

• We now know where the PPI drugs bind in the
  channel. Can we improve this binding affinity?

• S-S formation is a different binding mode and
  must be to Cys.813. Is there chemistry to improve
  this step? What about Cys. 822?

Cys-822
1.
2.
Binding to HK-ATPase
Chemical Conversion (Acidic canalicular space of
the parietal cell or elsewhere)
3.
Pantoprazole (Protonix)
S-S Maturation
Cys-813
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New Inhibitor Binding
Phenyl-imidizole compounds as investigative lead
compounds for new class of inhibitors (Lead
compounds from Altana).
Byk59
Byk73
99 binds with 1000 fold higher affinity than 73.
Byk84
Byk99
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New Inhibitor Binding
Docking determined the lowest energy site
consistent with mutagenesis for both the position
of the binding site and the orientation of the
phenyl ring.

• Byk99 and Byk73 both found a similar site in the
  centre of the channel.

• But consistent with experimental binding assays
  is that Byk99 binds with lower energy (-10.4 vs
  -8.2 kcal/mol).

I816

• Byk73 is shifted to accommodate the extra methyl
  group (I816).

Munson, K., Law, RJ., Sachs, G. (2006) Analysis
of the Gastric H,K ATPase E2P Conformation for
Ion pathways and Inhibitor Binding Sites
(Submitted to Biochemistry).
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New Inhibitor Binding non-competitive binding
site
Docking found site on the outside of the protein.

• Consistent with the data indicating the presence
  of a non-competitive binding site (DG -7.4
  kcal/mol 5.9mM (6.3mM for nc binding))

Cys 822

• Appears to be the site that gives maturation to
  Cys. 822

• Mechanism of inhibition maybe via restriction of
  relative motion of TM helices

Munson, K., Law, RJ., Sachs, G. (2006) Analysis
of the Gastric H,K ATPase E2P Conformation for
Ion pathways and Inhibitor Binding Sites
(Submitted to Biochemistry).
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Conclusions Future Work

• We can define the entry pathway and occlusion
  site for K. But how does it exit?

• We can define the E2-Kocc state and see the
  conformational change. But how does ATP drive
  the change from E1 to E2-P?

• What is the proton pump mechanism? H or H3O?

• We understand the mechanism of PPI drug action.
  But can we improve it?

• We found the non-competitive inhibitor site but
  what is the mechanism?

• We know the binding site for the new inhibitor
  class. Can we design better drugs?