Tailoring Drug Development in the Cholinergic Nervous System through Structural Templates

Three targets: Nicotinic Acetylcholine Receptors (nAChR) ~15 subunits in various pentameric permutations Acetylcholinesterase (AChE) Alternatively spliced single gene Neuroligin and Neurexin Synaptic adhesion molecules

in situ, Click-Chemistry Leads to Drug Candidates

(1)

in situ

Azide-Alkyne Cycloaddition on WT AChBPs (2)

in situ

Azide-Alkyne Cycloaddition on MT AChBPs Triazole Regioisomer Identification Cu ++ /Ru ++ catalyzed lead synthesis in microarray AChBP H 3 -Radioligand Binding Assay (SPA), K d X-Ray Structure with AChBPs Interaction Energy with crystallographic model Binding poses, potential interacting residues Whole Cell Binding Assay with nAChRS (SPA), K d Concentration dependence functional responses Receptor Subtype Occupation Receptor Selectivity Agonism (EC 50 ) or Antagonism (K A )

Novel Drug Discovery & Development

• • • • • • • Synthesis by in situ freeze-frame, click chemistry Physical analysis of lead complexes (binding energy, conformational changes, X-ray crystallography) Catalytic synthesis at the milligram level (refinement of SAR is done in microarrays and only leads are taken forward).

Quantitate affinity and activity in vitro Assay cellular toxicity and efficacy Efficacy in intact animals Pharmacokinetic studies of disposition in the target tissues (CNS) and plasma

Mechanism and Therapeutic Indications

Acetylcholinesterase

•

Mechanism

Antidotal Action at CNS and skeletal muscle targets • Scavenging in environment, portals of entry & circulation

Therapeutic Indications

Organophosphate pesticide toxicity Nerve agents in terrorism

Nicotinic Acetylcholine Receptor

•

Mechanism

Agonist, Partial Agonist and Antagonist Activity at nAChRs.

Allosteric Modifiers at nAChRs •

Therapeutic Indications

Disorders of nervous system development & aging dementias Tobacco cessation

Click Chemistry TSRI K. Barry Sharpless* Valery Fokin* Rakesh Sit* Timo Weide Joseph Fotsing Neil Grimster M.G. Finn, Georgia Tech* Kasia Kaczanowska* Gabi Amitai (IIBR) Acetylcholine Binding Protein Scott Hansen Todd Talley Ryan Hibbs Zoran Radic ’* Ákos Nemecz* John Yamauchi* AChE and AChBP Crystal Structures Scott Hansen Ryan Hibbs Todd Talley Akos Nemecz Kasia Kaczanowska* Zoran Radic ’* Jean-Pierre Changeux* Nan Arunrungvichian* Bill Chen* Andy McCammon(UCSD) Jon Lindstrom (Penn) David Johnson (UC, Riverside) Steve Sine (Mayo, Rochester) Pascale Marchot (Marseille)* Yves Bourne (Marseille)* Michal Harel (Weizmann)* John Casida (UC, Berkeley) Jordi Molgo (Gif-sur-Yvette) Mike McIntosh (Univ. of Utah) Toto Olivera (Univ. of Utah) Pharmacokinetics & Disposition Zoran Radic* Jeremiah Momper* Edmund Capparelli* Manjunatha Kini (NUS)

Active center gorge Peripheral site Active center

CNS H 3 C O P F O CH 3 CH 3 + AChE-OH AChE-O O P O CH 3 CH 3 CH 3

Blood-Brain Barrier

Plasma H 3 C O P F O CH 3 CH 3 + AChE-OH AChE-O O P O CH 3 CH 3 CH 3 + R N OH R N-O O P O CH 3 CH 3 CH 3 + AChE-OH

Reactivation of native peripheral AChE

Oxime-assisted Catalytic OP hydrolysis in Plasma

Alveolar Membrane

Site of Exposure H 3 C O P F O CH 3 CH 3

CNS H 3 C O P F O CH 3 CH 3 + AChE-OH AChE-O O P O CH 3 CH 3 CH 3

Blood-Brain Barrier

Plasma H 3 C O P F O CH 3 CH 3 + AChE-OH AChE-O O P O CH 3 CH 3 CH 3

HON N

Reactivation of native peripheral AChE

Oxime-assisted Catalytic OP hydrolysis in Plasma

Alveolar Membrane

Site of Exposure H 3 C O P F O CH 3 CH 3

H 3 C O P F O CH 3 CH 3 + AChE-OH H 3 C O P F O CH 3 CH 3 + AChE-OH AChE-O O P + O CH 3 R N CH 3

HON

R CH 3 O N-O P O CH 3 CH 3 +

NRR'H

AChE -OH

HN

H +

+ HON HN O

CNS

NRR' O

Oxime Reactivation of native AChE in CNS

Blood-Brain Barrier

Plasma

NRR'

AChE-O O P O CH 3 CH 3 CH 3

HON HN NRR'H

H +

+ HON HN O O

Reactivation of native peripheral AChE

Oxime-assisted Catalytic OP hydrolysis in Plasma

Alveolar Membrane

Site of Exposure H 3 C O P F O CH 3 CH 3

AChBP: a structural surrogate of the nAChR Ligand Binding Domain

5 subunits each of which have: I.

A large extra cellular domain II.

III.

4 membrane-spanning regions α β combinations make up most neuronal receptors Hansen, S. et. al. 2005 Brejc, K. et. al. 2001

Nicotine Anabaseine Epibatidine Cytisine Anatoxin-a d-Tubocurarine MLA SPX GYM DMXBA Tropisetron Lobeline Sazetidine A

a-

Conotoxin Waglerin Granisetron

a-

cobratoxin