# Presentation: 144689

- Adaptations in a Membrane Enzyme that
- Terminates Endocannabinoid Signaling

- Benjamin Cravatt
- The Scripps Research Institute

- _Exogenous_
- 9-THC
-    (Gaoni & Mechoulam 1965)

- Exogenous Versus Endogenous Cannabinoids

- _Endogenous_
-    Anandamide
-     (Devane et al. 1992)

*_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ *

*• *Active ingredient of marijuana
*•  *Agonist for a brain G-protein
- coupled receptor (CB1)

- •  Induces *intense *behavioral
- effects in rodents
- •  Behavioral effects *absent*
- in CB1 knockout mice

- • Natural brain lipid (fatty acid amide)
*• *Binds and activates CB1 receptor *in vitro*;
- produced in response to a variety of neural
- stimuli (electrical, chemical, behavioral)

- •  Induces *weak and transient* behavioral
- effects in rodents
- •  Behavioral effects *still occur* in CB1
- knockout mice

**Notes:**
• Comment on the fact that anandamide belongs to a family of fatty acid amides

- Cannabinoid Signaling in the CNS

- _Behavioral Effects_
- _of CB1 Activation_
-    - hypomotility
-    - analgesia
-    - catalepsy
-    - hypothermia

- The Enzymatic Inactivation of Fatty Acid Amides (FAAs)

- •  Brain extracts rapidly hydrolyze 14C-FAAs to their parent fatty acids
- _Fatty acid amide hydrolase (FAAH)_

* **- integral membrane protein*
* **- abundant in brain & liver*

**Notes:**
• Reiterate that there exists a family of FAAs that all can be hydrolyzed by this activity in vitro

•  However, in vivo is this really a single enzyme dedicated to the regulation of this class of signaling lipids

- •  FAAH belongs to a large enzyme class
- termed the *amidase signature* (AS) family

- FAAH and the Amidase Signature Family

- •  Until recently, little was known about
- AS enzyme structure and mechanism

- •  More than 100 distinct AS enzymes
- identified to date

- 	1)  Structural/Biophysical Studies
- 	2)  Enzymological Studies
- 	3)  Transgenic/Physiological Studies

- Experimental Approaches for the Study of FAAH

- 	1)  Structural/Biophysical Studies
- 	2)  Enzymological Studies
- 	3)  Transgenic/Physiological Studies

- Experimental Approaches for the Study of FAAH

- •  Viable, healthy, fertile

- Summary of FAAH KO Mice

- •  Exhibit exaggerated CB1-dependent behavioral responses to anandamide

- •  Have ~100 fold lower anandamide
-    hydrolase activity

- Cravatt et al. *PNAS *2001

- •  Possess highly elevated endogenous CNS levels of anandamide (and other
- fatty acid amides) that correlate with increased analgesia (CB1-dependent)

- Increased Brain Levels of Endogenous
- Fatty Acid Amides in FAAH KO Mice

- The Reduced Pain Behavior in FAAH KO Mice is
*Reversed * by the CB1 Antagonist SR141716A

- •  Anandamide levels and activity are primarily regulated *in vivo* by
-    a single degradative enzyme (FAAH)

- Summary of FAAH KO Mice

- •  FAAH sets an endogenous cannabinoid tone that modulates
-    mammalian pain perception (as well as possibly other neurobehaviors)

* **How are the levels of anandamide and related fatty acid amides*
* **so tightly controlled by a single enzyme?*

- Cravatt et al. *PNAS *2001

- 	1)  Structural/Biophysical Studies
- 	2)  Enzymological Studies
- 	3)  Transgenic/Physiological Studies

- Experimental Approaches for the Study of FAAH

- Generation of a Transmembrane Domain-
- Deleted FAAH (TM-FAAH)

- Biochemical Characterization of TM-FAAH

- •TM-FAAH is *catalytically active*, but _still_ acts like an integral membrane protein

- •  Produced in an *E. coli* expression system
-         -large quantities of pure TM-FAAH
-         protein (2-5 mg/L culture)

- Characterization of a Recombinant TM-FAAH

- •  TM-FAAH exists primarily as a single
- 11 S detergent-protein complex in solution

- • Crystals of a TM-FAAH
-   -  Diffract to 2.8 Å resolution
-   -  Only obtain crystals in the presence
-   of an active site-directed inhibitor:
- 	methoxy arachidonyl
- 	fluorophosphonate (MAFP)
-   -  Detergents required throughout

- Towards an X-Ray Structure of a FAAH-Inhibitor Complex

**Notes:**
• Emphasize that without MAP, no crystals.  Burley comments that top priority is chemical ligands to stabilize protein structures.

• LDAO - lauryl dimethyl amine oxide

- MAFP is a Covalent Inhibitor of FAAH

- •  As a result of a shared catalytic mechanism, nearly all serine hydrolases
- are irreversibly inhibited by *fluorophosphonate *(FP) reagents

- X-Ray Structure of a FAAH-Arachidonyl
- Phosphonate Complex

**Notes:**
• Phase solved by a combination of multiple isomorphous replacement with anomalous dispersion (MIRAS) and multiwavelength anomalous dispersion (MAD) strategies, as well as a heavy reliance on NCS.
• Twisted beta sheet

- Composition of the Active Site and Substrate
- Binding Pocket of FAAH

**Notes:**
•  Focus on hydrophobic active site that includes an isoleucine residue identified by crosslinking and mutagenesis to be involved in acyl chain
Selectivity

•  unusual triad

- Composition of the Active Site and Substrate
- Binding Pocket of FAAH

**Notes:**
•  amphipathic channel from surface to substrate channel - aspartate and arginine at surface
• cytosolic port bifuracted with trp blocking one access port

- Composition of the Membrane-Binding Interface of FAAH

- Surface Representation of the Membrane-Binding
- Interface of FAAH

- The Membrane-Binding
- Interface of FAAH
- Resembles Those of
- Other Monotopic Integral
- Membrane Enzymes

**Notes:**
•  All conduct chemistry on very hydrophobic substrates that are presumably embedded in the lipid bilayer of the cell

- Predicted Location of N-Terminal TM Domain of FAAH

**Notes:**
• Unlike PS and SC, FAAH possess N-terminal TM domain -why? Exon 1...

- A Comparison of the Structures of FAAH and the Soluble
- AS Enzyme Malonamidase (MAE2)

**Notes:**
•  Despite using roughly the same face for dimerization, the two enzymes exhibit different relative monomer orientations

- Opposite Relative Active Site Orientations for
- MAE2 and FAAH

**Notes:**
•  Antiparallel and parallel arrangement of active site entrances for MAE2 and FAAH respectively
• may permit both subunits to function concurrently by recruiting substrates from same membrane
•  also, could reinforce membrane binding properties of each monomer

- A Comparison of the Structures of FAAH and the Soluble
- AS Enzyme Malonamidase (MAE2)

- •  FAAH appears to interact with cell membranes
-    through at least two domains:

- Conclusions

- 	 1) *N*-terminal transmembrane helix

- 	2) A helix-turn-helix monotopic insertion motif

- •  FAAH possesses a bifurcated active site

-  Can the unusual structure of FAAH’s active site be exploited for
-    the design of potent and selective inhibitors?

*Does FAAH need to be associated with*
*membranes to function in vivo?*

**Notes:**
•  Mention desire to create soluble variant of FAAH to test whether needs to be on membranes to function as fatty acid amide hydrolase

- •  Matt Patricelli				• Michele McKinney
- •  Michael Bracey				• Kyle Chiang
- •  Michael Hanson				• Aron Lichtman
- •  Greg Hawkins 				• Ray Stevens
- •  Donmienne Leung 				• Dale Boger
- •  Kim Masuda 				• Jack Sipe

- Acknowledgements

- •  NIH (NIDA and NIMH) 		• NSF
- •  Searle Scholars Program 		• Baxter Foundation
- •  Activx Biosciences		        	• Skaggs Institute for Chemical Biology

- _Funding Sources_

- How Might a FAAH Inhibitor Differ in Pharmacological
- Properties from a Cannabinoid Agonist?

- _Behavioral Effects_
- _of CB1 Activation_
-    - hypomotility
-    - analgesia
-    - catalepsy
-    - hypothermia

- Ca2+ channels

**Notes:**
• Behavioral effects of THC - beneficial effects include - reduce pain - from a therapeutic perspective, never been an issue about what cannabinoids can do

- How Might a FAAH Inhibitor Differ in Pharmacological
- Properties from a Cannabinoid Agonist?

- _Behavioral Effects_
- _of CB1 Activation_
-    - hypomotility
-    - analgesia
-    - catalepsy
-    - hypothermia

- Ca2+ channels

**Notes:**
• problem is that with beneficial activities, you also get unwanted side effects, like motility and cognitive dysfunction, likely the result of the broad distribution of CB1 in our brains - thus the simultaneous activation of CB1 in neural pathways in several brain regions makes pure CB1 agonists a tough sell

- How Might a FAAH Inhibitor Differ in Pharmacological
- Properties from a Cannabinoid Agonist?

- _Behavioral Effects_
- _of CB1 Activation_
-    - hypomotility
-    - analgesia
-    - catalepsy
-    - hypothermia

- Ca2+ channels

- How Might a FAAH Inhibitor Differ in Pharmacological
- Properties from a Cannabinoid Agonist?

- _Behavioral Effects_
- _of FAAH Inactivation?_
-    - hypomotility
-    - analgesia
-    - catalepsy
-    - hypothermia

- Ca2+ channels

**Notes:**
•  However, we and others have proposed that by blocking FAAH one may be able to achieve the beneficial effects of cannabinoids without side effects - why?  Because endocannabinoids are produced in an activity-dependent manner, augmentation of their levels and activity by blockade of FAAH could lead to a subset of the behaviors observed with CB1 agonists. For example, in diseases like chronic pain, where specific neural circuits are hyperactive, one might expect that endocannabinoids would be produced at high levels in these pathways, and accordingly FAAH inhibitors would exert particularly significant effects in these behaviors

- Emerging Therapeutic Indications for FAAH Inhibitors

- Ca2+ channels

-   Pharmacological Effects
-      _of FAAH Inhibitors:_
- •  Analgesia in acute and
- neuropathic pain models
- 		(Piomelli, BMS)
- • Anxiolytic activity
- 		(Piomelli)

**Notes:**
• carbamate inhibitors - so, like most hydrolase inhibitors, they contain a key mechanism-based component and in this particular case, they are irreversible

- Composition of the Membrane-Binding Interface of FAAH

- A Comparison of the Structures of FAAH and the Soluble
- AS Enzyme Malonamidase (MAE2)

- Affinity Purification of Fatty Acid Amide Hydrolase (FAAH)