Annexos

ENDOCANNABINOID/PARENT FATTY ACID, LOW-ENERGY CONFORMATIONS

Fatty Acid Nomenclature

There are two accepted shorthand notation systems for long-chain unsaturated fatty acids in use in the fatty acid literature. In one system, the acyl moiety 20:4, !^5,8,11,14 indicates the presence of a 20-carbon atom chain; four cis homoallylic double bonds, with the double bonds at C5–C6, C8–C9, C11–C12, and C14–C15 (see drawing of AEA (1), for numbering system). In a second system, the acyl moiety (20:4, n-6) indicates the presence of a 20-carbon atom chain; four cis homoallylic double bonds with the last sp² hybridized carbon being the sixth carbon (i.e., C15) from the last carbon of the acyl chain (C20).

Arachidonic Acid and Related Fatty Acids: Conformational Analysis

Conformational analysis of highly exible ligands such as the endogenous cannabinoids is a challenging task due to the large number of conformations available to the ligand. The AA acyl chain of AEA and of 2-AG contains three different types of C#C bonds:Csp2#Csp2, C_sp2#Csp3, and C_sp3#Csp3, of which the last two types permit bond rotation and thus control the conformational changes possible for the molecule.

C_sp2–C_sp3 Bond Rotation

The C5 to C15 portion of the AA acyl chain of AEA and of 2-AG contains four cis homoallylic double bonds (i.e., cis double bonds separated by methylene carbons). One important feature of this chain is the great torsional mobility about the two torsion angles involving each methylene carbon between adjacent pairs of double bonds (vinyl groups) in the acyl chain (for example, the C8–C9–C10–C11 and C9–C10–C11–C12 torsion angles, for which rotation would occur about the C9–C10 and C10–C11 bonds. See drawing of AEA (1) for the numbering system). This involves rotation about C_sp2–C_sp3 bonds. Rabinovich and Ripatti (Rabinovich and Ripatti, 1991) reported that polyunsaturated acyl chains in which double bonds are separated by one methylene group (as in AA) are characterized by the highest equilibrium exibility compared with other unsaturated acyl chains. Rich(Rich, 1993) reports that a broad domain of low-energy conformational freedom exists for these C–C bonds. Feller and coworkers have reported that the rotational barriers for isomerization around methylenes connect-ing the vinyl groups in DHA (22:6, !4,7,10,13,16,19) are extremely low (<1 kcal/mol) (Feller et al., 2002).

Additionally, the torsional minima for the rotatable bonds in DHA are very broad. Results of the biased sampling phase from conformational memories (CM) calculations of AA are consistent with all of these results (Barnett-Norris et al., 1998), as they reveal a relatively broad distribution of populated torsional space about the classic skew angles of #119°(s$) and 119°(s) for the

C8–C9–C10–C11 torsion angle in AEA, for example. Thus, C_sp2–C_sp3 bond rotation introduces great

exibility into an acyl chain, and regions of acyl chains that contain two torsion angles involving a methylene carbon between adjacent pairs of double bonds (vinyl groups) are regions that can introduce curvature in the overall acyl chain shape (Barnett-Norris, Hurst, Lynch et al., 2002). The fact that the AEA acyl chain contains four cis double bonds separated by methylene carbons (like its parent AA) suggests that AEA is engineered to easily adapt and change shape. This may facilitate AEA interaction with targets of importance.

C_sp3–C_sp3 Bond Rotation

The C2 to C4 and C16 to C20 portion of the acyl chain of AEA and 2-AG, as well as their parent fatty acid AA, is saturated (i.e., contains C_sp3–C_sp3 bonds). Feller and co-workers have reported that in contrast to the low rotational energy barrier for rotation about the C_sp2–C_sp3 in docosahexaenoic acid (DHA 22:6, !4,7,10,13,16,19) (<1 kcal/mol), the rotational barrier for rotation about the C_sp3–C_sp3 bonds in the saturated portion of the DHA acyl chain is about 3.5 kcal/mol (Feller et al., 2002).

Rich reported similar results for AA. These results indicated that for saturated acyl chains, freedom of motion is restricted to trans (±180°) or gauche (±60°) congurations (Rich, 1993). Results of the biased sampling phase from CM calculations of AA are consistent with these results (Barnett-Norris et al., 1998), as they reveal a relatively narrow distribution of populated torsional space at 180° (trans), 60°, and #60° (gauche), with 180° being the highest populated torsion angle. These results indicate that saturated fatty acids or saturated portions of fatty acid chains are not very

What shapes are the acyl chain in AEA and its analogs likely to adopt? Discussed in the following subsections are both computational and experimental results that assess what conformations can be adopted by AEA and related molecules as a function of the environment. Figure 2.3 illustrates the general shapes reported in the literature to be adopted by AA (9) and its derivatives.

CALCULATIONS IN VACUO

The two most commonly used methods in the literature for studying exible ligands such as AA and AEA have been the Monte Carlo and molecular dynamics (MD) techniques. In their Monte Carlo study, Rabinovich and Ripatti (Rabinovich and Ripatti, 1991) found that polyunsaturated fatty acids whose double bonds are separated by one methylene carbon assume an extended (angle-iron) conformation when all molecules are efciently packed below the phase-transition temper-ature. MD or molecular dynamics/simulated annealing (MD/SA) studies (in vacuo) of AA (9) and of other polyunsaturated fatty acids related to AA have been published by several groups FIGURE 2.3 The general shapes/conformers commonly identied for arachidonic acid in structural and computational studies: (A) the angle-iron or extended conformation; (B) the hairpin or U-shaped con-formation; (C) the J-shaped concon-formation; and (D) the helical conformation. Beneath the label for each conformation is a summary of torsion angle values beginning with %I = C1–C2–C3–C4 and ending with

%17 = C17–C18–C19–C20 torsion angle of AA (see structure 9 for numbering system). The letter codes correspond to the following ideal torsion angles: cis, C (% = 0°); gauche, g^# (% = 60°) and g⁺ (% = #60°);

skew, s (% = 120 °) and s$ (% = #120 °); and trans, t (% = 180 °). (Barnett-Norris J, Guarnieri F, Hurst DP, and Reggio PH [1998] Exploration of biologically relevant conformations of anandamide, 2-arachi-donylglycerol, and their analogues using conformational memories. J Med Chem 41: 4861–72. With permission.)

(Bonzom et al., 1997; Corey et al., 1983; Corey et al., 1979; Leach and Prout, 1990; Rabinovich and Ripatti, 1991; Rich, 1993; Wilson et al., 1988). These computational studies of AA have primarily found looped or back-folded conformations to be low-energy conformers of AA. Rich conducted a quenched MD study of AA in vacuo (Rich, 1993). The two lowest enthalpy conformers found for AA were J-shaped conformers in which the carboxylic acid group is in close proximity to the C14–C15 & bond. This same J-shape was reported by Corey and co-workers (1983) as one type of low-energy minimum identied in their conformational analysis of AA. Corey suggested that such a J-shaped conformation in solution would be energetically favorable and would be consistent with the chemistry of peroxyarachidonic acid for which an internal epoxidation leads to 14, 15-epoxyarachidonic acid (Corey et al., 1979).

Using the A* algorithm followed by minimization in the BatchMin module of MacroModel, Leach and Prout (Leach and Prout, 1990) found right- and left-handed looped or “helical-like”

conformations of AA to be its lowest energy conformations. A simulated annealing study of AA identied a looped conformation (Wilson et al., 1988), while a MD study in vacuo found low-energy structures of AA to be hairpin, helical, and crown shaped (i.e., U-shaped conformers in which the ends of the chain come close to each other) (Bonzom et al., 1997). Lopez and co-workers also employed MD simulations in their study of AA, nding two low-energy conformations, one of which was U-shaped and was proposed as the bioactive conformation at its cyclooxygenase binding site (Lopez M. et al., 1993). Bonechi and co-workers performed an in vacuo Monte Carlo conformational analysis of AEA. Results indicated that the dominant conformation of AEA was a hybrid of which the extended and U-shaped forms are the limiting structures (Bonechi et al., 2001).

CALCULATIONS IN NONPOLAR (CHCl₃) VS. POLAR (AQUEOUS) ENVIRONMENTS

A new conformational analysis method, CM (Guarnieri and Weinstein, 1996; Guarnieri and Wilson, 1995), was employed by Barnett-Norris and co-workers (1998) to study the conformations of AA in solvent. The CM techniqueemploys multiple Monte Carlo simulated annealing (MC/SA) random walks using the MM3 force eld and the generalized Born/surface area (GB/SA) continuum solvation model for chloroform or water as implemented in the MacroModel molecular modeling package (Mohamadi et al., 1990).CM has been shown to achieve complete sampling of the conformational space of highly exible molecules, to converge in a very practical number of steps, and to be capable of overcoming energy barriers efciently (Guarnieri and Weinstein, 1996).The program, XCluster in MacroModel, is used to group the conformers generated by CM into families of like conformation called clusters. Conformers are grouped according to their increasing RMS deviation from the rst structure output at 310 K. Because XCluster rearranges the conformers so that the RMS deviation between the nearest neighbors is minimized, any large jump in RMS deviation is indicative of a large conformational change and hence identies a new conformational family or cluster (Cls).

Conformational families that are identied by CM to be highly populated are those that have low free energies, whereas conformations that outlie these major groupings are those whose free energies are not sufciently low enough to be visited frequently during the simulation.

CM calculations reported by Barnett-Norris and co-workers (1998) identied both extended conformers and folded or U-shaped conformers to be major conformational families of AA. The more compact (U-shaped) structure was found to predominate in water, whereas the extended shape was found to predominate in CHCl₃ (and in vacuo). The existence of J-shaped conformers as suggested in the literature (Corey et al., 1983; Corey et al., 1979; Rich, 1993)was conrmed by CM.

In this case, the J-shaped family was found to comprise a smaller but still signicant conformational family of AA. These results suggest that the CM method resulted in a much broader sampling of the conformational space of AA than had been performed previously using MD or MD/SA techniques (Applegate and Glomset, 1986; Bonzom et al., 1997; Corey et al., 1983; Leach and Prout, 1990;

Lopez M. et al., 1993; Rich, 1993; Wilson et al., 1988). As discussed above, these previous studies of AA found primarily only compact structures (Us, Js, and helicals) as energy minima. Although no

other theoretical studies of AA in solvent have been reported, the trends in the CM results reported by Barnett-Norris et al. (1998) for AA in CHCl₃ and in H₂O are consistent with the idea that in H₂O, AA would minimize the exposure of its hydrophobic portions by forming a more compact (U)-shape.

Barnett-Norris and co-workers reported CM results for a series of fatty acid ethanolamides, including AEA [22:4, !7,10,13,16 (10);20:4, !^5,8,11,14 (AEA; 1);20:3, !^8,11,14 (11); 20:2, !^11,14 (12)]

(Barnett-Norris, Hurst, Lynch et al., 2002) in CHCl₃. CM studies indicated that each analog could form both U-/J-shaped and extended families of conformers. For the analogs with three or four homoallylic double bonds, the U- or J-shaped family was higher in population (see Figure 2.4).

When the number of homoallylic double bonds decreased to two, a shift in the population was seen, with the extended conformer family now higher in population for the 20:2, !^11,14 (12) analog.

In addition, radius of curvature calculations for the U- or J-shaped family of each analog indicated that the radius increased as the number of double bonds decreased. The average radii of curvature (with their 95% condence intervals) were found to be 4.0 Å (3.6–4.5) for 22:4, !^7,10,13,16; 4.0 Å (3.7–4.2) for 20:4, !^5,8,11,14; 4.4 Å (4.1–4.7) for 20:3, !^8,11,14; and 5.8 Å (5.3–6.2) for 20:2, !^11,14. These calculations suggested that for fatty acid acyl chains that contain homoallylic double bonds, chains with decreasing amounts of unsaturation tend to show a decreasing tendency to form folded structures but still tend to curve in the acyl chain regions in which unsaturation is present.

CALCULATIONS IN PROTEIN ENVIRONMENTS

A recent modeling study of the binding of AA in the cyclooxygenase active site of mouse pros-taglandin endoperoxide synthase-2 (COX-2) predicts that AA will orient in a kinked or L-shaped conformation. In this conformation, the carboxylate moiety binds to Arg¹²⁰, while the %-end is positioned above Ser⁵³⁰ in a region termed the top channel (Rowlinson et al., 1999). In their theoretical conformational analysis of the complex of AA with "-tocopherol, Shamovsky and Yarovskaya (Shamovsky and Yarovskaya, 1990)found that AA assumes an extended conformation.

A computer docking study of (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (14) in a computer model of the transmembrane helix (TMH) bundle of CB₁ (activated state, R*) suggested that this molecule adopts a tightly curved U-shaped conformation at CB₁, and suggested that the TMH 2-3-6-7 region is the endocannabinoid binding region at CB₁ (Barnett-Norris, Hurst, Lynch et al., 2002). A subsequent docking study of AEA in a computer model of the CB₁ activated state suggested that AEA adopts a tightly curved conformation as well, to bind in the TMH 2-3-6-7 region of CB₁. The orientation of AEA was slightly different from that of (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (14) mentioned earlier, due to the differences in the head groups of these two ligands (McAllister et al., 2003).

EXPERIMENTAL X-RAY CRYSTAL STRUCTURES: ARACHIDONIC ACID AND ARACHIDONIC ACID DERIVATIVES

The X-ray crystal literature is now replete with numerous experimental examples of the natural plasticity of the AA acyl chain. In its X-ray crystal structure, AA has been found to exist in an extended (angle-iron) conformation in which double bonds 1 and 3 and double bonds 2 and 4 are coplanar, while the planes of adjacent double bonds are perpendicular to one another (see Figure 2.5A) (Ernst et al., 1979; Rich, 1993). An X-ray crystal study of arachidonate ion com-plexed with adipocyte-lipid-binding protein revealed that AA binds within the ' barrel cavity of this protein. Here the carboxylate group of AA is engaged in a strong electrostatic interaction with Arg¹²⁶, Tyr¹²⁸, and Arg¹⁰⁶ through an intervening water molecule, while the arachidonate acyl chain assumes a hairpin (i.e., U-shaped) conformation for which the rst double bond of AA is distorted out of the plane formed by the other three double bonds (see Figure 2.5B) (LaLonde et al., 1994). In the 3 Å resolution structure of prostaglandin H synthase-1 (Figure 2.5C), AA

adopts an extended L-shaped conformation that positions the 13proS hydrogen of AA for abstrac-tion by Tyr³⁸⁵, the likely radical donor at this enzyme’s active site (Malkowski et al., 2000). In the recently elucidated X-ray crystal structure of FAAH, the AA derivative, methoxy arachidonyl phosphonate (MAP) is covalently bound to Ser 241 (Bracey et al., 2002) and the conformation of the arachidonyl chain can best be described as helical (see Figure 2.5D).

FIGURE 2.4 Conformational memories (CM) results for a series of fatty acid ethanolamides [22:4, !^7,10,13,16 (10);20:4, !^5,8,11,14 (AEA, 1);20:3, !^8,11,14 (11); and 20:2, !^11,14 (12)] in CHCl3. (From Barnett-Norris J, Hurst DP, Lynch DL, Guarnieri F, Makriyannis A, and Reggio PH [2002] Conformational memories and the endocannabinoid binding site at the cannabinoid CB1 receptor. J Med Chem 45: 3649–59. With permission.) CM studies indicated that each analog could form both U- or J-shaped and extended families of conformers.

For the analogs with three or four homoallylic double bonds (1, 10, 11), the U- or J-shaped family was higher in population. When the number of homoallylic double bonds decreased to two, a shift in the population was seen, with the extended conformer family now being higher in population for the 20:2, !^11,14 analog (12).

EXPERIMENTAL NMR SOLUTION STUDIESOF AEA AND AEA ANALOGS

Bonechi and co-workers have assessed the solution conformation of AEA in DMSO-d₆ at 298 K using ¹H and ¹³C NMR combined with Monte Carlo calculations (Bonechi et al., 2001). These investigators found that the acyl chain of AEA was predominantly in an extended conformation in solution, whereas the head group of AEA was involved in an intramolecular hydrogen bond between the hydroxyl group hydrogen and the oxygen of the carboxamide group. The study of the dynamic properties of AEA in solution showed clearly that the molecule is in fast motion conditions and that only the C18, C19, and C20 carbons (see drawing of AEA (1) for numbering system) show values of the dynamic parameter (C, indicating the presence of a free tumbling motion in the terminal part of the acyl chain.

The CM calculations reported by Barnett-Norris and co-workers (1998) discussed earlier, identied both extended conformers and folded or U-shaped conformers to be major conformational families of AA. The more compact (U-shaped) structure was found to predominate in water, whereas the extended shape was found to predominate in CHCl₃ (and in vacuo). These results were taken to be consistent with the idea that in H₂O, AA would minimize the exposure of its hydrophobic FIGURE 2.5 X-ray crystal structure results for arachidonic acid (AA) and its derivatives alone or complexed with various proteins. (A) In its x-ray crystal structure, AA (9) has been found to exist in an extended (angle-iron) conformation in which double bonds 1 and 3 and double bonds 2 and 4 are coplanar, whereas the planes of adjacent double bonds are perpendicular to one another. (From Ernst J, Sheldrick WS, and Fuhrop JH [1979] The structures of the essential unsaturated fatty acids. Crystal structure of linoleic acid and evidence for the crystal structures of alpha-linoleic and arachidonic acid. Z. Naturforsch 34b: 706–711; Rich MR [1993]

Conformational analysis of arachidonic and related fatty acids using molecular dynamics simulations. Biochim Biophys Acta 1178: 87–96.) (B) An X-ray crystal study of arachidonate ion (AA) complexed with adipocyte-lipid-binding protein revealed that the arachidonate acyl chain assumes a hairpin (i.e., U-shaped) conformation for which the rst double bond of AA is distorted out of the plane formed by the other three double bonds.

(From LaLonde JM, Levenson MA, Roe JJ, Bernlohr DA, and Banaszak LJ [1994] Adipocyte lipid-binding protein complexed with arachidonic acid. Titration calorimetry and X-ray crystallographic studies. J Biol Chem 269: 25339–47.) (C) In the 3 Å resolution structure of prostaglandin H synthase-1, AA adopts an extended L-shaped conformation. (From Malkowski MG, Ginell SL, Smith WL, and Garavito RM [2000] The productive conformation of arachidonic acid bound to prostaglandin synthase. Science 289: 1933–7.) (D) In the recent X-ray crystal structure of FAAH, the AA derivative, methoxy arachidonyl phosphonate (MAP) is covalently bound to Ser241 (From Bracey MH, Hanson MA, Masuda KR, Stevens RC, and Cravatt BF [2002]

Structural adaptations in a membrane enzyme that terminates endocannabinoid signaling. Science 298:

1793–6.) and the conformation of the arachidonyl chain can best be described as helical.

portions by forming a more compact (U-shape) conformation. Van der Stelt and co-workers (2002) recently studied the solution conformation of a conjugated/hydroxylated analog of AEA, 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid in D₂O and in CDCl₃. Analysis of ¹H NMR spectra revealed that the acyl chain of 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid did not assume notably different conformations in the polar (water) vs. nonpolar (chloroform) environments. This result is likely because the location of the conjugation (C11 = C12 # C13 = C14; which introduces rigidity into the acyl chain) is in the region of the acyl chain that would need to curve to permit the adoption of a more compact (U-shape) conformation in water.

CALCULATIONS IN A LIPID ENVIRONMENT

In order to explore the behavior of AEA in a lipid environment, we recently undertook MD simu-lations of AEA in a dioleoylphosphatidylcholine (DOPC) bilayer (see Figure 2.6) (Reggio et al., 2003; Lynch and Reggio, 2005). A fully hydrated DOPC bilayer (28 waters/lipid) was built in order to generate a reasonable representation for the lipid bilayer. The calculations were run using an NPAT ensemble and the CHARMM27 set of all atom parameters designed for lipid simulations (Feller et al., 2002). Four AEAs were placed in the equilibrated bilayer, and the system was heated to 310 K and equilibrated. Two separate 5 nsec NPAT trajectories were simulated.

These simulations revealed that AEA assumes an extended conformation in the bilayer, with its C16–C20 alkyl tail portion (located near the bilayer center) capable of considerable motion.

During the simulation, AEA assumed many of the conformations discussed above for AA, including

FIGURE 2.6 Conformations adopted by anandamide (AEA) in a dioleoylphoshatidylcholine (DOPC) bilayer during a molecular dynamics (MD) simulation. (From Reggio P, Hurst DP, Ballesteros JA, and Lynch DL [2003] The CB1 V6.43/I6.46 groove is optimally placed in the lipid bilayer to recognize endocannabinoids in lipid, in 2003 Symposium on the Cannabinoids, p. 8, International Cannabinoid Research Society, Cornwall, Ontario, Canada.) This simulation employed a fully hydrated DOPC bilayer (28 waters/lipid) and the CHARMM27 set of all atom parameters designed for lipid simulations. (From Feller SE, Gawrisch K, and MacKerell AD, Jr. [2002] Polyunsaturated fatty acids in lipid bilayers: intrinsic and environmental contributions to their unique physical properties. J Am Chem Soc 124: 318–26.) AEA was placed in the equilibrated bilayer, and the system was heated to 310 K and equilibrated for 200 psec using an NVT ensemble. The simulation then was switched to NPT ensemble conditions (P = 1 atm, T = 310 K) and run for 2.5 nsec. This simulation revealed that the ethanolamide head group is located at the same depth as the DOPC glycerol group and overlaps substantially with the choline group of DOPC. AEA assumes an extended conformation in the bilayer, with its C16–C20 alkyl tail portion (located near the bilayer center) capable of considerable motion. During the simulation, AEA assumed many of the conformations discussed in this chapter for AA, including extended and J-shaped conformations.

extended and J-shaped conformations (see Figure 2.6). The ethanolamide head group was located at the same depth as the glycerol group and overlapped substantially with the choline group of DOPC. The radial distribution function of various lipid functional groups with the C2 hydrogens

extended and J-shaped conformations (see Figure 2.6). The ethanolamide head group was located at the same depth as the glycerol group and overlapped substantially with the choline group of DOPC. The radial distribution function of various lipid functional groups with the C2 hydrogens