Binding of 13-Amidohuprines to Acetylcholinesterase: Exploring the

J. Med. Chem. 2006, 49, 6833-6840

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Binding of 13-Amidohuprines to Acetylcholinesterase: Exploring the Ligand-Induced Conformational Change of the Gly117-Gly118 Peptide Bond in the Oxyanion Hole Pelayo Camps,† Elena Go´mez,†,‡ Diego Munõz-Torrero,*,† Albert Badia,§ Maria Victo`ria Clos,§ Carles Curutchet,|| Jordi Munõz-Muriedas,|| and Francisco Javier Luque*,|| Laboratori de Quı´mica Farmace` utica (Unitat Associada al CSIC), Facultat de Farma` cia, UniVersitat de Barcelona, AV. Diagonal 643, E-08028, Barcelona, Spain, Departament de Farmacologia, de Terape` utica i de Toxicologia, Facultat de Medicina, UniVersitat Auto` noma de Barcelona, E-08193-Bellaterra, Barcelona, Spain, and Departament de Fisicoquı´mica, Facultat de Farma` cia, UniVersitat de Barcelona, AV. Diagonal 643, E-08028, Barcelona, Spain ReceiVed August 4, 2006

The acetylcholinesterase (AChE) inhibitory activity of a series of 13-amido derivatives of huprine Y, designed to enlarge the occupancy of the catalytic binding site by mimicking the piridone moiety present in (-)huperzine A, has been assessed. Although both 13-formamido and 13-methanesulfonamido derivatives are more potent human AChE inhibitors than tacrine and (-)-huperzine A, none of them equals the potency of huprine Y. Molecular modeling studies show that the two derivatives effectively trigger the Gly117-Gly118 conformational flip induced upon binding of (-)-huperzine A, leading to a similar pattern of interactions as that formed by the pyridone amido group of (-)-huperzine A. The detrimental effect on the binding affinity relative to the 13-unsubstituted huprine could be ascribed to a sizable deformation cost associated with the ligand-induced peptide flip. This finding can be interpreted as a mechanism selected by evolution to ensure the preorganization of the functionally relevant oxyanion hole in the binding site of AChE, where residues Gly117 and Gly118 play a relevant role in mediating substrate recognition. Introduction Huprines have recently emerged as a novel class of highly potent acetylcholinesterase (AChE) inhibitors conceived as (-)huperzine A-tacrine hybrids.1-6 The most interesting member is (-)-huprine Y,3 (3; Figure 1), whose IC50 is 640- and 810fold lower than that of the parent tacrine and (-)-huperzine A, respectively, and 19-fold lower than that of donepezil, i.e., the most potent compound among the currently approved antiAlzheimer AChE inhibitors. Moreover, (-)-huprine Y binds to human AChE with one of the highest affinities reported for a reversible inhibitor (KI ) 33 pM), it being 940-, 140-, and 30fold more potent than tacrine, (-)-huperzine A, and donepezil, respectively.3 Finally, it is able to cross the blood-brain barrier in ex vivo studies4 and exhibits muscarinic M1 agonistic activity7 and neuroprotective effects through antagonism of glutamate NMDA receptors both in vitro and in vivo,8 which constitute important added values for an anti-Alzheimer drug candidate. Huprines were designed by combining the 4-aminoquinoline unit of tacrine and the carbobicyclic moiety of (-)-huperzine A.1 The success of such a conjunctive approach was confirmed by inspection of the X-ray structures of the complexes of Torpedo californica AChE (TcAChE) with tacrine (PDB ID 1ACJ),9 (-)-huperzine A (PDB ID 1VOT),10 and (-)-huprine X (the 9-ethyl analogue of huprine Y; PDB ID 1E66),11 which showed that huprines truly bind to AChE as huperzine A-tacrine hybrids. Thus, the 4-aminoquinoline system of (-)-huprine X occupies the same binding site as the corresponding moiety of tacrine and retains the most relevant interactions that mediate * To whom correspondence should be addressed. Phone: int. code + 34 + 934024533/ 934024557. Fax: int. code + 34 + 934035941/ 934035987. E-mail: [email protected] (D.M.-T.), [email protected] (F.J.L.). † Laboratori de Quı´mica Farmace ` utica, Universitat de Barcelona. ‡ Current address: Parc Cientı´fic de Barcelona, Universitat de Barcelona, Josep Samitier 1-5, E-08028, Barcelona, Spain. § Universitat Auto ` noma de Barcelona. | Departament de Fisicoquı´mica, Universitat de Barcelona.

Figure 1. Structures of (()-huprine Y, the 13-amidohuprines (()4a,b-6a,b, the 13-aminohuprines (()-7a,b, their parent compounds tacrine and (-)-huperzine A, and byproducts (()-8 and (()-9 isolated during the preparation of amidohuprines.

the binding of tacrine to AChE, such as the hydrogen bond of the protonated quinoline nitrogen atom with His440; the watermediated contacts of the exocyclic amino group with Asp72, Tyr121, Ser122, and Tyr334; and finally the stacking of the 4-aminoquinoline unit against the indole and benzene rings of

10.1021/jm060945c CCC: $33.50 © 2006 American Chemical Society Published on Web 10/19/2006

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Figure 2. (Left) Representation of the relative orientation of (-)huprine X (colored by atom) and (-)-huperzine A (orange, except nitrogen and oxygen atoms, which are shown in blue and red) obtained by superposition of the corresponding X-ray structures of TcAChE complexed to (-)-huprine X (1E66) and (-)-huperzine A (1VOT). The two diastereomeric arrangements (a, b) of the amido group in 13-amidohuprines are also shown. (Right) Location of residues Trp84 and Phe330 in the active site, as well as Gly117 and Gly118 in the oxyanion hole. Besides the different orientation adopted by the Phe330 benzene ring, binding of (-)-huperzine A is characterized by the conformational change in the Gly117-Gly118 peptide bond. For the sake of clarity, hydrogen atoms are not displayed, except those linked to position 13 of huprine.

Trp84 and Phe330. Noteworthy, this latter interaction forces the side chain of Phe330, which is characterized by a torsional angle around the CR-Cβ bond of around +160° in the complexes with tacrine and (-)-huprine X,9,11 to adopt a different conformation with regard to that observed in the TcAChE-(-)-huperzine A complex (torsional angle of around -170°).10 Similarly, the carbobicyclic fragment of (-)-huprine X fills the same hydrophobic pocket occupied by the corresponding moiety in (-)-huperzine A, thus showing the same interactions observed for the unsaturated three-carbon bridge in this latter compound. Moreover, the cation-π interaction formed by the bridgehead protonated amino group in (-)huperzine A with Trp84 is maintained to some extent by the interaction formed with the protonated quinoline unit in (-)huprine X. Overall, the higher affinity and inhibitory potency of huprines can be ascribed to the larger occupancy of the AChE active site compared to either tacrine or (-)-huperzine A. Superposition of the X-ray structures 1ACJ, 1VOT, and 1E66 suggested new chemical modifications that might enhance the occupancy of the AChE binding site. The sole zone of the AChE catalytic binding site not occupied by (-)-huprine X relative to the parent compounds, tacrine and (-)-huperzine A, is the side fitted by the pyridone ring of (-)-huperzine A, which is close to the methylene bridge of the carbobicyclic system of huprines (see Figure 2, left). Since the pyridone CdO and N-H groups in (-)-huperzine A form hydrogen bonds (mediated by a water molecule in the latter case) with Tyr130 and Glu199, respectively, it might be hypothesized that the introduction of an amido group at the methylene bridge of huprines should mimic these interactions, which should enhance the binding affinity of the resulting 13-amidohuprines. Noteworthy, inspection of the X-ray structures of the TcAChE-(-)-huperzine A10 and TcAChE-(-)-huprine X11 complexes reveals a relevant difference in the conformation of the Gly117-Gly118 peptide bond in the oxyanion hole. Thus, the conformation of the Gly117-Gly118 peptide bond in the apo form of the enzyme and in their complexes with either tacrine or (-)-huprine X is characterized by torsional angles of around -94° (φ117), -167° (ψ117), +133° (φ118), and +30° (ψ118). In contrast, the corresponding torsional angles in the TcAChE-(-)-huperzine A complex are -139.2°, +59.7°, -82.9°, and +77.6°, which reveals the occurrence of a drastic change in the Gly117-Gly118 peptide bond upon binding of (-)-huperzine A to AChE (Figure

Camps et al.

2, right). Such a peptide flip permits minimization of the steric clash that would occur between the carbonyl group in the piridone ring of (-)-huperzine A and the backbone carbonyl group of Gly117 in the apo enzyme (the O‚‚‚O distance estimated from superposition of the 1VOT and 1E66 X-ray structures is approximately 2.6 Å). Moreover, the Gly117Gly118 conformational flip triggered by (-)-huperzine A is also found in other AChE complexes of (-)-huperzine A related compounds.12 On the basis of the hypothesis that 13-amidohuprines should be endowed with enhanced binding affinity relative to the parent huprine, recently we carried out the synthesis of the 13formamido-, 13-acetamido-, and 13-methanesulfonamidoderivatives of huprine Y with the two possible diastereomeric arrangements at position 13 [compounds (()-4a,b-6a,b, Figure 1].13,14 This study pursues to determine the AChE inhibitory potency of these 13-amidohuprines, which in turn should depend on (i) their ability to mimic the conformational change of the Gly117-Gly118 peptide bond induced upon binding of (-)huperzine A to AChE and (ii) the expected gain in binding affinity due to the formation of the additional (-)-huperzine A pyridone-like interactions at the oxyanion hole. To address these questions, herein we describe the pharmacological evaluation and molecular modeling study of the 13-amidohuprines 4a,b6a,b. The results are discussed in light of the functional role played by Gly117 and Gly118 in the oxyanion hole of the AChE enzyme. Results Pharmacological Data. To determine the influence of the amido moiety on the binding affinity of huprines, the inhibitory activity on AChE from bovine (bAChE) and human (hAChE) erythrocytes was determined using Ellman’s method.15 The inhibitory activity was determined for the racemic 13-amidohuprines due to the complexity of their synthetic sequence,13,14 which precluded the obtention of great amounts of the different 13-amidohuprines and the resolution of their enantiomers. Owing to the tight-binding character exhibited by huprines, which display higher inhibitory activity after incubation of the enzyme with the inhibitor,4 the time dependence of the AChE inhibition by 13-amidohuprines was also determined after an incubation period of 45 min. In advanced AD patients, AChE activity is greatly reduced in specific brain regions, while butyrylcholinesterase (BChE) activity increases, perhaps compensating in part for AChE action.16 The increasing importance of BChE in the hydrolysis of acetylcholine as the ratio AChE/ BChE gradually decreases in these patients suggests that inhibition of BChE might be valuable in the search for antiAlzheimer agents. Consequently, the inhibitory activity on human serum BChE (hBChE) was also assayed by the same method. Table 1 reports the inhibitory activity on bAChE, hAChE, and hBChE determined for the racemic 13-amidohuprines 4a,b6a,b, the synthetic intermediate 13-aminohuprines 7a,b, and the huprine-like analogues 8 and 9, obtained as byproducts during the preparation of the 13-amidohuprines. Regarding the bAChE inhibitory activity, the results point out a clear stereochemical preference, since derivatives of the a series are more potent than their counterparts of the b series. Thus, after incubation of the enzyme with the inhibitor, the two most potent inhibitors, 4a and 6a, are 8.5- and 63-fold more potent than their counterparts in the b series. Interestingly, the 13-amidohuprines 4a and 6a exhibit a time-dependent increase in inhibitory potency (1.9and 3-fold increase after incubation), in agreement with the

Binding of 13-Amidohuprines to Acetylcholinesterase

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Table 1. Pharmacological Data of Tacrine, (-)-Huperzine A, (()-Huprine Y, the 13-Amidohuprines (()-4a,b-6a,b, the 13-Aminohuprines (()-7a,b, and Compounds (()-8 and (()-9a IC50 (nM) compound (()-4a (()-4b (()-5a (()-5b (()-6a (()-6b (()-7a (()-7b (()-8 (()-9 tacrine‚HCl (-)-huperzine A (()-huprine Y‚HCl

bovine AChE 0-min incub

45-min incub

161 ( 16 741 ( 15 1687 ( 206 3585 ( 215 118 ( 7.0 1370 ( 400 389 ( 32 482 ( 61 5684 ( 259 1317 ( 70 130 ( 10 74.0 ( 5.50 4.23 ( 0.86

84 ( 6.3b 717 ( 42 1367 ( 142 1334 ( 159b 39 ( 2.3b 2450 ( 1470 447 ( 24 478 ( 64 4380 ( 582 304 ( 14b nd nd nd

human AChE

human BChE

98 ( 13 ndc nd nd 40.5 ( 53 nd 225 ( 21 nd nd nd 205 ( 18 260 ( 18 0.78 ( 0.02

>10 000 3980 ( 420 8490 ( 840 >10 000 >10 000 nd 6480 ( 340 601 ( 56 >10 000 9220 ( 2500 43.9 ( 17 >10 000 236 ( 44

a Values expressed as mean ( standard error of the mean of at least four experiments. IC 50 inhibitory concentration (nM) of AChE (from bovine or human erythrocytes) or BChE (from human serum) activity. b P < 0.05 vs 0-min incubation (Student’s t-test). c Not determined.

trends shown by huprines,4 which suggests that a reversible tight-binding process takes place. This mechanism could result in a longer period of action without the toxic effect of irreversible inhibitors, although more studies would be necessary to confirm this hypothesis. The most potent compounds, formamide 4a and methanesulfonamide 6a, have IC50 values in the nanomolar range, the acetamide derivatives 5, as well as the 13-amino derivatives 7 and the huprine-like analogues 8 and 9 being clearly less potent. The results also show that the inhibitory activity of 4a and 6a is increased in the human enzyme by factors of 1.6 and 2.9, as already reported for huprines.4 These compounds are more potent hAChE inhibitors than tacrine (2.1- and 5.1-fold more potent) and (-)-huperzine A (2.7- and 6.4-fold more potent). However, none of these compounds equals the potency of the racemic parent huprine Y, which is 38- and 28-fold more potent than 4a and 6a toward bAChE and 126- and 52-fold more potent, respectively, toward hAChE. Finally, all of the tested compounds exhibit low human BChE inhibitory activity, and particularly, 4a and 6a are highly selective for human AChE vs BChE inhibition. Molecular Modeling Studies. Contrary to the expectations from our working hypothesis, the introduction of an amido group at position 13 of huprine Y turned out to be detrimental for the AChE inhibitory activity. The pharmacological data seemed to question the ability of formamido and methanesulfonamido moieties to trigger the conformational flip in the Gly117-Gly118 peptide bond induced upon binding of (-)-huperzine A. To explore this possibility, a series of molecular dynamics (MD) simulations was carried out to determine the binding mode of the most potent 13-amidohuprines 4a and 6a. Since previous studies have established that the eutomer of huprines is the levorotatory enantiomer having the (7S,11S)configuration,4,11,17 13-amidohuprines were modeled in this enantiomeric form. Taking advantage of the X-ray structure of the TcAChE-(-)-huprine X complex,11 compounds 4a and 6a were placed at the catalytic binding site following the binding mode of huprine X. This procedure not only permits us to retain the pattern of interactions that mediate the binding of huprine X but even more importantly to locate those water molecules that assist through water-mediated contacts the binding of the inhibitor, thus avoiding the risk of artifactual changes in the arrangement of the 13-amidohuprines in the active site. Moreover, since huprine X largely overlaps (-)-huperzine A when the peptide backbones of 1E66 and 1VOT are superimposed

(see Figure 2), this procedure is also useful to examine the orientation of the amido group at the binding site. In fact, the stereochemical preference of 4a and 6a relative to 4b and 6b (see above) can be justified by the steric hindrance between the amido moiety and the Trp84 side chain in the diastereomers of the b series (see Figure 2), which would impede the correct positioning of the inhibitor at the catalytic binding site. Taking advantage of the limited conformational flexibility of the formamido and methanesulfonamido derivatives of huprines, a systematic exploration was performed to determine the conformational preferences of 4a and 6a. The free energy differences between conformers in aqueous solution were determined by combining the relative stabilities obtained from single-point MP2/6-31G(d) computations using the geometries optimized at the HF/6-31G(d) level and the relative hydration free energies computed using the MST HF/6-31G(d) version18 of the PCM continuum model19 (see Experimental Section and Supporting Information). The conformational analysis of 4a revealed that the trans orientation of the formamido group is favored over the cis one by ∼1 kcal/mol in aqueous solution, which agrees with the available experimental data.20,21 However, it is worth noting that such conformational preference is opposite to the cis arrangement shown by the pyridone amido moiety of (-)-huperzine A, which might limit the ability of 4a to induce the flip of the Gly117-Gly118 peptide bond. In contrast, the cislike and translike arrangements of the methanesulfonamido group in 6a have similar stabilities, which reflects the larger conformational flexibility of the methanesulfonamido unit compared to the formamido one. Two 10-ns MD simulations were carried out by placing 4a (in the trans conformation) in the binding site of the Phe330fTyr mutant enzyme taken from X-ray structures of TcAChE complexed to (-)-huprine X (1E66)11 and (-)-huperzine A (1VOT),10 respectively (hereafter the terms 1E66 and 1VOT will be used to denote the two starting structures used in MD simulations). Replacement of Phe330 by Tyr was motivated by the fact that this is the sole mutation between TcAChE and human AChE enzymes at the catalytic site. This strategy, which has been used by other groups,22,23 was confirmed by previous MD simulations which established that Tyr occupies a position very similar to that of Phe330 in the binding site of AchE-huprine complexes.4,17 Inspection of the data collected in Table 2 points out that the binding mode of 4a is very similar in the two cases and reflects the main features of the interaction of (-)-huprine X bonded

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Table 2. Selected Geometrical Parameters for the Binding Mode of 4a and 6a Obtained from Molecular Dynamics Simulations structural parameter RMSD d(Trp84) R(Trp84) d(Tyr330) R(Tyr330) d(NH‚‚‚O-His440)b d(NH‚‚‚O-Glu199)c d(NH2‚‚‚O-Tyr330)d γ(C116-N117-CR-C117)e γ(N117-CR-C117-N118) γ(C117-N118-CR-C118) γ(N118-CR-C118-N119)

4a

6a

1VOT

1E66

1VOT

1E66

0.9 4.47 (0.11) +12.7 (4.8) 5.48 (0.13) +23.2 (7.8) 2.79 (0.08) 5.11 (0.22) 3.31 (0.24) +159.1 (8.4) +86.0 (9.9) -79.9 (10.6) +81.1 (8.0)

0.7 4.60 (0.11) +11.2 (4.8) 5.73 (0.13) +12.1 (5.5) 2.88 (0.11) 4.50 (0.16) 3.20 (0.16) +177.1 (7.3) +99.8 (8.5) -83.7 (8.8) +81.6 (8.1)

0.9 4.49 (0.12) +13.7 (4.3) 5.75 (0.13) +18.9 (7.2) 2.81 (0.08) 4.42 (0.22) 3.34 (0.21) +179.5 (8.3) +89.3 (9.7) -80.6 (8.5) +83.2 (7.1)

0.6 4.35 (0.11) +12.8 (4.8) 6.11 (0.15) +15.1 (6.5) 2.96 (0.13) 4.66 (0.20) 3.35 (0.20) -121.9 (11.1) -60.8 (10.3) +47.0 (12.2) +32.9 (10.1)

a Positional root-mean square deviation (RMSD; in Å) determined for the backbone atoms in the mobile region. Distances (d) are given in Å. Angles (R) and backbone dihedrals (γ) are given in degrees. b Distance from the protonated quinoline nitrogen atom. c Distance from the formamide nitrogen atom. d Distance from the exocyclic amino nitrogen atom. e The corresponding dihedral angles in the X-ray structures are -139.2°, +59.7°, -82.9°, and +77.6° for 1VOT and -98.4°, -172.9°, +139.2°, and +31.1° for 1E66.

Figure 3. Representation of the main interactions found between the formamido group of 4a in the binding site of the Phe330fTyr mutant in 1VOT (left) and 1E66 (right) MD simulations (colored by atom). Water molecules are shown as red spheres. Hydrogen-bond contacts are shown by dotted lines. The trace of the corresponding residues in the X-ray structure of TcAChE-(-)-huperzine A complex (1VOT) is shown in orange. For the sake of clarity, hydrogen atoms are not displayed.

to TcAChE.11 In the 1VOT MD simulation the conformation of the Gly117-Gly118 peptide bond remained stable along the whole trajectory with backbone torsional angles similar to those found in the 1VOT X-ray structure (Table 2; see Supporting Information). Remarkably, even though in the starting structure of the 1E66 MD simulation the formamido (Od)C-H group was oriented toward the Gly117 CdO group, the Gly117Gly118 peptide bond varied from the original crystallographic conformation found in 1E66 at the beginning of the simulation and adopted an arrangement similar to that found in the 1VOT complex (Table 2; see Supporting Information). Despite the similar arrangement adopted by 4a in the two MD simulations, there were differences in the interactions formed by the formamido group, which also differ from those found for the pyridone amido moiety in (-)-huperzine A. In this latter case the pyridone N-H group forms water-mediated contacts with both Glu199 carboxylate and Gly117 N-H groups, and the pyridone CdO unit is hydrogen-bonded to the Tyr130 hydroxyl group. In the 1VOT MD simulation (Figure 3; see also Supporting Information), two hydrogen-bonded water molecules link the Glu199 carboxylate and Gly117 N-H groups. Indeed, insertion of the second water molecule hinders the formation of the hydrogen bond between the formamido CdO group and Tyr130, while favors temporary water-mediated contacts with the CdO group of Trp84 and the side chain amide group of Gln69. In contrast, only one water molecule is found

to link Glu199 and Gly117 in the 1E66 MD simulation, while the direct interaction between the formamido CdO group and Tyr130 was more stable, though transient water-mediated contacts with the Trp84 CdO group were also detected (Figure 3; see also Supporting Information). In the two MD simulations, the orientation of the formamido group impedes the formation of the hydrogen bond between the formamido N-H group and the closest water molecule. Finally, it is worth noting that the backbone of Tyr116-Gly119 is stabilized by the same array of interactions found in the TcAChE-(-)-huperzine A complex, such as the contacts between the CdO group of Gly117 and the N-H units of Gly119 and Ala201, and between the N-H and CdO groups of Gly118 and the CdO and N-H units of Ser122 (see Figure 3). To further explore the structural plasticity of the Gly117Gly118 peptide, two additional 10-ns MD simulations were performed for 6a bonded to the Phe330fTyr TcAChE mutant built up from the 1VOT and 1E66 X-ray structures. As noted above, both cislike and translike conformations have similar stabilities in aqueous solution (see Supporting Information). Binding of 6a with the methanesulfonamido group in the cislike conformation, which would mimic the piridone cis-amido unit in (-)-huperzine A, would give rise to a steric clash with the backbone carbonyl group of Gly117 in the apo enzyme [as in the case of the binding of (-)-huperzine A], thus favoring the conformational change of the Gly117-Gly118 peptide bond. However, whether such a conformational flip could be promoted upon binding of 6a with the methanesulfonamido group in the translike conformation was not evident. Therefore, instead of using the cislike arrangement of the methanesulfonamido group, the inhibitor was initially placed in the translike conformation. In the two MD simulations the gross structural features of the binding mode of 6a reflect the main interactions observed for (-)-huprine X bonded to TcAChE11 (see Table 2). In the 1VOT MD simulation the Gly117-Gly118 peptide bond remained unaltered along the whole trajectory, and the backbone dihedral angles resembled those found in the 1VOT X-ray structure. Indeed, in the last 1.5 ns the methanesulfonamido group varied its conformation and adopted a cislike structure similar to that found in the piridone ring of (-)-huperzine A. However, in the 1E66 MD simulation the conformation of the Gly117-Gly118 peptide was different from that seen in both 1VOT and 1E66 X-ray structures, which suggests that the larger steric hindrance exerted by the methanesulfonamido unit relative to the formamido one prevents the complete relaxation of the peptide skeleton observed for 4a.


Figure 4. Representation of the main interactions found between the methanesulfonamido group (in the cislike arrangement) of 6a in the binding site of the Phe330fTyr mutant in the 1VOT MD simulation (colored by atom; a water molecule is shown as a red sphere). Hydrogen-bond contacts are shown by dotted lines. The trace of the corresponding residues in the X-ray structure of TcAChE-(-)huperzine A complex (1VOT) is shown in orange. For the sake of clarity, hydrogen atoms are not displayed.

In the final structure obtained in the 1VOT MD simulation (Figure 4; see also Supporting Information), the methanesulfonamido group mimics the interactions formed by the pyridone moiety of (-)-huperzine A in the 1VOT X-ray structure. Thus, the methanesulfonamido N-H group is hydrogenbonded to a water molecule, which in turn contacts both Glu199 carboxylate and Gly117 N-H groups. Moreover, one of the SdO groups is hydrogen-bonded to Tyr130, while the other one contacts the N-H group of Gly118, which in turn is at hydrogen-bond distance from the CdO group of Ser122. Moreover, the backbone of Tyr116-Gly119 is stabilized by a network of interactions that closely reflects that seen in the TcAChE-(-)-huperzine A complex. The preceding results point out that both formamido and methanesulfonamido units can trigger the conformational flip of the Gly117-Gly118 peptide bond, though the latter accommodates the array of interactions that mediate binding of the pyridone ring of (-)-huperzine A better than the former, as expected from the larger conformational flexibility of the methanesulfonamido moiety. Accordingly, 13-amidohuprines 4a and 6a can be considered effective hybrids between huprine Y and (-)-huperzine A. Even though the introduction of formamido and methanesulfonamido groups enables the corresponding 13-amidohuprines to fill a larger fraction of the catalytic binding site, it can be speculated that the detrimental effect on the AChE inhibitory potency can be ascribed to a sizable deformation cost of the Gly117-Gly118 peptide bond, which would make the net balance of the different factors that mediate the binding affinity unfavorable relative to the parent compound, (-)-huprine Y. In an attempt to gain insight into the preceding hypothesis, a comparison of the different contributions that mediate the binding affinity, even at a semiquantitative level, can be valuable. Since the substrate concentration in the AChE inhibitory assays (performed at 298 K) was the same for all the compounds, the experimental differences in binding free energy can be estimated from the IC50 data.24 From the data collected in Table 1, the binding affinity for hAChE of the formamido and methanesulfonamido derivatives relative to huprine Y can be estimated to be +2.8 and +2.3 kcal/mol, which compare with the relative binding affinites determined from the IC50 data measured for bAChE (without previous incubation of the enzyme with the inhibitor; +2.2 and +2.0 kcal/mol, respectively). Although these differences in binding affinity are determined from the inhibitory activity of the racemic compounds, it is reasonable to expect that they reflect the corresponding differences for the eutomers,


since the eutomer of huprine Y is roughly 2-fold more potent than the racemic compound, and a similar ratio is found on average for the other six huprines which have already been prepared in enantiopure form (see Supporting Information).3,4 The binding free energy of 4a and 6a to the enzyme was estimated from MM-PB/SA calculations using the protocol established in previous studies for the binding of a set of huprines with reduced conformational flexibility to bAChE (see Experimental Section).25 The binding free energy determined in this way accounts for the interaction energy between inhibitor and enzyme, and the desolvation cost associated with the formation of the inhibitor-enzyme complex. Moreover, for 6a the change in internal energy associated with the change between translike and cislike conformations was also considered. According to these calculations, 4a and 6a should bind to the enzyme around 0.6 and 1.2 kcal/mol more favorably than (-)huprine Y, which likely reflects the gain in interaction energy due to the additional contacts formed by the formamido and methanesulfonamido groups. However, these estimates do not include the entropic penalty arising from the selection of a single conformation of the 13-amidohuprine and the deformation cost of the Gly117-Gly118 conformational flip, which should amount to around 3.0 and 3.4 kcal/mol for the binding of 4a and 6a to bAChE and hAChE, respectively. The former contribution can be estimated from the inhibition constants of the transition state analogue inhibitor m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone (TMTFA) and its noncyclic analogue CF3CO(CH2)3N+Me3, which are 1.3 and 110 fM, respectively.26,27 Assuming that the two inhibitors have a common binding mode and that the difference in binding affinity comes from the entropic cost, freezing of each rotatable bond would contribute ∼0.6 kcal/mol, a value in agreement with the entropic cost of a rotatable bond derived from the analysis of the binding affinities for a variety of ligand-receptor complexes (from 0.3 to 0.8 kcal/mol per rotatable bond in the ligand).28,29 Accordingly, the deformation cost associated with the Gly117-Gly118 conformational flip can be estimated to be around 2.4 kcal/mol. Despite the semiquantitative character of the preceding analysis, it can be suggested that accommodation of the ligandinduced Gly117-Gly118 conformational flip seen in the TcAChE(-)-huperzine A complex has a net destabilizing effect on the binding affinity of the 13-amidohuprines analogues investigated here. In fact, such an effect must also operate in the binding of (-)-huperzine A. As noted by Fuxreiter and Warshel, the catalytic effect in AChE comes from the electrostatic stabilization associated with the preorganized polar environment.30 In particular, inspection of the X-ray structure of TcAChETMTFA complex shows that the Gly117-Gly118 peptide bond retains a conformation close to that found in the apo form and in the complex with (-)-huprine X (the backbone dihedral angles adopt the values -104.5°, -167.5°, +140.8°, and +35.8°).31 By adopting this conformation, the carbonyl group of the oxyanion of the tetrahedral adduct formed upon attack of the Ser200 hydroxyl group is stabilized with the N-H functions of Gly118 and Gly119, which constitute the oxyanion hole. This motif, which has also been identified in MD simulations of the natural substrate acetylcholine bonded to AChE,30,32 has been shown to be the main chain contribution to the overall catalytic effect of the enzyme.30 It can be hypothesized that the deformation cost associated with the Gly117-Gly118 rearrangement can be regarded as a mechanism selected by evolution to ensure the preorganization of the functionally relevant oxyanion hole in the binding site of AChE. In contrast, the large conformational flexibility of

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Phe330, which is reflected in the existence of three main conformations identified from inspection of the X-ray crystallographic structures of TcAChE complexes with several inhibitors,33 can be ascribed to the minor contribution played by this residue in mediating substrate binding to AChE. Conclusion The introduction of formamido and methanesulfonamido groups in the structure of huprine Y leads to the full occupancy of the AChE binding sites recognized by their parent compounds, tacrine and (-)-huperzine A. In fact, these groups effectively mimic the piridone amido moiety of (-)-huperzine A in triggering the conformational flip in the Gly117-Gly118 peptide bond induced upon binding of (-)-huperzine A. However, though the corresponding 13-amidohuprines are up to ∼3- and ∼6-fold more potent human AChE inhibitors than tacrine and (-)-huperzine A, they are less potent than the 13unsubstituted compound (huprine Y), which seems to stem from a sizable deformation cost associated with the flip of the Gly117Gly118 peptide bond. The failure of this strategy can be ascribed to the functional role played by Gly117 and Gly118 in the AChE oxyanion hole, since they play a relevant role in mediating substrate recognition. These results suggest that the inclusion of ligand-induced fit effects in drug design studies should take advantage of flexible regions not directly involved in functional roles of the target. Experimental Section Biochemical Studies. AChE inhibitory activity was evaluated spectrophotometrically at 25 °C by the method of Ellman,15 using AChE from bovine or human erythrocytes and acetylthiocholine iodide (0.53 or 0.27 mM for bovine and human AChE, respectively) as substrate. The reaction took place in a final volume of 3 mL of 0.1 M phosphate-buffered solution pH 8.0, containing 0.025 units of AChE and 333 µM 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB) solution used to produce the yellow anion of 5-thio-2-nitrobenzoic acid. Inhibition curves were performed in duplicate by incubating with at least eight concentrations of inhibitor for 15 min. One duplicate sample without inhibitor was always present to yield 100% AChE activity. The reaction was stopped by the addition of 100 µL of 1 mM eserine, and the color production was measured at 414 nm. In some experiments the time-dependence of the inhibitory processes was determined for the compounds with at least eight increasing concentrations of inhibitor after an incubation period of 45 min. BChE inhibitory activity determinations were carried out similarly, using 0.035 unit of human serum BChE and 0.56 mM butyrylthiocholine, instead of AChE and acetylthiocholine, in a final volume of 1 mL. Data from concentration-inhibition experiments of the inhibitors were calculated by nonlinear regression analysis, using the GraphPad Prism program package (GraphPad Software, San Diego, CA), which gave estimates of the IC50 (concentration of drug producing 50% of enzyme activity inhibition). Results are expressed as mean ( SEM of at least four experiments performed in duplicate. DTNB, acetylthiocholine, butyrylthiocholine, and the enzymes were purchased from Sigma, and eserine was from Fluka. Conformational Analysis. The conformational preferences in aqueous solution of the formamido and methanesulfonamido groups in compounds 4a and 6a were determined by combining the free energy differences determined in the gas phase from MP2/6-31G(d) computations using the geometries optimized at the HF/ 6-31G(d) level, and the relative free energies of hydration obtained from the MST HF/6-31G(d) version18 of the PCM continuum model.19 A systematic exploration at the HF/6-31G(d) level was performed by changing the torsional angle around the bonds C13-Namido (in 4a and 6a) and Namido-S (in 6a) by increments of 60° and excluding those conformers where a steric clash was observed. The geometry of the local minimum energy structures was then fully optimized.

Camps et al.

The minimum energy nature of all the stationary points located from HF/6-31G(d) geometry optimizations was verified from the analysis of the vibrational frequencies, which were positive in all cases. The free energy differences in the gas phase were determined by adding the zero-point energy, thermal, and entropic contributions (at 298 K) to the MP2/6-31G(d) relative energies. All calculations were performed using a locally modified version of Gaussian 03.34 Besides the conformers shown in the Supporting Information, other conformers of 4a and 6a were located, but they were not considered due to the large destabilization (i.e., by more than 4 kcal/mol) relative to the most stable conformer. Molecular Modeling Methods. The simulation system was defined following the protocol used in our previous studies on AChE-ligand complexes,4,17 which is briefly summarized here. The simulation system was based on the X-ray crystallographic structures of the AChE complexes with huprine X (1E66)11 and (-)huperzine A (1VOT),10 which were used as templates to position the 13-amidohuprines derivatives in the catalytic binding site. The enzyme was modeled in its physiologically active form with neutral His440 and deprotonated Glu327, which form together with Ser200 the catalytic triad. The standard ionization state at neutral pH was considered for the rest of ionizable residues with the exception of Asp392 and Glu443, which were neutral, and His471, which was protonated, according to previous numerical titration studies.35 Finally, Phe330 was replaced by Tyr to reflect the binding site sequence in bovine and human AChE. According to the basicity of the quinoline ring,36 the huprine derivatives were modeled in the protonated state. Starting simulation systems were defined by placing the inhibitor at the catalytic binding site of the AChE enzyme taken from X-ray structures 1E66 and 1VOT. In this latter structure the side chain of Tyr330 was rotated to maintain the planarity of the benzene ring of Tyr330, which was stacked against the 4-aminoquinoline ring of the amidohuprine in order to reproduce the binding mode observed for (-)-huprine X.11 The system was hydrated by centering a sphere of 40 Å of TIP3P37 water molecules at the inhibitor, paying attention to filling the position of crystallographic waters inside the binding cavity. The parm98 file of the AMBER force field38 was used to describe the enzyme. The charge distribution of the inhibitor was determined from fitting to the HF/6-31G(d) electrostatic potential using the RESP procedure,39 and the van der Waals parameters were taken from those defined for related atoms in AMBER force field. Torsional parameters for the methanesulfonamido group were taken from those reported in our previous studies.40 The final model system was partitioned into a mobile region and a rigid region. The former included the inhibitor, all the protein residues containing at least one atom within 14 Å from the inhibitor, and all the water molecules, while the rest of atoms defined the rigid part. The system was energy minimized and equilibrated using the AMBER program.41 First, all hydrogen atoms were minimized for 1000 steps of steepest descent. Next, the position of water molecules was relaxed for 2000 steps of steepest descent followed by 3000 steps of conjugate gradient. Finally, the whole system, including both mobile and rigid parts, was energy minimized for 2000 steps of steepest descent plus 3000 steps of conjugate gradients. At this point, the rigid part of the system was kept frozen and started the thermalization of the mobile part by running five 20-ps molecular dynamics (MD) simulations to increase the temperature up to 298 K. Subsequently, a 10-ns MD simulation was carried out. SHAKE was used to maintain all the bonds at their equilibrium distances, which allowed use of an integration time step of 2 fs. A cutoff of 11 Å was used for nonbonded interactions. In all cases the positional root-mean square deviation determined for the backbone and heavy atoms in the mobile region with regard to the corresponding X-ray crystallographic structure was in the range from 0.6 to 1.3 Å (see Supporting Information). The characterization of the structural features that mediate the binding to the enzyme was determined by averaging the geometrical parameters for the snapshots (saved every picosecond) sampled along the last 4 ns of the MD simulation.



MM-PB/SA Computations. The difference in binding free energy of compounds 4a and 6a was determined using the Molecular Mechanics-Poisson Boltzmann/Solvent Accessible (MMPB/SA) protocol used in previous studies for a series of huprines with limited conformational flexibility.25 According to this protocol, the relative binding free energy was computed from the addition of electrostatic and nonelectrostatic contributions (see eq 1). ∆∆Gbinding ) ∆∆Gele + ∆∆Gn-ele

(1)

The electrostatic term was computed by adding the solventscreened electrostatic interaction between ligand and enzyme, and the corresponding change in the desolvation free energy of the ligand upon binding. According to this procedure, the desolvation cost of the enzyme was assumed to be common for all the complexes between AChE and the inhibitors, which is justified by the fact that all of them share a common chemical skeleton and occupy the same position in the binding site, while providing a substantial saving in computer time. The different electrostatic contributions were determined from a finite-difference solution of the PB equation.42 The solvent was represented as a continuum with a dielectric constant of 78.4, and the protein and the inhibitor were treated as cavities with a dielectric constant of 1 containing fixed partial charges on their atoms. The dielectric boundary was defined using a 1.4-Å probe sphere and atomic van der Waals radii taken from the AMBER force field. A grid of 1.5 Å/point was used in PB calculations, which were carried out with the CMIP program.43 The nonelectrostatic term was determined from the addition of two contributions, which account for the van der Waals interaction energy between enzyme and inhibitor, and the change in the nonelectrostatic component of the solvation free energy. The former was computed using the van der Waals parameters in the AMBER force field, while the latter was estimated using an empirical relationship with the change in the solvent accessible surface of ligand and receptor upon binding. A value of 5.4 cal/(mol Å2) was adopted for the microscopic surface tension of all parts of the surface.44 MM-PB/SA computations were performed for a set of 50 structures of the enzyme-inhibitor complex taken from the last 500 ps of the MD simulation. Prior to the calculations, the complexes were energy-minimized to eliminate bad contacts (water molecules, inhibitor, and residues in the mobile part were minimized in a sequential way for a total of 2000 steps; at the end, all water molecules were removed). On the basis of our previous studies,25 the relative binding affinity was determined from the most favorable binding free energies determined for each inhibitor.

Acknowledgment. Financial support from Direccioń General de Investigacioń of Ministerio de Ciencia y Tecnologıá and FEDER (projects CTQ2005-02192/BQU, SAF2002-00049 and CTQ2005-09365) and Comissionat per a Universitats i Recerca of the Generalitat de Catalunya (projects 2005-SGR00180 and 2001-SGR00216) is gratefully acknowledged. Supporting Information Available: Conformational analysis of compounds 4a and 6a; plots showing the time dependence of (i) the positional root-mean-square deviation of backbone and heavy atoms along the molecular dynamics simulations, (ii) torsional angles of the Gly117-Gly118 peptide bond, and (iii) selected geometrical parameters corresponding to specific interactions between the inhibitor and the AChE catalytic binding site, together with superposition of snapshots taken every nanosecond along the MD simulation; AChE inhibitory activities of the racemic mixtures and the eutomer for a series of huprine derivatives. This material is available free of charge via the Internet at http://pubs.acs.org.

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