Computational Analysis of Plasmepsin IV Bound to an Allophenylnorstatine Inhibitor

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FEBS Letters 580 (2006) 5910–5916

Computational analysis of plasmepsin IV bound to an allophenylnorstatine inhibitor

Hugo Gutie´rrez-de-Tera´na,1, Martin Nervalla, Ben M. Dunnb, Jose C. Clementeb, Johan A˚ qvista,* a Department of Cell and Molecular Biology, Uppsala University, BMC, P.O. Box 596, 751 24 Uppsala, Sweden b Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, P.O. Box 100245, Gainesville, FL 32610-0245, USA

Received 12 September 2006; revised 18 September 2006; accepted 26 September 2006

Available online 5 October 2006

Edited by Peter Brzezinski

edge about protein–ligand interactions, and sometimes also a Abstract The plasmepsin proteases from the malaria parasite Plasmodium falciparum are attracting attention as putative drug quantitative energetic description of relevant interactions targets. A recently published crystal structure of Plasmodium responsible for binding affinity. One example of such efforts malariae plasmepsin IV bound to an allophenylnorstatine inhib- is the structure based design of antimalarial drugs that inhibit itor [Clemente, J.C. et al. (2006) Acta Crystallogr. D 62, 246– the aspartic proteases of the Plasmodium species known as 252] provides the first structural insights regarding interactions plasmepsins (PM) [1–3]. of this family of inhibitors with plasmepsins. The compounds in This family of enzymes, together with cysteine proteases this class are potent inhibitors of HIV-1 protease, but also show and metalloproteases, are responsible for the degradation of nM binding affinities towards plasmepsin IV. Here, we utilize hemoglobin by the parasite, which occurs in the acidic food automated docking, molecular dynamics and binding free energy vacuole during the intraerythrocytic stage of the life cycle calculations with the linear interaction energy LIE method to [4]. Four plasmepsins exist in the food vacuole of Plasmodium investigate the binding of allophenylnorstatine inhibitors to plasmepsin IV from two different species. The calculations yield falciparum, the most lethal species and responsible for the excellent agreement with experimental binding data and provide majority of the malaria related deaths, named PfPM I to new information regarding protonation states of active site resi- PfPM IV [5]. All plasmepsins are aspartic proteases except dues as well as conformational properties of the inhibitor com- for the histo-aspartic protease (HAP, or PfPM III) [6] and plexes. share more than 68% of sequence identity [7]. Among them, Ó 2006 Federation of European Biochemical Societies. Pub- PfPM IV is the ortholog of the unique plasmepsin present in lished by Elsevier B.V. All rights reserved. the food vacuole of the other three species infecting man (Plasmodium malariae, PmPM IV; Plasmodium vivax, PvPM Keywords: Malaria; Plasmepsin; Enzyme inhibitors; Linear IV; and Plasmodium ovale, PoPM IV) [8]. The identity among interaction energy method PM IV from these different species is between 70% and 87% [7]. Therefore, there is a growing interest in the structural characterization of this class of enzymes to aid structure- based drug design. There exist crystal structures of PfPM IV (PDB entry 1LS5) [9] and PvPM IV (PDB entry 1QS8) 1. Introduction [10] in complex with the canonical peptide-mimetic inhibitor pepstatin A. Very recently, a low resolution crystal structure Structure-based drug design strongly relies on the availabil- of PmPM IV in complex with an allophenylnorstatine inhib- ity of three-dimensional molecular structures of the biological itor was published (PDB code 2ANL) [11]. The allophenyl- targets of interest. The methods for structure determination norstatine (Apn) series of inhibitors were initially developed provide experimental data (i.e., electron density maps, NMR by Kiso and co-workers towards HIV protease [12]. The spectra) that need to be processed in order to obtain final potential application of this chemical scaffold for develop- molecular models. It follows that structural and computational ment of new plasmepsin ligands was soon appreciated [13], analysis of the reported structures can provide deeper knowl- and considerable efforts have been devoted to lead optimiza- tion towards these biological targets [3,14]. The binding mode of this class of compounds was previously hypothesized by molecular modeling [3], but these predictions seem incompat- *Corresponding author. Fax: +46 18 530396. ible with the new crystal structure of the PmPM IV–KNI764 ˚ E-mail address: [email protected] (J. Aqvist). complex [11]. Here, we present a computational analysis of 1 Present address: Department of Pharmacology, Faculty of Phar- the binding mode proposed by Clemente and co-workers macy, University of Santiago de Compostela, Campus Universitario [11] for the Apn inhibitor KNI764, extending their model Sur, 15708 Santiago de Compostela, Spain. to other Apn inhibitors and to the corresponding P. falciparum enzyme (PfPM IV). We find that consistence with experi- Abbreviations: Apn, allophenylnorstatine; LIE, linear interaction energy; MD, molecular dynamics; PmPM IV, Plasmodium malariae pla- mental data can be found only after some considerations smepsin IV; PfPM IV, Plasmodium falciparum plasmepsin IV; RMSD, that affect the detailed nature of ligand–protein binding root mean square deviation contacts.

0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.09.057 H. Gutie´rrez-de-Tera´n et al. / FEBS Letters 580 (2006) 5910–5916 5911

2. Methods (mol A˚ 2). In the case of the crystallized complex (2ANL), all ligand heavy atoms were also subjected to the same restraint as the protein All molecular alignments, manipulation of chemical structures and atoms. The production phase followed for 0.3–1.2 ns making use of the generation of figures were done with the software Pymol v0.99 [15]. the SHAKE algorithm [28] for the treatment of the bond lengths and collecting energies at regular intervals of 15 fs. Stability was addressed by comparing the average potential energy values of the first 2.1. Automated docking and second halves of the data collection period, which yields the esti- Automated docking exploration was performed with GOLD version mated error in the calculation of the corresponding potential energy. 3.0.1 [16], allowing full flexibility for the ligand while keeping the pro- Error estimates in the calculated free energies were obtained by adding tein fixed (i.e., only rotation of the protein hydroxyl groups was con- the errors in the potential energy from both the water and protein sim- sidered). The docking exploration consisted of 20 independent runs ulations, properly scaled through the use of the LIE a and b factors. with each compound, using the default genetic algorithm (GA) search Trajectory lengths yielding errors in the potential energies not larger parameters. Both scoring functions implemented in the program than 1 kcal/mol were generally accepted, since the computed error in (Chemscore and Goldscore) [17] were considered in parallel runs. the calculated free energy of binding then is less than 1 kcal/mol. The automated assignment of protein and ligand atom types was The MD sampling of the free ligand was again done in a TIP3P checked in order to ensure a correct treatment of the potential hydro- water sphere of the same radius as in the corresponding protein simu- gen bond donors and acceptors. In all cases, the protein model used lation. The sphere was equilibrated with a 10.2 ps MD run at 300 K, was PmPM IV (PDB entry 2ANL). This choice was based on the with positional restraints on the heavy atoms of the ligand (200 kcal/ observation that there is conformational variability of plasmepsins (mol A˚ 2) for the first 0.2 ps and 10 kcal/(mol A˚ 2) in the following depending on the inhibitor bound [18–20]. Therefore, the conforma- 10 ps). MD simulation then followed for at least 1.5 ns under the same tion adopted upon binding to KNI764 is the most reasonable protein conditions as for the bound state, but keeping the initial position of the structure for the purpose of docking a set of Apn inhibitors. The dock- central atom of the ligand fixed with 10 kcal/(mol A˚ 2) to ensure homo- ing into PfPM IV was done by superimposing the resulting inhibitor- geneous solvation. 2ANL complex on the crystal structure of PfPM IV (PDB entry 1LS5). Docking solutions were selected for post-processing only if they satisfied the following conditions: (i) The existence of at least one interaction between the allophenylnorstatine hydroxyl with the catalytic 3. Results aspartates and (ii) the best ranked representative of the cluster should be among the 25% top scoring solutions (clustering was done accord- The recent crystal structure of PmPM IV in complex with ing to a 1.5 A˚ RMSD clustering criterion). KNI764 [11] was analyzed by MD simulations, and the binding free energy of the inhibitor was estimated with the LIE 2.2. Molecular dynamics (MD) and binding free energy calculations Binding affinities were calculated using the linear interaction energy method. The protonation state of the catalytic aspartates (LIE) method, described in detail elsewhere [21,22]. Basically, this ap- was initially retrieved from earlier works [2,29], with the pro- proach estimates the ligand free energy of binding from the difference ton located on the oxygen of Asp34 (Od2) closest to Asp214 in the ligand-surrounding interaction energies between its bound and (hereafter referred to as model AspH34, see Fig. 1A). The pro- free state. The relationship between the ligand intermolecular inter- posed inhibitor position remained stable along the simulation, action energies and the free energy of binding is given by the equation: and up to eight hydrogen bonds (H-bonds) are detected be- vdW el DGbind ¼ aDhV ls iþbDhV lsiþc ð1Þ tween the ligand and protein (see Fig. 1C). Despite this exten- vdW el sive H-bond network, the calculated electrostatic component where V ls and V ls denote, respectively, the Lennard-Jones and electrostatic interactions between the ligand and its surroundings (l s). of the binding free energy remained positive (unfavorable). These interactions are evaluated as energy averages (denoted by the The reason for this unexpected result was found in the low open brackets) from separate MD simulations of the free (solvated absolute energy of interaction of the ligand with the negatively in water) and bound states. The difference (D) between such averages for each type of potential is scaled by different coefficients (see Ref. charged Asp214, which in previous calculations [2,29] was in [22]), giving the polar and non-polar contributions to the binding free the range of 20 to 30 kcal/mol (Gutie´rrez-de-Terań and energy. For the non-polar contribution, this coefficient has been empir- Nervall, unpublished observations), while in the present case ically set to a = 0.181, while for the polar contribution the scaling fac- the aforementioned interaction energy was only about tor is dependent on the chemical nature of the ligand. For ligands with one hydroxyl group (KNI727), the value is b = 0.37, while for the 5 kcal/mol. The estimated free energy of binding was there- ligands with two or more hydroxyls the value is b = 0.33 [22]. fore dominated by the hydrophobic contribution and the MD simulations were done using the program Q [23] and the OPLS calculated value is shifted by about 6 kcal/mol from the exper- force field there implemented [24]. Parameters for the ligands which imental value (see Table 1). Since the estimation of absolute were not included in our implementation of the force field were derived binding affinities with the LIE method may require the inclu- manually, following the rules of the original OPLS force field. The system was solvated with a simulation sphere of TIP3P waters [25] sion of a constant term, which is dependent on the nature of of radius 20 A˚ , centered on the carbon of the transition state mimic hy- the protein [30], other PM IV inhibitors were examined for rel- droxyl. The water surface of this sphere was subjected to radial and ative affinities. We selected KNI577, a much less potent inhib- polarization restraints [26] in order to mimic bulk water at the sphere itor of PmPM IV (K =3lM) [11], since the small structural boundary. Non-bonded interaction energies were calculated up to a i 10 A˚ cutoff, except for the ligand atoms for which no cutoff was used. difference with KNI764 made it straightforward to perform Beyond the cutoff, long-range electrostatics were treated with the local manual docking into the 2ANL crystal structure. Additionally, reaction field (LRF) multipole expansion method [27]. Protein atoms the ortholog enzyme in P. falciparum (PfPM IV) was explored outside the simulation sphere were restrained to their initial positions, with another pair of inhibitors presenting a significant differ- and only interacted with the system through bonds, angles and tor- ence in the experimental affinity for this enzyme [3]: sions. For the ligand–protein simulations, a heating and equilibration procedure was applied before the data collection phase. The equilibra- KNI10006 (IC50 = 15 nM) and KNI727 (IC50 = 740 nM). tion protocol started with 1000 steps MD using very short time step The chemical structures of the four inhibitors are shown in (0.2 fs) at 1 K temperature, coupled to a strong bath (0.2 fs bath cou- Fig. 2. Initially, manual docking was done by structural super- pling) with positional restraints on heavy atoms. Then the system was position of 2ANL onto 1LS5 followed by manual modification gradually heated up to 300 K, relaxing the temperature bath coupling 0 to 100 fs and increasing the timestep to 1.5 fs. During this equilibration of the corresponding P2 and P2 chains of KNI764. In neither process positional restraints on the protein heavy atoms and the cen- of the two PM IV enzymes (P. falciparum or P. malariae) did tral atom of the ligand were gradually released from 200 to 0 kcal/ we find any correlation with the relative affinities for each pair 5912 H. Gutie´rrez-de-Terań et al. / FEBS Letters 580 (2006) 5910–5916

Fig. 1. Top: initial H-bond network of 2ANL complex with a proton located on Asp34 (A) or Asp214 (B). Bottom: average structures after MD sampling of the two models: AspH34 (C) or AspH214 (D).

Table 1 According to the 2ANL crystal structure, the pair distances Experimental and calculated free energies of binding (in kcal/mol) of between the Od atoms of the catalytic aspartates and the het- the four PMIV–KNI complexes eroatoms of the ligand (the transition state mimic hydroxyl obs a calc calc Complex DGbind DGbind AspH214 DGbind AspH34 and the adjacent oxygen O23) are compatible with different KNI764–PmPMIV 9.6b 9.8 ± 0.7d 3.0 ± 0.4 hypotheses for the protonation state of the catalytic aspartates. KNI577–PmPMIV 7.6b 7.0 ± 0.8 4.3 ± 0.8 Our first model (AspH34) was unsuccessful in reproducing c KNI10006–PfPMIV 10.7 14.1 ± 0.4 5.9 ± 0.2 absolute as well as relative binding affinities. Consequently, KNI727–PfPMIV 8.4c 11.9 ± 0.5 5.9 ± 0.6 the next step was to explore alternative protonation states. a The binding free energy calculated from experimentally determined Fig. 1B shows how the location of the proton on the most ex- K ’s using DG0 RT ln K . i bind; exp ¼ i posed oxygen of Asp214 (hereafter referred to as the AspH214 bValues from Ref. [11]. cValues from Ref. [3]. model) can lead to a strong H-bond network between the Apn dAverage value from two MD simulations: starting from the crystal- core and the catalytic center. Moreover, the electronic repul- lographic coordinates and from the GOLD predicted pose. sion between a negatively charged aspartate and the carbonyl of the P10 amide bond (O23), present in the previous AspH34 model, is not observed and instead O23 accepts two H-bonds from AspH214 and Thr217. of inhibitors (see Table 1). In all cases, an unusually low con- In order to check the reliability of this hypothesis, we re- tribution from the electrostatic component of the binding free peated MD simulations of the 2ANL structure according to energy was observed. In particular, the electrostatic interac- the AspH214 model. This time, the estimation of the free en- tions of the ligand with the catalytic aspartates were rather ergy of binding was extremely accurate, and some interesting small (see Table 2). conformational rearrangements of the ligand and protein side- Attempts to re-dock KNI764 into PmPM IV resulted in the chains were observed (see Fig. 1D). The P2 amide bond flips at identification of several binding modes. Only the pose ranked the very beginning of the simulation, remaining stable in the #7 using the Goldscore function showed some similarity to the new position for the rest of the simulation. This allowed the crystallographic pose, with an RMSD of 2.7 A˚ . The binding formation of H-bonds in P2 with Ser79 in the flap and mode previously hypothesized by Nezami et al. [3] was among Thr217, after some reorientation of the sidechain of the latter the top ranked solutions (#1 with Chemscore and #3 with residue. Asp214 flips 90° to avoid electrostatic repulsion with Goldscore). This position involves a 180° turn along the Asp34, maintaining an H-bond to the carbonyl in the P10 prime–non-prime axis with respect to the binding mode shown amide bond, while the hydroxyl group of the inhibitor H- in the crystal structure. However, this orientation of the ligand bonds to the negatively charged Asp34. The interactions at is hardly compatible with the 3.3 A˚ electron density map of the the prime side remain unchanged with respect to the previously PmPM IV-KNI764 complex deposited in the PDB [11]. described simulation using the AspH34 model. As can be H. Gutie´rrez-de-Terań et al. / FEBS Letters 580 (2006) 5910–5916 5913

Fig. 2. Structures of the four PMIV inhibitors considered in this work.

Table 2 Interaction energies (kcal/mol) between the catalytic aspartates and the ligand el el a el b Complex hV lAsp’si DDGAsp’s DGbind AspH214 AspH34 AspH214 AspH34 KNI764–PmPMIV 27 16.5 3.9 4.9 +2.0 KNI577–PmPMIV 20 21.5 0.5 1.8 +0.1 KNI10006–PfPMIV 32 20.5 3.8 7.9 0.3 KNI727–PfPMIV 35 24 3.6 6.5 0.5 aDiﬀerence in the electrostatic component of the binding free energy due to interactions of the ligand with the catalytic aspartates, calculated as: el el el DDGAsp’s ¼ bðhV lAsp’siAspH214 hV lAsp’siAspH34Þ. Note that a negative sign indicates favored binding for the AspH214 model. b el el el Electrostatic component of the total binding free energy, calculated as DG ¼ bðhV lsip hV lsiwÞ.

The AspH214 model was subsequently used for new docking explorations. Interestingly, both Goldscore and Chemscore functions showed high sensitivity to this change, and the native pose was now found in the first (Chemscore) and second (Goldscore) ranking position. The RMSD with respect to the crystallographic pose was 1.0 and 0.9 A˚ , respectively. Interest- ingly, the amide bond in P2 was flipped in agreement with the MD simulations of the 2ANL complex. We repeated the MD simulation of PmPM IV-KNI764 complex, using the ligand conformation predicted by GOLD. The predicted free energy of binding was very accurate again (DGbind,calc = 10.0 kcal/ mol) and the superposition of the average structure of the complex with the electron density map of 2ANL is depicted in Fig. 4. The orientation of the sidechains of both Asp214 and Thr217 this time shows good agreement with the electron density, although a slight deviation of the 2-methylbenzyl ring Fig. 3. Plot of the difference in accumulated average non-bonded with respect to the mesh can be observed. ligand–protein interactions between the two trajectories of the Docking of the less active ligand KNI577 pointed in the KNI764–PmPMIV complex (using the AspH214 and AspH34 models). same direction, and again both scoring functions identified A more negative value indicates favored interactions for the AspH214 the same binding mode as described above for KNI764. MD model. Dashed lines account for electrostatic interaction energy, while solid lines indicates non-polar energy. The residues responsible of the simulation of this binding orientation of KNI577 demon- biggest differences are indicated. strated the stability of the complex, with the calculated value for the free energy being very close to the experimental value. The major differences with respect to KNI764 were found in appreciated in Fig. 3 and Table 2, the AspH214 model allowed the electrostatic interactions with Asp214 and Thr217 (see for stronger electrostatic interactions between ligand and pro- Table 3), which are weakened when the small isobutyl side- tein, since the H-bond network is more complete than in the chain is located in the contiguous S2 site, thus allowing more AspH34 model. flexibility on the non-prime side of the ligand. 5914 H. Gutie´rrez-de-Terań et al. / FEBS Letters 580 (2006) 5910–5916

Fig. 4. Left: average structure from MD simulation of KNI764 (cyan) in complex with PfPMIV (yellow). The average structure is superimposed on ˚ the 3.3 A resolution electron density of 2ANL. The map is a 2Fo Fc electron density map calculated from the 2ANL dataset with the ligand omitted and is displayed at 1.0 r. The average structure from the MD shows a perfect match with the electron density, apart from the 2-methylbenzyl group in P2. Right: superposition of the MD averaged structure of KNI764 (cyan) onto the crystal structure conformation from 2ANL (green). The major diﬀerence is the ﬂipped amide bond in the P1–P2 link.

Table 3 Experimental and calculated energetics (kcal/mol) of PMIV inhibition using the AspH214 model obs a calc d Complex DGbind (kcal/mol) DGbind (kcal/mol) Ligand-surrounding interactions (kcal/mol) vdw el vdw el hV ls ip hV lsip hV ls iw hV lsiw KNI764–PmPMIV 9.6b 9.8 ± 0.7d 68.1 ± 0.5 76.1 ± 1.3 42.7 ± 0.5 61.2 ± 0.7 KNI577–PmPMIV 7.6b 7.0 ± 0.8 67.2 ± 0.8 62.6 ± 1.4 39.3 ± 0.2 56.6 ± 0.5 KNI10006–PfPMIV 10.7c 14.1 ± 0.4 77.2 ± 0.1 93.3 ± 0.7 42.9 ± 0.0 69.4 ± 0.6 KNI727–PfPMIV 8.4c 11.9 ± 0.5 74.4 ± 0.4 70.4 ± 0.4 44.9 ± 0.1 52.7 ± 0.7 a 0 The binding free energy calculated from experimentally determined Ki’s using DGbind; exp ¼ RT ln Ki. bValues from Ref. [11]. cValues from Ref. [3]. dAverage value from two MD simulations: starting from the crystallographic coordinates and from the GOLD predicted pose.

Analysis of PfPM IV inhibitors were subsequently repeated the protein, compared to the smaller isobutyl in KNI727 (see using the AspH214 model for the P. falciparum protein. First, Table 3). As was the case with PmPM IV, the comparison of docking of KNI10006 and KNI727 was carried out using both the AspH34 and AspH214 models clearly suggest that the the 1LS5 and 2ANL structures. The same binding mode dis- AspH214 model accounts for stronger interactions of the cussed above for KNI764 and KNI577 in PmPM IV was found inhibitor with the catalytic site (see Table 2). among the top scoring poses of KNI727 (i.e., good superposi- It must be noted that, in the case of PfPM IV, it is not pos- tion with the conformation of KNI764 in 2ANL but with the sible to estimate absolute binding free energies, since only the P2 amide bond flipped). Not surprisingly, a better superposi- IC50 values have been reported in this case [3].IC50 values for tion with the crystallographic pose of KNI764 was achieved competitive inhibitors, as those used in this study, in general if 2ANL was used as the rigid protein structure. Docking of are weaker in magnitude than Ki values because the concentra- KNI10006 into 2ANL identified the same binding orientation, tion and affinity of the substrate for the enzyme are not explic- although slightly displaced with respect to the native pose of itly considered in the assay setup and IC50 calculations [31]. KNI764 (RMSD for backbones atoms was 1.3 A˚ ). On the con- Therefore, the calculated binding free energies are expected trary, when the 1LS5 protein structure was used no analogous to be more negative than the corresponding values obtained pose could be identified. For the following MD simulations, from IC50 experiments. Furthermore, earlier calculations on the starting position of the ligand in the protein binding pocket PfPMIV inhibitors have indicated that the constant c term in should be accurate enough so that the values averaged during Eq. (1) is positive and about 1.5 kcal/mol for PfPMIV, while the MD sampling can converge within reasonable calculation it is zero in PfPMII [29]. The average overestimation of time. Therefore, we retrieved the best pose found in the dock- 3 kcal/mol in our calculations is consistent with these observa- ing exploration of 2ANL-KNI727 and used it for MD simulations. tions with PfPM IV. To do this, superposition of the protein structures and, in the case of KNI10006, mutation of the P2 sidechain were performed. Again, the calculated affinities using 4. Discussion the AspH214 model could explain the experimental difference in affinity of the two ligands. The only structural difference be- The protonation state of the catalytic aspartates in aspartic tween this pair of molecules, the P2 sidechain, was identified as proteases is an ambiguous and unsolved question. Several the key to explain the difference in affinity: the aminoindanol studies have addressed this point using different computational group present in KNI10006 contributes to an enhancement [32–36] and experimental [37–39] approaches. It can be con- of both electrostatic and non-electrostatic interactions with cluded that no unique model exists and the location of the pro- H. Gutie´rrez-de-Terań et al. / FEBS Letters 580 (2006) 5910–5916 5915 ton(s) is dependent on the presence and type of inhibitor bound. As an example of the ambiguous location of the proton, two high resolution crystal structures of the HIV-1 protease in complex with allophenylnorstatine inhibitors suggest different protonation states. On the one hand, in the complex with KNI764, the proton is located in Od2 of Asp25B [38], in agreement with previous NMR studies [37]. On the other hand, the recent crystal structure of the same protein in complex with a phenylnorstatine peptidic inhibitor showed a com- pletely different scenario [39]. In this case, the central carbon bearing the hydroxyl group of the inhibitor had the opposite (R) stereochemistry as compared to KNI compounds (S) and the proton was found on Asp25A; more surprisingly, the electron density suggested the existence of a second proton shared by the two catalytic residues, resembling that proposed for the free enzyme [40]. In previous works [1,2], we have defined AspH34 molecular models of the Plasmodium falciparum plasmepsins II and IV, Fig. 5. Superposition of the structures of the four PMIV–KNI with the proton located on the most buried oxygen (Od1)of complexes, as average coordinates of the MD trajectories sampled Asp34. This representation allowed for an optimum H-bond- for binding free energy calculations: KNI764–PmPMIV (green), ing network with inhibitors sharing an ethylenediol transition KNI577–PmPMIV (cyan), KNI10006–PfPMIV (magenta), KNI727– PfPMIV (yellow). state mimicking group, leading to interpretative and explana- tory binding models. However, recent analysis of the two protonation models in PfPM IV in complex with pepstatin A did not point to any preferred location of the proton [29]. for a more conserved pattern of interactions with the flap. The application of the AspH34 protonation model to the re- Aspartic proteases generally show a double H-bond pattern cently reported structure of the PmPMIV–KNI764 complex in between the P2 and P20 carbonyl oxygens and the flap loop(s) binding affinity calculations was inconsistent with the experi- [41]. However, the 2ANL structure lacks the S2-flap interac- mental data. The reason was found in the electrostatic repul- tion, and the authors already noted this significant difference sion between partial negative charges of Asp214 and in the S2 conformation of KNI764 with respect to the confor- carbonyl of the amide bond in P1. This result was also ob- mation adopted in HIV-1 protease [11]. tained with the other three PMIV–KNI complexes examined. The present computational study provides complementary Additionally, the automated docking program GOLD, which energetic and structural data for a recent crystal structure of is generally successful in the re-docking of ligands onto the a plasmepsin in complex with inhibitor. The information corresponding native crystal structures (including several gained about the protonation state of the catalytic aspartates aspartic proteases) [16] failed in the present case. Conversely, and the position of the amide bond in P2 turned out to be GOLD successfully identified the native pose of the ligand as essential in order to reproduce the experimental binding data the top ranked solution when the location of the proton was (see [42] for a related case in trypsin). Using the LIE method changed (AspH214 model). The calculated affinities using the with the standard parameterization and the slightly modified LIE approach were extraordinarily close to the experimental model of KNI inhibitors bound to PM IV, we could predict ones for the pair of PmPM IV inhibitors (KNI764 and the binding free energy differences of two pairs of inhibitors KNI577), and also the relative affinities for the two PfPM IV bound to two different orthologs of the PM IV enzyme (i.e., complexes (KNI727 and KNI10006) were very well repro- PmPM IV and PfPM IV). This result clearly supports the duced. robustness of the binding mode proposed in this work for The simulations also identified an important difference with PM IV–KNI complexes. We suggest that the molecular inter- respect to the 2ANL binding mode, located in the S2 site, and actions described here for the binding mode of the KNI series in all cases was the amide bond in P2 flipped with respect to the of inhibitors to PMIV enzymes should be considered for fur- proposed binding mode in 2ANL, leading to a better H-bond ther structure-based design efforts. network with the S2 pocket (Ser79, Gly216). This was found not only using MD relaxation of the initial crystal structure, Acknowledgement: This work was supported by a grant from the but also the automated docking explorations were consistent Swedish Foundation for Strategic Research (SSF/Rapid). with this conformational change. As can be appreciated in Fig. 5, the binding mode of the four complexes is much conserved, and in all cases is the double hydrogen bond with the References flap attained. Structural superposition based on the ligand [1] Ersmark, K., Feierberg, I., Bjelic, S., Hulten, J., Samuelsson, B., and the catalytic aspartates of our refined binding mode of Aqvist, J. and Hallberg, A. (2003) C-2-symmetric inhibitors of PmPM IV–KNI764 with the crystal structure of HIV-1– Plasmodium falciparum plasmepsin II: synthesis and theoretical KNI764 complex [38] revealed some interesting features. First, predictions. Bioorg. Med. Chem. 11, 3723–3733. the protonated aspartate Asp25B in HIV PR is aligned with [2] Ersmark, K., Nervall, M., Gutie´rrez-de-Terań, H., Hamelink, E., Janka, L.K., Clemente, J.C., Dunn, B.M., Gogoll, A., Samuels- AspH214 in PM IV, giving additional support to our proton- son, B., Aqvist, J. and Hallberg, A. (2006) Macrocyclic inhibitors ation model. Second, the conformational change in the P2 of the malarial aspartic proteases plasmepsin I, II, and IV. Bioorg. chain observed in our MD and docking simulations allows Med. Chem. 14, 2197–2208. 5916 H. Gutie´rrez-de-Terań et al. / FEBS Letters 580 (2006) 5910–5916

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