Exploiting structural dynamics to design open-flap inhibitors of

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Exploiting structural dynamics to design openflap inhibitors of malarial aspartic proteases Raitis Bobrovs, Kristaps Jaudzems, and Aigars Jirgensons J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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Journal of Medicinal Chemistry

Perspective

Exploiting structural dynamics to design open-flap inhibitors of malarial aspartic proteases Raitis Bobrovs*, Kristaps Jaudzems, Aigars Jirgensons Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga, LV1006, Latvia

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Abstract Malaria is a life-threatening infectious disease caused by Plasmodium parasites. Plasmepsins – proteolytic enzymes of the parasite – have been considered as promising targets for the development of antimalarial drugs. To date, much knowledge has been obtained regarding the interactions of inhibitors with plasmepsins, as well as the structure-activity relationships of the inhibitors. The discovery and characterization of the plasmepsin inhibitors that bind in open flap conformation have led to several inhibitor classes that show high selectivity over other human aspartic proteases. This perspective addresses the flexibility of the plasmepsins that leads to inhibitor binding to the open flap conformation, summarizes known non-peptidomimetic plasmepsin inhibitors, and discusses the role of the inhibitor flap pocket substituent.


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1

Introduction

Plasmodium falciparum is the most dangerous of plasmodium species causing severe malaria, a lifethreatening disease transmitted through the bites of infected mosquitos, which caused an estimated 219 million cases and 435 000 deaths in 2017.1 Considerable success in diminishing the malaria burden has been achieved by the introduction of vector control programs and increased availability of drugs. However, the emergence of insecticide resistance in the malaria vectors2 and all-drug resistant parasite strains3,4 calls for the development of new efficacious agents to replace the current frontline drugs. Plasmepsins (plms) are aspartic proteases of the malaria parasites which have been considered as potential antimalarial drug targets for more than two decades.5,6 A total of ten plm isoforms (plm I-X) have been identified in P. falciparum. Plms I-IV, V, IX and X are expressed during the asexual blood stage of the parasite lifecycle, and have different functions: plm I-IV are the digestive (food) vacuole plasmepsins responsible for the processing of hemoglobin to amino acids;7 plm V is the endoplasmic reticulum protease which recognizes the conserved PEXEL motif and licenses effector proteins for export;8,9 plms IX and X are maturases essential for malaria parasite egress and invasion of erythrocytes.10–12 Plm VI-VIII are expressed in other stages of the parasite lifecycle which makes them less attractive as drug targets and, consequently, their functions have been less investigated.13–15 The known plm isoforms, their function, localization and importance for P. falciparum parasite survival are summarized in Table 1. Table 1. Function, localization and importance of the known plasmepsin isoforms.

Plasmepsin

Function

Localization

Necessity for the P. falciparum parasite

plm I

Hemoglobin degradation

Digestive vacuole

Not essential for parasite survival16–19

plm II


Digestive vacuole




plm III


Digestive vacuole


plm IV


Digestive vacuole


plm V

Protein export Endoplasmic reticulum

Essential9

plm VI

Not known

Expressed in the exoerythrocytic stages

Not known (essential role in P. berghei within the mosquito stage)14

plm VII

Not known


Not known (not essential role in P. berghei)15

plm VIII

Not known


Not known (essential role in P. berghei within the mosquito stage)20

plm IX

Potentially maturation of rhoptry proteins

Bulbs of rhoptry secretory organelles

Essential10–12

plm X

Maturation of Exoneme secretory the subtilisin- vesicles like serine protease SUB1

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Essential10–12

The digestive vacuole plms (particularly plm II) are the most studied isoforms owing to the availability of recombinant proteins and crystal structures.21–24 However, plms I-IV are not essential, but merely contribute to the fitness of the parasite.17 Plm V, IX and X have gained more attention in recent years and were found to be critical for parasite survival.8–12 Nevertheless, several inhibitors of plm I-IV exhibit potent cell-based antimalarial activities,10,25,26 likely by co-inhibiting the other plms expressed in the blood stage. Therefore, the digestive plasmepsins continue to be used as readily accessible model proteins for antimalarial drug discovery. To date, much knowledge has been obtained regarding the interactions of inhibitors with plasmepsins as well as the structure-activity relationships of the inhibitors. This has been summarized in several review articles aimed at advancing our understanding on various aspects of plasmepsin inhibitor design.24,27–33


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However, most of these studies and reviews focus on the prototypical plm II and neglect the role of protein conformational flexibility. Here we review the current state of plasmepsin inhibitor development from a structural dynamics perspective, with an aim of understanding the consequences of different enzyme configurations adopted upon inhibitor binding, and to highlight the possibilities to use this information for selective inhibitor design.

2

Structure of plasmepsins

Figure 1. Ribbon representation of the three-dimensional structure of: (A) superimposed plm I, plm II and plm IV (PDB ID: 3QS134, 1SME22, 1LS535, respectively); (B) plm III (histoaspartic protease HAP, PDB ID: 3FNT35); (C) plm V (PDB ID: 4ZL48). The Asp-dyad residues, (and histidine in the case of plm III), are displayed as sticks. The N-terminal domain is colored blue, C-terminal domain – salmon pink, the cross-linking 6-β-sheet domain – orange; flap loop (residues 72-85 in plm II) – red; flexible loop (residues 291-297 in plm II) – green. The additional helix bundle and loop in plm V is colored cyan. (D) Structure-based sequence alignment of plm I-V, plm IX and plm X active site residues (plm II numbering). Green – hydrophobic, red – acidic, blue – basic, and orange – other residues. ACS Paragon Plus 5 Environment


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Out of the ten plm isoforms identified in P. falciparum, crystal structures are available for plm I, II, IV, V and the closely related plm III (histoaspartic protease (HAP)). All plms with known crystal structures share high structural similarity (Figure 1, RMSD of backbone atoms between complexes with peptidomimetic inhibitors is ~1.1-2.3 Å). The only substantial difference between them is the presence of an additional helix bundle in the C-terminal domain and a 19-residue loop in the N-terminal domain of plm V (colored in cyan in Figure 1.C). Plm II has been by far the most studied isoform, since it can be easily produced in large quantities in E. coli and refolded after purification into an enzymatically active conformation.21 For this reason, the majority of the structure-based inhibitor design has been focused on the inhibition of plm II. However, due to high structural and active-site-sequence similarity between plm isoforms (see Figure 1.D, more on active site homology in section 2.2.1), it is expected that information obtained on the inhibition of this enzyme isoform could be easily translated to other plasmepsins that are believed to be more essential for the parasite survival (e.g. plm IX and X10–12). Plm V, which is also considered an important drug target,8 share less similarity with other plms (Figure 1.D), therefore, more extensive inhibitor substituent adjustments might be necessary to inhibit this plm isoform. The structure of plasmepsins consists of three distinct regions – two topologically similar N- and Cterminal domains (blue and salmon in Figure 1, respectively) and a 6-β-sheet domain that connects these two lobes (orange in Figure 1). The active site of the enzyme is located at the contact of the N- and Cterminal domains and contains two aspartic acid residues (Asp34 and Asp214 in plm II), a proton donor and acceptor, forming the catalytic dyad during cleavage of the peptide bond. Detailed catalytic mechanism of aspartic proteases has been described elsewhere.36,37 In plm III (HAP), one of the catalytic aspartates is replaced with histidine (Figure 1.B). Similar to other aspartic acid proteases,38 the Nterminal domain of plasmepsins contains a single long β-hairpin structure, usually referred to as a flap or flap loop (Lys72–Phe85 in plm II), that lies perpendicularly over the aspartic dyad (red in Figure 1). On


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the opposite side of the flap loop (in C-terminal domain) is another flexible flap-like loop structure (Gly291-Pro297 in plm II), also referred to as a proline-rich loop in other aspartic proteases22 (green in Figure 1). This is a highly conserved region among food vacuole plasmepsins, and it has been suggested by Liu et al.39 that it interacts with the hydrophobic hemoglobin surface residues and plays an essential role in the first step of hemoglobin cleavage. The majority of the available plm crystal structures are from complexes where the flap loop is closed over the active site. However, several crystal structures with non-peptidomimetic inhibitors are also available. For these, it is characteristic that the flap loop is moved away from the active site (PDB ID: 4Z2240, 2BJU23 and related 2IGX, 2IGY41). This indicates that the flap loop is a highly mobile region playing an important role in the substrate binding, and its dynamic properties could be used in the design of potent and selective inhibitors.

2.1 Comparison of plasmepsin II and human cathepsin D

Figure 2. Superimposed three-dimensional structures of plm II (green) and cat D (blue) complexes with pepstatin A (PDB ID: 1SME and 1LYB, respectively). Protein backbones are shown as ribbons, pepstatin A and side chains of differing amino acid residues are shown as sticks.


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The structural comparison of plm II with several human aspartic proteases has revealed that cathepsin D (cat D) is structurally the most similar of human aspartic proteases.42 Cat D is quite similar to the plms in several regions, including the flap, active site, and the 6-β-sheet domain. However, there are rather important differences in the flexible loop region, which is a characteristic structural feature of eukaryotic aspartic proteases.43 This loop includes three consecutive proline residues in cat D and renin, whereas in plm II one proline is substituted with valine, the loop is much shorter, and also its conformation is such that produces a more open binding site. The flap loop tip residue in the cat D is Gly79, whereas in plm II it is Val78. On the opposite side of the binding site there is Met309 in cat D and Leu292 in plm II. These pairs of amino acid residues are capable of coming in contact, thus effectively wrapping the flap around inhibitors or substrates. The Val-Leu pair in plm II is slightly bulkier and more hydrophobic than the cat D counterpart, thus, hydrophobic inhibitors capable of exploiting these differences could be preferred for the plm II inhibition. The conformation of the substrate analogue (pepstatin A) is similar when bound to cat D and plm II, with slight conformation differences in the S4 and S3’ regions, due to Met307 and Ile311 in cat D being substituted by Ile290 and Phe294 in plm II (see Figure 2). These substitutions and differences in the flexible loop results in a slightly more spacious active site and larger solvent accessible area in plm II than in cat D.

2.2 Dynamics of plasmepsins


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Figure 3. Superimposition of plm II crystal structures with various flap and flexible loop conformations. Red – closed-flap conformation in complex with peptidomimetic inhibitor pepstatin A (1SME). Green, orange and blue – open-flap conformation plm II in complex with non-peptidomimetic open-flap inhibitors; PDB IDs: 2BJU, 4Z22 and 2IGY, respectively. The Asp-dyad residues, Asp34 and Asp214, are displayed as sticks.

Crystal structures of various plasmepsin complexes and apo proteins have shown a high degree of enzyme flexibility, which is essential for the recognition of and binding to different sequences within the hemoglobin molecule.44 Conformational changes in the flap and flexible loop regions are particularly prominent (Figure 3), as they presumably are regulating the access of the substrate to the enzyme. Flap dynamics is a distinctive motion observed among several aspartic proteases, such as HIV protease,45,46 renin,47 β-secretase (BACE),48–51 cathepsins,52 plasmepsins. To date, nearly all of the studies on the flap dynamics of plasmepsins have been computational. Extensive reviews on dynamic features of the flap regions of aspartic proteases from molecular simulation perspective are provided by McGillewie et al.53 and Mahanti et al.54 The only experimental data on flap dynamics in plms come from X-ray crystallography studies, where the flap loop fluctuation is observed as a poorly defined electron density and increased temperature displacement factors B for the flap loop residues.34 Variations in the flap loop mobility have also been observed within the same crystal structure, where the electron density map is well defined for one chain, but is indistinguishable for the other (chain A and C in plm II-PG418 complex (PDB ID: 4Y6M55); chain A and B in plm I complex with peptidomimetic inhibitor (PDB ID: 3QS1)).


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Figure 4. Ribbon representation of the three-dimensional structure of plm II (PDB ID: 1SME). The Aspdyad residues, Asp34 and Asp214, are displayed as grey sticks; the residues that are conserved across the malarial strains, but not in human aspartic proteases are displayed as purple sticks. The flap loop (residues 72-85) is colored red; the flexible loop (residues 291-297) is colored green.

To understand how the observed increased mobility in the flap loop region could be used in selective inhibitor design most effectively, Valiente et al.56 identified active site residues that are conserved across plm isoforms (plm I, II and IV), but not in human aspartic proteases (cat D and cat E), indicating that these residues might play an important role in plasmepsin function57 (Figure 4). Seven amino acids Tyr17, Val105, Thr108, Leu191, Leu242, Gln275, and Thr298 (numbering according to plm II) were identified. Two of these amino acids – Val105 and Thr108 – are located in the flap pocket depth, where they interact only with functional groups of the non-peptidomimetic inhibitors that bind to plm in its open-flap conformation (PDB ID: 2BJU). The rest of the conserved residues - Tyr17, Leu191, Leu242, Gln275, and Thr298 – belong to well-defined pockets lining the binding site cavity. This information ACS Paragon Plus 10 Environment

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was further combined with MD simulations to predict the functionally significant residues in plasmepsins, and to identify interactions that might be essential for effective plm inhibitor design. Their MD studies of apo plm II, plm II-pepstatin A (PDB ID: 1XDH) and plm II-non-peptidomimetic inhibitor (PDB ID: 2BJU) complexes revealed that pepstatin A- bound plm exhibits the lowest fluctuations in the flap loop region, since flap loop residues are interacting with the inhibitor. In semiopen and open conformations, that were observed throughout MD simulations of apo and nonpeptidomimetic inhibitor bound plm II complexes, no additional interactions were present that could stabilize the flap loop, leading to higher flexibility in the flap loop region. In flexible loop region, however, fluctuations for plm II-pepstatin A complex were the highest among all systems studied. Similar MD studies on plm II and plm IV complexes with peptide substrate IEFLRL revealed that plm IV had lower fluctuations in the flap region compared to plm II. These studies indicate that flap loop dynamics is greatly affected by the interactions between the inhibitor and flap loop residues. The flap loop is stabilized in plm complexes with peptidomimetic inhibitors, whereas binding of non-peptidomimetic inhibitors does not result in considerable flap loop stabilization. Therefore, enzyme dynamics should be taken into account when designing plm inhibitors, nonpeptidomimetic inhibitors in particular. Moreover, if open-flap inhibitors are considered, one should target flap pocket residues Val105 and Thr108, as these residues are specific for plasmepsins and not human aspartic proteases, and could advance the design of potent and selective plm inhibitors.


2.2.1

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Hydrogen bonding network in active site of plms

Figure 5. Hydrogen bonding network in the active site of closed-flap (A, PDB ID: 1SME) and open-flap (B, PDB ID: 2BJU) plm II. The flap loop is colored red. Pepstatin A bound to closed-flap plm II is colored green and depicted in cartoon representation. The residue side chains (and Asn39 main chain) involved in the hydrogen bonding network are displayed as sticks. Hydrogen bonds are indicated by yellow dashed lines. Non-polar hydrogens are omitted for clarity.

Around the same time, Friedman and Caflisch58 used MD simulations to study the flexibility of plm II and pro-plm II, as well as the conversion from pro-plm II to plm II. While the main focus here was on ACS Paragon Plus 12 Environment

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the possible conversion mechanism from pro-plm II to plm II, some interesting findings on flap dynamics and interactions between plm II and substrate were revealed. As expected, the MD simulations confirmed the considerable flexibility of the flap loop, with the distance between the nitrogen atom of Val78 (plm II numbering) at the tip of the flap and the catalytic residues varying between 7.3 Å and 14.0 Å (weighted average of 9.5 Å) for the apo protein (PDB ID: 1LF435). A much wider distance range of 7.3 Å to 17.8 Å (weighted average distance of 11.4 Å, PDB ID: 2BJU) was observed for the simulations started from the open-flap structure. Just like in the simulations described by Valiante et al., the closed-flap plm II with a bound peptidomimetic inhibitor (PDB ID: 1LF259) was more rigid as the presence of the inhibitor restrained the motions of the flap. Further, their research was focused on the analysis of hydrogen bonding between Tyr77 in the flap loop and Trp41, which is known to affect the flexibility and presumably the binding mode of inhibitors to aspartic proteases.60 The analysis of crystal structures showed that the hydrogen bond between the hydroxyl group of Tyr77 and indole NH of the Trp41 is part of a linear hydrogen bond chain, which is characteristic for the closed-flap conformation (Figure 5.A). Rotation of the Trp41 side chain disrupts this hydrogen bond structure (Figure 5.B) and makes the flap more mobile, which might be essential for substrate binding. Importantly, this flap loop conformation with a missing hydrogen bond between Trp41 and Tyr77 is observed in crystal structures with open-flap inhibitors (2BJU, 4Z22, 2IGX and its analogues). In studies with human aspartic protease – BACE,61 it was found that the side chain of the flap residue Tyr71 spontaneously flipped its orientation during MD simulations, causing a rupture of the Trp–Tyr hydrogen bond that stabilizes the flap loop. Such a flipping of the Tyr77 side chain was not observed in the simulations of mature plm II, regardless of the initial orientation of Tyr77 and Trp41, however, spontaneous formation of a Trp–Tyr hydrogen bond occurred in some MD simulations of pro-plm. The formation of this Trp–Tyr hydrogen bond was also observed in simulations of cleaved pro-plm, where the initial hydrogen bond between Trp41 and Ser37 was disrupted, and, almost concomitantly, the indole


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ring flipped its orientation to form a stable hydrogen bond with Tyr77. Their studies were not able to indicate whether the reorientation of Trp41 and Tyr77 is a prerequisite for the formation of the active site, since the integrity of the active site was maintained in the absence of the Trp41–Tyr77 hydrogen bond. Nevertheless, the hydrogen bond between Trp41–Tyr77 side chains is present in the majority of the crystal structures of plasmepsins, including the apo enzymes. This implies that in the mature protein the closed-flap conformation is more likely to have the hydrogen bond formed between Trp41–Tyr77 side chains.

Figure 6. Hydrogen bonding network in the active site of closed-flap plm complexes: (A) plm III (PDB ID: 3FNT); (B) plm V (PDB ID: 4ZL4); and (C) plm IX and X (homology model based on PDB ID: 1SME). Protein backbone is depicted in cartoon representation, flap loop is colored red, substrate analogue – green. The residue side chains involved in the hydrogen bonding network are displayed as sticks. Hydrogen bonds are indicated by yellow dashed lines. Non-polar hydrogens are omitted for clarity.

The structure based sequence alignment of all plasmepsins (Figure 1.D) suggests that the hydrogen bonding network observed in plm I, II and IV cannot be formed in plm IX and X, since flap loop Tyr77 (plm II numbering) is substituted with Phe in plm IX and X (see active site of the plm X homology model in Figure 6.C). The lack of hydrogen bond forming group at this position means that flap loop cannot be “locked” in closed flap conformation as effectively as in the case of plm I, II and IV, and, presumably, would occupy open flap conformation more easily. This implies that plm IX and X might have slightly ACS Paragon Plus 14 Environment

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different mechanism of action that does not require the presence of this hydrogen bond and/or slightly different functions then the digestive plasmepsins. There are slight differences in this hydrogen bonding network also in plm III and plm V. Crystal structures of plm V (PDB ID: 4ZL4, 6C4G) reveals that Trp41 is replaced with Ser that does not appear to be involved in this hydrogen bonding network. Nevertheless, flap loop Tyr139 is interacting with Ser83 and Ser85 through a coordinated water molecule (Figure 6.B).8,62 This arrangement leads to hydrogen bonding network that is rather similar to that in plm II. The flap loop Tyr77 of the plm II has been substituted with Ser7563 in plm III. While it might be possible that Ser75 is involved in the hydrogen bonding network in unbound state, in complex with pepstatin A this hydrogen bond is not formed (Figure 6.A). This implies that the open flap conformation for plm III might be accessible more easily than in digestive plasmepsins.


2.2.2

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Characterization of the plasmepsin flap loop dynamics

Figure 7. (A) Schematic representation of the parameters used to define the flap loop motion: the distance between the flap loop tip (Cα of Val78) and flexible loop hinge residue (Cα of Leu292) d1, the dihedral angle φ, and the TriCα angles, θ1 and θ2. Flap loop (residues 72-85) is colored red; the flexible loop (residues 291-297) is colored green. Val78, Leu292 and aspartic dyad Asp34 and Asp214 residue sidechains are displayed as sticks. (B) Flap loop conformation in crystal structure 2BJU (orange) and the flap loop in twisted conformation (red). Twisted loop conformation was generated by performing MD simulation as described in the original article64.

Soliman et al. have also studied flap loop dynamics of P. falciparum plm II,65 plm I-IV in comparison with plm V (homology model),64 plm V,66 as well as plm IX in comparison with plm X (homology ACS Paragon Plus 16 Environment

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models).67 MD simulations of these apo systems once again confirmed that all plasmepsins are highly flexible, and that most of the flexibility is observed within the flap and flexible loop regions. However, besides the previously described flap loop opening and closing movement that accounts for most of the flap fluctuations, an additional flap loop “twisting” motion was identified. To describe this additional flap twisting motion, comprehensive flap loop dynamics characterization parameters were proposed: a) the distance, d1, between the flap tip and the flexible loop region; b) the dihedral angle, φ, to account for the twisting motion; and c) the TriCα angles, θ1 and θ2, that define the angles between flap and flexible loop tips and aspartates (Figure 7.A). This extends the previous description of the flap dynamics and flap loop position which was limited to a distance between the flap loop tip (Cα of Val78 in plm II) and the flexible loop (Cα of Leu292) or the catalytic aspartates. It is known from crystallographic data that distance d1 for the closed-flap conformation (bound with peptidomimetic ligands) typically is ~10-12 Å (PDB ID: 1SME, 4CKU68, 1XE6), whereas in the openflap conformation, when the enzyme is bound with non-peptidomimetic inhibitors, this distance increases up to 13.9 Å in 2BJU, 17.2 Å in 2IGY, and 18.5 Å in 4Z22. The apo proteins occupy a semi-open conformation with distance d1 in range from ~12-14 Å (PDB ID: 1LF4, 3F9Q59, 5BWY69). Their MD simulations of plm I–V, as well as homology models of plm V, IX and X indicated that apo plasmepsins transitions between open and semi-open conformations with an average distance d1 ~10 to 14 Å for plm I–V, and considerably higher for plm IX (21.3 Å) and X (16.1 Å) (Table 2.). The higher average distance d1 for plm IX and X implies that these enzymes might be more prone to binding with open-flap inhibitors compared to plm II, and possibly could accommodate bulkier inhibitors. However, these results need to be taken with caution due to the use of homology models for the study. The flap loops of plm IV, V and IX also seem to move apart the most, opening up to d1max of 19.6, 27.4 and 20.7 Å, respectively. A slightly lower range of movement was observed for plm I and plm II with a d1 of 15.1 and 16.8 Å. These findings are relevant for inhibitor development in the sense that known plm II


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open-flap inhibitors should not have spatial restrictions to bind to the more flexible plm IX and X, which are believed to be vital enzymes for parasite survival. The authors indicated that the homology models of plm IX and X displayed erratic behavior in comparison to plm I–V, however, a similar trend in the fluctuation in key residues was observed, with slightly greater flexibility in the aspartic residues, flap tip and hinge regions. In addition, the calculated potential energies and observed RMSF values for plm IX and X homology models suggested that plm IX is notably more stable than plm X. This finding was supported by a more prominent “twisting” motion in plm IX as opposed to plm X. Table 2. Distance d1 and twisting range Δφ by which the flap loop moves during MD simulations.

plm d1average / Å I 9.7 II 13.2 III (HAP) 11.9 IV 14.0 V homology 13.6 V 13.4 (10.1 bound) IX homology 21.3b X homology 16.1b a No values given in original article b

d1min / Å 5.9 8.9 8.6 7.5 6.5 -a (7.7 bound) 13.5 10.9

d1max / Å 15.1 16.8 17.4 19.6 20.7 17.1 (13.7 bound) 27.4b 20.5b

Δd / Å 9.2 7.9 8.7 12.2 14.3 -a (6.0 bound) 13.8 9.6

Δφ / ° 39.0 40.0 44.5 45.4 44.2 -a 31.5 25.5

We assume, that there is an error in the original article, with interchanged average and maximum values

The analysis of flap-loop twisting motion throughout the MD simulations revealed that more open plm conformations are accompanied by pronounced flap-loop twisting (Figure 7.B). The flap loop twisting results in a conformation where the flap and flexible loop regions move apart opening the active site. Such flap loop opening and twisting away from the active site leads to a conformation where the flap and S1/S3 pockets are connected, and ligands can easily access the active site of the enzyme from the side of the S1/S3 pocket. As expected, only minor flap loop twisting and opening was observed for ligand bound ACS Paragon Plus 18 Environment

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plasmepsins, since flap loop residues were involved in interactions with the ligand. Plm III, plm IV and plm V showed the widest twisting range (φ, see Table 2), whereas, plm IX and plm X showed less definitive twisting events in the flap-structure, and the twisting observed was more gradual and less erratic.

Altogether, the studies reviewed in this section suggest that the differences in the flap loop amino acid sequence and flap loop mobility between plm isoforms and human aspartic proteases could be exploited to design selective plm inhibitors. Recent plm inhibitor development23,40,70 has indicated that one of the most effective ways to develop plm selective inhibitors is to design inhibitors that bind the open-flap conformation. In the following section we will review the current state in the development of nonpeptidomimetic open-flap inhibitors that are designed to bind the open-flap conformation of plasmepsins, where the flap loop is moved away from the catalytic site.

3

Non-peptidomimetic open-flap inhibitors

The first plasmepsin inhibitors were substrate-based peptidomimetics, which showed high inhibition potency, but were non-selective with respect to the majority of other aspartic proteases, including human enzymes (e.g., cat D and E that are used to degrade dysfunctional human hemoglobin). Peptidomimetic inhibitors form complexes with the closed-flap conformation of plasmepsins, where the flap loop tip is covering the aspartic dyad and bound inhibitor. Such a flap-closed complex was first observed in the crystal structure of plm II–pepstatin A complex published in 199622 (PDB ID: 1SME). Since this was the only crystal structure available at that time, early structure-based drug design attempts were mostly limited to peptidomimetic inhibitors. First attempts to design non-peptidomimetic inhibitors were based on an assumption that plm II would also bind in open-flap conformation, as the sequentially and structurally similar human aspartic protease ACS Paragon Plus 19 Environment


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– renin, that had a crystal structure in open-flap conformation available (PDB ID: 1PR8, withdrawn from the PDB database for unknown reasons).71 By combining the information from both, plm II closed-flap and renin open-flap conformation crystal structures, attempts were made by researchers from F. Hoffman-La Roche;72 Actelion Pharmaceuticals Ltd;73–76 and F. Diederich et al.77 to design inhibitors that would bind to open-flap plm II. These studies resulted in the discovery of several inhibitor classes that show high selectivity over human aspartic proteases. The selectivity is attributed to the ability of ligands to bind to open-flap conformation in which an additional hydrophobic flap pocket is formed. Cathepsins either do not have or do not easily adapt the open-flap conformation,78 which determines the poor binding of the open-flap plm inhibitors. Factors determining whether the ligand binds to open- or closed-flap conformation are not yet clear. It has been speculated that the flap pocket is opened and shaped by n-alkyl substituents in an appropriate position.70 Earlier attempts to design hydrophobic pocket substituents at the enzyme active site, including plasmepsins, have been summarized by Zürcher and Diederich.78 However, the studies of the 2aminoquinazolin-4(3H)-one based inhibitors40 suggest that the presence of the hydrophobic flap pocket substituent is not a prerequisite, since this open-flap inhibitor class showed acceptable potency even without a flap pocket substituent (PDB ID: 4Z22). In the following sub-sections, we will summarize the known non-peptidomimetic plasmepsin inhibitors that bind to plms in their open-flap conformation, with the most attention devoted to the ligand structureactivity relationship of the flap pocket substituents.


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3.1 Piperidine based plm inhibitors 3.1.1

4-Arylpiperidine scaffold

Figure 8. General structure of piperidine based plm inhibitor 1 patented by F. Hoffmann-LaRoche; General structure and schematic binding mode of a 4-arylpiperidine based plm inhibitor 2; Example of a potent 4-arylpiperidine based plm inhibitor 3. Catalytic aspartic dyad residues are colored red; interactions between aspartic dyad and ligand core are indicated by grey dashed lines. The flap pocket substituent is indicated by a green rectangle; the S1/S3 pocket substituent – by a grey rectangle.

One of the first patented non-peptidomimetic inhibitor series was based on 3,4-disubstituted piperidine core, originating from known open-flap renin inhibitors.71,72,79–81 Since renin and plasmepsins share high sequence similarity (~35%), it was assumed that some of the known renin inhibitors should inhibit generally less specific plm II. Screening of the F. Hoffman-La Roche in-house renin inhibitor library identified 3,4-disubstituted piperidines as potent plm II inhibitors (Figure 8, general structure 1). The 3,4-disubstituted piperidine inhibitors were tailored for plm inhibition by using computer program GrowMol, which generates libraries of structures complementary to an enzyme active site, and can be used to identify known “non-peptide peptidomimetics” that bind to a structurally distinct aspartic peptidase active site conformation.82 The patent application describing the plm II inhibitors specifies 3ACS Paragon Plus 21 Environment


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substituted 4-phenyl-piperidine 2 as a preferred ligand core with aryl/heteroaryl group bearing electrondonating substituents as R2 substituent, and methoxyaryl groups as substituent R1. The optimization of parent compound lead to inhibitor that shows high cell-based activity against chloroquine resistant (K1) as well as chloroquine sensitive (NF54) strains of P. falciparum,72 however, no data on plm II inhibition were provided. The most potent inhibitors (3) displayed parasite growth inhibition activity in the double digit nM range. According to Boss et al.27, the X-ray crystal structure of a plm II complex with 4-phenyl-piperidine type inhibitor exists, however, it is not publicly available, nevertheless, the structural similarity to 4aminopiperidines (vide infra) with a known plm II complex crystal structure, allows an assumption that the piperidine group is involved in hydrogen bonding with the aspartic dyad and the benzene ring of 4arylpiperidine scaffold interacts with Tyr77, Trp41 and/or Phe111 at the flap pocket “entrance”. Substituent R2 is expected to bind in the flap pocket, whereas substituent R1 in the S1/S3 pocket. 3.1.2

Aryl-tetrahydropyridine scaffold


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Figure 9. (A) General structure of aryl-tetrahydropyridine based plm inhibitor 4 patented by Actelion Pharmaceuticals Ltd; General structure and schematic binding mode of aryl-tetrahydropyridine based plm inhibitor 5. Catalytic aspartic dyad residues are colored red; interactions between the aspartic dyad and ligand core are indicated with grey dashed lines. The flap pocket substituent is indicated by a green rectangle; S1/S3 pocket substituent – by a grey rectangle. (B) SARs for the flap pocket substituent of aryl-tetrahydropyridine based inhibitor. Plm II inhibition activity is indicated as either class A (IC50 < 100 nM) or class B (100 nM < IC50 < 10 µM).

Aryl-tetrahydropyridines (Figure 9.A, general structure 4) were patented by Actelion Pharmaceuticals Ltd.76 Just like 4-arylpiperidines, these aryl-tetrahydropyridine based inhibitors were originally designed as renin inhibitors,83 and were found to inhibit plm II as well. The patent application describes 19 plm inhibitors with various flap and S1/S3 substituents, and their plm II inhibition activities are indicated as either class A (IC50 < 100 nM) or class B (100 nM < IC50 < 10 µM). In both positions, substituted benzene rings were installed. From these, the 2-bromo-5-fluoro-phenoxy group was found to be the preferred flap pocket substituent as this motif was present in 9 out of the 19 examples described in the application. From the activity data given, it could be concluded that smaller hydrophobic substituents are preferred (Figure 9.B).


3.1.3

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4-Aminopiperidine scaffold

Figure 10. (A) General structure of amino-aza-cycloalkane based plm inhibitor 7 patented by Actelion Pharmaceuticals Ltd; General structure and schematic binding mode of 4-aminopiperidine based plm inhibitor 8. Catalytic aspartic dyad residues are colored red; interactions between aspartic dyad and ligand core are indicated by grey dashed lines. The flap pocket substituent is indicated by a green rectangle; S1’ and S1/S3 pocket substituents – by grey rectangles. (B) SARs for the flap pocket substituent of 4-aminopiperidine based plm inhibitors. aDifferent values in Boss et al. 200074 and Boss et al. 2003.27 (C) Crystal structure of 4-aminopiperidine based plm inhibitor bound in the open flap conformation of ACS Paragon Plus 24 Environment

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plm II (PDB ID: 2BJU). Inhibitor and flap residues closer than 4 Å from the inhibitor are shown as sticks; flap loop residues are colored red.

Actelion Pharmaceuticals Ltd have patented another inhibitor class based on piperidine core.73,74,84 This ligand class was identified in a high throughput FRET assay, where 50000 compounds were screened for plm II inhibition activity. The patent application covers an ethylene diamine-based inhibitor scaffold, including 4-aminopiperidines (Figure 10.A, 7). Screening identified compounds which inhibited plm II at low µM concentrations, and structural analysis of these compounds revealed some common features: ethylene diamine core unit; tertiary amine functionality; two rather lipophilic substituents connected to the 2nd end of the ethylene diamine unit; and the presence of 4-pentyl-benzoyl substituent 8.28,41,85–87 Optimization of the hits led to inhibitors with plm II inhibition potency in low nM concentrations (down to IC50 =56 nM) and good selectivity over cat D (Scat D/plm II=82). While SAR data provides ambiguous conclusions about the most optimal R2 and R3 substituents, it was evident that the most optimal substituent at R1 position was 4-pentyl-benzoyl moiety. Crystal structure of the complex of 4aminopiperidine based inhibitor 9 (R1 = 4-pentyl-benzoyl group) with plm II (PDB ID: 2BJU)23 showed a unique binding mode that was not observed before for any plasmepsin inhibitors (see Figure 10.C), however, previously were predicted based on structurally similar renin–inhibitor complexes.77 The inhibitor binds to plm II in open-flap conformation, where the flap loop is moved away from the aspartic dyad, and ligand piperidine nitrogen is engaged in a strong hydrogen bonding with the catalytic water. Identified HTS hits were optimized using structure based drug design, and focused on the substituents binding in the flap and S1/S3 pockets.74 Various combinations with linear aliphatic and 4-benzoyl substituted aliphatic groups as flap pocket substituents, and 4-biphenylyl, 4-benzyloxy-benzyl and 3,4bis-benzyloxybenzyl groups as S1/S3 pocket substituents were tested (Figure 10.B). The highest plm II inhibitory potency was achieved with a 4-pentyl-benzoyl group as a flap substituent (IC50 values in nM


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range vs μM range for other flap pocket substituents). Inhibitory potency for the compounds with an nalkyl flap pocket substituent was consistently poor. The SARs with respect to S1/S3 pocket substituent was rather flat for the series of analogues. The 4-biphenyl unit was selected as the most optimal S1/S3 substituent with potentially better metabolic stability compared to benzyloxybenzyl groups. Further studies73,84 indicated that no significant improvement in plm II inhibitory activity can be achieved by replacing the second aryl moiety in the S1/S3 pocket substituent R2 with a heteroaryl ring like pyrimidine, pyridine or thiophene (IC50 values were in range 30-200 nM). This observation lead to the conclusion that S1/S3 pocket substituents could be used to optimize the physicochemical properties and/or selectivity of 4-aminopiperidine based inhibitors and not to improve the potency. Additional studies,27 however, indicated that electron-rich aryl units like 4-methoxy-phenyl or 3,4-dimethoxyphenyl at this position seem to be favored over the electron-poor heteroaryl systems. The S1’ pocket substituent SAR studies showed that the most potent compounds are obtained when the benzyl group at the piperidine nitrogen is replaced with a very lipophilic 3-methyl-butyl or 3,3-dimethylbutyl group.27 In general, the presence of aliphatic groups at this position seems to be favored over the aromatic group, and presence of the polar groups (e.g. pyridyl, pyrimidinyl, hydroxy-phenyl or imidazolyl) at this position decreased the ligand potency considerably. Notably, the selectivity of the 4aminopiperidine based inhibitors towards plm II vs cat D is high, although the activity SAR for cat D inhibition seems to follow the same principles as for the inhibition of plm II.


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3.2 Bioisosteres of piperidine based plm inhibitors

Figure 11. (A) Examples of bioisosteric piperidine replacement. Introduction of cyclic hydrated α,αdifluoroketone 10 and azole functionality 11. (B) Acyclic α,α-difluoroketones 12 as transition state isosteres. (C) SARs for flap the pocket substituent in azole-based plm inhibitors. Catalytic aspartic dyad residues are colored red; interactions between aspartic dyad and ligand core are indicated by grey dashed lines. Flap pocket substituent is indicated by a green rectangle; S1’ and S1/S3 pocket substituents – by grey rectangles.

3.2.1

α,α-Difluoroketone hydrate based inhibitors

Bioisosteric substitution is a common approach for improving ligand activity, selectivity and/or physicochemical properties. It was proposed by F. Diederich and co-workers88 to swap piperidine moiety of the above discussed inhibitors with cyclic hydrated α,α-difluoroketone functionality (Figure 11.A). This was based on the known acyclic hydrated α,α-difluoroketones that have been introduced as a transition state isosteres into renin inhibitors (Figure 11.B, 12),89–91where the geminal diol mimics the ACS Paragon Plus 27 Environment


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transition state for amide cleavage. α,α-Difluoro-cyclopentanone and α,α-difluoro-cyclohexanone were used as cores to create inhibitors bearing the substituents addressing the flap and S1/S3 pockets of plms. Utilizing the information from previously identified plm inhibitors, a naphthyl group was chosen as an S1/S3 pocket substituent, and a 4-pentylphenyl group as a flap pocket substituent. The amide connector was optimized to direct these substituents into their respective pockets. This resulted in ligands that show IC50 values for plm II inhibition at the single digit micromolar range (down to 7 μM for compound 10), whereas, their potency for plm IV inhibition was substantially weaker (>100 μM). The selectivity vs cat D was high, as expected for open-flap inhibitors with a 4-pentylphenyl group as a flap pocket substituent. No additional fine tuning was performed for the flap pocket substituent. 3.2.2

Azole-based plasmepsin inhibitors

Another bioisosteric substitution in a piperidine based plm inhibitor was proposed by Kinena et al. (Figure 11.A, 11).92 The amide function in parent compound 8 was replaced by 1,2,3-triazole, isoxazole, imidazole and pyrrole leading to inhibitors that showed plm II inhibition potency in μM range. The triazole scaffold based inhibitors were further developed since they showed slightly higher potency and were easier to synthesize. Further, the S1’ pocket substituent – piperidine moiety, and distance between the heterocyclic ligand core and the amino function was optimized. It was determined that a two carbon atom linker between the triazole and the amino function provides proper positioning of the protonated amino nitrogen above the negatively charged Asp214 residue, and at the same time ensures concomitant hydrogen bonding with both Asp214 and Asp34 through water bridge. The S1’ pocket substituent – piperidine moiety – was replaced by different N-alkyl substituents, and it was determined that inhibitors bearing N,N-diethylamino and pyrrolidine groups at this position show the highest potency. The basic flap pocket substituent optimization indicated that the shortening of the alkyl-phenyl chain (from n-pentyl to methyl) results in a significant potency drop, and introduction of an additional CH2 linker between triazole and phenyl groups reduces inhibitor potency 10-fold (Figure 11.C). The ACS Paragon Plus 28 Environment

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introduction of a chlorine atom at the o-position did not affect plm II inhibition. The best triazole-based inhibitors showed submicromolar potency toward plm II. These studies suggest that amide function in the parent 4-aminopiperidine inhibitor is not crucial for the inhibitory activity, rather it serves as a structural backbone element.

3.3 Bi-cyclic amine based inhibitors 3.3.1

Diazabicyclononene scaffold

Figure 12. (A) General structure of diazabicyclononene based plm inhibitors 14 patented by Actelion Pharmaceuticals Ltd; General structure and schematic binding mode of diazabicyclononene based plm inhibitor 15. Catalytic aspartic dyad residues are colored red; interactions between aspartic dyad and ligand core are indicated by grey dashed lines. Flap pocket substituent is indicated by a green rectangle; S1’ and S1/S3 pocket substituents – by grey rectangles. (B) SARs for flap pocket substituent of ACS Paragon Plus 29 Environment


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diazabicyclononene based inhibitor. Plm II inhibition activity is indicated as either class A (IC50 < 10 nM), class B (10 nM < IC50 < 100 nM) or class C (100 nM < IC50 < 10 µM).

Diazabicyclononene based plm inhibitors (Figure 12.A) were initially patented as renin inhibitors93 and only later were found to be also plm inhibitors.75 The patent application describes a total of 90 compounds, and their plm II inhibition activities are given as either class A (IC50 < 10 nM), class B (10 nM < IC50 < 100 nM), or class C (100 nM < IC50 < 10 µM). The representatives of this inhibitor class seem to be substantially more potent against plm II than previously described tetrahydropyridine based compounds, however, the substantial drawback for their potential use as antimalarials is rather complex synthesis. There was no clear SAR for the flap pocket substituents observed (Figure 12.B), however 2-bromo-5fluoro-phenoxy moiety as R1 substituent is likely preferred for diazabicyclononene inhibitors, as it is present in the majority of class A inhibitors (not shown here). Various ester derivatives as R3 substituents seem to be well tolerated, while no clear preference for any specific substituent was observed. As for the S1/S3 pocket substituent, various di-substituted amines were tested, and the highest activity was observed for the compounds bearing small (methyl, cyclopropyl) alkyl group as one amine substituent, and substituted benzene ring with methyl or ethyl linker as second one. These inhibitors were also tested for biological activity against the human cat D, cat E and renin, however, no data were provided in the patent.


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3.3.2


Azabicycloheptane scaffold

Figure 13. (A) General structure and schematic binding mode of 7-azabicyclo[2.2.1]heptane 17, 11azatricyclo[6.2.1.02,7]undeca-2,4,6-triene 18, 2nd generation 7-azabicyclo[2.2.1]heptane based “diamine clamp” 19, and 3rd generation “diamine clamp” 20 plm inhibitors. Catalytic aspartic dyad residues are colored red; interactions between aspartic dyad and ligand core are indicated by grey dashed lines. Flap pocket substituent is indicated by a green rectangle; S1’ and S1/S3 pocket substituents – by grey rectangles. SARs for the flap pocket substituent of 11-azatricyclo[6.2.1.02,7]undeca-2,4,6-triene (B) and 2nd generation 7-azabicyclo[2.2.1]heptane based “diamine clamp” (C) based plm inhibitors. ACS Paragon Plus 31 Environment


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Another class of non-peptidomimetic inhibitors, based on the 7-azabicyclo[2.2.1]heptane scaffold (Figure 13.A, 17) was introduced by F. Diederich et al.77,94 This class of inhibitors was designed de novo using the hybrid plm model which was based on the X-ray crystal structure of plm II with pepstatin A (PDB ID: 1SME)22, in which conformational changes observed in renin complexed to 4-arylpiperidine inhibitors71 had been introduced. This unveiled the unexpected flexibility of the enzyme, and allowed to model a more open conformation of the active site with an unlocked hydrophobic flap pocket. The inhibitor based on a 7-azabicyclo[2.2.1]heptane scaffold, in its protonated form, formed an ionic hydrogen bond with the aspartic dyad, whereas the R1 (1,3-benzothiazole) substituents occupied the hydrophobic flap pocket and R2 (naphthyl residue), a spacious apolar S1/S3 pocket (or S2’ pocket, depending on the optical antipode of the inhibitor), respectively. The most active plm II inhibitors of this class showed IC50 values in a low μM range. The 7-azabicyclo[2.2.1]heptane based inhibitors were further developed by modifying the central scaffold into a more rigid 11-azatricyclo[6.2.1.02,7]undeca-2(7),3,5-triene 18.95 These modifications, however, did not result in better plm II inhibition potency (IC50 = 2 μM) or selectivity over cat D (Scat D/plm IV=3). Since the new scaffold was more extended, a shorter and less flexible linker was installed between the central scaffold and 1,3-benzothiazole moiety. Flap pocket substituent variations (Figure 13.B) indicated that presence of a larger substituent is required for inhibitor activity, and installation of a chlorine atom at the 5th position of 1,3-benzothiazole slightly increases the inhibitor potency. The presence of the ether group in the flap pocket linker to some extent affected only the inhibition of plm IV, whereas replacement of 1,3-benzothiazole with phenyl-1,3,4-oxadiazole group reduced inhibitory potency more than 20 times. Later development of this inhibitor class resulted in a slightly different inhibitor core, where an additional amino group was installed in the 2nd position of the bi-cycle scaffold, providing an additional attachment point for the inhibitor core, resulting in a hydrogen-bonded “diamine clamp” around the aspartic dyad ACS Paragon Plus 32 Environment

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(Figure 13.A, 19).96 The sulfone moiety was installed in the 3rd position of the scaffold to decrease the basicity of the amino groups, as well as to direct the flap pocket substituent into the pocket in optimal angle. Changes in the ligand core and introduction of 4-hexylbenzyl group as a flap pocket substituent noticeably increased ligand potency (IC50 up to 45 nM for plm II, and 10 nM for plm IV) and selectivity over cat D (Scat D/plm IV=90). The flap pocket substituent was further optimised,70,78,97 while keeping the azanorbornane core and the residue targeting the S1/S3 sub-pocket constant (Figure 13.C). Systematic variation of the length of the alkyl chain from n-butyl to n-undecyl was performed as well as the terminal cycloalkyl residues were introduced. These studies showed a clear correlation in a homologous series of n-alkyl chains for both plm II and IV. It was determined that the ideal chain length is different for plm II and IV. The appropriate flap pocket n-alkyl substituent length for this class of inhibitors corresponded to n-hexyl to n-octyl for plm II and n-hexyl for plm IV. Not only does plm IV prefer shorter chains, the decrease in the activity was also much stronger for plm IV than for plm II when exceeding the optimal chain length. These results suggested that plm IV, has a shorter flap pocket with a smaller binding volume if compared to plm II although no crystal structure in open-flap conformation is known for plm IV to confirm this. According to their molecular modelling studies, the n-heptyl substituent is the longest one that fits in its all-anti conformation into the flap pocket of plm II. Ligands with longer n-octyl and n-nonyl chains give similar IC50 values, however, they have to reduce their length by folding to fit into the flap pocket. Series with cyclo-alkyl substituents showed a clear trend for both plm II and plm IV – the bigger the substituent at the vector end, the lower the inhibitor potency. This implies that there is only limited space at the “end” of the flap pocket, and that the cavity cannot easily reorganize to accommodate bulkier substituents. It is worth noting that these observations on the flap pocket substituent “efficiency” are in agreement with findings of Mecozzi and Rebek,98 stating that inclusion complexes in confined cavities feature the highest stability when the guest occupies ~55 % of the available space within a host.


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Further research99 focused on the design and introduction and of the S1’ pocket substituent (compound 20). The installation of a small S1’ pocket substituent, such as cycloalkylaminomethyl, in 6th position of the 7-azanorbornane scaffold yielded highly potent and selective plm inhibitors. Most of the inhibitors from this series displayed inhibitory activity in the nanomolar range against plm I, II, and IV. Installation of the S1’ substituent gave the highest binding affinity gain for plm IV (IC50 values in low nanomolar range).The flap pocket substituent was also slightly modified, however, no explanation was given why 4-pentylbenzyl moiety was swapped for a 4-butoxybenzyl group.

3.4 2-Aminoquinazolin-4(3H)-one based inhibitors


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Figure 14. (A) Structure and schematic binding mode of 2-aminoquinazolin-4(3H)-one fragment 23 that was identified as a plm binder in NMR screening. 1st (24) and 2nd (25) generation of 2-aminoquinazolin4(3H)-one based inhibitors. Catalytic aspartic dyad residues are colored red; interactions between aspartic dyad and ligand core are indicated by grey dashed lines. Flap pocket substituent is indicated by a green rectangle; S1’ pocket substituent – by grey rectangles. SARs for the flap pocket substituent of 1st generation (B) and 2nd generation (C) 2-aminoquinazolin-4(3H)-one based plm inhibitors. (D) Crystal structure of 2-aminoquinazolin-4(3H)-one based plm inhibitor bound to the open flap conformation of plm II (PDB ID: 4Z22). Inhibitor and flap pocket surface residues are shown as sticks; flap loop residues are colored red.

2-Aminoquinazolin-4(3H)-ones as a potent and selective plm inhibitors were identified by Rasina et al.40,100 during their NMR-based fragment screening against plm II. The screening resulted in 10 compounds that showed measurable activity in an enzymatic assay. 2-Aminoquinazolin-4(3H)-one scaffold was selected as the most promising (Figure 14.A, 23), however, the binding mode of the identified fragment was unknown as all attempts to co-crystallize it with plm II failed. A hint for the initial optimization of the 2-aminoquinazolin-4(3H)-one was provided by NMR competition experiments. It was found that 2-aminoquinazolin-4(3H)-one and another fragment bind in close proximity (an interligand NOE in 1H−1H NOESY spectrum), which suggested introducing the phenyl group at the 7-position of the 2-aminoquinazolin-4(3H)-one. This provided an inhibitor with 10-fold increased potency against plm II (2.3 μM). The binding mode of this inhibitor was modelled using open- and closed-flap plm structures (PDB ID: 2BJU and 1LEE59, respectively). A plausible binding mode was produced in openflap conformation, with hydrogen-bonds between the N1 and 2-amino groups of the 2-aminoquinazolin4(3H)-one and the catalytic Asp34−Asp214 dyad. According to the modelled binding mode, lipophilic substituent was introduced at the 5th position of the THF group, providing inhibitor 24 which could be co-crystallized with plm II. X-Ray crystal structure of the complex was solved (PDB ID: 4Z22), ACS Paragon Plus 36 Environment

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confirming the binding mode (see Figure 14.D). The crystal structure revealed a widely open, unoccupied flap pocket, that was targeted by a linear aliphatic substituent of limited length installed at the 7-phenyl group, as known from the previous studies.23,70,97 The introduction of n-butyl and n-pentyl groups at the para- position of the 7-phenyl group improved the plm II inhibitory activity 2-fold and 3-fold, respectively (Figure 14.B). Although these modifications were beneficial, the increase in potency was less pronounced compared to other known non-peptidomimetic inhibitors. The ester group at the terminal position of the alkyl chain was also tolerated, whereas the other types of substituents led to less potent plm II inhibitors. While the introduction of the hydrophobic flap pocket substituents yielded inhibitors with high potency, these inhibitors had problems with increased lipophilicity (clogP > 5, LipE < 1). The activity against digestive plasmepsin subtypes (plm I, II, IV) was similar for nearly all inhibitors, except for the inhibitor bearing 3-phenylpropyl group as a flap pocket substituent that showed remarkable plm IV specificity (see Figure 14.B). The molecular modelling studies suggested that the flap pocket of plm IV is slightly more spacious in depth than that of plm II, due to a Thr108 to Ala108 mutation, and this might be the reason why the inhibitor bearing bulkier 3-phenylpropyl group as a flap pocket substituent, binds more effectively to plm IV compared to inhibitors bearing smaller linear alkyl groups. 2-Aminoquinazolin-4(3H)-one based plm inhibitors showed acceptable selectivity over cat D (>10 times). In cell-based assays, selected compounds, including plm IV selective inhibitor, showed growth inhibition of P. falciparum 3D7 with EC50 ∼ 1 μM. The correlation with the enzymatic assay allowed speculation that nondigestive plasmepsins structurally similar to plm IV (e.g., plm IX and X) could be the main targets to achieve parasite growth inhibition. Further 2-aminoquinazolin-4(3H)-one based inhibitor optimization was focused on reducing inhibitor lipophilicity and increasing selectivity over cat D.25 The strategy for reducing the inhibitor lipophilicity was to install hydrophilic substituents in positions that are facing polar protein surfaces or are solventexposed. Installation of polar substituents at the R2 position (Figure 14.A, 25) did not considerably


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change the plm inhibition potency as expected by their situation in an area exposed to the solvent, however, selectivity over cat D increased up to 100-fold. Next, the necessity for the phenyl substituent at the THF group was evaluated, as removal of this group would reduce the lipophilicity of the inhibitor. Molecular docking and dynamics studies suggested that the presence of the phenyl substituent in this position affects inhibitor mobility in the binding pocket. However, the importance of the Ph-THF group in compounds also containing a flap-pocket substituent may be diminished due to a larger entropic penalty associated with constraining the ligand flexibility and/or due to inability of the ligand to maintain an optimal binding pose when both terminal groups are locked in hydrophobic pockets (flap pocket and S1’ sub-pocket). These considerations implied that introduction of flap pocket substituent at 7-postion of 2-aminoquinazolin-4(3H)-one would allow to remove phenyl group from THF ring without notably impairing plasmepsin inhibitory potency. Several flap pocket substituents were tested for compounds bearing solvent exposed carboxyethyl and carboxyphenyl R2 groups (see Figure 14.C for carboxyethyl substituted ligands). The overall plm inhibition potency for 2nd generation inhibitors was ~10 times higher than for the 1st generation, however no changes in relative potency for inhibitors with various flap pocket substituents were observed. Taken these findings together, inhibitors were developed which showed plm II and IV inhibitory potency in low nanomolar range and remarkable selectivity against cat D (>500). These modifications were also beneficial to decrease the lipophilicity and increase the aqueous solubility of 2-aminoquinazolin-4(3H)one based inhibitors.


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3.5 Amino alcohol based inhibitors

Figure 15. General structure 28 and schematic binding mode of amino alcohol based plm inhibitors. Examples of potent amino alcohol based plm inhibitors (29, 30). Catalytic aspartic dyad residues are colored red; interactions between aspartic dyad and ligand core are indicated by grey dashed lines. Flap pocket substituent is indicated by a green rectangle; S1/S3 and S1’/S2 pocket substituents – by grey rectangles.

Combined fragment-based virtual screening, enzymatic assay and common substructure search performed by Friedman and Caflisch101 identified 13 plm II inhibitors (IC5010 nM) were identified by HTVS using models developed from plm II and cat D crystal structures in complex with pepstatin-A (PDB ID: 1M43 and 1LYB105). The results of enzyme inhibition assays were in-line with docking predictions, as both aspartic proteases were inhibited by acridinyl compounds in the nanomolar range. Molecular modelling suggested that the hydrazine core of the inhibitors acts as a transition state mimetic and interacts with the aspartic dyad through charged hydrogen bonds. The acridinyl moiety of the inhibitors was predicted to interact with S2’/S3’ pocket residues in cat D and the S1/S3 pocket in plm II. Thus, the acridinyl moiety was modelled to bind at opposite sides of the scissile bond in the substrate binding cleft of both proteases. Further studies by Azim et al.106 indicated additional two symmetric hydrazine based and four hydrazide based plm II inhibitors (Figure 16.A, 32 and 33). The enzyme inhibition assays demonstrated that these compounds are equally potent against both proteases, at low micromolar concentrations (IC50 from 1.0 to 2.5 μM). The binding mode of the two additionally identified hydrazines was expected to be similar to that of 9-hydrazinylacridine derivatives 31, whereas for hydrazides 33 no specific interactions were


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observed in addition to ligand core interactions with the aspartic dyad. Therefore, it is very likely that these inhibitors might have selectivity issues. There were no indications that these inhibitors would bind to open-flap conformation.⁠

4.2 Benzimidazole based inhibitors

Figure 17. Benzimidazole scaffold based plm inhibitors. Catalytic aspartic dyad residues are colored red; interactions between aspartic dyad and ligand core are indicated by grey dashed lines. The flap pocket substituent is indicated by a green rectangle; S1/S3 pocket substituent – by a grey rectangle.

Another in-house library screening campaign by Azim et al.107 identified benzimidazole-based compounds (Figure 17) as plm II and cat D inhibitors. Enzyme inhibition potency was determined to be in the low micromolar range (2–48 μM), however, there was no correlation between the inhibitor activity in the enzymatic assay and that in the cell-based anti-malarial assay. Two binding modes were predicted for these inhibitors, with one of them being considerably more favored (for 9 out of 11 identified compounds). In the preferred hypothetical binding mode 34, imidazole protonated nitrogen interacts with catalytic Asp214 and the carbonyl group of the acetophenone points toward the catalytic Asp34. This binding mode enables pyridine moiety interaction with S1’ pocket residues and points the acetophenone group toward the flap pocket. Despite the fact that this class of inhibitors was not expected to bind to plm in the open-flap conformation, the flap pocket substituent - acetophenone was fine-tuned (various small hydrophilic substituents and phenyl group were installed). The SAR analysis indicated that the presence of the substituent at the para position of acetophenone ring generally increases the inhibitor potency, and ACS Paragon Plus 42 Environment

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the introduction of the phenyl group lead to the most potent antiplasmodial inhibitor. This indicates that installation of an appropriate substituent at the para position of acetophenone ring might open the flap pocket and increase potency and selectivity of the inhibitor.

4.3 Diphenylurea based inhibitors

Figure 18. Diphenylurea scaffold based plm inhibitors. Catalytic aspartic dyad residues are colored red; interactions between aspartic dyad and ligand core are indicated by grey dashed lines. S2’ pocket substituent is indicated by a grey rectangle.

Diphenylurea derivatives were identified as potent plm II inhibitors by Jiang et al.108 through screening an in-house compound library. The screening identified 9 potent and selective (over cat D) compounds (Ki from 0.05 to 0.68 μM.) bearing a diphenylurea core (Figure 18), however, they showed poor inhibition of P. falciparum growth in vitro. All diphenylurea derivatives showing plm II inhibition potency contained phenoxyl and a sulfonic acid group. This suggests that diphenylurea with a phenoxyl side chain and sulfonic acid group near the urea carbonyl group might be the functional substructures responsible for plasmepsin inhibition. To understand the binding mode of this class of inhibitors, the diphenylurea derivatives were docked in plm II structures from P. falciparum (PDB ID: 1SME) and P. vivax (PDB ID: 1QS8105). It was concluded that diphenylurea mimics the core region of pepstatin A, while the phenoxy side chain occupies the S2’ pocket. Docking studies suggested that diphenylurea derivatives do not bind to plm in an open-flap conformation, however, one should keep in mind that docking experiments were performed for closed-flap plm II. Although all 9 discovered compounds inhibited plm II equally, only the basic 37 was a potent inhibitor of parasite growth.


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Urea and thiourea derivatives were also identified as potent plm inhibitors by Degliesposti et al.109, by performing an extensive analysis of the large scale screening campaign WISDOM110 results. The enzymatic inhibitory potency of urea and thiourea derivatives were in the nanomolar range. In addition to urea and thiourea derivatives, Degliesposti et al. also identified N-alkoxyamidines, guanidines and amides as potential plm inhibitors. Since only closed-flap plm structures were used in the WISDOM screening campaign, none of these compounds are expected to bind in open-flap conformation.

4.4 Azacyclic plm inhibitors

Figure 19. Azacyclic scaffold based plm inhibitors. Catalytic aspartic dyad residues are colored red; interactions between aspartic dyad and ligand core are indicated by grey dashed lines. Substituents occupying S2’ and S1/S3 pockets are indicated by grey rectangles. Potential flap pocket substituents are indicated by a green dashed line.

The idea of azacycles, containing basic amino functionality, as a core element for the inhibitor design was further investigated by Diedrich W.E. and co-workers. They proposed an inhibitor class that features a 2,3,4,7-tetrahydro-1H-azepine scaffold (Figure 19, 38).111 The amino group of the azepine moiety interacts through charge-assisted hydrogen bonds with the aspartic dyad, whereas, the substituent in the 3rd and 5th positions are oriented toward the S1 and S2’ pockets, respectively. The ester functionality in the 5th position substituent forms a hydrogen bond to the flap residue Ser79 (plm II), whereas its aromatic function occupies the hydrophobic S1 pocket. The carbonyl function in the substituent at the 3rd position forms a hydrogen bond to the flap backbone NH group of Val78 (plm II), and the corresponding R1 group


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occupies the S2’ pocket. The most active inhibitors of this class showed plm II and plm IV inhibition potency in low micromolar range, and good selectivity over cat D (Scat D/plm II) >50. There were no indications that this class of inhibitors would bind to plm in open-flap conformation. Further exploration of the possible use of azacycles in plm inhibitor design resulted in plm inhibitors featuring a pyrrolidine scaffold 39 as a core element.112 Such compounds, originally designed for HIV-1 protease inhibition,113 showed activity in the low micromolar range. The proposed binding mode of the pyrrolidine-based inhibitors is identical to that of azepine-based plm inhibitors. For the 3S,4Spyrrolidinediol-based inhibitors, the substituents in 3rd and 4th position are directed into S1 and S2’ pockets as in the parent compound. Additional aromatic groups were introduced in the 3S,4Sdiaminopyrrolidine (Figure 16.D, 40) with the intention of filling the S1’ and, potentially, the flap pocket. The most active pyrrolidine-based inhibitors showed poor selectivity over cat D.

4.5 Aminohydantoin based inhibitors

Figure 20. Aminohydantoin scaffold based plm inhibitors. Catalytic aspartic dyad residues are colored red; interactions between aspartic dyad and ligand core are indicated by grey dashed lines. Substituents occupying S1’, S2’ and S1/S3 pockets are indicated by grey rectangles.


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Just like some of the previously described inhibitors, aminohydantoins (Figure 20, 41) were originally designed to inhibit another aspartic protease – β-secretase (BACE).114,115 The loose sequence homology between BACE and plms, and the fact that there are several X-ray crystal structures that demonstrate the key interactions between the protonated aminohydantoin core and the aspartic acid residues of the BACE active site encouraged Meyers et al.116 to develop the aminohydantoins as plm inhibitors. They performed an aminohydantoin core substructure search on the Tres Cantos antimalarial compound set (TCAMS)117 that had their antimalarial activity in P. falciparum 3D7 infected red blood cells determined within a GlaxoSmithKline screening campaign. From the TCAMS collection, 35 aminohydantoin based compounds (general structure 42) were identified, with antimalarial activities ranging from 140 to 1500 nM. The most potent compound from the TCAMS collection 43 showed a more modest P. falciparum 3D7 EC50 of 2.76 μM in their tests. Interestingly, the plm II inhibition potency was high (IC50 = 12 nM) for this compound, whereas inhibition of plm V was rather low (IC50 = 1 μM). The available β-secretase-aminohydantoin crystal structures118,119 indicated that these inhibitors bind to the semi-open conformation with two phenyl groups occupying S2’ and S1/S3 pockets. The R1 substituent of the identified aminohydantoins was expected to bind in S1’ pocket, and was optimized first, while keeping R2 and R3 substituents constant (p-methoxyphenyl groups). The installation of small alkyl groups, such as methyl, ethyl, and propyl, resulted in size-dependent loss in potency, whereas larger hydrophobic groups, such as isopropyl, benzyl, phenethyl, were rather well tolerated. Moreover, this series indicated that incorporation of a bulky group at this position enhances antimalarial potency while reducing inhibition of human aspartic proteases, such as cat D, cat E and BACE. The most potent cyclohexyl analogue 44 was selected for further optimization studies. It is worth noting that the inhibitor bearing pyran group at R1 position was equally potent, while reducing the lipophilicity of the compound by 2 orders of magnitude. The SARs for the R2 and R3 substituents were rather flat, and slight


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improvement in plm II and plm IV inhibition activity was achieved only when 3-pyridyl ring was installed as a S1/S3 pocket substituent. These studies indicated that there is a clear correlation between the inhibition of plms and P. falciparum 3D7 malaria strains, however, correlation toward some specific plm subtype was determined only later,10 when plm X was identified as the main target (or one of them) of the aminohydantoins.

5

Conclusions

Figure 21. Schematic representation of preferred plasmepsin flap pocket substituent (plm II numbering). Catalytic aspartic dyad residues are colored red; electrostatic interactions between aspartic dyad and ligand core are indicated by grey dashed lines; hydrophobic interaction by a green dashed line. The flap pocket is indicated as a green rectangle; the flap pocket substituent – a dark grey rectangle.

Currently there are no cheap, safe and effective treatments for malaria. Plasmepsins have been identified, characterized, and validated as potential antimalarial drug targets for some time, however, no drugs targeting these aspartic proteases have reached the market yet. Early plasmepsin inhibitor design was mostly focused on peptidomimetics, which generally led to compounds with low selectivity toward plasmepsins compared to other aspartic proteases. These compounds typically were also of high molecular weight and lipophilicity. Due to these drawbacks, non-peptidomimetic inhibitors are currently considered more promising. The first non-peptidomimetic plm inhibitors were discovered nearly two ACS Paragon Plus 47 Environment


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decades ago, when known renin inhibitors were tested for plm inhibition activity. These studies resulted in several potent piperidine and bi-cyclic amine-based inhibitor classes that showed high selectivity over human aspartic proteases due to their unusual binding pose. The selectivity over human cat D/E was attributed to differences in enzyme flexibility which allows plasmepsins to bind the inhibitors in openflap conformation where an additional hydrophobic pocket is formed, whereas cathepsins either do not have or do not easily reach an open-flap conformation. Evidence of the open-flap plm conformation encouraged plm dynamics and flexibility studies. It was determined that the flap loop is the most mobile region of the enzyme, and its flexibility is affected by hydrogen bonding between Tyr77 in the flap loop and Trp41 in the flap pocket. This interaction is part of the hydrogen bonding network which is necessary to keep the integrity of the binding site, and is usually observed in closed-flap plm structures. Rotation of the Trp41 disrupts this hydrogen bond network and makes the flap more mobile. Such disrupted hydrogen bond conformation is observed in crystal structures with open-flap inhibitors, and it is believed that such a state might be essential for substrate binding. Simultaneous flap loop opening and twisting could be essential for the flap substituent binding in the flap pocket, as it creates a situation where the flap substituent can enter the flap pocket from the S2’ pocket side. The precise mechanism of enzyme reorganization for inhibitor binding in open-flap conformation is not yet known. However, it has been speculated that the flap pocket opening is governed by the hydrophobic, preferably n-alkyl, substituent in appropriate position. Studies of the 2-aminoquinazolin-4(3H)-one based inhibitors, however, suggest that the presence of the hydrophobic flap pocket substituent might not be a prerequisite, since this inhibitor class binds to open-flap conformation even without a flap pocket substituent. Examination of the crystal structure of 2-aminoquinazolin-4(3H)-one complex with plm II hints that inhibitor binding in open-flap conformation might be facilitated by the phenyl ring of the inhibitor which is situated next to the aromatic residues Trp41, Tyr77 and Phe111 located at the flap ACS Paragon Plus 48 Environment

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pocket entrance. Installation of additional substituents at the para position of the phenyl ring can further improve the potency and selectivity. The analysis of known open-flap plm inhibitors reveals a pattern that supports this assumption – nearly all (except amino alcohols) open-flap plm inhibitors have a phenyl ring situated ~8-9 Å away from the catalytic aspartic acid residues (Figure 21). Filling the rest of the pocket with a hydrophobic substituent improves the potency and selectivity of the inhibitors by introducing hydrophobic interactions with flap pocket depth residues (Pro43, Val105 and Thr108) and reducing the penalty associated with hydration of the predominantly hydrophobic pocket. A few inhibitors lacking the phenyl ring at the entrance of the flap pocket also bind to the open-flap conformation, however, their activity is typically several magnitudes lower and greatly varies from one inhibitor to other. Latest studies indicate that the preferred flap pocket substituent depends on the plm isoform. Studies by F. Diederich et al. showed70 that the optimal flap pocket substituent is an n-alkyl chain, and that the preferred substituent for plm II was an n-heptyl group, whereas for plm IV – an n-hexyl group. Their results also indicated that the decrease of the inhibitory potency when exceeding the optimal chain length was much stronger for plm IV than for plm II. Studies by Rasina et al. also indicated that inhibitor selectivity towards the desired plm isoform could be tuned using the flap pocket substituent. The preferred flap pocket substituent for both plm II and plm IV was an n-pentyl group, however, installation of a 3-phenylpropyl group as a flap pocket substituent offered an opportunity to achieve noticeable plm IV specificity without decreasing overall plm IV inhibition potency. Substantial progress has been achieved in understanding the dynamics of the plasmepsins, and possible ways to exploit the plm flap loop dynamics to design selective plm inhibitors. Filling the hydrophobic flap pocket provides significant binding energy, and is an effective way to achieve highly selective plm inhibitors, therefore, we believe that this design principle is a premise for potential plm targeting antimalarials.


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Author information Corresponding Author Raitis Bobrovs. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all the authors. All the authors have given approval to the final version of the manuscript.

Acknowledgement This work was supported by the European Regional Development Fund (Agreement No. 1.1.1.1/16/A/290).

Author biographies Raitis Bobrovs studied chemistry at the University of Latvia and obtained his PhD degree in 2015. His BSc, MSc and PhD research project were supervised by Prof. Andris Actiņš, and focused on the crystallization and physicochemical characterization of polymorphic pharmaceutical systems. Recently he have joined Department of Physical Organic Chemistry at Latvian Institute of Organic Synthesis (LIOS) where he focuses on the computational modelling of protein ligand interactions, with an aim to advance the development of plasmepsin inhibitors. During his research career he have performed research at Liverpool John Moores University with Linda Seton, KTH Royal Institute of Technology with Rossen Apostolov, and University College London with Matteo Salvalaglio. Kristaps Jaudzems studied chemical technology at the Riga Technical University (RTU) and obtained his Bachelor’s degree in 2006, followed by a Master’s degree in 2008. During his PhD studies in chemistry, also at RTU, he spent two years at the Scripps Research Institute as an external graduate


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student with Prof. Kurt Wüthrich. He received his PhD in 2011 under supervision of Prof. Edvards Liepinsh. Subsequently, he was senior researcher at the Latvian Institute of Organic Synthesis (LIOS). From 2015 to 2017 he was a Marie Skłodowska-Curie fellow at the Institut des Sciences Analytiques in Lyon. Since 2017, he has been group leader at LIOS and associate professor at the University of Latvia. His work focuses on NMR-based structural biology and biophysical techniques for drug discovery. Aigars Jirgensons obtained his PhD. degree in 2000 from University of Latvia working under supervision of Dr. Valerjans Kauss at the Latvian Institute of Organic Synthesis (LIOS), Riga. In 2002, he performed post-doctoral studies in the group of Prof. Pellicciari, University of Perugia, Italy. Currently he is scientific director and head of the group at LIOS and professor at Riga Technical University. His research is focused on the discovery of antimalarial and antibacterial agents as well as the development of novel synthetic methodology.

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Exploiting structural dynamics to design open-flap inhibitors of

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