Development of Novel Peptidyl Nitriles Targeting Rhodesain and Falcipain-2 for the Treatment of Sleeping Sickness and Malaria

In recent decades, neglected tropical diseases and poverty-related diseases have become a serious health problem worldwide. Among these pathologies, human African trypanosomiasis, and malaria present therapeutic problems due to the onset of resistance, toxicity problems and the limited spectrum of action. In this drug discovery process, rhodesain and falcipain-2, of Trypanosoma brucei rhodesiense and Plasmodium falciparum, are currently considered the most promising targets for the development of novel antitrypanosomal and antiplasmodial agents, respectively. Therefore, in our study we identified a novel lead-like compound, i.e., inhibitor 2b, which we proved to be active against both targets, with a Ki = 5.06 µM towards rhodesain and an IC50 = 40.43 µM against falcipain-2.


Introduction
Neglected tropical diseases (NTDs) and poverty-related diseases (PRDs) include a group of infections predominately occurring in tropical areas affecting the most disadvantaged communities, in particular, women and children [1].One of the most important NTDs is human African trypanosomiasis (HAT) [2], while malaria is the most widespread PRD in the world [3].
HAT (also known as sleeping sickness) is a parasitic disease widespread in sub-Saharan Africa, caused by trypanosome parasites, and transmitted by tsetse fly of Glossina genus.Two subspecies of Trypanosoma brucei cause this disease: T. b. gambiense in West Africa and T. b. rhodesiense in East Africa [2].
HAT is characterized by two main stages: in the first stage, also known as the hemolymphatic stage, a bloodstream invasion by the parasite induces fever and muscle aches; if untreated, the first stage can evolve into the neurological one, in which the penetration of the central nervous system by trypanosomes induces neurological disturbances, sleep disorders, and finally death.Malaria is transmitted through the bites of infected female mosquitoes of the Anopheles genus.Five Plasmodium parasite species cause malaria in humans.In particular, P. falciparum is the most dangerous and lethal species; it predominates in sub-Saharan Africa and is common in other tropical regions.P. vivax is the dominant malaria parasite in many countries outside of sub-Saharan Africa.A large number of other malaria species can infect humans; some of them are P. malariae, P. ovale, and P. knowlesi [3].
The most common symptoms of malaria are fever, chills, and headache.According to the latest World Malaria Report from the World Health Organization (WHO), there were an estimated 249 million cases of malaria in 2022, leading to 608,000 deaths in 2023 [3].Available antitrypanosomal and antimalarial agents have multiple limitations.The initial HAT treatment included only four drugs, among which three, i.e., suramin, pentamidine, and melarsoprol, were developed more than 60 years ago.Suramin and pentamidine are active on the hemolymphatic stage of the disease, while melarsoprol and eflornithine act in the neurological stage [4].
Melarsoprol, the most toxic of these compounds, is a trivalent arsenical and, unfortunately, it causes reactive encephalopathy in 5-10% of treated patients, with 1-5% mortality [4], Nifurtimox, a 5-nitrofuran with antifungal properties, formally approved for the American trypanosomiasis, is used off-label, in combination with eflornithine, for the treatment of the gambiense trypanosomiasis (g-HAT) [5].
Almost all drugs are administered parenterally, so there is always the problem of compliance.Fexinidazole was recently introduced into therapy and orally administered; however, it was approved for g-HAT only, so for the most severe form of HAT, namely the rhodesiense form (r-HAT), apart from the toxic melarsoprol, currently there are no antytripanosomal drugs [6].Artemisinin-based combination therapies (ACTs) are the mainstays for the treatment of uncomplicated malaria.This therapy includes two antimalarial agents with different mechanisms of action: artemether/lumefantrine, artesunate/amodiaquine (ASAQ), dihydroartemisinin/piperaquine, artesunate/pyronaridine (AS-pyronaridine), and artesunate/sulfadoxine/pyrimethamine.Severe malaria is treated with intravenous artesunate followed by a full course of an ACT once the patient can tolerate oral medicines [7].
Unfortunately, malaria remains an enormous problem, and its treatment and control are seriously challenged by the emergence of drug resistance.Partial resistance to artemisinins emerged in southeast Asia early this century and, more recently, emergence of different mutations that are validated markers of resistance has been documented in multiple places in eastern Africa, including Rwanda, Uganda, Tanzania, Ethiopia, and Eritrea [8].
In the present scenario, there is an urgent need to develop new drugs, ideally directed against novel targets, for the treatment of HAT and malaria.In this context, we focused our attention on inhibitors of the parasite cathepsin-L-like cysteine proteases rhodesain of T. b. rhodesiense and falcipain-2 (FP-2) of P. falciparum.
Rhodesain is a clan CA, family C1 (papain family) cysteine protease, essential for the parasite's survival [9,10].It is involved in parasite invasiveness, allowing it to cross the blood-brain barrier (BBB), leading to the neurological stage of HAT.To evade the host immune response, rhodesain takes part in the turnover of variant surface glycoproteins of the trypanosome coat and the degradation of human immunoglobulin [11,12].
In the present study, we designed a new series of peptidyl nitrile-based inhibitors.A very active area of pharmaceutical research concerns the design of peptidomimetics as new therapeutic agents, as they show good stability against enzymatic degradation and 3 of 18 good oral bioavailability.The use of conformationally constrained peptidomimetics should provide selectivity towards specific proteases.

Results and Discussion Chemistry
The designed peptidomimetics 1-4a-b (Figure 1) are characterized by a nitrile group as an electrophilic warhead able to interact with the catalytic cysteine of the target proteases FP-2 and rhodesain, to give a reversible thioimidate adduct.At the P3 site, an unsubstituted indole or a 2-or 3-methyl or 5-fluoro substituted indole scaffold was introduced within a characteristic peptide sequence.The choice of this scaffold was based on consistent data showing that several inhibitors containing this nucleus had marked antiplasmodial activity [32].At the P2 site, a glycine or a β-alanine residue was introduced to evaluate the optimal distance between the indole scaffold and the warhead for the accommodation in the protease pockets.At the P1 site, a homophenylalanine (hPhe) residue, a preferential amino acid for the recognition of the inhibitors by both FP-2 and rhodesain, was introduced.In addition, we also developed a second series of inhibitors, 5-8a-b (Figure 1), bearing at the P1 site a cyclopropyl carbonitrile residue present in the macrolactam co-crystallized with rhodesain (PDB: 6EXO) [33], to evaluate the impact of this structural modification on rhodesain inhibition.In the present study, we report the synthesis of these new compounds, investigation of activities against rhodesain and FP-2, and docking studies.
In the present study, we designed a new series of peptidyl nitrile-based inhibitors.A very active area of pharmaceutical research concerns the design of peptidomimetics as new therapeutic agents, as they show good stability against enzymatic degradation and good oral bioavailability.The use of conformationally constrained peptidomimetics should provide selectivity towards specific proteases.

Chemistry
The designed peptidomimetics 1-4a-b (Figure 1) are characterized by a nitrile group as an electrophilic warhead able to interact with the catalytic cysteine of the target proteases FP-2 and rhodesain, to give a reversible thioimidate adduct.At the P3 site, an unsubstituted indole or a 2-or 3-methyl or 5-fluoro substituted indole scaffold was introduced within a characteristic peptide sequence.The choice of this scaffold was based on consistent data showing that several inhibitors containing this nucleus had marked antiplasmodial activity [32].At the P2 site, a glycine or a β-alanine residue was introduced to evaluate the optimal distance between the indole scaffold and the warhead for the accommodation in the protease pockets.At the P1 site, a homophenylalanine (hPhe) residue, a preferential amino acid for the recognition of the inhibitors by both FP-2 and rhodesain, was introduced.In addition, we also developed a second series of inhibitors, 5-8a-b (Figure 1), bearing at the P1 site a cyclopropyl carbonitrile residue present in the macrolactam co-crystallized with rhodesain (PDB: 6EXO) [33], to evaluate the impact of this structural modification on rhodesain inhibition.In the present study, we report the synthesis of these new compounds, investigation of activities against rhodesain and FP-2, and docking studies.The synthesis of the P3-P2 synthons was realized starting from differently substituted indole scaffolds [32], which were N-alkylated with ethyl-2-bromoacetate 13a or ethyl-3bromopropionate 13b to obtain intermediates 14-17a-b.Alkaline hydrolysis of the ester function afforded the corresponding carboxylic acids 18-21a-b (Scheme 1).The final products 1-8a-b were obtained via coupling reactions between the carboxylic acids 18-21a-b and the (S)-2-amino-4-phenylbutanitrile, which was obtained The synthesis of the P3-P2 synthons was realized starting from differently substituted indole scaffolds [32], which were N-alkylated with ethyl-2-bromoacetate 13a or ethyl-3bromopropionate 13b to obtain intermediates 14-17a-b.Alkaline hydrolysis of the ester function afforded the corresponding carboxylic acids 18-21a-b (Scheme 1).
In the present study, we designed a new series of peptidyl nitrile-based inhibitors.A very active area of pharmaceutical research concerns the design of peptidomimetics as new therapeutic agents, as they show good stability against enzymatic degradation and good oral bioavailability.The use of conformationally constrained peptidomimetics should provide selectivity towards specific proteases.

Chemistry
The designed peptidomimetics 1-4a-b (Figure 1) are characterized by a nitrile group as an electrophilic warhead able to interact with the catalytic cysteine of the target proteases FP-2 and rhodesain, to give a reversible thioimidate adduct.At the P3 site, an unsubstituted indole or a 2-or 3-methyl or 5-fluoro substituted indole scaffold was introduced within a characteristic peptide sequence.The choice of this scaffold was based on consistent data showing that several inhibitors containing this nucleus had marked antiplasmodial activity [32].At the P2 site, a glycine or a β-alanine residue was introduced to evaluate the optimal distance between the indole scaffold and the warhead for the accommodation in the protease pockets.At the P1 site, a homophenylalanine (hPhe) residue, a preferential amino acid for the recognition of the inhibitors by both FP-2 and rhodesain, was introduced.In addition, we also developed a second series of inhibitors, 5-8a-b (Figure 1), bearing at the P1 site a cyclopropyl carbonitrile residue present in the macrolactam co-crystallized with rhodesain (PDB: 6EXO) [33], to evaluate the impact of this structural modification on rhodesain inhibition.In the present study, we report the synthesis of these new compounds, investigation of activities against rhodesain and FP-2, and docking studies.The synthesis of the P3-P2 synthons was realized starting from differently substituted indole scaffolds [32], which were N-alkylated with ethyl-2-bromoacetate 13a or ethyl-3bromopropionate 13b to obtain intermediates 14-17a-b.Alkaline hydrolysis of the ester function afforded the corresponding carboxylic acids 18-21a-b (Scheme 1).The final products 1-8a-b were obtained via coupling reactions between the carboxylic acids 18-21a-b and the (S)-2-amino-4-phenylbutanitrile, which was obtained according to the procedure that we recently reported [18], or the commercially available 1-aminocyclopropanocarbonitrile 23 (Scheme 2).
according to the procedure that we recently reported [18], or the commercially available 1-aminocyclopropanocarbonitrile 23 (Scheme 2).All compounds 1-8a-b were tested against recombinant rhodesain [34] by using Cbz-Phe-Arg-AMC as a fluorogenic substrate (Table 1).First, a preliminary screening at a fixed inhibitor concentration of 100 µM was performed.All compounds 1-8a-b were tested against recombinant rhodesain [34] by using Cbz-Phe-Arg-AMC as a fluorogenic substrate (Table 1).First, a preliminary screening at a fixed inhibitor concentration of 100 µM was performed.according to the procedure that we recently reported [18], or the commercially available 1-aminocyclopropanocarbonitrile 23 (Scheme 2).All compounds 1-8a-b were tested against recombinant rhodesain [34] by using Cbz-Phe-Arg-AMC as a fluorogenic substrate (Table 1).First, a preliminary screening at a fixed inhibitor concentration of 100 µM was performed.An equivalent volume of DMSO was used as the negative control, and E-64, an irreversible inhibitor of clan CA family C1 cysteine proteases, was used as the positive control.

Cmpds
For compounds that inhibited the enzyme activity at least 50% at 10 µM, continuous assays were performed at seven different concentrations to determine dissociation constants.
The best inhibitory activity was obtained for compounds having the 2-methyl substituted indole nucleus at the P3 site and a hPhe residue at the P1 site (i.e., 2a and 2b; Table 1), which is the preferential amino acid for the recognition of the inhibitor by rhodesain.Compound 2b, bearing a β-alanine residue at the P2 site, showed a slightly better enzymatic affinity (K i = 5.06 µM) than the glycine derivative 2a (K i = 6.56 µM), as shown in Table 1.
Compound 6b was also active, with a K i value of 8.18 µM.This compound contains a 2-methyl substituted indole nucleus at the P3 site, a β-alanine at the P2 site, and a cyclopropryl carbonitrile residue at the P1 site present, which as previously mentioned, is present in the macrolactam inhibitor with which the rhodesain was co-crystallized.
In the case of compounds containing a hPhe residue at P1 and a glycine at P2, the order of reactivity was 2-methyl substituted indole (i.e., 2a) > 3-methyl substituted indole (i.e., 3a) > unsubstituted indole (i.e., 1a), with K i values of 6.56 µM, 10.31 µM, and 13.11 µM, respectively.In contrast, compounds with a 5-fluoro-indole were inactive.All compounds were also tested against FP-2, and in this case inhibitor 2b was the only active compound, with an IC 50 of 40.43 µM (Table 1).All the active compounds were tested against T. b. brucei, and the only active compound was inhibitor 2b, with an IC 50 of 12 µM at both 24 h and 48 h of incubation (Figure 2).Bloodstream T. brucei parasites were incubated in the presence of various concentrations of the tested compounds and after 24 h or 48 h cell viability was determined using the ATPlite (PerkinElmer, Waltham, MA, USA) assay.Chlorhexidine served as positive control.Data represent mean ± SD of two independent experiments, except for those of 24 h treatment with chlorhexidine, which represent data from a single experiment.Cell viability data was normalized to the untreated controls and analyzed using GraphPad Prism.Whenever possible, data were fitted by log(inhibitor) vs response nonlinear fit (solid lines) and IC 50 values were calculated.
To gain deeper insight into the binding interactions of the newly discovered rhodesain inhibitors, molecular modeling studies were conducted.Specifically, docking experiments employed the X-ray structure of rhodesain bound to its inhibitor K11002 (PDB code: 2P86) [35].Considering the ability of the inspected ligands to form a reversible covalent adduct with the enzyme, a covalent docking protocol was adopted, employing the flexible side-chain method available within the AutoDock4 (AD4, v4.2.6) docking software.We chose to include 2b in this investigation, since it is the most active rhodesain inhibitor among those displaying inhibitory properties and antitrypanosomal activity (Table 1).Thus, 2b was covalently docked into the X-ray structure of rhodesain after simulating a modified Pinner reaction between the SH group of residue C25 and the nitrile warhead [36], resulting in a thioimidate group responsible for the reversible covalent bond (Figure 3).To further refine the predicted binding geometry, a 500 ns long molecular dynamics (MD) simulation was performed on the 2b docked complex.The results of the MD simulation suggested a preferential binding orientation, with the adopted conformation of 2b (Figure 4A) fairly stable throughout the simulation time, as demonstrated by the root mean square deviation (RMSD) calculated for the ligand's atoms over time (Figure 4B).The MD studies highlighted that 2b can form a second H-bond with the backbone CO of D161 thanks to its thioimidate moiety, while losing the interaction with G66.Additionally, 2b seemed to point its 2-methyl group towards nearby L67 and M68 in the S2 pocket.Interestingly, the role of the 2-methyl indole substitution was also underscored by The ligand is represented as orange sticks.Rhodesain is represented as white sticks and ribbons, and a blue surface.H-bonds are represented as green dashed lines.Pocket locations are indicated by red labels.
In the lowest energy conformation predicted by AD4, the ligand was located in the enzyme binding site at a position resembling the experimental binding position of the co-crystallized ligand.Specifically, the hPhe residue was pointing toward the enzyme S1' pocket with its aromatic ring positioned near H162 and W184 of the same pocket, where charge-transfer interactions can be established.The absence of the hPhe residue appeared to be detrimental for activity, with decreased rhodesain inhibition for compounds featuring the cyclopropyl moiety (compounds 5 to 8 in Table 1).In fact, the chemical nature of the latter did not permit it to efficiently contact the S1' pocket.It is worth noting that the nature of the thioimidate group permitted the formation of a H-bond with the backbone NH of G163 residue G163.The CO group of the P2 residue contacted the backbone NH of G66, while the NH of the same residueP1 site formed H-bonds with the backbone CO of D161.The presence of two methylene groups in the P2 region of the ligand seemed to better orient the attached indole ring towards the S2 pocket, where it established hydrophobic interactions with L67, M68, and A138.Of note, it has been reported that A138 limits the space available in this pocket [10,35].
To further refine the predicted binding geometry, a 500 ns long molecular dynamics (MD) simulation was performed on the 2b docked complex.The results of the MD simu-lation suggested a preferential binding orientation, with the adopted conformation of 2b (Figure 4A) fairly stable throughout the simulation time, as demonstrated by the root mean square deviation (RMSD) calculated for the ligand's atoms over time (Figure 4B).To further refine the predicted binding geometry, a 500 ns long molecular dynamics (MD) simulation was performed on the 2b docked complex.The results of the MD simulation suggested a preferential binding orientation, with the adopted conformation of 2b (Figure 4A) fairly stable throughout the simulation time, as demonstrated by the root mean square deviation (RMSD) calculated for the ligand's atoms over time (Figure 4B).The MD studies highlighted that 2b can form a second H-bond with the backbone CO of D161 thanks to its thioimidate moiety, while losing the interaction with G66.Additionally, 2b seemed to point its 2-methyl group towards nearby L67 and M68 in the S2 pocket.Interestingly, the role of the 2-methyl indole substitution was also underscored by the generally lower rhodesain inhibition of compounds devoid of this group (1b) or featuring the same group in position 3 of the same ring (3b).

Chemistry
All reagents and solvents were obtained from commercial suppliers and were used without any further purification.Melting points were determined in open capillary tubes on a Stuart Scientific melting point apparatus SMP3 and were uncorrected.To determine the purity of compounds elemental analyses were carried out on a C. Erba Model 1106 (Elemental Analyser for C, H, and N) instrument, and the obtained results were within ± 0.4% of the theoretical values.Merck silica gel 60 F254 plates were used for analytical TLC; flash column chromatography was performed on Merck silica gel (200-400 mesh). 1 H and 13 C and NMR spectra were recorded on a Varian 500 MHz spectrometer equipped with a The MD studies highlighted that 2b can form a second H-bond with the backbone CO of D161 thanks to its thioimidate moiety, while losing the interaction with G66.Additionally, 2b seemed to point its 2-methyl group towards nearby L67 and M68 in the S2 pocket.Interestingly, the role of the 2-methyl indole substitution was also underscored by the generally lower rhodesain inhibition of compounds devoid of this group (1b) or featuring the same group in position 3 of the same ring (3b).

Chemistry
All reagents and solvents were obtained from commercial suppliers and were used without any further purification.Melting points were determined in open capillary tubes on a Stuart Scientific melting point apparatus SMP3 and were uncorrected.To determine the purity of compounds elemental analyses were carried out on a C. Erba Model 1106 (Elemental Analyser for C, H, and N) instrument, and the obtained results were within ± 0.4% of the theoretical values.Merck silica gel 60 F254 plates were used for analytical TLC; flash column chromatography was performed on Merck silica gel (200-400 mesh). 1 H and 13 C and NMR spectra were recorded on a Varian 500 MHz spectrometer equipped with a ONE_NMR probe and operating at 499.74 and 125.73 MHz for 1 H and 13 C, respectively.We used the residual signal of the deuterated solvent as an internal standard.Splitting patterns are described as singlet (s), doublet (d), doublet of doublet (dd), triplet (t), quartet (q), multiplet (m), or broad singlet (bs). 1 H and 13 C NMR chemical shifts (δ) are expressed in ppm and coupling constants (J) are given in Hz.All 1 H-and 13 C-NMR spectra are reported in the Supporting Information (see Figures S1-S48).

FP-2 Inhibition
Compounds were tested for inhibition of recombinant FP-2, which was expressed and purified as previously reported [37].Stock solutions of the compounds, substrate, and the positive control E64 (Sigma Aldrich, St. Louis, MO, USA) were prepared at 10 mM in 100% DMSO.The compounds were incubated in 96-well white flat-bottom plates with 30 nM recombinant FP-2 for 10 min at room temperature in an assay buffer of 100 mM sodium acetate, pH 5.5, with 5 mM DTT.After incubation, the fluorogenic substrate Cbz-Leu-Arg-AMC (R&D Systems, Minneapolis, MN, USA) was added at a concentration of 25 µM in a final assay volume of 200 µL.Fluorescence was monitored with a Varioskan Flash (ThermoScientific, Waltham, MA, USA) with excitation 355 nm and emission 460 nm.The IC 50 values were calculated using GraphPad Prism v. 6 (GraphPad Software Inc., San Diego, CA, USA)) based on a sigmoidal dose response curves.

Docking Experiments
AutoDock4 (AD4) was employed for the molecular docking calculations [40].To model the formation of the covalent adduct between the ligand and the protein, the "flexible side chain method" was used [41].In the preparation phase, the ligand was modeled within the Maestro suite to incorporate two additional atoms (a sulfur and a carbon atom) to be aligned with the C25 residue of the protein.The X-ray structure of rhodesain (PDB code: 2P86) underwent preliminary adjustments for docking purposes by using the Protein Preparation Wizard integrated into the Schrödinger suite [42].Alignment of the ligand with the reactive cysteine was facilitated by scripts available from the AD4 website.Protein grid maps, employing ligand atom types as probes, were generated with AutoGrid4 (v 4.2.6)software.The dimensions of the enzyme grid box were set at 60 Å × 60 Å × 60 Å with a 0.375 Å spacing, centered on the coordinates of the native cocrystallized ligand.The docking calculations were executed with flexibility allowed for the modified cysteine/ligand residue.The Lamarckian genetic algorithm (LGA) was used for these docking simulations, encompassing 100 LGA runs.All other settings were maintained at their default values.The docking results were subsequently grouped based on the root mean square deviation (RMSD) criterion, whereby solutions differing by less than 2.0 Å were considered part of the same cluster.The ranking of these clusters was determined based on the calculated lowest free energy of binding (∆GAD4).Visualization of the results was carried out using the UCSF Chimera X (version 1.7.1)software [43].

Molecular Dynamics Experiments
The ligand-protein complex for 2b obtained from the docking experiments was used to construct a molecular dynamics system that was solvated in water within an orthorhombic box with a buffer distance of 10 Å.Additionally, the overall charge of the system was automatically neutralized by Maestro's System Builder utility [44].The salt concentration was set to 0.15 M NaCl.The chosen force field for constructing the system was OPLS3 [45].
The Desmond module within the Schrodinger suite was used for a 500 ns long MD simulation.NPT was chosen as the experimental environment (constant pressure of 1.01325 bar and constant temperature of 300 K).In detail, the temperature was controlled using the Nosé-Hoover thermostat with a 1.0 ps relaxation time [46,47].The pressure was controlled using the Martyna-Tobias-Klein barostat with an isotropic coupling style and a 2.0 ps relaxation time [48,49].The cutoff distance for short-range nonbonded interactions was 9 Å.To minimize the computation time, nonbonded forces were calculated using a RESPA integrator where the short-range forces were updated every two steps [50], and the long-

18 Figure 2 .
Figure 2.Antitrypanosomal activity of compounds 1a, 2a, 2b, 3a, 6b, as well as chlorhexidine.Bloodstream T. brucei parasites were incubated in the presence of various concentrations of the tested compounds and after 24 h or 48 h cell viability was determined using the ATPlite (PerkinElmer, Waltham, MA, USA) assay.Chlorhexidine served as positive control.Data represent mean ± SD of two independent experiments, except for those of 24 h treatment with chlorhexidine, which represent data from a single experiment.Cell viability data was normalized to the untreated controls and analyzed using GraphPad Prism.Whenever possible, data were fitted by log(inhibitor) vs response nonlinear fit (solid lines) and IC50 values were calculated.

Figure 2 .
Figure 2. Antitrypanosomal activity of compounds 1a, 2a, 2b, 3a, 6b, as well as chlorhexidine.Bloodstream T. brucei parasites were incubated in the presence of various concentrations of the tested

18 Figure 3 .
Figure 3. Predicted binding mode of 2b in complex with rhodesain (PDB code: 2P86) (ligand as orange sticks, protein as white sticks and ribbons and blue surface).H-bond interactions are represented as green dashed lines.Enzyme pockets are indicated by red labels.

Figure 4 .
Figure 4. (A) Representative frame MD calculated for the 2b/rhodesain complex (ligand as orange sticks, protein as white sticks and ribbons and blue surface).H-bond interactions are represented as green dashed lines.Enzyme pockets are indicated by red labels.(B) Root mean square deviation (RMSD) over time for 2b.

Figure 3 .
Figure 3. Predicted binding mode of 2b in complex with rhodesain (PDB code: 2P86) (ligand as orange sticks, protein as white sticks and ribbons and blue surface).H-bond interactions are represented as green dashed lines.Enzyme pockets are indicated by red labels.

Figure 3 .
Figure 3. Predicted binding mode of 2b in complex with rhodesain (PDB code: 2P86) (ligand as orange sticks, protein as white sticks and ribbons and blue surface).H-bond interactions are represented as green dashed lines.Enzyme pockets are indicated by red labels.

Figure 4 .
Figure 4. (A) Representative frame MD calculated for the 2b/rhodesain complex (ligand as orange sticks, protein as white sticks and ribbons and blue surface).H-bond interactions are represented as green dashed lines.Enzyme pockets are indicated by red labels.(B) Root mean square deviation (RMSD) over time for 2b.

Figure 4 .
Figure 4. (A) Representative frame MD calculated for the 2b/rhodesain complex (ligand as orange sticks, protein as white sticks and ribbons and blue surface).H-bond interactions are represented as green dashed lines.Enzyme pockets are indicated by red labels.(B) Root mean square deviation (RMSD) over time for 2b.

Table 1 .
Activity against rhodesain and FP-2 of compounds 1

Table 1 .
Activity against rhodesain and FP-2 of compounds 1

. n Rhodesain Percentage of Inhibition at 100 µM or K i µM
n.i.: no inhibition.