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.

Influence of amino acid size at the P3 position of N-Cbz-tripeptide Michael acceptors targeting falcipain-2 and rhodesain for the treatment of malaria and human african trypanosomiasis.

In recent decades, several structure-activity relationship (SAR) studies provided potent inhibitors of the cysteine proteases falcipain-2 (FP-2) and rhodesain (RD) from Plasmodium falciparum and Trypanosoma brucei rhodesiense, respectively. Whilst the roles of the warhead and residues targeting the P1 and P2 pockets of the proteases were extensively investigated, the roles of the amino acids occupying the S3 pocket were not widely assessed. Herein we report the synthesis and biological evaluation of a set of novel Michael acceptors bearing amino acids of increasing size at the P3 site (1a-g/2a-g, SPR20-SPR33) against FP-2, RD, P. falciparum, and T. brucei. Overall, the Michael acceptors bearing small amino acids at the P3 site exhibited the most potent inhibitory properties towards FP-2. In contrast, analogues with bulky residues at the P3 position were very potent rhodesain inhibitors. In cell based assays, single-digit micromolar EC50 values against the two protozoa were observed. These findings can be a starting point for the development of peptide-based FP-2 and RD inhibitors.

In silico investigation of falcipain-2 inhibition by hybrid benzimidazole-thiosemicarbazone antiplasmodial agents: A molecular docking, molecular dynamics simulation, and kinetics study.

The emergence of artemisinin-resistant variants of Plasmodium falciparum necessitates the urgent search for novel antimalarial drugs. In this regard, an in silico study to screen antimalarial drug candidates from a series of benzimidazole-thiosemicarbazone hybrid molecules with interesting antiplasmodial properties and explore their falcipain-2 (FP2) inhibitory potentials has been undertaken herein. FP2 is a key cysteine protease that degrades hemoglobin in Plasmodium falciparum and is an important biomolecular target in the development of antimalarial drugs. Pharmacokinetic properties, ADMET profiles, MM/GBSA-based binding free energies, reaction mechanisms, and associated barrier heights have been investigated. DFT, molecular dynamics simulation, molecular docking, and ONIOM methods were used. From the results obtained, four 4N-substituted derivatives of the hybrid molecule (E)-2-(1-(5-chloro-1H-benzo[d]imidazol-2-yl)ethylidene)hydrazine-1-carbothioamide (1A) denoted 1B, 1C, 1D, and 1E are drug-like and promising inhibitors of FP2, exhibiting remarkably small inhibitory constants (5.94 × 10-14 - 2.59 × 10-04 [Formula: see text]M) and favorable binding free energies (-30.32 to -17.17 kcal/mol). Moreover, the ONIOM results have revealed that 1B and possibly 1C and 1D may act as covalent inhibitors of FP2. The rate-determining step of the thermodynamically favorable covalent binding mechanism occurs across a surmountable barrier height of 24.18 kcal/mol in water and 28.42 kcal/mol in diethyl ether. Our findings are useful for further experimental investigations on the antimalarial activities of the hybrid molecules studied.

New insights of falcipain 2 structure from Plasmodium falciparum 3D7 strain.

Malaria identifies as a tropical hallmark, conforming to the burgeoning notion of escalating drug resistance among virulent strains, with the burdensome Plasmodium falciparum under its wing. The cysteine protease Falcipain-2 (FP2) is released in the parasite's food vacuole in the trophozoite stage and contributes to disease progression through its hemoglobinase activity. In the present study, we have determined the crystal structure of FP2 from a drug resistant P. falciparum 3D7 strain. FP2-3D7 sequence has detected four amino acid variants, R12K, E14 N, P100T and G102D, in the mature domain of the protease, when compared with other reported structures. FP2-3D7 protease has been crystallized in the presence of two inhibitors E-64 and Iodoacetamide, which diffracted up to 3.5 Å and 3.4 Å respectively. Structural analyses of the mature domain helped unveil two solvent-exposed pockets with bound ligands where one is structurally homologous to the allosteric binding site of human Cathepsin-K and thus, could be exploited for designing allosteric modifiers of FP2. The structure has also revealed (poly)ethylene glycol molecules along the catalytic cleft, providing interesting insights for exploring FP2 as a chemotherapeutic target and for PEGylation in drug delivery. The side-chains of P2 and P3 residues of E-64 also adopt different rotamer conformations, compared with previously reported structure, emphasizing strain-specific multiple binding-modes of active-site targeted inhibitors. Docking studies, along with normal mode analyses, highlight the mode of hemoglobin binding and the active/inactive switch in hemoglobinase activity, conjecturing the formation of a stable dimeric state with a symmetry related copy in crystal packing.

In silico identification of noncompetitive inhibitors targeting an uncharacterized allosteric site of falcipain-2.

Falcipain-2 (FP-2) is a Plasmodium falciparum hemoglobinase widely targeted in the search for antimalarials. FP-2 can be allosterically modulated by various noncompetitive inhibitors that have been serendipitously identified. Moreover, the crystal structures of two inhibitors bound to an allosteric site, termed site 6, of the homolog enzyme human cathepsin K (hCatK) suggest that the equivalent region in FP-2 might play a similar role. Here, we conduct the rational identification of FP-2 inhibitors through virtual screenings (VS) of compounds into several pocket-like conformations of site 6, sampled during molecular dynamics (MD) simulations of the free enzyme. Two noncompetitive inhibitors, ZINC03225317 and ZINC72290660, were confirmed using in vitro enzymatic assays and their poses into site 6 led to calculated binding free energies matching the experimental ones. Our results provide strong evidence about the allosteric inhibition of FP-2 through binding of small molecules to site 6, thus opening the way toward the discovery of new inhibitors against this enzyme.

Quinoline carboxamide core moiety-based compounds inhibit P. falciparumfalcipain-2: Design, synthesis and antimalarial efficacy studies.

Targeting Falcipain-2 (FP2) for the development of antimalarials is a promising and established concept in antimalarial drug discovery and development. FP2, a member of papain-family cysteine protease of the malaria parasite Plasmodium falciparum holds an important role in hemoglobin degradation pathway. A new series of quinoline carboxamide-based compounds was designed, synthesized and evaluated for antimalarial activity. We integrated molecular hybridization strategy with in-silico drug design to develop FP2 inhibitors. In-vitro results of FP2 inhibition by Qs17, Qs18, Qs20 and Qs21 were found to be in low micromolar range with IC50 4.78, 7.37, 2.14 and 2.64 µM, respectively. Among the 25 synthesized compounds, four compounds showed significant antimalarial activities. These compounds also depicted morphological and food-vacuole abnormalities much better than that of E-64, an established FP2 inhibitor. Overall these aromatic substituted quinoline carboxamides can serve as promising leads for the development of novel antimalarial agents.

X-ray Structure Determination, Antioxidant Voltammetry Studies of Butein and 2',4'-Dihydroxy-3,4-dimethoxychalcone. Computational Studies of 4 Structurally Related 2',4'-diOH Chalcones to Examine Their Antimalarial Activity by Binding to Falcipain-2.

Malaria is a huge global health burden with resistance to currently available medicines resulting in the search for newer antimalarial compounds from traditional medicinal plants in malaria-endemic regions. Previous studies on two chalcones, homobutein and 5-prenylbutein, present in E. abyssinica, have shown moderate antiplasmodial activity. Here, we describe results from experimental and computational investigations of four structurally related chalcones, butein, 2',4'-dihydroxy-3,4-dimethoxychalcone (DHDM), homobutein and 5-prenylbutein to elucidate possible molecular mechanisms by which these compounds clear malaria parasites. The crystal structures of butein and DHDM show that butein engages in more hydrogen bonding and consequently, more intermolecular interactions than DHDM. Rotating ring-disk electrode (RRDE) voltammetry results show that butein has a higher antioxidant activity towards the superoxide radical anion compared to DHDM. Computational docking experiments were conducted to examine the inhibitory potential of all four compounds on falcipain-2, a cysteine protease that is involved in the degradation of hemoglobin in plasmodium-infected red blood cells of the host. Overall, this work suggests butein as a better antimalarial compound due to its structural features which allow it to have greater intermolecular interactions, higher antioxidant activity and to create a covalent complex at the active site of falcipain-2.

Identification of antimalarial leads with dual falcipain-2 and falcipain-3 inhibitory activity.

Falcipains (FPs), cysteine proteases in the malarial parasite, are emerging as the promising antimalarial drug targets. In order to identify novel FP inhibitors, we generated a pharmacophore derived from the reported co-crystal structures of inhibitors of Plasmodium falciparum Falcipain-3 to screen the ZINC library. Further, the filters were applied for dock score, drug-like characters, and clustering of similar structures. Sixteen molecules were purchased and subject to in vitro enzyme (FP-2 and FP-3) inhibition assays. Two compounds showed in vitro inhibition of FP-2 and FP-3 at low µM concentration. The selectivity of the inhibitors can be explained based on the predicted interactions of the molecule in the active site. Further, the inhibitors were evaluated in a functional assay and were found to induce morphological changes in line with their mode of action arresting Plasmodium development. Compound 15 was most potent inhibitor identified in this study.

Identification and structure-activity relationship (SAR) studies of carvacrol derivatives as potential anti-malarial against Plasmodium falciparum falcipain-2 protease.

In an effort to develop a potent anti-malarial agent against Plasmodium falciparum, a structure-guided virtual screening using an in-house library comprising 652 compounds was performed. By docking studies, we identified two compounds (JMI-105 and JMI-346) which formed significant non-covalent interactions and fit well in the binding pocket of PfFP-2. We affirmed this observation by MD simulation studies. As evident by the biochemical analysis, such as enzyme inhibition assay, Surface Plasmon Resonance (SPR), live-cell imaging and hemozoin inhibition, JMI-105 and JMI-346 at 25 µM concentration showed an inhibitory effect on purified PfFP-2. JMI-105 and JMI-346 inhibited the growth of CQS (3D7; IC50 = 8.8 and 13 µM) and CQR (RKL-9; IC50 = 14.3 and 33 µM) strains of P. falciparum. Treatment with compounds resulted in defect in parasite growth and development. No significant hemolysis or cytotoxicity towards human cells was observed suggesting that these molecules are non-toxic. We pursued, structural optimization on JMI-105 and in the process, SAR oriented derivatives (5a-5l) were synthesized and evaluated for growth inhibition potential. JMI-105 significantly decreased parasitemia and prolonged host survival in a murine model with P. berghei ANKA infection. The compounds (JMI-105 and JMI-346) against PfFP-2 have the potential to be used as an anti-malarial agent.

A Novel Series of [1,2,4]Triazolo[4,3-a]Pyridine Sulfonamides as Potential Antimalarial Agents: In Silico Studies, Synthesis and In Vitro Evaluation.

For the development of new and potent antimalarial drugs, we designed the virtual library with three points of randomization of novel [1,2,4]triazolo[4,3-a]pyridines bearing a sulfonamide fragment. The library of 1561 compounds has been investigated by both virtual screening and molecular docking methods using falcipain-2 as a target enzyme. 25 chosen hits were synthesized and evaluated for their antimalarial activity in vitro against Plasmodium falciparum. 3-Ethyl-N-(3-fluorobenzyl)-N-(4-methoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine-6-sulfonamide and 2-(3-chlorobenzyl)-8-(piperidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one showed in vitro good antimalarial activity with inhibitory concentration IC50 = 2.24 and 4.98 µM, respectively. This new series of compounds may serve as a starting point for future antimalarial drug discovery programs.

Targeting malaria and leishmaniasis: Synthesis and pharmacological evaluation of novel pyrazole-1,3,4-oxadiazole hybrids. Part II.

In continuance with earlier reported work, an extension has been carried out by the same research group. Mulling over the ongoing condition of resistance to existing antimalarial agents, we had reported synthesis and antimalarial activity of certain pyrazole-1,3,4-oxadiazole hybrid compounds. Bearing previous results in mind, our research group ideated to design and synthesize some more derivatives with varied substitutions of acetophenone and hydrazide. Following this, derivatives 5a-r were synthesized and tested for antimalarial efficacy by schizont maturation inhibition assay. Further, depending on the literature support and results of our previous series, certain potent compounds (5f, 5n and 5r) were subjected to Falcipain-2 inhibitory assay. Results obtained for these particular compounds further strengthened our hypothesis. Here, in this series, compound 5f having unsubstituted acetophenone part and a furan moiety linked to oxadiazole ring emerged as the most potent compound and results were found to be comparable to that of the most potent compound (indole bearing) of previous series. Additionally, depending on the available literature, compounds (5a-r) were tested for their antileishmanial potential. Compounds 5a, 5c and 5r demonstrated dose-dependent killing of the promastigotes. Their IC50 values were found to be 33.3 ±â€¯1.68, 40.1 ±â€¯1.0 and 19.0 ±â€¯1.47 µg/mL respectively. These compounds (5a, 5c and 5r) also had effects on amastigote infectivity with IC50 of 44.2 ±â€¯2.72, 66.8 ±â€¯2.05 and 73.1 ±â€¯1.69 µg/mL respectively. Further target validation was done using molecular docking studies. Acute oral toxicity studies for most active compounds were also performed.

Virtual design of novel Plasmodium falciparum cysteine protease falcipain-2 hybrid lactone-chalcone and isatin-chalcone inhibitors probing the S2 active site pocket.

We report computer-aided design of new lactone-chalcone and isatin-chalcone (HLCIC) inhibitors of the falcipain-2 (PfFP-2). 3D models of 15 FP-2:HLCIC1-15 complexes with known observed activity (IC50exp) were prepared to establish a quantitative structure-activity (QSAR) model and linear correlation between relative Gibbs free energy of enzyme:inhibitor complex formation (ΔΔGcom) and IC50exp: pIC50exp = -0.0236 × ΔΔGcom+5.082(#); R2 = 0.93. A 3D pharmacophore model (PH4) derived from the QSAR directed our effort to design novel HLCIC analogues. During the design, an initial virtual library of 2621440 HLCIC was focused down to 18288 drug-like compounds and finally, PH4 screened to identify 81 promising compounds. Thirty-three others were added from an intuitive substitution approach intended to fill better the enzyme S2 pocket. One hundred and fourteen theoretical IC50 (IC50pre) values were predicted by means of (#) and their pharmacokinetics (ADME) profiles. More than 30 putative HLCICs display IC50pre 100 times superior to that of the published most active training set inhibitor HLCIC1.

Peptidomimetic nitrile inhibitors of malarial protease falcipain-2 with high selectivity against human cathepsins.

Falcipain-2 (FP2) is an essential enzyme in the lifecycle of malaria parasites such as Plasmodium falciparum, and its inhibition is viewed as an attractive mechanism of action for new anti-malarial agents. Selective inhibition of FP2 with respect to a family of human cysteine proteases (that include cathepsins B, K, L and S) is likely to be required for the development of agents targeting FP2. Here we describe a series of P2-modified aminonitrile based inhibitors of FP2 that provide a clear strategy toward addressing selectivity for the P. falciparum and show that it can provide potent FP2 inhibitors with strong selectivity against all four of these human cathepsin isoforms.

Synthesis of novel benzimidazole acrylonitriles for inhibition of Plasmodium falciparum growth by dual target inhibition.

Antimalarial drug resistance has emerged as a threat for treating malaria, generating a need to design and develop newer, more efficient antimalarial agents. This research aimed to identify novel leads as antimalarials. Dual receptor mechanism could be a good strategy to combat developing drug resistance. A series of benzimidazole acrylonitriles containing 18 compounds were designed, synthesized and evaluated for cytotoxicity, heme binding, ferriprotoporphyrin IX biomineralisation inhibition, and falcipain-2 enzyme assay. Furthermore, in silico docking and MD simulation studies were also performed.The tests revealed quite encouraging results. Three compounds, viz. R-01 (0.69 µM), R-04 (1.60 µM), and R-08 (1.61 µM), were found to have high antimalarial activity. These compounds were found to be in bearable cytotoxicity limits and their biological assay suggested that they had inhibitory activity against falcipain-2 and hemozoin formation. The docking revealed the binding mode of benzimidazole acrylonitrile derivatives and MD simulation studies revealed that the protein-ligand complex was stable. The agents exhibit good hemozoin formation inhibition activity and, hence, may be utilized as leads to design a newer drug class to overcome the drug resistance of hemozoin formation inhibitors such as chloroquine.

Predicting binding modes of reversible peptide-based inhibitors of falcipain-2 consistent with structure-activity relationships.

Falcipain-2 (FP-2) is a major hemoglobinase of Plasmodium falciparum, considered an important drug target for the development of antimalarials. A previous study reported a novel series of 20 reversible peptide-based inhibitors of FP-2. However, the lack of tridimensional structures of the complexes hinders further optimization strategies to enhance the inhibitory activity of the compounds. Here we report the prediction of the binding modes of the aforementioned inhibitors to FP-2. A computational approach combining previous knowledge on the determinants of binding to the enzyme, docking, and postdocking refinement steps, is employed. The latter steps comprise molecular dynamics simulations and free energy calculations. Remarkably, this approach leads to the identification of near-native ligand conformations when applied to a validation set of protein-ligand structures. Overall, we proposed substrate-like binding modes of the studied compounds fulfilling the structural requirements for FP-2 binding and yielding free energy values that correlated well with the experimental data. Proteins 2017; 85:1666-1683. © 2017 Wiley Periodicals, Inc.

Docking, synthesis and antimalarial activity of novel 4-anilinoquinoline derivatives.

A series of 4-anilinoquinoline triazine derivatives were designed, synthesized and screened for in vivo antimalarial activity against a chloroquine-sensitive strain of Plasmodium berghei. The compounds were further subjected to in vitro antimalarial activity against chloroquine-resistant W2 strain of Plasmodium falciparum and ß-haematin inhibition studies. All the compounds exhibited in vivo antimalarial activity better than that shown by the standard drug, chloroquine. Twelve out of fifteen compounds showed better inhibition than that of chloroquine against chloroquine-resistant W2 strain of Plasmodium falciparum. Ten compounds showed ß-haematin inhibition, better than that of chloroquine, with IC50 values in the range of 18-25µM. One compound, 3k, was found to be better than artemisinin against W2 strain of Plasmodium falciparum and also displayed the best ß-haematin inhibitory activity, thereby becoming eligible to be explored as a potential lead for antimalarial chemotherapy.

Novel series of 1,2,4-trioxane derivatives as antimalarial agents.

Among three series of 1,2,4-trioxane derivatives, five compounds showed good in vitro antimalarial activity, three compounds of which exhibited better activity against P. falciparum resistant (RKL9) strain than the sensitive (3D7) one. Two best compounds were one from aryl series and the other from heteroaryl series with IC50 values of 1.24 µM and 1.24 µM and 1.06 µM and 1.17 µM, against sensitive and resistant strains, respectively. Further, trioxane derivatives exhibited good binding affinity for the P. falciparum cysteine protease falcipain 2 receptor (PDB id: 3BPF) with well defined drug-like and pharmacokinetic properties based on Lipinski's rule of five with additional physicochemical and ADMET parameters. In view of having antimalarial potential, 1,2,4-trioxane derivative(s) reported herein may be useful as novel antimalarial lead(s) in the discovery and development of future antimalarial drug candidates as P. falciparum falcipain 2 inhibitors against resistant malaria.

Identification of Tight-Binding Plasmepsin II and Falcipain 2 Inhibitors in Aqueous Extracts of Marine Invertebrates by the Combination of Enzymatic and Interaction-Based Assays.

Natural products from marine origin constitute a very promising and underexplored source of interesting compounds for modern biotechnological and pharmaceutical industries. However, their evaluation is quite challenging and requires specifically designed assays to reliably identify the compounds of interest in a highly heterogeneous and interfering context. In the present study, we describe a general strategy for the confident identification of tight-binding protease inhibitors in the aqueous extracts of 62 Cuban marine invertebrates, using Plasmodium falciparum hemoglobinases Plasmepsin II and Falcipain 2 as model enzymes. To this end, we first developed a screening strategy that combined enzymatic with interaction-based assays and then validated screening conditions using five reference extracts. Interferences were evaluated and minimized. The results from the massive screening of such extracts, the validation of several hits by a variety of interaction-based assays and the purification and functional characterization of PhPI, a multifunctional and reversible tight-binding inhibitor for Plasmepsin II and Falcipain 2 from the gorgonian Plexaura homomalla, are presented.

Comparative molecular field analysis and molecular docking studies on novel aryl chalcone derivatives against an important drug target cysteine protease in Plasmodium falciparum.

The computational studies namely molecular docking simulations and Comparative Molecular Field Analysis (CoMFA) are executed on series of 52 novel aryl chalcones derivatives using Plasmodium falciparum cysteine proteases (falcipain - 2) as vital target. In the present study, the correlation between different molecular field effects namely steric and electrostatic interactions and chemical structures to the inhibitory activities of novel aryl chalcone derivatives is inferred to perceive the major structural prerequisites for the rational design and development of potent and novel lead anti-malarial compound. The apparent binding conformations of all the compounds at the active site of falcipain - 2 and the hydrogen-bond interactions which could be used to modify the inhibitory activities are identified by using Surflex-dock study. Statistically significant CoMFA model has been developed with the cross-validated correlation coefficient (q(2)) of 0.912 and the non-cross-validated correlation coefficient (r(2)) of 0.901. Standard error of estimation (SEE) of 0.210, with the optimum number of components is ten. The predictability of the derived model is examined with a test set consists of sixteen compounds and the predicted r(2) value is found to be 0.924. The docking and QSAR study results confer crucial suggestions for the optimization of novel 1,3-diphenyl-2-propen-1-one derivatives and synthesis of effective anti- malarial compounds.

Bistacrine derivatives as new potent antimalarials.

Linking two tacrine molecules results in a tremendous increase of activity against Plasmodia in comparison to the monomer. This finding prompted the synthesis of a library of monomeric and dimeric tacrine derivatives in order to derive structure-activity relationships. The most active compounds towards chloroquine sensitive Plasmodium strain 3D7 and chloroquine resistant strain Dd2 show IC50 values in the nanomolar range of concentration, low cytotoxicity and target the cysteine protease falcipain-2, which is essential for parasite growth.