Plasmodium falciparum proteases as new drug targets with special focus on metalloproteases.

Malaria poses a significant global health threat particularly due to the prevalence of Plasmodium falciparum infection. With the emergence of parasite resistance to existing drugs including the recently discovered artemisinin, ongoing research seeks novel therapeutic avenues within the malaria parasite. Proteases are promising drug targets due to their essential roles in parasite biology, including hemoglobin digestion, merozoite invasion, and egress. While exploring the genomic landscape of Plasmodium falciparum, it has been revealed that there are 92 predicted proteases, with only approximately 14 of them having been characterized. These proteases are further distributed among 26 families grouped into five clans: aspartic proteases, cysteine proteases, metalloproteases, serine proteases, and threonine proteases. Focus on metalloprotease class shows further role in organelle processing for mitochondria and apicoplasts suggesting the potential of metalloproteases as viable drug targets. Holistic understanding of the parasite intricate life cycle and identification of potential drug targets are essential for developing effective therapeutic strategies against malaria and mitigating its devastating global impact.

Identification of 3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine Derivatives as Novel Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase.

Plasmodium falciparum's resistance to available antimalarial drugs highlights the need for the development of novel drugs. Pyrimidine de novo biosynthesis is a validated drug target for the prevention and treatment of malaria infection. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the oxidation of dihydroorotate to orotate and utilize ubiquinone as an electron acceptor in the fourth step of pyrimidine de novo biosynthesis. PfDHODH is targeted by the inhibitor DSM265, which binds to a hydrophobic pocket located at the N-terminus where ubiquinone binds, which is known to be structurally divergent from the mammalian orthologue. In this study, we screened 40,400 compounds from the Kyoto University chemical library against recombinant PfDHODH. These studies led to the identification of 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine and its derivatives as a new class of PfDHODH inhibitor. Moreover, the hit compounds identified in this study are selective for PfDHODH without inhibition of the human enzymes. Finally, this new scaffold of PfDHODH inhibitors showed growth inhibition activity against P. falciparum 3D7 with low toxicity to three human cell lines, providing a new starting point for antimalarial drug development.

Dephospho-CoA kinase, a nuclear-encoded apicoplast protein, remains active and essential after Plasmodium falciparum apicoplast disruption.

Malaria parasites contain an essential organelle called the apicoplast that houses metabolic pathways for fatty acid, heme, isoprenoid, and iron-sulfur cluster synthesis. Surprisingly, malaria parasites can survive without the apicoplast as long as the isoprenoid precursor isopentenyl pyrophosphate (IPP) is supplemented in the growth medium, making it appear that isoprenoid synthesis is the only essential function of the organelle in blood-stage parasites. In the work described here, we localized an enzyme responsible for coenzyme A synthesis, DPCK, to the apicoplast, but we were unable to delete DPCK, even in the presence of IPP. However, once the endogenous DPCK was complemented with the E. coli DPCK (EcDPCK), we were successful in deleting it. We were then able to show that DPCK activity is required for parasite survival through knockdown of the complemented EcDPCK. Additionally, we showed that DPCK enzyme activity remains functional and essential within the vesicles present after apicoplast disruption. These results demonstrate that while the apicoplast of blood-stage P. falciparum parasites can be disrupted, the resulting vesicles remain biochemically active and are capable of fulfilling essential functions.

Nucleotide analogues containing a pyrrolidine, piperidine or piperazine ring: Synthesis and evaluation of inhibition of plasmodial and human 6-oxopurine phosphoribosyltransferases and in vitro antimalarial activity.

Parasites of the Plasmodium genus are unable to produce purine nucleotides de novo and depend completely on the salvage pathway. This fact makes plasmodial hypoxanthine-guanine-(xanthine) phosphoribosyltransferase [HG(X)PRT] a valuable target for development of antimalarial agents. A series of nucleotide analogues was designed, synthesized and evaluated as potential inhibitors of Plasmodium falciparum HGXPRT, P. vivax HGPRT and human HGPRT. These novel nucleoside phosphonates have a pyrrolidine, piperidine or piperazine ring incorporated into the linker connecting the purine base to a phosphonate group(s) and exhibited a broad range of Ki values between 0.15 and 72 µM. The corresponding phosphoramidate prodrugs, able to cross cell membranes, have been synthesized and evaluated in a P. falciparum infected human erythrocyte assay. Of the eight prodrugs evaluated seven exhibited in vitro antimalarial activity with IC50 values within the range of 2.5-12.1 µM. The bis-phosphoramidate prodrug 13a with a mean (SD) IC50 of 2.5 ± 0.7 µM against the chloroquine-resistant P. falciparum W2 strain exhibited low cytotoxicity in the human hepatocellular liver carcinoma (HepG2) and normal human dermal fibroblasts (NHDF) cell lines at a concentration of 100 µM suggesting good selectivity for further structure-activity relationship investigations.

Discovery of new non-pyrimidine scaffolds as Plasmodium falciparum DHFR inhibitors by fragment-based screening.

In various malaria-endemic regions, the appearance of resistance has precluded the use of pyrimidine-based antifolate drugs. Here, a three-step fragment screening was used to identify new non-pyrimidine Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors. Starting from a 1163-fragment commercial library, a two-step differential scanning fluorimetry screen identified 75 primary fragment hits. Subsequent enzyme inhibition assay identified 11 fragments displaying IC50 in the 28-695 µM range and selectivity for PfDHFR. In addition to the known pyrimidine, three new anti-PfDHFR chemotypes were identified. Fragments from each chemotype were successfully co-crystallized with PfDHFR, revealing a binding in the active site, in the vicinity of catalytic residues, which was confirmed by molecular docking on all fragment hits. Finally, comparison with similar non-hit fragments provides preliminary input on available growth vectors for future drug development.

Discovery of Traditional Chinese Medicine Derived Compounds as Wild Type and Mutant Plasmodium falciparum Dihydrofolate Reductase Inhibitors: Induced Fit Docking and ADME Studies.

BACKGROUND: In a bid to come up with effective compounds as inhibitors for antimalarial treatment, we built a library of 2,000 traditional Chinese medicine(TCM)-derived compounds retrieved from TCM Database@Taiwan. METHODS: The active sites of both the wild type and mutant Plasmodium falciparum dihydrofolatereductase (pfDHFR) were explored using computational tools. pfDHFR, one of the prime drug targets in the prevention of malaria infection induced by the female anopheles mosquito has continued to offer resistance to drugs (antifolates) due to mutation in some of the key amino acid residues crucial for its inhibition. RESULTS: We utilized virtual throughput screening and glide XP docking to screen the compounds, and 8 compounds were found to have promising docking scores with both the wild type and mutant pfDHFR. They were further subjected to Induce Fit Docking (IFD) to affirm their inhibitory potency. The ADME properties and biological activity spectrum of the compounds were also considered. The inhibition profile of the compounds revealed that a number of compounds formed intermolecular interactions with ASP54, ILE14, LEU164, SER108/ASN108, ARG122 and ASP58. Most of the compounds can be considered as drug candidates due to their antiprotozoal activities and accordance with the Lipinski's Rule of Five (ROF). CONCLUSION: The outcome of the present study should further be investigated to attest the efficacy of these compounds as better drug candidates than the antifolates.

Plasmodium falciparum purine nucleoside phosphorylase as a model in the search for new inhibitors by high throughput screening.

Studies have shown that inhibition of Plasmodium falciparum Purine Nucleoside Phosphorylase (PfPNP) blocks the purine salvage pathway in vitro and in vivo. In this study, PfPNP was evaluated as a model in the search for new inhibitors using surface plasmon resonance (SPR). Its expression, purification, oligomeric state, kinetic constants, calorimetric parameters and kinetic mechanisms were obtained. PfPNP was immobilized on a CM5 sensor chip and sensorgrams were produced through binding the enzyme to the substrate MESG and interactions between molecules contained in 10 fractions of natural extracts. The oligomeric state showed that recombinant PfPNP is a hexamer. The true steady-state kinetic parameters for the substrate inosine were: KM 17 µM, kcat 1.2 s-1, VMax 2.2 U/mg and kcat/KM 7 × 10-4; for MESG they were: KM 131 µM, kcat 2.4 s-1, VMax 4.4 U/mg and kcat/KM 1.8 × 10-4. The thermodynamic parameters for the substrate Phosphate were: ΔG - 5.8 cal mol-1, ΔH - 6.5 cal mol-1 and ΔS - 2.25 cal mol-1/degree. The ITC results demonstrated that the binding of phosphate to free PfPNP led to a significant change in heat and association constants and thermodynamic parameters. A sequential ordered mechanism was proposed as the kinetic mechanism. Three plant extracts contained molecules capable of interacting with PfPNP, showing different levels of affinity. The identification of plant extract fractions containing molecules that interact with recombinant PfPNP using SRP validates this target as a model in the search for new inhibitors. In this study, we showed for the first time the true steady-state kinetic parameters for reactions catalyzed by PfPNP and a model using PfPNP as a target for High-throughput Screening for new inhibitors through SPR. This knowledge will allow for the development of more efficient research methods in the search for new drugs against malaria.

The cytochrome bc1 complex as an antipathogenic target.

The cytochrome bc1 complex is a key component of the mitochondrial respiratory chains of many eukaryotic microorganisms that are pathogenic for plants or humans, such as fungi responsible for crop diseases and Plasmodium falciparum, which causes human malaria. Cytochrome bc1 is an enzyme that contains two (ubi)quinone/quinol-binding sites, which can be exploited for the development of fungicidal and chemotherapeutic agents. Here, we review recent progress in determination of the structure and mechanism of action of cytochrome bc1 , and the associated development of antimicrobial agents (and associated resistance mechanisms) targeting its activity.

Identification of Plasmodium dipeptidyl aminopeptidase allosteric inhibitors by high throughput screening.

Dipeptidyl aminopeptidases (DPAPs) are cysteine proteases that cleave dipeptides from the N-terminus of protein substrates and have been shown to play important roles in many pathologies including parasitic diseases such as malaria, toxoplasmosis and Chagas's disease. Inhibitors of the mammalian homologue cathepsin C have been used in clinical trials as potential drugs to treat chronic inflammatory disorders, thus proving that these enzymes are druggable. In Plasmodium species, DPAPs play important functions at different stages of parasite development, thus making them potential antimalarial targets. Most DPAP inhibitors developed to date are peptide-based or peptidomimetic competitive inhibitors. Here, we used a high throughput screening approach to identify novel inhibitor scaffolds that block the activity of Plasmodium falciparum DPAP1. Most of the hits identified in this screen also inhibit Plasmodium falciparum DPAP3, cathepsin C, and to a lesser extent other malarial clan CA proteases, indicating that these might be general DPAP inhibitors. Interestingly, our mechanism of inhibition studies indicate that most hits are allosteric inhibitors, which opens a completely new strategy to inhibit these enzymes, study their biological function, and potentially develop new inhibitors as starting points for drug development.

Effect of Dihydroartemisinin on Plasmodium NADH-Dependent Glutamate Synthase: The Implication in Malaria Management.

Artemisinin and its analogues (ARTs) are currently the most effective anti-malarial drugs, but the precise mechanism of action is still highly controversial. Effects of ARTs on Plasmodium genes expression are studied in our Lab. The overexpression of an interesting amidotransferase, NADH-dependent glutamate synthase (NADH-GltS) was found in treated by dihydroartemisinin (DHA). The increased expression occurred not only from global transcriptomics analysis on the human malaria parasite Plasmodium falciparum (P. falciparum) 3D7 and gene expression screening on all of iron-sulphur cluster proteins from P.f. 3D7 in vitro but also from Plasmodium berghei (P. berghei) ANKA in mice. Influence of DHA on NADH-GltS was specifically at trophozoite stage of P. falciparum and in a dose-dependent manner below the effective doses. L-glutamine (Gln) and L-glutamate (Glu) are the substrate and product of NADH-GltS respectively. Azaserine (Aza) is specific inhibitor for NADH-GltS. Experimental data showed that Glu levels were significantly decreasing with DHA dose increasing but NADH-GltS enzyme activities were still remained at higher levels in parasites, and appropriate amount of exogenous Glu could significantly reduce anti-malarial action of DHA but excessive amount lost the above effect. Aza alone could inhibit proliferation of P. falciparum and had an additive effect in combination with DHA. Those results could suggest that: Glutamate depletion is one of the anti-malarial actions of DHA; overexpression of NADH-GltS would be a feedback pattern of parasite itself due to glutamate depletion, but not a direct action of DHA; the "feedback pattern" is one of protective strategies of Plasmodium to interfere with the anti-malarial actions of DHA; and specific inhibitor for NADH-GltS as a new type of anti-malarial agents or new partner in ACT might provide a potential.

Virtual Screening, Molecular Dynamics and ADME-Tox Tools for Finding Potential Inhibitors of Phosphoglycerate Mutase 1 from Plasmodium falciparum.

BACKGROUND: Nowadays, malaria is still one of the most important and lethal diseases worldwide, causing 445,000 deaths in a year. Due to the actual treatment resistance, there is an emergency to find new drugs. OBJECTIVE: The aim of this work was to find potential inhibitors of phosphoglycerate mutase 1 from P. falciparum. RESULTS: Through virtual screening of a chemical library of 15,123 small molecules, analyzed by two programs, four potential inhibitors of phosphoglycerate mutase 1 from P. falciparum were found: ZINC64219552, ZINC39095354, ZINC04593310, and ZINC04343691; their binding energies in SP mode were -7.3, -7.41, -7.4, and -7.18 kcal/mol respectively. Molecular dynamic analysis revealed that these molecules interact with residues important for enzyme catalysis and molecule ZINC04343691 provoked the highest structural changes. Physiochemical and toxicological profiles evaluation of these inhibitors with ADME-Tox method suggested that they can be considered as potential drugs. Furthermore, analysis of human PGAM-B suggested that these molecules could be selective for the parasitic enzyme. CONCLUSION: The compounds reported here are the first selective potential inhibitors of phosphoglycerate mutase 1 from P. falciparum, and can serve as a starting point in the search of a new chemotherapy against malaria.

Target Validation and Identification of Novel Boronate Inhibitors of the Plasmodium falciparum Proteasome.

The Plasmodium proteasome represents a potential antimalarial drug target for compounds with activity against multiple life cycle stages. We screened a library of human proteasome inhibitors (peptidyl boronic acids) and compared activities against purified P. falciparum and human 20S proteasomes. We chose four hits that potently inhibit parasite growth and show a range of selectivities for inhibition of the growth of P. falciparum compared with human cell lines. P. falciparum was selected for resistance in vitro to the clinically used proteasome inhibitor, bortezomib, and whole genome sequencing was applied to identify mutations in the proteasome ß5 subunit. Active site profiling revealed inhibitor features that enable retention of potent activity against the bortezomib-resistant line. Substrate profiling reveals P. falciparum 20S proteasome active site preferences that will inform attempts to design more selective inhibitors. This work provides a starting point for the identification of antimalarial drug leads that selectively target the P. falciparum proteasome.

Specific Stereoisomeric Conformations Determine the Drug Potency of Cladosporin Scaffold against Malarial Parasite.

The dependence of drug potency on diastereomeric configurations is a key facet. Using a novel general divergent synthetic route for a three-chiral center antimalarial natural product cladosporin, we built its complete library of stereoisomers (cladologs) and assessed their inhibitory potential using parasite-, enzyme-, and structure-based assays. We show that potency is manifest via tetrahyropyran ring conformations that are housed in the ribose binding pocket of parasite lysyl tRNA synthetase (KRS). Strikingly, drug potency between top and worst enantiomers varied 500-fold, and structures of KRS-cladolog complexes reveal that alterations at C3 and C10 are detrimental to drug potency whereas changes at C3 are sensed by rotameric flipping of glutamate 332. Given that scores of antimalarial and anti-infective drugs contain chiral centers, this work provides a new foundation for focusing on inhibitor stereochemistry as a facet of antimicrobial drug development.

Coptisine, a natural alkaloid from Coptidis Rhizoma, inhibits plasmodium falciparum dihydroorotate dehydrogenase.

Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) is a promising drug target for antimalarial chemotherapy. In our continuous efforts to develop more potent PfDHODH inhibitors, a unique library of active ingredients from traditional Chinese medicine (TCM) has been collected and was screened in this study. Through the initial screening, we found that coptisine, a natural alkaloid from TCM Coptidis Rhizoma, was a novel and potent inhibitor of PfDHODH with an IC50 value of 1.83 ± 0.08 µm. At the same time, enzyme kinetic analysis using Lineweaver-Burk plot indicated that coptisine is an uncompetitive inhibitor for PfDHODH. Thermal shift assay and molecular docking simulation research reveal that coptisine is capable of binding with PfDHODH. Moreover, coptisine exhibits weak inhibition activity against human DHODH, indicating that coptisine is a selective inhibitor of PfDHODH. Taken together, our study highlights the potential of active ingredients in TCM as valuable resource for discovering novel chemical scaffolds for PfDHODH.

Pyrazole Schiff Base Hybrids as Anti-Malarial Agents: Synthesis, In Vitro Screening and Computational Study.

BACKGROUND: Malaria is one of the most vital infectious diseases caused by protozoan parasites of the Plasmodium genus. As P. falciparum, the cause of most of the severe cases of malaria, is increasingly resistant to available drugs such as amodioquine, chloroquine, artemisinin, and antifolates, there is an urgent need to identify new targets for chemotherapy. OBJECTIVE: This study screened novel pyrazole derivatives carrying iminium & benzothiazole group for antimalarial potential against P. falciparum chloroquine sensitive (3D7) strain. MATERIALS & METHODS: Several pyrazole schiff base hybrids with a wide range of substitution have been synthesized via condensation of substituted aniline with substituted 4-formylpyrazole and evaluated for their in vitro antimalarial activity against asexual blood stages of human malaria parasite, Plasmodium falciparum. The interaction of these conjugate hybrids was also investigated by molecular docking studies in the binding site of P. falciparum cystein protease falcipain-2. The pharmacokinetic properties were also studied using ADME prediction. RESULTS: Among all compounds, 6bf and 6bd were found to be potential molecules with EC50 1.95µg/ml and 1.98µg/ml respectively. Docking study results reveal that the pyrazole schiff base derivatives occupy the PfFP binding sites and they show good interactions with significant values of binding energies. CONCLUSION: We provide evidence which implicates pyrazole Schiff base hybrids as potential prototypes for the development of antimalarial agents.

Identification of Collateral Sensitivity to Dihydroorotate Dehydrogenase Inhibitors in Plasmodium falciparum.

Drug resistance has been reported for every antimalarial in use highlighting the need for new strategies to protect the efficacy of therapeutics in development. We have previously shown that resistance can be suppressed with a population biology trap: by identifying situations where resistance to one compound confers hypersensitivity to another (collateral sensitivity), we can design combination therapies that not only kill the parasite but also guide its evolution away from resistance. We applied this concept to the Plasmodium falciparum dihydroorotate dehydrogenase ( PfDHODH) enzyme, a well validated antimalarial target with inhibitors in the development pipeline. Here, we report a high-throughput screen to identify compounds specifically active against PfDHODH resistant mutants. We additionally perform extensive cross-resistance profiling allowing us to identify compound pairs demonstrating the potential for mutually incompatible resistance. These combinations represent promising starting points for exploiting collateral sensitivity to extend the useful lifespan of new antimalarial therapeutics.

High throughput in silico identification and characterization of Plasmodium falciparum PRL phosphatase inhibitors.

Kinases and phosphatases are involved in many essential processes in Plasmodium lifecycle. Among the identified 67 Plasmodium falciparum phosphatases, Phosphatase of Regenerating Liver (PRL) family protein homolog, PfPRL, is an essential parasite tyrosine phosphatase. PfPRL is shown to be prenylated, secreted, and involved in the host invasion process. In the present study, a structure-based high throughput in silico screening of PfPRL binders, using ChEMBL-NTD compounds lead to the identification of nine compounds based on binding energy, Lipinski rule of five, and QED score. The most of the shortlisted compounds are known to inhibit parasite growth at a concentration (EC50) ≤2 µm in in vitro P. falciparum culture assays. MD simulations were carried out on the shortlisted nine potential enzyme-inhibitor complexes to analyze specificity, stability, and to calculate the free binding energies of the complexes. The study identifies PfPRL as one of the potential drug targets for selected ChEMBL-NTD compounds that may be exploited as a scaffold to develop novel antimalarials.

In silico 3-D structure prediction and molecular docking studies of inosine monophosphate dehydrogenase from Plasmodium falciparum.

Growing resistance in malarial parasites, particularly in Plasmodium falciparum needs a serious search for the discovery of novel drug targets. Inosine monophosphate dehydrogenase (IMPDH) is an important target for antimalarial drug discovery process in P. falciparum for the treatment of malaria. In the absence of x-ray crystal structure of this enzyme, homology modeling proved to be a reasonable alternate to study substrate binding mechanisms of this enzyme. In this study, a 3-D homology model for P. falciparum IMPDH was constructed taking human IMPDH (PDB code 1NF7) as template. Furthermore, an in-silico combinatorial library of ribavirin (RVP) derivatives (1347 molecules) was designed and virtually screened for ligands having selectively greater binding affinity with Plasmodium falciparum IMPDH relative to human IMPDH II. A total of five Ribavirin derivatives were identified as having greater binding affinity (-126 to -108Kcal/mol and -9.4 to -8.6Kcal/mol) with Plasmodium falciparum IMPDH. These five inhibitors should be used as selective and potent for Plasmodium falciparum IMPDH. Such type of study will provide information to synthetic medicinal chemist to enhance the potential of compounds (RVP derivatives) as chemotherapeutic agents to fight against the increasing burden of malarial infections.

Multicomplex-based pharmacophore modeling coupled with molecular dynamics simulations: An efficient strategy for the identification of novel inhibitors of PfDHODH.

Enormous efforts have been made in the past to identify novel scaffolds against the potential therapeutic target, Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH). Fourteen different organic molecules have been crystallized to understand the structural basis of the inhibition. However, the pharmacophoric studies carried out so far, have not exploited all the structural information simultaneously to identify the novel inhibitors. Therefore, an attempt was made to construct the pharmacophore hypotheses from the available PfDHODH structural proteome. Among the generated hypotheses, a representative hypothesis was employed as a primary filter to list the molecules with complimentary features accountable for inhibition. Moreover, the auxiliary evaluations of the filtered molecules were accomplished via docking and drug-likeness studies. Subsequently, the stability of the protein-ligand complex was evaluated by using molecular dynamics simulations (MDs). The molecular details of binding interactions of the potential hits were compared with the highly active experimental structure (5FI8) to seek the more potent candidates that can be targeted against PfDHODH. Overall, the combination of screening and stability procedures resulted in the identification of three potent candidates. The drug-likeness of these molecules lie within the acceptable range and consequently increased the opportunities for their development to new anti-malarials.