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Cyclic nucleotide-dependent phosphodiesterases (PDEs) play essential roles in regulating the malaria parasite life cycle, suggesting that they may be promising antimalarial drug targets. PDE inhibitors are used safely to treat a range of noninfectious human disorders. Here, we report three subseries of fast-acting and potent Plasmodium falciparum PDEβ inhibitors that block asexual blood-stage parasite development and that are also active against human clinical isolates. Two of the inhibitor subseries also have potent transmission-blocking activity by targeting PDEs expressed during sexual parasite development. In vitro drug selection experiments generated parasites with moderately reduced susceptibility to the inhibitors. Whole-genome sequencing of these parasites detected no mutations in PDEβ but rather mutations in downstream effectors: either the catalytic or regulatory subunits of cyclic adenosine monophosphate-dependent protein kinase (PKA) or in the 3-phosphoinositide-dependent protein kinase that is required for PKA activation. Several properties of these P. falciparum PDE inhibitor series make them attractive for further progression through the antimalarial drug discovery pipeline.

The enzyme deoxyhypusine synthase (DHS) catalyzes the first step in the post-translational modification of the eukaryotic translation factor 5A (eIF5A). This is the only protein known to contain the amino acid hypusine, which results from this modification. Both eIF5A and DHS are essential for cell viability in eukaryotes, and inhibiting DHS is a promising strategy to develop new therapeutic alternatives. DHS proteins from many are sufficiently different from their human orthologs for selective targeting against infectious diseases; however, no DHS inhibitor selective for parasite orthologs has previously been reported. Here, we established a yeast surrogate genetics platform to identify inhibitors of DHS from Plasmodium vivax, one of the major causative agents of malaria. We constructed genetically modified Saccharomyces cerevisiae strains expressing DHS genes from Homo sapiens (HsDHS) or P. vivax (PvDHS) in place of the endogenous DHS gene from S. cerevisiae. Compared with a HsDHS complemented strain with a different genetic background that we previously generated, this new strain background was ~60-fold more sensitive to an inhibitor of human DHS. Initially, a virtual screen using the ChEMBL-NTD database was performed. Candidate ligands were tested in growth assays using the newly generated yeast strains expressing heterologous DHS genes. Among these, two showed promise by preferentially reducing the growth of the PvDHS-expressing strain. Further, in a robotized assay, we screened 400 compounds from the Pathogen Box library using the same S. cerevisiae strains, and one compound preferentially reduced the growth of the PvDHS-expressing yeast strain. Western blot revealed that these compounds significantly reduced eIF5A hypusination in yeast. The compounds showed antiplasmodial activity in the asexual erythrocyte stage; EC50 in high nM to low μM range, and low cytotoxicity. Our study demonstrates that this yeast-based platform is suitable for identifying and verifying candidate small molecule DHS inhibitors, selective for the parasite over the human ortholog., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fernandes Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A series of novel 4-acetyl-1,3,4-oxadiazole derivatives was designed and synthesized for their biological evaluation in vitro against Trypanosoma cruzi (T. cruzi) and Leishmania mexicana (L. mexicana). Additionally, all compounds were evaluated by molecular docking on the cruzain of T. cruzi (TcCz) and the cysteine protease B (CPB) of L. mexicana (LmCPB) to know their potential mechanism of binding. Compound OX-12 had better trypanocidal activity against NINOA (IC 50 =10.5 μM) and A1 (IC 50 =21.7 μM) T. cruzi strains that reference drug benznidazole (IC 50 =30.3 μM and 39.8 μM, respectively). Compound OX-2 had the best biological activity against L. mexicana in M379 (IC 50 =11.9 μM) and FCQEPS (IC 50 =34.0 μM) strains that the reference drug glucantime (IC 50 >120 μM). All the compounds showed important interactions with residues on the active site of TcCz (Gly66, Trp26, Leu67, and Ala138) and LmCPB (Gly67, Asn62, Leu68, and Ala140). Finally, the molecular dynamics simulations of the compound OX-12 shown moderate stability from 40-115 ns with an RMSD value of 6.5 Å. Meanwhile, compound OX-2 showed a minor stability in complex with CPB from 25-200 ns of simulation (RMSD<9 Å). These results encourage to develop more potent and efficient trypanocidal and leishmanicidal agents using the 1,3,4-oxadiazole scaffold., (© 2024 Wiley-VCH GmbH.)

The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti-Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis., (© 2024 The Protein Society.)

The onset of resistance to artemisinin for malaria treatment has stimulated the quest for novel antimalarial drugs. Herein, the gold(III) coordination complexes Aubipy [Au(bipy)Cl] + (bipy = 2,2'-bipyridine), Auphen [Au(phen)Cl] + (phen = phenanthroline), Auterpy [Au(terpy)Cl] 2+ (terpy = 2,2';6',2″-terpyridine), and corresponding hydrolyzed species, have been investigated as inhibitors of the Plasmodium falciparum aquaglyceroporin (PfAQP) protein by computational methods. Through an in-silico approach using an Umbrella Sampling protocol to sample how Aubipy, Auphen, and Auterpy permeate through the PfAQP, their permeability coefficients were estimated using the Inhomogeneous Solubility Diffusion (ISD) model with promising results. The efficacy of the gold complexes was then probed by an in vitro assay testing the growth inhibition in chloroquine sensitive and resistant P. falciparum strains. In accordance with the computational data, Auterpy achieved the highest efficiency with an IC 50 in the nanomolar range (590 nM) on resistant strain cultures, additionally revealing a good selectivity as compared to its activity against the human aquaglyceroporin 3., Competing Interests: Declarations. Conflict of interest: Ricardo J. Ferreira and Johan Wennerberg are currently employed by Red Glead Discovery AB, Lund, Sweden. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Consent for publication: Not applicable. Ethics approval and consent to participate: Not applicable., (© 2024. The Author(s), under exclusive licence to Society for Biological Inorganic Chemistry (SBIC).)

Leishmaniasis is a neglected tropical disease and is one of the major causes of mortality in poverty-stricken areas. A limited chemotherapeutics arsenal is available to tackle this deadly infection. Thus, identifying novel potent scaffolds using innovative strategies is the need of the hour. High-throughput screening (HTS) is a critical technique that can accelerate the process of drug discovery by evaluating millions of drug-like molecules using various automation tools and biological assays. In the present study, we have employed the HTS strategy to identify potent hits against Leishmania donovani sterol C-24 methyltransferase (LdSMT) from the in-house ChemBridge library. Firstly, a robust dataset was prepared with previously reported sterol C-24 methyltransferase inhibitors, belonging to diverse structural classes. Then, ligand-based virtual screening using similarity search was performed to screen the ChemBridge library having ∼20,000 molecules. This computational approach yielded 81 candidate compounds, which were selected for further molecular docking and biological evaluation. Anti-leishmanial assays revealed that out of 81 molecules, seven showed potential parasitic killing. Three molecules namely IIIM-CB-14, IIIM-CB-29, and IIIM-CB-45 were the most potent ones with 50 % inhibitory concentration (IC 50 ) of 5.76, 8.08, and 10.64 µg/mL, respectively. SEM analyses suggest that these potent hits cause considerable morphological alterations. ADME studies of the potent hit molecules indicate that all the hits have considerable drug-likeness properties. Further, molecular dynamics studies were also performed to check the stable confirmation of LdSMT protein with the top two hits (IIIM-CB-14 and IIIM-CB-45). Thus, the present study harnesses computational and experimental approaches to unravel potent anti-leishmanial scaffolds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Malaria affects almost 250 million people annually and continues to be a significant threat to global public health. Infection with protozoan parasites from the genus Plasmodium causes malaria. The primary treatment for malaria is artemisinin-based combination therapies (ACTs). The spread of ACT-resistant parasites has undermined efforts to control and eradicate malaria. Thus, it is crucial to identify new targets for the development of novel antimalarial drugs. Phosphate is an essential nutrient for all cells. The Plasmodium falciparum genome encodes a single sodium-coupled inorganic phosphate transporter named PfPiT that is essential for parasite proliferation in the asexual blood stage. Thus, PfPiT inhibitors may be promising antimalarial drugs. Like Plasmodium, yeast requires phosphate to grow. We developed a Saccharomyces cerevisiae based growth assay to identify inhibitors of PfPiT. Genome editing was used to create a yeast strain where PfPiT was the only phosphate transporter. Using a radioactive [ 32 P]phosphate uptake assay, the measured phosphate K m for PfPiT in yeast was 56 ± 7 μM in 1 mM NaCl at pH 7.4. The K m decreased to 24 ± 3 μM in 25 mM NaCl consistent with it being a Na +  coupled cotransporter. Conditions under which yeast growth was dependent on phosphate uptake mediated by PfPiT were identified and a 22-h growth assay was developed to screen for PfPiT inhibitors. In a screen of 21 compounds, two compounds were identified that inhibited the growth of the PfPiT strain but not that of the parental strain expressing Pho84, one of the five endogenous yeast phosphate transporters. Radioactive phosphate uptake experiments confirmed inhibition of phosphate uptake by the two compounds. The growth inhibition assay provides a simple and inexpensive approach to screen a large compound library for PfPiT inhibitors that may serve as starting points for the development of novel antimalarial drugs., Competing Interests: Declaration of competing interest The authors report no conflicts of interest relevant to this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Toxoplasmosis is a widespread parasitic disease, caused by Toxoplasma gondii, that affects nearly one-third of the human population. The lack of effective treatments drives the demand for novel anti-toxoplasmosis therapeutic options. In the present study, we used computational approaches and experimental validation to identify therapeutic inhibitors of toxoplasmosis. Initially, using the structure of the co-crystallized ligand of T. gondii calcium-dependent protein kinase 1 (TgCDPK1), we retrieved 3000 compounds from the database of COCONUT (COlleCtion of Open Natural ProdUcTs). These compounds were docked against the crystal structure of TgCDPK1 on the Glide Ligand Docking panel of Maestro 12.5 (Schrödinger Suite 2020-3). Based on the docking scores, we assessed promising molecules for toxicity potential on the ProTox-II online server, while the ADME profiling was done on the SwissADME server. Following the computational studies, we selected nine promising compounds for experimental validation against T. gondii in vitro. Of the compounds, C4, C5, C6, and C8 exhibited dose-dependent anti-T. gondii action with EC 50 values ranging from 3.3 to 120.2 μg/mL. Host toxicity profiling revealed differential cytotoxic action with a selectivity index (SI) of <1 for the compounds except C5, which had an SI of 1.8. To validate our screening assay, we used sulfadiazine, a standard drug for toxoplasmosis and showed that it inhibited parasite growth. Further experiments showed that C5, an imidazole-based natural compound, has strong but reversible anti-parasitic action that peaks within the first 8 h. In addition, C5 exhibited similar toxic tendencies towards T. gondii within (intracellular) and outside (extracellular) the host, suggesting it likely has a parasite target(s). C5 showed no effect on host invasion but strongly impeded parasite replication and growth, thereby affecting the T. gondii lytic cycle. Furthermore, C5 treatment raised the reactive oxygen species level, but this may be a secondary effect because augmentation with Trolox antioxidant failed to block C5 anti-T. gondii action. In addition, molecular dynamics simulations of C5 and TgCDPK1 complex revealed relative stability within 100 ns run time. Collectively, our findings support the potential of imidazole-based compounds as novel, alternative anti-parasitic agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The protozoan protein kinase calcium-dependent protein kinase 1 (CDPK1) has emerged as a potential therapeutic target for the treatment of cryptosporidiosis. A focused screen of known kinase inhibitors identified a pyridopyrimidinone as a new chemotype of Cryptosporidium parvum (Cp) CDPK1 inhibitors. Structural comparison of CpCDPK1 to two representative human kinases, RIPK2 and Src, revealed differences in the positioning of the αC-helix that was used in the design of a potent pyridopyrimidinone-based CpCDPK1 inhibitor 7 (a.k.a. UH15-16, IC 50 = 10 nM), which blocked the growth of three C. parvum strains (EC 50 = 12-40 nM) as well as C. hominis (EC 50 = 85 nM) in HCT-8 host cells. Pharmacokinetic and tissue distribution analyses indicated that 7 had low systemic exposure after oral administration, but high gastrointestinal concentration, as well as good Caco-2 cell permeability. Finally, 7 demonstrated partial efficacy in an IL-12 knock-out mouse model of acute cryptosporidiosis., Competing Interests: Declaration of Competing Interest WCVV is an owner/officer of ParaTheraTech Inc, a company which is seeking to bring bumped kinase (e.g. CDPK1) inhibitors to the animal health market. All other authors declare no interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Metacaspases are a distinct class of cysteine proteases predominantly found in plants, fungi, and protozoa, crucial for regulating programmed cell death (PCD). They possess unique structural features and differ markedly from caspases in their activation mechanisms and substrate specificities, with a notable preference for binding basic residues in substrates. In this study, we introduced vanillin-derived oximic compounds to explore their pharmaceutical potential. We evaluated these compounds for their inhibitory effects on TbMCA2, a metacaspase in Trypanosoma brucei, identifying AO-7, AO-12, and EO-20 as promising inhibitors. AO-12 showed significant potential as a non-competitive inhibitor with notable IC 50 values. Molecular docking studies were also conducted to evaluate the binding affinity of these compounds for TbMCA2. This research is particularly relevant given the urgent need for more effective and less toxic treatments for trypanosomiasis, a parasitic disease caused by trypanosomes. The absence of available vaccines and the limitations imposed by drug toxicity underscore the importance of these findings. Our study represents a significant advancement in developing therapeutic agents targeting metacaspases in trypanosomatids and highlights the necessity of understanding metacaspase regulation across various species. It provides valuable insights into inhibitor sensitivity and potential species-specific therapeutic strategies. In conclusion, this research opens promising avenues for novel therapeutic agents targeting metacaspases in trypanosomatids, addressing a critical gap in combating neglected diseases associated with these pathogens. Further research is essential to refine the efficacy and safety profiles of these compounds, aiming to deliver more accessible and effective therapeutic solutions to populations afflicted by these debilitating diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Malaria which is mainly caused by Plasmodium falciparum parasite remains a devastating public health concern, necessitating the need to develop new antimalarial agents. P. falciparum heat shock protein 90 (Hsp90), is indispensable for parasite survival and a promising drug target. Inhibitors targeting the ATP-binding pocket of the N-terminal domain have anti-Plasmodium effects. We proposed a de novo active learning (AL) driven method in tandem with docking to predict inhibitors with unique scaffolds and preferential selectivity towards PfHsp90. Reference compounds, predicted to bind PfHsp90 at the ATP-binding pocket and possessing anti-Plasmodium activities, were used to generate 10,000 unique derivatives and to build the Auto-quantitative structures activity relationships (QSAR) models. Glide docking was performed to predict the docking scores of the derivatives and > 15,000 compounds obtained from the ChEMBL database. Re-iterative training and testing of the models was performed until the optimum Kennel-based Partial Least Square (KPLS) regression model with a regression coefficient R2 = 0.75 for the training set and squared correlation prediction Q2 = 0.62 for the test set reached convergence. Rescoring using induced fit docking and molecular dynamics simulations enabled us to prioritize 15 ATP/ADP-like design ideas for purchase. The compounds exerted moderate activity towards P. falciparum NF54 strain with IC50 values of ≤ 6μM and displayed moderate to weak affinity towards PfHsp90 (KD range: 13.5-19.9μM) comparable to the reported affinity of ADP. The most potent compound was FTN-T5 (PfN54 IC50:1.44μM; HepG2/CHO cells SI≥ 29) which bound to PfHsp90 with moderate affinity (KD:7.7μM), providing a starting point for optimization efforts. Our work demonstrates the great utility of AL for the rapid identification of novel molecules for drug discovery (i.e., hit identification). The potency of FTN-T5 will be critical for designing species-selective inhibitors towards developing more efficient agents against malaria., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Matlhodi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The dual-target inhibitors (ZINC000008876351 and ZINC000253403245) were identified by utilizing an advanced computational drug discovery method by targeting two critical enzymes such as FeSODA (Iron superoxide dismutase) and TryR (Trypanothione reductase) within the antioxidant defense system of Leishmania donovani (Ld). In vitro enzyme inhibition kinetics reveals that both the compound's ability to inhibit the function of enzyme LdFeSODA and LdTryR with inhibition constant (Ki) value in the low μM range. Flow cytometry analysis, specifically at IC 50 and 2X IC 50 doses of both the compounds, the intracellular ROS was significantly increased as compared to the untreated control. The compounds ZINC000253403245 and ZINC000008876351 exhibited strong anti-leishmanial activity in a dose-dependent manner against both the promastigote and amastigote stages of the parasite. The data indicate that these molecules hold promise as potential anti-leishmanial agents for developing new treatments against visceral leishmaniasis, specifically targeting the LdFeSODA and LdTryR enzymes. Additionally, the in vitro MTT assay shows that combining these compounds with miltefosine produces a synergistic effect compared to miltefosine alone. This suggests that the compounds can boost miltefosine's effectiveness by synergistically inhibiting the growth of L. donovani promastigotes. Given the emergence of miltefosine resistance in some Leishmania strains, these findings are particularly significant., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Chagas disease is caused by the eukaryote parasite Trypanosoma cruzi . Current treatment exhibits limited efficacy and selenium-based compounds emerged as promising candidates for new therapies which is surpassing its bioisoster, sulfur. We designed new thiosemicarbazones, thiazoles, selenosemicarbazones and selenazoles, using isosteric substitution. We synthesized 57 new chalcogen compounds which were evaluated against T. cruzi , C2C12 cells and cruzain, the main target of this parasite. Additionally, human cathepsin L, was tested for selectivity. Three compounds were selected, based on their activity against the intracellular amastigotes (EC 50 < 1 μM, SI > 10) and cruzain (IC 50 < 100 nM, SI > 5.55) which compared favorably with the approved drug, Benznidazole, and the well-established cruzain inhibitor K777. Seleno-compounds demonstrated enhanced activity and selenazoles showed a decrease in selenium-associated toxicity. Compound 4-methyl-2-(2-(1-(3-nitrophenyl)ethylidene)hydrazineyl)-1,3-selenazole ( Se 2h ) emerged as a promising candidate, and its binding to cruzain was investigated. Pharmacokinetic assessment was conducted, showing a favorable profile for subsequent in vivo assays.

Malaria still causes over 600,000 deaths annually, with rising resistance to frontline drugs by Plasmodium falciparum increasing this number each year. New medicines with novel mechanisms of action are, therefore, urgently needed. In this work, we solved the cocrystal structure of the essential malarial kinase Pf CLK3 with the reversible inhibitor TCMDC-135051 ( 1 ), enabling the design of covalent inhibitors targeting a unique cysteine residue (Cys368) poorly conserved in the human kinome. Chloroacetamide 4 shows nanomolar potency and covalent inhibition in both recombinant protein and P. falciparum assays. Efficacy in parasites persisted after a 6 h washout, indicating an extended duration of action. Additionally, 4 showed improved kinase selectivity and a high selectivity index against HepG2 cells, with a low propensity for resistance (log MIR > 8.1). To our knowledge, compound 4 is the first covalent inhibitor of a malarial kinase, offering promising potential as a lead for a single-dose malaria cure.

Leishmania, a protozoan parasite, is responsible for significant morbidity and mortality worldwide, manifesting as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases pose a substantial burden, especially in impoverished regions with limited access to effective medical treatments. Current therapies are toxic, have low efficacy, and face growing resistance. Understanding the metabolic pathways of Leishmania, particularly those differing from its host, can unveil potential therapeutic targets. In this study, we investigated the acetyl-CoA synthetase (ACS) enzyme from Leishmania infantum (LiAcs1), which, unlike many organisms, also exhibits acetoacetyl-CoA synthetase (KBC) activity. This dual functionality is unique among acyl-CoA/NRPS/luciferase superfamily enzymes and crucial for the parasite's reliance on leucine catabolism, energy production, and sterol biosynthesis. Our biochemical characterization of LiAcs1 revealed its ability to utilize both acetate and acetoacetate substrates. Additionally, LiAcs1 displayed a distinct CoA substrate inhibition pattern, partially alleviated by acetoacetate. Structural analysis provided insights into the substrate binding flexibility of LiAcs1, highlighting a more promiscuous substrate pocket compared to other ACS- or KBC-specific enzymes. Substrate mimetics elucidated its ability to accommodate both small and large AMP-ester derivatives, contributing to its dual ACS/KBC functionality. These findings not only advance our understanding of Leishmania metabolism but also present LiAcs1 as a promising drug target. The dual functionality of LiAcs1 underscores the potential for developing selective inhibitors that could disrupt critical metabolic pathways across Leishmania spp. as it appears this enzyme is highly conserved across this genus. This paves the way for developing novel effective treatments against this devastating disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Visceral leishmaniasis is a life-threatening parasitic disease, but current antileishmanial drugs have severe drawbacks. Antifungal azoles inhibit the activity of cytochrome P450 (CYP) 51 enzymes which are responsible for removing the C14α-methyl group of lanosterol, a key step in ergosterol biosynthesis in Leishmania. However, they exhibit varying degrees of antileishmanial activities in culture, suggesting the existence of unrecognized molecular targets. Our previous study reveals that, in Leishmania, lanosterol undergoes parallel C4- and C14-demethylation to form 4α,14α-dimethylzymosterol and T-MAS, respectively. In the current study, CYP5122A1 is identified as a sterol C4-methyl oxidase that catalyzes the sequential oxidation of lanosterol to form C4-oxidation metabolites. CYP5122A1 is essential for both L. donovani promastigotes in culture and intracellular amastigotes in infected mice. CYP5122A1 overexpression results in growth delay, increased tolerance to stress, and altered expression of lipophosphoglycan and proteophosphoglycan. CYP5122A1 also helps to determine the antileishmanial effect of antifungal azoles in vitro. Dual inhibitors of CYP51 and CYP5122A1 possess superior antileishmanial activity against L. donovani promastigotes whereas CYP51-selective inhibitors have little effect on promastigote growth. Our findings uncover the critical biochemical and biological role of CYP5122A1 in L. donovani and provide an important foundation for developing new antileishmanial drugs by targeting both CYP enzymes., (© 2024. The Author(s).)

The natural product 9-methoxystrobilurin G (9MG) from Favolaschia spp basidiomycetes is a potent and selective antimalarial. The mechanism of action of 9MG is unknown. We induced 9MG resistance in Plasmodium falciparum 3D7 and Dd2 strains and identified mutations associated with resistance by genome sequencing. All 9MG-resistant clones possessed missense mutations in the cytochrome b (CYTB) gene, a key component of mitochondrial complex III. The mutations map to the quinol oxidation site of CYTB, which is also the target of antimalarials such as atovaquone. In a complementary approach to identify protein targets of 9MG, a photoactivatable derivative of 9MG was synthesized and applied in chemoproteomic-based target profiling. Three components of mitochondrial complex III (QCR7, QCR9, and COX15) were specifically enriched consistent with 9MG targeting CYTB and complex III function in P. falciparum . Inhibition of complex III activity by 9MG was confirmed by ubiquinone cytochrome c reductase assay using P. falciparum extract. The findings from this study may be useful for developing novel antimalarials targeting CYTB.

Inhibition of the lactate transporter PfFNT is a valid novel mode of action against malaria parasites. Current pyridine-substituted pentafluoro-3-hydroxy-pent-2-en-1-ones act as substrate analogs with submicromolar EC 50 in vitro, and >99.7% activity in mice. The recently solved structure of a PfFNT-inhibitor complex visualized the binding mode. Here, we extended the inhibitor layout by series of amine- and anilide-linked pyridine p-substituents to generate additional interactions in the cytoplasmic vestibule. Virtual docking indicated hydrogen bonding to Tyr31 and Ser102. Fluorescence cross-correlation spectroscopy yielded respectively enhanced target affinity. Strikingly, the in vitro activity increased by 1 order of magnitude to 14.8 nM at negligible cytotoxicity. While p -amine substitutions were rapidly metabolized, the more stable p -acetanilide cleared 99.7% of parasites at 4 × 50 mg kg -1 in a mouse malaria model. Future stabilization of the p-substitution against metabolism may translate the gain in in vitro potency to the in vivo situation.

Since Chagas disease, melioidosis, and Legionnaires' disease are all potentially life-threatening infections, there is an urgent need for new treatment strategies. All causative agents, Trypanosoma cruzi , Burkholderia pseudomallei , and Legionella pneumophila , express a virulence factor, the macrophage infectivity potentiator (MIP) protein, emerging as a promising new therapeutic target. Inhibition of MIP proteins having a peptidyl-prolyl isomerase activity leads to reduced viability, proliferation, and cell invasion. The affinity of a series of pipecolic acid-type MIP inhibitors was evaluated against all MIPs using a fluorescence polarization assay. The analysis of structure-activity relationships led to highly active inhibitors of MIPs of all pathogens, characterized by a one-digit nanomolar affinity for the MIPs and a very effective inhibition of their peptidyl-prolyl isomerase activity. Docking studies, molecular dynamics simulations, and quantum mechanical calculations suggest an extended σ-hole of the meta -halogenated phenyl sulfonamide to be responsible for the high affinity.

The methyl-d-erythritol phosphate (MEP) pathway has emerged as an interesting target in the fight against antimicrobial resistance. The pathway is essential in many human pathogens, including Plasmodium falciparum ( Pf ), but is absent in human cells. In the present study, we report on the discovery of a new chemical class targeting IspD, the third enzyme in the pathway. Exploration of the structure-activity relationship yielded inhibitors with potency in the low-nanomolar range. Moreover, we investigated the whole-cell activity, mode of inhibition, metabolic, and plasma stability of this compound class, and conducted in vivo pharmacokinetic profiling on selected compounds. Lastly, we disclosed a new mass spectrometry (MS)-based enzymatic assay for direct IspD activity determination, circumventing the need for auxiliary enzymes. In summary, we have identified a readily synthesizable compound class, demonstrating excellent activity and a promising profile, positioning it as a valuable tool compound for advancing research on IspD.

Plasmodium parasite resistance to antimalarial drugs is a serious threat to public health in malaria-endemic areas. Compounds that target core cellular processes like translation are highly desirable, as they should be capable of killing parasites in their liver and blood stage forms, regardless of molecular target or mechanism. Assays that can identify these compounds are thus needed. Recently, specific quantification of native Plasmodium berghei liver stage protein synthesis, as well as that of the hepatoma cells supporting parasite growth, was achieved via automated confocal feedback microscopy of the o-propargyl puromycin (OPP)-labeled nascent proteome, but this imaging modality is limited in throughput. Here, we developed and validated a miniaturized high content imaging (HCI) version of the OPP assay that increases throughput, before deploying this approach to screen the Pathogen Box. We identified only two hits; both of which are parasite-specific quinoline-4-carboxamides, and analogs of the clinical candidate and known inhibitor of blood and liver stage protein synthesis, DDD107498/cabamiquine. We further show that these compounds have strikingly distinct relationships between their antiplasmodial and translation inhibition efficacies. These results demonstrate the utility and reliability of the P. berghei liver stage OPP HCI assay for the specific, single-well quantification of Plasmodium and human protein synthesis in the native cellular context, allowing the identification of selective Plasmodium translation inhibitors with the highest potential for multistage activity., Competing Interests: The authors declare no conflict of interest.

Protein arginine methyltransferases (PRMTs) play critical roles in Plasmodium falciparum , a protozoan causing the deadliest form of malaria, making them potential targets for novel antimalarial drugs. Here, we screened 11 novel PRMT inhibitors against P. falciparum asexual growth and found that onametostat, an inhibitor for type II PRMTs, exhibited strong antimalarial activity with a half-maximal inhibitory concentration (IC 50 ) value of 1.69 ± 0.04 µM. In vitro methyltransferase activities of purified PfPRMT5 were inhibited by onametostat, and a shift of IC 50 to onametostat was found in the PfPRTM5 disruptant parasite line, indicating that PfPRTM5 is the primary target of onametostat. Consistent with the function of PfPRMT5 in mediating symmetric dimethylation of histone H3R2 (H3R2me2s) and in regulating invasion-related genes, onametostat treatment led to the reduction of H3R2me2s level in P. falciparum and caused the defects on the parasite's invasion of red blood cells. This study provides a starting point for identifying specific PRMT inhibitors with the potential to serve as novel antimalarial drugs., Competing Interests: The authors declare no conflict of interest.

The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB's low specificity for Tv20S compared to the human proteasome and demonstrate CP-17's higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis., (© 2024. The Author(s).)

Toxoplasmosis persists as a prevalent disease, facing challenges from parasite resistance and treatment side effects. Consequently, identifying new drugs by exploring novel protein targets is essential for effective intervention. Cyclosporin A (CsA) possesses antiparasitic activity against Toxoplasma gondii, with cyclophilins identified as possible targets. However, CsA immunosuppressive nature hinders its use as an antitoxoplasmosis agent. Here, we evaluate the potential of three CsA derivatives devoid of immunosuppressive activity, namely, NIM811, Alisporivir, and dihydrocyclosporin A to target a previously characterized cyclophilin from Toxoplasma gondii (TgCyp23). We determined the X-ray crystal structures of TgCyp23 in complex with the three analogs and elucidated their binding and inhibitory properties. The high resolution of the structures revealed the precise positioning of ligands within the TgCyp23 binding site and the details of protein-ligand interactions. A comparison with the established ternary structure involving calcineurin indicates that substitutions at position 4 in CsA derivatives prevent calcineurin binding. This finding provides a molecular explanation for why CsA analogs can target Toxoplasma cyclophilins without compromising the human immune response., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Plasmodium vivax is a major cause of malaria, which poses an increased health burden on approximately one third of the world's population due to climate change. Primaquine, the preferred treatment for P. vivax malaria, is contraindicated in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, a common genetic cause of hemolytic anemia, that affects ∼2.5% of the world's population and ∼8% of the population in areas of the world where P. vivax malaria is endemic. The Seattle Structural Genomics Center for Infectious Disease (SSGCID) conducted a structure-function analysis of P. vivax N-myristoyltransferase (PvNMT) as part of efforts to develop alternative malaria drugs. PvNMT catalyzes the attachment of myristate to the N-terminal glycine of many proteins, and this critical post-translational modification is required for the survival of P. vivax. The first step is the formation of a PvNMT-myristoyl-CoA binary complex that can bind to peptides. Understanding how inhibitors prevent protein binding will facilitate the development of PvNMT as a viable drug target. NMTs are secreted in all life stages of malarial parasites, making them attractive targets, unlike current antimalarials that are only effective during the plasmodial erythrocytic stages. The 2.3 Å resolution crystal structure of the ternary complex of PvNMT with myristoyl-CoA and a novel inhibitor is reported. One asymmetric unit contains two monomers. The structure reveals notable differences between the PvNMT and human enzymes and similarities to other plasmodial NMTs that can be exploited to develop new antimalarials., (open access.)

There is an urgent need to develop new, safer, and more effective drugs against Chagas disease (CD) as well as related kinetoplastid diseases. Targeting and inhibiting the Trypanosoma cruzi proteasome has emerged as a promising therapeutic approach in this context. To expand the chemical space for this class of inhibitors, we performed virtual screening campaigns with emphasis on shape-based similarity and ADMET prioritization. We describe the ideation and application of robustly validated shape queries for these campaigns, which furnished 44 compounds for biological evaluation. Five hit compounds demonstrated in vitro antitrypanosomal activity by potential inhibition of T. cruzi proteasome and notable chemical diversities, particularly, LCQFTC11. Structural insights were achieved by homology modeling, sequence/structure alignment, proteasome-species comparison, docking, molecular dynamics, and MMGBSA binding affinity estimations. These methods confirmed key interactions as well as the stability of LCQFTC11 at the β4/β5 subunits' binding site of the T. cruzi proteasome, consistent with known inhibitors. Our results warrant future assay confirmation of our hit as a T. cruzi proteasome inhibitor. Importantly, we also shed light into dynamic details for a proteasome inhibition mechanism that shall be further investigated. We expect to contribute to the development of viable CD drug candidates through such a relevant approach., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Malaria is a disease caused mostly by Plasmodium falciparum, affects millions of people each year. The kinases are validated targets for malaria infection. In this study, we investigate for real and hypothetical compounds that can inhibit cyclic guanosine monophosphate (CGMP)-dependent protein kinase using molecular docking via combined similarity analysis, molecular dynamics simulations, quantitative structure activity relationship (QSAR). Using Tanimoto similarity scores, ∼8.4 million compounds were screened. Compounds that have at least 70% similarity are used in further analysis. These compounds are assessed by means of docking, MMBPSA, MMGBSA and ANI&#95;LIE. Based on consensus of different free energy methods and docking we revealed two potential inhibitors that can be useful for treatment of malaria. Apart from screening of real compounds, we have also selected the 10 most plausible hypothetical compounds by performing QSAR. By QSAR proposed pharmacophores, we generated over 247 hypothetical compounds and among them 19 molecules with lower QSAR predicted IC50 values and high docking scores were selected for further analysis. We selected the top 10 inhibitor candidates and performed MD simulations for free energy calculations like the protocol applied for real compounds. According to the free energy calculations, we suggest 2 real (C 34 H 29 F 5 N 8 O 4 S and C 30 H 27 F 2 N 7 O 2 S 2 , PubChem IDs: 140564801 and 89035196, respectively) and 2 hypothetical (C 23 H 27 FN 6 O 2 S, MOL3 and C 23 H 25 FN 6 O 2 S, MOL4) compounds that can be effective inhibitors against the protein kinase of Plasmodium falciparum.Communicated by Ramaswamy H. Sarma.

Chagas disease has grown into a serious public health threat, with a high morbidity rate, major social impact, and global neglect. Therapeutic adhesion, unwanted side-effects, and resistance make its present therapy ineffective. Discovery of more effective drugs is hence needed. Using natural compounds conjointly with computational methods helps better to find promising compounds, speeding up drug discovery process and reducing its cost. In the present study, a docking protocol against cruzipain (PDB: 3l06), an important druggable target, was applied to a library of 50 sorted natural compounds. Compounds were further analysed for binding mode and interactions with cruzipain active site, conformational alignment studies and in-silico pharmacokinetic studies so as to predict their plausible anti-cruzipain mechanism. The results provided computational insights into the molecular interaction of naturals against T. cruzi cruzipain. Study also lead to identification of Hinokiflavone; BA = -10.2 kcal mol -1 as reasonably promising potential natural cruzipain inhibitor.

Cryptosporidium parvum (C. parvum), a protozoan parasite, is known to induce significant gastrointestinal disease in humans. Lactate dehydrogenase (LDH), a protein of C. parvum, has been identified as a potential therapeutic target for developing effective drugs against infection. This study utilized a computational drug discovery approach to identify potential drug molecules against the LDH protein of C. parvum. In the present investigation, we conducted a structure-based virtual screening of 55 phytochemicals from the Syzygium aromaticum (S. aromaticum). This process identified four phytochemicals, including Gallotannin 23, Eugeniin, Strictinin, and Ellagitannin, that demonstrated significant binding affinity and dynamic stability with LDH protein. Interestingly, these four compounds have been documented to possess antibacterial, antiviral, anti-inflammatory, and antioxidant properties. The docked complexes were simulated for 100 ns using Desmond to check the dynamic stability. Finally, the free binding energy was computed from the last 10ns MD trajectories. Gallotannin 23 and Ellagitannin exhibited considerable binding affinity and stability with the target protein among all four phytochemicals. These findings suggest that these predicted phytochemicals from S. aromaticum could be further explored as potential hit candidates for developing effective drugs against C. parvum infection. The in vitro and in vivo experimental validation is still required to confirm their efficacy and safety as LDH inhibitors., Competing Interests: Declaration of competing interest The author(s) declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

The majority of anti-leishmanial drugs used for treating trypanosomatid parasites help to reduce human morbidity and mortality. However, parasites have developed drug resistance, which has made it challenging to treat leishmaniasis. Therefore, new drugs and drug targets need to be identified. Protein synthesis is a crucial anabolic mechanism necessary for parasite survival. Histidyl-tRNA synthetase (HisRS) is an essential enzyme that is required for histidine incorporation into proteins. Recent studies on HisRS have shown differences between trypanosomatid HisRS and human HisRS, which could lead to the development of trypanosomatid HisRS structure-based inhibitors. This study aims to determine the role of L. donovani HisRS (LdHisRS) in parasite growth and virulence in vitro using RNAi. The silencing effect of LdHisRS expression was determined using qPCR. The results showed that after 24 and 48 h of incubation with 90 ng siRNAs, LdHisRS mRNA expression levels were significantly reduced by ∼3.14-fold and ∼3.90-fold, respectively. SiRNA-treated parasites also exhibited ∼46.6 % delayed growth and ∼47 % reduced virulence. Additionally, homology modeling, virtual screening, and molecular docking studies were performed with potential inhibitors that have significant suppressive activity in bacteria, fungi, and viruses. Halofuginone was found to have the best binding affinity of -9.09 kcal/mol as a potent inhibitor against LdHisRS. The molecular dynamics (MD) results showed that halofuginone could interact with the various active site segments, potentially blocking substrate access. The data on gene silencing through siRNA suggests that LdHisRS is essential for the parasite's growth and survival. The computational findings could lead to the development of a potent ligand (halofuginone) as a future anti-leishmanial drug, paving the way for an effective therapeutic treatment., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Vikas Kushwaha reports financial support was provided by University Grant Commission, Government of India under the D.S. Kothari Post-Doctoral Fellowship (Grant number.F.4-2/2006 (BSR)/BL/18–19/0387). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

RNA editing pathway is a validated target in kinetoplastid parasites ( Trypanosoma brucei , Trypanosoma cruzi , and Leishmania spp.) that cause severe diseases in humans and livestock. An essential large protein complex, the editosome, mediates uridine insertion and deletion in RNA editing through a stepwise process. This study details the discovery of editosome inhibitors by screening a library of widely used human drugs using our previously developed in vitro biochemical Ribozyme Insertion Deletion Editing (RIDE) assay. Subsequent studies on the mode of action of the identified hits and hit expansion efforts unveiled compounds that interfere with RNA-editosome interactions and novel ligase inhibitors with IC 50 values in the low micromolar range. Docking studies on the ligase demonstrated similar binding characteristics for ATP and our novel epigallocatechin gallate inhibitor. The inhibitors demonstrated potent trypanocidal activity and are promising candidates for drug repurposing due to their lack of cytotoxic effects. Further studies are necessary to validate these targets using more definitive gene-editing techniques and to enhance the safety profile.

Cruzipain (CZP), the major cysteine protease present in T. cruzi , the ethiological agent of Chagas disease, has attracted particular attention as a therapeutic target for the development of targeted covalent inhibitors (TCI). The vast chemical space associated with the enormous molecular diversity feasible to explore by means of modern synthetic approaches allows the design of CZP inhibitors capable of exhibiting not only an efficient enzyme inhibition but also an adequate translation to anti- T. cruzi activity. In this work, a computer-aided design strategy was developed to combinatorially construct and screen large libraries of 1,4-disubstituted 1,2,3-triazole analogues, further identifying a selected set of candidates for advancement towards synthetic and biological activity evaluation stages. In this way, a virtual molecular library comprising more than 75 thousand diverse and synthetically feasible analogues was studied by means of molecular docking and molecular dynamic simulations in the search of potential TCI of CZP, guiding the synthetic efforts towards a subset of 48 candidates. These were synthesized by applying a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) centered synthetic scheme, resulting in moderate to good yields and leading to the identification of 12 hits selectively inhibiting CZP activity with IC 50 in the low micromolar range. Furthermore, four triazole derivatives showed good anti- T. cruzi inhibition when studied at 50 μM; and Ald-6 excelled for its high antitrypanocidal activity and low cytotoxicity, exhibiting complete in vitro biological activity translation from CZP to T. cruzi . Overall, not only Ald-6 merits further advancement to preclinical in vivo studies, but these findings also shed light on a valuable chemical space where molecular diversity might be explored in the search for efficient triazole-based antichagasic agents.

The human malaria parasite Plasmodium falciparum (P. falciparum) continues to pose a significant public health challenge, leading to millions of fatalities globally. Halofuginone (HF) has shown a significant anti-P. falciparum effect, suggesting its potential as a therapeutic agent for malaria treatment. In this study, we synthesized a photoaffinity labeling probe of HF to identify its direct target in P. falciparum. Our results reveal that ubiquitin carboxyl-terminal hydrolase 3 (PfUCHL3) acts as a crucial target protein of HF, which modulates parasite growth in the intraerythrocytic cycle. In particular, we discovered that HF potentially forms hydrogen bonds with the Leu10, Glu11, and Arg217 sites of PfUCHL3, thereby inducing an allosteric effect by promoting the embedding of the helix 6' region on the protein surface. Furthermore, HF disrupts the expression of multiple functional proteins mediated by PfUCHL3, specifically those that play crucial roles in amino acid biosynthesis and metabolism in P. falciparum. Taken together, this study highlights PfUCHL3 as a previously undisclosed druggable target of HF, which contributes to the development of novel anti-malarial agents in the future., (© 2024 Wiley-VCH GmbH.)

Background: Leishmaniasis is a deadly protozoan parasitic disease and a significant health problem in underdeveloped and developing countries. The global spread of the parasite, coupled with the emergence of drug resistance and severe side effects associated with existing treatments, has necessitated the identification of new and potential drugs., Objective: This study aimed to identify promising compounds for the treatment of leishmaniasis by targeting two essential enzymes of Leishmania donovani: trypanothione reductase (Try-R) and trypanothione synthetase (Try-S)., Methods: High-throughput virtual and in vitro screening of in-house and commercial databases was conducted. A pharmacophore model with seven features was developed and validated using the Guner-Henery method. The pharmacophore-based virtual screening yielded 690 hits, which were further filtered through Lipinski's rule, ADMET analysis, and molecular docking against Try-R and Try-S. Molecular dynamics studies were performed on selected compounds, and in vitro experiments were conducted to evaluate their activity against the promastigote and amastigote forms of L. donovani., Results: The virtual screening and subsequent analysis identified 33 promising compounds. Molecular dynamics studies of two compounds (comp-1 and comp-2) demonstrated stable binding interactions with the target enzymes and high affinity. In vitro experiments revealed that 13 compounds exhibited moderate activity against both the promastigote (IC 50 , 41 µM-76 µM) and the amastigote (IC 50 , 44 µM-72 µM) forms of L. donovani. Compounds 1 and 2 showed the highest percent inhibition and the lowest IC 50 values., Conclusion: The identified compounds demonstrated significant inhibitory activity against Leishmania donovani and stable interactions with target enzymes. These findings suggest that the compounds could serve as promising leads for developing new treatments for leishmaniasis., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

In the absence of an efficacious vaccine, chemotherapy remains crucial to prevent and treat malaria. Given its key role in haemoglobin degradation, falcilysin constitutes an attractive target. Here, we reveal the mechanism of enzymatic inhibition of falcilysin by MK-4815, an investigational new drug with potent antimalarial activity. Using X-ray crystallography, we determine two binary complexes of falcilysin in a closed state, bound with peptide substrates from the haemoglobin α and β chains respectively. An antiparallel β-sheet is formed between the substrate and enzyme, accounting for sequence-independent recognition at positions P2 and P1. In contrast, numerous contacts favor tyrosine and phenylalanine at the P1' position of the substrate. Cryo-EM studies reveal a majority of unbound falcilysin molecules adopting an open conformation. Addition of MK-4815 shifts about two-thirds of falcilysin molecules to a closed state. These structures give atomic level pictures of the proteolytic cycle, in which falcilysin interconverts between a closed state conducive to proteolysis, and an open conformation amenable to substrate diffusion and products release. MK-4815 and quinolines bind to an allosteric pocket next to a hinge region of falcilysin and hinders this dynamic transition. These data should inform the design of potent inhibitors of falcilysin to combat malaria., (© 2024. The Author(s).)

The Plasmodium falciparum mitochondrial electron transport chain (mETC) is responsible for essential metabolic pathways such as de novo pyrimidine synthesis and ATP synthesis. The mETC complex III (cytochrome bc 1 complex) is responsible for transferring electrons from ubiquinol to cytochrome c and generating a proton gradient across the inner mitochondrial membrane, which is necessary for the function of ATP synthase. Recent studies have revealed that the composition of Plasmodium falciparum complex III (PfCIII) is divergent from humans, highlighting its suitability as a target for specific inhibition. Indeed, PfCIII is the target of the clinically used anti-malarial atovaquone and of several inhibitors undergoing pre-clinical trials, yet its role in parasite biology has not been thoroughly studied. We provide evidence that the universally conserved subunit, PfRieske, and the new parasite subunit, PfC3AP2, are part of PfCIII, with the latter providing support for the prediction of its divergent composition. Using inducible depletion, we show that PfRieske, and therefore, PfCIII as a whole, is essential for asexual blood stage parasite survival, in line with previous observations. We further found that depletion of PfRieske results in gametocyte maturation defects. These phenotypes are linked to defects in mitochondrial functions upon PfRieske depletion, including increased sensitivity to mETC inhibitors in asexual stages and decreased cristae abundance alongside abnormal mitochondrial morphology in gametocytes. This is the first study that explores the direct role of the PfCIII in gametogenesis via genetic disruption, paving the way for a better understanding of the role of mETC in the complex life cycle of these important parasites and providing further support for the focus of antimalarial drug development on this pathway.

Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Å resolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-β-thia and β-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors., (© 2024 The Author(s).)

Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles ( 1 ) and 2-guanidino benzimidazoles ( 2 ), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of Tb DHFR and Tb PTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent ( K i = 9 nM) and highly selective Tb DHFR inhibitor, 6000-fold over Tb PTR1 and 394-fold over h DHFR. The 5,6-dimethyl tricyclic analogue 1g , despite showing a lower potency and selectivity profile than 2g , shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.

Plasmodium falciparum subtilisin-like serine protease 1 (PfSUB1) is essential for egress of invasive merozoite forms of the parasite, rendering PfSUB1 an attractive antimalarial target. Here, we report studies aimed to improve drug-like properties of peptidic boronic acid PfSUB1 inhibitors including increased lipophilicity and selectivity over human proteasome (H20S). Structure-activity relationship investigations revealed that lipophilic P 3 amino acid side chains as well as N -capping groups were well tolerated in retaining PfSUB1 inhibitory potency. At the P 1 position, replacing the methyl group with a carboxyethyl substituent led to boralactone PfSUB1 inhibitors with remarkably improved selectivity over H20S. Combining lipophilic end-capping groups with the boralactone reduced the selectivity over H20S. However, compound 4c still showed >60-fold selectivity versus H20S and low nanomolar PfSUB1 inhibitory potency. Importantly, this compound inhibited the growth of a genetically modified P. falciparum line expressing reduced levels of PfSUB1 13-fold more efficiently compared to a wild-type parasite line.

Bromodomains are structural folds present in all eukaryotic cells that bind to other proteins recognizing acetylated lysines. Most proteins with bromodomains are part of nuclear complexes that interact with acetylated histone residues and regulate DNA replication, transcription, and repair through chromatin structure remodeling. Bromodomain inhibitors are small molecules that bind to the hydrophobic pocket of bromodomains, interfering with the interaction with acetylated histones. Using a fluorescent probe, we have developed an assay to select inhibitors of the bromodomain factor 2 of Trypanosoma cruzi ( Tc BDF2) using fluorescence polarization. Initially, a library of 28,251 compounds was screened in an endpoint assay. The top 350-ranked compounds were further analyzed in a dose-response assay. From this analysis, seven compounds were obtained that had not been previously characterized as bromodomain inhibitors. Although these compounds did not exhibit significant trypanocidal activity, all showed bona fide interaction with Tc BDF2 with dissociation constants between 1 and 3 µM validating these assays to search for bromodomain inhibitors., Competing Interests: The authors declare no conflict of interest.

Owing to its life cycle involving multiple hosts and species-specific biological complexities, a vaccine against Plasmodium , the causative agent of Malaria remains elusive. This makes chemotherapy the only viable means to address the clinical manifestations and spread of this deadly disease. However, rapid surge in antimalarial resistance poses significant challenges to our efforts to eliminate Malaria since the best drug available to-date; Artemisinin and its combinations are also rapidly losing efficacy. Sodium ATPase ( Pf ATP4) of Plasmodium has been recently explored as a suitable target for new antimalarials such as Cipargamin . Prior studies showed that multiple compounds from the Medicines for Malaria Venture (MMV) chemical libraries were efficient Pf ATP4 inhibitors. In this context, we undertook a structure- based virtual screening approach combined to Molecular Dynamic (MD) simulations to evaluate whether new molecules with binding affinity towards Pf ATP4 could be identified from the Pandemic Response Box (PRB), a 400-compound library of small molecules launched in 2019 by MMV. Our analysis identified new molecules from the PRB library that showed affinity for distinct binding sites including the previously known G358 site, several of which are clinically used anti-bacterial (MMV1634383, MMV1634402), antiviral (MMV010036, MMV394033) or antifungal (MMV1634494) agents. Therefore, this study highlights the possibility of exploiting PRB molecules against Malaria through abrogation of Pf ATP4 activity.Communicated by Ramaswamy H. Sarma.

Plasmodium falciparum aminoacyl tRNA synthetases (PfaaRSs) are potent antimalarial targets essential for proteome fidelity and overall parasite survival in every stage of the parasite's life cycle. So far, some of these proteins have been singly targeted yielding inhibitor compounds that have been limited by incidences of resistance which can be overcome via pan-inhibition strategies. Hence, herein, for the first time, we report the identification and in vitro antiplasmodial validation of Mitomycin (MMC) as a probable pan-inhibitor of class 1a (arginyl(A)-, cysteinyl(C), isoleucyl(I)-, leucyl(L), methionyl(M), and valyl(V)-) PfaaRSs which hypothetically may underlie its previously reported activity on the ribosomal RNA to inhibit protein translation and biosynthesis. We combined multiple in silico structure-based discovery strategies that first helped identify functional and druggable sites that were preferentially targeted by the compound in each of the plasmodial proteins: Ins1-Ins2 domain in Pf-ARS; anticodon binding domain in Pf-CRS; CP1-editing domain in Pf-IRS and Pf-MRS; C-terminal domain in Pf-LRS; and CP-core region in Pf-VRS. Molecular dynamics studies further revealed that MMC allosterically induced changes in the global structures of each protein. Likewise, prominent structural perturbations were caused by the compound across the functional domains of the proteins. More so, MMC induced systematic alterations in the binding of the catalytic nucleotide and amino acid substrates which culminated in the loss of key interactions with key active site residues and ultimate reduction in the nucleotide-binding affinities across all proteins, as deduced from the binding energy calculations. These altogether confirmed that MMC uniformly disrupted the structure of the target proteins and essential substrates. Further, MMC demonstrated IC 50  < 5 μM against the Dd2 and 3D7 strains of parasite making it a good starting point for malarial drug development. We believe that findings from our study will be important in the current search for highly effective multi-stage antimalarial drugs., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum ( Pf A-M1) and Plasmodium vivax ( Pv A-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets Pf A-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on Pf A-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of Pf A-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy., Competing Interests: CG, MC, GS, RE, TM, CW, ND, NV, CM, NC, SD, SW, SD, VA, TD, PS, SM, DC No competing interests declared, (© 2024, Giannangelo, Challis et al.)

Aminoacylation by tRNA synthetase is a crucial part of protein synthesis and is widely recognized as a therapeutic target for drug development. Unlike the arginyl-tRNA synthetases (ArgRSs) reported previously, here, we report an ArgRS of Leishmania donovani (LdArgRS) that can follow the canonical two-step aminoacylation process. Since a previously uncharacterized insertion region is present within its catalytic domain, we implemented the splicing by overlap extension PCR (SOE-PCR) method to create a deletion mutant (ΔIns-LdArgRS) devoid of this region to investigate its function. Notably, the purified LdArgRS and ΔIns-LdArgRS exhibited different oligomeric states along with variations in their enzymatic activity. The full-length protein showed better catalytic efficiency than ΔIns-LdArgRS, and the insertion region was identified as the tRNA binding domain. In addition, a benzothiazolo-coumarin derivative (Comp-7j) possessing high pharmacokinetic properties was recognized as a competitive and more specific inhibitor of LdArgRS than its human counterpart. Removal of the insertion region altered the mode of inhibition for ΔIns-LdArgRS and caused a reduction in the inhibitor's binding affinity. Both purified proteins depicted variances in the secondary structural content upon ligand binding and thus, thermostability. Apart from the trypanosomatid-specific insertion and Rossmann fold motif, LdArgRS revealed typical structural characteristics of ArgRSs, and Comp-7j was found to bind within the ATP binding pocket. Furthermore, the placement of tRNA Arg near the insertion region enhanced the stability and compactness of LdArgRS compared to other ligands. This study thus reports a unique ArgRS with respect to catalytic as well as structural properties, which can be considered a plausible drug target for the derivation of novel anti-leishmanial agents., (© 2024 Federation of European Biochemical Societies.)

Background Objectives: The battle against malaria has witnessed remarkable progress in recent years, characterized by increased funding, development of life-saving tools, and a significant reduction in disease prevalence. Yet, the formidable challenge of drug resistance persists, threatening to undo these gains., Methods: To tackle this issue, it is imperative to identify new effective drug candidates against the malaria parasite that exhibit minimal toxicity. This study focuses on discovering such candidates by targeting PfRecQ1, also known as PfBLM, a vital protein within the malaria parasite Plasmodium falciparum . PfRecQ1 plays a crucial role in the parasite's life cycle and DNA repair processes, making it an attractive drug development target. The study employs advanced computational techniques, including molecular modeling, structure-based virtual screening (SBVS), ADMET profiling, molecular docking, and dynamic simulations., Results: The study sources ligand molecules from the extensive MCULE database and utilizes strict filters to ensure that the compounds meet essential criteria. Through these techniques, the research identifies MCULE-3763806507-0-9 as a promising antimalarial drug candidate, surpassing the binding affinity of potential antimalarial drugs. However, it is essential to underscore that drug-like properties are primarily based on in silico experiments, and wet lab experiments are necessary to validate these candidates' therapeutic potential., Interpretation Conclusion: This study represents a critical step in addressing the challenge of drug resistance in the fight against malaria., (Copyright © 2024 Copyright: © 2024 Journal of Vector Borne Diseases.)

Toxoplasma gondii , an important opportunistic pathogen, underscores the necessity of developing novel therapeutic drugs and identifying new drug targets. Our findings indicate that the half-maximal inhibitory concentrations (IC 50 ) of KU60019 and CP466722 (abbreviated as KU and CP) against T. gondii are 0.522 μM and 0.702 μM, respectively, with selection indices (SI) of 68 and 10. Treatment with KU and CP affects the in vitro growth of T. gondii , inducing aberrant division in the daughter parasites. Transmission electron microscopy reveals that KU and CP prompt the anomalous division of T. gondii , accompanied by cellular enlargement, nuclear shrinkage, and an increased dense granule density, suggesting potential damage to parasite vesicle transport. Subsequent investigations unveil their ability to modulate the expression of certain secreted proteins and FAS II (type II fatty acid synthesis) in T. gondii , as well as including the dot-like aggregation of the autophagy-related protein ATG8 (autophagy-related protein 8), thereby expediting programmed death. Leveraging DARTS (drug affinity responsive target stability) in conjunction with 4D-Label-free quantitative proteomics technology, we identified seven target proteins binding to KU, implicated in pivotal biological processes such as the fatty acid metabolism, mitochondrial ATP transmission, microtubule formation, and Golgi proteins transport in T. gondii . Molecular docking predicts their good binding affinity. Furthermore, KU has a slight protective effect on mice infected with T. gondii . Elucidating the function of those target proteins and their mechanism of action with ATM kinase inhibitors may potentially enhance the treatment paradigm for toxoplasmosis.

Aminoacyl-tRNA synthetases (aaRSs) play a central role in the translation of genetic code, serving as attractive drug targets. Within this family, the lysyl-tRNA synthetase (LysRS) constitutes a promising antimalarial target. ASP3026, an anaplastic lymphoma kinase (ALK) inhibitor was recently identified as a novel Plasmodium falciparum LysRS (PfLysRS) inhibitor. Here, based on cocrystal structures and biochemical experiments, we developed a series of ASP3026 analogues to improve the selectivity and potency of LysRS inhibition. The leading compound 36 showed a dissociation constant of 15.9 nM with PfLysRS. The inhibitory efficacy on PfLysRS and parasites has been enhanced. Covalent attachment of L-lysine to compound 36 resulted in compound 36K3, which exhibited further increased inhibitory activity against PfLysRS but significantly decreased activity against ALK. However, its inhibitory activity against parasites did not improve, suggesting potential future optimization directions. This study presents a new example of derivatization of kinase inhibitors repurposed to inhibit aaRS., (© 2024. The Author(s).)

Palmitoylation is an essential post-translational modification in Leishmania donovani , catalyzed by enzymes called palmitoyl acyl transferases (PATs) and has an essential role in virulence. Due to the toxicity and promiscuity of known PAT inhibitors, identification of new molecules is needed. Herein, we identified a specific novel de novo peptide inhibitor, PS1, against the PAT6 Leishmania donovani palmitoyl acyl transferase ( Ld PAT6). To demonstrate specific inhibition of Ld PAT6 by PS1, we employed a bacterial orthologue system and metabolic labeling-coupled click chemistry where both Ld PAT6 and PS1 were coexpressed and displayed palmitoylation suppression. Furthermore, strong binding of the Ld PAT6-DHHC domain with PS1 was observed through analysis using microscale thermophoresis, ELISA, and dot blot assay. PS1 specific to Ld PAT6 showed significant growth inhibition in promastigotes and amastigotes by expressing low cytokines levels and invasion. This study reveals discovery of a novel de novo peptide against Ld PAT6-DHHC which has potential to block survivability and infectivity of L. donovani .

Leishmaniasis is a neglected tropical disease; there is currently no vaccine and treatment is reliant upon a handful of drugs suffering from multiple issues including toxicity and resistance. There is a critical need for development of new fit-for-purpose therapeutics, with reduced toxicity and targeting new mechanisms to overcome resistance. One enzyme meriting investigation as a potential drug target in Leishmania is M17 leucyl-aminopeptidase (LAP). Here, we aimed to chemically validate LAP as a drug target in L. major through identification of potent and selective inhibitors. Using RapidFire mass spectrometry, the compounds DDD00057570 and DDD00097924 were identified as selective inhibitors of recombinant Leishmania major LAP activity. Both compounds inhibited in vitro growth of L. major and L. donovani intracellular amastigotes, and overexpression of Lm LAP in L. major led to reduced susceptibility to DDD00057570 and DDD00097924, suggesting that these compounds specifically target Lm LAP. Thermal proteome profiling revealed that these inhibitors thermally stabilized two M17 LAPs, indicating that these compounds selectively bind to enzymes of this class. Additionally, the selectivity of the inhibitors to act on Lm LAP and not against the human ortholog was demonstrated, despite the high sequence similarities LAPs of this family share. Collectively, these data confirm Lm LAP as a promising therapeutic target for Leishmania spp. that can be selectively inhibited by drug-like small molecules.

To combat the global burden of malaria, development of new drugs to replace or complement current therapies is urgently required. Here, we show that the compound MMV1557817 is a selective, nanomolar inhibitor of both Plasmodium falciparum and Plasmodium vivax aminopeptidases M1 and M17, leading to inhibition of end-stage hemoglobin digestion in asexual parasites. MMV1557817 can kill sexual-stage P. falciparum , is active against murine malaria, and does not show any shift in activity against a panel of parasites resistant to other antimalarials. MMV1557817 -resistant P. falciparum exhibited a slow growth rate that was quickly outcompeted by wild-type parasites and were sensitized to the current clinical drug, artemisinin. Overall, these results confirm MMV1557817 as a lead compound for further drug development and highlights the potential of dual inhibition of M1 and M17 as an effective multi-species drug-targeting strategy.IMPORTANCEEach year, malaria infects approximately 240 million people and causes over 600,000 deaths, mostly in children under 5 years of age. For the past decade, artemisinin-based combination therapies have been recommended by the World Health Organization as the standard malaria treatment worldwide. Their widespread use has led to the development of artemisinin resistance in the form of delayed parasite clearance, alongside the rise of partner drug resistance. There is an urgent need to develop and deploy new antimalarial agents with novel targets and mechanisms of action. Here, we report a new and potent antimalarial compound, known as MMV1557817 , and show that it targets multiple stages of the malaria parasite lifecycle, is active in a preliminary mouse malaria model, and has a novel mechanism of action. Excitingly, resistance to MMV15578117 appears to be self-limiting, suggesting that development of the compound may provide a new class of antimalarial., Competing Interests: The authors declare no conflict of interest.