Structural Analysis of Plasmodium falciparum Hexokinase Provides Novel Information about Catalysis Due to a Plasmodium-Specific Insertion.

The protozoan parasite Plasmodium falciparum is the causative pathogen of the most severe form of malaria, for which novel strategies for treatment are urgently required. The primary energy supply for intraerythrocytic stages of Plasmodium is the production of ATP via glycolysis. Due to the parasite's strong dependence on this pathway and the significant structural differences of its glycolytic enzymes compared to its human counterpart, glycolysis is considered a potential drug target. In this study, we provide the first three-dimensional protein structure of P. falciparum hexokinase (PfHK) containing novel information about the mechanisms of PfHK. We identified for the first time a Plasmodium-specific insertion that lines the active site. Moreover, we propose that this insertion plays a role in ATP binding. Residues of the insertion further seem to affect the tetrameric interface and therefore suggest a special way of communication among the different monomers. In addition, we confirmed that PfHK is targeted and affected by oxidative posttranslational modifications (oxPTMs). Both S-glutathionylation and S-nitrosation revealed an inhibitory effect on the enzymatic activity of PfHK.

Chemistry of polyhalogenated nitrobutadienes, 17: Efficient synthesis of persubstituted chloroquinolinyl-1H-pyrazoles and evaluation of their antimalarial, anti-SARS-CoV-2, antibacterial, and cytotoxic activities.

A series of 26 novel 1-(7-chloroquinolin-4-yl)-4-nitro-1H-pyrazoles bearing a dichloromethyl and an amino or thio moiety at C3 and C5 has been prepared in yields up to 72% from the reaction of 1,1-bisazolyl-, 1-azolyl-1-amino-, and 1-thioperchloro-2-nitrobuta-1,3-dienes with 7-chloro-4-hydrazinylquinoline. A new way for the formation of a pyrazole cycle from 3-methyl-2-(2,3,3-trichloro-1-nitroallylidene)oxazolidine (6) is also described. In addition, the antimalarial activity of the synthesized compounds has been evaluated in vitro against the protozoan malaria parasite Plasmodium falciparum. Notably, the 7-chloro-4-(5-(dichloromethyl)-4-nitro-3-(1H-1,2,4-triazol-1-yl)-1H-pyrazol-1-yl)quinoline (3b) and 7-chloro-4-(3-((4-chlorophenyl)thio)-5-(dichloromethyl)-4-nitro-1H-pyrazol-1-yl)quinoline (9e) inhibited the growth of the chloroquine-sensitive Plasmodium falciparum strain 3D7 with EC50 values of 0.2 ± 0.1 µM (85 ng/mL, 200 nM) and 0.2 ± 0.04 µM (100 ng/mL, 200 nM), respectively. Two compounds (3b and 10d) have also been tested for anti-SARS-CoV-2, antibacterial, and cytotoxic activity.

Targeting spectrin redox switches to regulate the mechanoproperties of red blood cells.

The mechanical properties of red blood cells (RBCs) are fundamental for their physiological role as gas transporters. RBC flexibility and elasticity allow them to survive the hemodynamic changes in the different regions of the vascular tree, to dynamically contribute to the flow thereby decreasing vascular resistance, and to deform during the passage through narrower vessels. RBC mechanoproperties are conferred mainly by the structural characteristics of their cytoskeleton, which consists predominantly of a spectrin scaffold connected to the membrane via nodes of actin, ankyrin and adducin. Changes in redox state and treatment with thiol-targeting molecules decrease the deformability of RBCs and affect the structure and stability of the spectrin cytoskeleton, indicating that the spectrin cytoskeleton may contain redox switches. In this perspective review, we revise current knowledge about the structural and functional characterization of spectrin cysteine redox switches and discuss the current lines of research aiming to understand the role of redox regulation on RBC mechanical properties. These studies may provide novel functional targets to modulate RBC function, blood viscosity and flow, and tissue perfusion in disease conditions.

The C-terminus of Sudan ebolavirus VP40 contains a functionally important CXnC motif, a target for redox modifications.

The Ebola virus matrix protein VP40 mediates viral budding and negatively regulates viral RNA synthesis. The mechanisms by which these two functions are exerted and regulated are unknown. Using a high-resolution crystal structure of Sudan ebolavirus (SUDV) VP40, we show here that two cysteines in the flexible C-terminal arm of VP40 form a stabilizing disulfide bridge. Notably, the two cysteines are targets of posttranslational redox modifications and interact directly with the host`s thioredoxin system. Mutation of the cysteines impaired the budding function of VP40 and relaxed its inhibitory role for viral RNA synthesis. In line with these results, the growth of recombinant Ebola viruses carrying cysteine mutations was impaired and the released viral particles were elongated. Our results revealed the exact positions of the cysteines in the C-terminal arm of SUDV VP40. The cysteines and/or their redox status are critically involved in the differential regulation of viral budding and viral RNA synthesis.

Prominent role of cysteine residues C49 and C343 in regulating Plasmodium falciparum pyruvate kinase activity.

The protozoan parasite Plasmodium falciparum causes the most severe form of malaria and is highly dependent on glycolysis. Glycolytic enzymes were shown to be massively redox regulated, inter alia via oxidative post-translational modifications (oxPTMs) of their cysteine residues. In this study, we identified P. falciparum pyruvate kinase (PfPK) C49 and C343 as amino acid residues essentially involved in maintaining structural and functional integrity of the enzyme. The mutation of these cysteines resulted in an altered substrate affinity, lower enzymatic activities, and, as studied by X-ray crystallography, conformational changes within the A-domain where the substrate binding site is located. Although the loss of a cysteine evoked an impaired catalysis in both mutants, the effects observed for mutant C49A were more severe: multiple conformational changes, caused by the loss of two hydrogen bonds, impeded proper substrate binding and thus the transfer of phosphate upon catalysis.

Comparative Venom Proteomics of Iranian, Macrovipera lebetina cernovi, and Cypriot, Macrovipera lebetina lebetina, Giant Vipers.

Envenoming by Macrovipera lebetina subspecies causes severe life-threatening difficulties for people living in North Africa and the Middle East. To better understand the pathophysiology of envenoming and improve patient management, knowledge about the venom components of the subspecies is essential. Here, the venom proteomes of Macrovipera lebetina lebetina from Cyprus and Macrovipera lebetina cernovi from Iran were characterized using RP-HPLC separation of the crude venom proteins, SDS-PAGE of fractionated proteins, and LC-MS/MS of peptides obtained from in-gel tryptic digestion of protein bands. Moreover, we also used high-resolution shot-gun proteomics to gain more reliable identification, where the whole venom proteomes were subjected directly to in-solution digestion before LC-HR-MS/MS. The data revealed that both venoms consisted of at least 18 protein families, of which snake venom Zn2+-dependent metalloprotease (SVMP), serine protease, disintegrin, phospholipase A2, C-type lectin-like, and L-amino acid oxidase, together accounted for more than 80% of the venoms' protein contents. Although the two viper venoms shared mostly similar protein classes, the relative occurrences of these toxins were different in each snake subspecies. For instance, P-I class of SVMP toxins were found to be more abundant than P-III class in the venoms of M. l. cernovi compared to M. l. lebetina, which gives hints at a more potent myonecrotic effect and minor systemic hemorrhage following envenoming by M. l. cernovi than M. l. lebetina. Moreover, single-shot proteomics also revealed many proteins with low abundance (<1%) within the venoms, such as aminopeptidase, hyaluronidase, glutaminyl-peptide cyclotransferase, cystatin, phospholipase B, and vascular endothelial growth factor. Our study extends the in-depth understanding of the venom complexity of M. lebetina subspecies, particularly regarding toxin families associated with envenoming pathogenesis and those hard-detected protein classes expressed in trace amounts.

1,2,4-Triazole-3-thione compounds with a 4-ethyl alkyl/aryl sulfide substituent are broad-spectrum metallo-ß-lactamase inhibitors with re-sensitization activity.

Metallo-ß-lactamases (MBLs) are important contributors of Gram-negative bacteria resistance to ß-lactam antibiotics. MBLs are highly worrying because of their carbapenemase activity, their rapid spread in major human opportunistic pathogens while no clinically useful inhibitor is available yet. In this context, we are exploring the potential of compounds based on the 1,2,4-triazole-3-thione scaffold as an original ligand of the di-zinc active sites of MBLs, and diversely substituted at its positions 4 and 5. Here, we present a new series of compounds substituted at the 4-position by a thioether-containing alkyl chain with a carboxylic and/or an aryl group at its extremity. Several compounds showed broad-spectrum inhibition with Ki values in the µM to sub-µM range against VIM-type enzymes, NDM-1 and IMP-1. The presence of the sulfur and of the aryl group was important for the inhibitory activity and the binding mode of a few compounds in VIM-2 was revealed by X-ray crystallography. Importantly, in vitro antibacterial susceptibility assays showed that several inhibitors were able to potentiate the activity of meropenem on Klebsiella pneumoniae clinical isolates producing VIM-1 or VIM-4, with a potentiation effect of up to 16-fold. Finally, a selected compound was found to only moderately inhibit the di-zinc human glyoxalase II, and several showed no or only moderate toxicity toward several human cells, thus favourably completing a promising behaviour.

Synthesis and Evaluation of Antimycobacterial and Antiplasmodial Activities of Hirsutellide A and Its Analogues.

Hirsutellide A is nature-derived cyclic hexadepsipeptide with reported antimycobacterial and antiplasmodial activities. To verify its structure, hirsutellide A was synthesized following a solution-phase peptide synthesis approach. A detailed analysis of the 1H and 13C NMR spectra of the synthesized compound revealed structural variation from what had been originally assigned for hirsutellide A, despite the use of identical building blocks. This variation occurred at the two allo-Ile moieties. To investigate the structure-activity relationship, the depsipeptide and peptide analogues of hirsutellide A were prepared and tested for antimycobacterial and antiplasmodial activities. The compounds displayed antiplasmodial potency against Plasmodium falciparum 3D7 while showing weak or no activity against Mycobacterium tuberculosis H37Rv. The drug-likeness of the series was assessed through in vitro absorption, distribution, metabolism, and excretion (ADME) profiling, revealing systematic differences between the pharmacokinetic properties of cyclic hexapeptides and hexadepsipeptides.