Reinvestigation of diphenylmethylpiperazine analogues of pyrazine as new class of Plasmodial cysteine protease inhibitors for the treatment of malaria.

Malaria eradication is still a global challenge due to the lack of a broadly effective vaccine and the emergence of drug resistance to most of the currently available drugs as part of the mainline artemisinin-based combination therapy. A variety of experimental approaches are quite successful in identifying and synthesizing new promising pharmacophore hybrids with distinct mechanisms of action. Based on our recent findings, the current study demonstrates the reinvestigation of a series of diphenylmethylpiperazine and pyrazine-derived molecular hybrids. Pyrazine-derived molecular hybrids were screened to investigate the antiplasmodial activity on drug-susceptible Pf3D7 and drug-resistant PfW2 strains. The selected compounds were shown to be potent dual inhibitors of cysteine protease PfFP2 and PfFP3. Time-course parasitic development study demonstrated that compounds were able to arrest the growth of the parasite at the early trophozoite stage. The compounds did not show hemolysis of red blood cells and showed selectivity to the parasite compared with the mammalian Vero and A5489 cell lines. The study underlined HR5 and HR15 as a new class of Plasmodial falcipain inhibitors with an IC50 of 6.2 µM and 5.9 µM for PfFP2 and 6.8 µM and 6.4 µM for PfFP3, respectively. Both compounds have antimalarial efficacy with IC50 values of 3.05 µM and 2.80 µM for the Pf3D7 strain, and 4.35 µM and 3.39 µM for the PfW2 strain, respectively. Further structural optimization may turn them into potential Plasmodial falcipain inhibitors for malaria therapeutics.

Plasmodium falciparum contains functional SCF and CRL4 ubiquitin E3 ligases, and CRL4 is critical for cell division and membrane integrity.

Protein ubiquitination is essential for cellular homeostasis and regulation of several processes, including cell division and genome integrity. Ubiquitin E3 ligases determine substrate specificity for ubiquitination, and Cullin-RING E3 ubiquitin ligases (CRLs) make the largest group among the ubiquitin E3 ligases. Although conserved and most studied in model eukaryotes, CRLs remain underappreciated in Plasmodium and related parasites. To investigate the CRLs of human malaria parasite Plasmodium falciparum, we generated parasites expressing tagged P. falciparum cullin-1 (PfCullin-1), cullin-2 (PfCullin-2), Rbx1 (PfRbx1) and Skp1 (PfSkp1). PfCullin-1 and PfCullin-2 were predominantly expressed in erythrocytic trophozoite and schizont stages, with nucleocytoplasmic localization and chromatin association, suggesting their roles in different cellular compartments and DNA-associated processes. Immunoprecipitation, in vitro protein-protein interaction, and ubiquitination assay confirmed the presence of a functional Skp1-Cullin-1-Fbox (PfSCF) complex, comprising of PfCullin-1, PfRbx1, PfSkp1, PfFBXO1, and calcyclin binding protein. Immunoprecipitation, sequence analysis, and ubiquitination assay indicated that PfCullin-2 forms a functional human CRL4-like complex (PfCRL4), consisting of PfRbx1, cleavage and polyadenylation specificity factor subunit_A and WD40 repeat proteins. PfCullin-2 knock-down at the protein level, which would hinder PfCRL4 assembly, significantly decreased asexual and sexual erythrocytic stage development. The protein levels of several pathways, including protein translation and folding, lipid biosynthesis and transport, DNA replication, and protein degradation were significantly altered upon PfCullin-2 depletion, which likely reflects association of PfCRL4 with multiple pathways. PfCullin-2-depleted schizonts had poorly delimited merozoites and internal membraned structures, suggesting a role of PfCRL4 in maintaining membrane integrity. PfCullin-2-depleted parasites had a significantly lower number of nuclei/parasite than the normal parasites, indicating a crucial role of PfCRL4 in cell division. We demonstrate the presence of functional CRLs in P. falciparum, with crucial roles for PfCRL4 in cell division and maintaining membrane integrity.

Development of diphenylmethylpiperazine hybrids of chloroquinoline and triazolopyrimidine using Petasis reaction as new cysteine proteases inhibitors for malaria therapeutics.

Malaria is a widespread infectious disease, causing nearly 247 million cases in 2021. The absence of a broadly effective vaccine and rapidly decreasing effectiveness of most of the currently used antimalarials are the major challenges to malaria eradication efforts. To design and develop novel antimalarials, we synthesized a series of 4,7-dichloroquinoline and methyltriazolopyrimidine analogues using a multi-component Petasis reaction. The synthesized molecules (11-31) were screened for in-vitro antimalarial activity against drug-sensitive and drug-resistant strains of Plasmodium falciparum with an IC50 value of 0.53 µM. The selected compounds were screened to evaluate in-vitro and in-silico enzyme inhibition efficacy against two cysteine proteases, PfFP2 and PfFP3. The compounds 15 and 17 inhibited PfFP2 with an IC50 = 3.5 and 4.8 µM, respectively and PfFP3 with an IC50 = 4.9 and 4.7 µM, respectively. Compounds 15 and 17 were found equipotent against the Pf3D7 strain with an IC50 value of 0.74 µM, whereas both were displayed IC50 values of 1.05 µM and 1.24 µM for the PfW2 strain, respectively. Investigation of effect of compounds on parasite development demonstrated that compounds were able to arrest the growth of the parasites at trophozoite stage. The selected compounds were screened for in-vitro cytotoxicity against mammalian lines and human red-blood-cell (RBC), which demonstrated no significant cytotoxicity associated with the molecules. In addition, in silico ADME prediction and physiochemical properties supported the drug-likeness of the synthesized molecules. Thus, the results highlighted the diphenylmethylpiperazine group cast on 4,7-dichloroquinoline and methyltriazolopyrimidine using Petasis reaction may serve as models for the development of new antimalarial agents.

A comparative study of antibody response, virus neutralization efficiency & metabolites in SARS-CoV-2-infected adults & children.

Background & objectives: COVID-19 has been a global pandemic since early 2020. It has diverse clinical manifestations, but consistent immunological and metabolic correlates of disease severity and protection are not clear. This study was undertaken to compare seropositivity rate, antibody levels against nucleocapsid and spike proteins, virus neutralization and metabolites between adult and child COVID-19 patients. Methods: Plasma samples from naïve control (n=14) and reverse transcription (RT)-PCR positive COVID-19 participants (n=132) were tested for reactivity with nucleocapsid and spike proteins by ELISA, neutralization of SARS-CoV-2 infectivity in Vero cells and metabolites by [1]H nuclear magnetic resonance (NMR) spectroscopy. Results: An ELISA platform was developed using nucleocapsid and spike proteins for COVID-19 serosurvey. The participants showed greater seropositivity for nucleocapsid (72%) than spike (55.3%), and males showed higher seropositivity than females for both the proteins. Antibody levels to both the proteins were higher in intensive care unit (ICU) than ward patients. Children showed lower seropositivity and antibody levels than adults. In contrast to ICU adults (81.3%), ICU children (33.3%) showed lower seropositivity for spike. Notably, the neutralization efficiency correlated with levels of anti-nucleocapsid antibodies. The levels of plasma metabolites were perturbed differentially in COVID-19 patients as compared with the naive controls. Interpretation & conclusions: Our results reflect the complexity of human immune response and metabolome to SARS-CoV-2 infection. While innate and cellular immune responses are likely to be a major determinant of disease severity and protection, antibodies to multiple viral proteins likely affect COVID-19 pathogenesis. In children, not adults, lower seropositivity rate for spike was associated with disease severity.

Inhalation dose due to residential radon and thoron exposure in rural areas: a case study at Erravalli and Narasannapet model villages of Telangana state, India.

Exposure to indoor radon has been identified as a cause of lung cancer. The corresponding inhalation radiation dose received is an important parameter in estimating the risk of cancer due to the inhalation of radon. The present investigation is aimed at the estimation of the radiation dose due to radon, its isotopes, and progeny to the public residing in dwellings constructed in model villages of Telangana state, India. The indoor activity concentrations of radon and thoron were measured using pin-hole dosimeters. The measured activities along with appropriate dose conversion and occupancy factors were used in the estimation of the dose received by the dwellers. The doses estimated were compared with those to inhabitants of control dwellings. The estimated doses received by the public due to radon were found to be 1.54 ± 0.60 mSv and 1.51 ± 1.20 mSv, in the investigated model houses and in the control dwellings, respectively. Correspondingly, radiation doses due to thoron were found to be 1.08 ± 0.81 mSv and 1.44 ± 1.04 mSv, respectively. It is concluded that the model dwellings pose no extra radiation burden to the public.

The p38 MAP kinase inhibitor, PD 169316, inhibits flagellar motility in Leishmania donovani.

Mitogen-activated protein kinases (MAPKs) have been demonstrated to regulate flagellar/ciliary motility of spermatozoa and miracidia of Schistosoma mansoni. However, the role of MAPKs in mediating flagella-driven motility of Leishmania donovani is unexplored. We investigated the function of MAPKs in motility regulation of L. donovani using pharmacological inhibitors and activators of various MAPKs and fast-capture videomicroscopy. Our studies have revealed that the inhibitor of p38 MAPK, PD 169316, significantly affected various motility parameters such as flagellar beat frequency, parasite swimming speed, waveform of the flagellum and resulted in reduced parasite motility. Together, our results suggest that a MAPK, similar to human p38 MAPK, is implicated in flagellar motility regulation of L. donovani.

Characterization of ciliobrevin A mediated dynein ATPase inhibition on flagellar motility of Leishmania donovani.

Axonemal dyneins are members of AAA+ proteins involved in force generation and are responsible for flagellar motility in eukaryotes. In this study, we characterized the effects of ciliobrevin A (CbA), a dynein ATPase inhibitor, on flagella driven motility of the protozoan parasite Leishmania donovani. Using fast-capture video microscopy, we observed that CbA decreased flagellar beat frequency of swimming parasites in a concentration-dependent manner. Beat frequency of live and reactivated L. donovani decreased by approximately 89% and 41% respectively in the presence of 250µM CbA. This inhibition was lost when CbA was removed, suggesting its effects were reversible. CbA also altered wavelength and amplitude of the flagellum of live parasites. Waveform analysis of live and reactivated L. donovani revealed that CbA significantly affected flagellar waveform by inducing non-uniform bends with the flagellum beating away from the cell axis. These results suggest that CbA sensitive dynein ATPases possibly are responsible for power generation and waveform maintenance of the flagellum of L. donovani. This ability to inhibit axonemal dyneins also emphasizes the use of dynein inhibitors as valuable tools in studying functional roles of axonemal dyneins of flagellated eukaryotes.