Mechanistic and structural insights into vitamin B2 metabolizing enzyme riboflavin kinase from Leishmania donovani.

Leishmania donovani relies on specific vitamins and cofactors crucial for its survival and pathogenesis. Tailoring therapies to disrupt these pathways offers a promising strategy for the treatment of Visceral Leishmaniasis. Current treatment regimens are limited due to drug resistance and high costs. The dependency of Leishmania parasites on Vitamin B2 and its metabolic products is not known. In this study, we have biochemically and biophysically characterized a Vitamin B2 metabolism enzyme, riboflavin kinase from L. donovani (LdRFK) which converts riboflavin (vitamin B2) into flavin mononucleotide (FMN). Sequence comparison with human counterpart reflects 31.58 % identity only, thus opening up the possibility of exploring it as drug target. The rfk gene was cloned, expressed and the recombinant protein was purified. Kinetic parameters of LdRFK were evaluated with riboflavin and ATP as substrates which showed differential binding affinity when compared with the human RFK enzyme. Thermal and denaturant stability of the enzyme was evaluated. The rfk gene was overexpressed in the parasites and its role in growth and cell cycle was evaluated. In the absence of crystal structure, homology modelling and molecular dynamic simulation studies were performed to predict LdRFK structure. The data shows differences in substrate binding between human and parasite enzyme. This raises the possibility of exploring LdRFK for specific designing of antileishmanial molecules. Gene disruption studies can further validate its candidature as antileishmanial target.

A comprehensive review on the current status of CRISPR based clinical trials for rare diseases.

A considerable fraction of population in the world suffers from rare diseases. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its related Cas proteins offer a modern form of curative gene therapy for treating the rare diseases. Hereditary transthyretin amyloidosis, hereditary angioedema, duchenne muscular dystrophy and Rett syndrome are a few examples of such rare diseases. CRISPR/Cas9, for example, has been used in the treatment of ß-thalassemia and sickle cell disease (Frangoul et al., 2021; Pavani et al., 2021) [1,2]. Neurological diseases such as Huntington's have also been focused in some studies involving CRISPR/Cas (Yang et al., 2017; Yan et al., 2023) [3,4]. Delivery of these biologicals via vector and non vector mediated methods depends on the type of target cells, characteristics of expression, time duration of expression, size of foreign genetic material etc. For instance, retroviruses find their applicability in case of ex vivo delivery in somatic cells due to their ability to integrate in the host genome. These have been successfully used in gene therapy involving X-SCID patients although, incidence of inappropriate activation has been reported. On the other hand, ex vivo gene therapy for ß-thalassemia involved use of BB305 lentiviral vector for high level expression of CRISPR biological in HSCs. The efficacy and safety of these biologicals will decide their future application as efficient genome editing tools as they go forward in further stages of human clinical trials. This review focuses on CRISPR/Cas based therapies which are at various stages of clinical trials for treatment of rare diseases and the constraints and ethical issues associated with them.

Unraveling Amentoflavone's Therapeutic Potential in Alzheimer's Disease: A Preclinical Assessment.

Alzheimer's disease is one of the neurodegenerative diseases which causes cognition deficit. There are currently few medications available to treat Alzheimer's disease, even though researchers have devoted a great deal of time studying the condition and offering many benefits. Thus, only a few drugs are available for the treatment of Alzheimer's disease. Amentoflavone is a dietary component found in many plants and herbs that has several health advantages. Amentoflavone has demonstrated strong protective benefits against a range of brain illnesses in preclinical trials, most frequently in Alzheimer's disease. Amentoflavone, a biflavonoid, can be identified in a variety of herbs upon isolation. Considering the beneficial properties of this compound, this review emphasizes the pharmacological effects and botanical sources of amentoflavone, as well as the compound's benefits and possible applications in the treatment of Alzheimer's disorders.

Clotrimazole causes membrane depolarization and induces sub G0 cell cycle arrest in Leishmania donovani.

Clotrimazole is an FDA approved drug and is widely used as an antifungal agent. An extensive body of research is available about its mechanism of action on various cell types but its mode of killing of Leishmania donovani parasites is unknown. L. donovani causes Visceral Leishmaniasis which is a public health problem with limited treatment options. Its present chemotherapy is expensive, has adverse effects and is plagued with drug resistance issues. In this study we have explored the possibility of repurposing clotrimazole as an antileishmanial drug. We have assessed its efficacy on the parasites and attempted to understand its mode of action. We found that it has a half-maximal inhibitory concentration (IC50) of 35.75 ± 1.06 µM, 12.75 ± 0.35 µM and 73 ± 1.41 µM in promastigotes, intracellular amastigotes and macrophages, respectively. Clotrimazole is 5.73 times more selective for the intracellular amastigotes as compared to the mammalian cell. Effect of clotrimazole was reduced by ergosterol supplementation. It leads to impaired parasite morphology. It alters plasma membrane permeability and disrupts plasma membrane potential. Mitochondrial function is compromised as is evident from increased ROS generation, depolarized mitochondrial membrane and decreased ATP levels. Cell cycle analysis of clotrimazole treated parasites shows arrest at sub-G0 phase suggesting apoptotic mode of cell death.

Pyridoxal kinase gene deletion leads to impaired growth, deranged redox metabolism and cell cycle arrest in Leishmania donovani.

Pyridoxal kinase (PdxK) is a vitamin B6 salvage pathway enzyme which produces pyridoxal phosphate. We have investigated the impact of PdxK deletion in Leishmania donovani on parasite survivability, infectivity and cellular metabolism. LdPdxK mutants were generated by gene replacement strategy. All mutants showed significant reduction in growth in comparison to wild type. For PdxK mediated biochemical perturbations, only heterozygous mutants and complementation mutants were used as the growth of null mutants were compromised. Heterozygous mutant showed reduction invitro infectivity and higher cytosolic and mitochondrial ROS levels. Glutathione levels decreased significantly in heterozygous mutant indicating its involvement in cellular oxidative metabolism. Pyridoxal kinase gene deletion resulted in reduced ATP levels in parasites and arrest at G0/G1 phase of cell cycle. All these perturbations were rescued by PdxK gene complementation. This is the first report to confirm that LdPdxK plays an indispensable role in cell survival, pathogenicity, redox metabolism and cell cycle progression of L. donovani parasites. These results provide substantial evidence supporting PdxK as a therapeutic target for the development of specific antileishmanial drug candidates.

Role of Alpha-7-Nicotinic Acetylcholine Receptor in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder affecting millions worldwide. One of the leading hypotheses for the underlying cause of AD is a reduction in nicotinic receptor levels in the brain. Among the nicotinic receptors, the alpha-7-nicotinic acetylcholine receptor (α7nAChR) has received particular attention due to its involvement in cognitive function.α7nAChR is a ligand-gated ion channel that is primarily found in the hippocampus and prefrontal cortex, areas of the brain responsible for learning, memory, and attention. Studies have shown that α7nAChR dysfunction is a key contributor to the pathogenesis of AD. The receptor is involved in regulating amyloidbeta (Aß) production, a hallmark of AD pathology. Many drugs have been investigated as α7nAChR agonists or allosteric modulators to improve cognitive deficits in AD. Clinical studies have shown promising results with α7nAChR agonists, including improved memory and cognitive function. Although several studies have shown the significance of the α7 nAChR in AD, little is known about its function in AD pathogenesis. As a result, in this review, we have outlined the basic information of the α7 nAChR's structure, functions, cellular responses to its activation, and its role in AD's pathogenesis.

Use of Eravacycline for Acinetobacter baumannii Infections: A Case Series.

This case series describes the clinical course of 10 patients who received eravacycline antimicrobial therapy for a variety of different Acinetobacter baumannii infection types at a community care hospital.

Analytical method development, validation, and out-of-specification investigations for polyethylene glycol.

This work was motivated by the United States Pharmacopeia monograph modernization initiative and European directorate for the quality of medicines and healthcare. An out-of-specification (OOS) occurrence prompted OOS investigations for the cGMP (cGMP refers to the Current Good Manufacturing Practice regulations enforced by the FDA) release testing of residual ethylene oxide in polyethylene glycol (PEG) according to the standards detailed within the United States Pharmacopeia/National Formulary (USP-NF) for polyethylene glycol, and the European Pharmacopoeia (Ph. Eur.) for macrogols. During the OOS root cause investigations, we observed a PEG degradant and identified it as methyl formate, which co-elutes with ethylene oxide using the GC (gas chromatography) methods published in current USP-NF and Ph. Eur. for PEGs. To address this, a novel method utilizing static headspace gas chromatography with flame ionization detection (HS-GC-FID) was developed to test various grades of PEGs for the presence of residual ethylene oxide, along with other process related impurities and degradation products. With this new method, ethylene oxide, a known genotoxic impurity and IARC (International Agency for Research on Cancer) Class 1 human carcinogen, can be well resolved from methyl formate, a newly identified and comparatively innocuous degradant derived from PEG. The currently available official compendial methods are unable to separate the specific impurities in PEGs. However, the new GC method presented in this paper has been shown to have a high and unique selectivity, allowing for baseline separations (chromatographic resolutions greater than 1.5) of all related impurities in PEGs, which is a challenging task. Additionally, this method has been found to be stability-indicating based on a forced degradation study. During stress stability study of PEGs involving acid, alkali, heat, light, and oxidation, some other PEG degradants, including formaldehyde, were identified. It is possible that formaldehyde undergoes a disproportionation reaction known as the Cannizzaro reaction, leading to the formation of formic acid and methanol. The formic acid then undergoes an esterification reaction with methanol to produce methyl formate. To the best of our knowledge, this is the first stability-indicating HS-GC-FID method that accomplishes this separation in a single run, and it was successfully validated as per ICH (the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guidelines. Therefore, this method is suitable for the conduct of cGMP batch release and stability testing of PEGs in regulated quality control (QC) laboratories.

Insights into Leishmania donovani potassium channel family and their biological functions.

Leishmania donovani is the causative organism for visceral leishmaniasis. Although this parasite was discovered over a century ago, nothing is known about role of potassium channels in L. donovani. Potassium channels are known for their crucial roles in cellular functions in other organisms. Recently the presence of a calcium-activated potassium channel in L. donovani was reported which prompted us to look for other proteins which could be potassium channels and to investigate their possible physiological roles. Twenty sequences were identified in L. donovani genome and subjected to estimation of physio-chemical properties, motif analysis, localization prediction and transmembrane domain analysis. Structural predictions were also done. The channels were majorly α-helical and predominantly localized in cell membrane and lysosomes. The signature selectivity filter of potassium channel was present in all the sequences. In addition to the conventional potassium channel activity, they were associated with gene ontology terms for mitotic cell cycle, cell death, modulation by virus of host process, cell motility etc. The entire study indicates the presence of potassium channel families in L. donovani which may have involvement in several cellular pathways. Further investigations on these putative potassium channels are needed to elucidate their roles in Leishmania. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03692-y.

Molecular Docking and Dynamic Simulation to Identify α7nAChR Binding Affinity of Flavonoids for the Treatment of Alzheimer's Disease.

BACKGROUND: Alpha-7-nicotinic acetylcholine receptor (α7nAChR), a ligand-gated ion channel is one of the important parts of the cholinergic pathway in the brain and has a remarkable role in Alzheimer's disease (AD). It has been documented that the modulation of α7nAChR with the help of phytoconstituent can be helpful in the treatment of AD. METHOD: The binding efficacy of fifty flavonoids was evaluated for human α7nAChR using molecular docking. The best two flavonoids shortlisted from docking analysis were then subjected to molecular dynamic simulations for 100 ns to analyze conformational binding stability with the target protein. Further, the druggability of the selected flavonoids was checked using in silico ADMET studies. RESULT: The top two flavonoids selected based on binding affinity toward the binding site of α7nAChR from molecular docking were amentoflavone (-9.1 kcal/mol) and gallocatechin (-8.8 kcal/mol). The molecular dynamics simulation revealed that amentoflavone and gallocatechin have a stable state during overall simulation time, lesser root mean deviation (RMSD) and root mean square fluctuation (RMSF), and complex of both compounds with protein is stable until 100 ns. CONCLUSION: The two flavonoids amentoflavone and gallocatechin are potential lead molecules that could be utilized as effective agonists of α7nAChR to combat Alzheimer's disease. Future in vitro and in vivo analyses are required to confirm their effectiveness.

Highly variable hearing loss due to POU4F3 (c.37del) is revealed by longitudinal, frequency specific analyses.

Genotype-phenotype correlations add value to the management of families with hereditary hearing loss (HL), where age-related typical audiograms (ARTAs) are generated from cross-sectional regression equations and used to predict the audiogram phenotype across the lifespan. A seven-generation kindred with autosomal dominant sensorineural HL (ADSNHL) was recruited and a novel pathogenic variant in POU4F3 (c.37del) was identified by combining linkage analysis with whole exome sequencing (WES). POU4F3 is noted for large intrafamilial variation including the age of onset of HL, audiogram configuration and presence of vestibular impairment. Sequential audiograms and longitudinal analyses reveal highly variable audiogram features among POU4F3 (c.37del) carriers, limiting the utility of ARTAs for clinical prognosis and management of HL. Furthermore, a comparison of ARTAs against three previously published families (1 Israeli Jewish, 2 Dutch) reveals significant interfamilial differences, with earlier onset and slower deterioration. This is the first published report of a North American family with ADSNHL due to POU4F3, the first report of the pathogenic c.37del variant, and the first study to conduct longitudinal analysis, extending the phenotypic spectrum of DFNA15.

Bilateral chancre of the tongue: An unusual presentation of primary syphilis.

Syphilis is a sexually transmitted infectious disease caused by the spirochete bacterium Treponema pallidum. A characteristic lesion of primary syphilis is chancre. It can develop over genital or extra genital sites, depending on the site of contact with the infectious agent. Cases of oral syphilis have been on the rise in the previous two decades, probably because of the involvement of the oral cavity in sexual practices. We here report an unusual case of primary syphilis who presented with a painless indurated oral ulcer over the lateral borders of the tongue.

Leishmania donovani 6-phosphogluconolactonase: Crucial for growth and host infection?

The hexose monophosphate shunt is a crucial pathway in a variety of microorganisms owing to its vital metabolic products and intermediates such as NADPH, ribose 5-phosphate etc. The enzyme 6-phosphogluconolactonase catalyses the second step of this pathway, converting 6-phosphogluconolactone to 6-phosphogluconic acid. This enzyme has been known to have a significant involvement in growth, pathogenesis and sensitivity to oxidative stress in bacterial and protozoal pathogens. However, the functional role of kinetoplastid Leishmania donovani 6-phospohogluconolactonase (Ld6PGL) remains unexplored. L. donovani is the second largest parasitic killer and causative organism of life threatening visceral leishmaniasis. To understand its possible functional role in the parasite, the alleles of Ld6PGL were sequentially knocked-out followed by gene complementation. The Ld6PGL mutant cell lines showed decrease in transcriptional and translational expression as well as in the enzyme activity. In case of Ld6PGL null mutants, approximately 2-fold reduction was observed in growth. The null mutants also showed ∼38% decrease in infectivity, which recovered to ∼15% on complementation. Scanning electron microscopy showed a marked decrease in flagellar length in the knockout parasites. When treated with the standard drug miltefosine, the mutant strains had no significant change in the drug sensitivity. However, the Ld6PGL mutants were more susceptible to oxidative stress. Our findings suggest that 6PGL is required for parasite growth and infection, but it is not essential.

Outbreak of colistin resistant, carbapenemase (bla NDM, bla OXA-232) producing Klebsiella pneumoniae causing blood stream infection among neonates at a tertiary care hospital in India.

Infections caused by multi-drug resistant Klebsiella pneumoniae are a leading cause of mortality and morbidity among hospitalized patients. In neonatal intensive care units (NICU), blood stream infections by K. pneumoniae are one of the most common nosocomial infections leading to poor clinical outcomes and prolonged hospital stays. Here, we describe an outbreak of multi-drug resistant K. pneumoniae among neonates admitted at the NICU of a large tertiary care hospital in India. The outbreak involved 5 out of 7 neonates admitted in the NICU. The antibiotic sensitivity profiles revealed that all K. pneumoniae isolates were multi-drug resistant including carbapenems and colistin. The isolates belonged to three different sequence types namely, ST-11, ST-16 and ST-101. The isolates harboured carbapenemase genes, mainly bla NDM-1, bla NDM-5 and bla OXA-232 besides extended-spectrum ß-lactamases however the colistin resistance gene mcr-1, mcr-2 and mcr-3 could not be detected. Extensive environmental screening of the ward and healthcare personnel led to the isolation of K. pneumoniae ST101 from filtered incubator water, harboring bla NDM-5, bla OXA-232 and ESBL genes (bla CTX-M) but was negative for the mcr genes. Strict infection control measures were applied and the outbreak was contained. This study emphasizes that early detection of such high-risk clones of multi-drug resistant isolates, surveillance and proper infection control practices are crucial to prevent outbreaks and further spread into the community.

Visceral leishmaniasis in the COVID-19 pandemic era.

Visceral l eishmaniasis (VL), also known as kala-azar, had once been targeted for elimination in 2020, which now has been shifted to 2030. The year 2020 was also the year in which the world was gripped by the coronavirus disease 2019 (COVID-19) pandemic. This review sheds light on the impact of COVID-19 on VL elimination programmes and the increasing incidences of COVID-19/VL cases. Lockdowns were imposed worldwide that led to the suspension of surveys, active case finding and mass drug administration, which are important activities to manage neglected tropical diseases. Healthcare machinery was redirected to control the pandemic and acute resource shortages were seen. Budget cuts from funding agencies and donors also came as a severe blow. Priority changes for manufacturers of drugs and diagnostic kits have also exacerbated the situation. Cases where patients were co-infected with VL and COVID-19 were reported across various settings and in people of various age groups, posing unprecedented challenges in diagnosis and treatment. Concerted efforts from all stakeholders are required to understand and deal with the impact that this pandemic has had on VL.

Targeting Parallel Topology of G-Quadruplex Structures by Indole- Fused Quindoline Scaffolds.

Preferential stabilization of G-quadruplex (G4) structures using small-molecule ligands has emerged as an effective approach to develop anticancer drugs. Herein, we report the synthesis of three indole-fused quindoline derivatives with varying lengths of side chains (InqEt1, InqEt2, and InqPr2) as selective ligands for promoter G4 structures. The ligands stabilize the parallel topology of c-MYC and c-KIT1 promoter G4 DNAs over telomeric and duplex DNAs, as evident from the circular dichroism melting and polymerase stop-assay experiments. The lead ligand, InqPr2, downregulates the gene expression of c-MYC and c-KIT in HeLa and HepG2 cells, respectively, leading to apoptotic cell death. Molecular modeling and dynamics studies support the 2:1 binding stoichiometry revealed from the Job plot analysis and show the ligand's structural features that enable the preferential binding to the parallel G4 structures over other topologies. Our studies show that indole-fused quindoline derivatives can be harnessed as new molecular scaffolds for selective targeting of parallel G4 topologies.

Molecular explorations of the Leishmania donovani 6-phosphogluconolactonase enzyme, a key player in the pentose phosphate pathway.

The enzymes of the pentose phosphate pathway are vital to survival in kinetoplastids. The second step of the pentose phosphate pathway involves hydrolytic cleavage of 6-phosphogluconolactone to 6-phosphogluconic acid by 6- phosphogluconolactonase (6PGL). In the present study, Leishmania donovani 6PGL (Ld6PGL) was cloned and overexpressed in bacterial expression system. Comparative sequence analysis revealed the conserved sequence motifs, functionally and structurally important residues in 6PGL family. In silico amino acid substitution study and interacting partners of 6PGL were predicted. The Ld6PGL enzyme was found to be active in the assay and in the parasites. Specificity was confirmed by Western blot analysis. The ∼30 kDa protein was found to be a dimer in MALDI, glutaraldehyde crosslinking and size exclusion chromatography studies. Kinetic analysis and structural stability studies of Ld6PGL were performed with denaturants and at varied temperature. Computational 3D Structural modelling of Ld6PGL elucidates that it has a similar α/ß hydrolase fold structural topology as in other members of 6PGL family. The three loops are found in extended form when the structure is compared with the human 6PGL (Hs6PGL). Further, enzyme substrate binding mode and its mechanism were investigated using the molecular docking and molecular simulation studies. Interesting dynamics action of substrate 6-phosphogluconolactone was observed into active site during MD simulation. Interesting differences were observed between host and parasite enzyme which pointed towards its potential to be explored as an antileishmanial drug target. This study forms the basis for further analysis of the role of Ld6PGL in combating oxidative stress in Leishmania.

An insight into the morphology of DNA compaction induced by homobinuclear Ru(II) polypyridyl complexes.

Binuclear Ru(II) polypyridyl complexes [Ru2(NN)4(BIPMB)]4+ (1-4), where N-N = 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido [3,2-d:2',3'-f] quinoxaline (dpq), and dipyrido[3,2-a:2',3'-c]phenazine (dppz), have been synthesized using suitable precursors and bridging ligand (BIPMB), where BIPMB = 3,3'-bis-{(imidazol-1-yl)-[4,5-f]-1,10-phenanthroline) methyl}-1,1'-biphenyl. The binding mode and affinity of complexes 1-4 with Calf Thymus DNA (CT-DNA) were determined by absorption and steady-state fluorescence spectroscopy. The decrease in viscosity of CT-DNA on sequential addition of these complexes indicated DNA condensation and the result was corroborated by circular dichroism (CD). The nanosized globular aggregates of CT-DNA induced by complexes 1-4 were observed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. The gel electrophoretic mobility studies revealed the small orderly particles of supercoiled plasmid pBR322 DNA induced by these complexes. Additionally, the morphology and size of compact plasmid DNA condensates were studied by DLS and atomic force microscopy (AFM). The complexes were moderately cytotoxic against MCF-7 cells.

A Core-Linker-Polyamine (CLP) Strategy Enables Rapid Discovery of Antileishmanial Aminoalkylquinolinecarboxamides That Target Oxidative Stress Mechanism.

A Core-Linker-Polyamine (CLP) strategy has been exploited to develop new antileishmanial agents. It involves the linker-based assembly of alkyl-polyamine side chain as a potential pharmacophore motif with a privileged heterocyclic motif, 4-arylquinoline. A series of aminoalkyl 4-arylquinoline-2-carboxamides and their analogs were synthesized and tested against L. donovani promastigotes. Among all synthesized derivatives, 10 compounds showed significant antipromastigote activities with more efficacy (IC50 : 4.75-8 µM) than an antileishmanial oral drug Miltefosine (IC50 : 8.9±1.55 µM). Most active aminoalkyl-quinoline-carboxamides 9 a and 9 b, displayed negligible cytotoxicity towards human monocytic (THP-1) macrophages. The compounds show antileishmanial activity by generating mitochondrial superoxide radicals. However, they did not show interference with trypanothione reductase, a redox enzyme of Leishmania. Significant change in the morphology of the L. donovani promastigote by the compounds was observed. The Structure-activity relationship analysis suggest the pharmacophoric importance of alkylpolyamine and carboxamide motifs. In silico evaluation indicated that the investigated active molecules 9 a and 9 b possess important drug-likeness, physicochemical and pharmacokinetic-relevant properties.

Identification of 2-arylquinazolines with alkyl-polyamine motifs as potent antileishmanial agents: synthesis and biological evaluation studies.

2-Arylquinazolines with a range of alkyl polyamines as side chain/ring functional motifs at the 4th-position were considered for antileishmanial study with the rationale that related heterocyclic scaffolds and polyamine functionalities are present in drugs, clinical trial agents, natural products and anti-parasitic/leishmanial agents. Synthesis involves construction of the 2-arylquinazolin-4-one ring and deoxyamination via deoxychlorination followed by SNAr-based amination or a methodology of SNAr-deoxyamination driven by BOP-mediated hydroxyl-activation. Various alkyl-polyamines important for activities were incorporated. A total of 26 compounds were prepared and screened against Leishmania donovani (Ld) promastigote cells using the MTT assay. Most of the investigated series of compounds showed characteristic leishmanicidal properties. Several compounds showed pronounced leishmanicidal activities (IC50: 5-6.5 µM) with higher efficiency than the antileishmanial drug miltefosine (IC50: 10.5 µM), and relatively less cytotoxicity to macrophage host cells (SI: 9.27-13.5) compared to miltefosine (SI: 3.42). Important pharmacophoric skeletons were identified.