Virtual screening and multilevel precision-based prioritisation of natural inhibitors targeting the ATPase domain of human DNA topoisomerase II alpha

Human DNA topoisomerase II alpha (hTopIIα) is a classic chemotherapeutic drug target. The existing hTopIIα poisons cause numerous side effects such as the development of cardiotoxicity, secondary malignancies, and multidrug resistance. The use of catalytic inhibitors targeting the ATP-binding cavity of the enzyme is considered a safer alternative due to the less deleterious mechanism of action. Hence, in this study, we carried out high throughput structure-based virtual screening of the NPASS natural product database against the ATPase domain of hTopIIα and identified the five best ligand hits. This was followed by comprehensive validation through molecular dynamics simulations, binding free energy calculation and ADMET analysis. On stringent multilevel prioritization, we identified promising natural product catalytic inhibitors that showed high binding affinity and stability within the ligand-binding cavity and may serve as ideal hits for anticancer drug development. Communicated by Ramaswamy H. Sarma</p

Inhibition of IKKβ by celastrol and its analogues – an <i>in silico</i> and <i>in vitro</i> approach

<p><b>Context:</b> Alzheimer’s disease (AD) is the most common form of dementia affecting the aged population and neuroinflammation is one of the most observed AD pathologies. NF-κB is the central regulator of inflammation and inhibitor κB kinase (IKK) is the converging point in NF-κB activation. Celastrol is a natural triterpene used as a treatment for inflammatory conditions.</p> <p><b>Objective:</b> This study determines the neuroprotective and inhibitory effect of celastrol on amyloid beta_1-42 (Aβ_1-42) induced cytotoxicity and IKKβ activity, respectively.</p> <p><b>Materials and methods:</b> Retinoic acid differentiated IMR-32 cells were treated with celastrol (1 μM) before treatment with Aβ_1-42 (IC₃₀ 10 μM) for 24 h. The cytotoxicity and IKK phosphorylation were measured by MTT and western blotting analysis, respectively. We screened 36 celastrol analogues for the IKKβ inhibition by molecular docking and evaluated their drug like properties to delineate the neuroprotective effects.</p> <p><b>Results:</b> Celastrol (1 μM) inhibited Aβ_1-42 (10 μM) induced IκBα phosphorylation and protected IMR-32 cells from cell death. Celastrol and 25 analogues showed strong binding affinity with IKKβ as evidenced by strong hydrogen-bonding interactions with critical active site residues. All the 25 analogues displayed strong anti-inflammatory properties but only 11 analogues showed drug-likeness. Collectively, molecule 15 has highest binding affinity, CNS activity and more drug likeness than parent compound celastrol.</p> <p><b>Discussion and conclusion:</b> The decreased expression of pIκBα in celastrol pretreated cells affirms the functional representation of inhibited IKKβ activity in these cells. The neuroprotective potentials of celastrol and its analogues may be related to IKK inhibition.</p

Biochemical and biophysical characterization of biosynthetic arginine decarboxylase from <i>Thermus thermophilus</i>

The biosynthetic arginine decarboxylase in Thermus thermophilus is responsible for producing spermidine, a polyamine with numerous biological applications in humans. The arginine decarboxylase has significant applications in biotechnology industries, suggesting the need to evaluate its biochemical and biophysical characteristics at the molecular level. In this study, both in vitro and in silico methods were employed to investigate the structural and functional behavior of the arginine decarboxylase protein. In in vitro, MALDI-TOF, size exclusion, and assay studies were performed to examine the nature and activity of the protein. The MALDI-TOF analysis confirmed the purified protein as biosynthetic arginine decarboxylase. The assay results revealed that the Pyridoxal 5’-Phosphate (PLP) cofactor plays a crucial role in enhancing enzyme activity by producing agmatine (a by-product of spermidine). Further, optimum enzyme activity was observed at 50 °C, suggesting the extremophilic nature of the enzyme. Unlike other proteins, this enzyme displayed optimal activity at both acidic and basic pH, demonstrating its sensitivity to pH changes. Furthermore, the addition of divalent ions like Mg 2+ increased the rate of reaction. In in silico, structure modeling, and comparative molecular dynamics simulation studies were used to assess the protein stability and behavior at different pH and temperature conditions. The findings of this study could be applied to improve enzyme production in the industry. Communicated by Ramaswamy H. Sarma</p

Synthesis and Characterization of a Novel Peptide Targeting Human Tenon Fibroblast Cells To Modulate Fibrosis: An Integrated Empirical Approach

Fibrosis is the primary factor influencing the prognosis of glaucoma post-trabeculectomy surgery, an eye condition characterized by increased intraocular pressure (IOP). Despite advancements in surgical procedures and aftercare, it continues to be a serious impediment. During the clinical intervention of scarring, fibrosis is managed by using topical application of combined antifibrotic drugs (mitomycin C). But still, scarring remains a key problem due to minimal drug penetration and nonbioavailability. In this study, we synthesized a cell-specific peptide for modulating scarring in human tenon fibroblasts undergoing epithelial–mesenchymal transition (EMT). The peptide was also conjugated with mitomycin C in order to investigate the effect of the drug conjugation on human tenon fibroblasts from the nanofiber composite system and to evaluate the fibrosis process. Peptide VRF2019 was identified using a subtractive proteomics approach, including solubility, cell penetration, and amphipathic properties. The peptide structure was determined using circular dichroism spectroscopy. The peptide and drug was conjugated using N-ethyl-N′-(3-(dimethylamino)propyl) carbodiimide/N-hydroxysuccinimide (EDC-NHS) chemistry, and the conjugation efficiency was evaluated using high-pressure liquid chromatography. The conjugated product and polycaprolactone (PCL) were electrospun to form a composite nanofiber, which was tested for cytotoxicity and drug release on human tenon fibroblast cells. The modeled VRF2019 peptide structure formed an α-helical structure with all residues spanning the allowed regions of the Ramachandran plot. Subsequent molecular dynamics simulations also demonstrated its membrane penetration potential. The peptide uptake was also studied in human tenon fibroblast cells. High-pressure liquid chromatography (HPLC) and mass spectrometry measurements confirmed peptide–drug conjugation and stability. Furthermore, scanning electron microscopy (SEM) investigation revealed the structure and size of the PCL composite nanofiber. We infer from early research that the PCL composite nanofiber matrix can greatly increase drug delivery and bioavailability

Data_Sheet_1_Structural analysis and molecular dynamics simulation studies of HIV-1 antisense protein predict its potential role in HIV replication and pathogenesis.docx

The functional significance of the HIV-1 Antisense Protein (ASP) has been a paradox since its discovery. The expression of this protein in HIV-1-infected cells and its involvement in autophagy, transcriptional regulation, and viral latency have sporadically been reported in various studies. Yet, the definite role of this protein in HIV-1 infection remains unclear. Deciphering the 3D structure of HIV-1 ASP would throw light on its potential role in HIV lifecycle and host-virus interaction. Hence, using extensive molecular modeling and dynamics simulation for 200 ns, we predicted the plausible 3D-structures of ASP from two reference strains of HIV-1 namely, Indie-C1 (subtype-C) and NL4-3 (subtype-B) so as to derive its functional implication through structural domain analysis. In spite of sequence and structural differences in subtype B and C ASP, both structures appear to share common domains like the Von Willebrand Factor Domain-A (VWFA), Integrin subunit alpha-X (ITGSX), and ETV6-Transcriptional repressor, thereby reiterating the potential role of HIV-1 ASP in transcriptional repression and autophagy, as reported in earlier studies. Gromos-based cluster analysis of the centroid structures also reassured the accuracy of the prediction. This is the first study to elucidate a highly plausible structure for HIV-1 ASP which could serve as a feeder for further experimental validation studies.</p

Understanding variable disease severity in X-linked retinoschisis: Does RS1 secretory mechanism determine disease severity?

<div><p>X-linked retinoschisis (XLRS) is a retinal degenerative disorder caused by mutations in <i>RS1</i> gene leading to splitting of retinal layers (schisis) which impairs visual signal processing. Retinoschisin (RS1) is an adhesive protein which is secreted predominantly by the photoreceptors and bipolar cells as a double-octameric complex. In general, XLRS patients show wide clinical heterogeneity, presenting practical challenges in disease management. Though researchers have attempted various approaches to offer an explanation for clinical heterogeneity, the molecular basis has not been understood yet. Therefore, this study aims at establishing a link between the phenotype and genotype based on the molecular mechanism exerted by the mutations. Twenty seven XLRS patients were enrolled, of which seven harboured novel mutations. The mutant constructs were genetically engineered and their secretion profiles were studied by <i>in vitro</i> cell culture experiments. Based on the secretory profile, the patients were categorized as either secreted or non-secreted group. Various clinical parameters such as visual acuity, location of schisis, foveal thickness and ERG parameters were compared between the two groups and control. Although the two groups showed severe disease phenotype in comparison with control, there was no significant difference between the two XLRS groups. However, the secreted group exhibited relatively severe disease indications. On the other hand molecular analysis suggests that most of the <i>RS1</i> mutations result in intracellular retention of retinoschisin. Hence, clinical parameters of patients with non-secreted profile were analyzed which in turn revealed wide variability even within the group. Altogether, our results indicate that disease severity is not merely dependent on secretory profile of the mutations. Thus, we hypothesize that intricate molecular detail such as the precise localization of mutant protein in the cell as well as its ability to assemble into a functionally active oligomer might largely influence disease severity among XLRS patients.</p></div

Representative clinical images and data of XLRS patients.

<p>(A) Fundus exhibiting spoke wheel pattern like schisis at the macula (indicated by arrow). (B) Optical coherence tomography showing splitting of the inner retinal layers. (C) Electroretinogram showing reduced waveforms of rod and cone responses, a negative b-wave pattern noted on standard combined response (circled).</p

List of XLRS patients and their clinical characteristics.

<p>List of XLRS patients and their clinical characteristics.</p

Clinical data of XLRS patients showing non-secreted RS1 profile.

<p>Clinical data of XLRS patients showing non-secreted RS1 profile.</p

Comparison of ERG parameters between control samples and XLRS patients showing non-secreted or secreted or protein profile.

<p>Comparison of ERG parameters between control samples and XLRS patients showing non-secreted or secreted or protein profile.</p