Vesicle-Encapsulated Rolipram (PDE4 Inhibitor) and Its Anticancer Activity.

Vesicular carriers of drugs are popular for specific targeting and delivery. The most popular vesicles among these are liposomes. However, they suffer from some inherent limitations. In this work, alternative vesicles with enhanced stability, i.e., niosomes and bilosomes have been prepared, characterized, and their delivery efficiency studied. Bilosomes have the additional advantage of being able to withstand the harsh environment of the gastrointestinal tract (GIT). The taurine-derived bile salt (NaTC) was incorporated into the bilosome bilayer. The inspiration behind NaTC insertion is the recent reports on antiaging action and immune function of taurine. Fluorescence probing was used to study the vesicle environment. The entrapment and subsequent release of the important cAMP-specific PDE4 inhibitor/drug Rolipram, which has antibreast cancer properties, was assessed on the breast cancer cell line MCF-7. Rolipram has important therapeutic applications, one of the most significant in recent times being the treatment of Covid-19-triggered pneumonia and cytokine storms. As for cancer chemotherapy, the localization of drug, targeted delivery, and sustained release are extremely important issues, and it seemed worthwhile to explore the potential of the bilosomes and niosomes to entrap and release Rolipram. The important finding is that niosomes perform much better than bilosomes in the hormone-responsive breast cancer mileau MCF-7. Moreover, there was a 4-fold decrease in the IC50 of Rolipram encapsulated in niosomes compared to Rolipram alone. On the other hand, bilosome-encapsulated Rolipram shows higher IC50 value. The results can be further understood by molecular docking studies.

Molecular characterization of the unusual peptide CORO1C-47aa encoded by the circular RNA and docking simulations with its binding partner.

Circular RNAs, which have covalently closed ends, are in the class of non-coding RNAs. Recent studies reveal that they are associated with various biochemical pathways. One such involvement of circular RNAs is in the onset of different types of cancers. Though the circular RNAs are known as non-coding RNAs, some of them are found to possess the capacities to code for proteins. One such circular RNA is hsa-circ-0000437 which is known to code for a short peptide referred to as CORO1C-47aa. The peptide has anti-angiogenic activity and is associated with the prevention of endometrial cancer. The peptide binds to the PAS-B domain of the Aryl hydrocarbon Receptor Nuclear Translocator (ARNT). However, till date only the amino acid sequence of the peptide is known and no structural details of the peptide are available. Therefore, in this work, our aim was to predict how the peptide would fold and what could be its possible ligand binding sites. We used computational tools to determine the structure of the peptide refined further by molecular dynamics simulations. We then performed molecular docking simulations of the peptide with its known binding partner ARNT to gain an insight into the modes of binding as the process is associated with endometrial cancer. The possible ligand binding sites along-with the natures of the possible other different ligands of the peptide were analyzed further. From this structure function analysis study, we tried to elucidate the plausible mechanism of the involvements of the peptide in the onset of endometrial cancer. This is the first report on the structural characterization of the peptide and its modes of interactions with the partner protein ARNT. This study may therefore be useful in determining the structures of new drug candidates for the treatment of endometrial cancer.

Analysis of the structural dynamics of the mutations in the kinase domain of PINK1 protein associated with Parkinson's disease.

Parkinson's disease (PD) is a very common neurodegenerative disorder and is considered to be one of the most severe disorders worldwide. Mutations in some PD causing genes are responsible for the early onset of the disease. Pathogenic variants in parkin, PINK1 and DJ1 genes can cause early-onset of PD. Many PINK1 gene mutations have been reported, but not all variants are pathogenic. The gene product of PINK1, also known as PINK1 protein, has 581 amino acid residues in it. Several different mutations are present throughout the kinase domain of PINK1 protein. In this work, we used in silico approaches to analyze the different types of mutations that are distributed in the kinase domain of the PINK1 protein. Based on our results, we categorized the mutations as high, moderate and low pathogenic variants. Furthermore, we performed molecular dynamics simulations of the pathogenic PINK1 variants to decipher their possible impacts on the structure and made a comparison with the wild type PINK1. In conclusion, we suggested the possible mechanistic roles of the pathogenic variants of PINK1 kinase domain that can affect its function. These pathogenic variants are the causative agents of early onset of PD called autosomal recessive Parkinson disease.

Study of the Effects of Nicotine and Caffeine for the Treatment of Parkinson's Disease.

Parkinson's disease (PD) is considered to be a highly severe neurological disorder. PD occurs due to a decrease in dopamine production by the degeneration of dopamine-secreting neurons. Genetic mutations, environmental toxins and lifestyle are some of the risk factors of the progressive neurodegenerative disorder PD. Parkin protein, which is encoded by the PARK gene, is one of the important proteins, which is one of the causative agents. The Parkin protein has several mutations which lead to the development of the disease. Apart from PD, the mutations in Parkin also showed to be responsible for the onset of diseases like cancers. It is reported that the E28K mutation in the Ubl domain of parkin is highly deleterious and responsible for the onset of melanoma. This necessitates the development of new therapeutics against PD. Molecules like levodopa, carbidopa, monoamine oxidase type B inhibitors (MBO inhibitors), dopamine agonists, anticholinergics and amantadine are some commonly used drugs used to treat PD. Recently, there have been increasing evidence which shows that cigarette smoking and consumptions of coffee and tea could have important roles in modulating the risk of PD. Therefore, we planned to analyse the molecular mechanism of the binding interactions of nicotine, caffeine and the polyphenol ( -)-epigallocatechin-3-gallate (EGCG) from green tea with Parkin protein to predict their therapeutic potentials in PD targeting the E28K mutation. We focused on E28K mutant of Parkin as this mutant form of parkin has been shown to be the most pathogenic one. We could identify the potential therapeutic aspects of these natural products to prevent the onset of PD. This work may therefore be considered to be the first of its kind which would take into consideration the environmental toxicological approach in designing natural product inhibitors against the onset of PD.

Amelioration of oxidative stress mediated inflammation and apoptosis in pancreatic islets by Lupeol in STZ-induced hyperglycaemic mice.

BACKGROUND: Type 1 Diabetes mellitus initiates by loss of pancreatic activity which affects other major organs leading to multi-organ failure. Lupeol, a novel phytochemical, is emerging as a potent bioactive molecule. However, the effect of lupeol on hyperglycaemia is not clearly understood. This study delivers an elaborate vision towards the detailed molecular pathway of lupeol against STZ induced diabetic difficulties of the pancreas. METHOD: The current experiments were designed to focus on the ameliorative effect of the triterpene in combating oxidative damage on the pancreas in a preclinical streptozotocin induced mouse model. After diabetic induction, the animals were subjected to administration with 75 mg kg-1 body weight of lupeol, thrice a week for 7 weeks. Histological measurements were done to investigate the anatomy of the pancreas as well as molecular mechanisms were explored. RESULTS: The compound was found to regulate several hyperglycaemic and oxidative stress related markers. Lupeol treatment also reversed the expression levels of inflammatory cytokines (TNF-α and IL-1ß) as well as attenuated the NF-κB mediated inflammatory and extrinsic apoptotic pathway. DISCUSSION: These findings in preclinical streptozotocin induced in vivo mouse model strongly suggest the discovery of novel properties of lupeol against oxidative stress in pancreatic ß cells by regulating the NF-κB and extrinsic apoptotic pathway.

Mutational Impact on "in-Between-Ring" (IBR) Domain of PARKIN on Protein Stability and Function.

Mutations in parkin, which is encoded by the PARK2 gene, are associated with a rare form of Parkinson's disease called autosomal recessive juvenile parkinsonism (ARJP). Parkin is a member of RBR family of E3 ubiquitin ligase. Parkin contains a RING1-In-Between-Ring (IBR)-RING2 motif. The IBR domain is located at the C-terminal end of the parkin protein. Two zinc-binding sites are present in the IBR domain which shows zinc ion-dependent folding and maintains the orientation and geometry of RING domains. So, mutation in a zinc-binding region can be responsible for improper folding of parkin protein, which eventually affects the protein structure and function. Abnormalities in parkin protein increase the aggregation of mis-folded proteins in the brain cell. As a consequence, cellular toxicity occurs. The IBR domain also interacts with UbcH7 and UbcH8 proteins belonging to E2 protein family and facilitates synphilin-1, Sept5, and SIM2 protein ubiquitination. It is reported that missense mutation in parkin protein are responsible for autosomal recessive juvenile Parkinson disease. In this work, we first collected the missense mutations in the IBR domain from literature and sequence databases. Then, using various computational tools, we predicted their pathogenicity and involvements in causing possible changes in various protein properties. Evolutionary conservation of amino acids, solvent accessible surface areas, the physico-chemical properties, and changes of protein structure were analyzed. We, for the first time, analyzed the effects of these mutations in parkin to decipher the plausible molecular mechanism of Parkinson's disease.

Structural analysis of the effects of mutations in Ubl domain of Parkin leading to Parkinson's disease.

Parkinson's disease (PD) is characterized by progressive death of dopamine producing neurons in the substantia nigra pars compacta of the mid brain. Dysfunction of an E3 ligase protein, Parkin, encoded by PARK2 gene, results in accumulation of misfolded proteins in brain cells which lead to the onset of PD. Parkin is a multi-domain protein consisting of N-terminal ubiquitin-like domain (Ubl) followed by RING0, RING1, In Between Ring (IBR) domain and RING2 domain which is present at the C-terminal end of Parkin protein. Ubl domain is the smallest domain of Parkin and is involved in the binding of Parkin with E2 protein molecule required for proper Ubiquitination and functioning of proteins in the brain. Mutations in the Parkin protein are known to be associated with protein dysfunction leading to PD. This study aims to decipher the characteristics and effects of the different mutations in the Ubl domain by an in-silico analysis. The mutations were collected from PDmutDB and COSMIC databases. The pathogenic impacts of amino-acid mutations on Ubl structure and function were analysed by using various computational tools. Due to lack of proper full-chain structure of the Ubl domain, a homology model of the domain was reconstructed using Discovery Studio 2.5 (DS 2.5) software suite. We found that the mutations A31D, A46P, C59F, A46T, E28K, E49K, R42P, R42S, and Q63K were the most deleterious ones which might be associated with the onset of PD. In order to study the dynamic behaviour of the Parkin Ubl domain in cellular environment, molecular dynamics (MD) simulations were carried out using the wild-type and mutant Ubl domains. Our analyses could predict the cellular dynamics of the mutations and therefore might help in predicting the hitherto unknown molecular mechanism of the disease onset and designing precision medicine for the treatment of PD.

Structure based virtual screening of natural products to disrupt the structural integrity of TRAF6 C-terminal domain homotrimer.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an E3 ligase which takes part in different cellular pathways. TRAF6 is seen to be highly expressed in various cancers and most importantly is known to drive cancer metastasis. This makes TRAF6 a potential therapeutic target. In our previous studies, we observed that the C-terminal domain of TRAF6 forms a mushroom shaped trimer structure. Lys340 and Glu345 were identified to be the most critical residues in the trimer interface. In this current work, we screened for more than 14000 small molecules derived from various natural sources and they were screened against TRAF6 C-terminal trimer interaction interface to prevent the formation of the interface. All the obtained molecules were tested for their drug-likeliness properties. The ligands which qualified the filter were considered for protein-ligand docking or structure based virtual screening in GOLD 5.2. Pose selection was carried out on the basis of GoldScore and ChemScore function of GOLD 5.2. Top 20 molecules binding the TRAF6 trimeric interface were tested for their ADME properties. From the top 20 molecules, top 3 ligands were chosen based on their abilities to pass the maximum numbers of ADME filters.