Molecular Framework of Mouse Endothelial Cell Dysfunction during Inflammation: A Proteomics Approach.

A key aspect of cytokine-induced changes as observed in sepsis is the dysregulated activation of endothelial cells (ECs), initiating a cascade of inflammatory signaling leading to leukocyte adhesion/migration and organ damage. The therapeutic targeting of ECs has been hampered by concerns regarding organ-specific EC heterogeneity and their response to inflammation. Using in vitro and in silico analysis, we present a comprehensive analysis of the proteomic changes in mouse lung, liver and kidney ECs following exposure to a clinically relevant cocktail of proinflammatory cytokines. Mouse lung, liver and kidney ECs were incubated with TNF-α/IL-1ß/IFN-γ for 4 or 24 h to model the cytokine-induced changes. Quantitative label-free global proteomics and bioinformatic analysis performed on the ECs provide a molecular framework for the EC response to inflammatory stimuli over time and organ-specific differences. Gene Ontology and PANTHER analysis suggest why some organs are more susceptible to inflammation early on, and show that, as inflammation progresses, some protein expression patterns become more uniform while additional organ-specific proteins are expressed. These findings provide an in-depth understanding of the molecular changes involved in the EC response to inflammation and can support the development of drugs targeting ECs within different organs. Data are available via ProteomeXchange (identifier PXD031804).

Molecular Subtypes As Emerging Predictors of Clinicopathological Response to Neoadjuvant Chemotherapy (NACT) in Locally Advanced Breast Cancer (LABC): A Single-Centre Experience in Western India.

INTRODUCTION: Locally advanced breast cancer (LABC) is a subset of breast cancer characterized by the most advanced breast tumours in the absence of distant metastasis. Treatment of LABC has evolved from a single modality treatment to multimodality management. Neoadjuvant chemotherapy (NACT) is increasingly being used to treat patients with LABC. This study assessed tumour response after NACT using clinical changes, Response Evaluation Criteria in Solid Tumors (RECIST) criteria and pathological report. METHODOLOGY: This study was a prospective as well as retrospective observational study carried out in the department of general surgery, Dr. Sampurnanand Medical College, Jodhpur. All the patients admitted with stage III (IIIA, IIIB, IIIC) were included in the study after obtaining approval from the institutional ethical committee. Clinical response was assessed by RECIST criteria (clinical complete response (cCR), clinical partial response (cPR), clinical progressive disease (cPD), and clinical stable disease (cSD)) and pathological response by histopathological report (pCR). Response of various molecular subtypes was noted. RESULTS: Among 31 patients included in the study, cCR observed in 22.58% cases, cPR observed in 61.29% cases while cPD and cSD seen in 3.22% and 12.90% cases, respectively. Pathological complete response (pCR) observed in 19.35% cases. Favourable response seen with human epidermal growth factor receptor 2 (HER2) overexpression (cCR = 50%, pCR = 37.50%) followed by triple negative (cCR = 25%, pCR = 25%) molecular subtypes. CONCLUSIONS: It can be concluded that molecular subtype determination helps in deciding treatment protocol in patients with LABC with HER2 overexpression and triple-negative breast cancers having a better clinicopathological response to NACT than luminal subtypes. NACT results in downstaging of tumours, thus, help in achieving surgically clear margins and elimination of micrometastases which decreases the recurrence rates and morbidity/mortality of patients.

Evaluating Drug Deposition Patterns from Turbuhaler® in Healthy and Diseased Lung Models of Preschool Children.

The efficacy of pediatric oral drug delivery using dry powder inhalers, such as Turbuhaler®, is dependent on the age and health of the test subjects. The available clinical data for these studies is scant and rarely provide correlations between the health condition and the regional lung deposition. In particular, the data and the correlations for pre-school children are minimal. Deposition simulations were performed using the newly developed Quasi-3D whole lung model to analyze the effect of health conditions on the regional lung deposition from the Turbuhaler® in 3-year-old children. The healthy lung model was created from CT scan data. Cystic-fibrosis models were created by uniformly constricting the airways to various degrees. The simulated drug deposition outcomes were validated against the available experimental data. The results show that, while the dose deposited in the lungs exhibits minor variations, the Peripheral:Central (P/C) ratio is strongly affected by both the health condition and the inflow variations. The above ratio is reduced by ~30% for the severely diseased case, compared to its healthy counterpart, for the same inhalation profile. This indicates that lower doses reach the peripheral lung, in pediatric cystic-fibrosis subjects, thus requiring a larger therapeutic dose.

Immediate rehabilitation of a rheumatoid arthritis patient with single-piece implants.

INTRODUCTION AND IMPORTANCE: The aim of this article is to report the long-term outcome of full mouth rehabilitation with single piece, smooth surface implants following immediate loading protocol on a patient suffering with RA and severe unilateral condylar resorption. CASE PRESENTATION: Here, we present a challenging case of a patient suffering from Rheumatoid Arthritis who was stabilized and completed successfully with a 4 year follow-up period. Prosthetic management optimized the inter-occlusal relationship to maintain both function and esthetic integrity. Single piece implants are designed to engage and take support from the cortical bone low in metabolic activities thus promoting the force transmission through apical threads that are engaged in the cortical bone. DISCUSSION: Rheumatoid Arthritis [RA] is an auto-immune inflammatory condition in which the inflamed and hypertrophic synovial membrane grows into the articulation surfaces. The Temporomandibular Joints [TM] are frequently involved in rheumatoid arthritis. According to the literature on RA, due to frequent periodontitis, decreased salivary secretion, medication, as well as decrease in bone regenerative potential, RA is often considered as a relative contraindication in the use of implants. Atrophic jaws and cases with comorbidities like osteoporosis, diabetes, rheumatoid arthritis, periodontally infected cases are restored with high success by single piece smooth surface. CONCLUSION: To the best of our knowledge, this may be the first case of immediate functional loading by bi-cortical single piece implants.

Natural-Product-Inspired Compounds as Countermeasures against the Liver Carcinogen Aflatoxin B1.

Aflatoxin B1 (AfB1) ranks among the most potent liver carcinogens known, and the accidental or intentional exposure of humans and livestock to this toxin remains a serious global threat. One protective measure that had been proposed is employing small-molecule therapeutics capable of mitigating the toxicity of AfB1; however, to date, these efforts have had little clinical success. To identify molecular scaffolds that reduce the toxicity of AfB1, we developed a cell-based high-throughput high-content imaging assay that enabled our team to test natural products (pure compounds, fractions, and extracts) for protection of monolayers and spheroids composed of HepG2 liver cells against AfB1. The spheroid assay showed notable potential for further development, as it afforded greater sensitivity of HepG2 cells to AfB1, which is believed to better mimic the in vivo response of hepatocytes to the toxin. One of the most bioactive compounds to arise from this investigation was alternariol-9-methyl ether (1, purified from an Alternaria sp. isolate), which inspired the synthesis and testing of several structurally related molecules. Based on these findings, it is proposed that several types of natural and synthetic polyarene molecules that have undergone oxidative functionalization (e.g., compounds containing 3-methoxyphenol moieties) are promising starting points for the development of new agents that protect against AfB1 toxicity.

A physicochemical descriptor-based scoring scheme for effective and rapid filtering of kinase-like chemical space.

BACKGROUND: The current chemical space of known small molecules is estimated to exceed 1060 structures. Though the largest physical compound repositories contain only a few tens of millions of unique compounds, virtual screening of databases of this size is still difficult. In recent years, the application of physicochemical descriptor-based profiling, such as Lipinski's rule-of-five for drug-likeness and Oprea's criteria of lead-likeness, as early stage filters in drug discovery has gained widespread acceptance. In the current study, we outline a kinase-likeness scoring function based on known kinase inhibitors. RESULTS: The method employs a collection of 22,615 known kinase inhibitors from the ChEMBL database. A kinase-likeness score is computed using statistical analysis of nine key physicochemical descriptors for these inhibitors. Based on this score, the kinase-likeness of four publicly and commercially available databases, i.e., National Cancer Institute database (NCI), the Natural Products database (NPD), the National Institute of Health's Molecular Libraries Small Molecule Repository (MLSMR), and the World Drug Index (WDI) database, is analyzed. Three of these databases, i.e., NCI, NPD, and MLSMR are frequently used in the virtual screening of kinase inhibitors, while the fourth WDI database is for comparison since it covers a wide range of known chemical space. Based on the kinase-likeness score, a kinase-focused library is also developed and tested against three different kinase targets selected from three different branches of the human kinome tree. CONCLUSIONS: Our proposed methodology is one of the first that explores how the narrow chemical space of kinase inhibitors and its relevant physicochemical information can be utilized to build kinase-focused libraries and prioritize pre-existing compound databases for screening. We have shown that focused libraries generated by filtering compounds using the kinase-likeness score have, on average, better docking scores than an equivalent number of randomly selected compounds. Beyond library design, our findings also impact the broader efforts to identify kinase inhibitors by screening pre-existing compound libraries. Currently, the NCI library is the most commonly used database for screening kinase inhibitors. Our research suggests that other libraries, such as MLSMR, are more kinase-like and should be given priority in kinase screenings.

Exploring polypharmacology using a ROCS-based target fishing approach.

Polypharmacology has emerged as a new theme in drug discovery. In this paper, we studied polypharmacology using a ligand-based target fishing (LBTF) protocol. To implement the protocol, we first generated a chemogenomic database that links individual protein targets with a specified set of drugs or target representatives. Target profiles were then generated for a given query molecule by computing maximal shape/chemistry overlap between the query molecule and the drug sets assigned to each protein target. The overlap was computed using the program ROCS (Rapid Overlay of Chemical Structures). We validated this approach using the Directory of Useful Decoys (DUD). DUD contains 2950 active compounds, each with 36 property-matched decoys, against 40 protein targets. We chose a set of known drugs to represent each DUD target, and we carried out ligand-based virtual screens using data sets of DUD actives seeded into DUD decoys for each target. We computed Receiver Operator Characteristic (ROC) curves and associated area under the curve (AUC) values. For the majority of targets studied, the AUC values were significantly better than for the case of a random selection of compounds. In a second test, the method successfully identified off-targets for drugs such as rimantadine, propranolol, and domperidone that were consistent with those identified by recent experiments. The results from our ROCS-based target fishing approach are promising and have potential application in drug repurposing for single and multiple targets, identifying targets for orphan compounds, and adverse effect prediction.

Novel and selective DNA methyltransferase inhibitors: Docking-based virtual screening and experimental evaluation.

The DNA methyltransferase (DNMT) enzyme family consists of four members with diverse functions and represents one of the most promising targets for the development of novel anticancer drugs. However, the standard drugs for DNMT inhibition are non-selective cytosine analogues with considerable cytotoxic side-effects that have been developed several decades ago. In this work, we conducted a virtual screening of more than 65,000 lead-like compounds selected from the National Cancer Institute collection using a multistep docking approach with a previously validated homology model of the catalytic domain of human DNMT1. Experimental evaluation of top-ranked molecules led to the discovery of novel small molecule DNMT1 inhibitors. Virtual screening hits were further evaluated for DNMT3B inhibition revealing several compounds with selectivity towards DNMT1. These are the first small molecules reported with biochemical selectivity towards an individual DNMT enzyme capable of binding in the same pocket as the native substrate cytosine, and are promising candidates for further rational optimization and development as anticancer drugs. The availability of enzyme-selective inhibitors will also be of great significance for understanding the role of individual DNMT enzymes in epigenetic regulation.

Discovery of a novel protein kinase B inhibitor by structure-based virtual screening.

Protein kinase B (PKB/AKT) is a promising and attractive therapeutic target in anticancer drug development. Herein, we report the findings of virtual screening for novel ATP-competitive inhibitors of AKT-2 using 2D- and 3D-similarity searching and sequential molecular docking with two crystal structures of AKT-2. Our multistep approach led to the identification of a low micromolar AKT-2 inhibitor (IC(50)=1.5 microM) with a novel scaffold. The experimentally validated inhibitor represents the starting point for an optimization program.

Molecular modeling and molecular dynamics studies of hydralazine with human DNA methyltransferase 1.

DNA methyltransferases (DNMTs) are a family of enzymes that methylate DNA at the C5 position of cytosine residues, and their inhibition is a promising strategy for the treatment of various developmental and proliferative diseases, particularly cancers. In the present study, a binding model for hydralazine, with a validated homology model of human DNMT, was developed by the use of automated molecular docking and molecular dynamics simulations. The docking protocol was validated by predicting the binding mode of 2'-deoxycytidine, 5-azacytidine, and 5-aza-2'-deoxycytidine. The inhibitory activity of hydralazine toward DNMT may be rationalized at the molecular level by similar interactions within the binding pocket (e.g., by a similar pharmacophore) as established by substrate-like deoxycytidine analogues. These interactions involve a complex network of hydrogen bonds with arginine and glutamic acid residues that also play a major role in the mechanism of DNA methylation. Despite the different scaffolds of other non-nucleoside DNMT inhibitors such as procaine and procainamide, the current modeling work reveals that these drugs exhibit similar interactions within the DNMT1 binding site. These findings are valuable in guiding the rational design and virtual screening of novel DNMT inhibitors.