Identification of a new and diverse set of Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng) inhibitors using structure-based virtual screening: Experimental validation and molecular dynamics studies.

Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng), a key DNA repair enzyme, represents an attractive target for the design of new antimycobacterial agents. However, only a limited number of weak MtUng inhibitors are reported, primarily based on the uracil ring, and hence, lack diversity. We report the first structure-based virtual screening (SBVS) using three separate libraries consisting of uracil and non-uracil small molecules, together with the FDA-approved drugs. Twenty diverse virtual hits with the highest predicted binding were procured and screened using a fluorescence-based assay to evaluate their potential to inhibit MtUng. Several of these molecules were found to inhibit MtUng activity at low mM and µM levels, comparable to or better than several other reported Ung inhibitors. Thus, these molecules represent a diverse set of scaffolds for developing next-generation MtUng inhibitors. The most active uracil-based compound 5 (IC50 = 0.14 mM) was found to be âˆ¼ 15-fold more potent than the positive control, uracil. The binding stability and conformation of compound 5 in complex with the enzyme were further confirmed using molecular dynamics simulation.

Essential and selective role of SNX12 in transport of endocytic and retrograde cargo.

The endosomal protein-sorting machineries play vital roles in diverse physiologically important cellular processes. Much of the core membrane-sorting apparatus is conserved in evolution, such as retromer, which is involved in the recycling of a diverse set of cargoes via the retrograde trafficking route. Here, in an RNAi-based loss-of-function study, we identified that suppression of SNX12 leads to a severe blockage in CIM6PR (also known as IGF2R) transport and alters the morphology of the endocytic compartments. We demonstrate that SNX12 is involved in the early phase of CIM6PR transport, and mediates receptor recycling upstream of the other well-established SNX components of retromer. Ultra-structural analysis revealed that SNX12 resides on tubulo-vesicular structures, despite it lacking a BAR domain. Furthermore, we illustrate that SNX12 plays a key role in intraluminal vesicle formation and in the maturation of a subpopulation of early endosomes into late endosomes, thereby regulating selective endocytic transport of cargo for degradation. This study therefore provides evidence for the existence of early endosomal subpopulations that have differential roles in the sorting of the cargoes along endocytic degradative pathways.

It's not who you know, but who you are: Explaining income gaps of stigmatized-caste business owners in India.

Scholars across disciplines and around the world have diverted research attention to rising income inequalities across groups and strategies to reduce them. The literature has broadly identified human capital and social capital as two potential tools to facilitate economic mobility and to reduce inequalities. However, it is not known whether these tools work equally well for stigmatized groups, particularly in societies with systemic inequalities. Analyzing data from a pan-India survey, we show that business owners from stigmatized groups (i.e., Dalits in India, who are stigmatized as untouchables) experience a business income gap of around 16% compared to others, including those business owners who are from communities that are disadvantaged but are not similarly stigmatized. We find that, instead of being reduced, this gap in fact increases at higher levels of social capital, especially bridging social capital, illustrating the social processes of stigmatization that limit the benefits that Dalits can reap from social capital. By contrast, Dalits can reap similar income benefits as others from human capital. Our results show that human capital helps stigmatized groups mitigate the implications of stigma, but social capital does not.

Insights into cargo sorting by SNX32 and its role in neurite outgrowth.

Sorting nexins (SNX) are a family of proteins containing the Phox homology domain, which shows a preferential endo-membrane association and regulates cargo sorting processes. Here, we established that SNX32, an SNX-BAR (Bin/Amphiphysin/Rvs) sub-family member associates with SNX4 via its BAR domain and the residues A226, Q259, E256, R366 of SNX32, and Y258, S448 of SNX4 that lie at the interface of these two SNX proteins mediate this association. SNX32, via its PX domain, interacts with the transferrin receptor (TfR) and Cation-Independent Mannose-6-Phosphate Receptor (CIMPR), and the conserved F131 in its PX domain is important in stabilizing these interactions. Silencing of SNX32 leads to a defect in intracellular trafficking of TfR and CIMPR. Further, using SILAC-based differential proteomics of the wild-type and the mutant SNX32, impaired in cargo binding, we identified Basigin (BSG), an immunoglobulin superfamily member, as a potential interactor of SNX32 in SHSY5Y cells. We then demonstrated that SNX32 binds to BSG through its PX domain and facilitates its trafficking to the cell surface. In neuroglial cell lines, silencing of SNX32 leads to defects in neuronal differentiation. Moreover, abrogation in lactate transport in the SNX32-depleted cells led us to propose that SNX32 may contribute to maintaining the neuroglial coordination via its role in BSG trafficking and the associated monocarboxylate transporter activity. Taken together, our study showed that SNX32 mediates the trafficking of specific cargo molecules along distinct pathways.

Crystal structures of non-uracil ring fragments in complex with Mycobacterium tuberculosis uracil DNA glycosylase (MtUng) as a starting point for novel inhibitor design: A case study with the barbituric acid fragment.

Uracil DNA glycosylase (UDG or Ung) is a key enzyme involved in uracil excision from the DNA as a repair mechanism. Designing Ung inhibitors is thus a promising strategy to treat different cancers and infectious diseases. The uracil ring and its derivatives have been shown to inhibit Mycobacterium tuberculosis Ung (MtUng), resulting from specific and strong binding with the uracil-binding pocket (UBP). To design novel MtUng inhibitors, we screened several non-uracil ring fragments hypothesised to occupy MtUng UBP due to their high similarity to the uracil structural motif. These efforts have resulted in the discovery of novel MtUng ring inhibitors. Here we report the co-crystallised poses of these fragments, confirming their binding within the UBP, thus providing a robust structural framework for the design of novel lead compounds. We selected the barbituric acid (BA) ring as a case study for further derivatisation and SAR analysis. The modelling studies predicted the BA ring of the designed analogues to interact with the MtUng UBP much like the uracil ring. The synthesised compounds were screened in vitro using radioactivity and a fluorescence-based assay. These studies led to a novel BA-based MtUng inhibitor 18a (IC50 = 300 µM) displaying ∼24-fold potency over the uracil ring.

Screening of phytochemicals as potential anti-breast cancer agents targeting HER2: an in-silico approach.

Breast cancer is the most common cancer among women around the world. Human Epidermal growth factor Receptor-2 (HER2) is a membrane tyrosine kinase overexpressed in 30% of human breast cancers; thus, it serves as an important drug target. Currently available HER2 inhibitor lapatinib targets the ATP binding site of the cytoplasmic kinase domain, blocking autophosphorylation and activation of HER-2. However, it causes side effects like diarrhea, nausea, rash and possible liver toxicity. As phytochemicals have fewer side effects and are relatively affordable, they offer an effective alternative. Hence, we aimed to identify potential phytochemicals that could act as HER2 inhibitors employing computational methods such as molecular docking, molecular dynamic simulation, and ADMET prediction. Out of 1500 phytochemicals docked to the ATP binding site of the HER2 kinase domain, luxenchalcone, rhinacanthin Q, subtrifloralacton D, and 7,7″-dimethyllanaraflavone exhibited higher binding affinity than the reference inhibitor and satisfied the Lipinski's rule of five. Analysis of molecular dynamics simulation trajectory showed that Rhinacanthin Q, subtrifloralacton D, and 7,7″-dimethyllanaraflavone formed a stable and compact complex without vast conformational fluctuations. MM/PBSA binding free energy analysis revealed that Rhinacanthin Q, subtrifloralacton D, and 7,7″-dimethyllanaraflavone have high binding affinity to HER2. Therefore, Rhinacanthin Q, subtrifloralacton D, and 7,7″-dimethyllanaraflavone could be potential bioactive molecules to act as inhibitor of HER2 protein. Eventually, experimental studies are needed to evaluate the potentials of these phytochemicals further. The development of drug for HER2 positive breast cancer could be accelerated with the findings of our research. Communicated by Ramaswamy H. Sarma.

Use of a molecular beacon based fluorescent method for assaying uracil DNA glycosylase (Ung) activity and inhibitor screening.

Uracil DNA glycosylases are an important class of enzymes that hydrolyze the N-glycosidic bond between the uracil base and the deoxyribose sugar to initiate uracil excision repair. Uracil may arise in DNA either because of its direct incorporation (against A in the template) or because of cytosine deamination. Mycobacteria with G, C rich genomes are inherently at high risk of cytosine deamination. Uracil DNA glycosylase activity is thus important for the survival of mycobacteria. A limitation in evaluating the druggability of this enzyme, however, is the absence of a rapid assay to evaluate catalytic activity that can be scaled for medium to high-throughput screening of inhibitors. Here we report a fluorescence-based method to assay uracil DNA glycosylase activity. A hairpin DNA oligomer with a fluorophore at its 5' end and a quencher at its 3' ends was designed incorporating five consecutive U:A base pairs immediately after the first base pair (5' C:G 3') at the top of the hairpin stem. Enzyme assays performed using this fluorescent substrate were seen to be highly sensitive thus enabling investigation of the real time kinetics of uracil excision. Here we present data that demonstrate the feasibility of using this assay to screen for inhibitors of Mycobacterium tuberculosis uracil DNA glycosylase. We note that this assay is suitable for high-throughput screening of compound libraries for uracil DNA glycosylase inhibitors.

Plasticity, ligand conformation and enzyme action of Mycobacterium smegmatis MutT1.

Mycobacterium smegmatis MutT1 (MsMutT1) is a sanitation enzyme made up of an N-terminal Nudix hydrolase domain and a C-terminal domain resembling a histidine phosphatase. It has been established that the action of MutT1 on 8-oxo-dGTP, 8-oxo-GTP and diadenosine polyphosphates is modulated by intermolecular interactions. In order to further explore this and to elucidate the structural basis of its differential action on 8-oxo-NTPs and unsubstituted NTPs, the crystal structures of complexes of MsMutT1 with 8-oxo-dGTP, GMPPNP and GMPPCP have been determined. Replacement soaking was used in order to ensure that the complexes were isomorphous to one another. Analysis of the structural data led to the elucidation of a relationship between the arrangements of molecules observed in the crystals, molecular plasticity and the action of the enzyme on nucleotides. The dominant mode of arrangement involving a head-to-tail sequence predominantly leads to the generation of NDPs. The other mode of packing arrangement appears to preferentially generate NMPs. This work also provides interesting insights into the dependence of enzyme action on the conformation of the ligand. The possibility of modulating the enzyme action through differences in intermolecular interactions and ligand conformations makes MsMutT1 a versatile enzyme.

EarBit: Using Wearable Sensors to Detect Eating Episodes in Unconstrained Environments.

Chronic and widespread diseases such as obesity, diabetes, and hypercholesterolemia require patients to monitor their food intake, and food journaling is currently the most common method for doing so. However, food journaling is subject to self-bias and recall errors, and is poorly adhered to by patients. In this paper, we propose an alternative by introducing EarBit, a wearable system that detects eating moments. We evaluate the performance of inertial, optical, and acoustic sensing modalities and focus on inertial sensing, by virtue of its recognition and usability performance. Using data collected in a simulated home setting with minimum restrictions on participants' behavior, we build our models and evaluate them with an unconstrained outside-the-lab study. For both studies, we obtained video footage as ground truth for participants activities. Using leave-one-user-out validation, EarBit recognized all the eating episodes in the semi-controlled lab study, and achieved an accuracy of 90.1% and an F1-score of 90.9% in detecting chewing instances. In the unconstrained, outside-the-lab evaluation, EarBit obtained an accuracy of 93% and an F1-score of 80.1% in detecting chewing instances. It also accurately recognized all but one recorded eating episodes. These episodes ranged from a 2 minute snack to a 30 minute meal.