Ultrasensitive Detection of MCF-7 Cells with a Carbon Nanotube-Based Optoelectronic-Pulse Sensor Framework

Biosensors are of vital significance for healthcare by supporting the management of infectious diseases for preventing pandemics and the diagnosis of life-threatening conditions such as cancer. However, the advancement of the field can be limited by low sensing accuracy. Here, we altered the bioelectrical signatures of the cells using carbon nanotubes (CNTs) via structural loosening effects. Using an alternating current (AC) pulse under light irradiation, we developed a photo-assisted AC pulse sensor based on CNTs to differentiate between healthy breast epithelial cells (MCF-10A) and luminal breast cancer cells (MCF-7) within a heterogeneous cell population. We observed a previously undemonstrated increase in current contrast for MCF-7 cells with CNTs compared to MCF-10A cells with CNTs under light exposure. Moreover, we obtained a detection limit of ∼1.5 × 10^{3} cells below a baseline of ∼1 × 10^{4} cells for existing electrical-based sensors for an adherent, heterogeneous cell population. All-atom molecular dynamics (MD) simulations reveal that interactions between the embedded CNT and cancer cell membranes result in a less rigid lipid bilayer structure, which can facilitate CNT translocation for enhancing current. This as-yet unconsidered cancer cell-specific method based on the unique optoelectrical properties of CNTs represents a strategy for unlocking the detection of a small population of cancer cells and provides a promising route for the early diagnosis, monitoring, and staging of cancer

Diversity-oriented synthesis as a tool for identifying new modulators of mitosis.

The synthesis of diverse three-dimensional libraries has become of paramount importance for obtaining better leads for drug discovery. Such libraries are predicted to fare better than traditional compound collections in phenotypic screens and against difficult targets. Herein we report the diversity-oriented synthesis of a compound library using rhodium carbenoid chemistry to access structurally diverse three-dimensional molecules and show that they access biologically relevant areas of chemical space using cheminformatic analysis. High-content screening of this library for antimitotic activity followed by chemical modification identified 'Dosabulin', which causes mitotic arrest and cancer cell death by apoptosis. Its mechanism of action is determined to be microtubule depolymerization, and the compound is shown to not significantly affect vinblastine binding to tubulin; however, experiments suggest binding to a site vicinal or allosteric to Colchicine. This work validates the combination of diversity-oriented synthesis and phenotypic screening as a strategy for the discovery of biologically relevant chemical entities.This is the author's accepted manuscript. The final version was published in Nature Communications here: http://www.nature.com/ncomms/2014/140117/ncomms4155/full/ncomms4155.html#affil-auth

Cyclisation strategies for stabilising peptides with irregular conformations

Cyclisation is a common synthetic strategy for enhancing the therapeutic potential of peptide-based molecules. While there are extensive studies on peptide cyclisation for reinforcing regular secondary structures such as α-helices and β-sheets, there are remarkably few reports of cyclising peptides which adopt irregular conformations in their bioactive target-bound state. In this review, we highlight examples where cyclisation techniques have been successful in stabilising irregular conformations, then discuss how the design of cyclic constraints for irregularly structured peptides can be informed by existing β-strand stabilisation approaches, new computational design techniques, and structural principles extracted from cyclic peptide library screening hits. Through this analysis, we demonstrate how existing peptide cyclisation techniques can be adapted to address the synthetic design challenge of stabilising irregularly structured binding motifs

Targeted covalent inhibitors of MDM2 using electrophile-bearing stapled peptides.

Herein, we describe the development of a novel staple with an electrophilic warhead to enable the generation of stapled peptide covalent inhibitors of the p53-MDM2 protein-protein interaction (PPI). The peptide developed showed complete and selective covalent binding resulting in potent inhibition of p53-MDM2 PPI.Royal Society, Trinity College, A*STAR (IAF-PP H17/01/a0/010

Targeting cancer addiction for SALL4 by shifting its transcriptome with a pharmacologic peptide

Sal-like 4 (SALL4) is a nuclear factor central to the maintenance of stem cell pluripotency and is a key component in hepatocellular carcinoma, a malignancy with no effective treatment. In cancer cells, SALL4 associates with nucleosome remodeling deacetylase (NuRD) to silence tumor-suppressor genes, such as PTEN. Here, we determined the crystal structure of an amino-terminal peptide of SALL4(1-12) complexed to RBBp4, the chaperone subunit of NuRD, at 2.7 Å, and subsequent design of a potent therapeutic SALL4 peptide (FFW) capable of antagonizing the SALL4-NURD interaction using systematic truncation and amino acid substitution studies. FFW peptide disruption of the SALL4-NuRD complex resulted in unidirectional up-regulation of transcripts, turning SALL4 from a dual transcription repressor-activator mode to singular transcription activator mode. We demonstrate that FFW has a target affinity of 23 nM, and displays significant antitumor effects, inhibiting tumor growth by 85% in xenograft mouse models. Using transcriptome and survival analysis, we discovered that the peptide inhibits the transcription-repressor function of SALL4 and causes massive up-regulation of transcripts that are beneficial to patient survival. This study supports the SALL4-NuRD complex as a drug target and FFW as a viable drug candidate, showcasing an effective strategy to accurately target oncogenes previously considered undruggable

Development of Cell-Permeable, Non-Helical Constrained Peptides to Target a Key Protein-Protein Interaction in Ovarian Cancer.

There is a lack of current treatment options for ovarian clear cell carcinoma (CCC) and the cancer is often resistant to platinum-based chemotherapy. Hence there is an urgent need for novel therapeutics. The transcription factor hepatocyte nuclear factor 1β (HNF1β) is ubiquitously overexpressed in CCC and is seen as an attractive therapeutic target. This was validated through shRNA-mediated knockdown of the target protein, HNF1β, in five high- and low-HNF1β-expressing CCC lines. To inhibit the protein function, cell-permeable, non-helical constrained proteomimetics to target the HNF1β-importin α protein-protein interaction were designed, guided by X-ray crystallographic data and molecular dynamics simulations. In this way, we developed the first reported series of constrained peptide nuclear import inhibitors. Importantly, this general approach may be extended to other transcription factors

Discovery of a small-molecule binder of the oncoprotein gankyrin that modulates gankyrin activity in the cell.

Gankyrin is an ankyrin-repeat oncoprotein whose overexpression has been implicated in the development of many cancer types. Elevated gankyrin levels are linked to aberrant cellular events including enhanced degradation of tumour suppressor protein p53, and inhibition of gankyrin activity has therefore been identified as an attractive anticancer strategy. Gankyrin interacts with several partner proteins, and a number of these protein-protein interactions (PPIs) are of relevance to cancer. Thus, molecules that bind the PPI interface of gankyrin and interrupt these interactions are of considerable interest. Herein, we report the discovery of a small molecule termed cjoc42 that is capable of binding to gankyrin. Cell-based experiments demonstrate that cjoc42 can inhibit gankyrin activity in a dose-dependent manner: cjoc42 prevents the decrease in p53 protein levels normally associated with high amounts of gankyrin, and it restores p53-dependent transcription and sensitivity to DNA damage. The results represent the first evidence that gankyrin is a "druggable" target with small molecules.The work was supported by a grant from the Development Gap Fund (MRC Technology), a research grant from the Isaac Newton Trust, Cambridge and from the CORE charity. LSI acknowledges the support of a Senior Fellowship from the Medical Research Foundation. TR holds a Royal Society University Research Fellowship. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ ERC grant agreement n° [279337/DOS]

Web usability studies for popular websites

This report provides details of my Final Year Project from August 2013 to March 2014. The aim of this project was to conduct usability studies of popular websites, mainly with the use of an eye tracker. A number of objectives were set, including the identification of a website that best satisfied both web advertisers and web users. Six participants were instructed to complete 3 different experiments. Eye tracking metrics and eye tracking visualizations were used in conjunction with conventional usability practices for a thorough assessment of the results from the experiments. Based on results from the experiments, Facebook was determined to be the website that best satisfies web advertisers. YouTube was determined to be the website that best satisfies web users. Overall, YouTube was determined to be the website that provides maximum satisfaction to both web advertisers and web users. The features of the website that contributed to this result are discussed and a number of key observations are obtained.Bachelor of Engineering (Computer Science

The use of chlorobenzene as a probe molecule in molecular dynamics simulations

We map ligand binding sites on protein surfaces in molecular dynamics simulations using chlorobenzene as a probe molecule. The method was validated on four proteins. Two types of affinity maps that identified halogen and hydrophobic binding sites on proteins were obtained. Our method could prove useful for the discovery and development of halogenated inhibitors.Accepted versio

Inhibiting S100B(ββ) for Activating Wild-Type p53: Design of Stapled Peptides

S100B(ββ) is a member of the S100B protein family and is distributed in a cell-specific manner. Its levels are elevated in several cancers such as malignant melanoma and correlate directly with poor prognosis in patients. S100B(ββ) directly interacts with the tumor suppressor p53, inhibiting tetramerization and protein kinase C-dependent phosphorylation, consequently decreasing p53 DNA binding and transcriptional activity, and preventing apoptosis. Thus, S100B(ββ) is being pursued as a target for therapeutic inhibition. However, development of small molecule inhibitors targeting p53-interactions has met with limited success. In this work, we present a set of designed stapled peptide inhibitors of S100B(ββ), guided by the structure of the C-terminal domain of p53 complexed with S100B(ββ). We further modified a tightly binding stapled peptide with imaging agents and propose these as potential diagnostic agents to detect S100B(ββ) as a biomarker.NRF (Natl Research Foundation, S’pore)ASTAR (Agency for Sci., Tech. and Research, S’pore)EDB (Economic Devt. Board, S’pore)Published versio