News Article | April 19, 2017
More effective cancer treatments are likely to emerge from the drug development pipeline. Cancer drug discovery hinges on identifying and characterizing binding pockets in target proteins. Typically, this evaluation uses computational techniques that rely on static protein structures. However, proteins have an inherent flexibility that causes a tendency to change shape upon contact with the drugs. Certain binding pockets remain undetectable unless they interact with an appropriate substance and, therefore, are missed by conventional simulations. These hidden pockets, however, are usually water-repelling or hydrophobic sites that only open when there are low polarity substances. To tackle this, Yaw Sing Tan and Chandra Verma from the Bioinformatics Institute have developed a probe-based method called ligand-mapping molecular dynamics (LMMD). They used this technique to seek hidden binding pockets in the anticancer target protein MDM2. The resulting predictions were experimentally validated by long-standing collaborators from A*STAR's p53 Laboratory and Institute of Chemical and Engineering Sciences as well as structural biologists from Newcastle University, UK. Tan explains that initially he had designed this probe-based method for another target protein and successfully used it to find a hidden binding pocket that stayed closed in conventional simulations. "We then decided to apply this approach to MDM2 to see if we could discover any previously unknown binding sites that could enhance the potency of existing MDM2 inhibitors," he adds. Using benzene molecules as hydrophobic pocket detection probes, the researchers computationally identified two new binding sites on MDM2. "We were excited to see that these sites lie very close to the binding pocket of the tumor suppressor protein p53," says Tan. Furthermore, the researchers expect the newly found sites to lead to more potent stapled peptides—these are amino acid helices chemically stabilized by a hydrocarbon chain that have recently emerged as powerful p53 activators. Consequently, they created stapled peptides from analogs known to tightly bind MDM2 and reactivate p53, and determined the affinity of these peptides to MDM2. Their simulations showed that the peptides bound MDM2 more strongly than p53 in the pockets and matched biophysical and X-ray crystallography experiments. "This method could be used to interrogate other anticancer protein targets to uncover novel binding sites that could be targeted for inhibition," says Tan. The team is now working to expand the reach of LMMD probes to other ligand types. Explore further: Novel use of fluorescent probe may lead to future anti-cancer drugs More information: Yaw Sing Tan et al. Benzene Probes in Molecular Dynamics Simulations Reveal Novel Binding Sites for Ligand Design, The Journal of Physical Chemistry Letters (2016). DOI: 10.1021/acs.jpclett.6b01525
Ramalingam B.,Neuros |
Sana B.,p53 Laboratory |
Seayad J.,Neuros |
Ghadessy F.J.,p53 Laboratory |
Sullivan M.B.,Institute of High Performance Computing of Singapore
RSC Advances | Year: 2017
Laccase-catalysed oligomerisation of dimeric β-O-4 linked lignin model compounds was studied in detail to understand the oligomerisation process by monitoring the reaction progress using high performance liquid chromatography (HPLC) and mass spectroscopy (MS). The initial oxidation intermediates of oligomerisation were isolated for the first time and characterised by spectroscopic methods sucessfully. The experimental observations indicated that C5-C5′ biphenyl linkages, one of the most thermodynamically stable linkages present in the native lignin, are formed exclusively during the early stage of the oligomerisation process. The experimental observations were supported by density functional theory (DFT) calculations of relative free energies of possible products. The C5-C5′ biphenyl tetramer is the thermodynamically more favoured product compared to the C5-O-C4′ product by a free energy difference of 10.0 kcal mol−1 in water. Among the various linking possibilities for further formation of hexamers, the thermodynamically more stable product with a similar C-C linkage is proposed as a plausible structure based on the mass of the hexamer isolated and DFT calculations. The current study demonstrates that laccase catalyzes the oligomerisation more preferentially than oxidative bond cleavage in β-O-4 linkages and that product formation is likely controlled by the thermodynamic stability of the resultant oligomers. © The Royal Society of Chemistry.
Tan B.X.,P53 Laboratory |
Khoo K.H.,P53 Laboratory |
Lim T.M.,National University of Singapore |
Lane D.P.,P53 Laboratory
Oncotarget | Year: 2014
Although p53 is found mutated in almost 50% of all cancers, p53 mutations in leukaemia are relatively rare. Acute myeloid leukaemia (AML) cells employ other strategies to inactivate their wild type p53 (WTp53), like the overexpression of the p53 negative regulators Mdm2 and Mdm4. As such, AMLs are excellent candidates for therapeutics involving the reactivation of their WTp53 to restrict and destroy cancer cells, and the Mdm2 antagonist nutlin-3 is one such promising agent. Using AML cell lines with WTp53, we identified stable and high levels of p53 in the OCI/AML-2 cell lines. We demonstrate that this nutlin-3 sensitive cell line overexpressed Mdm4 to sequester, stabilise and inhibit p53 in the cytoplasm. We also show that elevated Mdm4 competed with Mdm2-p53 interaction and therefore extended p53 half-life while preventing p53 transcriptional activity. Our results provide biochemical evidence on the dynamics of the p53-Mdm2-Mdm4 interactions in affecting p53 levels and activity, and unlike previously reported findings derived from genetically manipulated systems, AML cells with naturally high levels of Mdm4 remain sensitive to nutlin treatment. Key Points Endogenously high levels of Mdm4 inhibit and sequester p53 in AML. High levels of Mdm4 do not block function of Mdm2 inhibitors in AML.
Goh A.M.,p53 Laboratory |
Coffill C.R.,Institute of Molecular and Cell Biology |
Lane D.P.,p53 Laboratory
Journal of Pathology | Year: 2011
Mutations in the TP53 (p53) gene are present in a large fraction of human tumours, which frequently express mutant p53 proteins at high but heterogeneous levels. The clinical significance of this protein accumulation remains clouded. Mouse models bearing knock-in mutations of p53 have established that the mutant p53 proteins can drive tumour formation, invasion and metastasis through dominant negative inhibition of wild-type p53 as well as through gain of function or 'neomorphic' activities that can inhibit or activate the function of other proteins. These models have also shown that mutation alone does not confer stability, so the variable staining of mutant proteins seen in human cancers reflects tumour-specific activation of p53-stabilizing pathways. Blocking the accumulation and activity of mutant p53 proteins may thus provide novel cancer therapeutic and diagnostic targets, but their induction by chemotherapy may paradoxically limit the effectiveness of these treatments. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Hu K.,CAS Hefei Key Laboratory of Materials for Energy Conversion |
Schmidt N.W.,University of California at Los Angeles |
Zhu R.,CAS Hefei Key Laboratory of Materials for Energy Conversion |
Jiang Y.,CAS Hefei Key Laboratory of Materials for Energy Conversion |
And 8 more authors.
Macromolecules | Year: 2013
Polymeric synthetic mimics of antimicrobial peptides (SMAMPs) have recently demonstrated similar antimicrobial activity as natural antimicrobial peptides (AMPs) from innate immunity. This is surprising, since polymeric SMAMPs are heterogeneous in terms of chemical structure (random sequence) and conformation (random coil), in contrast to defined amino acid sequence and intrinsic secondary structure. To understand this better, we compare AMPs with a "minimal" mimic, a well-characterized family of polydisperse cationic methacrylate-based random copolymer SMAMPs. Specifically, we focus on a comparison between the quantifiable membrane curvature generating capacity, charge density, and hydrophobicity of the polymeric SMAMPs and AMPs. Synchrotron small-angle X-ray scattering (SAXS) results indicate that typical AMPs and these methacrylate SMAMPs generate similar amounts of membrane negative Gaussian curvature (NGC), which is topologically necessary for a variety of membrane-destabilizing processes. Moreover, the curvature generating ability of SMAMPs is more tolerant of changes in the lipid composition than that of natural AMPs with similar chemical groups, consistent with the lower specificity of SMAMPs. We find that, although the amount of NGC generated by these SMAMPs and AMPs are similar, the SMAMPs require significantly higher levels of hydrophobicity and cationic charge to achieve the same level of membrane deformation. We propose an explanation for these differences, which has implications for new synthetic strategies aimed at improved mimesis of AMPs. © 2013 American Chemical Society.
Wong J.S.,P53 Laboratory |
Wong J.S.,A-Life Medical |
Warbrick E.,A-Life Medical |
Vojtesk B.,Masaryk Memorial Cancer Institute |
And 2 more authors.
Oncotarget | Year: 2013
c-Met is a tyrosine receptor kinase which is activated by its ligand, the hepatocyte growth factor. Activation of c-Met leads to a wide spectrum of biological activities such as motility, angiogenesis, morphogenesis, cell survival and cell regeneration. c-Met is abnormally activated in many tumour types. Aberrant c-Met activation was found to induce tumour development, tumour cell migration and invasion, and the worst and final step in cancer progression, metastasis. In addition, c-Met activation in cells was also shown to confer resistance to apoptosis induced by UV damage or chemotherapeutic drugs. This study describes the development of monoclonal antibodies against c-Met as therapeutic molecules in cancer treatment/diagnostics. A panel of c-Met monoclonal antibodies was developed and characterised by epitope mapping, Western blotting, immunoprecipitation, agonist/antagonist effect in cell scatter assays and for their ability to recognise native c-Met by flow cytometry. We refer to these antibodies as Specifically Engaging Extracellular c-Met (seeMet). seeMet 2 and 13 bound strongly to native c-Met in flow cytometry and reduced SNU-5 cell growth. Interestingly, seeMet 2 binding was strongly reduced at 4°C when compared to 37°C. Detail mapping of the seeMet 2 epitope indicated a cryptic binding site hidden within the c-Met a-chain.
Lane D.P.,p53 Laboratory |
Cheok C.F.,p53 Laboratory |
Brown C.J.,p53 Laboratory |
Madhumalar A.,Agency for Science, Technology and Research Singapore |
And 2 more authors.
Cell Cycle | Year: 2010
The p53 protein and its negative regulator the ubiquitin E3 ligase Mdm2 have been shown to be conserved from the T. adhaerens to man. In common with D. melanogaster and C. elegans, there is a single copy of the p53 gene in T. adhaerens, while in the vertebrates three p53-like genes can be found: p53, p63 and p73. The Mdm2 gene is not present within the fully sequenced and highly annotated genomes of C. elegans and D. melanogaster. However, it is present in Placazoanand the presence of multiple distinct p53 genes in the Sea anemone N. vectensis led us to examine the genomes of other phyla for p53 and Mdm2-like genes. We report here the discovery of an Mdm2-like gene and two distinct p53-like genes in the Arachnid loxodes scapularis (Northern Deer Tick). The two predicted Deer Tick p53 proteins are much more highly related to the human p53 protein in sequence than are the fruit fly and nematode proteins. One of the Deer Tick genes encodes a p53 protein that is initiated within the DNA binding domain of p53 and shows remarkable homology to the newly described N-terminally truncated delta isoforms of human and zebrafish p53. © 2010 Landes Bioscience.
Tay Y.,p53 Laboratory |
Ho C.,p53 Laboratory |
Droge P.,p53 Laboratory |
Ghadessy F.J.,p53 Laboratory
Nucleic acids research | Year: 2010
In vitro compartmentalization (IVC) was employed for the first time to select for novel bacteriophage lambda integrase variants displaying significantly enhanced recombination activity on a non-cognate target DNA sequence. These variants displayed up to 9-fold increased recombination activity over the parental enzyme, and one mutant recombined the chosen non-cognate substrate more efficiently than the parental enzyme recombined the wild-type DNA substrate. The in vitro specificity phenotype extended to the intracellular recombination of episomal vectors in HEK293 cells. Surprisingly, mutations conferring the strongest phenotype do not occur in the lambda integrase core-binding domain, which is known to interact directly with cognate target sequences. Instead, they locate to the N-terminal domain which allosterically modulates integrase activity, highlighting a previously unknown role for this domain in directing integrase specificity. The method we describe provides a robust, completely in vitro platform for the development of novel integrase reagent tools for in vitro DNA manipulation and other biotechnological applications.
Goh W.,p53 Laboratory |
Lane D.,p53 Laboratory |
Ghadessy F.,p53 Laboratory
Cell Cycle | Year: 2010
The p53 tumor suppressor plays a critical role in cancer biology, functioning as a transcription factor capable of directing cell fate. It interacts with specific DNA response elements (REs) to regulate the activity of target genes. We describe here a novel, non-radioactive assay to measure p53-DNA binding which involves the sequential use of in vitro transcription/ translation (IVT), immunoprecipitation and real-time PCR. The method reliably enables the detection of sequencespecific DNA binding of full-length p53 at low concentrations of physiologically relevant REs (<5 nM). Furthermore, we demonstrate multiplexing of 4 different REs in a single binding reaction. The use of IVT precludes the requirement for purified protein, enabling rapid characterization of the binding properties of p53 variants. Uniquely, it also offers the opportunity to add compounds during translation that might modulate and activate p53. When compared to prevailing protein-DNA binding assays, this method exhibits comparable or higher sensitivity, in addition to an expansive dynamic range afforded by the use of real-time PCR. A further extrapolation of its utility is demonstrated when the addition of a peptide known to activate p53 increased its binding to a consensus RE, consistent with published data. © 2010 Landes Bioscience.
Nirantar S.R.,P53 Laboratory |
Ghadessy F.J.,P53 Laboratory
Proteomics | Year: 2011
Emulsion technology has been successfully applied to the fields of next-generation high-throughput sequencing, protein engineering and clinical diagnostics. Here, we extend its scope to proteomics research by developing and characterizing a method, termed iCLIP (in vitro compartmentalized linkage of interacting partners), which enables genes encoding interacting protein pairs to be linked in a single segment of DNA. This will facilitate archiving of the interactomes from library versus library two-hybrid screens as libraries of linked DNAs. We further demonstrate the ability to interrogate a model yeast two-hybrid iCLIP library for interactants by "PCR-pulldown," using a primer specific to a gene of interest along with a universal primer. iCLIP libraries may also be subjected to high-throughput sequencing to generate interactome information. The applicability of the technique is also demonstrated in the related context of the bacterial two-hybrid system. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.