Full Schedule

Gillian Murphy, Ph.D.
Professor, Department of Oncology, University of Cambridge, UK

A novel strategy for targeting metalloproteinases in cancer

Epithelial tumours evolve in a multi-step manner, involving both inflammatory and mesenchymal cells. Although intrinsic factors drive malignant progression, the influence of the micro-environment of neoplastic cells is a major feature of tumorigenesis. Extracellular proteinases, notably the metalloproteinases, are key players in the regulation of this cellular environment, acting as major effectors of both cell-cell and cell-extracellular matrix (ECM) interactions. They are involved in modifying ECM integrity, growth factor availability and the function of cell surface signalling systems, with consequent effects on cellular differentiation, proliferation and apoptosis.This has made metalloproteinases important targets for therapeutic interventions in cancer and small molecule inhibitors focussed on chelation of the active site zinc and binding within the immediate active site pocket were developed.  These were not successful in early clinical trials due to the relative lack of specificity and precise knowledge of the target proteinase(s) in specific cancers. We can now appreciate that it is essential that we understand the relative roles of the different enzymes (of which there are over 60) in terms of their pro and anti tumour activity and their precise sites of expression The next generations of metalloproteinase inhibitors need the added specificity that might be gained from an understanding of the structure of individual active sites and the role of extra catalytic domains in substrate binding and other aspects of their biology. We have prepared scFv antibodies to the extra catalytic domains of two membrane metalloproteinases, MMP-14 and ADAM17, that play key roles in the tumour microenvironment. Our rationale and experiences with these agents will be presented in more detail.

Deepthy Menon, Ph.D.
Associate Professor, Centre for Nanosciences & Molecular Medicine, Health Sciences Campus, Amrita University, Kochi, India

Nanobioengineering of implant materials for improved cellular response and activity

Deepthy Menon, Divyarani V V, Chandini C Mohan, Manitha B Nair, Krishnaprasad C & Shantikumar V Nair

Abstract

Current trends in biomaterials research and development include the use of surfaces with topographical features at the nanoscale (dimensions < 100 nm), which influence biomolecular or cellular level reactions in vitro and in vivo. Progress in nanotechnology now makes it possible to precisely design and modulate the surface properties of materials used for various applications in medicine at the nanoscale. Nanoengineered surfaces, owing to their close resemblance with extracellular matrix, possess the unique capacity to directly affect protein adsorption that ultimately modulates the cellular adhesion and proliferation at the site of implantation. Taking advantage of this exceptional ability, we have nanoengineered metallic surfaces of Titanium (Ti) and its alloys (Nitinol -NiTi), as well as Stainless Steel (SS) by a simple hydrothermal method for generating non-periodic, homogeneous nanostructures. The bio- and hemocompatibility of these nanotextured metallic surfaces suggest their potential use for orthopedic, dental or vascular implants. The applicability of nanotextured Ti implants for orthopedic use was demonstrated in vivo in rat models, wherein early-stage bone formation at the tissue-implant interface without any fibrous tissue intervention was achieved. This nanoscale topography also was found to critically influence bacterial adhesion in vitro, with decreased adherence of staphylococcus aureus. The same surface nanotopography also was found to provide enhanced proliferation and functionality of vascular endothelial cells, suggesting its prospective use for developing an antithrombotic stent surface for coronary applications. Clinical SS & NiTi stents were also modified based on this strategy, which would offer a suitable solution to reduce the probability of late stent thrombosis associated with bare metallic stents. Thus, we demonstrate that nanotopography on implant surfaces has a critical influence on the fate of cells, which in turn dictates the long term success of the implant.

Acknowledgement: Authors gratefully acknowledge the financial support from Department of Biotechnology, Government of India through the Bioengineering program.

Manzoor K, Ph.D.
Professor, Centre for Nanoscience & Molecular Medicine, Amrita University

Targeting aberrant cancer kinome using rationally designed nano-polypharmaceutics

Manzoor Koyakutty, Archana Ratnakumary, Parwathy Chandran, Anusha Ashokan, and Shanti Nair

`War on Cancer’ was declared nearly 40 years ago. Since then, we made significant progress on fundamental understanding of cancer and developed novel therapeutics to deal with the most complex disease human race ever faced with. However, even today, cancer remains to be the unconquered `emperor of all maladies’. It is well accepted that meaningful progress in the fight against cancer is possible only with in-depth understanding on the molecular mechanisms that drives its swift and dynamic progression. During the last decade, emerging new technologies such as nanomedicine could offer refreshing life to the `war on cancer’ by way of providing novel methods for molecular diagnosis and therapy.

In the present talk, we discuss our approaches to target critically aberrant cancer kinases using rationally designed polymer-protein and protein-protein core-shell nanomedicines. We have used both genomic and proteomic approaches to identify many intimately cross-linked and complex aberrant protein kinases behind the drug resistance and uncontrolled proliferation of refractory leukemic cells derived from patients. Small molecule inhibitors targeted against oncogenic pathways in these cells were found ineffective due to the involvement of alternative survival pathways. This demands simultaneous inhibition more than one oncogenic kinases using poly-pharmaceutics approach. For this, we have rationally designed core-shell nanomedicines that can deliver several small molecules together for targeting multiple cancer signalling. We have also used combination of small molecules and siRNA for combined gene silencing together with protein kinase inhibition in refractory cancer cells. Optimized nanomedicines were successfully tested in patient samples and found enhanced cytotoxicity and molecular specificity in drug resistant cases.

Nano-polypharmaceutics represents a new generation of nanomedicines that can tackle multiple cancer mechanisms simultaneously. Considering the complexity of the disease, such therapeutic approaches are not simply an advantage, but indispensable.

Acknowledgements:
We thank Dept. of Biotechnology and Dept. Of Science and Technology,Govt. of India for the financial support through `Thematic unit of Excellence in Medical NanoBiotechnology’ and `Nanomedicine- RNAi programs’.

Rajasekhar Chekkara, Venkata Reddy Gorla and Sobha Rani Tenkayala

Pharmacophore modeling, atom-based 3D-QSAR and molecular docking studies on Pyrimido[5,4-e][1,2,4]triazine derivatives as PLK 1 inhibitors

Polo-like kinase 1 (PLK1) is a significant enzyme with diverse biological actions in cell cycle progression, specifically mitosis. Suppression of PLK1 activity by small molecule inhibitors has been shown to inhibit cancer, being BI 2536 one of the most potent active inhibitor of PLK1 mechanism. Pharmacophore modeling, atom-based 3D-QSAR and molecular docking studies were carried out for a set of 54 compounds belonging to Pyrimido[5,4-e][1,2,4]triazine derivatives as PLK1 inhibitors. A six-point pharmacophoremodel AAADDR, with three hydrogen bond acceptors (A), two hydrogen bond donors (D) and one aromatic ring (R) was developed by Phase module of Schrdinger suite Maestro 9. The generated pharmacophore model was used to derive a predictive atom-based 3D quantitative structure-activity relationship analysis (3D-QSAR) model for the training set (r2 = 0.88, SD = 0.21, F = 57.7, N = 44) and for test set (Q2 = 0.51, RMSE = 0.41, PearsonR = 0.79, N = 10). The original set of compounds were docked into the binding site of PLK1 using Glide and the active residues of the binding site were analyzed. The most active compound H18 interacted with active residues Leu 59, Cys133 (glide score = −10.07) and in comparison of BI 2536, which interacted with active residues Leu 59, Cys133 (glide score = −10.02). The 3D-QSAR model suggests that hydrophobic and electron-withdrawing groups are essential for PLK1 inhibitory activity. The docking results describes the hydrogen bond interactions with active residues of these compounds. These results which may support in the design and development of novel PLK1 inhibitors.

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Sudarslal S, Ph.D.
Associate Professor, School of Biotechnology, Amrita University

Electrospray ionization ion trap mass spectrometry for cyclic peptide characterization

There has been considerable interest in the isolation and structural characterization of bioactive peptides produced by bacteria and fungi. Most of the peptides are cyclic depsipeptides characterized by the presence of lactone linkages and β-hydroxy fatty acids. Occurrence of microheterogeneity is another remarkable property of these peptides. Even if tandem mass spectrometers are good analytical tools to structurally characterize peptides and proteins, sequence analysis of cyclic peptides is often ambiguous due to the random ring opening of the peptides and subsequent generation of a set of linear precursor ions with the same m/z. Here we report combined use of chemical derivatization and multistage fragmentation capability of ion trap mass spectrometers to determine primary sequences of a series of closely related cyclic peptides.

Ravindra Gudihal, Suresh Babu C V

Bioanalytical Characterization of Therapeutic Proteins

The characterization of therapeutic proteins such as monoclonal antibody (mAb) during different stages of manufacturing is crucial for timely and successful product release. Regulatory agencies require a variety of analytical technologies for comprehensive and efficient protein analysis. Electrophoresis-based techniques and liquid chromatography (LC) either standalone or coupled to mass spectrometry (MS) are at the forefront for the in-depth analysis of protein purity, isoforms, stability, aggregation, posttranslational modifications, PEGylation, etc. In this presentation, a combination of various chromatographic and electrophoretic techniques such as liquid-phase isoelectric focusing, microfluidic and capillary-based electrophoresis (CE), liquid chromatography (LC) and combinations of those with mass spectrometry techniques will be discussed. We present a workflow based approach to the analysis of therapeutic proteins. In successive steps critical parameters like purity, accurate mass, aggregation, peptide sequence, glycopeptide and glycan analysis are analyzed. In brief, the workflow involved proteolytic digestion of therapeutic protein for peptide mapping, N-Glycanase and chemical labeling reaction for glycan analysis, liquid-phase isoelectric focusing for enrichment of charge variants followed by a very detailed analysis using state of the art methods such as CE-MS and LC-MS. For the analysis of glycans, we use combinations of CE-MS and LC-MS to highlight the sweet spots of these techniques. CE-MS is found to be more useful in analysis of highly sialylated glycans (charged glycans) while nano LC-MS seems to be better adapted for analysis of neutral glycans. These two techniques can be used to get complementary data to profile all the glycans present in a given protein. In addition, microfluidic electrophoresis was used as a QC tool in initial screening for product purity, analysis of papain digestion fragments of mAb, protein PEGylation products, etc. The described workflow involves multiple platforms, provides an end to end solution for comprehensive protein characterization and aims at reducing the total product development time.

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