Upinder S. Bhalla, Ph.D.
Professor & Dean, NCBS, Bengaluru, India
Watching the network change during the formation of associative memory
The process of learning is measured through behavioural changes, but it is of enormous interest to understand its cellular and network basis. We used 2-photon imaging of hippocampal CA1 pyramidal neuron activity in mice to monitor such changes during the acquisition of a trace conditioning task. One of the questions in such learning is how the network retains a trace of a brief conditioned stimulus (a sound), until the arrival of a delayed unconditioned stimulus (a puff of air to the eye). During learning, the mice learn to blink when the tone is presented, well before the arrival of the air puff.
The mice learnt this task in 20-50 trials. We observed that in this time-frame the cells in the network changed the time of their peak activity, such that their firing times tiled the interval between sound and air puff. Thus the cells seem to form a relay of activity. We also observed an evolution in functional connectivity in the network, as measured by groupings of correlated cells. These groupings were stable till the learning protocol commenced, and then changed. Thus we have been able to observe two aspects of network learning: changes in activity (relay firing), and changes in connectivity (correlation groups).
Terry Hermiston, Ph.D.
Vice President, US Biologics Research Site Head, US Innovation Center Bayer Healthcare, USA
ColoAd1 – An oncolytic adenovirus derived by directed evolution
Attempts at developing oncolytic viruses have been primarily based on rational design. However, this approach has been met with limited success. An alternative approach employs directed evolution as a means of producing highly selective and potent anticancer viruses. In this method, viruses are grown under conditions that enrich and maximize viral diversity and then passaged under conditions meant to mimic those encountered in the human cancer microenvironment. Using the “Directed Evolution” methodology, we have generated ColoAd1, a novel chimeric oncolytic adenovirus. In vitro, this virus demonstrated a >2 log increase in both potency and selectivity when compared to ONYX-015 on colon cancer cells. These results were further supported by in vivo and ex vivo studies. Importantly, these results have validated this methodology as a new general approach for deriving clinically-relevant, highly potent anti-cancer virotherapies. This virus is currently in clinical trials as a novel treatment for cancer.
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’.
Lalitha Subramanian, Ph.D.
Chief Scientific Officer & VP, Services at Scienomics, USA
Nanoscale Simulations – Tackling Form and Formulation Challenges in Drug Development and Drug Delivery
Lalitha Subramanian, Dora Spyriouni, Andreas Bick, Sabine Schweizer, and Xenophon Krokidis Scienomics
The discovery of a compound which is potent in activity against a target is a major milestone in Pharmaceutical and Biotech industry. However, a potent compound is only effective as a therapeutic agent when it can be administered such that the optimal quantity is transported to the site of action at an optimal rate. The active pharmaceutical ingredient (API) has to be tested for its physicochemical properties before the appropriate dosage form and formulation can be designed. Some of the commonly evaluated parameters are crystal forms and polymorphs, solubility, dissolution behavior, stability, partition coefficient, water sorption behavior, surface properties, particle size and shape, etc. Pharmaceutical development teams face the challenge of quickly and efficiently determining a number of properties with small quantities of the expensive candidate compounds. Recently the trend has been to screen these properties as early as possible and often the candidate compounds are not available in sufficient quantities. Increasingly, these teams are leveraging nanoscale simulations similar to those employed by drug discovery teams for several decades. Nanoscale simulations are used to predict the behavior using very little experimental data and only if this is promising further experiments are done. Another aspect where nanoscale simulations are being used in drug development and drug delivery is to get insights into the behavior of the system so that process failures can be remediated and formulation performance can be improved. Thus, the predictive screening and the in-depth understanding leads to experimental efficiency resulting in far-reaching business impacts.
With specific examples, this talk will focus on the different types of nanoscale simulations used to predict properties of the API in excipients and also provide insight into system behavior as a function of shelf life, temperature, mechanical stress, etc.
Gokul Das, Ph.D.
Co-Director, Breast Disease Site Research Group, Roswell Park Cancer Institute, Buffalo, NY
Probing Estrogen Receptor−Tumor Suppressor p53 Interaction in Cancer: From Basic Research to Clinical Trial
Tumor suppressor p53 and estrogen receptor have opposite roles in the onset and progression of breast cancer. p53 responds to a variety of cellular of stresses by restricting the proliferation and survival of abnormal cells. Estrogen receptor plays an important role in normal mammary gland development and the preservation of adult mammary gland function; however, when deregulated it becomes abnormally pro-proliferative and greatly contributes to breast tumorigenesis. The biological actions of estrogens are mediated by two genetically distinct estrogen receptors (ERs): ER alpha and ER beta. In addition to its expression in several ER alpha-positive breast cancers and normal mammary cells, ER beta is usually present in ER alpha-negative cancers including triple-negative breast cancer. In spite of genetically being wild type, why p53 is functionally debilitated in breast cancer has remained unclear. Our recent finding that ER alpha binds directly to p53 and inhibits its function has provided a novel mechanism for inactivating genetically wild type p53 in human cancer. Using a combination of proliferation and apoptosis assays, RNAi technology, quantitative chromatin immunoprecipitation (qChIP), and quantitative real-time PCR (qRT-PCR), in situ proximity ligation assay (PLA), and protein expression analysis in patient tissue micro array (TMA), we have demonstrated binding of ER alpha to p53 and have delineated the domains on both the proteins necessary for the interaction. Importantly, ionizing radiation inhibits the ER-p53 interaction in vivo both in human cancer cells and human breast tumor xenografts in mice. In addition, antiestrogenstamoxifen and faslodex/fulvestrant (ICI 182780) disrupt the ER-p53 interaction and counteract the repressive effect of ER alpha on p53, whereas 17β-estradiol (E2) enhances the interaction. Intriguingly, E2 has diametrically opposite effects on corepressor recruitment to a p53-target gene promoter versus a prototypic ERE-containing promoter. Thus, we have uncovered a novel mechanism by which estrogen could be providing a strong proliferative advantage to cells by dual mechanisms: enhancing expression of ERE-containing pro-proliferative genes while at the same time inhibiting transcription of p53-dependent anti-proliferative genes. Consistently, ER alpha enhances cell cycle progression and inhibits apoptosis of breast cancer cells. Correlating with these observations, our retrospective clinical study shows that presence of wild type p53 in ER-positive breast tumors is associated with better response to tamoxifen therapy. These data suggest ER alpha-p53 interaction could be one of the mechanisms underlying resistance to tamoxifen therapy, a major clinical challenge encountered in breast cancer patients. We have launched a prospective clinical trial to analyze ER-p53 interaction in breast cancer patient tumors at Roswell Park Cancer Institute. Our more recent finding that ER beta has opposite functions depending on the mutational status of p53 in breast cancer cells is significant in understanding the hard-to-treat triple-negative breast cancer and in developing novel therapeutic strategies against it. Our integrated approach to analyze ER-p53 interaction at the basic, translational, and clinical research levels has major implications in the diagnosis, prognosis, and treatment of breast cancer.
Akhilesh Pandey, Ph.D.
Professor, Johns Hopkins University School of Medicine, Baltimore, USA
A draft map of the human proteome
We have generated a draft map of the human proteome through a systematic and comprehensive analysis of normal human adult tissues, fetal tissues and hematopoietic cells as an India-US initiative. This unique dataset was generated from 30 histologically normal adult tissues, fetal tissues and purified primary hematopoietic cells that were analyzed at high resolution in the MS mode and by HCD fragmentation in the MS/MS mode on LTQ-Orbitrap Velos/Elite mass spectrometers. This dataset was searched against a 6-frame translation of the human genome and RNA-Seq transcripts in addition to standard protein databases. In addition to confirming a large majority (>16,000) of the annotated protein-coding genes in humans, we obtained novel information at multiple levels: novel protein-coding genes, unannotated exons, novel splice sites, proof of translation of pseudogenes (i.e. genes incorrectly annotated as pseudogenes), fused genes, SNPs encoded in proteins and novel N-termini to name a few. Many proteins identified in this study were identified by proteomic methods for the first time (e.g. hypothetical proteins or proteins annotated based solely on their chromosomal location). We have generated a catalog of proteins that show a more tissue-restricted pattern of expression, which should serve as the basis for pursuing biomarkers for diseases pertaining to specific organs. This study also provides one of the largest sets of proteotypic peptides for use in developing MRM assays for human proteins. Identification of several novel protein-coding regions in the human genome underscores the importance of systematic characterization of the human proteome and accurate annotation of protein-coding genes. This comprehensive dataset will complement other global HUPO initiatives using antibody-based as well as MRM mass spectrometry-based strategies. Finally, we believe that this dataset will become a reference set for use as a spectral library as well as for interesting interrogations pertaining to biomedical as well as bioinformatics questions.