Aug
13
Tue
2013
Invited Talk: A cost-effective approach to Protein Structure-guided Drug Discovery: Aided by Bioinformatics, Chemoinformatics and computational chemistry @ Sathyam Hall
Aug 13 @ 11:15 am – 11:40 am

kalKal Ramnarayan, Ph.D.
Co-founder President & Chief Scientific Officer, Sapient Discovery, San Diego, CA, USA


A cost-effective approach to Protein Structure-guided Drug Discovery: Aided by Bioinformatics, Chemoinformatics and computational chemistry

With the mapping of the human genome completed almost a decade ago, efforts are still underway to understand the gene products (i.e., proteins) in the human biological and disease pathways.  Deciphering such information is very important for the discovery and development of small molecule drugs as well as protein therapeutics for various human diseases for which no cure exists.  As an example, with more than 500 members, the kinase family of protein targets continues to be an important and attractive class for drug discovery.  While how many of the members in this family are actually druggable is still to be established, there are several ongoing efforts on this class of proteins across a broad spectrum of disease categories.  Even though in general the protein structural topology might looks similar, there are issues with respect selectivity of identified small molecule inhibitors when, the lead molecule discovery is carried out at the ATP binding site.  As an added complexity, allosteric modulators are needed for some of the members, but the actual site for such modulation on the protein target can not resolved with uncertainty.  In this presentation we will describe a bioinformatics and computational based platform for small molecule discovery for protein targets that are involved in protein-protein interactions as well as targets like kinases and phosphatases.  We will describe a computational approach in which we have used an informatics based platform with several hundred kinases to sort through in silico and identify inhibitors that are likely to be highly selective in the lead generation phase.  We will discuss the implication of this approach on the drug discovery of the kinase and phosphatase classes in general and independent of the disease category.

 

Invited Talk: Pertubation of DNA topology in mycobacteria @ Acharya Hall
Aug 13 @ 11:50 am – 12:12 pm

NagarajaV. Nagaraja Ph.D.
Professor, Indian Institute of Science, Bengaluru, India


Perturbation of DNA topology in mycobacteria

To maintain the topological homeostasis of the genome in the cell, DNA topoisomerases catalyse DNA cleavage, strand passage and rejoining of the ends. Thus, although they are essential house- keeping enzymes, they are the most vulnerable targets; arrest of the reaction after the first trans-esterification step leads to breaks in DNA and cell death.  Some of the successful antibacterial or anticancer drugs target the step ie arrest the reaction or stabilize the topo -DNA covalent complex. I will describe our efforts in this direction – to target DNA gyrase and also topoisomerase1 from mycobacteria. The latter, although essential, has no inhibitors described so far. The new inhibitors being characterized are also used to probe topoisomerase control of gene expression.

In the biological warfare between the organisms, a diverse set of molecules encoded by invading genomes target the above mentioned most vulnerable step of topoisomerase  reaction, leading to the accumulation of double strand breaks. Bacteria, on their part appear to have developed defense strategies to protect the cells from genomic double strand breaks. I will describe a mechanism involving three distinct gyrase interacting proteins which inhibit the enzyme in vitro. However, in vivo all these topology modulators protect DNA gyrase from poisoning effect by sequestering the enzyme away from DNA.

Next, we have targeted a topology modulator protein, a nucleoid associated protein(NAP) from Mycobacterium tuberculosis to develop small molecule inhibitors by structure based design. Over expression of HU leads to alteration in the nucleoid architecture. The crystal structure of the N-terminal half of HU reveals a cleft that accommodates duplex DNA. Based on the structural feature, we have designed inhibitors which bind to the protein and affect its interaction with DNA, de-compact the nucleoid and inhibit cell growth. Chemical probing with the inhibitors reveal the importance of HU regulon in M.tuberculosis.

Invited Talk: Probing Estrogen Receptor – Tumor Suppressor p53 Interaction in Cancer: From Basic Research to Clinical Trial @ Acharya Hall
Aug 13 @ 3:26 pm – 3:57 pm

gokuldasGokul 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.

 

Aug
14
Wed
2013
Delegate Talk: Intrinsic modulation of cytokine response by mycobacteria @ Acharya Hall
Aug 14 @ 11:35 am – 11:45 am
Delegate Talk: Intrinsic modulation of cytokine response by mycobacteria @ Acharya Hall | Vallikavu | Kerala | India

Sukhithasri V, Nisha N, Vivek V and Raja Biswas


The host innate immune system acts as the first line of defense against invading pathogens. During an infection, the host innate immune cells recognize unique conserved molecules on the pathogen known as Pathogen Associated Molecular Patterns (PAMPs). This recognition of PAMPs helps the host mount an innate immune response leading to the production of cytokines (Akira et al. 2006). Peptidoglycan, one of the most conserved and essential component of the bacterial cell wall is one such PAMP. Peptidoglycan is known to have potent proinflammatory properties (Gust et al. 2007). Host recognize peptidoglycan using Nucleotide oligomerization domain proteins (NODs). This recognition of peptidoglycan activates the NODs and triggers downstream signaling leading to the nuclear translocation of NF-κB and production of cytokines (McDonald et al. 2005). Pathogenic bacteria modify their peptidoglycan as a strategy to evade innate immune recognition, which helps it to establish infection in the host. These peptidoglycan modifications include O-acetylation and N-glycolylation of muramic acid and N-deacetylation of N-acetylglucosamine (Davis et al. 2011). Modification of mycobacterial peptidoglycan by N-glycolylation prevents the catalytic activity of lysozyme (Raymond et al. 2005). Additionally, mycobacterial peptidoglycan is modified by amidation for unknown reasons.

Here, we have investigated the role of amidated peptidoglycan in Mycobacterium sp in modulating the innate immune response. We isolated amidated peptidoglycan from Mycobacterium sp and non-amidated peptidoglycan from Escherichia coli. We made a comparative analysis of the cytokine response produced on stimulation of innate immune cells by peptidoglycan from E. Coli and Mycobacterium sp. Macrophages and whole blood were treated with peptidoglycan and the cytokines secreted into spent medium and plasma respectively were analyzed using ELISA. Our results show that peptidoglycan from Mycobacterium sp is less effective in stimulating innate immune cells to produce cytokines. This intrinsic modulation of the cytokine response suggests that mycobacteria modify their peptidoglycan by amidation to evade innate immune response.