Advances in Research
Fishing in Nature’s Molecular pools
Nature is a treasure trove of potential drugs. The human world is thriving with diseases and applications of every kind. How do we mix and match? Where do we begin our search? We could, for example, start with 100s of different cell lines and begin our test in a systematic manner, cataloging them as we go. Or we could take a bag full of tissues and expose them to the drugs all at once! And here we bring you, the zebrafish screen! A whole organism screen with hundreds of transparent larvae, where the tissues are tagged with fluorescent proteins so you can visually inspect their size, shape, growth and death. Zebrafish embryos offer a first-stop-screening for many organ systems at once. So, can we use zebrafish and go rummaging in the natural world for more and more drugs?
Is there a why not? Let us take the example of the wonder-drug Taxol (now Paclitaxel), a highly successful treatment for various kinds of cancer. Taxol was discovered in the 1960s as part of a US government initiative to find treatments for cancer from the natural world. So, where was the problem? Taxol came from the bark of the Pacific Yew tree, not a commonly found species; and 12 trees had to be killed to yield 1 gm of Taxol!
So, what is the alternative? What if we could synthesize compounds found in the nature? It is not as easy as it sounds. The chemical space that is currently being explored by synthetic routes for drug discovery is but a tiny one. Natural products are complex, multi-ringed structures, very intractable to synthesis in the laboratory. But by bringing the possibility of synthesis to structures inspired by nature we could find a middle ground to play.
As part of efforts that started 9 years ago at bringing together chemists and biologists we recently embarked on this project with the synthetic chemist Dr D Srinivasa Reddy, at the CSIR-NCL. Dr Reddy’s lab offered us a bag full, or rather box full of vials with synthetic products inspired from the natural world. We had our own multi-well plates full of zebrafish larvae, colour-coded for heart and liver and vasculature. When the fish met the compounds, many ended in tragedy. But a very small number showed very interesting and tantalizing phenotypes.
Of these, one had a very potent inhibitory affect on the sprouting of new blood vessels, a process called angiogenesis. Angiogenesis is a critical process for development of the organism, since blood vessels are important for the transport of nutrients and oxygen to the rapidly dividing embryonic cells. So is it critical for the other rapidly dividing cell type that wreaks havoc in our bodies, the cancer cells. We xenografted human cancer cells into the fish embryo to test if the inhibitor will impede their growth. Early fish embryos do not reject such grafts because their adaptive immunity is still developing. These grafted cells secrete growth factors that seduce blood vessels to abandon their straight and narrow path and steer towards the hungry cancer cells. Treatment with our nature-inspired-synthetic molecule could stop them in their tracks, which could lead to tumour growth arrest eventually.
Perhaps, using a combination of natural product resource, synthetic organic chemistry and zebrafish embryonic assays we maybe able to dive into nature’s treasure trove repeatedly and come up with powerful molecules for the drug cabinet.
Muthukumarasamy KM, Handore KL, Kakade DN, Shinde MV, Ranjan S, Kumar N, Sehrawat S, Sachidanandan C, Reddy DS.
Org Biomol Chem. 2016 Jan 27;14(5):1569-78. doi: 10.1039/c5ob01594d.
Heart disease is the leading cause for death in men and women. Early detection of heart attacks can be saving. In a collaborative work, Deepika Bhatnagar and her colleagues have developed a sensor to detect the antigen cardiac-Troponin I (cTnI). The technique uses Graphene quantum dots conjugated to antibody anti-cardiac Troponin I and fluorescence resonance energy transfer (FRET). The sensor is highly specific and shows negligible response to non-specific antigens. The sensor displayed a linear response to cTnI from 0.001 to 1000 ng/mL with a limit of detection of 0.192 pg/mL.
Bhatnagar D, Kumar V, Kumar A, Kaur I. Graphene quantum dots FRET based sensor for early detection of heart attack in human. Biosens Bioelectron. 2016 May 15;79:495-9. doi: 10.1016/j.bios.2015.12.083. Epub 2015 Dec 25. PubMed PMID: 26748366.
Skin is the largest organ of the human body. Its large surface area and easy accessibility as well as the advantage of easy monitoring of the after-effects of treatment makes it an attractive choice for delivery of different kinds of molecules for both pharmaceutical (i.e. as a drug) and cosmeceutical (i.e. for skin beautification) purpose. Two modes of delivery are predominant: topical (to skin) or transdermal (through skin) - depending upon the final destination of these molecules. Topical delivery is required when one attempts to treat skin diseases e.g. psoriasis, atopic dermatitis, vitiligo or melanoma or for treatment of conditions like wounds or sunburns or even for application of cosmetics. Transdermal delivery is required when one needs to send drugs through the skin into the dermis in order to go into circulation and reach some other affected organ. However drug delivery to and through the skin poses its own challenges. Skin allows favorable entry of small molecules which are less than 500Da in size and lipophilic in nature that can passively diffuse whereas all others usually need active carriers or physical interventions for transport. The complex skin architecture makes it very difficult for large biomolecules to pass to or through the skin. In most cases, harsh physical or chemical techniques are used for drug delivery to skin leading to toxic effects. We have developed an amphipathic peptide that can not only penetrate the uncompromised skin and efficiently enter skin cells but can also deliver plasmid DNA efficiently by forming nanometric complexes without any additional physical or chemical intervention. We observe efficient gene expression up to the highly proliferating basal layer of the skin without observing any adverse reactions or toxic effects after delivery of reporter plasmids. Our data indicates that entry mechanism possibly involves reversible modulation of junction proteins in the skin along with transient changes in skin structure. This peptide is likely to also find use as an efficient transporter of therapeutic nucleic acids and possibly other drugs to skin.
Manika Vij, Poornemaa Natarajan, Bijay Ranjan Pattnaik, Shamshad Alam, Nidhi Gupta, Deenan Santhiya, Rajpal Sharma, Archana Singh, Kausar Mahmood Ansari, Rajesh S. Gokhale, Vivek T. Natarajan, Munia Ganguli.
Non-invasive topical delivery of plasmid DNA to the skin using a peptide carrier.
J Control Release; January; 2016
doi: 10.1016/j.jconrel.2015.12.017. Epub 2015 Dec 14.
Polyketide Quinones Are Alternate Intermediate Electron Carriers during Mycobacterial Respiration in Oxygen-Deficient Niches
Tuberculosis (TB) is one of the leading causes of infectious disease mortality. India is the highest TB burden country in the world with about 25% of the global TB burden (TB India 2014, Revised National TB Control Programme Annual Status Report). It is generally believed that one third of the world population is latently infected with Mycobacterium tuberculosis (Mtb), the causative organism of TB. This latent bacterium gets reactivated upon immune system weakening that can happen during several other disease conditions. The asymptomatic latent population is extremely difficult to diagnose and there is no effective drug for this growth form of Mtb. In the latent state the bacteria undergo substantial metabolic remodeling whose mechanistic understanding is vague. Although the metabolic pathways are slowed down, the bacteria have to sustain a threshold supply of ATP. Mtb has the genomic repertoire to modulate the respiratory pathways but the mechanism of this modulation is poorly understood. Oxygen level changes are known to affect the respiratory electron transport chain and bacteria are known to utilize alternate electron carriers and acceptors at different oxygen partial pressure.
Several Gram-negative bacteria are known to switch between ubiquinone (CoQ) to menaquinone (MK) with decreasing oxygen concentrations, this mode of exchange between isoprenoid quinones is absent in Gram-positive species. Surprisingly, the only known respiratory electron carrier quinone from mycobacteria, MK, is markedly downregulated in hypoxic condition. We recently identified a novel electron shuttling molecule, polyketide quinone (PkQ), in mycobacteria that support bacterial survival in oxygen-depleted habitats. Unlike canonical respiratory quinones that are derived from isoprenoid pathway, these molecules are produced by a polyketide synthase based pathway. PkQs function as electron carriers in the respiratory electron transport chain. This biosynthetic system is conserved in all mycobacteria as well as several Actinobacteria and a few Proteobacteria, suggesting a key role of these quinones in bacterial adaptability.
Amitesh Anand, Priyanka Verma, Anil Kumar Singh, Sandeep Kaushik, Rajesh Pandey, Ce Shi, Harneet Kaur, Manbeena Chawla, Chandra Kumar Elechalawar, Dhirendra Kumar, Yong Yang, Neel S. Bhavesh, Rajkumar Banerjee, Debasis Dash, Amit Singh, Vivek T. Natarajan, Anil K. Ojha, Courtney C. Aldrich & Rajesh S. Gokhale
Polyketide Quinones Are Alternate Intermediate Electron Carriers during Mycobacterial Respiration in Oxygen-Deficient Niches
Molecular Cell, November, 2015
What ails India? The twitter hashtag #whatailsindia is full of issues related to every sphere of life, however, little is known about the medical ailments of Indians. Simple questions like the main reasons for patients to visit doctors or the typical time of onset of chronic ailments, like obstructive airway disease, diabetes, cardiovascular disease, remain unanswered. The POSEIDON study (Salvi et al Lancet Global Health, Dec 2015) examined the reasons why 204,912 patients visited 7400 general practitioners, in 880 locations spanning all of India, in a single day. This is the first large scale practice-based point prevalence study from India and provides an overview of the disease burden from the medical practitioner point of view. Network based analysis of this large dataset permitted an understanding of the connectivity between ailments and diseases. Changes in network structure, when broken down into decades of age, permitted visualization of age-related trends in disease burden of Indians. We found that in this microcosm of Indian healthcare, respiratory symptoms were the most frequent reasons to visit doctors, while hypertension was the most frequent disease requiring treatment. Importantly, more than a fifth of the hypertensives were less than 40 years old, with metro cities showing greater hypertension burden. There were strong connections between diabetes and cardiovascular disease with a temporal lag of about one decade (diabetes followed by CVD). Non-metro cities, had a higher burden of obstructive airway disease (OAD) and in secondary analyses, utilizing open governance data, we have been able to infer an increase in OAD when LPG penetrance is low. These findings have important implications for multiple government initiatives ranging from national health programs to the “Give It Up” initiative. Further, an editorial on the research paper states that "such high rates of hypertension in younger people has important implications for premature death and disability in the most productive years of life, with economic effects that would extend to the families supported by these people". Together these emphasize the importance of ongoing research at IGIB on understanding the genesis of cardio-respiratory diseases. POSEIDON represents only the first medical snapshot of the problems faced by Indians, and it is our hope that follow-up studies and prospective medical data collection from the IGIB-HP eHealth Centers (Agrawal et al, PLoS Medicine, June 2013) will give us a more clear data-driven understanding of what ails India.
Wound healing is a normal biological process whereby the injured tissue regains its normal function through precisely and highly programmed overlapping phases. Any interference in any one or more stages of these events causes delayed/impaired wound. Several pathophysiological conditions play critical roles in delaying the process of wound repair. Diabetes is a highly prevalent chronic metabolic disorder characterized by high levels of blood glucose and delayed wound healing is one of the major complications associated with diabetes that quite often leads to traumatic lower extremity amputations and chronic wound ulcers. We have been trying to study factors behind the delayed wound closure during diabetes and have identified an altered miRNA signature during such delayed wound closure. This was accompanied by decreased levels of Dicer, the enzyme that is responsible for miRNA biogenesis. Also several wound healing specific genes were altered in the unhealed wound during diabetes. Results suggest that there might be a correlation between the altered miRNA signature and altered wound healing genes that might be responsible for prolonging wound closure during diabetes. In keratinocyte cells, Dicer inhibition prevented cell migration and affected wound closure in-vitro. Results suggest a potential role of miRNAs and Dicer during delayed wound closure and offer promise to address the issue of impaired wound healing. (Molecular Medicine. 2015, doi: 10.2119/molmed.2014.00186)
Research Scholar, CSIR-IGIB.....
Currency notes are extensively exchanged among individuals in a population and have been studied for their ability to carry and transmit infectious agents. Generally microbial culture-based approaches were used to characterize organisms on currency notes. Culture-based are limited by the conditions and culturing media, which differ widely among different organisms. Previous approaches used 16S ribosomal RNA typing to examine taxonomy of the genetic material isolated. The high-throughput DNA sequencing approach offers advantages in providing additional information on the gene repertoire of organisms on the fomite. This information could provide insights into the catalog of antibiotic resistance genes harboured by these organisms. We show success of our approach by characterizing the microbes, including pathogens on currency notes and additionally provide insights into the antibiotic resistance gene repertoire of the microbes.
Jalali S, Kohli S, Latka C, Bhatia S, Vellarikal SK, Sivasubbu S, Scaria V, Ramachandran S.
Screening currency notes for microbial pathogens and antibiotic resistance genes using a shotgun metagenomic approach.
PLoS One. 2015 Jun 2;10(6):e0128711. doi: 10.1371/journal.pone.0128711. eCollection 2015
Abhay Sharma, Scientist, CSIR-IGIB.....
It is an interesting time in biology. New discoveries about non-genetic inheritance is calling for integration of physiology, epigenetics, molecular biology, and genomic networks in the evolutionary theory so much so that Lamarck and Darwin must be turning over in their graves. We reported first evidence of transgenerational spermatogenic inheritance of induced traits, attempted to integrate diverse findings relevant in non-genetic transmission leading to synthesis of a new concept "evolutionary transgenerational systems biology" later recast as unified theory of biology.
Suhani Nagpal, Research Scholar, CSIR-IGIB.....
Proteins are workhorses of a cell and they achieve their correct function through a well-defined fold also known as native state. The consequence of improper fold or unfolding leads to toxic effects, which could lead to diseases such as Alzheimer’s, Huntington’s etc. Most proteins require assistance of molecular chaperones for proper folding. These chaperones are helper proteins, which provide an environment for the proteins to accurately fold into their native structure. So far the focus has been to study structural changes of chaperone. However, little is known about the non-native states.
The Free Energy landscape of DapA. At the bottom most level is the 'native' ensemble. The physical interactions of the amino acid residues of this ensemble is displayed in the network graphs. Dark purple nodes represent highly interacting amino acid residues whereas light coloured nodes represent sparsely interacting residues. The partially unfolded ensembles appear at the top 2 levels. It is evident that in these ensembles, the interactions are reduced compared to the 'native' state, which is densely connected.
Trayambak Basak and Ajay Bhat, Research Scholars, CSIR-IGIB.....
Proteomics has great potential in discovering potential biomarkers for several diseases that could aid in diagnosis and management of diseases. Blood plasma is usually used as it remains in contact with nearly all cells of the organism and is also easily accessible. After immuno-depletion of the abundant proteins, identification of low abundant proteins that are differentially expressed in diseased conditions compared to healthy controls is achieved through various quantitative techniques.
In-depth comparative proteomic analysis of yeast proteome using iTRAQ and SWATH based MS.
Mol. Biosystems, 2015, 11(8), 2135-43. (Cover Page article)