Stereoselective Organic Synthesis and Translational Chemical Biology

Part 1: Stereoselective Organic Synthesis - Developing Methods to Building a Natural Product-inspired Chemical Toolbox

Our quest to undertaking signaling pathway-based targets is challenging the current thinking of building a chemical toolbox that is traditionally enriched with heterocyclic compounds or compounds, in general, dominated by sp2 bonds. One of the main limitations with the current approaches is that they are biased to search for inhibitors of enzymes (i.e. the luxury of deep and well-defined pockets which is generally not the case for protein-protein interactions (PPIs)). Over the years, natural products from various sources have demonstrated a proven track record to function as the modulators of protein-protein interactions, and, in general, signaling pathways, but often they are hard to placing onto the drug discovery journey. To us, they serve as an excellent source of inspiration in developing novel synthesis approaches allowing us building a chemical toolbox with compounds that can be classified as natural product-like and hybrid natural products. Several features in our design and synthesis program are unique and highly attractive, and, these are: (i) natural product-inspired 3D architectures, (ii) compounds are more close to bioactive natural products with sufficient complexity, (iii) practical methods that utilize modern organic synthesis for obtaining compounds in sufficient amounts and in a timely manner, (iv) functionalized macrocyclic compounds (for mapping a large surface area, in particular for crucial for PPIs), and (v) amenable to developing medicinal chemistry programs on first generation hits. During the past nearly 2 decades, we have developed several methods for obtaining polycyclic and macrocyclic compounds having indole- and tetrahydroquinoline-inspired alkaloids, benzofuran- and benzopuran-inspired polyphenolics. The past few years (see our recent publications), we reported several practical approaches to the fragments of bioactive natural products/compounds such as eribulin, rapamycin, epothilone/ixabepilone, treprostinil, latrunclins and geldanamycin and thoroughly utilized them to building a macrocyclic diversity-based chemical toolbox.

Part 2: Translational Chemical Biology
Project 1 : Chemical Biology Approaches to Study the Differentiation of Human Stem Cells to Neurons by Novel, Natural Product-Inspired Compounds

Collaborative Team Members:

  • Subhadra Dravida, Founder and CEO, Transcell Biologics Hyderabad Anand Ram Soorneedi, Research Scientist, Transcell Biologics Hyderabad (expertise and role - mesenchymal stem cells from healthy donors and neuro-patients, development of assays for trans-differentiation of stem cells to neurons by small molecules, validation by specific biomarkers)
  • Kaveh Mashayekhi, CSO, Sandor Life Sciences Hyderabad Madhu Mohan, Research Scientist, Sandor Life Sciences Hyderabad (expertise and role - genomic studies, next generation sequencing, single cell genomics)
  • N. Sadananda Singh, School of Biology, IISER TVM (expertise and role - CRISPR/Cas9 technology for target ID and validation; addressing mechanistic questions)
  • Kiranam Chatti, Biology Dept, DRILS Hyderabad (expertise and role - small molecule-biotin conjugate approaches to target ID and validation; addressing mechanistic questions)
  • Amitabha Majumdar, Wellcome-DBT Intermediate Fellow, NCCS Pune (expertise and role - chemical neurobiology; various drosophila and yeast-based assays for Huntington's)
  • Steve Haggarty, MGH Boston (expertise and role - chemical neurobiology; various neuro-patient-derived, Induced Pluripotent Stem Cell-based cellular assays for Alzheimer's/dementia)
  • Satish Srinivas Kitambi, Karolinska Institute, Sweden (expertise and role - In vivo evaluation of chemical toolbox to identify novel neuroprotective agents for Alzheimer’s
  • Hari Narayana Rao, Reliance Life Sciences, Mumbai (expertise and role - animal model studies)

The proposed integrated, multidisciplinary research program aims at developing chemical biology approaches to study the trans-differentiation of human mesenchymal stem cells to neurons by novel, natural product-inspired small molecules. Through creating a highly interconnected team culture and working with different skill-sets, we plan enhancing our current understanding of various factors playing key roles in this process. Following the discovery of novel small molecules causing this trans-differentiation, serious efforts will be made to addressing the mechanistic questions involving: genomics studies (i.e. next generation sequencing and single cell genomics), CRISPR-Cas9 studies and small molecule-biotin conjugates (for target identification and validation). To further expand the scope of active compounds identified in differentiation, these novel small molecules will then be thoroughly tested for their ability to function, either as neuroprotective or neurogenesis agents in various neurological disorders (for example, studies related to Huntington and Alzheimers's/dementia). Finally, the lead compounds identified through our deep chemical biology studies will then be subjected to a series of neuro-disease specific, animal model studies for building a solid foundation of the translational research path.

To achieve these objectives, our highly integrated team comprises skills-sets and leadership in (i) modern and stereoselective organic synthesis, (ii) isolation of mesenchymal stem cells from healthy donors (source - umbilical cord tissue and blood) and neuro-patients (source - blood), (iii) development of various cellular assays to study the trans-differentiation of stem cells to neurons, (iv) validation of neuronal cells by electrophysiology studies (v) validation of neuronal cells by specific biomarkers using RT-PCR methods, (vi) validation of biomarkers using genomic tools - next generation sequencing and by the techniques of single cell genomic studies, (vii) target validation and identification by CRISP-Cas9 studies and small molecule-biotin conjugates for pull-down experiments, (viii) evaluation of active small molecules in various neuro-assays specific for Huntington’s and Alzheimer's/dementia and, finally, (ix) the extension of our cellular studies to various neuro-disease-related animal model studies.

The foundation of this comprehensive program was initiated a few years ago where our early work identified several novel natural product-inspired small molecules capable of trans-differentiating human mesenchymal stem cells (from healthy donors) to neurons. These findings were further validated by the formation of neuronal cells by specific biomarkers, such as agrin, nestin and RTN4. The initial results obtained to date have allowed us to file 4 patent applications (Indian patents filed; work is in progress for US patent applications), submit 1 paper for publication (Jan 2017) and prepare 3 more publications that are in progress.

Our early success through working at the interface of modern organic synthesis and human stem cell biology (isolation and development of various cellular assays) has allowed us establishing a solid foundation in this program. Moreover, this further led us providing an excellent opportunity to bring other competencies for taking this program to a different translational level with a strong scientific base. To our knowledge, this has not been achieved to date, even at the international level. No single research group can achieve these objectives, and typically, the lack of integrated deeper research efforts, often results in a fragmented translational output with a little serious impact! The successful outcome of this program is highly likely to play a seminal role for our younger generation to build highly integrated teams for undertaking complex and challenging research problems. Not only will these efforts lead to publishing high quality papers but given the translational research of this program, this will also lead to building a strong IP portfolio, eventually leading to next generation highly innovative spin-outs, that we are all striving for in India!

Project 2 : Patient-derived Cancer Drug Discovery and Natural Product-inspired Small Molecules

Collaborative Team Members:

  • Subhadra Dravida, PhD, CEO and Founder Transcell Biologics Hyderabad, India
  • Dr. A. Rajeshwar Laparoscopic Surgeon & Cancer Specialist Assistant Professor in Surgical Oncology MNJ Institute of Oncology - Regional Cancer Centre, Hyderabad
  • Kiranam Chatti, Biology Department Dr. Reddy's Institute of Life Sciences, Hyderabad
  • Harinarayana Rao, Reliance Life sciences Dhirubhai Life Sciences Research Center, Navi Mumbai

The proposed program aims at utilizing our first generation, novel, natural product-inspired, small molecules identified by our group, as the selective killer (tested by cell viability, toxicology and selectivity) and the promoters of apoptosis (caspase 3 activation assay) in patient-derived brain glioblastoma cells (published work from Arya group, 2016). By using this knowledge and experience, we are now aiming for designing highly selective, next generation natural product-inspired compounds that would go through an extensive cell viability testing, caspase 3 activation assay for apoptosis in 24 different primary cancer cells and tumorspheres (enriched with cancer stem cells) from a panel of patient-derived samples (from Brain, Breast and Head and Neck Cancer) before and after the xenograft studies. In addition to this, we will also be testing our compounds for their cell migration inhibitory properties utilizing these different cell lines. Finally, the highly selective compounds after going through extensive toxicology and selectivity studies, along with the SAR program, will then be subjected to patient-derived tumor xenograft-based, in vivo studies.

Using a diverse panel of patient-derived primary cancer cells, tumorspheres (enriched in cancer stem cells) for three different cancers - Brain, Breast and Head and Neck, and also with primary cells after the tumor xenograft studies, this approach places us in a unique position for exploring the scope of tumor heterogeneity and complexity for the generation of highly efficacious and selective, next-generation, anti-cancer drug candidates.

Our program that brings together (i) modern organic synthesis / medicinal chemistry (expertise with DRILS), (ii) Indian patient-derived various tumor banking with primary cancer cells and tumorspheres (expertise with Transcell Biologics, Hyderabad), (iii) patient-derived xenograft (expertise with Reliance Life Sciences, Mumbai), and finally, (iv) cell biology expertise with a variety of apoptosis and cell migration assays (expertise with Transcell Biologics and DRILS Biology), is unprecedented. Our early work done together as a team, also places us in a highly advantageous situation, for taking these challenges that are hitherto not possible to achieve in a single academic and/or industrial setting. Our early published work (Asian JOC 2016) at the interface of natural product-inspired synthesis and patient-derived screening for cell viability and apoptosis led the identification of a novel small molecule as the highly selective promoter of apoptosis in glioblastoma cells. As an extension to this study, we established a team in India, and are now, aiming to identify novel small molecules as highly selective anti-cancer agents after going through extensive testing (for apoptosis and cell migration) with Indian patient-derived primary cancer cells and tumorspheres for three categories - Brain, Breast and Head and Neck Cancer. In addition, these cell lines will also be utilized in patient-derived tumor xenograft studies which would then allow evaluating a variety of cancer cells that would be obtained from the xenograft work.

Click Here to download slides highlight our research vision and objectives.

Selected References

  • Prabhat Arya, Doug T. H. Chou and Myung-Gi Baek.  Diversity-based Organic Synthesis in the era of Genomics and Proteomics.  Angew. Chem. Int. Ed., 2001, 40, 339-346
  • Prabhat Arya and Myung-Gi Baek.  Natural Product-like, Chiral Derivatives by Solid Phase Synthesis.  Curr. Opin. Chem. Biol., 2001, 5, 292-301
  • Prabhat Arya, Reni Joseph and Doug T. H. Chou.  Toward High-throughput Synthesis of Complex Natural Product-like Compounds in the Genomics and Proteomics Age.  Chemistry & Biology, 2002, 9, 145-156
  • P. Arya, S. Quevillon, R. Joseph, C.-Q. Wei, Z. Gan, M. Parisien, E. Sesmilo, P. T. Reddy, Z.-X. Chen, P. Durieux, D. Laforce, L.-C. Campeau, S. Khadem, S. Couve-Bonnaire, R. Kumar, U. Sharma, D. M. Leek, M. Daroszewska and M. L. Barnes.  Towards the Library Generation of Natural Product-like Polycyclic Derivatives by Stereocontrolled Diversity-Oriented Synthesis.  Pure & Appl. Chem., 2005, 77, 163-178
  • Zhonghong Gan, P. Thirupathi Reddy, Sophie Quevillon, Samuel Couve-Bonnaire and Prabhat Arya.  Stereocontrolled, Solid Phase Synthesis of a 90 Membered Library of Indoline Alkaloid-like Polycycles from an Enantioenriched Aminoindoline Scaffold.  Angew. Chem. Int. Ed., 2005, 44, 1366-1368.
  • Prabhat Arya, Reni Joseph, Zhonghong Gan and Bojana Rakic.  Exploring New Chemical Space by Stereocontrolled Diversity Oriented Synthesis.  Chemistry & Biology, 2005, 12, 163-180
  • Ayub Reayi and Prabhat Arya.  Natural Product-like Chemical Space:  Search for Chemical Dissectors of Macromolecular Interactions. Curr. Opin. Chem. Biol., 2005, 9, 240-247 (Combinatorial Chemistry Theme Issue- Editors: Prabhat Arya and Hans Joerg Roth).
  • Jyoti P. Nandy, Michael Prakesch, Shahriar Khadem, P. Thirupathi Reddy, Utpal Sharma and Prabhat Arya.  Advances in Solution and Solid Phase Synthesis toward the Generation of Natural Product-like Libraries.  Chem. Rev. 2009, 109(5), 1999-2060
  • 14-Membered Macrocyclic Ring-derived Toolbox:  The Identification of Small Molecule Inhibitors of Angiogenesis and an Early Embryo Development in Zebrafish Assays.  Madhu Aeluri, Chinmoy Pramanik, Lakshindra Chetia, Naveen Kumar Mallurwar, Sridhar Balasubramanian, Gayathri Chandrasekar, Satish Srinivas Kitambi, and Prabhat Arya. Org. Lett. 2013, 15(3), 436-439 
  • Macrocyclic Glycohybrid Toolbox Identifies Novel Anti-angiogenesis Agents from Zebrafish Assay.  Bhanudas Dasari, Srinivas Jogula, Ramdas Borhade, Sridhar Balasubramanian, Gayathri Chandrasekar, Satish Srinivas Kitambi, and Prabhat Arya. Org. Lett. 2013, 15(3), 432-435
  • Tetrahydroquinoline-derived Macrocyclic Toolbox Identifies Novel Anti-angiogenesis Agents and Inhibitors of an Early Embryo Development in Zebrafish Assays.  Shiva Krishna Reddy Guduru,Srinivas Chamakuri, Gayathri Chandrasekar, Satish Srinivas Kitambi and Prabhat Arya.  ACS Med. Chem. Lett., 2013, 4, 666-670
  • Small Molecule Modulators of Protein-Protein Interactions: Selected Case Studies. Madhu Aeluri, Srinivas Chamakuri, Bhanudas Dasari, Shiva Krishna Reddy Guduru, Ravikumar Jimmidi, Srinivas Jogula and Prabhat Arya.  Chem. Rev. 2014, 114, 4640-4690 (for a theme topic: Chemical Biology of Protein-Protein Interactions)
  • Rethinking Drug Discovery.  Invited article for Magazine, Pharma Focus Asia.  Subhadraw Dravida and Prabhat Arya.  2014, Vol 21, Nov Issue.
  • A Divergent Approach to Building Latrunculin Family-derived, Hybrid Macrocyclic Toolbox.M.Aeluri, M.; B. Dasari, P. Arya. Org. Lett. 2015, 17(3), 472-475.
  • Practical Stereoselective Synthesis of Eribulin Fragment toward Building a Hybrid Macrocyclic Toolbox.  Ravkumar Jimmidi, Shiva Krishna Reddy Guduru and P. Arya. Org. Lett. 2015, 17(3), 468-471.
  • Stereoselective Synthesis of Rapamycin Fragment to Build a Macrocyclic Toolbox. Shiva Krishna Reddy Guduru, Ravikumar Jimmidi, Girdhar Singh Deora and Prabhat Arya. Org. Lett. 2015, 17(3), 480-483.
  • A Macrocyclic Small Molecule Having Cyclosporin A-Like Activity Prevents the Induction of Mitochondrial Permeability Transition and Cytochrome c.  Govardhan K. Shroff,  Mithila Sawant,  Ravikumar, Jimmidi, Girdhar Singh Deora, Sandhya Sitasawad, Kiranam Chatti, Prabhat Arya and Prasenjit Mitra.  2015, submitted.
  • Synthesis and Biological Evaluation of Rapamycin-derived, Next Generation Small Molecules.  Shiva Krishna Reddy Guduru and Prabhat Arya. 2016, submitted.
  • Stereoselective Synthesis of C27-C35 Eribulin Fragment for Building A Macrocyclic Diversity. Saidulu Konda, Naveen Kumar Mallurwar, Mahender Khatravath, Pallavi Rao, Shivashankar Sripally, Javed Iqbal and Prabhat Arya. 2016, accepted for the journal, Synthesis, special issue on "target oriented synthesis of complex molecules".
  • A Modular Approach to Building 17- and 18-Membered Macrocyclic Diversity from Eribulin C14-C21 Fragment. Naveen Kumar Mallurwar, Saidulu Konda, Mahender Khatravath, Pallavi Rao, Shivashankar Sripally, Javed Iqbal and Prabhat Arya. 2016, submitted.
  • Synthesis of C1-C10 Eribulin Fragment and its Analogues for Building A Diverse Set of Macrocycles. Mahender Khatravath, Saidulu Konda, Naveen Kumar Mallurwar, Pallavi Rao, Shivashankar Sripally, Javed Iqbal and Prabhat Arya. 2016, accepted.
  • Building A Macrocyclic Toolbox from Epothilone Fragment Identifies Actin Binders and Novel Promoters of Apoptosis in Patient-derived Brain Tumor Cells. Bhanudas Dasari, Temesgen Fufa, Madhu Aeluri, Girdhar Singh Deora, Frank Gaunitz, Satish Srinivas Kitambi  and Prabhat Arya. 2016, submitted.
  • Synthesis of Geldanamycin-Inspired Macrocyclic Compounds with the Goal of Identifying a New Family of HSP90 Binders. Srinivas Jogula, Srinivas Chamakuri, Sri Krishna Varma Penmetsa, Girdhar Singh Deora and Prabhat Arya. 2016, submitted.
  • Small molecule inhibition of pro-apoptotic Bax and Bak promotes long term cell survival and protects primary neurons from excitotoxicity. Xin Niu, Hetal Brahmbhatt, Philipp Mergenthaler, Zhi Zhang, Jing Sang, Wibke Diederich, Eve Wong, Weijia Zhu, Justin Pogmore, Jyoti P. Nandy, Maragani Satyanarayana, Ravi K. Jimmidi, Prabhat Arya, Brian Leber, Jialing Lin, Carsten Culmsee, Jing Yi  and David W. Andrews. 2016 submitted.