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PhD Research | Summary

Nitric Oxide Donor

Nitric oxide (NO) is an important gaseous signaling molecule, which is produced in nearly all cells and mediates numerous cellular processes including vasodilation, neurotransmission and immune response. The relationship between NO and cancer is complex and is largely dependent on the location, concentration, duration of release as well as the presence or lack thereof of other reactive entities. Since NO is a short lived, highly reactive, highly diffusible molecule, cancer targeted delivery of NO and real-time monitoring of NO in cells is challenging. Thus, a strategy where a small molecule can generate NO in a controlled manner to the cancer cells selectively along with a fluorescence reporter for NO would be useful for NO based cancer therapy. As a proof-of-concept, we first designed and synthesized FLUORO/NO, a new class of triggerable NO donors with an in-built fluorescence reporter. Upon activation by an esterase enzyme the compound produces NO as well as a fluorescence signal simultaneously. Cellular studies with a FLUORO/NO derivative revealed a dose-dependent enhancement of NO as well as fluorescence. The trigger that we had is esterase, which is present in nearly all cells, and therefore FLUORO/NO would be suitable for a range of cell biology studies (ChemBioChem, 2017, 18, 1529-1534). Next, in order to deliver NO selectively to cancer cells, a second stimulus for activation was chosen: hydrogen peroxide (H2O2), a reactive oxygen species (ROS). As ROS is frequently found to be elevated in rapidly dividing cells such as cancers, H2O2 has been previously used to specifically activate prodrugs and latent fluorophores (as imaging agents) in cancers. Boronate ester is known to react with H2O2 to produce an alcohol; hence this functional group was chosen as the metabolic stimulus. We designed and synthesized a series of arylboronate ester based diazeniumdiolates (BORO/NO). Having established that BORO/NO derivatives are capable of generating NO when triggered by H2O2 (Organic Letters, 2014, 16, 2610-2613), next we synthesized Thera/NO, a H2O2 activated NO donor with fluorescence reporter. Upon activation by H2O2 in buffer, a nearly quantitative correlation between fluorescence signal and NO generation was observed. When encountered with cellular situations with varying ROS levels, Thera/NO is observed to preferentially generate NO in cell lines with elevated ROS levels. Together, we have developed a convenient tool to enhance NO selectively in cancer cells and allows real-time monitoring of NO release (Chemical Communications, 2017, 53, 13352-13355). Further adaption of this technology to better direct NO to cancers is possible. The versatility of this scaffold for real-time monitoring of NO released was demonstrated with two distinct triggers, and can, in principle, be extended to other stimuli of interest as well.

Figure: (a) Stimuli responsive nitric oxide generation by a NO donor requires a secondary probe for detection of NO produced. (b) Incorporation of a latent fluorophore in the donor eliminates the need for the secondary probe for monitoring of NO. (c) FLUORO/NO, an esterase activated NO donor with a fluorescence reporter and Thera/NO, H2O2 activated NO donor with a fluorescence reporter.

Reactive Oxygen Species (ROS)

In addition to NO, a number of other redox active reactive species have important biological roles. Controlled generation as well as reliable detection of these species is challenging. In order to assess the generality of the method developed herein, we aimed to develop a ROS generator with a fluorescence reporter. As a proof of concept, first we have designed an esterase activated ROS generator without fluorescence reporter (MGR-1). Upon activation of MGR-1 by esterase, produces JCHD, which dissociates at pH 7.4 to generate ROS. Next, the ability of MGR-1 to produce ROS and oxidize phosphatidylserine was shown with different mammalian cells in collaboration with Dr. Siddhesh Kamat (IISER Pune). Finally, using a chemical genetic screen, ABHD12 was identified as an oxidized phosphatidylserine lipase in mammalian cells (Nature Chemical Biology, 2019, 15, 169-178). (MGR1 is commercially available now in Sigma-Merck).

Having established that MGR-1 is an efficient ROS generator within cells, next we designed ROS generator with a fluorescence reporter (2). In the presence of esterase, an increase in fluorescence as well as ROS was observed. Using independent assays, the cell permeability and ROS generating capability was demonstrated. This tool would be useful to deliver and real-time monitor ROS generation in cells without the need for secondary assays (Manuscript under preparation). Further modification of the trigger can facilitate directed delivery of ROS. Also worked on secondary projects with my colleagues in the group: 1) Hydrogen Sulfide (H2S) Donors: (a) Esterase Activated Carbonyl Sulfide/Hydrogen Sulfide (H2S) Donors (Organic Letters, 2017, 19, 62-65), (b) Carbonyl Sulfide (COS) Donor induced protein persulfidation protects against oxidative stress (Chemistry-An Asian Journal, 2019, 14, 4717-4724) 2) Sulfur Dioxide (SO2) Donors: Esterase Sensitive Self- Immolative Sulfur Dioxide Donors (Organic Letters, 2018, 20, 4-7), 3) Targeted Drug delivery: Targeted Antibacterial Activity Guided by Bacteria-Specific Nitroreductase Catalytic Activation to Produce Ciprofloxacin (Bioconjugate Chemistry, 2019, 30, 751-759), 4) NO Donors: a) Nitroreductase (NTR) Activated NO Donor with a Fluorescence Reporter, b) BODIPY Based Visible Light Activated NO Donors. And also I have synthesized number of fluorescent probes for a) Nitric Oxide (NO), b) Hydrogen peroxide (H2O2), c) Galactosidase, d) Carbon Monoxide (CO).

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