Development of photopharmacophores for light mediated control of metalloenzymes in in vitro and in vivo systems




Aggarwal, Kanchan

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Biological systems consist of complex and highly dynamic networks of molecular processes carried out by the enzymes. About one third of the enzymes need a metal cofactor for their activity, and hence, are called as metalloenzymes. One such metalloenzyme is carbonic anhydrase (CA), a Zn²⁺ containing protein, that catalyzes the reversible hydration of carbon dioxide, thereby playing an important role in acid base homeostasis. There are multiple isoforms of CA and their activity, localization, and expression level vary in different cell types and also differ between normal and disease states. CA is known to be overexpressed in hypoxia, which leads to the greater pH imbalance providing a positive pathway for the cancerous tumors to metastasize. Therefore, it is imperative to get precise on-demand control over these isoforms without perturbing the whole system in order to understand their role in biological systems, especially in the therapeutic context. Light serves as a wonderful external trigger due to these advantages: non-invasive; easy to control the wavelength, duration, and intensity of light; and exhibits high spatiotemporal precision. My doctoral research focuses on developing photopharmacophores for light mediated control of CA in in vitro and in vivo systems. We designed carbonic anhydrase azobenzene photoswitch (CAP-1) that can isomerize between two isomeric forms, trans and cis. This probe contains a metal binding group which interacts with the Zn²⁺ in the active site only in trans form, hence, inhibiting the protein activity. We further tuned the design with photoswitch CAP-5, which allowed the visible light mediated bidirectional control over CA in an isolated protein, intact live cells, and in vivo with zebrafish during embryo development. Additionally, we explored the prospect of photoregulation of a specific membrane-associated isoform of CA (CAIX) by modifying CAP-F5 to confine the probe to extracellular matrix. Correspondingly, we used the photoremovable protecting group (PPG) to achieve spatiotemporal control over the mitochondrial isoform of carbonic anhydrase (CAV). The probe PCE-M consists of three parts: inhibitor, PPG and a mitochondrial-directing group. The photocleaving process is triggered with 410 nm light to get free inhibitor that can inhibit CAV without interference from other isoforms of CA.



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