Synthesis and Characterization of Histone Deacetylase (HDAC) Inhibitors and PROTACs

Abstract

Histone deacetylases (HDACs) are a class of enzymes that remove acyl groups from ε-N-lysine residues of histone and non histone proteins. There are 18 human HDAC isoforms, of which 11 have a Zn2⁺ ion in their catalytic center. They are grouped into different classes according to their shape, size, and substrate specificity. An overexpression or malfunction of HDACs is observed in various diseases like cancer, inflammatory processes or neurodegenerative disorders. Currently there are four FDA-approved HDAC inhibitors (HDACi) for the treatment of hematological cancers. However, all approved HDACi exhibit severe side effects, which often lead to treatment interruption. The development of more isoform selective HDACi holds the promise of reducing side effects and a safer drug profile. Moreover, prodrugs, which selectively release the drug in the target tissue, have been used effectively in the past to circumvent unwanted side effects. <br /> In the first project (chapter 2), new HDAC6 selective inhibitors were developed. Based on a previously published crystal structure, docking studies were performed to explore the binding site of the catalytic domain 2 (CD2) of HDAC6. Through an efficient four step protocol, incorporating a multicomponent synthesis, 13 inhibitors were synthesized and assessed for their in vitro HDAC inhibitory activity. The two most potent and selective inhibitors were further tested for their influence on the NLRP3 inflammasome activation and their potential use against NLRP3 inflammasome driven diseases. <br /> In the second project (chapter 3), a set of HDACi containing a substituted hydrazide as the zinc-binding group (ZBG) was developed. 11 HDACi were synthesized through a three step protocol and tested for their inhibitory activity against multiple HDAC isoforms. The most potent compounds exhibited low nanomolar activity against the class I HDAC isoforms HDAC1-3 with single digit activity against HDAC3. Those compounds were further evaluated for their antiproliferative activity against the native and cisplatin resistant tumor cell lines A2780 and Cal27. The hit compounds completely reversed cisplatin resistance and showed very strong synergism with cisplatin, according to Chou-Talalay and LOEWE model, which was further confirmed in apoptosis and caspase 3/7 activation assays. <br /> In the third project (chapter 4), the application of targeted protein degradation of HDACs utilizing proteolysis targeting chimeras (PROTACs) was explored. A small set of VHL recruiting HDAC degrading PROTACs was synthesized. To this end the synthesis of the VHL ligand was optimized to enhance the purification efficiency and yields. Altogether, six PROTACs featuring variations in their spacer structure were synthesized and investigated for their degradation efficacy of HDAC1-4 and 6. The PROTACs present promising lead structures for the further development of class I HDAC degraders as an alternative to the traditional small molecule approach. <br /> In the fourth project (chapter 5), the utilization of photoactivatable HDAC prodrugs was explored. The ZBG, as the central warhead, of the previously published HDACi DDK137 was masked with a light-cleavable moiety. The derived prodrug exhibited only low inhibitory activity against HDAC1 and HDAC6 as desired. After irradiation with UV light of 365 nm for 10 min, the inhibitory activity was comparable to the parent compound. In a cellular environment, the prodrugs showed antiproliferative activity only after irradiation with light.