Design, Synthesis, and Biological Characterization of Largazole Analogues

Histone deacetylases (HDACs) have been shown to play key roles in tumorigenesis, and

have been validated as effective enzyme target for cancer treatment. Largazole, a marine natural

product isolated from the cyanobacterium Symploca, is an extremely potent HDAC inhibitor that

has been shown to possess high differential cytotoxicity towards cancer cells along with excellent

HDAC class-selectivity. However, improvements can be made in the isoform-selectivity and

pharmacokinetic properties of largazole.

In attempts to make these improvements and furnish a more efficient biochemical probe

as well as a potential therapeutic, several largazole analogues have been designed, synthesized,

and tested for their biological activity. Three different types of analogues were prepared. First,

different chemical functionalities were introduced at the C2 position to probe the class Iselectivity profile of largazole. Additionally, docking studies led to the design of a potential

HDAC8-selective analogue. Secondly, the thiol moiety in largazole was replaced with a wide

variety of othe zinc-binding group in order to probe the effect of Zn2+ affinity on HDAC

inhibition. Lastly, three disulfide analogues of largazole were prepared in order to utilize a

different prodrug strategy to modulate the pharmacokinetic properties of largazole.

Through these analogues it was shown that C2 position can be modified significantly

without a major loss in activity while also eliciting minimal changes in isoform-selectivity. While

the Zn2+-binding group plays a major role in HDAC inhibition, it was also shown that the thiol

can be replaced by other functionalities while still retaining inhibitory activity. Lastly, the use of

a disulfide prodrug strategy was shown to affect pharmacokinetic properties resulting in varying

functional responses in vitro and in vivo.

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Largazole is already an impressive HDAC inhibitor that shows incredible promise.

However, in order to further develop this natural product into an anti-cancer therapeutic as well as

a chemical probe, improvements in the areas of pharmacokinetics as well as isoform-selectivity

are required. Through these studies we plan on building upon existing structure–activity

relationships to further our understanding of largazole’s mechanism of inhibition so that we may

improve these properties and ultimately develop largazole into an efficient HDAC inhibitor that

may be used as an anti-cancer therapeutic as well as a chemical probe for the studying of

biochemical systems.

Structural and Functional Analysis of the Caspase –dependent and –independent Domains of the X-linked Inhibitor of Apoptosis Protein in Inflammatory Breast Cancer Tumor Biology

Inflammatory breast cancer (IBC) is an extremely rare but highly aggressive form of breast cancer characterized by the rapid development of therapeutic resistance leading to particularly poor survival. Our previous work focused on the elucidation of factors that mediate therapeutic resistance in IBC and identified increased expression of the anti-apoptotic protein, X-linked inhibitor of apoptosis protein (XIAP), to correlate with the development of resistance to chemotherapeutics. Although XIAP is classically thought of as an inhibitor of caspase activation, multiple studies have revealed that XIAP can also function as a signaling intermediate in numerous pathways. Based on preliminary evidence revealing high expression of XIAP in pre-treatment IBC cells rather than only subsequent to the development of resistance, we hypothesized that XIAP could play an important signaling role in IBC pathobiology outside of its heavily published apoptotic inhibition function. Further, based on our discovery of inhibition of chemotherapeutic efficacy, we postulated that XIAP overexpression might also play a role in resistance to other forms of therapy, such as immunotherapy. Finally, we posited that targeting of specific redox adaptive mechanisms, which are observed to be a significant barrier to successful treatment of IBC, could overcome therapeutic resistance and enhance the efficacy of chemo-, radio-, and immuno- therapies. To address these hypotheses our objectives were: 1. to determine a role for XIAP in IBC pathobiology and to elucidate the upstream regulators and downstream effectors of XIAP; 2. to evaluate and describe a role for XIAP in the inhibition of immunotherapy; and 3. to develop and characterize novel redox modulatory strategies that target identified mechanisms to prevent or reverse therapeutic resistance.

Using various genomic and proteomic approaches, combined with analysis of cellular viability, proliferation, and growth parameters both in vitro and in vivo, we demonstrate that XIAP plays a central role in both IBC pathobiology in a manner mostly independent of its role as a caspase-binding protein. Modulation of XIAP expression in cells derived from patients prior to any therapeutic intervention significantly altered key aspects IBC biology including, but not limited to: IBC-specific gene signatures; the tumorigenic capacity of tumor cells; and the metastatic phenotype of IBC, all of which are revealed to functionally hinge on XIAP-mediated NFκB activation, a robust molecular determinant of IBC. Identification of the mechanism of XIAP-mediated NFκB activation led to the characterization of novel peptide-based antagonist which was further used to identify that increased NFκB activation was responsible for redox adaptation previously observed in therapy-resistant IBC cells. Lastly, we describe the targeting of this XIAP-NFκB-ROS axis using a novel redox modulatory strategy both in vitro and in vivo. Together, the data presented here characterize a novel and crucial role for XIAP both in therapeutic resistance and the pathobiology of IBC; these results confirm our previous work in acquired therapeutic resistance and establish the feasibility of targeting XIAP-NFκB and the redox adaptive phenotype of IBC as a means to enhance survival of patients.