Highly-simplified, functional bryostatin analogs through design and efforts toward a biorelevant model for allosteric regulation of protein kinase C [electronic resource]
Keywords: Chemistry.

The marine-derived macrolide bryostatin 1 has garnered significant interest both for its synthetic complexity and its therapeutically relevant activities. Indeed, bryostatin has been evaluated in nearly 40 clinical trials for three distinctly different and highly impactful indications: cancer, Alzheimer's disease, and HIV. These evaluations have yet to result in an approval, however, as a result of a dose-limiting toxicity of myalgia and the lack of a reliable supply of the natural product. Both of these deficiencies can be solved through the recognition that the natural product itself is not necessarily the optimized therapeutic candidate, rather it may serve as a framework for the design of simpler analogs that recapitulate or even improve upon the function of the parent scaffold. Precluding a fully-informed design strategy for bryostatin-inspired drug candidates is the general lack of clarity surrounding the exogenous regulation of the its putative intracellular candidate, protein kinase C (PKC). PKC is a central component of cellular signal transduction in both normal and diseased states, and the prevalence of compensatory mechanisms complicates the ability to predict phenotypic outcomes from simple analysis of ligand affinities. Additionally, PKC signaling occurs at the membrane, with its activation relying on the insertion of an allosteric regulatory domain, the C1 domain, into the phospholipid bilayer after association with the secondary messenger diacylglycerol, a role mimicked by bryostatin. The membrane-associative nature has prevented structural analysis of these complexes, thus molecular-level detail that could begin to deconvolute the link between structure and function is thus far not available. The work presented herein seeks to address the above issues both through the design of simplified analogs that improve synthetic accessibility and through the solid-state REDOR NMR investigations on ligand-PKC-membrane complexes. Chapter 1 provides the competitive landscape for therapeutic efforts centered on modulation of PKC. This chapter details the various methods for exogenous PKC regulation, covering direct kinase site inhibition, C1 domain-based allosteric control (such as bryostatin-based strategies), and disruption of protein-protein interactions. These approaches vary greatly in their level of clinical evaluation. Some potentially promising scaffolds have yet to move beyond basic in vitro assays while others have advanced all the way to clinical approval. The current status of each as well as the historical perspective for each class of candidate is described. Chapter 2 represents the approaches to address the supply issue. While industry is likely capable of developing a sufficient supply of the natural compound based on the most recent total syntheses, preparing a more accessible compound with the same or better efficacy as bryostatin would reduce the barrier to advancement. Two strategies are detailed, a salicylate-derived scaffold and a C19 ketal-based scaffold, though the chapter primarily focuses on the former as the latter did not succumb to synthesis in the initial investigations. The parent salicylate system was obtained in just 23 steps, nearly half of that required to prepare the natural scaffolds. Additionally, a variant of this system containing a C7' aryl bromide was shown to be amenable to final step diversification via Suzuki coupling conditions. The resultant library of analogs is still undergoing evaluation, though preliminary results suggest it has applications as a biochemical tool (anilino-substituted analogs operate as solvatochromic dyes) and in PKC-independent treatment of Chikungunya virus. Chapter 3 addresses the lack of structural detail on ligand-PKC complexes. While several X-ray crystallography and solution state NMR studies of PKC C1 domains have been reported, dating back to the mid-1990s, few of these have incorporated ligand components. Even fewer introduce lipids, despite the fact that the membrane plays a crucial role in PKC activation. Solid-state REDOR NMR is expected to be uniquely well-suited to generate atomic level detail on these types of tertiary complexes. It has previously been shown to operate in membranous systems and to provide atomic-level detail on non-traditional ligand-substrate complexes. The efforts in this chapter demonstrate that, when using a phorbol diacetate model system, REDOR NMR can recapitulate in silico-predicted distances between strategically placed non-natural isotopic labels. Additionally, the design and synthesis of suitably-labeled bryostatin analogs is described. REDOR NMR studies are underway for one such compound, representing the first structural interrogation of a bryostatin-PKC complex. As this intraligand analysis, as well as future analyses of interactions involving the C1 domain and membrane components, will eventually inform molecular dynamics simulations, this work lays the foundation for the most biorelevant structural model of PKC activation. Given that the Wender group is actively pursuing advanced animal model data for a variety of indications, this work could also be coupled with these phenotypic results to provide the most robustly-informed design strategy to date. Chapter 4 represents a departure from this PKC-centric theme but is still driven by the principles of using synthesis and molecular level modifications to impact therapeutically relevant systems. RNAi therapies have unmatched potential in terms of selectivity in drug development as they lead to formal protein inhibition through post-trancriptional control over expression. Unfortunately, as these strategies depend on polyanionic siRNA sequences, their delivery into the cell presents a major barrier to their clinical development. The Wender group previously developed guanidinium-rich, amphipathic co-oligomers for the complexation, delivery and release of siRNA, the synthesis of which was enabled through an organocatalytic ring-opening polymerization methodology developed in the Waymouth group. However, this system used an entirely non-natural backbone that could draw concerns with respect to metabolism and excretion. This chapter details preliminary efforts toward developing a fully biocompatible variation on this initial report by exchanging the backbone with a glycerol-derived system and demonstrating that the new scaffold is still effective in delivering siRNA.