Computational and synthetic efforts towards bryostatin 1 and bryostatin analogs [electronic resource]
Keywords: Chemistry.

Bryostatin 1 is a marine natural product that has been of great interest to chemists and clinicians due to its highly complex structure and its remarkable biological activity. Bryostatin has been investigated for the treatment of many indications, most notably cancer, HIV, and Alzheimer's disease, collectively for which it has been entered into over 40 clinical trials. Notwithstanding the immense potential impact of bryostatin's biological activity, its current supply is nearly exhausted and future supply is uncertain. All bryostatin that has been used clinically has been sourced from one GMP isolation in 1991, and all subsequent efforts to isolate more (through isolation from source organism, aquaculture, engineered biosynthesis, or total synthesis) have been unsuccessful or not scalable. One solution to bryostatin's supply problem is the design of a shorter, supply-impacting synthesis. If accomplished, this solution would allow for a rapid replenishment of bryostatin's supply and an immediate clinical impact. A second solution to bryostatin's supply problem is the design of new bryostatin analogs. Because bryostatin was not optimized for its therapeutic use in humans, new analogs can be designed that retain or even improve upon bryostatin's biological activity while also reducing its immense complexity. Such an effort, however, is complicated by the fact that there exists little structural information about bryostatin's target, protein kinase C (PKC), in its active, membrane-associated state. Thus, a substantial portion of the work described here has used both molecular dynamics (MD) simulations and solid state NMR experiments to more fully understand the structure and function of membrane-associated PKC. Chapter 1 provides a survey of the structure, function, and membrane interactions of PKC. This chapter contains a brief overview of the different PKC isoforms, their various functions within the cell, and the biological indications that are tied to PKC regulation (such as cancer, HIV, and Alzheimer's disease). It examines the bryostatin analogs that have been synthesized in order to target these indications. Of particular emphasis is that design of new PKC activators has been complicated by the fact that while a few X-ray and NMR structures of PKC fragments exist, there are no structures of membrane-associated PKC. The importance of the membrane in PKC function is described, as are the efforts thus far to examine the role of the membrane in the activity of PKC activators. Chapter 2 details the use of molecular dynamics (MD) simulations in elucidating the membrane-associated structure of ligand-bound PKC. These simulations examine how different PKC activators differentially position the ligand-bound PKC complex in the membrane, and the role of waters and lipid headgroups at the interface of the membrane and cytosol. These simulations also provide an explanation for why bryostatin's northern region is important to its activity despite not being in contact with the binding pocket, thus providing a hypothesis for future design of new bryostatin analogs. Chapter 3 details the synthesis of a new library of greatly simplified bryostatin analogs, and the development and use of a new assay to test the PKC binding affinity of these and other compounds across all conventional and novel PKC isoforms. These greatly simplified compounds remove bryostatin's complex northern region entirely by replacing the A- and B-rings with a short diester chain, thus reducing a 20-membered macrocycle to a 14-membered one, and are synthesized in only 19 linear steps (20 total). It is also shown that while some compounds in this library bind to PKC as strongly as bryostatin 1 across all isoforms, others exhibit unprecedented selectivity between conventional and some novel PKCs. Chapter 4 addresses the lack of any existing experimental membrane-associated structure of the PKC-ligand complex. This problem is addressed through the use of solid-state REDOR NMR studies, in which interatomic distances are measured between different isotopes. These experiments used an isotopically-labeled bryostatin analog bound to the PKCδ C1b domain in the presence of phospholipid vesicles. In doing so, this represents the first experimental determination of the bound conformation of any PKC activator in a phospholipid membrane. These experiments are coupled with MD simulations to use the measured interatomic distances to construct a full picture of the ensemble of conformations that exist in this PKC-ligand-membrane complex. Chapter 5 details the total synthesis of bryostatin 1. Through the collaborative work of 8 co-workers in the Wender lab, we have accomplished the shortest reported synthesis of bryostatin 1 at 19 linear steps (29 total). My key contributions to this collaborative effort are highlighted. This short synthesis is scalable and has thus far produced more than 2 grams of bryostatin 1. This chapter also describes how such a synthesis fundamentally alters the landscape of bryostatin supply; all bryostatin that had ever been used in the clinic was from one GMP isolation in 1991 and is almost entirely exhausted. Subsequent efforts to isolate bryostatin and replenish this supply have proven either unsuccessful or not scalable. Our accomplishment in producing a short, scalable synthesis breaks through this barrier and finally provides a new, renewable source of bryostatin 1.