Adam J. Schrier

Designed, synthetically accessible bryostatin analogues potently induce activation of latent HIV reservoirs in vitro

Bryostatin is a unique lead in the development of potentially transformative therapies for cancer, Alzheimer’s disease, and the eradication of HIV/AIDS. However, the clinical use of bryostatin has been hampered by its limited supply, difficulties in accessing clinically-relevant derivatives, and side effects. Herein, we address these problems through the step-economical syntheses of seven members of a new family of designed bryostatin analogues utilizing a highly convergent Prins-macrocyclization strategy. We also demonstrate for the first time that such analogues effectively induce latent HIV activation in vitro with potencies similar to or better than bryostatin. Significantly, these analogues are up to 1000-fold more potent in inducing latent HIV expression than prostratin, the current clinical candidate for latent virus induction. This study provides the first demonstration that designed, synthetically-accessible bryostatin analogues could serve as superior candidates for the eradication of HIV/AIDS through induction of latent viral reservoirs in conjunction with current antiretroviral therapy.

Efficient Synthetic Access to a New Family of Highly Potent Bryostatin Analogues via a Prins-Driven Macrocyclization Strategy

The step-economical synthesis of a new class of bryostatin analogues that contain the complete oxycarbocyclic core ring system of the bryostatin natural products is reported. These agents are convergently assembled via a highly efficient, functional-group-tolerant, and stereoselective Prins-driven macrocyclization. These tetrahydropyranyl B-ring analogues are among our most potent and efficacious analogues to date, exhibiting nanomolar and picomolar activities in protein kinase C affinity assays as well as in cellular antiproliferation assays.

Design, synthesis, and evaluation of potent bryostatin analogs that modulate PKC translocation selectivity

Modern methods for the identification of therapeutic leads include chemical or virtual screening of compound libraries. Nature’s library represents a vast and diverse source of leads, often exhibiting exquisite biological activities. However, the advancement of natural product leads into the clinic is often impeded by their scarcity, complexity, and nonoptimal properties or efficacy as well as the challenges associated with their synthesis or modification. Function-oriented synthesis represents a strategy to address these issues through the design of simpler and therefore synthetically more accessible analogs that incorporate the activity-determining features of the natural product leads. This study illustrates the application of this strategy to the design and synthesis of functional analogs of the bryostatin marine natural products. It is specifically directed at exploring the activity-determining role of bryostatin A-ring functionality on PKC affinity and selectivity. The resultant functional analogs, which were prepared by a flexible, modular synthetic strategy, exhibit excellent affinity to PKC and differential isoform selectivity. These and related studies provide the basic information needed for the design of simplified and thus synthetically more accessible functional analogs that target PKC isoforms, major targets of therapeutic interest.

Simplified Bryostatin Analogues Protect Cells from Chikungunya Virus-Induced Cell Death

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus showing a recent resurgence and rapid spread worldwide. While vaccines are under development, there are currently no therapies to treat this disease, except for over-the-counter (OTC) analgesics, which alleviate the devastating arthritic and arthralgic symptoms. To identify novel inhibitors of the virus, analogues of the natural product bryostatin 1, a clinical lead for the treatment of cancer, Alzheimer’s disease, and HIV eradication, were investigated for in vitro antiviral activity and were found to be among the most potent inhibitors of CHIKV replication reported to date. Bryostatin-based therapeutic efforts and even recent anti-CHIKV strategies have centered on modulation of protein kinase C (PKC). Intriguingly, while the C ring of bryostatin primarily drives interactions with PKC, A- and B-ring functionality in these analogues has a significant effect on the observed cell-protective activity. Significantly, bryostatin 1 itself, a potent pan-PKC modulator, is inactive in these assays. These new findings indicate that the observed anti-CHIKV activity is not solely mediated by PKC modulation, suggesting possible as yet unidentified targets for CHIKV therapeutic intervention. The high potency and low toxicity of these bryologs make them promising new leads for the development of a CHIKV treatment.

In vivo activation of latent HIV with a synthetic bryostatin analog effects both latent cell "kick" and "kill" in strategy for virus eradication

The ability of HIV to establish a long-lived latent infection within resting CD4+ T cells leads to persistence and episodic resupply of the virus in patients treated with antiretroviral therapy (ART), thereby preventing eradication of the disease. Protein kinase C (PKC) modulators such as bryostatin 1 can activate these latently infected cells, potentially leading to their elimination by virus-mediated cytopathic effects, the host’s immune response and/or therapeutic strategies targeting cells actively expressing virus. While research in this area has focused heavily on naturally-occurring PKC modulators, their study has been hampered by their limited and variable availability, and equally significantly by sub-optimal activity and in vivo tolerability. Here we show that a designed, synthetically-accessible analog of bryostatin 1 is better-tolerated in vivo when compared with the naturally-occurring product and potently induces HIV expression from latency in humanized BLT mice, a proven and important model for studying HIV persistence and pathogenesis in vivo. Importantly, this induction of virus expression causes some of the newly HIV-expressing cells to die. Thus, designed, synthetically-accessible, tunable, and efficacious bryostatin analogs can mediate both a “kick” and “kill” response in latently-infected cells and exhibit improved tolerability, therefore showing unique promise as clinical adjuvants for HIV eradication.

Bryostatin analogue-induced apoptosis in mantle cell lymphoma cell lines

The anti-cancer effects of bryostatin-1, a potent diacylglycerol analogue, have traditionally been attributed to its action on protein kinase C. However, we previously documented apoptosis in a B non-Hodgkin lymphoma cell line involving diacylglycerol analogue stimulation of Ras guanyl-releasing protein, a Ras activator, and Bim, a proapoptotic Bcl-2 family protein. To further explore the role of Bim, we examined several Bim-deficient B non-Hodgkin lymphoma cells for their responses to pico, a synthetic bryostatin-1-like compound. The Bim(-) mantle cell lymphoma cell lines Jeko-1, Mino, Sp53, UPN1, and Z138 and the Bim(+) cell line Rec-1, as well as the Burkitt lymphoma cells lines BL2 (Bim(-)) and Daudi (Bim(+)), were examined for their response to pico using assays for proliferation and apoptosis as well as biochemical methods for Ras guanyl-releasing proteins and Bcl-2 family members. With the exception of UPN1, mantle cell lymphoma cell lines underwent pico-induced apoptosis, as did BL2. In some cases, hallmarks of apoptosis were substantially diminished in the presence of mitogen-activated protein kinase kinase inhibitors. Pico treatment generally led to increased expression of proapoptotic Bik, although the absolute levels of Bik varied considerably between cell lines. A pico-resistant variant of Z138 exhibited decreased Bik induction compared to parental Z138 cells. Pico also generally decreased expression of anti-apoptotic Bcl-XL and Mcl1. Although, these changes in Bcl-2 family members seem unlikely to fully account for the differential behavior of the cell lines, our demonstration of a potent apoptotic process in most cell lines derived from mantle cell lymphoma encourages a re-examination of diacylglycerol analogues in the treatment of this subset of B non-Hodgkin lymphoma cases.

Characterization of designed, synthetically accessible bryostatin analog HIV latency reversing agents.

HIV latency in resting CD4+ T cell represents a key barrier preventing cure of the infection with antiretroviral drugs alone. Latency reversing agents (LRAs) can activate HIV expression in latently infected cells, potentially leading to their elimination through virus-mediated cytopathic effects, host immune responses, and/or therapeutic strategies targeting cells actively expressing virus. We have recently described several structurally simplified analogs of the PKC modulator LRA bryostatin (termed bryologs) designed to improve synthetic accessibility, tolerability in vivo, and efficacy in inducing HIV latency reversal. Here we report the comparative performance of lead bryologs, including their effects in reducing cell surface expression of HIV entry receptors, inducing proinflammatory cytokines, inhibiting short-term HIV replication, and synergizing with histone deacetylase inhibitors to reverse HIV latency. These data provide unique insights into structure-function relationships between A- and B-ring bryolog modifications and activities in primary cells, and suggest that bryologs represent promising leads for preclinical advancement.