Important strides are being made in understanding the effects of structural features of bryostatin1, a candidate therapeutic agent for cancer and dementia, in conferring its potency toward protein kinaseC and the unique spectrum of biological responses that it induces. A critical pharmacophoric element in bryostatin1 is the secondary hydroxy group at the C26 position, with a corresponding primary hydroxy group playing an analogous role in binding of phorbol esters to protein kinase C. Herein, we describe the synthesis of a bryostatin homologue in which the C26 hydroxy group is primary, as it is in the phorbol esters, and show that its biological activity is almost indistinguishable from that of the corresponding compound with a secondary hydroxy group.

To investigate the cellular distribution of tumor‐promoting vs. non‐tumor‐promoting bryostatin analogues, we synthesized fluorescently labeled variants of two bryostatin derivatives that have previously shown either phorbol ester‐like or bryostatin‐like biological activity in U937 leukemia cells. These new fluorescent analogues both displayed high affinity for protein kinase C (PKC) binding and retained the basic properties of the parent unlabeled compounds in U937 assays. The fluorescent compounds showed similar patterns of intracellular distribution in cells, however; this argues against an existing hypothesis that various patterns of intracellular distribution are responsible for differences in biological activity. Upon further characterization, the fluorescent compounds revealed a slow rate of cellular uptake; correspondingly, they showed reduced activity for cellular responses that were only transient upon treatment with phorbol ester or bryostatin 1.

As an initial step in designing a simplified bryostatin hybrid molecule, three bryostatin analogues bearing a diacylglycerol lactone-based C-ring, which possessed the requisite pharmacophores for binding to protein kinase C (PKC) together with a modified bryostatin-like A- and B-ring region, were synthesized and evaluated. Merle 46 and Merle 47 exhibited binding affinity to PKC alpha with Ki values of 7000 ± 990 and 4940 ± 470 nM, respectively. Reinstallation of the trans-olefin and gem-dimethyl group present in bryostatin 1 in Merle 48 resulted in improved binding affinity, 363 ± 42 nM. While Merle 46 and 47 were only marginally active biologically, Merle 48 showed sufficient activity on the U937 cells to confirm that it was PMA-like for growth and attachment, as predicted by the substitution pattern of its A- and B-rings.

Bryostatin 1, a complex macrocyclic lactone, is the subject of multiple clinical trials for cancer chemotherapy. Although bryostatin 1 biochemically functions like the classic mouse skin tumor promoter phorbol 12‐myristate 13‐acetate (PMA) to bind to and activate protein kinase C, paradoxically, it fails to induce many of the typical phorbol ester responses, including tumor promotion. Intense synthetic efforts are currently underway to develop simplified bryostatin analogs that preserve the critical functional features of bryostatin 1, including its lack of tumor promoting activity. The degree to which bryostatin analogs maintain the unique pattern of biological behavior of bryostatin 1 depends on the specific cellular system and the specific response. Merle 23 is a significantly simplified bryostatin analog that retains bryostatin like activity only to a limited extent. Here, we show that in mouse epidermal cells the activity of Merle 23 was either similar to bryostatin 1 or intermediate between bryostatin 1 and PMA, depending on the specific parameter examined. We then examined the hyperplastic and tumor promoting activity of Merle 23 on mouse skin. Merle 23 showed substantially reduced hyperplasia and was not tumor promoting at a dose comparable to that for PMA. These results suggest that there may be substantial flexibility in the design of bryostatin analogs that retain its lack of tumor promoting activity. © 2016 Wiley Periodicals, Inc.

We describe a convergent synthesis of a bryostatin analogue in which the natural A- and B-ring pyrans have been replaced by phenyl rings. The new analogue exhibited PMA like behavior in cell assays, but failed to maintain high affinity binding for PKC, despite retaining an unaltered C-ring 'binding domain'

Bryostatin 1, a complex macrocyclic lactone isolated from Bugula neritina, has been the subject of multiple clinical trials for cancer. Although it functions as an activator of protein kinase C (PKC) in vitro, bryostatin 1 paradoxically antagonizes most responses to the prototypical PKC activator, the phorbol esters. The bottom half of the bryostatin 1 structure has been shown to be sufficient to confer binding to PKC. In contrast, we have previously shown that the top half of the bryostatin 1 structure is necessary for its unique biological behavior to antagonize phorbol ester responses. Neristatin 1 comprises a top half similar to that of bryostatin 1 together with a distinct bottom half that confers PKC binding. We report here that neristatin 1 is bryostatin 1-like, not phorbol ester-like, in its biological activity on U937 promyelocytic leukemia cells. We conclude that the top half of the bryostatin 1 structure is largely sufficient for bryostatin 1-like activity, provided the molecule also possesses an appropriate PKC binding domain.

A convergent synthesis of a des-B-ring bryostatin analogue is described. This analogue was found to undergo an unexpected ring expansion of the bryolactone core to generate the corresponding 21-membered macrocycle. The parent analogue and the ring-expanded product both displayed nanomolar binding affinity for PKC. Despite containing A-ring substitution identical to that of bryostatin 1 and displaying bryostatin-like biological function, the des-B-ring analogues displayed a phorbol-like biological function in cells. These studies shed new light on the role of the bryostatin B-ring in conferring bryo-like biological function to bryostatin analogues.

Abstract Protein kinase C (PKC) is differentially regulated in a range of cancers and has become an attractive therapeutic target. A number of natural compounds exist that have been found to regulate PKCs, and the biological responses of each compound can vary. The PKC activator PMA is an established tumor promoter, while others, such as the bryostatins, are non-tumor promoting and have inhibitory effects on the PKC pathway. Because of its effects on the PKC pathway, bryostatin 1 is currently in clinical trials as an anti-cancer agent. Limited availability and the difficulty involved in synthesizing bryostatin 1 make its use as an anti-cancer agent less attractive. Merle 23, a simplified synthetic analog of bryostatin 1, could be an alternative to bryostatin 1 if it acted similarly. Initial investigations of Merle 23 have revealed biological responses both similar to bryostatin 1 and similar to PMA, depending on the system. In U937 leukemia cells Merle 23 acts similar to PMA, and in the LNCaP prostate cancer cell line it behaves more like bryostatin 1. Its behavior in mouse skin is of particular interest, since this is the system in which the tumor promoting activity of PMA and the anti-tumor promoting activity of bryostatin 1 have been characterized. The activity of Merle 23 was compared to PMA and bryostatin 1 in mouse epidermal cells and mouse skin. In mouse primary keratinocytes PMA produces a prolonged morphological response, while the morphological response to bryostatin 1 is transient. The response to Merle 23 is intermediate, but more similar to bryostatin 1. When applied simultaneously, Merle 23, like bryostatin 1, is able to protect the cells from the morphological response induced by PMA. In the PKC pathway, all three compounds promote similar levels of initial PKCδ activation and ERK1/2 phosphorylation. By 24 hrs, however, the responses differ. At high concentrations, bryostatin 1 protects PKCδ from down regulation whereas Merle 23 does not. Bryostatin 1 is also much more potent for down regulation of PKCα, whereas Merle 23 and PMA are similar. The inflammatory response was measured by monitoring the transcript levels of various genes involved in inflammation. PMA induced the largest increase in transcript levels, while Merle 23 was more similar to bryostatin 1. Short-term in vivo analysis reveals no epidermal thickening in response to low doses of Merle 23 whereas the corresponding dose of PMA induced significant hyperplasia. The in vivo analysis of Merle 23 is ongoing. The results presented suggest that Merle 23 behaves more similarly to bryostatin 1 in the mouse epidermal cells at the level of biological response, raising the possibility that it may likewise be non-promoting. The results emphasize that the structure activity relations responsible for the distinct behavior between PMA and bryostatin 1 are distinct for different cellular systems and different biological endpoints. Citation Format: Jessica Kelsey, Noemi Kedei, Christophe Cataisson, Mark Petersen, Stuart Yuspa, Gary Keck, Peter Blumberg. Bryostatin 1 and the simplified analog Merle 23 have similar and opposing properties on mouse epidermal cells and mouse skin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1642. doi:10.1158/1538-7445.AM2014-1642

Protein kinase C (PKC), a validated therapeutic target for cancer chemotherapy, provides a paradigm for assessing structure–activity relations, where ligand binding has multiple consequences for a target. For PKC, ligand binding controls not only PKC activation and multiple phosphorylations but also subcellular localization, affecting subsequent signaling. Using a capillary isoelectric focusing immunoassay system, we could visualize a high resolution isoelectric focusing signature of PKCδ upon stimulation by ligands of the phorbol ester and bryostatin classes. Derivatives that possessed different physicochemical characteristics and induced different patterns of biological response generated different signatures. Consistent with different patterns of PKCδ localization as one factor linked to these different signatures, we found different signatures for activated PKCδ from the nuclear and non-nuclear fractions. We conclude that the capillary isoelectric focusing immunoassay system may provide a window into the integrated consequences of ligand binding and thus afford a powerful platform for compound development.

Abstract Bryostatin 1 (bryo 1) is a promising cancer chemotherapeutic agent with a unique mechanism of action. Whereas bryo 1 binds to and activates protein kinase C (PKC) like the phorbol esters, it paradoxically antagonizes most phorbol ester responses. Previously, we compared the changes in transcription in response to the phorbol ester phorbol 12-myristate 13-acetate (PMA), to bryo 1, and to the synthetic bryo 1 derivative Merle 23 in the human cancer cell lines LNCaP and U937. Bryo1 failed to induce a typical phorbol ester biological response in either cell line, whereas the bryo 1 analog Merle 23 resembled PMA in the U937 cells and bryo 1 in the LNCaP cells. In both cell lines, bryo 1 differed from PMA in inducing a shorter duration of the transcriptional responses for a panel of PKC-regulated genes as quantitated by qPCR. Merle 23, in contrast, induced a pattern very similar to that of PMA. Here, we analyzed by microarray the genome wide transcriptional changes in LNCaP and U937 cells treated with maximally effective doses of PMA, bryo 1 and Merle 23 for 6 and 8 hours, respectively. Several thousand genes were significantly up- or down-regulated by PMA (3131 and 3206), bryo 1 (1569 and 2454) and Merle 23 (3533 and 3207) compared to control in LNCaP and U937 cells, respectively. As seen previously for selected genes, the magnitude of the changes was much greater for LNCaP cells than for U937 cells. The great majority of the genes followed a pattern in which bryo 1 induced changes similar to PMA but to a significantly smaller extent (3096 and 1610 genes significantly different in LNCaP and U937 cells), whereas Merle 23 was very similar to PMA (only 230 and 30 genes different between them). A detailed qPCR analysis (dose and time courses) of representative outlier genes revealed that the shorter duration of bryo 1 induced response was responsible for the differences between bryo 1 and PMA effect in all cases. The dose response experiments did not reveal a difference in bryo 1 potency linked to any specific pattern of response. Analysis of transcription factors revealed overrepresentation of the same families of transcription factors (13 in the LNCaP cells; 12 in the U937 cells) among the genes differentially regulated by PMA and bryo 1. These included AP2F, E2F, EGRF, KLF, NRF1, SP1F. However, the target genes of the transcription factor families were largely different between the two cell lines. The analysis of 45 signaling pathways using reporter assays identified significant changes in 7 pathways induced both by PMA and bryo 1 at 6 hours. While the analysis of the microarray data is ongoing, we conclude that early turn-off of the induced responses by bryo 1 is the central feature accounting for its difference from PMA in its transcriptional response. Understanding the basis for this transiency of response may reveal a more general strategy for inhibition of PKC pathways. Citation Format: Noemi Kedei, Aleksandra M. Michalowski, Mark E. Petersen, Gary E. Keck, Peter M. Blumberg. Comparison of transcriptional response to phorbol ester, bryostatin 1, and bryostatin analogue in LNCaP and U937 cells provides insight into their differential mechanism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2101. doi:10.1158/1538-7445.AM2013-2101

Abstract Tumor promoting phorbol esters, such as PMA, are potent activators of the PKC pathway. The bryostatins, marine natural products, activate PKCs similarly to phorbol esters but paradoxically inhibit most phorbol ester responses, including skin tumor promotion. Because of this inhibitory activity on the PKC pathway, bryostatin 1 has been the subject of extensive clinical trials. Serious impediments to clinical development, however, have been the scarcity of natural supplies coupled with great structural complexity, making synthesis problematic. Recent synthetic advances are now yielding simplified structural derivatives. Here, we compare the behavior of the bryostatin derivative Merle 23 with that of bryostatin 1 and phorbol 12-myristate 13-acetate (PMA) in mouse epidermal cells. While having similar binding activity on PKC as does bryostatin 1, Merle 23 behaves like PMA rather than like bryostatin 1 in U937 human leukemia cells but conversely behaves more like bryostatin 1 in the human prostate cancer cell line LNCaP. As a first step in assessing whether Merle 23 possesses or lacks skin tumor promoting activity, its activity in mouse primary keratinocyte cultures was assessed. PMA induces a prominent, prolonged morphological response in keratinocytes. Bryostatin 1 induces a similar initial response, which is however very transient. The morphological response to Merle 23 is more prolonged than for bryostatin 1 but much shorter than for PMA. For initial activation of PKCδ or induction of ERK1/2 phosphorylation, all three ligands show similar activity. By 24 hrs, however, bryostatin 1 is much more potent for down regulation of PKCα, whereas Merle 23 and PMA are similar. For PKCδ, bryostatin 1 at higher concentrations protects PKCδ from down regulation whereas Merle 23 does not. Transcriptional responses of various inflammation genes were monitored by qPCR. PMA causes the greatest increase in transcript levels for all genes investigated. In many cases bryostatin 1 induces a smaller response and the response to Merle 23 generally resembles that of bryostatin 1. While these studies are ongoing, they suggest that Merle 23 largely behaves like bryostatin 1 in the mouse epidermal cells. Our findings emphasize the important role of cellular context in determining the pattern of behavior of Merle 23. An important implication is that different cellular environments reveal different structure activity relations for bryostatin 1-like behavior. Finally, our studies raise the possibility that Merle 23, which is significantly simplified relative to bryostatin 1 in its upper two rings, may be non-promoting, at least in the mouse skin tumor promotion system. Citation Format: Jessica Kelsey, Noemi Kedei, Christophe Cataisson, Mark Petersen, Stuart Yuspa, Gary Keck, Peter Blumberg. Comparison of the activity of the bryostatin derivative Merle 23 with that of bryostatin 1 and phorbol ester in mouse epidermal cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2454. doi:10.1158/1538-7445.AM2013-2454

The bryostatins are a group of 20 macrolides isolated by Pettit and co-workers from the marine organism Bugula neritina. Bryostatin 1, the flagship member of the family, has been the subject of intense chemical and biological investigations due to its remarkably diverse biological activities, including promising indications as therapy for cancer, Alzheimer's disease, and HIV. Other bryostatins, however, have attracted far less attention, most probably due to their relatively low natural abundance and associated scarcity of supply. Among all macrolides in this family, bryostatin 7 is biologically the most potent protein kinase C (PKC) ligand (in terms of binding affinity) and also the first bryostatin to be synthesized in the laboratory. Nonetheless, almost no biological studies have been carried out on this agent. We describe herein the total synthesis of bryostatin 7 based on our pyran annulation technology, which allows for the first detailed biological characterizations of bryostatin 7 with side-by-side comparisons to bryostatin 1. The results suggest that the more easily synthesized and less lipophilic bryostatin 7 may be an effective surrogate for bryostatin 1.

Steigende Überlebensraten bei steigender Anzahl der Krebserkrankungen haben zu einem zunehmenden Bewusstsein für die Lebensqualität nach dem Überstehen der Krankheit geführt. Eine Langzeitfolge der onkologischen Therapie kann die dauerhafte Infertilität männlicher Patienten sein. Obwohl schonendere Behandlungsstrategien in den letzten Jahren eine Verringerung der Hodenschädigung und des damit verbundenen Fertilitätsverlusts zeigen, ist es oft schwierig, den Grad der Schädigung vor der Behandlung einzuschätzen. Deshalb sollte dem an Krebs erkrankten Mann mit nicht abgeschlossener Familienplanung durch rechtzeitige Fertilitätsprotektion die Chance auf eine spätere Vaterschaft bewahrt bleiben. Als etablierte Methode der ersten Wahl steht die Kryokonservierung der Spermien zur Realisierung eines Kinderwunsches für postpubertäre Männer zur Verfügung. Neuere Techniken zum Fertilitätserhalt wie die Entnahme von Hodengewebe zur späteren Keimzelltransplantation oder Autotransplantation sind in der Entwicklung. Durch die zu erwartende klinische Etablierung dieser Methoden im Forschungsstadium könnte gerade für präpubertäre männliche Patienten durch rechtzeitige jetzige Entnahme von Hodengewebe mit Kryokonservierung und Langzeitlagerung eine Chance für eine spätere Vaterschaft erhalten werden.

The role of the C(8) gem-dimethyl group in the A-ring of bryostatin 1 has been examined through chemical synthesis and biological evaluation of a new analogue. Assays for biological function using U937, K562, and MV4-11 cells as well as the profiles for downregulation of PKC isozymes revealed that the presence of this group is not a critical determinant for the unique pattern of biological activity of bryostatin.

Bryostatin 1, like the phorbol esters, binds to and activates protein kinase C (PKC) but paradoxically antagonizes many but not all phorbol ester responses. Previously, we have compared patterns of biological response to bryostatin 1, phorbol ester, and the bryostatin 1 derivative Merle 23 in two human cancer cell lines, LNCaP and U937. Bryostatin 1 fails to induce a typical phorbol ester biological response in either cell line, whereas Merle 23 resembles phorbol ester in the U937 cells and bryostatin 1 in the LNCaP cells. Here, we have compared the pattern of their transcriptional response in both cell lines. We examined by qPCR the transcriptional response as a function of dose and time for a series of genes regulated by PKCs. In both cell lines bryostatin 1 differed primarily from phorbol ester in having a shorter duration of transcriptional modulation. This was not due to bryostatin 1 instability, since bryostatin 1 suppressed the phorbol ester response. In both cell lines Merle 23 induced a pattern of transcription largely like that of phorbol ester although with a modest reduction at later times in the LNCaP cells, suggesting that the difference in biological response of the two cell lines to Merle 23 lies downstream of this transcriptional regulation. For a series of bryostatins and analogs which ranged from bryostatin 1-like to phorbol ester-like in activity on the U937 cells, the duration of transcriptional response correlated with the pattern of biological activity, suggesting that this may provide a robust platform for structure activity analysis.

Phorbol 12‐myristate 13‐acetate (PMA) and bryostatin 1 are both potent protein kinase C (PKC) activators. In LNCaP human prostate cancer cells, PMA induces tumor necrosis factor alpha (TNFα) secretion and inhibits proliferation; bryostatin 1 does not, and indeed blocks the response to PMA. This difference has been attributed to bryostatin 1 not localizing PKCδ to the plasma membrane. Since phorbol ester lipophilicity influences PKCδ localization, we have examined in LNCaP cells a series of phorbol esters and related derivatives spanning some eight logs in lipophilicity (logP) to see if any behave like bryostatin 1. The compounds showed marked differences in their effects on proliferation and TNFα secretion. For example, maximal responses for TNFα secretion relative to PMA ranged from 97 % for octyl‐indolactam V to 24 % for phorbol 12,13‐dibenzoate. Dose–response curves ranged from monophasic for indolactam V to markedly biphasic for sapintoxin D. The divergent patterns of response, however, correlated neither to lipophilicity, to plasma membrane translocation of PKCδ, nor to the ability to interact with model membranes. In U937 human leukemia cells, a second system in which PMA and bryostatin 1 have divergent effects, viz. PMA but not bryostatin 1 inhibits proliferation and induces attachment, all the compounds acted like PMA for proliferation, but several induced a reduced level or a biphasic dose–response curve for attachment. We conclude that active phorbol esters are not all equivalent. Depending on the system, some might partially resemble bryostatin 1 in their behavior; this encourages the concept that bryostatin‐like behavior may be obtained from other structural templates.

Bryostatin 1 has attracted considerable attention both as a cancer chemotherapeutic agent and for its unique activity. Although it functions, like phorbol esters, as a potent protein kinase C (PKC) activator, it paradoxically antagonizes many phorbol ester responses in cells. Because of its complex structure, little is known of its structure-function relations. Merle 23 is a synthetic derivative, differing from bryostatin 1 at only four positions. However, in U-937 human leukemia cells, Merle 23 behaves like a phorbol ester and not like bryostatin 1. Here, we characterize the behavior of Merle 23 in the human prostate cancer cell line LNCaP. In this system, bryostatin 1 and phorbol ester have contrasting activities, with the phorbol ester but not bryostatin 1 blocking cell proliferation or tumor necrosis factor alpha secretion, among other responses. We show that Merle 23 displays a highly complex pattern of activity in this system. Depending on the specific biological response or mechanistic change, it was bryostatin-like, phorbol ester-like, intermediate in its behavior, or more effective than either. The pattern of response, moreover, varied depending on the conditions. We conclude that the newly emerging bryostatin derivatives such as Merle 23 provide powerful tools to dissect subsets of bryostatin mechanism and response.