Nancy E. Lewin

Residues in the second cysteine-rich region of protein kinase C delta relevant to phorbol ester binding as revealed by site-directed mutagenesis.

Phorbol esters bind with high affinity to protein kinase C (PKC) isozymes as well as to two novel receptors, n-chimaerin and Unc-13. The cysteine-rich regions present in these proteins were identified as the binding sites for the phorbol ester tumor promoters and the lipophilic second messenger sn-diacylglycerol. A 50-amino-acid peptide comprising the second cysteine-rich region of PKC , expressed in Escherichia coli as a glutathione S-transferase (GST)-fusion protein, bound phorbol 12,13-dibutyrate (PDBu) with high affinity (K =0.8 nM). Using the cDNA of that cysteine-rich region as a template, a series of 37 point mutations was generated by site-directed mutagenesis, and the mutated proteins were analyzed quantitatively for binding of [H]PDBu and, as appropriate, for binding of the ultrapotent analog [H]bryostatin 1. Mutants displayed one of three patterns of behavior: phorbol ester binding was completely abolished, binding affinity was reduced, or binding was not significantly modified. As expected, five of the six cysteines as well as the two histidines involved in Zn coordination are critical for the interaction of the protein with the phorbol esters. In addition, mutations in several positions, including phenylalanine 3, tyrosine 8, proline 11, leucines 20, 21, and 24, tryptophan 21, glutamine 27, and valine 38 drastically reduced the interaction with the ligands. The effect of these mutations can be rationalized from the three-dimensional (NMR) structure of the cysteine-rich region. In particular, the C-terminal portion of the protein does not appear to be essential, and the loop comprising amino acids 20 to 28 is implicated in the binding activity.

Characterization of the Cysteine-rich Region of the Caenorhabditiselegans Protein Unc-13 as a High Affinity Phorbol Ester Receptor ANALYSIS OF LIGAND-BINDING INTERACTIONS, LIPID COFACTOR REQUIREMENTS, AND INHIBITOR SENSITIVITY

The Caenorhabditiselegans Unc-13 protein is a novel member of the phorbol ester receptor family having a single cysteine-rich region with high homology to those present in protein kinase C (PKC) isozymes and the chimaerins. We expressed the cysteine-rich region of Unc-13 in Escherichiacoli and quantitatively analyzed its interactions with phorbol esters and related analogs, its phospholipid requirements, and its inhibitor sensitivity. [3H]Phorbol 12,13-dibutyrate [3H]PDBu bound with high affinity to the cysteine-rich region of Unc-13 (K= 1.3 ± 0.2 nM). This affinity is similar to that of other single cysteine-rich regions from PKC isozymes as well as n-chimaerin. As also described for PKC isozymes and n-chimaerin, Unc-13 bound diacylglycerol with an affinity about 2 orders of magnitude weaker than [3H]PDBu. Structure-activity analysis revealed significant but modest differences between recombinant cysteine-rich regions of Unc-13 and PKC . In addition, Unc-13 required slightly higher concentrations of phospholipid for reconstitution of [3H]PDBu binding. Calphostin C, a compound described as a selective inhibitor of PKC, was also able to inhibit [3H]PDBu binding to Unc-13, suggesting that this inhibitor is not able to distinguish between different classes of phorbol ester receptors. In conclusion, although our results revealed some differences in ligand and lipid cofactor sensitivities, Unc-13 represents a high affinity cellular target for the phorbol esters as well as for the lipid second messenger diacylglycerol, at least in C. elegans. The use of phorbol esters or some “specific” antagonists of PKC does not distinguish between cellular pathways involving different PKC isozymes or novel phorbol ester receptors such as n-chimaerin or Unc-13.

Specific vanilloid responses in C6 rat glioma cells

Capsaicin and its ultrapotent analog resiniferatoxin (RTX) act through specific vanilloid receptors on sensory neurons. Here, we describe specific vanilloid responses in rat C6 glioma cells. Capsaicin and RTX stimulated 45Ca uptake in a similar fashion to that found for cultured rat dorsal root ganglion neurons (DRGs); this response was antagonized by the antagonists capsazepine and ruthenium red. As in DRGs, pretreatment of C6 cells with capsaicin or RTX produced desensitization to subsequent stimulation of 45Ca uptake. The potency for desensitization by RTX in the C6 cells corresponded to that for 45Ca uptake, whereas in DRGs it occurred at significantly lower concentrations corresponding to that for the high affinity [3H]RTX binding site. Consistent with this difference, in C6 cells we were unable to detect [3H]RTX binding. These characteristics suggest the presence of C-type but not R-type vanilloid receptors on C6 cells. After 2 day treatment, capsaicin but not RTX inhibited the proliferation and altered the differentiation of the cells and produced apoptosis. In the long term experiments, capsazepine, instead of antagonizing the effect of capsaicin, acted as an agonist. Moreover, capsazepine displayed these effects with higher potency than that of capsaicin. The different potencies and structure activity relations suggest a distinct mechanism for these long-term vanilloid effects. Our finding that C6 cells can respond directly to capsaicin necessitates a reevaluation of the in vivo pathway of response to vanilloids, and highlights the importance of the neuron-glial network.

Convergent assembly of highly potent analogues of bryostatin 1 via pyran annulation: bryostatin look-alikes that mimic phorbol ester function.

Highly potent bryostatin analogues which contain the complete bryostatin core structure have been synthesized using a pyran annulation approach as a key strategic element. The A ring pyran was assembled using a pyran annulation reaction between a C1-C8 hydroxy allylsilane and an aldehyde comprising C9-C13. This pyran was transformed to a new hydroxy allylsilane and then coupled with a preformed C ring aldehyde subunit in a second pyran annulation, with concomitant formation of the B ring. This tricyclic intermediate was elaborated to bryostatin analogues which displayed nanomolar to subnanomolar affinity for PKC, but displayed properties indistinguishable from a phorbol ester in a proliferation/attachment assay.

Wealth of opportunity - the C1 domain as a target for drug development.

The diacylglycerol-responsive C1 domains of protein kinase C and of the related classes of signaling proteins represent highly attractive targets for drug development. The signaling functions that are regulated by C1 domains are central to cellular control, thereby impacting many pathological conditions. Our understanding of the diacylglycerol signaling pathways provides great confidence in the utility of intervention in these pathways for treatment of cancer and other conditions. Multiple compounds directed at these signaling proteins, including compounds directed at the C1 domains, are currently in clinical trials, providing strong validation for these targets. Extensive understanding of the structure and function of C1 domains, coupled with detailed insights into the molecular details of ligand - C1 domain interactions, provides a solid basis for rational and semi-rational drug design. Finally, the complexity of the factors contributing to ligand - C1 domain interactions affords abundant opportunities for manipulation of selectivity; indeed, substantially selective compounds have already been identified.

Molecular modeling, total synthesis, and biological evaluations of C9-deoxy bryostatin 1.

The bryostatins are a family of natural products of marine origin that display both intriguing structural complexity and a fascinating profile of biological activity.[1] These materials were isolated (from Bugula neritina) and their structures determined through the pioneering work of Pettit and coworkers.[2] Subsequently, a monumental large scale collection and isolation effort managed to yield some 18 grams of bryostatin 1, the most abundant and now most thoroughly investigated member of this family, from some 13,000 kg of the source organism.[3] This world’s supply of material has supported numerous biological investigations and roughly 80 clinical trials against various cancers.[4] Recently, a clinical trial against Alzheimer’s disease has also commenced.[5]

Substitution on the A-Ring Confers to Bryopyran Analogues the Unique Biological Activity Characteristic of Bryostatins and Distinct From That of the Phorbol Esters

A close structural analogue of bryostatin 1, which differs from bryostatin 1 only by the absence of the C(30) carbomethoxy group (on the C(13) enoate of the B-ring), has been prepared by total synthesis. Biological assays reveal a crucial role for substitution in the bryostatin 1 A-ring in conferring those responses which are characteristic of bryostatin 1 and distinct from those observed with PMA.

The bryostatin 1 A-ring acetate is not the critical determinant for antagonism of phorbol ester-induced biological responses.

The contribution of the A-ring C(7) acetate to the function of bryostatin 1 has been investigated through synthesis and biological evaluation of an analogue incorporating this feature into the bryopyran core structure. No enhanced binding affinity for protein kinase C (PKC) was observed, relative to previously characterized analogues lacking the C(7) acetate. Functional assays showed biological responses characteristic of those induced by the phorbol ester PMA and distinctly different from those observed with bryostatin 1.

Binding of [3H]bryostatin 4 to protein kinase C

The bryostatins represent a unique class of activators of protein kinase C (PKC) which induce only a subset of the responses typical of the phorbol esters and block those responses to the phorbol esters which they themselves do not induce. To better understand the interaction of the bryostatins with PKC, we have synthesized [26-3H]bryostatin 4 and characterized its binding to PKC. [3H]Bryostatin 4 and [3H]phorbol 12,13-dibutyrate ([3H]PDBu) differed markedly in their binding to PKC reconstituted with phosphatidylserine (PS). The binding affinity of [3H]bryostatin 4 under these conditions was too high to measure and the rate of release of bound bryostatin was much slower than that of the phorbol esters, with a half-time of several hours. These properties caused bryostatin 1 to appear to inhibit [3H]PDBu binding under these conditions in a non-competitive fashion. Both the high potency and the slow rate of release of the bryostatins may contribute to their unique pattern of biological activity. By reconstituting PKC in a mixture of 1.5% Triton X-100:0.3% PS, we were able to establish reversible conditions for [3H]bryostatin 4 binding. Under these latter conditions, binding of [3H]bryostatin 4 was competitively inhibited by PDBu, consistent with both the bryostatin and phorbol esters binding to PKC in a qualitatively similar fashion. Binding affinities to PKC isozymes alpha, beta, and gamma were compared and little difference was found, suggesting that differential recognition by these isozymes does not account for the unique biological activity of the bryostatins.

Searching for Disease Modifiers—PKC Activation and HDAC Inhibition—A Dual Drug Approach to Alzheimer's Disease that Decreases Aβ Production while Blocking Oxidative Stress

A series of benzolactam compounds were synthesized, some of which caused a concentration‐dependent increase in sAPPα and decrease in Aβ production in the concentration range of 0.1–10 μM. Moreover, some compounds showed neuroprotective effects in the 10–20 μM range in the HCA cortical neuron model of oxidative stress and no toxicity in measurements of neuron viability by MTT assay, even at the highest concentrations tested (20 μM).