Noemi Kedei

Characterization of the Interaction of Ingenol 3-Angelate with Protein Kinase C

Ingenol 3-angelate (I3A) is one of the active ingredients in Euphorbia peplus, which has been used in traditional medicine. Here, we report the initial characterization of I3A as a protein kinase C (PKC) ligand. I3A bound to PKC-alpha in the presence of phosphatidylserine with high affinity; however, under these assay conditions, little PKC isoform selectivity was observed. PKC isoforms did show different sensitivity and selectivity for down-regulation by I3A and phorbol 12-myristate 13-acetate (PMA) in WEHI-231, HOP-92, and Colo-205 cells. In all of the three cell types, I3A inhibited cell proliferation with somewhat lower potency than did PMA. In intact CHO-K1 cells, I3A was able to translocate different green fluorescent protein-tagged PKC isoforms, visualized by confocal microscopy, with equal or higher potency than PMA. PKC-delta in particular showed a different pattern of translocation in response to I3A and PMA. I3A induced a higher level of secretion of the inflammatory cytokine interleukin 6 compared with PMA in the WEHI-231 cells and displayed a marked biphasic dose-response curve for the induction. I3A was unable to cause the same extent of association of the C1b domain of PKC-delta with lipids, compared with PMA or the physiological regulator diacylglycerol, and was able to partially block the association induced by these agents, measured by surface plasmon resonance. The in vitro kinase activity of PKC-alpha induced by I3A was lower than that induced by PMA. The novel pattern of behavior of I3A makes it of great interest for further evaluation.

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.

Interaction between Protein Kinase Cμ and the Vanilloid Receptor Type 1

The capsaicin receptor VR1 is a polymodal nociceptor activated by multiple stimuli. It has been reported that protein kinase C plays a role in the sensitization of VR1. Protein kinase D/PKCμ is a member of the protein kinase D serine/threonine kinase family that exhibits structural, enzymological, and regulatory features distinct from those of the PKCs, with which they are related. As part of our effort to optimize conditions for evaluating VR1 pharmacology, we found that treatment of Chinese hamster ovary (CHO) cells heterologously expressing rat VR1 (CHO/rVR1) with butyrate enhanced rVR1 expression and activity. The expression of PKCμ and PKCβ1, but not of other PKC isoforms, was also enhanced by butyrate treatment, suggesting the possibility that these two isoforms might contribute to the enhanced activity of rVR1. In support of this hypothesis, we found the following. 1) Overexpression of PKCμ enhanced the response of rVR1 to capsaicin and low pH, and expression of a dominant negative variant of PKCμ reduced the response of rVR1. 2) Reduction of endogenous PKCμ using antisense oligonucleotides decreased the response of exogenous rVR1 expressed in CHO cells as well as of endogenous rVR1 in dorsal root ganglion neurons. 3) PKCμ localized to the plasma membrane when overexpressed in CHO/rVR1 cells. 4) PKCμ directly bound to rVR1 expressed in CHO cells as well as to endogenous rVR1 in dorsal root ganglia or to an N-terminal fragment of rVR1, indicating a direct interaction between PKCμ and rVR1. 5) PKCμ directly phosphorylated rVR1 or a longer N-terminal fragment (amino acids 1–118) of rVR1 but not a shorter one (amino acids 1–99). 6) Mutation of S116A in rVR1 blocked both the phosphorylation of rVR1 by PKCμ and the enhancement by PKCμ of the rVR1 response to capsaicin. We conclude that PKCμ functions as a direct modulator of rVR1.

Arachidonyl dopamine as a ligand for the vanilloid receptor VR1 of the rat

The vanilloid receptor VR1 is a nonspecific Ca(2+) channel, expressed in sensory neurons in the peripheral nervous system and in various brain regions, which is believed to be an important molecular integrator of several chemical (acid, vanilloids) and physical stimuli (heat) that cause pain. Recently, several endogenous ligands for VR1 have been identified such as arachidonyl ethanolamide (anandamide) and the more potent arachidonyl dopamine (AA-DO). Here, we further characterize AA-DO as a ligand for rat VR1, heterologously expressed in CHO and HEK293 cells. AA-DO inhibited the binding of [3H]RTX to VR1 with a K(d) value of 5.49 +/- 0.68 microM and with positive cooperativity (p = 1.89 +/- 0.27), indicating that AA-DO was about 5-fold more potent than anandamide in this system. The K(d) (9.7 +/- 3.3 microM), and p values (1.54 +/- 0.04) for the binding of AA-DO to spinal cord membranes are in good correlation with the CHO-VR1 data. AA-DO stimulated 45Ca(2+) uptake on CHO-VR1 and HEK-VR1 cells with EC(50) values of 4.76 +/- 1.43 and 7.17 +/- 1.64 microM and Hill coefficients of 1.28 +/- 0.11 and 1.13 +/- 0.13, respectively, consistent with the binding measurements. In contrast to anandamide, AA-DO induced virtually the same level of 45Ca(2+) uptake as did capsaicin (90 +/- 6.6% in the CHO cells expressing VR1 and 89.3 +/- 9.4% in HEK293 cells expressing VR1). In a time dependent fashion following activation, AA-DO partially desensitized VR1 both in 45Ca(2+) uptake assays (IC(50) = 3.24 +/- 0.84 microM, inhibition is 68.5 +/- 6.85%) as well as in Ca(2+) imaging experiments (35.8 +/- 5.1% inhibition) using the CHO-VR1 system. The extent of desensitization was similar to that caused by capsaicin itself. We conclude that AA-DO is a full agonist for VR1 with a potency in the low micromolar range and is able to significantly desensitize the cells in a time and dose dependent manner.

Absolute quantitation of endogenous proteins with precision and accuracy using a capillary Western system.

Precise and accurate quantification of protein expression levels in a complex biological setting is challenging. Here, we describe a method for absolute quantitation of endogenous proteins in cell lysates using an automated capillary immunoassay system, the size-based Simple Western system (recently developed by ProteinSimple). The method was able to accurately measure the absolute amounts of target proteins at picogram or sub-picogram levels per nanogram of cell lysates. The measurements were independent of the cell matrix or the cell lysis buffer and were not affected by different antibody affinities for their specific epitopes. We then applied this method to quantitate absolute levels of expression of protein kinase C (PKC) isoforms in LNCaP and U937 cells, two cell lines used extensively for probing the downstream biological responses to PKC targeted ligands. Our absolute quantitation confirmed the predominance of PKCδ in both cells, supporting the important functional role of this PKC isoform in these cell lines. The method described here provides an approach to accurately quantitate levels of protein expression and correlate protein level with function. In addition to enhanced accuracy relative to conventional Western analysis, it circumvents the distortions inherent in comparison with signal intensities from different antibodies with different affinities.

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.

Evodiamine functions as an agonist for the vanilloid receptor TRPV1

Evodiamine, a quinozole alkaloid constituent of Evodia rutaecarpa, has been reported previously to induce several responses comparable to capsaicin in animal systems. Here, we characterize evodiamine as an agonist for rat TRPV1 expressed heterologously in CHO cells. Evodiamine bound to rat TRPV1 with a Ki of 5.95 +/- 0.87 microM, as measured by inhibition of [3H] RTX binding (capsaicin, Ki = 1.8 +/- 0.3 microM). Evodiamine was a full agonist for induction of 45Ca2+ uptake, with an EC50 of 856 +/- 43 nM (capsaicin, EC50 = 45 +/- 4 nM) and was competitively antagonized by capsazepine, as revealed by a Schild plot. The pattern of cellular response, as determined by calcium imaging, was similar to that with capsaicin and yielded an EC(50) of 1.03 +/- 0.21 [micro sign]M. Molecular modeling suggested a consistent pattern of overlap between evodiamine and TRPV1 agonists. We conclude that evodiamine represents a novel class of agonists for rat TRPV1, albeit 3-19-fold less potent than capsaicin, and thus represents a new potential class of lead molecules for drug development.