IMPORTANT NEW DATA:  4α-PDD Activation of TRPV4 Channels!  Long thought to be a biologically inactive or extremely weak phorbol ester analog (i.e., an ED50 >25 µM for binding to protein kinase C), 4α-PDD has now been shown to be a reasonably potent activator of two TRPV4 channels, namely human VRL-2 and murine TRP12 channels [H. Watanabe et al., J. Biol. Chem. 277:  13569-13577 (2002)].  The ED 50 of 4α-PDD for activation of the TRP12 channel was ~400 nM, and for increasing internal calcium levels in 1321N1 astrocytoma cells expressing human VRL-2, the ED50 of 4α-PDD was ~185 nM.  This work extends earlier results showing non-phorbol-ester-like effects of 4α-PDD [H. Reeve et al., Eur. J. Physiol. 429:  729-737 (1995)] and 4α-phorbol 12,13-dibutyrate (4 -PDBu) [D. Doerner et al., J. Neurosci. 10:  1699-1706 (1990)] on calcium currents.

Because 4α-PDD has few, if any, recognized biological effects at sub-micromolar concentrations other than these effects on TRPV4 channels, Watanabe et al. certainly seem justified in stating that "4α-PDD can be used as a robust and reliable tool to study several features of TRPV channels and to probe functional effects of the activation of this channel in in vivo systems".  That said, it is also important to note that absolute selectivity of 4α-PDD for activating TRPV4 channels has not been demonstrated.  Though 4α-PDD has been shown over the years to have little or no effect in a fairly wide range of biological assays, 4α-PDD might prove to have other, as-yet-unidentified activities if subjected to more extensive testing against various targets.

[As an aside, we point out that reference #25 in the Watanabe article, cited in support of the inactivity of 4α-PDD on PKC, appears not to contain any mention at all of 4α-PDD or other 4α-phorbol esters.  For the convenience of those who are preparing manuscripts dealing with 4α-phorbol esters, one or more appropriate references supporting the low activity of these compounds on PKC will be added to this LC Labs product description shortly.]

[Also, see below for an important note about nomenclature.  Technically, 4α-PDD is not a phorbol ester, it is a 4α-phorbol ester — a small but important distinction — and must always be specified as such to avoid confusion with the dramatically different properties of the phorbol esters.]

Surprisingly (in view of historical structure-activity data), PMA (phorbol 12-myristate 13-acetate), the classical nanomolar-potency PKC activator, was 10- to 50-fold weaker than 4α-PDD for activation of the TRPV4 channels.  If both PMA and 4α-PDD were targeting a PKC-related protein, via a mechanism fundamentally similar to that of classical PKC activation by phorbol esters, PMA would be expected to be many orders of magnitude more potent than 4α-PDD.  Watanabe et al. tested a wide range of PMA and 4α-PDD concentrations, and there appears to be no doubt that the relative potencies expected for PMA and 4α-PDD for classical PKC-related effects are strikingly reversed for the TRPV4 channel activation phenomenon.  Furthermore, in some assays PMA was merely a "partial agonist", showing only 50-65% of the response elicited by 4α-PDD.

The PMA/4α-PDD potency inversion in turn strongly suggests that 4α-PDD must be acting via a mechanism distinct from the classical interaction of a phorbol 12,13-diester with a phorbol ester/diacylglycerol-type receptor target, such as those found on the PKC family of proteins.  Given the long history and, until now, largely settled picture of the biological properties of phorbol and 4α-phorbol esters, this question of mechanism is of very high interest indeed, and the answer(s) might turn out to have a wide impact on several areas of pharmacology.

Watanabe et al. favor direct binding of 4α-PDD to the TRPV4 channels as the specific pharmacological mechanism underlying the effects they observed.  However, as of this writing (April 2003) no evidence for direct binding of PMA or 4α-PDD to TRPV4 channels appears to be available.  Lacking such evidence, the target question remains open, and serious consideration must also be given to other, indirect potential mechanisms.

PMA and 4α-PDD have close structural similarity, and the family of highly specific phorbol ester receptors, of known amino acid sequence pattern, have been extensively characterized.  Thus, in the absence of evidence to the contrary, "the usual suspect" for a target mediating the effects of 4α-PDD on TRPV4 would be a site similar to the well-known family of phorbol ester receptors, perhaps with some crucial amino acid residue differences that result in reversal of the usual comparative potencies of phorbol vs. 4α-phorbol esters for a given effect.  Such a target might occur either on a PKC-type protein or on some other, perhaps non-kinase, protein bearing a phorbol-type receptor.  Furthermore, the well-established ability of PKC to phosphorylate and modulate a number of ion channels supports suspicions that a phorbol ester-like compound such as 4α-PDD might be acting through a PKC-like entity to activate TRPV4 channels.

It appears that Watanabe et al. did not test the effects of any PKC inhibitors in their experimental systems.  If several kinase inhibitors known to be reasonably specific for PKC were to be tested and shown not to inhibit the 4α-PDD effects on TRPV4 channels, this would further support the direct binding hypothesis.  In this context it is important to note that some PKC isotypes are affected only weakly or not at all by common PKC inhibitors; such inhibitors are imperfect phamacological tools.  Also, even if inhibitor studies were to reasonably rule out the PKC family as a target site, there remain several other classes of cellular proteins that are not PKC's but nonetheless have functioning, high-affinity phorbol ester receptors.  These other phorbol ester receptor-bearing proteins could also be candidates for the actual target of 4α-PDD in the present case.

Clearly, if the amino acid sequences of the TRPV4 channels contain a region homologous to phorbol ester receptors, that would strongly support a direct binding mechanism for the effects of 4α-PDD on these channels.  For lack of space and time, our discussion here does not include any comparative sequence information about this obvious question.  If other investigators do make such comparisons and find an absence of the well-known phorbol ester receptor-like sequences in the TRPV4 channel family, such a result would then require that either a completely new sequence motif for binding 4α-phorbol-type compounds be present in TRPV4 channels, or that the mechanism of action of 4α-PDD on these channels be indirect.

With respect to the details of an indirect mechanism for the effects of 4α-PDD on TRPV4 channels, it is not difficult to imagine how 4α-PDD might interact with a PKC or similar phorbol ester receptor in a manner sufficiently different from that of the classical PMA-PKC interaction to elicit a profile of biological activities distinct from that of PMA, such as reported now in the TRPV4 studies of Watanabe et al.  Considerable evidence already exists indicating that even subtly different phorbol ester analogs can all bind to PKC family members but at the same time can differ from one another in terms of the cellular location to which the resulting binding complex is directed.  This topological control might be functioning in the case of 4α-PDD, wherein both PMA and 4α-PDD might bind to a PKC family member or other phorbol-receptor-containing protein but tend to direct the resulting complexes to different cellular locations, adjacent to, e. g., different kinase substrates.

There remains also the question of possible non-specific effects of 4α-PDD as an explanation for its effects on TRPV4 channels.  4α-PDD is a non-ionic detergent-like molecule having a polar head group and a long hydrophobic tail, and it might be expected to perturb membranes and ion channels in a non-specific manner.  Ironically, 4α-phorbol esters such as 4α-PDD have themselves long been used as "inactive negative controls" for potential detergent-like or other non-specific effects of the PKC-activating phorbol esters.  Hence, in the present case, one needs "a control for the control", i. e., an analog of 4α-PDD having similar hydrophobicity and detergent-like structure but which is inactive on all potentially relevant targets such as TRPV4 channels, PKC family members and other proteins bearing phorbol ester receptors.  LC Laboratories may be able to provide such a compound in the near future.

In pursuit of more details about the interaction of 4α-PDD with its biological target, one might hope to employ a ligand-protein binding interaction using a labeled 4α-PDD in a receptor binding protocol.  Unfortunately, this may prove to be quite difficult in the case of 4α-PDD because (i) it is not terribly potent (only about 400 nM, compared to ca. 1 nM potencies for phorbol ester binding to PKC), and (ii) it is highly hydrophobic.  The latter property is especially problematic because the typical phorbol ester/ receptor complex requires the presence of quite a bit of phospholipid, to which large of amounts of 4α-PDD would be expected to bind nonspecifically.  Taken together, this all suggests that the active 4α-PDD/ receptor complex involved in the present case, whether PKC-related or not, might not be measurable because of an overwhelming level of non-specific binding of labeled 4α-PDD.  This would obscure any specific binding in a typical preparation containing membrane lipids.

From the point of view of structure-activity relations, to date it appears that, among 4α-phorbols, only the effects of 4α-PDD on TRPV4 channels have been reported.  LC Laboratories also offers other 4α-phorbol diesters of varying hydrophobicity; these presumably can be used for structure-activity studies of the TRPV4 activation effect.  Specifically, we offer 4α-PMA (Cat. No. P-8880) and 4α-phorbol 12,13-dibutyrate (4α-PDBu; Cat. No. P-4678) , both of which (especially 4α-PDBu) are less hydrophobic than 4α-PDD.

These analogs of 4α-PDD have considerable potential utility.  The high hydrophobicity (high lipid partition coefficient) of 4α-PDD makes it quite soluble in cellular membrane compartments, and it is reliably presumed to be very difficult to wash this compound out of membrane preparations or cell cultures.  4α-PMA and 4α-PDBu may prove to be less potent than 4α-PDD, but if they retain sufficient potency vis-a-vis 4α-PDD, they might be preferable as research tools because of their enhanced potential to equilibrate among aqueous and lipid cellular compartments and to be washed out of experimental preparations.

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