NV-128
NV-128 is the third drug to emerge from the Novogen anti-cancer pipeline to be ear-marked for development.
NV-128 was selected from several hundred candidate compounds for further development, in part because it demonstrates considerably greater anti-cancer potency than phenoxodiol and triphendiol,
and in part because its mechanism of action appears to differ substantially from that of those other two drugs. Where triphendiol shows a modest degree of difference (TRAIL vs Fas death receptors) from phenoxodiol to warrant being regarded as a different drug with different clinical opportunities, NV-128 appears to have taken the program into even more different territory.
In keeping with the isoflavonoid family of anti-cancer drugs, NV-128 is broadly acting across a wide range of human cancer types and appears to be highly selective for cancer cells, with no known side-effects. What made it stand out in the initial screening process was that it was at least 10-fold more potent than phenoxodiol and triphendiol across almost all types of cancer.
It shows particularly strong anti-cancer activity against non-small cell lung cancer and ovarian cancer, and these appear to be the clinical targets that have been identified by the Company at this time for
regulatory approval.
Once the drug began to be looked at in some more detail, two interesting features emerged which have marked this drug as something special. The first is that it appears to be killing cancer cells in a caspase-independent manner via the mTOR pathway; the second feature is that it kills cancer stem cells.
The mTOR signalling pathway
Every year so year, a new signalling pathway becomes flavour of the month and becomes the main topic of conversation in laboratories and at cancer conferences. And biotech companies that just appen to have a drug in development that targets such pathways receive investor attention (unless you happen to be Novogen).
mTOR is the latest pathway to be so honoured.
To be fair, it is a pathway that appears to play a key role in cell survival and function, and almost certainly is involved in the cancer process, but as has proved to be the case with virtually all other signalling pathways that have enjoyed being flavour of the month, they are so complex and intricate that to target them in isolation without due regard to what is happening elsewhere in the cancer cell is being simplistic in the extreme.
Nevertheless, mTOR is relevant to understanding how NV-128 is working, so it is worth paying it some attention.
The following diagram shows very nicely just how complex the mTOR signalling pathway is. And it is worth bearing in mind that this represents only what we know about it (and that almost certainly will prove to be just a small amount), and does not take into account the inputs from and interactions with all the other equally important signalling pathways (like the sphingomyelin cycle) that contribute to cell survival, growth and function.
In trying to reduce this jumble to some sort of meaningful perspective, we need to start with some fundamentals.
The role of signalling pathways is to turn an external message into action. They are the link between the external stimuli that cells rely on to stay alive and to function, and the parts of the cell such as the nucleus (activation of genes) and the mitochondria (energy production) that enable to cell to undertake its functions.
The external messages are received by a rich array of protein receptors on the plasma membrane. Prominent among these signals are the so-called growth signals that drive a cell to survive, to divide, and to function. These growth signals include hormones (insulin, growth hormone, sex hormones etc) and growth factors and cytokines (Epidermal growth Factor, Vascular Endothelial growth Factor, interleukins etc), to name but a few of the many thousands of different messages coming into each cell.
Once activated, these growth receptors in turn activate a complex system of internal signal pathways known as signal transduction pathways. Each pathway comprises a chain of separate steps that might vary from several up to dozens of separate steps. Most of these steps are specific proteins (or enzymes known as kinases) that activate the next protein in the chain of command through the process of phosphorylation, whereby a phosphate group is attached to the protein, so making that protein biologically active
As you can see from the diagram above, signal transduction is an extraordinarily complex process, much like the wiring of a telephone exchange, with so much cross-wiring in place that no single pathway can be considered in isolation. Incoming signals are fed into key common points that then re-distribute those signals to a variety of other signalling pathways. Understanding just how a cell integrates and redistributes all these different signalling pathways remains as much a challenge to the molecular biologist as an understanding of the complexities of the universe is to the astronomer. Which is why it is simplistic in the extreme to isolate a single pathway or even a part of one pathway, and then design a drug to target that one isolated function, and believe that you will end up with a magic cancer bullet. That notion makes no allowance for the fact that carcinogenesis is almost certain to involve multiple pathways, or that the signalling wiring is so intricate and cross-wired that a
cancer cell cannot quickly learn to circumvent the blocked pathway.
But back to mTOR and where it fits into the NV-128 story.
mTOR first came to scientific attention with the development of the drug, rapamycin. Isolated in 1975 from a soil bacterium (Streptomyces) collected from Easter Island (local name Rapa Nui) and with a view to it being a new antibiotic, rapamycin (trade name Rapamune) was developed as an immunosuppressive drug for organ transplantation because of its ability to block the response of immune cells to foreign antigen. A couple of years later a closely related drug, FK506 (trade name Prograf), also isolated from Streptomyces, this time from soil in Japan, came along and followed the same development path as rapamycin. Both drugs remain widely used today in all forms of organ transplantation.
In looking at how both drugs worked (that is, how in an antibiotic role they killed bacteria, and how in their immunosuppressive role they switched off lymphocytes), researchers found that both drugs were binding to a protein inside the cell that subsequently was named FK506-binding protein 12 (FKBP12). The consequence of the drugs binding to this protein is that FKBP12 is prevented from binding to a second protein that became known as the mammalian target of rapamycin, shortened to mTOR. The joining of these two proteins leads to other proteins becoming involved, with the entire complex becoming known as the mTOR Complex (or mTORC), which acts in turn as a kinase for a number of downstream signalling pathways. Just to complicate things a little more, it subsequently was discovered that there were two distinct forms of mTORC. The original complex (that involves FKBP12) is now referred to as mTORC1. The more recently-discovered complex, mTORC2, has its own set of separate functions and is distinctive from mTORC1 in some important ways – first, it is not stimulated directly by growth receptors, and second, that it does not require the FKBP12 binding protein to be activated. rapamycin only very weakly inhibits mTORC2.
mTORC1
One of the main roles of this complex is to serve as a monitor of the body’s nutrient status and to regulate a cell’s activity based on that status.
mTORC1 responds directly to a range of growth factors and to nutrient receptors that are monitoring levels of glucose and amino acids in the cell’s environment. growth stimulation coming from high nutrient/energy levels and high hormone (insulin, growth hormone, sex hormones etc) activity activates mTORC1, while low nutrient/energy levels shut down its function, presumably as a basic survival tool in the event of low nutrient levels. The extension of life that animal research has shown comes from caloric restriction is thought to be associated with the shutdown of mTORC1.
Some other key functions of this complex in relation to cancer are as follows.
(a) Protein synthesis and cell division.
In the presence of adequate levels of amino acids, mTORC1 drives protein synthesis in the cell via activation of the enzyme, p70-S6 kinase. This is turn drives the cell cycle and cell division. Over-expression of p70-S6 activity has been shown to be associated with a number of different types of cancer.
(b) Autophagy.
mTORC1 regulates the process of autophagy. Autophagy is a form of self-cannibalism by the cell. It is distinct from apoptosis which ends in cell death. The purpose of autophagy is to degrade selective parts of the cell, thereby ensuring cell survival. In part, this is a routine housekeeping duty that removes worn-out organelles. But it also is a response to nutrient starvation.
Autophagy requires activation of autophagy proteins such as Beclin-1 and Bcl-2. These proteins normally are suppressed by mTORC1, hence typically are at low levels in cancer cells.
mTORC2
mTORC2 also appears to receive some direct input from the growth receptors and nutrient receptors as does mTORC1, but most of its activation comes as a result of being downstream of the all-important p13k/Akt signalling pathway. This is the same pathway through which another key signalling system (sphingosine kinase) is working, although their respective inputs are quite different. As
the following diagram shows, sphingosine kinase is upstream of Akt, while mTORC2 is downstream.
p13k is short for phosphoinositide 3-kinase. p13k is activated by a wide range of receptors responding to growth signals and nutrient levels (eg insulin receptor), responding by attaching a phosphate group to proteins known as phosphatidylinositols at the cell surface. This in turn activates Akt at the cell membrane, a key signalling point in the regulation of cell meytabolism, division and survival.
The role of mTORC2 is much less studied than that of mTORC1, but the following general points can be made even at this early stage of discovery.
(i) The functions of mTORC1 and mTORC2 cross-over to a certain degree. This appears to reflect a high degree of cross-communication between the two pathways, as well as direct inputs from the same receptors and the sharing of common downstream signalling pathways.
(ii) The difference between the two pathways appears to be one of preferential activity rather than independent activity. Thus, mTORC1 carries prime responsibility as a sensor of nutrient status, but mTORC2 also plays some role in this area. mTORC2 on the other hand appears to play a more dominant role in cell proliferation and differentiation.
(iii)A major role of mTORC2 appears to be that of regulating the production of the cell’s cytoskeleton …. the structures such as the microtubules and filaments that support all of the cell’s organelles.
mTOR- inhibiting drugs
It is the aberrant behaviour of incoming signalling pathways such as mTOR and sphingosine kinase and key exchange points such as p13k/Akt that underlie the development of most forms of cancer. Uncontrolled cell growth and mobility, the switching off of apoptotic mechanisms, the promotion of angiogenesis and the development of chemo-resistance, are all associated with over-activity of all three signalling pathways, making each of these three pathways of such considerable interest as targets for the development of new anti-cancer therapies.
Several different approaches have been adopted in the quest for a drug to inhibit mTOR pathways.
The first approach is to follow the lead of rapamycin. A number of drugs have been developed as mTOR inhibitors based on the original rapamycin macrolide structure. Temsirolimus (Torisel) and everolimus (Afinitor) have been approved for use in advanced renal cell carcinoma. A third, deforolimus, is in development.
The second approach is a drug temozolomide (XL765; Exelixis) that inhibits both p13k and mTOR. This drug is in Phase 2 studies currently.
It is interesting to note that the mTOR pathways are sensitive to natural events beyond rapamycin and low nutrient levels. mTORC1 is inhibited also by caffeine and the polyphenol, curcumin, the yellow pigment in the Indian spice, tumeric. Equally, mTORC2 is inhibited by resveratrol, a polyphenol found in red wine. [Note the ability of naturally-occurring polyphenols, a family of compounds to which isoflavones belong, to inhibit mTOR function.]
mTOR-inhibiting effects of NV-128
NV-128 is distinctive in being able to inhibit both mTORC1 and mTORC2. The consequences of this are full and effective prevention of cell division, the initiation of cell death, and the blocking of angiogenesis.
p70-S6 kinase activity is inhibited, halting protein synthesis and cell division.
Cell death then ensues by apoptosis, but not by the caspase-dependent mechanism that we see with phenoxodiol and triphendiol. The action of these two latter drugs is to allow death signals from the Fas and TRAIL death receptors to initiate caspase activity by nullifying the over-production of proteins that specifically block the death signals.
With NV-128, cell death is occurring not via the caspases, but via an extension of the autophagy process. As a result of mTOR inhibition, expression of the autophagy proteins, Beclin-1 and Bcl-2, is increased to the point that they are depolarising the mitochondrial membrane, causing the release from the mitochondria of the proteolytic enzyme, endonuclease G. This enzyme attaches itself to the nuclear membrane, auto-digesting the cell’s DNA, and resulting in cell death.
NV-128 vs other mTOR- inhibiting drugs
The mTOR-inhibiting action of NV-128 is distinguished from that of the rapamycin analogues and XL765 in 2 important ways.
1. The first is that those drugs are non-discriminatory. They rely on cancer cells being more susceptible to the drug than healthy cells because of the proportionally higher levels of mTOR activity in ancer cells. However, healthy cells are not immune to their inhibitory action as evidenced by the high level of adverse side-effects associated with both the rapamycin analogues and XL765.
2. The second is that those drugs may have incomplete actions, or even worse, unwelcome actions. In the case of the rapamycin analogues that only inhibit mTORC1, growing evidence (eg. Cancer Research 2008, 68:7409) is now pointing to the fact that inhibition of mTORC1 alone results in a compensatory activation of key signalling pathways such as Akt and ERK through various feedback mechanisms. The concern now being expressed is that initial tumor response with these drugs could be followed by enhanced tumor survival.
3. Information on XL765 has been less forthcoming. Claimed to be an inhibitor of p13k and mTOR,it is unclear what aspects of mTOR are being targeted. The suggestion that it is mTOR activity downstream of Akt suggests that it is targeting mTORC2, but that is not clear. If that is the case, then the same risk associated with incomplete inactivation of mTOR pathways as with the rapamycin analogues could apply to this drug.
NV-128 has significant advantages over these other mTOR-inhibitors in (a) providing concomitant inhibition of both mTORC1 and mTORC2, and (b) in selectively targeting these functions in cancer cells.
Follow-up thoughts
NV-128 is a chemical analogue of phenoxodiol and triphendiol. It therefore would be surprising to find that it worked in a completely different way based on completely different molecular targest to those two closely related drugs. And yet on the surface that appears to be the case.
I am not aware if the Company has looked to see if NV-128 is switching off a cancer cell’s proton pump or inhibiting sphingosine kinase activity, but my guess is that they have not. Equally, I am unaware if phenoxodiol or triphendiol have been checked to see what effect they have on mTOR function, but again I am guessing that they have not.
Two factors come to mind in pondering this. The first is that the naturally-occurring polyphenols, resveratrol and curcumin, can inhibit mTOR, but apparently not universally. That is, resveratrol inhibits mTORC2, while curcumin (and rapamycin) inhibit mTORC1. So there would appear to be a facility for molecules with a phenolic structure either to bind directly to proteins associated with the mTOR complex (such as FKBP12), or (as I think more likely) to be binding to and inhibiting a protein upstream of both mTORC1 and mTORC2.
The second point of interest is the role of mTOR in oxidative stress. Oxidative stress reflects a problem with a cell’s control over its redox (reduction-oxidation control mechanisms). Redox function
(of which the proton pump is an integral part) may well be the link that binds phenoxodiol, triphendiol and NV-128 in their primary target.
Cancer stem cells
The concept of cancer stem cells is relatively new and is the subject of ongoing debate . The concept goes something like this …. all tissues contain stem cells with the ability to differentiate into the full range of cell types found in any tissue, including blood vessels and supporting connective tissue….that cancers also contain stem cells which themselves are cancerous and which differentiate into the full spectrum of cells that go to make up a tumour…. that cancer stem cells are relatively resistant chemotherapy and radiotherapy, and that the oft-experienced outcome of chemotherapy of tumour regression followed by tumor re-growth is the result of a residual and impervious population of cancer stem cells.
The Yale University group headed by Professor Gil Mor has been active in the area of ovarian cancer stem cells. His group has identified a population of ovarian cancer cells that are highly resistant to conventional chemotherapy and which can differentiate into different cell types including blood vessels.
NV-128 has been shown to block this differentiation, as well as blocking cell division by the stem cells. The stem cells were shown to have up-regulated mTOR activity, and the ability of NV-128 to inhibit these cells was associated with inhibition of this activity.
It is too early in this matter to say much more than this, but it does represent a particularly exciting development and one that is sure to be keenly followed by the pharmaceutical industry.
SUMMARY
The best summary is probably contained in the news announced late last year that a combination of NV-128 and phenoxodiol delivers a potent synergistic effect in the laboratory. When tested against non
small cell lung (NSCL) cells, NV-128 and paclitaxel delivered significant synergy, while a combination of NV-128 and phenoxodiol delivered an even great anti-cancer effect.
The combination of two drugs offers the following advantages:
a unique ability between the two drugs to target the main signalling pathways (sphingosine kinase, Akt, mTOR) up-regulated in cancer cells;
a unique ability to induce apoptosis by both caspase-dependent and caspase-independent pathways, thereby overcoming problems associated with chemo-resistance;
a unique ability in addition to target cancer stem cells.
The combination of
NV-128 + phenoxodiol + a standard chemotherapy drug looks to me to be a remarkable prospect for cancer therapy.