Lesson 7: RB (Retinoblastoma), a children's eye cancer, could be treated by PAK1-blockers such as propolis

Loss or dysfunction of tumor suppressor RB gene causes a rare eye cancer, during an early childhood, called retinoblastoma (RB). So far no effective therapeutics has been developed. However, recently a team in Puerto Rico and USA jointly discovered the very first clue to the potentially effective RB therapy.  They found that the transcription factor RB represses the expression of oncogenic/ ageing PAK1 gene (1).  In other words, blocking PAK1 over-expressed by loss of RB in retinoblastoma could lead to suppression of the growth of this eye cancer.  In the past (more than a decade ago) , butyrate, a HDAC (histone deacetylase) inhibitor, was shown to suppress the growth of RB (retinoblastoma) cells in cell culture, most likely by down-regulating two oncogenic kinases, AKT and PAK1. However, the IC50 of butyrate against HDAC is very high (above 1 mM), and therefore has never been used clinically. I believe a variety of far more effective PAK1-blockers (synthetic or herbal) such as propolis would be useful for treatment of retinoblastoma and many other PAK1-dependent cancers without any side effect. 

References:

1. Sosa-García B¹, Vázquez-Rivera V¹, González-Flores JN¹, Engel BE², Cress WD², Santiago-Cardona PG¹. The Retinoblastoma Tumor Suppressor (RB) Transcriptionally Represses Pak1 in Osteoblasts. PLoS One. 2015 Nov 10;10(11):e0142406.

2015 Nobel Prize in Physiology/Medicine: Discovery of Ivermectin and Artemisinin against Tropical Parasites

It has been a rather rare event that the Nobel Foundation gave an award to biomedical scientists who contributed to either discovery or development of antibiotics (herbal remedy) such as penicillin and streptomycin.  Nevertheless, this year three scientists are going to share a Nobel prize in physiology/medicine for their discovery/development of ivermectin and artemisinin which kill tropical parasites such as malaria and pathological nematodes.  Youyou Tu (85) in China who discovered an anti-malaria herbal compound called artemisinin in 1970s shares a half of this prize, while Satoshi Omura (80) at Kitasato Institute in Japan and William Campbell (85) at Drew University in US, who discovered/developed an anti-nematode antibiotic called ivermectin in 1980s, share the remaining half of this prize. 

Quite interestingly, both artemisinin and ivermectin are PAK1-blockers, and are well known to suppress the growth of cancers, and many other PAK1-dependent diseases/disorders.  However, ivermectin appears not to pass BBB (blood brain barrier), and therefore would be useless for the therapy of brain tumors and neuronal diseases such as AD (Alzheimer’s disease).  Although the IC50 of artemisinin (AM) against cancer cells in cell culture is rather high, very recently a far more potent AM derivative was developed by a German group. This AM derivative kills cancer cells with IC50 around 10 nM (1). Thus, in the future, not only people living in tropical zones, but also those in the remaining areas could have a significant benefit from this potent anti-cancer AM derivative. 

References:

1.  Reiter C¹, Fröhlich T¹, Zeino M², Marschall M³, Bahsi H³, Leidenberger M⁴, Friedrich O⁴, Kappes B⁴, Hampel F¹, Efferth T⁵, Tsogoeva SB⁶. New efficient artemisinin derived agents against human leukemia cells, human cytomegalovirus and Plasmodium falciparum: 2nd generation 1,2,4-trioxane-ferrocene hybrids. Eur J Med Chem. 2015 ; 97:164-72.

 

Beta-Elemene : a herbal PAK1-blocker that up-regulates PAK1-interacting protein 1

  According to a recent paper by a Chinese group, beta-elemene from Curcuma increases the radio-sensitivity of cancer cells by blocking the oncogenic/ageing kinase PAK1 (1).  Both radiation and UV irradiation are known to activate PAK1, making cancer cells more resistant to any anti-cancer drugs. 

How does beta-elemene block PAK1?  They found that it up-regulates a PAK1-inhibitor called "PAK1-interacting protein 1" which binds the N-terminal 70 amino acid motif of PAK1 (2).  

Unfortunately, however, the IC50 of beta-elemene against cancer cells in cell culture is rather high, around 300 micro M. Thus, it is unlikely that be-elemene is clinically useful for cancer therapy.  Thus, another Chinese group developed a far more potent "dimethylpiperazine"  derivative beta-elemenecalled IIi whose IC50 against cancer cells in cell culture is around 3 micro M (3).

  
References:

1。Liu JS¹, Che XM¹, Chang S¹, Qiu GL¹, He SC¹, Fan L¹, Zhao W¹, Zhang ZL¹, Wang SF¹ β-elemene enhances the radiosensitivity of gastric cancer cells by inhibiting Pak1 activation. World J Gastroenterol. 2015 Sep 14; 21(34): 9945-56.

2. Xia C¹, Ma W, Stafford LJ, Marcus S, Xiong WC, Liu M. Regulation of the p21-activated kinase (PAK) by a human Gbeta -like WD-repeat protein, hPIP1. Proc Natl Acad Sci U S A. 2001;  98(11): 6174-9.

3.  Ding XF¹, Shen M, Xu LY, Dong JH, Chen G.

13,14-bis(cis-3,5-dimethyl-1-piperazinyl)-β-elemene, a novel β-elemene derivative, shows potent antitumor activities via inhibition of mTOR in human breast cancer cells. Oncol Lett. 2013; 5(5): 1554-1558.

Another "Holy Grail": A geo-specific esterase that cleaves the Arg-ester of ST2002 at position 3 or 9

Staurosporine (ST) is a highly cell permeable but non-specific kinase inhibitor. To make this compound highly specific for the oncogenic/ageing kinase PAK1, ST should bear a bulky side chain at position 3 or 9 to exclude all kinases except for PAK1. Furthermore, it has been known for more than a decade, OH at position 3 (or 9)=ST2001, but not both (ST2002), boosts the anti-PAK1 activity by 50 times (reaching IC50=1 nM). ST is not water-soluble, but if you attach the basic amino acids such as Arg and Lys, to position 3 or 9 (or both), it becomes water-soluble and more cell-permeable.  Considering these factors, we are currently designing a potent, water-soluble, highly cell-permeable PAK1-specific ST derivatives called "ST3009".

ST2001 was found in a marine organism more than a decade ago, but this organism has vanished from the ocean sadly.  Chemical synthesis of ST2001 is possible, but its yield is so low, and economically unfeasable. The major product of ST (chemical) hydroxylation is ST2002 in which both 3 and 9 is hydroxylated.  Unfortunately, ST2002 has no anti-PAK1 activity.

Thus, to make the ST3009 (golden egg) efficiently, we have to esterize ST2002 at both positions 3 and 9 chemically, making ST3003 first, and then cleave only one of these ester bonds geo-selectively by a specific esterase, eventually yielding the ST3009.  This sort of "geo-specific" esterase is our "Holy Grail" in the forthcoming ST3009 project.

Lesson 6: CK2 (Casein Kinase 2) is also essential for the activation of PAK1 in cells

At least three distinct Tyr-kinases (ETK, JAK2 and FYN) are essential for the activation of PAK1 in cells. These kinases act down-stream of the oncogenic RAS. Recently another kinase was found to activate PAK1 in a RAS-dependent manner, too. It is called CK2 (Casein Kinase 2) which phosphorylates PAK1 at Ser 223 (1). The CK2-PAK1 interaction requires another protein called CKIP1, a PH domain protein. Furthermore, it is also known that PI-3 kinase acts upstream of the oncogenic CKIP1-CK2-PAK1 pathway. Thus, RAS-PI-3 kinase-CKIP1-CK2-PAK1 cascade has been established. In other words, CK2 inhibitors would block PAK1.

So I wonder what sort of CK2 inhibitors have been developed till now. Among several CK2 inhibitors, "D11 " is among the most potent, with the IC50 around 5 nM in vitro (2). It  was recently developed by a Danish group led by Barbara Guerra, and inhibits the PAK1-dependent growth of pancreatic cancer cells with the IC50 around 50 micro M in cell culture. Another potent CK2 inhibitor called "TF" (IC50=50 nM in vitro) inhibits the growth of cancer cells with IC50 around 50 micro M. Thus, their cell-permeability is extremely poor. I wonder if there is any potent CK2 inhibitor(s)  inhibiting the growth of cancer cells with IC50 at low nM levels.

So far CX-4945 (developed by Cylene Pharmaceuticals in US) is the most potent among CK2 inhibitors, with IC50=1 nM (in vitro) but 1 micro M (in cell culture), and inhibits the growth of human pancreatic cancer grafted in mice by 93 % with the daily dose of 75 mg/kg orally (3).

If I understand correctly, Senhwa Biosciences in Taiwan/US is currently conducting the phase1/2 clinical trials of CX-4945 for cancer therapy in combination with gemcitabin and cisplatin, hinting that CX-4945 alone is not sufficient for the complete cure of cancers.

References:

1. Kim YB, Shin YJ, Roy A, Kim JH.　The Role of the Pleckstrin Homology Domain-Containing Protein CKIP-1 in Activation of p21-activated Kinase 1 (PAK1). J Biol Chem. 2015 Jul 9.

2. Guerra B, Hochscherf J, Jensen NB, Issinger OG. Identification of a novel potent, selective and cell permeable inhibitor of protein kinase CK2 from the NIH/NCI Diversity Set Library. Mol Cell Biochem. 2015 May 12.

3. Siddiqui-Jain A¹, Drygin D, Streiner N, Chua P,et al. CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy. Cancer Res. 2010; 70: 10288-98.
 

The Direct Target of CAPE: AKR1B10 ("Aldo-Keto-Reductase" ) that is essential for RAS/RAC/PAK1 signaling

CAPE (caffeic acid phenethyl ester), the major anti-cancer ingredient in propolis such as Bio 30, has been known to down-regulate RAC that is essential for the activation of the major oncogenic/ageing kinase PAK1. However, the direct target of CAPE remained unknown till recently. 

A few years ago, a group at Gifu College of Pharmacy in Japan identified AKR1B10 ("Aldo-Keto-Reductase" ) as the direct target of CAPE with the IC50=80 nM, and developed a more potent CAPE derivative (called "10C") with the IC50=6 nM (1).  AKR1B10 is essential for the activation of RAS and RAC, which eventually activate PAK1 in cells.  Thus, we wonder if "Nepofein" is more potent than the compound 10C or not in cell culture and in vivo. The cell-permeability of "compound 10C" is still very low, and the IC50 against cancer cell growth (or AKR1B10 ) in cell culture is around 300 nM, indicating that only 1/50 of this compound enters cells.

References:

1. Soda M, Hu D, Endo S, Takemura M, Li J, Wada R, Ifuku S, Zhao HT, El-Kabbani O, Ohta S, Yamamura K, Toyooka N, Hara A, Matsunaga T.

Design, synthesis and evaluation of caffeic acid phenethyl ester (CAPE) -based inhibitors targeting a selectivity pocket in the active site of human aldo-keto reductase 1B10. Eur J Med Chem. 2012 ; 48:321-9.

Nepofein: Nepodin-Caffeic Acid Ester for Therapy of Cancers and Diabetes?

We recently heard from an old Korean friend that a naphthalene called “Nepodin” (MW=216) from roots of an Okinawa weed is a potent anti-diabetic compound which activates the anti-oncogenic kinase AMPK (1).  It is effective at the daily dose of 2-10 mg/kg in mice. Interestingly, it was shown by a leading Korean pharmaceutical company (CKD) to be a potent anti-malaria drug as well (2).  

Almost all herbal AMPK activators are PAK1-blockers, and both diabetes (type 2) and malaria infection require the oncogenic/ageing kinase PAK1. Thus, it is not unreasonable to suspect that Nepodin also blocks PAK1. Interestingly, CAPE (caffeic acid phenethyl ester), the major anti-cancer/anti-PAK1 ingredient in propolis, is made simply by condensation of CA (caffeic acid) and phenethyl alcohol.  Thus, by analogy, it would be quite easy to synthesize a new ester called “Nepofein” by condensation of Nepodin and CA, and test its anti-cancer and anti-diabetic property, in which the CKD Pharm would be surely interested. http://www.ckdpharm.com

References:

1.   Ha BG, Yonezawa T, Son MJ, Woo JT, Ohba S, Chung UI, Yagasaki K. Antidiabetic effect of nepodin, a component of Rumex roots, and its modes of action in vitro and in vivo. Biofactors. 2014; 40(4): 436-47.

2.  Lee KH¹, Rhee KH. Antimalarial activity of nepodin isolated from Rumex crispus. Arch Pharm Res. 2013; 36(4):430-5.
 

Hunting (Edible) Starfishes for PAK1-blocking Saponins

Recently several anti-cancer saponins were isolated from marine organisms such as “edible” sea cucumbers and starfishes. One of these marine saponins is Frondoside A from sea cucumber (Cucumaria frondosa) which suppresses the growth of pancreatic cancer in vivo (xenograft in mice). Since the growth of pancreatic cancers depends on PAK1, and this saponin induces p21, a CDK-inhibiting protein, whose gene is activated by PAK1-blockers, it is most likely that Frondoside A (FRA) is a PAK1-blocker. Recently we confirmed that FRA indeed inhibits PAK1 directly.

More recently a Korean group found another anti-cancer saponin in methanol extract from an edible starfish grown in Vietnam coast line (1).  The IC₅₀ of this extract against cancer cells is around 1 ppm (1 micro g/ml). In other words, its anti-PAK index is around 100, several times more potent than Bio 30, a CAPE-based propolis. Furthermore, it inactivates the oncogenic kinase ERK, just downstream of PAK1. Thus, we recently started hunting starfishes along Okinawa coast line, screening for a similarly (or even more) potent anti-cancer (PAK1-blocking) saponin(s).

References:

1. Thao NP, Luyen BT, Kim EJ, Kang HK, Kim S, Cuong NX, Nam NH, Kiem PV, Minh CV, Kim YH. 　Asterosaponins from the Starfish Astropecten monacanthus suppress growth and induce apoptosis in HL-60, PC-3, and SNU-C5 human cancer cell lines. Biol Pharm Bull. 2014;　37(2):　315-21.
 

"Anti-PAK Index" : the International standard of PAK1-blocking herbal products such as propolis for quality control

There is a wide variety of edible PAK1-blocking herbal products such as propolis available on the market world-wide which could be very useful for improving our health and even therapy of cancer and many other PAK1-dependent diseases/disorders such as Alzheimer’s disease (1). However, unlike FDA-approved drugs, none of them is associated with any reliable international quality control standard such as IC₅₀ or ED (effective dose). For instance, the quality of propolis depends on the sources of plants where bees harvest from, and the actual content of PAK1-blocking ingredients such CAPE, apigenin, ARC (artepillin C) and propolin G (=nymphaeol C) in each propolis. However, since 1960s till present, the only available quality stardard used for propolis is just “the total flavonoid content” for CAPE-based propolis or the ARC content in Brazillian green propolis. If these “herbal” health-promoting products are regulated by a single reliable pharmacological quality standard, we could compare the quality or effectiveness from one sample to another quite objectively, regardless of their detailed content.

Hence, we would propose here for the fist time to use a universal standard called “Anti-PAK Index” which is the 100 X reciprocal of the IC₅₀ in ppm (micro g/ml). For instance, “Anti-PAK Index” of Bio 30 (CAPE-based propolis from New Zealand) , (propolin G-based) Okinawa propolis (OP) and (ARC-based ) Brazilian green propolis (GP) are 12.5, 8 and 1, respectively, since their IC₅₀ for A549 cancer cells are around 8, 12 and 100 ppm, respectively, whereas the “Anti-PAK Index” of the “pure” compound "Cucurbitacin" (CB) is around 1400, as the IC₅₀ for A549 is around 140 nM (0.07 ppm). The higher the Anti-PAK Index, the more potent a given sample. In other words, 1 mg of CB is equivalent to 112 mg of Bio 30 for therapy of cancers and many other PAK1-dependent diseases/disorders. This rough estimation is not far from the actual  in vivo data where the daily dose of  CB (1 mg/kg),  and that of Bio 30 (50 mg/kg) are their effective dose to suppress the PAK1-dependent growth of pancreatic cancers or NF tumors in mice (1), suggesting that their in vivo bioavailability is quite similar.

The only difference between these two is that Bio 30 has been available on the market world-wide for clinical uses for almost a decade, but CB is not as yet.  The only way for us to take CB is to eat the edible bitter melon (Goya) grown in Okinawa (which contains around 1 g of CB per kg) or drink Goya teas. Since roughly 90% of Goya is water, the “Anti-PAK Index”of Goya extract/tea could be around 14, pretty close to that of Bio 30.

We recently managed to synthesize a highly cell-permeable and water-soluble compound whose IC50 is around 24 nM (0.01 ppm) against the growth of A549 cancer cells (2). Its "Anti-PAK Index" is 10,000, several times more potent than CB, and we have filed a US patent on this new PAK1- blocker called "15K" for its clinical application (The related Japanese patent was granted). It is derived from an old FDA-approved synthetic anti-inflammatory pain killer (Ketorolac).  It passes the BBB (blood brain barrier), and could be useful for treating a variety of PAK1-dependent neuronal diseases/disorders such as brain tumors, AD (Alzheimer's disease), PD (Parkinson's disease), epilepsy, depression,  schizophrenia and even autism. Furthermore, it extends the healthy lifespan of C. elegans by 30% at 50 nM, clearly indicating that "15K"  causes no side effect!

I bet that leading pharmaceutical giants such as Pfeizer, Roche and Novartis, who are currently racing for developing potent PAK1-blockers which would be useful for clinical application, would be most likely keen to buy an exclusive license (worth more than 200 million USD) from this patent of ours. 

References:

１．Maruta H. Herbal therapeutics that block the oncogenic kinase PAK1: A practical approach towards PAK1-dependent diseases and longevity. Phytother Res. 2014; 28:656-672..

2.    Nguyen BC, Takahashi H, Uto Y, Shahinozzaman MD, Tawata S, Maruta H. Chemistry"-based highly potent PAK1-blocking cancer-killer. Eur J Med Chem. 2016 ; 126: 270-6.