Hiroshi Maruta was born as the eldest child between his father Masao and his mother Sumiko in Tokyo, Japan, on Novemer 08, 1942, during the WWII. His father (1906-1989) was a graduate from Kyoto University, and an expert in German literature and philosophy. His mother (1918-2014) was a graduate from Kobe Woman University, an American mission school, and an expert in English/ American literature. Owing to these parental western culture backgrounds, Hiroshi was destined to work mainly overseas for the most of his adult life. During the spring of 1953, when he was around 10, both he and two younger sisters of his were contracted with the post-war pandemic TB, and were forced to stay at home for 5 months for their recovery from this deadly TB. Oweing to PAS (para-aminosalicylate) provided by US GHQ (occupation forces) where his mother used to work as a translator, three children of hers were miraculously recovered from TB by the end of summer! This experience made a big impact on her eldest son’s choice of the life-long profession as a drug developer (pharmaceutical scientist) later. After he graduated from a local junior high-school in 1958, he successfully passed the entrance exam to the most prestigeous public high school “Hibiya High” in Tokyo. Dr. Susumu Tonegawa at MIT (1987 Nobel laureate in medicine) was his 3 year senior at this senior high school. After graduated from this high school, Hiroshi decided to study pharmaceutical sciences at Tokyo University, aiming to develop a new effective anti-cancer drug(s), free of any side effect, hopefully some days in the future…
Oveseas Adventure
Shortly after he got a Ph.D. in immunology at Tokyo Graduate School in 1972 (1) under the supervision of Prof. Den-ichi Mizuno (1920-2017), he spent 15 months as an instructor there, and then went to US with a NIH international postdoctoral fellowship via a large US contena ship called “Oregon”, across the Pacific Ocean. On this ship he was the sole Japanese passenger, and got a “free” English lesson every day from the remaining 12 American passengers and over 40 sailing crews during the “non-stop” 9 days voyage passing near Anchorage in Alaska (instead of Hawaii). Overseas he also acquired a “western” nickname “Charlie” after Charles Darwin, Charles Dickens and Charlie Chaplin. After landing on Seattle on August 3, 1973, he took an overnight Greyhound bus trip to Boulder in Coloradio across the Rockies. Charlie spent one year at University of Colorado, Boulder (1-mile city) under supervision of Prof. Lester Goldstein, learning how to transplant nuclei from one giant amoeba (Amoeba proteus) to another under dissecting microscope, in order to trace the fate (disassembly and reassembly) of “radioactively-lableled” nuclear envelope after its disapearance during cytokinesis in the absence of de novo protein synthesis (2). Among his major achievements there was to reach the very top of the highest peak of Rockies, Longs Peak (4346 meter high), about a half way to Mt. Everest, with a law-school student called Sanches from Nebraska, just before Charlie moved to the East Coast to join the then “world-leading” NIH (National Institutes of Health) in Bethesda, outskirts of Washington, D.C.
Discovery of PAK1 in Amoeba
Then there were more than 40 buildings over NIH campus. At the oldest Bld 3 (belonging to NHLBI), where Prof. Albert Szent-Gyorgyi (1937 Nobel laureate in Medicine) used to work on biochemisty of muscle contraction during 1948-1950 (3), under the supervision of Dr. Edward Korn, Charlie started working on another amoeba called Acanthamoeba castellanii, which can be mass-cultured in a synthetic medium (6 X 15 liters bottles), to obtain around 1 kg of the soil amoeba for studying biochemistry of non-muscle actin and myosins. In this amoeba, Tom Pollard, a postdoc from Harvard Medical School, found a very peculiar “single-headed” myosin (Myosin I), whose actin-activation of ATPase activity requires a “third” amoeba protein, called “cofactor” (4). Shortly after Tom left Dr. Korn’s lab at NIH, Charlie joined Edward Korn’s lab, and was assigned to identify this “mysterious” cofactor. However, due to the complexity in this simple amoeba, Charlie had to spend a few extra years before reaching this “cofactor”. First of all, he found in this amoeba a double-headed myosin (Myosin II) as well, which does not require the “cofactor” for its actin-activation (5). Furthermore, this amobae was found to contain several distinct "actin-gelling" proteins (actin-crosslinkers) called “Gelactins” (6), and Myosin II causes a shrinkage of this actin-gel in ATP-dependent manner (5, 6). More than two decades later, towards the end of 1990s, Charlie was surprised to find that a unique yellow pigment called MKT-077 (originally developed by Fuji-Film for color films) cross-links actin filaments and suppresses selectively the growth of pancreatic cancers (7).
Meanwhile, in 1975, Bob Adelstein’s team at NIH‘s clinical Bld 10, found the first myosin kinase in human platelets which phosphorylates the regulatory light chain of double-headed myosins (Myoisn II) in mammals (8). Around 1977, Charlie got an interesting clue that the amoeba “cofactor” binds ATP or ADP immobilized to agarose beads, as do myosin ATPases, but with clearly distinct affinity. Thus, he managed to purify the “cofactor” by ADP-agarose chromatography. However, the amoeba “cofactor” failed to phosphorylate the light chain of amoeba myosins. Instead, he found that this cofactor is a new kinase that phosphorylates the heavy chain of Myoisn I but not Myosin II (9). This phosphorylation is essential for the actin-activation of amoeba myosin I ATPase (9). 17 years later, in 1994, Ed Manser’s team in Singapore cloned the first PAK1 in mammals which is very similar to the amoeba Myosin I kinase in structure and function (10). Both mammalian PAK1 and amoeba kinase are activated by a couple of closely related GTPases/G proteins called RAC/ CDC42 (10, 11), which works downstream of an oncogene called RAS. PAK1 is ubiquitously present in any species of the animal kingdom from yeasts to human being, but totally absent in the plant kingdom as well as both bacteria and viruses.
“Pathogenic” Mammalian PAK1
Furthermore, five years later, Ed found another protein called PIX in mammals which is essential for the full activation of PAK1 in cells, in addition to RAC/CDC42 (12). He managed to locate the PIX-binding Pro-rich domain of 18 amino acids on PAK1 which is called “PAK18”, binding the SH3 domain of PIX (13). Meanwhile, Charlie worked at Max-Planck Institute in Munich, West Germany (1980-1984), and Yale University and UCSD (1985-1987), handling several other amoeba (14, 15), and eventually since 1988, moved to Australia, started working seriously on mammalian RAS oncogenes at Ludwig Institute for Cancer Research (LICR) in Melbourne, and around the turn of this century, managed to develope the highly basic cell-permeable PAK18 peptide derivative called “WR-PAK18” to block the PAK1-PIX interaction in living cells (13). Interestingly, this peptide inhibited RAS-induced malignant transformation of normal cells with IC50 around 5 micro M, without any effect on normal cell growth (13). This peptide provided the very first biochemical evidence for PAK1-dependency of RAS-based cancers, and opened the “new” era in quest of potent PAK1-blockers for cancer therapy and even longevity, as Charlie found later (around 2007) that PAK1-deficient mutant (RB689) of C. elegans, a tiny worm, lives 60% longer than the wild-type worms (16)!
PAK1-inhibitors: CEP-1347 and Merlin
Around the beginning of 1998, two interesting articles were published side by side (17, 18 but in different journals). One from Cepharon, suggesting that a chemical compound called CEP-1347 blocks a kinase called JNK which is responsible for PD (Parkinson’s Disease). The other from University of Washington (Seattle), proving that JNK is activated by PAK1. Charlie immediately suspected that the direct target of CEP-1347 is not JNK but PAK1. His team eventually proved this notion in 2002 (19). Furthermore, in 2004, his team identified that an NF2 gene product called “Merlin” also inhibits PAK1 directly (20). Since NF2 tumors in brain is caused by dysfunction of Merlin, in theory CEP-1347 could be a cure for NF2, in addition to PD. Unfortunately, however, clinical trials of CEP-1347 for PD was suddenly terminated at phase II, and this compound has never been marketed. Thus, his dream on NF2 therapy by CEP-1347 was suddenly evaporated! That is the reason why he suddenly switched his primary project to “herbal” PAK1-blockers for therapy of pancreatic cancers and NF (neuro-fibromatosis) for which no effective drug was available by then. The first herbal PAK1-blocker that he identified was in ethanol/warm water-extract of Sichuan pepper called “Hua Jiao” (21) which has been used as an essential seasoning for preparation of “Marba-Tofu” (bean curd) soup over 1000 years in China. Unfortunately, however, his ”herbal” project was not so popular within LICR which prefers “patentable” anti-cancer chemical compounds or monoclonals.
Propolis as PAK1-blocker
Thus, Chalie decided to retire from LICR in 2006, in order to keep working on PAK1-blocking herbs at NF clinical research group of UKE (Hamburg University Hospital) in Germany as a visiting professor with a DFG scholarship, and found that a bee product called “propolis” and its major ingredient called CAPE (caffeic acid phenethyl ester) are PAK1-blockers that suppress both growth and metastasis of NF tumors and even terminal pancreatic cancers clinically, and extend the healthy lifespan of C. elegans at least 30% (22). CAPE-rich propolis in particular Bio 30 (alcohol-free liquid) from New Zealand turned out to be very useful for the non-invasive therapy of NF tumors in brain as well (23). Since these PAK1-blockers suppress inflammation in general and stimulate our immune system (24, 25), they would be very useful for treating even COVID-19 patients suffering from fibrosis (inflammation in lungs) without any side effect (26). In addition to propolis, the 2015 Nobel-winning antibiotic called “Ivermectin” was identified as a PAK1-blocker by Charlie in 2009 (27), and has been proven clinically world-wide to reduce the death rate of COVID-19 patients in ICU from 20% to 3% (28)!
A Short List of PAK1-dependent Diseases/disorders
Since then, a series of genetic or biochemical studies have proven that PAK1 is responsible not only for solid-tumorgenesis, inflammation, bacterial or viral infection, but also for a wide variety of other diseases such as AD (Alzheimer's disease), PD, epilepsy, autism, depression, schizophrenia, hypertension, diabetes (type 2), obesity, hair-loss, melanogenesis, immuno-suppression, and eventually shortening lifespan.
Hibiscus Flowers for Longevity
Among these PAK1-blockers, Charlie recently learned an intrigue lesson from Hibiscus flowers. Back in 1835, Charles Darwin, the father of evolution theory by natural selection, found a charming 5 years old female giant turtle called “Harriet” in Galapagos Islands, and on his way back to England he stopped by Australia, celebrating his 27th birthday, and donated Harriet to Queensland Zoo in Brisbane. She survived 176 years in good health, and passed away peacefully there in 2006. Interestingly among her favourite diets were red Hibiscus flowers (29). Thus, Charlie and a few other curious fellows suspected that Hibiscus flowers might contribute to her “exordinary” longevity. In 2020, a German team indeed proved this notion: C. elegans fed with Hibiscus flower extract lived 30% longer than the control non-treated worms (30). This “red” extract (tea) contains anthocyanin and a few other PAK1 blockers (31).
“Chemical Evolution” of PAK1-blockers
Although a wide variety of natural PAK1-blockers are currently awailable in the market rather cheaply, many of them are poorly cell-permeable, mainly due to their COOH moiety. Thus, in 2015, Charlie organized a special PAK research team at Okinawa in order to conduct a “chemical evolution” of these natural or synthetic PAK1-blockers via a rather simple organic chemistry called “Click Chemistry” (CC) originally invented by Prof. Barry Sharpless (2000 Nobel laureate in Chemistry) at MIT in 2000. Firstly, two natural PAK1-blockers in propolis carrying COOH, Artepillin C (ARC) and Caffeic Acid (CA), were esterized by 2-azidoanisole via CC, being converted to 1,2,3-triazolyl esters called 15A and 15C. The cell-permeability of these esters is 100 and over 400 times higher than ARC and CA, respectively (32). Finally, an old PAK1-blocking synthetic pain-killer called Ketorolac carrying COOH was esterized via CC in a similar manner, and its cell-permability was boosted over 500 times (23). The resulting ester called “15K” suppressed almost completely both growth and metastasis of chemo-resistant human pancreatic cancer xenografted in mice even at a tiny daily dose as 0.1 mg/kg, and even 5 mg/kg it caused no side effect (34)! Eventually he managed to create a far more potent analog (isomer) of 15K called "15X" via an old fashioned alkyne-azido cycloaddition (AAC) called "Huisgen Reaction" with heat (instead of Cu ion) in 2021.
PAK1-blockers for Surviving the “Global-warming”
Again 15K (even at 10 nM) extended the healthy lifespan of C. elegans by 30% at 20oC, and boosted 10 times its heat-resistance at 35oC (35). Since we are currently suffering from so-called “global warming” mainly due to an excess of CO2 emmision from over-populated human society on this limited planet, Charlie predicts it most likely that 15K and many other PAK1-blockers would serve as the major effective means for “survival of the fittest” by natural/climatic selection…
Find Your Own “Everest”!
Far back towards the end of May in 1953, when Charlie was still forced to stay at home for his recovery from TB, his father showed him a newspaper article telling that Sir Edmund Hillary from New Zealand and Tenzin Norgay from Nepal managed to reach the very top of Mt. Everest (8850 m high) for the first time (36)! Then Charlie was encouraged by his father to find his own “Everest”, a "virgin" peak, which has never been conquered by anyone else. More than 6 decades later, the moment when Charlie managed to develope the extremely potent PAK1-blocker “15X”, he got a feeling that he finally conquered his own “Everest”!
2019 PAK Symposium (25th anniversary) in NYC
Back on November 6-9, 1998, Charlie organized and chaired a New York Academy of Sciences–sponsored international symposium on “Anti-Cancer Molecules: Structure, Function and Design” at Rockefeller University in NYC. Based on this experience, over two decades later, on October 12, 2019, celebrating 25th anniversary of the first mammalian PAK discovery, Charlie organized the first international PAK symposium, again in NYC. This symposium was entitled “Pathogenic roles of PAK1 including oncogenesis and ageing”, and chaired by Charlie and Ed Manser. Around ten speakers were invited around the world by PAK Research Foundation (37):
The Ultimate Dream: ST-3009
Back in 2001, Charlie used to work on a staurosporine (ST) derivative called ST-2001 derived from a marine organism with a German marine biologist in Guam Island, which is structurally related to CEP-1347 (with IC50 around 1 micro M against PAK1), but its pentose ring is replaced by hexose ring, and hydroxylated at position 3 of indolocarbazole ring. The antibiotic ST was originally isolated by Prof. Satoshi Omura (2015 Nobel laureate) at Kitasato Institute around 1977, but is a non-specific kinase inhibitor with IC50 around 50 nM. Interestingly, Charlie found that ST-2001 is far more potent against PAK1 and many other kinases with IC50 around 1 nM (19)! Furthermore, if it is further modified at position 9 of indolocarbazole ring (as is CEP-1347) with a bulky side chain or Arg, in theory, the resultant compound called ST-3009 would be highly specific for PAK1 (19). Unfortunately, this specific marine organism suddenly disappeared around the coast of Guam Island (probably due to the “global-warming”) and chemical hydroxylation of ST only at position 3 or 9 is very difficult (with a very low yield!). Usually both positions 3 and 9 would be simultaneously hydroxylated, and its anti-kinase activity is totally lost! Thus, Charlie planned to create ST-3009, first by (i) hydroxylating both positions, then (ii) esterizing with Arg, and finally (iii) cleaving one of the Arg ester bonds with a “stereo-specific” esterase (38). The "specific" reason for using Arg as the bulky side chain is to boost its "cell-permeability". For this “ST-3009” project, he needs a million dollars to purchase the very expensive ST from Kitasato Institute or Novartis, and he just hopes/ dreams that marketing of “15K” would eventually provide him with such a capital, some days in the future…
References:
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