Background: In mammals, adrenaline and ATP are life-essential vicinal diol and cis-diol functional groups. Here, we show that interactions between a safe organogermanium compound and these cis-diol compounds have the potential to regulate physiological functions. In addition, we represent a possible new druggable target for controlling the action of cis-diol compounds. Results: We analyzed a single crystal structure of organogermanium 3-(trihydroxygermyl)propanoic acid (THGPA), a hydrolysate of safe Ge-132, in complex with catecholamine (adrenaline and noradrenaline), and evaluated the affinity between several cis-diol compounds and THGPA by NMR. An in vitro study using normal human epidermal keratinocytes was performed to investigate the inhibition of cis-diol compound-stimulated receptors by THGPA. At high concentration, THGPA inhibited the calcium influx caused by adrenaline and ATP. Conclusion: This study demonstrates that THGPA can modify cis-diol-mediated cell-to-cell signaling.
The element germanium belongs to the group 14 carbon family and generally exhib-its 4 covalent bonds when incorporated into chemical compounds. Germanium is a met-alloid and can act as a semiconductor. The inorganic germanium compound GeO
2
exhibits an erythropoietic effect, as reported by Hammett and Nowrey [1] and Ham-mett et al. [2] in 1922. However, inorganic germanium can be hazardous. In the 1990s, GeO
2 erroneously sold as organogermanium
led to deaths by renal toxicity [3]. Sanai et al. described the toxicity of GeO
2 and deemed
Ge-132, poly-trans-[(2-carboxyethyl) ger-masesquioxane], safe to consume [4]. Exces-sive GeO
2 intake leads to lethal nephropathy
via accumulation in the nephrons of kidneys. Limited amounts of GeO
2 have physiological
functions, while an abundance can be lethal.Ge-132 is a water-soluble organogerma-
nium compound that has been deemed safe in multiple toxic evaluations [5]. An acute toxicity test in male dogs revealed an LD50 of 8500 mg/kg bodyweight. Ge-132 affects multiple physiological systems, including the immune response, and has anti-inflam-
matory, antiosteoporosis and antihyperten-sive effects, among others [5–10]. Our group previously revealed that Ge-132 induces antioxidant production and promotes heme synthesis [11,12]. Ge-132 was developed for medical use and has undergone multiple clinical evaluations. However, more recently, it has been categorized as a food supplement in Japan. Thus, Ge-132 is now mainly used to supplement food and cosmetics in Japan, China, Korea and USA.
When Ge-132 is used as a medical cream for peripheral burn care, pain decreases very quickly after treatment. Therefore, we were interested in the action of Ge-132 in the peripheral pain signaling system. Although the nociceptive effect of Ge-132 has been described using the acetic acid writhing method [13] and the tail–flick method [14] in mice, the mechanism of this pain inhibition remains unclear. In the recent literature, an extracellular ATP, a cis-diol compound, is well known as a nociceptive factor that can cause pain in the peripheral and central nerve systems [15]. When skin tissue is damaged by a peripheral burn, large quantities of ATP are
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Research Article Nakamura, Shimada, Takeda, Sato, Akiba & Fukaya
leaked from the injured cells. We believe that the inter-action between Ge-132 and leaked ATP can suppress the pain signal resulting from the burn.
Germanate interacts with d-fructose to promote the isomerization of d-glucose to d-fructose [16,17]. Recently, we reported an interaction between Ge-132 and certain monosaccharides [18]. GeO
2 also reacts with
phenylfluorone, which contains a catechol skeleton, and this reaction is employed in GeO
2 analyzes [19].
Complex formation between GeO2 and catechol com-
pounds has also been detected [20–22]. Furthermore, phenylfluorone can be used to detect Ge-132. Thus, Ge-132 can be chelated by catechol and other cis-diol compounds. Catechol compounds have important functions in humans. Specifically, some catechol-amines act as neurotransmitters or hormones [23–27]. Previously, Ge-132 was found to relieve the suppres-sive effect of catecholamines against intestinal peristal-sis [28]. This effect might be due to the formation of a chelate between Ge-132 and catecholamines. Cis-diol element-containing compounds are very important in mammals, particularly as components of some neu-rotransmitters. Several separation technics for cis-diol biomaterials have recently been reported based on a similar interaction by some organoboronates [29–31]. Every purified cis-diol biomolecule has been shown to be important to life. Therefore, we predict that Ge-132
can regulate the function of neurotransmitters and hormones possessing cis-diol structures.
In this study, we evaluated chelate formation between Ge-132 and adrenaline, the most well-known catecholamine. Crystals of the chelate complex were prepared, and structural analyzes were performed by X-ray diffraction and NMR. The rate of chelate forma-tion between Ge-132 and several cis-diol compounds, such as catecholamines (accurately vicinal diol; how-ever in this study we also express about chatechol as cis-diol) and nucleotides, was studied. We also evaluated the effect of Ge-132 on the application of adrenaline and ATP to human skin keratinocytes. Here, we dem-onstrate that the chelation of cis-diol neurotransmit-ters may regulate homeostasis and neurotransmission through the adjustment of receptor binding activity. This is the first report of a direct reaction between an organogermanium compound and active biological compounds based on structural information.
ResultsComplex of organogermanium with catecholamineGe-132 is a water-soluble organogermanium com-pound and is a polymer of 3-(trihydroxygermyl)pro-panoic acid, THGPA (Figure 1). THGPA can interact with cis-diol structures through its trihydroxy groups, and here, we evaluated the ability of THGPA to inter-act with several physiological cis-diol compounds. First, we evaluated the crystal formation of THGPA, a safe form of organogermanium, which possesses a trihydroxy structure, and adrenaline complexes. Equi-molar aqueous solutions of THGPA and adrenaline were combined. The resulting mixture had a pH of ∼4. Other solutions were pH adjusted with NaOH or HCl and did not form crystals of the desired complex. At lower pH values, as adjusted with HCl, only Ge-132 crystals were formed. The crystals of the THGPA and adrenaline complex were planar and colorless (Supplementary Figure 1). We speculated that THGPA and adrenaline were dehydrolyzed at their hydroxyl moieties. Moreover, we predicted that a pentacoordi-nate structure had formed via lactone ring formation (Figure 1). Crystals of THGPA/dl-adrenaline and THGPA/l-noradrenaline were analyzed using X-ray diffraction, and their structures were determined (ORTEP in Figure 2A–D). The molecular formulae of the two complex compounds are C
12 H
17Ge N O
6
(Mw. 343.9) and C11
H15
Ge N O6 (Mw. 329.9), respec-
tively. Dehydration resulted in bonding between the THGPA and adrenaline molecules. Two hydroxyl moi-eties from each compound formed a ring composed of C–C–O–Ge–O–. Moreover, two H
2O molecules
were formed (Figure 2B & D). Note that the complex
Key terms
Ge-132: Water-soluble organogermanium compound poly-trans-[(2-carboxyethyl)germasesquioxane](IUPAC name) is known by several names. In addition, 2-carboxyethylgermanium sesquioxide has been used in previous research reports. Ge-132 is one polymer form of (3-trihydroxygermyl)propanoic acid. Another polymer type is known as propagermanium, which differs from Ge-132 in function and use. Propagermanium is used as a drug component for treating chronic hepatitis. Repagermanium is the name of Ge-132 as a cosmetic use.
Cis-diols: Two vicinal surface OH groups are oriented in the same direction from a plane of bonding carbon in a ring structure. Sugars possess many cis-diol structures (and/or trans-diol structures). However, the hydroxyl groups of catechol and catechin are located on the flat plane of the benzene ring plate. For convenience in this article, we also defined these flat diol structures as cis-diols.
Hypervalent : In addition to transition metals, other types of atoms can possess extraordinary electron orbitals. In general, small atoms can form from one to four chemical bonds. However, large atoms with extraordinary orbitals can bind with more than five atoms. A molecule that contains these types of high coordinate bonds is known as a hypervalent coordinate molecule. In the case of organogermanium compounds, the molecule, which contains a germanium atom, can form between five and seven coordinate bonds. Such molecules are known as penta-, hexa- and hepta-coordinated germanium compounds, respectively.
Figure 1. Structural scheme of 3-(trihydroxygermyl)propanoic acid/dl-adrenaline complex. A schematic illustration of complex formation between 3-(trihydroxygermyl)propanoic acid (THGPA) and adrenaline is shown. The germanium atom has a vacant d-orbital, and we predicted that the complex compound would have a lactone ring.
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Ge-132 forms complexes with physiological cis-diol compounds Research Article
atoms were hypervalent (pentacoordinate). The propanoic group originating from THGPA formed a lactone ring (butyrolactone) and formed a coordinate bond with the vacant d-orbital of germanium, result-ing in the formation of a pentacoordinate germanium (V) atom. We speculated that a proton originating from the lactone ring might be found neighboring the germanium atom (Figure 1); however, the proton was actually bound to the nitrogen atom (Figure 2A & C). These data suggest that these complex molecules may have an electric polarization within each individual compound because germanium is negatively charged by the formation of a lactone ring; in addition, the protonation to nitrogen gives the germanium a posi-tive charge. The THGPA and dl-adrenaline com-pounds formed a complex with reverse chirality (d-form and l-form), found in an antiparallel posi-tion. This might originate from the polarization described above. The complex compounds lie in two layers (Supplementary Figure 3A), based on stacked benzene rings (Supplementary Figure 3B) that might form pi bonds. The distance between the two planes is 3.345 Å. For the complex formed by THGPA and l-noradrenaline, the 3D configuration of two complex molecules was twisted (Figure 2D). Two H
2O molecules
were observed per complex molecule.The IR spectra of the THGPA and adrenaline crys-
tals were measured by the KBr method. The spec-trum of the pentacoordinate compound was differ-ent from the spectra of the two original compounds (Supplementary Figure 4A). The strong absorbance at 800 cm-1 that originated from Ge–O–Ge in Ge-132 did not exist in the newly formed complex. Moreover, the strong absorbance at 1686 cm-1 that originated from C=O of a carboxyl radical in Ge-132 was shifted to 1622 cm-1. The shift may be related to the formation of a lactone ring.
The crystals of THGPA and adrenaline were soluble in methanol. However, their solubility in water was very low at room temperature. The dehydration of four hydroxyl moieties in the two original compounds led to a reduction of hydrophilicity. Therefore, structural studies and stability evaluations were conducted by dis-solution in D3-methanol. The 1H- and 13C-NMR CH-COSY spectra are shown in Supplementary Figure 4B. These signals did not change after 2 or 4 months, suggesting that this complex is stable at ambient conditions.
Affinity between organogermanium & catecholamine & mono nucleic acidUsing NMR, we investigated the interactions between THGPA and other physiologically relevant cis-diol compounds. At physiological pH (∼7.4–7.6), THGPA
and adrenaline exist as a mixture of the complex and its parental molecules. The NMR spectrum of THGPA and adrenaline in deuterium oxide (D
2O) is shown in
Figure 3A. The red signals in the proton NMR analysis originated from adrenaline, and the blue signals origi-nated from THGPA. Upon formation of the complex, the chemical shifts resembled the original shifts but in different environments. The signals of complex com-pounds are typically broader. The methylene neigh-boring the germanium gave three triplet signals at 1.7, 1.5 and 1.3 ppm that corresponded to complex a, the original and complex b, respectively. The ratio of the areas of these signals can be used to quantify com-plex formation. Many important cis-diol compounds exist that can interact with THGPA (Figure 3B). The affinities between THGPA and some cis-diol com-pounds are shown in Figure 3C. The complex forma-tion ratio was very high for the catecholamines, where noradrenaline had the highest, followed by adrena-line. The complex formation ratios of the ribose com-pounds adenosine and ATP were lower than those of the catecholamine compounds. Lower concentrations decreased the resulting amount of complex forma-tion. Due to the low concentrations of THGPA in our bodies, oral intake of Ge-132 is safe, although our experiments confirmed that interactions between THGPA and cis-diol compounds can occur. We uti-lized the inactive form of the compound 3-(trimeth-ylgermyl)propanoic acid (TMGPA) for affinity evalua-tion (Supplementary Figure 2). However, TMGPA did
OH
Adrenaline
THGPA
Vacant d-orbital
NH
OH
OHGe
OH
HO
HOOH
O
-2 H2O
GeO
OH
NH
O
O OOH
H+
-
Figure 2. ORTEP diagram of 3-(trihydroxygermyl)propanoic acid/catecholamine complexes. (A) ORTEP diagram of the crystal obtained from a mixed solution of THGPA and dl-adrenaline. The structure of one molecule is drawn, and the X-ray analysis revealed the lactone ring and NH2. (B) The complex compound had two crystallized water molecules. The symmetry of the complex compounds resulting from D formation and L formation is shown. (C) ORTEP figure of the crystal obtained from a mixed solution of THGPA and l-noradrenaline. (D) 3D diagram of the THGPA and l-noradrenaline complex compounds. The configuration of the two complex molecules was twisted. Two water molecules were present per complex molecule. THGPA: 3-(trihydroxygermyl)propanoic acid.
1236 Future Med. Chem. (2015) 7(10) future science group
Research Article Nakamura, Shimada, Takeda, Sato, Akiba & Fukaya
C6
C9C8
C10
C11C12
C7
C6
C5
C1C2
C3
C4
05
0102
03
04N1
Ge1
C4
C4
C4
C5C3
C2
C1
C5
C5
C6
C6
C8
C8
C9
C7C606
C8
C9
C9
C7
C7
06
C10
C10
C10
C11N1
01S
C11
C11
C12
C2
C3
C1
C2
C3
C1
03
03
04
04
0405
02
03
01
01
01
05
05
02
06Ge1
Ge1
Ge1
01S
N1
N1
02
A
B
C
D
Figure 3. Complex formations of 3-(trihydroxygermyl)propanoic acid with cis-diol compounds. (A) NMR spectrum of THGPA and dl-adrenaline in D2O. The pH was adjusted to 7.4 with NaOD. The signals revealed that a mixture of complex compound and parental compounds was present. The complex formation rate was calculated from the ratio of the areas of the complexed and free compounds. (B) The complex formation pattern of THGPA and the cis-diol compound. Typical biological cis-diol compounds are aligned. (C) The ratio of complex formation between THGPA and four types of biological cis-diol compounds is shown. Noradrenaline exhibits a high affinity for THGPA. However, TMGPA did not exhibit any complex formation with ATP. AD: Adrenaline; Ado: Adenosine; NA: Noradrenaline; THGPA: 3-(trihydroxygermyl)propanoic acid.
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Ge-132 forms complexes with physiological cis-diol compounds Research Article
Figure 4. Inhibition of calcium signaling by cis-diol binding by 3-(trihydroxygermyl)propanoic acid in NHEK cells. (A) The gene expression level of purinergic receptor subtypes in NHEK cells was evaluated by quantitative PCR. (B) The suppressive effect of THGPA on the signaling of adrenaline and ATP by human keratinocytes, NHEKs, was evaluated. A calcium ion indicator was incorporated into the cells, and calcium ion influx was measured by the acceptance of adrenaline and ATP. (C) NHEKs incorporated fluo-8AM responded to ATP stimulation. (D) Calcium ion influx was suppressed by the addition of THGPA. (E) The calcium ion influx triggered by adrenaline was suppressed with THGPA treatment. A high concentration of THGPA (5 mM) yielded significant suppression. (F) The calcium ion influx triggered by ATP was suppressed by THGPA treatment. A high concentration of THGPA (10 mM) suppressed calcium ion influx significantly. All data are displayed as mean ± SEM and were analyzed using ANOVA and Tukey’s test. *p < 0.05; **p < 0.01. ANOVA: Analysis of variance; NHEK: Normal human epidermal keratinocyte; SEM: Standard error of the mean; THGPA: 3-(trihydroxygermyl)propanoic acid.
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Research Article Nakamura, Shimada, Takeda, Sato, Akiba & Fukaya
1.5
1.0
Exp
ress
ion
rat
io a
gai
nst
Act
b
0.5
N.D
Purinergic receptor subtype
ATP addition 0 s
Adrenaline ATP
60 120
0 30 60
THGPA
Ca ion influx
Control (without THGPA)
90 120 150 180
100
80
60
40
20
0
** ***
(n = 6) (n = 8)
40
20
00 mM
THGPA2.5 mMTHGPA
5 mMTHGPA
0 mMTHGPA
1 mMTHGPA
10 mMTHGPA
Incr
easi
ng
in fl
uo
resc
ence
(%
)
Incr
easi
ng
in fl
uo
resc
ence
(%
)F
luo
resc
ent
inte
nsi
ty
Time (s)ATP addition 15 s
0.0p2x3 p2y1 p2y2 NHEK(F) cell
Medium+
THGPA(0 to 10 mM)
Adrenaline or ATP
Ca2+
A B
C D
E F
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Ge-132 forms complexes with physiological cis-diol compounds Research Article
not form complexes with ATP at a high concentration (100 mM), suggesting that the trihydroxy form is essential for this interaction.
Inhibition effect of THGPA addition for calcium ion influx by cis-diolsNormal human epidermal keratinocytes (NHEKs) have β-adrenergic receptors and P2X2/3 recep-tors. These receptors are G-protein coupled recep-tors and Ca2+ ion channels, respectively. It is known that NHEKs also contain P2Y1 and P2Y2 receptors, which are also categorized as G-protein coupled recep-tors. We evaluated the expression levels of P2 recep-tors. The mRNA expression levels of P2X3, P2Y1 and P2Y2 in NHEKs in normal culture conditions are shown in Figure 4A. P2X3 was not detected, whereas the expression of P2Y1 and P2Y2 was confirmed. The cell response to adrenaline and ATP administration was monitored with the calcium ion indicator Fluo-8 AM. To evaluate the effect of THGPA, the influx of calcium ions triggered by either adrenaline or ATP was measured (Figure 4B). Calcium influx by ATP addition to NHEKs was visualized and is shown in Figure 4C. The cells exhibited green fluorescence after ATP addi-tion. The addition of adrenaline to the cells led to an influx of calcium ions, and THGPA inhibited this response (Figure 4D). Treatment with 1 μM adrena-line triggered calcium ion influx into NHEK cells. The response times of calcium influx after the addi-tion of ATP and adrenaline were different. After the ATP addition, 15 s was a sufficient response time for emission (Figure 4C). However, the addition of adrena-line required a longer response time, and the maximal lighting by calcium influx was observed from a half minute to 1 min later (Figure 4D). THGPA inhibited the fluorescence intensity in a dose-dependent manner (Figure 4E). The percentage increases in fluorescence for the addition of 0, 2.5 and 5 mM THGPA were 41, 27 and 13%, respectively. Calcium ion influx was significantly inhibited by 5 mM THGPA (p < 0.01). Similar results were observed with the addition of ATP (Figure 4F). The increased fluorescence values obtained with THGPA additions of 0, 1 and 10 mM were 97, 75 and 32%, respectively. THGPA suppressed the cal-cium ion influx activated by ATP in a dose-dependent manner, and the difference between 0 and 10 mM was significant (p < 0.01). The difference between 1 and 10 mM was also significant (p < 0.05). THGPA sup-pressed the cell response to the acceptance of cis-diols at the high concentration.
DiscussionThis study supposes novel targets for the control of cell–cell communication by cis-diol compounds.
We confirmed that organogermanium can form com-plexes with important cis-diol compounds, including hormonal compounds such as adrenaline, noradrena-line and ATP. Today, many current drugs of clinical value are used to target receptors. Furthermore, most drugs act by blocking binding sites, thus acting as antagonists. Many cis-diol compounds have impor-tant roles in human physiology, including neurotrans-mitters such as catecholamines, ATP and adenosine, hormones such as catecholamines, genetic informa-tion such as RNA and coenzymes such as NAD and NADP (Figure 3B). These molecules share common fundamental structures such as catechol and ribose. Catecholamines are known as stress hormones, and they are released from the adrenal gland during psy-chological stress; moreover, an induction of hypergly-cemia by adrenaline has been reported [32]. Recently, the structures of β-adrenergic receptors bound to ago-nists were revealed [33]. Similarly, ATP is one of the most important components for life and is known as a biological energy compound [34,35]. However, the extracellular concentration of ATP is extremely low. Purine receptors have many roles, and some of these receptors sense the release of purines from punctured cells during pain signaling. ATP can act as a signal for cell damage. Extracellular ATP serves as a nocicep-tive signal in peripheral and central neuronal signaling through interactions with P2X2/3 and P2X3 or with P2X4, respectively [15,36–39]. Notably, the functions of both catecholamine and ATP are related to stress, and organogermanium, Ge-132, can interact with cis-diol compounds that have stress-related physiological func-tions. In general, a receptor is depolarized by ligand binding. Therefore, an excess of ligand compounds decreases the intensity of the cellular response. Hence, spontaneous stress and cell damage may decrease the ability of the cell to respond. We evaluated interac-tions between THGPA and cis-diol compounds and whether ligand trapping by complex formation with THGPA suppressed binding and cell response. We crystallized the complexes of THGPA with adrenaline and noradrenaline (Figure 2). The crystals contained pentacoordinated germanium with a lactone ring. Ger-manium compounds have a carboxyethyl moiety that
Key term
Cell–cell communication: Multicellular eukaryotes form complex organisms based on many cell types. The cells rely on many kinds of signals to inform each other of their various conditions. The cell-to-cell signal ligand binds to receptors on the cell surface, and the signal transits to another signal molecule within the cytoplasm, allowing the cell to respond to the outside condition. The cells utilize crosstalk for communication with each other, where ligands are used as signal molecules and specific receptors.
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Research Article Nakamura, Shimada, Takeda, Sato, Akiba & Fukaya
can form a lactone ring with pentacoordinated germa-nium in the formation of complexes. Moreover, if elec-tron-donating groups exist on the delta position of the organogermanium compound (e.g., lactamo-N-methyl ligands), a cyclic structure with pentacoordinated ger-manium can form. NMR analyzes in deuterium oxide (Figure 3A) suggest an equilibrium state between tetra-coordinate (THGPA) and pentacoordinate (THGPA/catecholamine complex). However, X-ray analysis revealed that complexes with cis-diol structures only have pentacoordinated atoms. Therefore, we speculate that triols bonded to germanium are close to a diol when the pentacoordinate is formed (Figure 2). The distances of triols are estimated at 2.78–2.95 Å for the tetracoordinate. However, the distance of cis-diol for the THGPA/adrenaline complex was 2.57 Å in this study, which is a shorter distance than in the other compounds. Therefore, the pentacoordinate position may be suitable for this short distance.
These types of complexes are usually hydrolyzed under hydrophobic conditions. Therefore, an equi-librium state exists between the complex and sub-strates. If the complex formation is extremely strong, the signal transduction of the adrenaline and ATP ligands is reduced by Ge-132 intake. However, this equilibrium may simulate a decrease in ligand con-centration. Although neurotransmitter and hormone signal transductions are essential molecules, an excess of these signals is not conducive to normal cell func-tion. In this case, Ge-132 may change the apparent signal concentration in the receptors. In this study, we mainly focused on adrenaline and ATP. Previous reports indicate that Ge-132 can relieve the effect of catecholamine in the intestine [28]. Moreover, Ge-132 is useful for pain relief, and Ge-132 decreased pain in terminal cancer patients in double-blind clinical tri-als [40]. Extracellular ATP, adenosine, and catechol-amine function in central or peripheral pain signal transduction or nociception [38,41–44]. Furthermore, in our previous study, THGPA suppressed a nociception threshold mediated P2X purinergic receptor by ATP signaling in mice (unpublished data). These facts suggest that interactions between THGPA and these compounds are involved in this pain relief. The inter-
action of THGPA with cis-diol compounds is of great interest because many kinds of cis-diol compounds are essential in mammals.
THGPA is a water-soluble compound and may not be readily incorporated into intercellular compart-ments. Therefore, a higher proportion of THGPA may be present in the extracellular space than in the intracellular fluid. For biological application, we dem-onstrate the inhibition of adrenaline and ATP binding to the receptors in Figure 4E & F. In this study, NHEKs expressed both P2Y1 and P2Y2 receptors in normal cul-ture conditions. Mechanical stimulation evokes Ca2+ waves mediated by ATP signaling through P2Y2 recep-tors, and Koizumi et al. reported that these Ca2+ waves, as pain signals, are transmitted to sensory neurons [45]. Therefore, THGPA may suppress this Ca2+ wave sig-naling via ATP to peripheral neurons. In reality, the acute pain of a burn wound is blocked by the applica-tion of THGPA. A similar inhibition is expected for other cis-diol compounds in the extracellular space if THGPA is distributed within the area at high concen-tration. The receptors for catecholamine and purines are rich in variety and can be found in multiple cell types. For example, adrenergic receptors α1, 2 and β1–3 and purine receptors P2X1–12 and P2Y1–13 have been known. These subtypes have different functions against the same compounds. When the cell membrane is harmed, ATP leakage occurs. Similarly, psychologi-cal or cold stress triggers the release of catecholamine. THGPA binds these cis-diol compounds and suppresses their binding to receptors. Ho et al. have reported that the intraperitoneal administration of Ge-132 lowered the mean arterial pressure in a dose-dependent manner in rat [46]. They supposed that Ge-132 acts by mediat-ing the catecholamines in the brain. In this study, we evaluated Ge-132 by using the NHEK model; how-ever, the same mechanism will be applicable to vas-cular smooth muscle cells. We speculate one aspect of Ge-132’s mechanism cis-diol compound mediation in our body, illustrating this concept schematically in Figure 5. Peripheral pain due to leaked ATP from injured cells is suppressed by THGPA trapping due to equilibrium complex formation. However, the forma-tion of complexes between THGPA and chatechol-amines inhibits vasoconstriction mediating α1 recep-tors. Moreover, it has been reported that noradrenaline enhanced P2X3 expression in dosal root ganglia in rat [43]. In this system, the pain signal mediates two different cis-diol molecules (noradrenaline and ATP), and both of them can interact with THGPA; therefore, THGPA treatment of peripheral skin may be beneficial for peripheral nociceptive pain. In addition, complex formation with adenosine, a metabolite of ATP, may inhibit its metabolism to inosine by adenosine deami-
Key term
Purinergic receptors: Extracellular ATP occurs in very low concentrations in extracellular spaces, and when it leaks from cells, the molecule plays a role in danger signaling. Purinergic receptors of the P2 type are receptors for ATP binding and mediate intercellular ATP signaling. P2 receptors are categorized into two types: the P2X type includes calcium ion channels, and P2Y, as G protein coupled receptors. Many subtypes exist for both of P2X and P2Y.
Figure 5. Image illustrating cis-diol trapping by Ge-132. The Ge-132 monomer, 3-(trihydroxygermyl)propanoic acid, can interact with catecholamines and nucleotides. The suppression of pain by leaking ATP signaling and the inhibition of acute high blood pressure by catecholamines are expected after 3-(trihydroxygermyl)propanoic acid application or intake. EKC: Epidermal keratinocyte; VEC: Vascular epithelial cell; VMC: Vascular muscle cell.
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nase. Multiple Ge-132 physiological functions have been reported, but the mechanism behind its action has not been determined. Differentiated cells have differ-ent receptor subtypes, and the same ligand can lead to different functions. Thus, the interaction of THGPA (Ge-132) with various cis-diol compounds may affect homeostasis, thereby explaining some of the functions of Ge-132. We anticipate that interactions with cis-diol compounds might be important. Different compounds can interact with cis-diol compounds, for example, such as phenyl borate and GeO
2. One borate compound,
Bortezomib, is used to inhibit the proteasome in mul-tiple myeloma. Recently, the inhibition of Bortezomib activity by epigallocatechin gallate was reported [47].
It has been suggested that the affinity for epigallocat-echin gallate can be explained by its interaction with cis-diols in the body. However, Bortezomib exhibits several severe side effects, and some boronic acids and GeO
2 are also reportedly toxic. Therefore, the affinity
of borate or GeO2 for cis-diols may be too strong for
use in physiological applications. THGPA has an equi-librium interaction with cis-diol compounds in hydro-philic conditions (in water). This equilibrium is shown as different spectra of 1H-NMR for the THGPA and adrenaline complex in Supplementary Figure 4B (part of CH COSY in D3-methanol) and Figure 3A (in D
2O).
The proton signals from 1.5 to 2.5 ppm originated from THGPA and were detected as different triplet signals
Terminalsensoryneuron
Cell injury
Pain signal
VMC
VEC
Leaked ATP
Layer of keratin
EKCEpiderm
O
O
ATP
ATP
Ge
P2X3
P2Y2
BloodCA GeInhibit
vasoconstriction
α1
O
O
C6
C9 C8
C10
C11C12
C7
C6C5
C1C2
C3
C4
05
0102
03
04 N1
Ge1
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Research Article Nakamura, Shimada, Takeda, Sato, Akiba & Fukaya
related to either free or complexed THGPA (Figure 3A). This hydrolysis equilibrium condition is related to the safety of Ge-132. THGPA possesses a second type of polymer structure, propagermanium, relative to Ge-132. Propagermanium is a CCR2 antagonist and is used as a drug for the treatment of chronic hepatitis. Recently, Yumimoto et al. revealed a cancer metastasis mechanism and the strong inhibition of cancer metas-tasis via propagermanium intake [48]. Although the structural mechanism of this inhibition has not been demonstrated, the interaction between THGPA and biological cis-diol compounds may affect CCR2 inhi-bition by propagermanium.
Although this study suggests that the physiological function of Ge-132 may be related to the interaction with bio active cis-diol compounds, the concentration needed for effectiveness was considerable. Therefore, a micro regional distributional study of THGPA in vivo will be important. Recently, our group has developed a micro analysis method of THGPA with structural information by LC/MS/MS [49]. We expect to carry out further studies on the affinity of THGPA and cis-diol compounds in detail using this LC/MS/MS anal-ysis method. In addition, we performed NHEK cell analyzes in normal conditions; therefore, a deviation from treatment by the complexes among THGPA and cis-diol compounds would be interesting to study in the future. In microglial cells of allodynia, the expression of P2X4 is very high [50]; hence, the effect of THGPA in such a model should also be revealed in the future.
ExperimentalChemicals & reagentsGe-132 (Lot 006316A; purity over 99%), also known as poly-trans-([2-carboxyethyl]germasesquioxane), was synthesized at the manufacturing plant of Asai Germanium Research Institute Co., Ltd. The synthe-sis procedure for Ge-132 used by the Asai Germanium Research Institute is as follows. Ingots of germanium (purity greater than 99.9999%) were milled in powder. The powdered germanium was reacted with hydrochlo-ride gas at high temperature, and trichlorogermane was produced. Next, the trichlorogermane was reacted with acrylic acid to form THGPA. The obtained 3-(trichlo-rogermyl)propanoic acid was hydrolyzed to THGPA and dried to a polymer, Ge-132. An inactive form of the compound, TMGPA, was synthesized by Toru Yoshihara at Asai Germanium Research Institute Co., Ltd. Adenosine, dl-epinephrine (adrenaline), l-nor-adrenaline and l-dopa (3-(3,4-dihydroxyphenyl)-l-alanine) were purchased from Wako Pure Chemical Industrial Co., Ltd. (Osaka, Japan). ATP was pur-chased from Oriental Yeast Co., Ltd. (Tokyo, Japan). D4-methanol and deuterium oxide (D
2O) were pur-
chased from Merck, and D3-methanol was purchased from Isotec Laboratories, Inc. (Champaign, IL, USA).
Preparation of the adrenaline & Ge-132 chelate complex crystalEqual moles each of adrenaline or noradrenaline and THGPA (0.5 mol of Ge-132) were reacted in 10 ml of pure water at room temperature. The solution was incubated at 4°C for several days. Single crystals began to form after half of a day. The crystals were observed with an optical microscope, and digital photographs were taken with a Moticam 2000 digital CCD scope (Shimadzu Corporation, Kyoto, Japan). The crystals were removed from the mother liquor, rinsed with pure water and dried. The crystals were subsequently used for structural analysis.
Structural analyzes of the complex crystalX-ray structural analysisA single crystal (∼0.2 mm) was used for X-ray struc-tural analysis with a Smart APEX II (Bruker AXS cor-porations, Billerica, MA, USA), which is a single crys-tal X-ray diffractometer with CCD detection. The data were processed with the APEX II software for reduc-tion and cell refinement. X-ray diffraction analyzes were deposited with the Cambridge Crystallographic Data Centre. Deposition numbers are CCDC- 894300 for Ge-132/dl-adrenaline and CCDC-894301 for Ge-132/l-noradrenaline. Free copies of the data can be obtained via the Cambridge Crystallographic Data Centre website [51] or from the Cambridge Crystal-lographic Data Centre, 12 Union Road, Cambridge, CB2 1EZ, UK (Fax: +44 1223 336033; e-mail: [email protected]).
FTIR analysis of the complex crystalsThe spectra were measured using an IR 4000 spec-trometer (Shimadzu) with the potassium bromide (KBr) method. A tablet was formed by adding 2 mg of pulverized crystals and 0.2 g of KBr to a press. The FTIR spectra were scanned from 4600 to 400 cm-1.
NMR analysis of the complex crystalsProton and 13C-NMR analyzes were conducted at 300 Hz (1H) and 75 MHz (13C) using a Gemini 300 spectrometer (Varian, Agilent Technologies, Inc., Santa Clara, CA, USA). Crystals of the THGPA and adrena-line complex were dissolved in 800 μl of D3-methanol at saturation. The methanol signal was adjusted to 3.4 ppm in the 1H-NMR study. Measurements were acquired at 25°C, with 16 transients for 1H analyzes and 40,000 transients for 13C analyzes. CH-COSY and DEPT analyzes were used to assign each signal. The complex stability was evaluated by the collection of 1H-
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Ge-132 forms complexes with physiological cis-diol compounds Research Article
NMR spectra over time. The assigned δ H signals are reported as follows (refer to Supplementary Figure 4B). A broad triplet at 1.77 ppm corresponds to a methy-lene (C1) neighboring germanium. A sharper triplet at 2.56 ppm confirms a methylene (C2) neighboring the ketone. Because Ge-132 does not dissolve in methanol, we posit that these are specific signals originating from THGPA incorporated into the complex with adrena-line. One other methylene (C11), originally from adren-aline, exhibits a doublet signal at 3.25 ppm. The bond between a methyl (C12) and the amine was detected as a singlet at 2.84 ppm. The hydrogen bonded to C10 was detected as a triplet signal at 4.88 ppm. Two hydrogen atoms bonded to C6 and C9 were detected at 6.75 ppm as a multiplet signal. The hydrogen bonded to C8 was detected at 6.88 ppm as a doublet signal. The assigned signals (refer to Supplementary Figure 4B) of δC are as follows: 16.6 (C1), 30.0 (C2), 33.8 (C12), 57.0 (C11), 70.4 (C10), 110.6 (C6), 112.5 (C9), 117.2 (C8), 131.5 (C7), 150.9 (C4), 151.4 (C5) and 181.2 (C3) ppm. The signals were assigned based on the known chemical shifts of adrenaline from the SDBS spectral database of organic compounds [52] (National Institute of Advanced Industrial Science and Technology, November 13 2014). Changes in spectral patterns were determined by comparing spectra acquired after 2 and 4 months at room temperature without inactive gas.
NMR analysis for affinity evaluationFor the affinity studies, each of adenosine, ATP, dl-adrenaline and l-noradrenaline, and THGPA were evaluated at equal molar amounts (0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250 and 500 mmol/l each) in D
2O. ATP
and TMGPA were also evaluated in D2O at 100 mmol/l
concentration each. For adrenaline, the 250 and 500 mmol/l solutions were too high (insoluble) to analyze. The solution was adjusted to pH 7.4 with NaOD. Mea-surements were acquired at 25°C, with 16 transients for 1H analyzes. The complex formation rates (CFR as%) were determined by the following formula (Equation 1):
CFR (%) = (areas of complex signals) × 100(area of original signal +areas of complex signals)
Equation 1
Application for cellular study using human keratinocytes in vitroGene expression analysis of P2 receptor in human keratinocytesNHEK cells were obtained from KURABO (Osaka, Japan). The cells were cultured in Humedia KB-2 (Kurabo) supplemented with insulin, hEGF, bovine pituitary extract and hydrocortisol. The purinergic receptor expression of NHEK cells was confirmed by quantitative PCR analysis. The cells were cultured in 12-well plates in Humedia KB-2. Confluent cells were lysed with 1 ml of Isogen (Nippon Gene Co., Tokyo, Japan), and then total RNA was extracted using the manufacturer’s protocol. One microgram of extracted total RNA was used as a template for cDNA synthe-sis via reverse transcription with an oligo dT primer and Super Script III (Invitrogen, Carlsbad, CA, USA). The synthesized cDNA was used for quantitative PCR analysis. Quantitative real-time PCR was performed for P2X3, P2Y1 and P2Y2 as target genes and for β-actin (ACTB) as a housekeeping gene. The PCR primer sets for each gene are listed in Table 1. The cDNA was amplified using SYBR Premix ExTaq II (Takara Bio, Ohtsu, Japan) on an Opticon 2 (Bio-Rad Laboratories, Hercules, CA, USA), which was pro-grammed for 95°C for 30 s, followed by 40 cycles of denaturation (95°C for 5 s), annealing and extension (60°C for 30 s). Each expression value was calculated according to the threshold cycle value, and the data are displayed as the ratio of expression of each gene to β-actin.
Calcium influx measurement in human keratinocytesA fluorescent reagent was used to measure the influx if calcium ions into cells. NHEK cells were cultured according to the supplier’s recommendations, as described above. The cells were harvested after treat-ment with trypsin and were prepared as a suspen-sion in culture medium to a final concentration of 6 × 105 cells/ml. Each well of a 96-well cell culture plate (black plate, catalog No.165305, Nunc, Thermo Fisher Scientific K.K., Yokohama, Japan) received 100 μl of cell solution. The cells were cultured for
Table 1. Primer sets for quantitative polymerase chain reactions for P2 receptor expression analysis.
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Research Article Nakamura, Shimada, Takeda, Sato, Akiba & Fukaya
4 days and subsequently used for calcium ion influx measurements in HHBS (1X Hank’s balanced salt solution with 20 mM HEPES buffer, pH 7.0). The calcium indicator reagent Fluo-8 AM (ABD Bioquest, Inc. CA, USA) was added to each well, followed by incubation at 37°C to permit the incorporation of the reagent. After 30 min, the solution was removed, and the wells were rinsed with phosphate-buffered saline. HHBS containing (3-trihydroxygermyl)propano-ate (THGPNa) (final concentration of 0, 2.5 and 5 mM) was added to the cells, which were incubated for 5 min at 37°C. Fluorescence was recorded at 490 nm excitation and 530 nm emission with a micro-injector equipped with an ARVO3 (Perkin Elmer, Inc., Waltham, MA, USA). Each well received 10 μl of adrenaline by injection (final concentration of 1 μM), and fluorescence was recorded each second after injection for 5 min. The effect of sodium THG-PNa addition was evaluated 1 min after the addition of adrenaline. Calcium influx after the addition of ATP was evaluated in the same way. The final ATP concentration was 10 μM, and the concentrations of THGPNa were 0, 1 and 10 mM.
Statistical testsThe data are presented as the mean ± standard error of the mean (SEM). Statistical significance was eval-uated for the in vitro study using a one-way analysis of variance (ANOVA) with Tukey’s multiple com-parisons, and p < 0.01 and p < 0.05 were considered significant for the cell experiments.
ConclusionThe structural details of the interaction between an organogermanium compound and active biologi-cal compounds were presented for the first time in this study. This finding accounts for the numerous physiological functions of Ge-132 while exhibiting low toxicity. Ge-132 is safe, and its effects are not potent. The low toxicity of THGPA may originate from its existence in equilibrium between bound and free ligand states. Moreover, we confirmed the inhibition of receptor binding of adrenaline and ATP by THGPA in vitro. However, the THGPA concen-
trations necessary to achieve significant inhibition were considerably high; therefore, whether effective cis-diol trapping can be achieved by THGPA may be highly restricted. Even so, the complex formed between THGPA and catecholamine may be benefi-cial for antioxidation applications in terms of mela-nin formation via catechol to quinone. Although the complex compound with an unstable catechol struc-ture, the crystals of THGPA/adrenaline were very stable at conventional atmosphere for a few months, and the associated NMR spectrum did not change.
Future perspectiveThe organogermanium compound, Ge-132, may represent a leading candidate for interaction with cis-diols toward establishing a novel type of medicine. In all likelihood, the affinity of Ge-132 with tar-geted cis-diols is too weak for use a strong, effective drug. However, when we see the various physiologi-cal effects of THGPA and its polymers, we estimate that controlling cis-diol compounds with specific interactions has potential as a novel drug target. In the future, we anticipate that novel medicines will modulate receptor binding via complex formation with cis-diols. We expect advances in the study of the biological application of organogermanium com-pounds, standing on the points of relation to cis-diol compounds.
Supplementary data To view the supplementary data that accompany this pa-
per please visit the journal website at: www.future-science.
com/10.4155/FMC.15.62
Financial & competing interests disclosureThe authors, excluding M Akiba and H Fukaya, were em-
ployed by the Asai Germanium Research Institute Co., Ltd.
The authors have no other relevant affiliations or financial
involvement with any organization or entity with a finan-
cial interest in or financial conflict with the subject matter
or materials discussed in the manuscript apart from those
disclosed.
No writing assistance was utilized in the production of
this manuscript.
Executive summary
• The biological cis-diol compounds are very important for life.• Safe organogermanium compound Ge-132, a polymer of 3-(trihydroxygermyl)propanoic acid (THGPA), can
interact with bioactive cis-diol compounds.• The affinities of THGPA and catecholamine are higher than that of THGPA and mono nucleic acids.• The complex crystal of THGPA/catecholamine has pentacoordinate germanium (V) and a lactone ring structure
of the propanoic moiety.• THGPA inhibits cell signaling mediating receptor for cis-diols by ligand trapping at high concentration.• The cell–cell signaling control by the ligand with cis-diol structure trapping may be a novel draggable target.
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Ge-132 forms complexes with physiological cis-diol compounds Research Article
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